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Sample records for immunosorbent assay employing

  1. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    Science.gov (United States)

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W

    2015-12-01

    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  2. Optimising automation of a manual enzyme-linked immunosorbent assay

    OpenAIRE

    Corena de Beer; Monika Esser; Wolfgang Preiser

    2011-01-01

    Objective: Enzyme-linked immunosorbent assays (ELISAs) are widely used to quantify immunoglobulin levels induced by infection or vaccination. Compared to conventional manual assays, automated ELISA systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.Design: The VaccZyme™ Human Anti-Haemophilus influenzae type B (Hib) kit (MK016) from The Binding Site Company was optimised to be used on an automated BioRad...

  3. Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    Manisha Sathe

    2016-09-01

    Full Text Available The effect of spacers and the enzyme-linked immunosorbent assay (ELISA formats on the functional parameters of assays such as lower detection limit, inhibitory concentration at 50 per cent (IC50, and specificity were studied. Enzyme conjugates having hydrophobic and hydrophilic spacers were prepared using O-isopropyl methylphosphonic acid (IMPA and horseradish peroxidase (HRP as an enzyme label. Comparison was made with reference to enzyme conjugate without any spacer. The present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate significantly improves the sensitivity of assays. An enhanced IC50 value achieved was 0.01 μg mL−1 for free antigen detection by direct immunoassay using hydrophilic spacers and precoating of ELISA plates by secondary antibody. The use of a hydrophilic spacer might have helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay.

  4. Immunometric Antibody Sandwich Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Kohl, Thomas O; Ascoli, Carl A

    2017-06-01

    The antibody sandwich enzyme-linked immunosorbent assay (ELISA) is the most commonly used assay for rapid and accurate detection of antigens. It displays greater sensitivity compared with the indirect ELISA and can be used to determine absolute antigen concentrations in unknown samples provided purified antigen standards are available, although it requires the use of two different antibodies. Briefly, wells are coated with antigen-specific capture antibody then incubated with samples containing unknown antigen. Washing removes unbound antigen and exogenous sample protein before incubation with a second antigen-specific detection antibody, washing, and reincubation with a reporter-labeled tertiary antibody. After tertiary antibody is washed off, substrate is added and hydrolysis is measured spectrophotometrically. The signal intensity is directly proportional to the concentration of the antigen in the test sample. © 2017 Cold Spring Harbor Laboratory Press.

  5. Optimising automation of a manual enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Corena de Beer

    2011-12-01

    Full Text Available Objective: Enzyme-linked immunosorbent assays (ELISAs are widely used to quantify immunoglobulin levels induced by infection or vaccination. Compared to conventional manual assays, automated ELISA systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.Design: The VaccZyme™ Human Anti-Haemophilus influenzae type B (Hib kit (MK016 from The Binding Site Company was optimised to be used on an automated BioRad PhD™ system in the Immunology Laboratory (National Health Laboratory Service in Tygerberg, South Africa.Methods: An automated ELISA system that uses individual well incubation was compared to a manual method that uses whole-plate incubation.Results: Results were calculated from calibration curves constructed with each assay. Marked differences in calibration curves were observed for the two methods. The automated method produced lower-than-recommended optical density values and resulted in invalid calibration curves and diagnostic results. A comparison of the individual steps of the two methods showed a difference of 10 minutes per incubation cycle. All incubation steps of the automated method were subsequently increased from 30 minutes to 40 minutes. Several comparative assays were performed according to the amended protocol and all calibration curves obtained were valid. Calibrators and controls were also included as samples in different positions and orders on the plate and all results were valid.Conclusion: Proper validation is vital before converting manual ELISA assays to automated or semi-automated methods. 

  6. Immunometric Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Kohl, Thomas O; Ascoli, Carl A

    2017-06-01

    The double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is preferentially used to determine the concentration of unknown antibody in a sample. Pure antigen is not required in this assay; however, the use of a reporter-labeled detection antibody is essential. The double-antibody sandwich ELISA is suitable for epitope mapping of different monoclonal antibodies that have been generated against a single antigen. First, plates are coated with a capture antibody specific for immunoglobulins generated by immunization of a host species. Next, the test antibody solution (e.g., serum) is incubated with the capture antibody to facilitate binding. The plates are washed to remove unbound antibody, and then antigen is added. The plates are washed again followed by the addition of an antigen-specific reporter-labeled antibody. Following incubation, unbound reporter antibody is washed off, and reporter-specific substrate is added. Reporter-mediated substrate hydrolysis is visualized and measured. The signal is proportional to the number of test antibodies present in the serum. © 2017 Cold Spring Harbor Laboratory Press.

  7. Establishment of an Enzyme-linked Immunosorbent Assay for Thyroglobulin Antibody

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Thyroglobulin is coated on the microtiter plate and labeled with horseradish peroxidase(HRP). Thetwo-step assay is established based on enzyme-linked immunosorbent assay(ELISA). TMB-H2O2 solution

  8. Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.

    Science.gov (United States)

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D; Thinon, Emmanuelle; Rodgers, Ursula R; Owens, Raymond J; Magee, Anthony I; Tate, Edward W

    2015-12-01

    Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

  9. Evaluation of an enzyme-linked immunosorbent assay for determination of porcine haptoglobin

    DEFF Research Database (Denmark)

    Petersen, H. H.; Nielsen, J. P.; Jensen, A.L.

    2001-01-01

    An enzyme-linked immunosorbent assay for quantification of haptoglobin in porcine serum was evaluated. Tbe detection limit when expressed as the estimated concentration of a blank sample was 0.0003 mg/ml. The precision of the assay was acceptable with intra-assay coefficients of variation below 4...

  10. Heterologous enzyme linked immunosorbent assay for measurement of testosterone in serum.

    Science.gov (United States)

    Shrivastav, Tulsidas G; Chaube, Shail K; Prasad, Pramod K V; Kariya, Kiran P; Kumar, Dinesh

    2012-01-01

    In steroid enzyme immunoassay (EIA), homologous and heterologous combinations of enzyme conjugate with immunogen influences labeled steroid recognition by antibodies that affect sensitivity of the assay. To develop testosterone enzyme linked immunosorbent assay (ELISA), antibodies were generated against testosterone-3-carboxymethyloxime-bovine serum albumin (T-3-CMO-BSA), testosterone-11-hemisuccinate-bovine serum albumin (T-11-HS-BSA), testosterone-17-hemisuccinate-bovine serum albumin (T-17-HS-BSA), testosterone-17-glucuronide-bovine serum albumin (T-17-G-BSA), and testosterone-19-carboxymethylether-bovine serum albumin (T-19-CME-BSA). Testosterone horseradish peroxidase (HRP) enzyme conjugate were prepared using carboxyl derivatives of 11-keto-testosterone (11-keto-T) and 1-dehydrotestosterone (1-Dehydro-T). Ten combinations of heterologous assays were evaluated. The data of the present study revealed that the use of the T-11-HS-BSA antibody in antigen plus site heterologous assay that employed 11-keto-testosterone-3-CMO-HRP as the label showed binding and displacement, and led to the development of sensitive and specific assay.

  11. Establishment of an Enzyme Linked Immunosorbent Assay for Neonatal Thyrotropin

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A sensitive and specific ELISA for neonatal thyrotropin(Neonatal TSH) is established with using twoanti-TSH monoclonal antibody. One of them is coated on the microtiter plate, the other is conjugate ofbiotin. The label is horseradish peroxidase(HRP) conjugate of streptavidin. TMB-H2O2 solution is used asthe substrate of HRP.The sensitivity of the assay is 0.5 mIU/L, the intra-assay CVs and the intre-assay

  12. Standardization of the dot enzyme-lynked immunosorbent assay (dot-ELISA for experimental plague

    Directory of Open Access Journals (Sweden)

    Silvia M. L. Montenegro

    1993-03-01

    Full Text Available A dot enzyme linked immunosorbent assay (dot-ELISA was previously developed to detect specific antibodies in rabbits sera immunized against FIA protein obtained from Yersina pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate. Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of FIA protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate.

  13. Validated enzyme-linked immunosorbent assay for determination of rosuvastatin in plasma at picogram level.

    Science.gov (United States)

    Darwish, Ibrahim A; Al-Obaid, Abdul-Rahman M; Al-Malaq, Hamoud A

    2013-05-01

    In this study, a highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed and validated for the determination of rosuvastatin (ROS) in plasma samples at picogram level. The assay employed a polyclonal antibody that specifically recognizes ROS with high affinity, and ROS conjugate of bovine serum albumin (ROS-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labelled second anti-rabbit IgG antibody (HRP-IgG) and 3,3`,5,5`-tetramethylbenzidine (TMB) as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the colour intensity in the assay wells. The assay limit of detection was 25 pg ml(-1) and the effective working range at relative standard deviations (RSD) of ≤ 5% was 40-2000 pg ml(-1). Analytical recovery of ROS from spiked plasma was 96.2 - 104.8 ± 2.12 - 5.42%. The precision of the assay was satisfactory; RSD was 2.47 - 4.46 and 3.24 - 5.27% for the intra- and inter-assay precision, respectively. The analytical procedure is convenient, and one can analyze ~ 200 samples per working day, facilitating the processing of large-number batch of samples. The proposed ELISA has a great value in routine analysis of ROS for its pharmacokinetic studies.

  14. Optimising automation of a manual enzyme-linked immunosorbent assay

    National Research Council Canada - National Science Library

    Corena de Beer; Monika Esser; Wolfgang Preiser

    2011-01-01

    .... Compared to conventional manual assays, automated ELISA systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.Design: The VaccZyme...

  15. Comparison of immunofluorescence and enzyme-linked immunosorbent assays for the serology of hantavirus infections.

    NARCIS (Netherlands)

    J. Groen (Jan); G. van der Groen (Guido); G. Hoofd; A.D.M.E. Osterhaus (Albert)

    1989-01-01

    textabstractThree enzyme-linked immunosorbent assay (ELISA) systems based upon different principles were developed for the serology of Hantaan virus infections and compared with an indirect immunofluorescence assay (IFA). The indirect IFA was carried out with gamma-irradiated Hantaan virus-infected

  16. Identification of the species origin of fresh meat using an enzyme-linked immunosorbent assay procedure.

    Science.gov (United States)

    Kang'ethe, E K; Jones, S J; Patterson, R L

    1982-11-01

    A modification of indirect enzyme-linked immunosorbent assay (ELISA) has been successfully applied to the detection of horse meat and beef. This technically simple assay requiring species specific antibody, conjugated enzyme anti-IgG and a polystyrene protein-binding solid phase, can be adapted for the identification of meat species in circumstances where laboratory facilities are minimal.

  17. Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces.

    OpenAIRE

    Eriksson, P V; di Paola, G N; Pasetti, M F; Manghi, M A

    1995-01-01

    An inhibition enzyme-linked immunosorbent assay was developed for Pseudomonas fluorescens enumeration of meat surfaces. The assay detected contamination levels as low as 3 x 10(5) bacteria per ml and could be completed within 4 h. It could be used as a framework for a test system for quantifying P. fluorescens spoilage in meat products.

  18. Indirect solid-phase immunosorbent assay for detection of arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A. (Institute of Poliomyelitis anU Viral Encephalities of the U.S.S.R. Academy of Medical Sciences, Moscow)

    1984-05-01

    Indirect enzyme-linked immunosorbent assay (ELISA) and solid phase radioimmunoassay (SPRIA) using either enti-human or anti-mouse IgG labelled with horseradish peroxidase and /sup 125/I, respectively, were developed for the detection of Junin, Machupo, Tacaribe, Amapari, Tamiami, Lassa and LCM arenaviruses. Both methods allow high sensitivity detection of arenavirus antigens and antibodies.

  19. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    Science.gov (United States)

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  20. Enzyme-linked immunosorbent assay antibody responses to a temperature-sensitive mutant of Pseudomonas aeruginosa.

    OpenAIRE

    Sordelli, D. O.; Rojas, R A; Cerquetti, M C; Hooke, A M; Degnan, P J; Bellanti, J A

    1985-01-01

    The serum immunoglobulin G and M responses induced by immunization of mice with temperature-sensitive mutant A/10/25 of Pseudomonas aeruginosa were evaluated by enzyme-linked immunosorbent assay. These antibody responses were immunotype specific, and the immunoglobulin G antibody level, although low, was still significant by day 52 after vaccination.

  1. Application of an Improved Enzyme-Linked Immunosorbent Assay Method for Serological Diagnosis of Canine Leishmaniasis

    NARCIS (Netherlands)

    N. Santarem; R. Silvestre; L. Cardoso; H. Schallig; S.G. Reed; A. Cordeiro-da-Silva

    2010-01-01

    Accurate diagnosis of canine leishmaniasis (CanL) is essential toward a more efficient control of this zoonosis, but it remains problematic due to the high incidence of asymptomatic infections. In this study, we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based techn

  2. The use of subcutaneous fat tissue for amyloid typing by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Olsen, K E; Sletten, K; Westermark, Per

    1999-01-01

    for typing the most common systemic amyloidoses of AL, AA, and transthyretin types by enzyme-linked immunosorbent assay (ELISA), using abdominal wall subcutaneous fat biopsy specimens. The method was tested on 21 abdominal fat biopsy specimens that were sent to the laboratory. Of these, 15 contained amyloid...

  3. An enzyme-linked immunosorbent assay for autoantibodies against the nuclear protein Scl-70

    DEFF Research Database (Denmark)

    Geisler, C; Høier-Madsen, M

    1985-01-01

    This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established...

  4. Comparison of five commercial anti-tetanus toxoid immunoglobulin G enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Perry, A L; Hayes, A J; Cox, H A; Alcock, F; Parker, A R

    2009-12-01

    Five commercially available enzyme-linked immunosorbent assays for the measurement of anti-tetanus toxoid immunoglobulin G (IgG) antibodies were evaluated for performance. The data suggest that there are manufacturer-dependent differences in sensitivity and accuracy for the determination of tetanus toxoid IgG antibodies that could result in different diagnostic interpretations.

  5. Predicting detection limits of enzyme-linked immunosorbent assay (ELISA) and bioanalytical techniques in general.

    Science.gov (United States)

    Zhang, Shiyun; Garcia-D'Angeli, Alexa; Brennan, Joseph P; Huo, Qun

    2014-01-21

    The detection limit is one of the most important performance parameters for bioanalytical techniques. Here we present a generic method to estimate the detection limit of biomolecular assays based on a step-by-step analysis of the assay procedure. Enzyme-linked immunosorbent assay (ELISA) is used here as an example; however, much of the information presented in this article may be applied to other types of biomolecular assays and analytical techniques. A clear understanding of what affects the detection limit can help researchers to evaluate different bio-analytical techniques properly, and to design better strategies to optimize and achieve the best analytical performance.

  6. Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies.

    Science.gov (United States)

    Suzuki, Miho; Udaka, Hikari; Fukuda, Takeshi

    2017-09-05

    An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Paper-based colorimetric enzyme linked immunosorbent assay fabricated by laser induced forward transfer.

    Science.gov (United States)

    Katis, Ioannis N; Holloway, Judith A; Madsen, Jens; Faust, Saul N; Garbis, Spiros D; Smith, Peter J S; Voegeli, David; Bader, Dan L; Eason, Robert W; Sones, Collin L

    2014-05-01

    We report the Laser Induced Forward Transfer (LIFT) of antibodies from a liquid donor film onto paper receivers for application as point-of-care diagnostic sensors. To minimise the loss of functionality of the active biomolecules during transfer, a dynamic release layer was employed to shield the biomaterial from direct exposure to the pulsed laser source. Cellulose paper was chosen as the ideal receiver because of its inherent bio-compatibility, liquid transport properties, wide availability and low cost, all of which make it an efficient and suitable platform for point-of-care diagnostic sensors. Both enzyme-tagged and untagged IgG antibodies were LIFT-printed and their functionality was confirmed via a colorimetric enzyme-linked immunosorbent assay. Localisation of the printed antibodies was exhibited, which can allow the creation of complex 2-d patterns such as QR codes or letters for use in a final working device. Finally, a calibration curve was determined that related the intensity of the colour obtained to the concentration of active antibodies to enable quantitative assessment of the device performance. The motivation for this work was to implement a laser-based procedure for manufacturing low-cost, point-of-care diagnostic devices on paper.

  8. Development of a sensitive and specific enzyme-linked immunosorbent assay for the determination of fludioxonil residues in fruit juices

    OpenAIRE

    Mercader Badia, Josep Vicent; Abad Fuentes, Antonio; Agulló, Consuelo; Abad Somovilla, Antonio; Esteve Turrillas, Francesc Albert

    2014-01-01

    Fludioxonil is a fungicide with a singular mode of action that is widely employed in the treatment of fruit crops. A competitive enzyme-linked immunosorbent assay (cELISA) has been developed and validated for the determination of residues of this fungicide in fruit juices. A collection of monoclonal antibodies (mAbs) against the fungicide fludioxonil has been produced and characterized using direct and indirect cELISAs. The high affinity achieved (IC50lower than 0.5 μg L-1) considerably impro...

  9. THE SPOROZOITE ENZYME-LINKED IMMUNOSORBENT ASSAY : APPLICATION IN MALARIA EPIDEMIOLOGY

    Directory of Open Access Journals (Sweden)

    Michael J. Bangs

    2012-09-01

    Full Text Available Recent biotechnological breakthroughs have led to the development of various methods for detection and identification of human pathogens in their vectors. Monoclonal antibodies produced against malaria sporozoite antigens have permitted the development of several sensitive, species specific immunological tests (IFA, IRMA, ELIS A. One of these, a two-site enzyme-linked immunosorbent assay (ELIS A has been developed as a useful epidemiological tool in the identification of malaria-infected mosquitoes. This method employs highly species specific monoclonal antibodies that recognize the repetitive immunodominant epitope of the circumsporozoite (CS protein. Monoclonal antibodies have been developed for all four species of human malaria The key feature of the ELISA technique is the use of an enzyme indicator for an immunological reaction. The antigen capture or "sandwich" ELISA configuration uses the purified monoclonal both as the solid phase and, conjugated to enzyme, as a marker for the presence of CS protein in a mosquito homogenate incubated in the wells of a microtitration plate. This technology has shown advantages over other methods for epidemiological data collection. Mosquitoes can be caught, dried and stored until a time convenient for examination. The sporozoite rate by Plasmodium species can be identified easily, and when combined with the man-biting rate provides the sporozoite inoculation rate, an important entomologic estimate of the number of potential infective bites a person could expect over a given period of time. Presently, mosquitoes can be tested individually or pooled up to 20 anophe lines. The assay is sensitive enough to detect 1 infected mosquito per pool or as few as 25 sporozoites per 50 pi of mosquito extract. Basic principles and procedures are covered concerning solid substrate, adsorption to solid substrate, buffers and wash solutions, conjugates and enzyme substrates. The advantages and limitations of this technique

  10. THE SPOROZOITE ENZYME-LINKED IMMUNOSORBENT ASSAY : APPLICATION IN MALARIA EPIDEMIOLOGY

    Directory of Open Access Journals (Sweden)

    Michael J. Bangs

    2012-09-01

    Full Text Available Recent biotechnological breakthroughs have led to the development of various methods for detection and identification of human pathogens in their vectors. Monoclonal antibodies produced against malaria sporozoite antigens have permitted the development of several sensitive, species specific immunological tests (IFA, IRMA, ELIS A. One of these, a two-site enzyme-linked immunosorbent assay (ELIS A has been developed as a useful epidemiological tool in the identification of malaria-infected mosquitoes. This method employs highly species specific monoclonal antibodies that recognize the repetitive immunodominant epitope of the circumsporozoite (CS protein. Monoclonal antibodies have been developed for all four species of human malaria The key feature of the ELISA technique is the use of an enzyme indicator for an immunological reaction. The antigen capture or "sandwich" ELISA configuration uses the purified monoclonal both as the solid phase and, conjugated to enzyme, as a marker for the presence of CS protein in a mosquito homogenate incubated in the wells of a microtitration plate. This technology has shown advantages over other methods for epidemiological data collection. Mosquitoes can be caught, dried and stored until a time convenient for examination. The sporozoite rate by Plasmodium species can be identified easily, and when combined with the man-biting rate provides the sporozoite inoculation rate, an important entomologic estimate of the number of potential infective bites a person could expect over a given period of time. Presently, mosquitoes can be tested individually or pooled up to 20 anophe lines. The assay is sensitive enough to detect 1 infected mosquito per pool or as few as 25 sporozoites per 50 pi of mosquito extract. Basic principles and procedures are covered concerning solid substrate, adsorption to solid substrate, buffers and wash solutions, conjugates and enzyme substrates. The advantages and limitations of this technique

  11. Diagnosis of internal acariasis with avidin-biotin system enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    Rong-Bo Zhang; Yong Huang; Chao-Pin Li; Yu-Bao Cui

    2004-01-01

    AIM: To explore the value of avidin-biotin system enzymelinked immunosorbent assay (ABC-ELISA) in diagnosis of intestinal acariasis.METHODS: Mite-specific IgG levels in serum of 48 patients with intestinal acariasis were measured with ABC-ELISA.The sensitivity of this method was compared with that of staphylococcal protein A enzyme-linked immunosorbent assay (SPA-ELISA).RESULTS: The positive rate of mite-specific IgG detected with ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48), respectively. The positive rate with ABCELISA was statistically higher than that with SPA-ELISA (X2=13.50, P<0.01).CONCLUSION: ABC-ELISA is an effective method for the diagnosis of intestinal acariasis.

  12. Evaluation of a Commercial Glycoprotein Enzyme-Linked Immunosorbent Assay for Measuring Vaccine Immunity to Varicella

    OpenAIRE

    Kim, Yun Hwa; Hwang, Ji Young; Shim, Hye Min; Lee, Eunsil; Park, Songyong; Park, Hosun

    2014-01-01

    Purpose To evaluate a recently marketed commercial glycoprotein enzyme-linked immunosorbent assay (gpEIA) kit, the VaccZyme™ VZV gpEIA, for measuring the immunity of varicella-vaccinated children. Materials and Methods We investigated the accuracy and reproducibility of the VaccZyme™ VZV gpEIA kit for the detection of antibodies to VZV. We also examined the sensitivity, specificity, and correlation between antibody titers calculated with gpEIA versus fluorescent antibody to membrane antigen (...

  13. Aspergillus Galactomannan Enzyme-Linked Immunosorbent Assay Cross-Reactivity Caused by Invasive Geotrichum capitatum

    Science.gov (United States)

    Giacchino, Mareva; Chiapello, Nadia; Bezzio, Stefania; Fagioli, Franca; Saracco, Paola; Alfarano, Alda; Martini, Vincenza; Cimino, Giuseppe; Martino, Pietro; Girmenia, Corrado

    2006-01-01

    We report three cases of invasive Geotrichum capitatum infection in patients with acute leukemia for which an enzyme-linked immunosorbent assay (ELISA) for Aspergillus galactomannan was positive, with no evidence of aspergillosis. Supernatants obtained from suspensions of 17 G. capitatum strains gave positive reactions with the Aspergillus galactomannan ELISA. These clinical and laboratory data seem to suggest that G. capitatum produces a soluble antigen that is cross-reactive with Aspergillus galactomannan. PMID:16954294

  14. Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Dziegiel, M; Borre, Mette; Jepsen, S

    1991-01-01

    A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P...... of the immunogenicity of a possible future P. falciparum vaccine utilizing epitopes from GLURP....

  15. The Establishment of an Enzyme-linked Immunosorbent Assay for Carcinoembryonic Antigen

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two anti-CEA monoclonal antibodies are used, one is coated on the microtiter plate, the other is labeled with horseradish peroxidase(HRP). The two-step assay is established based on enzyme-linked immunosorbent assay(ELISA). TMB-H2O2 solution is used as the substrate of HRP. The sensitivity of the assay is 0.4 μ g/L. The intra-assay CVs and the inter-assay CVs are lower than 10.0% and 15.0%, respectively. The analytical recoveries are ranged from 99.4% to 108.7%. The reference cut-off value of normal serum (n= 100 ) is 10.0 ng/L.

  16. Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays

    DEFF Research Database (Denmark)

    Holm, B. E.; Bergmann, Ann Christina; Hansen, Paul Robert

    2015-01-01

    In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme......-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies...... to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially...

  17. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ploug, M;

    1994-01-01

    variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer......Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal...... tissue. Recent studies have shown that a high uPA level in tumor extracts is in some cancers associated with poor prognosis. The present assay will now allow similar prognostic studies of uPAR levels....

  18. Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot (ELISPOT) assay.

    Science.gov (United States)

    Wang, Wei; Cheng, Tong; Ma, Ke; Xia, Dezhen; Wang, Yongmei; Liu, Jian; Du, Hailian; Shih, James Wai Kuo; Zhang, Jun; Zhao, Qinjian; Xia, Ningshao

    2013-03-01

    The baculovirus expression vector system (BEVS) is one of the most powerful methods for production of recombinant proteins for research or commercial purposes. Titration of viable virus in insect cell culture is often required when BEVS is used for basic research or bioprocessing. An enzyme-linked immunosorbent spot (ELISPOT) assay using monoclonal antibodies against the major capsid protein VP39 of both Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) was developed for baculovirus quantitation at 48h post-infection. The titer was determined by visualizing infected insect cells as blue spots and automated spot counting was achieved with ELISPOT hardware and software. Log-scale comparison of the results between the ELISPOT assay and a conventional end point dilution assay using a fluorescent marker showed a good correlation for both AcMNPV (R(2)=0.9980, pspot counting.

  19. Evaluation of a novel enzyme-linked immunosorbent assay for serological diagnosis of porcine proliferative enteropathy

    DEFF Research Database (Denmark)

    Boesen, Henriette Toft; Jensen, Tim Kåre; Møller, Kristian;

    2005-01-01

    A specific enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the porcine pathogen Lawsonia intracellularis was developed and evaluated using sera from naive, naturally infected as well as experimentally infected pigs. On the basis of 37 serum samples collected from experime...... was a specific and sensitive method for detecting specific antibodies, and may be a good alternative to the existing serological tests for L intracellularis. It may be usable for diagnosis of proliferative enteropathy and for determination of a herd's epidemiologic status....

  20. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay.

    OpenAIRE

    Castillo, R M; Grados, P; Carcamo, C.; Miranda, E; T Montenegro; Guevara, A.; Gilman, R H

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum immunoglobulin G antibodies in 65 patients infected with Hymenolepis nana and 30 noninfected patients. Antibody was detected in 51 of 65 (sensitivity, 79%) and 5 of 30 H. nana-negative patients (specificity, 83%). Nine patients infected with H. nana were treated with praziquantel (20 to 25 mg/kg of body weight). Antibody disappeared from the sera at 90 days in six patients, five of whom had eliminated H. nana. Antibod...

  1. The enzyme immunosorbent assay (ELISA) for serologic diagnosis of toxoplasmosis in pregnancy.

    Science.gov (United States)

    Luerti, M; Corticelli, C; Santini, A; Privitera, G; Nardi, G

    1984-01-01

    The enzyme-linked immunosorbent assay (ELISA) is simple, quick, and inexpensive, characteristics that make it particularly suitable for large screening programs. We compared the results obtained by ELISA with those obtained by immunofluorescent antibody test (IFAT), indirect hemoagglutination test (IHAT), and Sabin Feldman Dye test in a group of 105 pregnant women in the course of a screening for toxoplasmosis. Prevalence of IgG antibody titers were not significantly different. ELISA results were particularly close to those of the reference tests (IFAT and Dye test).

  2. Immunosorbent assay using gold colloid cluster technology for determination of IgEs in patients’ sera

    Directory of Open Access Journals (Sweden)

    Haifa Al-Dubai

    2010-10-01

    Full Text Available Haifa Al-Dubai1, Irene Lichtscheidl2, Martina Strobl1, Gisela Pittner1, Fritz Pittner11Department of Biochemistry, Max F Perutz Laboratories, University of Vienna, Vienna, Austria; 2Institute of Cell Imaging and Ultrastructure Research, Vienna, AustriaAbstract: This study focuses on the development of a sensitive and simple cluster-linked immunosorbent assay (CLISA using gold colloidal cluster labeling for determination of proteins such as antigens (Ags or antibodies (Abs. Abs for detection can be labeled with gold colloid clusters (GCCs. The Fc domain of the Abs binds to the clusters, and the Fab domain to the Ag on a nitrocellulose membrane or a microtiter plate as a support for dot-blotting. The signal of positive interaction between GCC-labeled Abs and its dotted Ag is detectable by the naked eye and can be quantified by comparison to a color scale prepared from a dilution series of known sample concentrations. The colored reaction product is stable for prolonged periods and does not fade, making this method a simple, fast, and convenient means for detection of Ag or Ab biorecognitions and an alternative to enzyme-linked immunosorbent assay. Several interactions between different Ags or Abs (eg, ß-lactoglobulin and solutions avoiding gold colloidal cluster flocculation (eg, using protein G were studied. CLISA was tested for other analytical purposes such as detection of IgEs in patients’ sera.Keywords: ELISA, allergen, patient sera, CLISA, immunoassay, ß-lactoglobulin

  3. Methodology for determination of plasma cortisol in fish using Competitive Enzyme-Linked Immunosorbent Assay (ELISA)

    DEFF Research Database (Denmark)

    Velasco-Santamaría, Yohana M.; Cruz-Casallas, Pablo E.

    2007-01-01

    Objective. To determine plasma cortisol procedure in fish using competitive enzymelinked immunosorbent assay (ELISA). Materials and methods. Two plasma samples of juveniles rainbow trout Oncorhynchus mykiss were analized by using ELISA human kit for cortisol assay. For standard curve calibration...... seven standard solutions of cortisol in human plasma (0, 20, 50, 100, 200, 400 and 800 ng.ml-1) were used. For the recovery test 50, 100 and 200 ng.ml-1 standard solutions of cortisol were used; for the linearity test four dilutions of the fish plasma samples (1/2, 1/4, 1/8 and 1/16) were prepared....... To each well fish plasma samples and standard solutions were added and its content were conjugated to peroxidase and subsequently enzyme substrate was added. The enzymatic reaction was stopped by addition of phosphoric acid 0.5 M and the absorbance was read at 450 nm. The accuracy of the pipetting...

  4. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    Science.gov (United States)

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  5. Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep.

    Science.gov (United States)

    Gurung, R B; Begg, D J; Purdie, A C; Eamens, G J; Whittington, R J

    2015-12-01

    An enzyme-linked immunosorbent assay (ELISA) was developed and optimised using a Mycobacterium avium subspecies paratuberculosis (MAP) antigen prepared from a C strain (316v) passed through a French press. The optimised assay was evaluated with a panel of sera from MAP infected (n = 66) and uninfected (n = 1,092) sheep. Animals in the MAP infected category were positive on either tissue culture or histopathology but were of unknown serum antibody status. The diagnostic performance and cost of the assay were compared with those of a commercial ELISA (IDEXX). At 99.8% diagnostic specificity the assay showed a diagnostic sensitivity of 23% (95% CI: 15.1-35.8) compared with 36.4% (95% CI: 25.8-48.4) for the commercial ELISA (McNemar's test: chi-square 5.82, p ELISA for an equivalent number of tests, the multiple depending on the number of plates processed per run. For flock-level surveillance, to account for the lower sensitivity of the 316v ELISA compared with the commercial ELISA, sample sizes would be increased but the test cost would still be lower. The 316v assay will be useful for diagnosis of Johne's disease in sheep flocks, particularly in developing countries where labour costs are low relative to the cost of consumables.

  6. A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins.

    Science.gov (United States)

    Geumann, Constanze; Grønborg, Mads; Hellwig, Michaela; Martens, Henrik; Jahn, Reinhard

    2010-07-15

    Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78ng/ml to 77pg/ml (or 74-0.1fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.

  7. Markers of human immunodeficiency virus infection in high-risk individuals seronegative by first generation enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Pedersen, C; Lindhardt, B O; Lauritzen, E;

    1989-01-01

    A total of 228 stored serum samples from 140 high risk individuals was examined for serological markers of human immunodeficiency virus (HIV) infection by second generation enzyme-linked immunosorbent assay, immunoblot, and HIV antigen assay. All the samples were negative in first generation enzyme...

  8. Identification of Hantavirus serotypes by testing of post-infection sera in immunofluorescence and enzyme-linked immunosorbent assays.

    NARCIS (Netherlands)

    J. Groen (Jan); H.G.M. Jordans; J.P.G. Clement; E.J.M. Rooijakkers; F.G.C.M. Uytdehaag (Fons); J.M. Dalrymple; G. van der Groen (Guido); A.D.M.E. Osterhaus (Albert)

    1991-01-01

    textabstractSerum samples were collected from 27 individuals who had been infected with a member of the genus Hantavirus in the Netherlands or Belgium during the last 15 years. These samples were tested in an immunofluorescence assay (IFA) and two enzyme-linked immunosorbent assay (ELISA) systems, u

  9. Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Toh, Saw Yi; Citartan, Marimuthu; Gopinath, Subash C B; Tang, Thean-Hock

    2015-02-15

    The application of antibodies in enzyme-linked immunosorbent assay (ELISA) is the basis of this diagnostic technique which is designed to detect a potpourri of complex target molecules such as cell surface antigens, allergens, and food contaminants. However, development of the systematic evolution of Ligands by Exponential Enrichment (SELEX) method, which can generate a nucleic acid-based probe (aptamer) that possess numerous advantages compared to antibodies, offers the possibility of using aptamers as an alternative molecular recognition element in ELISA. Compared to antibodies, aptamers are smaller in size, can be easily modified, are cheaper to produce, and can be generated against a wide array of target molecules. The application of aptamers in ELISA gives rise to an ELISA-derived assay called enzyme-linked apta-sorbent assay (ELASA). As with the ELISA method, ELASA can be used in several different configurations, including direct, indirect, and sandwich assays. This review provides an overview of the strategies involved in aptamer-based ELASA.

  10. Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods

    Directory of Open Access Journals (Sweden)

    Stef J. Koppelman

    2015-01-01

    Full Text Available Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested. The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame.

  11. Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods.

    Science.gov (United States)

    Koppelman, Stef J; Söylemez, Gülsen; Niemann, Lynn; Gaskin, Ferdelie E; Baumert, Joseph L; Taylor, Steve L

    2015-01-01

    Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested). The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame.

  12. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan

    2011-01-01

    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

  13. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Castillo, R M; Grados, P; Carcamo, C; Miranda, E; Montenegro, T; Guevara, A; Gilman, R H

    1991-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum immunoglobulin G antibodies in 65 patients infected with Hymenolepis nana and 30 noninfected patients. Antibody was detected in 51 of 65 (sensitivity, 79%) and 5 of 30 H. nana-negative patients (specificity, 83%). Nine patients infected with H. nana were treated with praziquantel (20 to 25 mg/kg of body weight). Antibody disappeared from the sera at 90 days in six patients, five of whom had eliminated H. nana. Antibody persisted in three patients in whom H. nana infection did not clear after treatment. The H. nana ELISA had a high rate of cross-reactions with sera from patients with cysticercosis (8 of 29 [28%]) and hydatidosis (8 of 23 [35%]). The ELISA for H. nana may be useful for defining the epidemiology of H. nana infections, especially in areas free from cysticercosis and hydatidosis. PMID:2007652

  14. Control panels of meat juice samples for a Salmonella enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Bak, H.; Sørensen, Vibeke

    2006-01-01

    In the Danish pig production system, an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies in meat juice is used for Salmonella surveillance. Quality control (QC) of this ELISA was previously based on repeated testing of control serum samples. The purpose of the study...... reported here was to collect, characterize, and implement a panel of meat juice pools for supplemental internal QC. Muscle samples for extraction of meat juice were collected from slaughter pigs of 5 herds infected with Salmonella spp. and from 4 herds without Salmonella infection. A QC panel with 39 pools...... of meat juice, yielding ELISA optical density (OD) values covering the full range of expected OD values, was prepared and tested repeatedly to determine mean and SD OD values. Each pool was tested twice on each microtitration plate, and the results were used to determine limits for validity of future...

  15. An enzyme-linked immunosorbent assay (ELISA) for methylphenidate (Ritalin ) in urine.

    Science.gov (United States)

    Lewis, Mark G; Lewis, John G; Elder, Peter A; Moore, Grant A

    2003-09-01

    A direct enyzme-linked immunosorbent assay (ELISA) for urinary immunoreactive methylphenidate (Ritalin), in which a standard 96-well microtiter plate is used, is described. For this ELISA, a methylphenidate-thyroglobulin conjugate is immobilized to the microtiter plate and competes with methylphenidate in the standard or urine sample for antibody-binding sites. After washing, the sheep methylphenidate antibody bound to immobilized methylphenidate is detected with peroxidase-labelled goat antisheep IgG. Following a further wash, tetramethylbenzidine is added, color is developed, and the plate is read at 450 nm on an ELISA plate reader. This method is unaffected by drugs of abuse and is suitable for routine use in the toxicology laboratory.

  16. Detection of TGEV Antibody by Enzyme-Linked Immunosorbent Assay Using Recombinant Nucleocapsid Proteins

    Institute of Scientific and Technical Information of China (English)

    YU Li-yun; HOU Xi-lin

    2005-01-01

    An enzyme linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichia coli was evaluated for its sensitivity and specificity for diagnosis of transmissible gastroenteritis virus (TGEV) infection.The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. The recombinant N protein migrated at 42 kDa and reacted with His6 tag specific monoclonal antibody by immunoblotting.Recombinant N protein ELISA (rnELISA) demonstrated 97.5% specificity among 80 TGEV-free individuals, and 97.3%sensitivity ranging among 110 clinical samples with TGEV. Taken together, these results indicated that nucleocapsid may be a useful antigen for the sera-diagnosis of TGEV and it was also suggested that the ELISA is a highly sensitive and specific test for detecting antibodies against TGEV.

  17. Effect of treatment on serum antibody to Hymenolepis nana detected by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Castillo, R M; Grados, P; Carcamo, C; Miranda, E; Montenegro, T; Guevara, A; Gilman, R H

    1991-02-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum immunoglobulin G antibodies in 65 patients infected with Hymenolepis nana and 30 noninfected patients. Antibody was detected in 51 of 65 (sensitivity, 79%) and 5 of 30 H. nana-negative patients (specificity, 83%). Nine patients infected with H. nana were treated with praziquantel (20 to 25 mg/kg of body weight). Antibody disappeared from the sera at 90 days in six patients, five of whom had eliminated H. nana. Antibody persisted in three patients in whom H. nana infection did not clear after treatment. The H. nana ELISA had a high rate of cross-reactions with sera from patients with cysticercosis (8 of 29 [28%]) and hydatidosis (8 of 23 [35%]). The ELISA for H. nana may be useful for defining the epidemiology of H. nana infections, especially in areas free from cysticercosis and hydatidosis.

  18. Detection of Ricin Contamination in Ground Beef by Electrochemiluminescence Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    David L. Brandon

    2011-04-01

    Full Text Available Ricin is a highly toxic protein present in the seeds of Ricinus communis (castor, grown principally as a source of high quality industrial lubricant and as an ornamental. Because ricin has been used for intentional poisoning in the past and could be used to contaminate food, there is a need for analytical methodology to detect ricin in food matrices. A monoclonal antibody-based method was developed for detecting and quantifying ricin in ground beef, a complex, fatty matrix. The limit of detection was 0.5 ng/g for the electrochemiluminescence (ECL method and 1.5 ng/g for enzyme-linked immunosorbent assay (ELISA. The detection of nanogram per gram quantities of ricin spiked into retail samples of ground beef provides approximately 10,000-fold greater sensitivity than required to detect a toxic dose of ricin (>1 mg in a 100 g sample.

  19. Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Wolf, Johannes; Lachmann, Ingolf; Wagner, Uta; Osman, Awad A; Mothes, Thomas

    2011-12-15

    Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.

  20. [Development of a specific and sensitive enzyme-linked immunosorbent assay for vindesine].

    Science.gov (United States)

    Nakano, Yukitaka; Saita, Tetsuya; Fujito, Hiroshi

    2012-01-01

    This paper reports a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of vindesine (VDS). Anti-VDS antibody was obtained by immunizing rabbits with VDS conjugated with bovine serum albumin using N-[β-(4-diazophenyl) ethyl] maleimide as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling VDS with horseradish peroxidase using N-(4-diazophenyl) maleimide. The detection limit of VDS by ELISA was approximately 24 pg/mL with 50-mL samples. This assay was specific for VDS and showed very slight cross-reactivity with other vinca alkaloids, vincristine (0.18%) and vinblastine (0.11%). The values for the VDS concentrations detected using this assay were comparable with those detected using HPLC. There was a good correlation between the values determined by the two methods. Moreover, ELISA was about 50-fold more sensitive in detecting VDS at lower concentrations. The sensitivity and specificity of ELISA should provide a useful tool for pharmacokinetic studies of VDS.

  1. Sensitive measurement of thrombopoietin by a monoclonal antibody based sandwich enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Folman, C C; von dem Borne, A E; Rensink, I H; Gerritsen, W; van der Schoot, C E; de Haas, M; Aarden, L

    1997-10-01

    In this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs). The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 +/- 0.8 A.U./ml, mean +/- sd) was lower than the concentration found in controls (11 +/- 8 A.U./ml, mean +/- sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma. We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels. In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

  2. Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

    1986-02-15

    A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D/sub 3/ and should be useful for the detection of antibodies to ligand-binding proteins in general.

  3. Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system.

    Science.gov (United States)

    Moorthy, Jaisree; Mensing, Glennys A; Kim, Dongshin; Mohanty, Swomitra; Eddington, David T; Tepp, William H; Johnson, Eric A; Beebe, David J

    2004-06-01

    A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.

  4. Determination of marbofloxacin residues in beef and pork with an enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sheng, Wei; Xia, Xunfeng; Wei, Keyi; Li, Ji; Li, Qing X; Xu, Ting

    2009-07-01

    Marbofloxacin is a fluoroquinolone veterinary antibiotic. An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of marbofloxacin using polyclonal antibody. The half-maximum inhibition concentrations (IC(50)) and limit of detection (LOD, calculated as IC(20)) of the ELISA for marbofloxacin in phosphate buffer were 4.6 and 0.6 ng/mL, respectively. The assay showed little cross-reactivity with marbofloxacin structural analogues, except for ofloxacin (148%). Matrixes from the extracts of beef and pork muscle have shown a significant influence on the ELISA. Standard curves of ELISA for marbofloxacin in the extracts of the appropriate marbofloxacin-free control muscles were used in the analysis of marbofloxacin in the animal muscles without any cleanup. The average recoveries of intra- and interassay for marbofloxacin from fortified muscle samples, at five concentrations of 10, 50, 100, 500, and 1000 ng/g, were 87-93 and 84-95%, respectively. The LOD of this assay for marbofloxacin in real muscle extracts was 0.8 ng/mL. A survey of 55 animal muscle samples purchased from local markets by the ELISA was conducted, and marbofloxacin was detected in one of them at a concentration of 22 ng/g. This positive sample was validated by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to be 28 ng/g of marbofloxacin.

  5. Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

    NARCIS (Netherlands)

    Groen, J; Van Dijk, G; Niesters, H G; Van Der Meijden, W I; Osterhaus, A D

    The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull

  6. Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA).

    Science.gov (United States)

    Neiser, Susann; Koskenkorva, Taija S; Schwarz, Katrin; Wilhelm, Maria; Burckhardt, Susanna

    2016-07-21

    Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may

  7. Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA

    Directory of Open Access Journals (Sweden)

    Susann Neiser

    2016-07-01

    Full Text Available Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR, the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3 and an enzyme-linked immunosorbent assay (ELISA were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000 does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the

  8. Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

    Science.gov (United States)

    Bian, Zhi-Ping; Li, Xiong-Zhi; Wu, Heng-Fang; Xu, Jin-Dan; Gu, Chun-Rong; Chen, Xiang-Jian; Yang, Di

    2016-04-01

    Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

  9. Enzyme-Linked Immunosorbent Assays for Detection of Equine Antibodies Specific to Sarcocystis neurona Surface Antigens†

    Science.gov (United States)

    Hoane, Jessica S.; Morrow, Jennifer K.; Saville, William J.; Dubey, J. P.; Granstrom, David E.; Howe, Daniel K.

    2005-01-01

    Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM. PMID:16148170

  10. Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

    Science.gov (United States)

    Hoane, Jessica S; Morrow, Jennifer K; Saville, William J; Dubey, J P; Granstrom, David E; Howe, Daniel K

    2005-09-01

    Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

  11. Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage.

    Science.gov (United States)

    Brasino, Michael; Lee, Ju Hun; Cha, Jennifer N

    2015-02-01

    For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented-by either chemical functionalization or genetic engineering-with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.

  12. Performance of a Pneumolysin Enzyme-Linked Immunosorbent Assay for Diagnosis of Pneumococcal Infections▿

    Science.gov (United States)

    del Mar García-Suárez, María; Cima-Cabal, María Dolores; Villaverde, Roberto; Espinosa, Emma; Falguera, Miquel; de Los Toyos, Juan R.; Vázquez, Fernando; Méndez, Francisco J.

    2007-01-01

    A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children. PMID:17728474

  13. Development of enzyme-linked immunosorbent assay for estimation of urinary albumin.

    Science.gov (United States)

    Shrivastav, Tulsidas G; Kariya, Kiran P; Prasad, Pramod K V; Chaube, Shail K; Kumar, Dinesh

    2014-01-01

    Yearly estimation of urinary albumin is a prerequisite for predicting renal status in Diabetes Type II patients with negative dipstick results for overt proteinuria. A simple, sensitive, and cost-effective enzyme linked immunosorbent assay (ELISA) for urinary albumin has been developed using human serum albumin antiserum (HSA-antiserum), HSA-biotin, and streptavidin-horseradish peroxidase (SA-HRP) conjugates. To the antibody-coated wells, 100 μL of HSA standards followed by 1:100 diluted urine samples in duplicate were added and then 50 μL of HSA-biotin conjugates was added in all the wells. 100 μL of SA-HRP was added after washing. Bound enzyme activity was measured by adding 100 μL TMB/H2O2. The analytical sensitivity and ED50 of the developed method was found to be 0.01 μg/mL and 0.35 μg/mL, respectively. The percent recovery of the HSA from exogenously spiked urine pools were in the range of 98.13-100.29%. The intra- and inter-assay coefficient of variation (CVs) ranged from 3.38-10.32 % and 4.22-11.01%, respectively. The antibody showed 4.4% and 3.2% cross reactivity with monkey and horse serum albumin, respectively. There was no cross reaction with human β2-microglobulin, γ-globulin, and haemoglobulin.

  14. Development of a specific and sensitive enzyme-linked immunosorbent assay for the quantification of imatinib.

    Science.gov (United States)

    Saita, Tetsuya; Shin, Masashi; Fujito, Hiroshi

    2013-01-01

    Imatinib is an oral tyrosine kinase inhibitor used for first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring targeting trough plasma levels of about 1000 ng/mL may help to optimize imantinib's therapeutic effect. This paper reports a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for a pharmacokinetic evaluation of imatinib. Anti-imatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin and succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. Enzyme labeling of imatinib with horseradish peroxidase was similarly performed using succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. A simple ELISA for imatinib was developed using the principle of direct competition between imatinib and the enzyme marker for anti-imatinib antibody which had been adsorbed by the plastic surface of a microtiter plate. Serum imatinib concentrations lower than 40 pg/mL were reproducibly measurable using the ELISA. This ELISA was specific to imatinib and showed very slight cross-reactivity (1.2%) with a major metabolite, N-desmethyl imatinib. Using this assay, drug levels were easily measured in the blood of mice after their oral administration of imatinib at a single dose of 50 mg/kg. The specificity and sensitivity of the ELISA for imatinib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of imatinib.

  15. Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

    Science.gov (United States)

    Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

    2016-01-01

    Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

  16. Evaluation of a ganglioside immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin.

    Science.gov (United States)

    Bäck, E; Svennerholm, A M; Holmgren, J; Möllby, R

    1979-12-01

    The GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA), an immunological method for detection of Escherichia coli heat-labile enterotoxin (LT), was quantitatively and qualitatively compared with the conventional adrenal cell test for the identification of LT-producing strains. A micromodification model of the assay was developed. Enterotoxin preparations from 120 E. coli isolates from individuals with diarrhea, which had been previously shown to be enterotoxigenic by the adrenal cell test, and from 44 control strains of E. coli were compared in parallel by the two methods. Quantitatively the covariation of the enterotoxin titers was highly significant (RS = 0.98, P less than 0.001), the GM1-ELISA being somewhat more sensitive than the adrenal cell test. The methodological error was less than 5% in both tests. Qualitatively the overall agreement for positive and negative reactions for the two methods was 89%. The GM1-ELISA is practical for routine use in the diagnosis of enterotoxigenic E. coli, especially in laboratories without facilities for cell culture.

  17. Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    SHENG Jianwu; HE Miao; YU Shaoqing; SHI Hanchang; QIAN Yi

    2007-01-01

    Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established and used to detect microcystin-LR (MC-LR) in drinking and surface waters.The concentration of coating antigen was 5 μg/mL,the dilution of monoclonal antibody MC10E7 was 1:3 000,the dilution of enzyme tracer (goat anti-mouse IgG-peroxidase) was 1:3 000,the standard concentration of MC-LR ranged from 0.001 μg/L to 30 μg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC) with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 μg/L and up to 5.1 μg/L.The quantitative detection range was from 0.03 μg/L to 3 μg/L,and the antibody had high specificity for [4-arginine]microcystins.It performed well in spite of the influence of the real samples.

  18. Comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    Science.gov (United States)

    Kösters, K; Riffelmann, M; Dohrn, B; von König, C H

    2000-05-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for evaluation of the kits. The specimens contained 20 paired serum samples from patients with clinical pertussis, 15 samples were from children vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven specimens were taken from an interlaboratory comparison of ELISAs, and there were three reference preparations from the Food and Drug Administration's (FDA's) Laboratory of Pertussis and from our laboratory. Reference values were obtained from the FDA or from results obtained with an in-house ELISA. Commercial ELISAs were compared with respect to their reproducibility and variability, their ability to detect significant titer rises in paired serum samples, their ability to detect an immune response after vaccination, and the comparability of semiquantitative and quantitative results. Reproducibility was generally good (>89%), intra-assay variation ranged from 2.4 to 28.7%, and indeterminate results were recorded in up to 18.5% of all specimens. Most kits correctly identified the antibody response to an acellular pertussis vaccine. None of the commercial kits identified all cases of pertussis correctly, and the sensitivity ranged between 60 and 95%. All five commercial ELISAs showed great discrepancies when comparing semiquantitative results and contained obviously different antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.

  19. The effects of detergent on the enzyme-linked immunosorbent assay (ELISA) of blood group substances.

    Science.gov (United States)

    McCabe, J P; Fletcher, S M; Jones, M N

    1988-04-06

    The detergents 1-0-n-octyl-beta-D-glucopyranoside (OBG) and sodium n-dodecyl sulphate (SDS) have been used to extract blood group substances from human erythrocyte membranes for detection by enzyme-linked immunosorbent assay (ELISA). The effect of detergent concentration on the extraction process and detection by ELISA have been investigated. Detergent extraction increased the ELISA response relative to response from membrane suspensions approximately 1000-fold. Optimum responses occurred using detergent concentrations near the critical micelle concentration (cmc) for OBG and below the cmc for SDS. High detergent concentrations interfered with the ELISA but this effect was reduced by dilution of the extracts before adsorption of antigen on the microtitre wells. The interference effects of detergent on ELISA were also investigated using ovarian cyst glycoproteins as antigen. It was found that detergents inhibit the assay at the initial stage by competing with antigens for adsorption sites on the microtitre well surface and that subsequent detergent can displace pre-bound antigen. The results are discussed in terms of detergent binding to proteins (and glycoproteins) in relation to free (unbound) detergent concentration.

  20. Determination of the folate content in cladodes of nopal (Opuntia ficus indica) by microbiological assay utilizing Lactobacillus casei (ATCC 7469) and enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ortiz-Escobar, Tania Breshkovskaya; Valverde-González, Maria Elena; Paredes-López, Octavio

    2010-05-26

    Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable.

  1. Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays.

    Science.gov (United States)

    Wu, John T Y; Dreger, Sally; Chow, Eva Y W; Bowlby, Evelyn E

    2002-10-01

    This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV-) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV- values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures.

  2. Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

    Science.gov (United States)

    Baldofski, Stefanie; Hoffmann, Holger; Lehmann, Andreas; Breitfeld, Stefan; Garbe, Leif-Alexander; Schneider, Rudolf J

    2016-11-01

    Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters.

  3. Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays

    Science.gov (United States)

    Wu, John T.Y.; Dreger, Sally; Chow, Eva Y.W.; Bowlby, Evelyn E.

    2002-01-01

    Abstract This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV−) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV− values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures. PMID:12418782

  4. Enzyme-linked immunosorbent assays using immune complexes for the diagnosis of tuberculosis.

    Science.gov (United States)

    Pereira Arias-Bouda, Lenka M; Kuijper, Sjoukje; van Deutekom, Henk; van Gijlswijk, Rob; Pekel, Inge; Jansen, Henk M; Kolk, Arend H J

    2003-12-01

    The serodiagnosis of tuberculosis has long been the subject of investigation, but we still lack a test with widespread clinical utility. The poor sensitivity and specificity of commercial assays precludes their use as the sole means of diagnosis. All of these assays use mycobacterial antigens adsorbed onto a surface. Little attention has been paid to changes in antigen conformation that may occur as a result of passive coating of these antigens to solid supports like polystyrene. Such changes may cause technical artifacts resulting in false-positive (FP) and false-negative (FN) reactions. We have developed two different enzyme-linked immunosorbent assay (ELISA) systems, in which human serum antibodies and target antigens of Mycobacterium tuberculosis are able to associate and dissociate freely in solution to form immune complexes. In one ELISA, rabbit antibodies against M. tuberculosis, passively coated in the ELISA wells, capture the immune complexes (ICs). In the other ELISA, the ICs are detected by these same rabbit antibodies but are first captured by passively coated goat anti-rabbit IgG. We have compared these two ELISA systems with an ELISA using M. tuberculosis antigens passively adsorbed to the solid polystyrene surface of the plate. We studied sera from 81 patients with tuberculosis and 47 healthy subjects. The differences between tuberculosis (TB) patients and healthy subjects were statistically significant in all three of our ELISA systems. However, the ELISA systems using soluble M. tuberculosis antigens distinguished better between TB patients and healthy subjects than the ELISA using surface-adsorbed M. tuberculosis antigens. We suggest that in the latter ELISA, passive adsorption of the target antigens induces conformational change, generating altered epitopes that are recognized by antibodies present in the serum from even healthy people. These altered conformational epitopes are recognized by antibodies that were originally evoked by antigens

  5. Development of an enzyme-linked immunosorbent assay for the veratrum plant teratogens: cyclopamine and jervine.

    Science.gov (United States)

    Lee, Stephen T; Panter, Kip E; Gaffield, William; Stegelmeier, Bryan L

    2003-01-29

    Veratrum californicum was responsible for large losses of sheep grazing high mountain ranges in central Idaho in the 1950s. Veratrum induces various birth defects including the cyclopic-type craniofacial defect (monkey-faced lambs) that is specifically induced in lambs after pregnant ewes grazed the plant on the 14th day of gestation. The steroidal alkaloids cyclopamine (1) and jervine (2) were isolated from Veratrum and shown to be primarily responsible for the malformations. Cyclopamine (1) and jervine (2) are potent teratogens that inhibit Sonic hedgehog (Shh) signaling during gastrulation-stage embryonic development, producing cyclopia and holoprosencephaly. Although losses to the sheep industry from Veratrum are now relatively infrequent, occasional incidents of toxicoses and craniofacial malformations are still reported in sheep and other species. However, the benefits to biomedical research using cyclopamine (1) as a tool to study human diseases have greatly expanded. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) to detect and measure cyclopamine (1) and jervine (2) was developed using polyclonal antibodies produced in ewes. The limits of detection of the assay were 90.0 and 22.7 pg for cyclopamine (1) and jervine (2), respectively. This assay was used for the detection and measurement of cyclopamine (1) spiked into sheep blood. The simple extraction-ELISA methods developed in this study demonstrate the potential of using these techniques for the rapid screening of biological samples to detect the presence and concentration of cyclopamine (1) and jervine (2) and will be beneficial to pharmacological studies and livestock diagnostics.

  6. Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum

    DEFF Research Database (Denmark)

    Klausen, Joan; Andresen, Lars Ole; Barfod, Kristen

    2001-01-01

    A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6...

  7. Extraction procedure for sulfachloropyridazine in porcine tissues and detection in a sulfonamide-specific enzyme-linked immunosorbent assay (ELISA)

    NARCIS (Netherlands)

    Cliquet, P.; Cox, E.; Haasnoot, W.; Schacht, B.; Goddeeris, B.M.

    2003-01-01

    Sulfonamide-specific polyclonal rabbit antibodies were obtained after immunization with a sulfathiazole derivative (N1-[4-(carboxymethyl)-2-thiazolyl]sulfanilamide = TS) coupled to keyhole lympet hemocyanin. Using these antibodies, two sulfonamide-specific enzyme-linked immunosorbent assays (ELISAs)

  8. Extraction procedure for sulfachloropyridazine in porcine tissues and detection in a sulfonamide-specific enzyme-linked immunosorbent assay (ELISA)

    NARCIS (Netherlands)

    Cliquet, P.; Cox, E.; Haasnoot, W.; Schacht, B.; Goddeeris, B.M.

    2003-01-01

    Sulfonamide-specific polyclonal rabbit antibodies were obtained after immunization with a sulfathiazole derivative (N1-[4-(carboxymethyl)-2-thiazolyl]sulfanilamide = TS) coupled to keyhole lympet hemocyanin. Using these antibodies, two sulfonamide-specific enzyme-linked immunosorbent assays (ELISAs)

  9. Evaluation of new transport medium for detection of herpes simplex virus by culture and direct enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ogburn, J R; Hoffpauir, J T; Cole, E; Hood, K; Michael, D; Nguyen, T; Raden, S; Raju, B; Reisinger, V; Oefinger, P E

    1994-12-01

    The transport medium Multi-Microbe Media (M4) was evaluated prospectively by culture and direct enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus from 473 specimens. In addition, 377 specimens in Bartels Viral Transport Medium were evaluated. By using culture as a "gold standard," the ELISA sensitivity was approximately 85%, while the specificities exceeded 96% for both media.

  10. An enzyme-linked immunosorbent assay for the detection of mouse polyomavirus-specific antibodies in laboratory mice.

    NARCIS (Netherlands)

    H.W.J. Broeders; J. Groen (Jan); A.D.M.E. Osterhaus (Albert); G. van Steenis (Bert)

    1994-01-01

    textabstractAn enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of IgM and IgG serum antibodies to mouse polyomavirus (MPV). To evaluate the potential of this ELISA for the screening of laboratory rodents, serum samples from specific pathogen free (SPF) BA

  11. Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein

    DEFF Research Database (Denmark)

    Rasmussen, M; Dahl, M; Buus, S;

    2014-01-01

    . We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide...

  12. Comparison of enzyme-linked immunosorbent assay test with immunoblot assay in the diagnosis of pemphigus in Indian patients

    Directory of Open Access Journals (Sweden)

    Khandpur Sujay

    2010-01-01

    Full Text Available Background: The diagnosis of pemphigus vulgaris (PV and pemphigus foliaceous (PF rests upon clinical, histological and immunofluorescence features. Enzyme-linked immunosorbent assay (ELISA test and immunoblot (IB assay have shown variable sensitivity and specificity. Aims: We compared the utility of ELISA and IB in pemphigus patients. Methods: Sixty-six pemphigus cases (PV-54, PF-12 and 72 controls (other vesicobullous disorders and healthy controls were inducted. ELISA for anti-Dsg 3 and 1 antibodies and IB assay were performed. Results: On ELISA, both mean anti-Dsg 1 and 3 titers were raised in PV and PF. Mean anti-Dsg 1 in mucocutaneous PV was significantly higher than in mucosal PV and mean anti-Dsg 3 was significantly raised in PV than in PF. Anti-Dsg 1 and 3 in the control group were negative. Sensitivity and specificity of ELISA in PV was 98.14% and 90.5% while in PF it was 91.6% and 61.1%, respectively.On IB in PV, 36 cases (66.67% showed the 130 kDa and 160 kDa antigen bands, 12 (22.2% only the 130 kDa and six (11.1% only the 160 kDa band. Eight of the nine pure mucosal cases (88.8% showed only the 130 kDa. In PF, only the 160 kDa antigen was detected. These antigens were not identified in the control group. Sensitivity and specificity of IB in PV was 88.9% and 100% and in PF it was 100% and 95.2%, respectively. Conclusion: Both tests could differentiate pemphigus from other dermatoses, including other blistering disorders. ELISA could not make a distinction between PV and PF or between the various clinical phenotypes of PV. IB differentiated between PV and PF and the different clinical variants of PV.

  13. Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and Haemophilus influenzae Type b

    OpenAIRE

    Pickering, Jerry W.; Martins, Thomas B.; Schroder, M. Carl; Hill, Harry R.

    2002-01-01

    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays ...

  14. Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Sails, A. D.; Bolton, F. J.; Fox, A. J.; Wareing, D. R. A.; Greenway, D. L. A.

    2002-01-01

    A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELIS...

  15. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...

  16. Diagnostic tests for amoebic liver abscess: comparison of enzyme - linked immunosorbent assay (Elisa and counterimmunoelectrophoresis (CIE

    Directory of Open Access Journals (Sweden)

    Marcos I. Restrepo

    1996-02-01

    Full Text Available The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoeletrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results iin both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100% than that of the CIE technique (66%.

  17. Evaluation of commercial enzyme-linked immunosorbent assays to identify psychedelic phenethylamines.

    Science.gov (United States)

    Kerrigan, Sarah; Mellon, Monica Brady; Banuelos, Stephanie; Arndt, Crystal

    2011-09-01

    The 2C, 2C-T, and DO series of designer drugs pose a number of challenges to forensic toxicology laboratories. Although these drugs are seized by law enforcement agencies throughout the United States, they are not readily detected in forensic toxicology laboratories. A systematic evaluation of the cross-reactivity of 9 commercial enzyme-linked immunosorbent assays (ELISAs) was conducted using 11 designer drugs. Cross-reactivity was measured towards 2,5-dimethoxy-4-bromophenethylamine (2C-B), 2,5-dimethoxyphenethylamine (2C-H), 2,5-dimethoxy4-iodophenethylamine (2C-I), 2,5-dimethoxy-4ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4isopropylthiophenethylamine (2C-T-4), 2,5-dimethoxy-4propylthiophenethylamine (2C-T-7), 2,5-dimethoxy-4bromoamphetamine (DOB), 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-iodoamphetamine (DOI), 2,5-dimethoxy-4-methylamphetamine (DOM), and 4methylthioamphetamine (4-MTA). Cross-reactivity towards the 2C, 2C-T, and DO series of psychedelic amphetamines was psychedelic phenethylamines makes it harder to detect these drugs using routine screening. As a consequence, laboratories that rely upon immunoassay rather than more broad spectrum chromatographic screening techniques, may fail to detect these powerful psychedelic substances.

  18. [An urease enzyme linked immunosorbent assay for detection of Helicobacter pylori infection].

    Science.gov (United States)

    Ding, S Z; Jia, B Q; Liu, X G

    1993-05-01

    A sensitive and specific serological diagnostic test for Helicobacter pylori infection has been developed and validated in 120 patients with dyspeptic symptoms undergoing endoscopy. This test is to use urease, a protein unique to H. pylori, as the basis for the enzyme linked immunosorbent assay (ELISA) that detects serum H. pylori urease antibodies. The ELISA mean optical density (OD) in H. pylori-positive group is higher than that in H. pylori-negative group (0.57 +/- 0.23 vs 0.24 +/- 0.15, P < 0.001), a cut-off 0.3 OD yields a sensitivity of 95% and a specificity of 93%. Serum absorption test showed that Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Yersinia enterocolotica, Pseudomonas aeruginosa cell lysate do not influence serum H. pylori urease antibody level, though they all have urease except E. coli. The result implied that H. pylori urease can be a good antigen to detect serum H. pylori antibody and it would be useful for epidemiological survey and routine diagnostic approach. Nearly half of the blood donors showed positive result with H. pylori urease antibody. It is suggested that H. pylori infection is quite common in the asymptomatic population.

  19. [Development of direct competitive enzyme-linked immunosorbent assay for the determination of domoic acid].

    Science.gov (United States)

    Wang, Qian; Cheng, Jin-Ping; Gao, Li-Li; Dong, Yu; Xi, Lei

    2012-02-01

    To develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid detection of domoic acid concentrations, HRP (horse radish peroxidase) was successfully linked to DA using EDC. The concentration of DA was quantitatively analyzed on the basic of the specific immune responses between the DA- HRP and the monoclonal antibodies made in advance. Calibration curve were established after the optimization of reaction conditions such as the type of blocking solution, the blocking time and the incubation temperature. The results show that, the best reaction condition of the direct competitive ELISA is 1% gelatin, blocking 1 h at 37 degrees C, incubating 1 h at 37 degrees C after the monoclonal antibodies added. The detect limit is 3.58 ng x mL(-1), the coefficient of variation between the holes is below 15%, and the recovery is 80% - 120%. The whole analysis process could be completed within 1.5 h. It meets the requirements of rapid and batch detection of domoic acid. The method will have broad development prospects.

  20. Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System.

    Science.gov (United States)

    Akama, Kenji; Shirai, Kentaro; Suzuki, Seigo

    2016-07-19

    Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

  1. Quantification of Sorafenib in Human Serum by Competitive Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Saita, Tetsuya; Yamamoto, Yuta; Noda, Satoshi; Shioya, Makoto; Hira, Daiki; Andoh, Akira; Morita, Shin-Ya; Terada, Tomohiro; Shin, Masashi

    2015-01-01

    The multikinase inhibitor sorafenib has been used in the treatment of hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. Here we have demonstrated the production of the first specific antibody against sorafenib. Anti-sorafenib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified 4-(4-aminophenoxy)-N-methyl-2-pyridinecarboxamide (AMPC) using the N-succinimidyl ester method. Enzyme labeling of sorafenib with horseradish peroxidase was similarly performed using carboxylic modified AMPC. A simple competitive enzyme-linked immunosorbent assay (ELISA) for sorafenib was developed using the principle of direct competition between sorafenib and the enzyme marker for anti-sorafenib antibody, which had been adsorbed by the plastic surface of a microtiter plate. Serum sorafenib concentrations lower than 0.04 µg/mL were reproducibly measurable using the ELISA. This ELISA was specific to sorafenib and showed very slight cross-reactivity (2.5%) with a major metabolite, sorafenib N-oxide. The values of serum sorafenib levels from 32 patients measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (Y=1.016X-0.137, r=0.979). The specificity and sensitivity of the ELISA for sorafenib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of sorafenib.

  2. Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus.

    Science.gov (United States)

    Li, Zhi; Zhang, Yanlong; Wang, Huiguo; Jin, Jinhua; Li, Wenzhe

    2013-10-01

    A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease.

  3. Enzyme-linked immunosorbent assay (ELISA) for antibodies against Campylobacter jejuni, and its clinical application.

    Science.gov (United States)

    Walder, M; Forsgren, A

    1982-12-01

    Antibody response to Campylobacter jejuni/coli (CJC) was investigated, using an enzyme-linked immunosorbent assay, ELISA. With a mixture of lipopolysaccharide from two CJC strains as antigen in ELISA, all 24 tested rabbit anti-CJC sera showed high antibody levels. However, only 70% of sera from patients with Campylobacter enteritis demonstrated an antibody response against the combined LPS antigen, using paired sera. In addition, the results obtained suggested non-specific binding of human immunoglobulin. When 24 formalinized whole CJC bacteria were used as antigen in ELISA, all corresponding rabbit antisera reacted with one strain (M 14). Essentially no unspecific binding of human immunoglobulin was obtained. Antibodies were detected in sera from healthy blood donors and at a lower level in sera from children, suggesting early immunization. In 67 enteritis patients with positive stool cultures for CJC, a significantly increased level of IgG antibodies could be detected in single or paired serum samples from 82% of the patients. An IgG titre increase occurred early in the course of infection, suggesting a boosting of an earlier immunization. IgM antibodies could be detected in the same sera in 77% of the patients. Considering both IgG and IgM analyses of the enteritis sera, 94% of the patients were positive in Campylobacter ELISA serology compared with only 5% of healthy controls.

  4. Enzyme-linked immunosorbent assay for Helicobacter pylori needs adjustment for the population investigated.

    Science.gov (United States)

    Hoang, Thi Thu Ha; Wheeldon, Thuc-Uyen; Bengtsson, Carina; Phung, Dac Cam; Sörberg, Mikael; Granström, Marta

    2004-02-01

    Helicobacter pylori infection and peptic ulcer disease are common in developing countries, e.g., Vietnam. An enzyme-linked immunosorbent assay (ELISA) for screening of patients and for seroepidemiology is a useful tool but needs to be validated in the population studied. We used in-house ELISA with sonicated Swedish and Vietnamese strains as antigens to measure immunoglobulin G antibodies after absorption with sonicated Campylobacter jejuni in sera from 270 H. pylori culture-confirmed peptic ulcer patients, 128 Swedish urea-breath test and immunoblot-positive healthy controls, and 432 Vietnamese immunoblot-positive population controls. Sonicated whole-cell antigen based on the local strains showed a significantly better performance. Immunoblot-positive peptic ulcer patients had significantly higher antibody concentrations than immunoblot-positive population controls, necessitating a lower cutoff level if serology is used for screening or epidemiological purposes. The study shows that the parameters of ELISA for H. pylori need to be adjusted for the population being investigated.

  5. An enzyme-linked immunosorbent assay for diagnosis of Fasciola gigantica infection in cattle and buffaloes.

    Science.gov (United States)

    Krishna Murthy, C M; Souza, Placid E D

    2015-12-01

    The enzyme-linked immunosorbent assay (ELISA) was evaluated for the diagnosis of Fasciola gigantica infection in cattle and buffaloes. The excretory-secretory (E-S Ag) antigen of F. gigantica adult flukes obtained after invitro incubation was used as an antigen. The test was conducted with 276 sera collected from cattle and buffaloes which included 22 sera each from naturally infected cattle and buffaloes (known positive serum) and with similar number of samples with healthy cattle and buffaloes (known negative serum). The positive results were observed in 18 and 19 of the sera from naturally infected cattle and buffaloes with sensitivity of 81.8 and 86.3 % respectively. Out of 188 serum samples which were found negative on faecal examination 32 (34 %) sera of cattle and 40 (42.5 %) sera of buffaloes were found positive by ELISA respectively. The sensitivity of the test was found to be 91.6 and 95.6 % in cattle and buffaloes respectively.

  6. Chromium functionalized diglyme plasma polymer coating enhances enzyme-linked immunosorbent assay performance.

    Science.gov (United States)

    Welch, Nicholas G; Madiona, Robert M T; Easton, Christopher D; Scoble, Judith A; Jones, Robert T; Muir, Benjamin W; Pigram, Paul J

    2016-11-10

    Ensuring the optimum orientation, conformation, and density of substrate-bound antibodies is critical for the success of sandwich enzyme-linked immunosorbent assays (ELISAs). In this work, the authors utilize a diethylene glycol dimethyl ether plasma polymer (DGpp) coating, functionalized with chromium within a 96 well plate for the enhanced immobilization of a capture antibody. For an equivalent amount of bound antibody, a tenfold improvement in the ELISA signal intensity is obtained on the DGpp after incubation with chromium, indicative of improved orientation on this surface. Time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS) and principal component analysis were used to probe the molecular species at the surface and showed ion fragments related to lysine, methionine, histidine, and arginine coupled to chromium indicating candidate antibody binding sites. A combined x-ray photoelectron spectroscopy and ToF-SIMS analysis provided a surface molecular characterization that demonstrates antibody binding via the chromium complex. The DGpp+Cr surface treatment holds great promise for improving the efficacy of ELISAs.

  7. DEVELOPMENT OF ENZYME-LINKAGE IMMUNOSORBENT ASSAY AGAINST TYPE B OF CLOSTRIDIUM BOTULINUM: A PRELIMINARY STUDY

    Directory of Open Access Journals (Sweden)

    S. N. Depamede

    2011-12-01

    Full Text Available Clostridium botulinum neurotoxin (BoNTs is one of the causes of economic loss in the livestock industry. This economic loss would be as a direct result when animals poisoned by BoNTs or indirectly when the livestock products are contaminated by BoNTs, which end up with the products are banned by authority. Therefore a routine surveillance of BoNTs in the farm and in livestock product processing industry is urgently needed. One of the most relatively quick and accurate methods to perform a routine detection of the presence of BoNTs is enzyme-linkage immunosorbant assay (ELISA. In this article we describe the results of the development of ELISA, using polyclonal antibodies against BoNTs-B produced locally. Antibodies were generated from six Balb/c mice with standard immunological methods. Mice were immunized three times for a period of 8 weeks with a commercial type B Clostridium botulinum toxoid at a dose of 100 ng per mouse per injection. The resulting antibody was purified by a combination of ammonium sulfate precipitation 50% (w/v technique and a protein A column method. The results of this preliminary study indicated that the developed ELISA method capable of detecting type B Clostridium botulinum toxin up to 1.0 ng/ml.

  8. A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

    Science.gov (United States)

    Chiang, S; Dar, A M; Goyal, S M; Sheikh, M A; Pedersen, J C; Panigrahy, B; Senne, D; Halvorson, D A; Nagaraja, K V; Kapur, V

    2000-07-01

    Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.

  9. Development of enzyme-linked immunosorbent assay for detecting Ornithobacterium rhinotracheale (ORT infection in chicken

    Directory of Open Access Journals (Sweden)

    Adin Priadi

    2006-10-01

    Full Text Available Ornithobacterium rhinotracheale (ORT has been recognized in chicken in Indonesia and incriminated as a possible additional causative agent in respiratory disease complex. An enzyme-linked immunosorbent assay (ELISA has been developed for the seroepidemiological study of ORT infection in chickens. Ten weeks old chickens are injected with 0.5 ml of killed O. rhinotracheale emulsified in Freund's complete adjuvant at a concentration of 109 CFU/ml. Hyperimmune sera and non-reactive control sera were used to standardized the ELISA for ORT infection. Optimum condition for the ORT ELISA was antigen dilution 1/800, serum dilution 1/100 and 1/4000 conjugate dilution. Optical density cut-off point was determined by using 31 serum samples from 2 broiler farms. Cut-off for negative serum was 0.27 (mean + 3 standard deviation. With these optima, 187 chicken sera from broiler, layer and broiler breeder farms were collected and screened. Seroconvertions were detected from broiler and layer farms in Magelang district, Central Java (Bojong I, Paremono, Bojong II, Keblukan and a broiler breeder farm in West Java. The seraconvertion were 0, 10, 94, 88 and 100 percents respectively. These figures show that the prevalence of O. rhinotracheale infection in chicken in layer and breeder farms were very high.

  10. Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay for the Analysis of Jasmonates in Plants

    Institute of Scientific and Technical Information of China (English)

    Aixing Deng; Weiming Tan; Suping He; Wei Liu; Tiegui Nan; Zhaohu Li; Baomin Wang; Qing X.Li

    2008-01-01

    Methyl jasmonate (MeJA) and its free-acid form,jasmonic acid (JA) are naturally occurring plant growth regulators widely distributed in higher plants.In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples,a monoclonal antibody (MAb) (designated as MAb 3E5D7C4B6) against MeJA was derived from a JAbovine serum albumin (BSA) conjugate as an immunogen.The antibody belongs to the IgG1 subclass with a κ type light chain and has a dissociation constant of approximately 6.07 x 10-9 M.MAb3E5D7C4B6 is very specific to MeJA.It was used to develop a direct competitive enzyme-linked immunosorbent assay (dcELISA),conventional and simplified indirect competitive ELISAs (icELISA).JA was derivatized into MeJA for the ELISA analysis.The IC50 value and detection range for MeJA were,respectively,34 and 4-257 ng/mL by the conventional icELISA,21 and 3-226 ng/mL by the simplified icELISA and 5.0 and 0.7-97.0 ng/mL by the dcELISA.The dcELISA was more sensitive than either the conventional or simplified icELISA.The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves.The increased jasmonates content indicated its role in response to the drought stress and pathogens.

  11. Diagnostic value of enzyme linked immuno-sorbent assay for cytomegalovirus disease.

    Directory of Open Access Journals (Sweden)

    Priya K

    2002-07-01

    Full Text Available BACKGROUND: Since interpretation of results of enzyme linked immuno-sorbent assay (ELISA for diagnosis of Cytomegalovirus (CMV infection in India is difficult, its diagnostic value required evaluation. AIMS: To evaluate the diagnostic value of ELISA against polymerase chain reaction (PCR in CMV disease. SETTINGS AND DESIGN: Results of ELISA test for CMV antibodies in CMV-DNA PCR positive and negative patients and normal healthy blood donors were analysed. METHODS AND MATERIAL: Anti-CMV antibodies were assayed by ELISA on the sera of 26 CMV PCR positive and 21 PCR negative patients and 35 normal healthy blood donors. STATISTICAL ANALYSIS: Chi square and Fischer exact test were used for statistical analysis. RESULTS: Anti-CMV antibodies (IgG or IgG and IgM were present in 20 (76.9% of 26 PCR positive and 13 (61.9% of 21 PCR negative patients. ELISA was negative in six (23.1% of 26 PCR positive patients. Of the 28 paediatric patients, ELISA was positive in 14 (73.7% of 19 PCR positive and three (33.3% of nine PCR negative patients showing a statistically significant difference (Chi square test, P value 0.038. Among the 19 patients having complications after organ transplant, ELISA showed anti-CMV antibodies in six (85.7% of seven PCR positive and 11 (91.7% of 12 PCR negative patients showing no significant difference. CMV-DNA was not detected in the buffy coat of 35 sero-positive blood donors. CONCLUSION: ELISA has no diagnostic value in the detection of CMV activation although it may help in the differential diagnosis of CMV infection in the paediatric age group.

  12. Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses.

    Science.gov (United States)

    Sudarshana, M R; Reddy, D V

    1989-10-01

    A penicillinase (PNC)-based, enzyme-linked immunosorbent assay (ELISA) was standardized to detect maize mosaic virus (MMV) in sorghum leaf extracts, peanut mottle virus (PMV) in pea leaf extracts, and tomato spotted wilt virus (TSWV) in peanut leaf extracts. Rabbit Fc-specific antibodies were conjugated with PNC by a single step glutaraldehyde bridge. Among several indicators tested, bromothymol blue (BTB) was found suitable for measuring PNC activity under simulated conditions. Two reagents, starch-iodine complex (SIC) and a mixed pH indicator, containing bromocresol purple and BTB (2:1) used earlier for the PNC-based ELISA, were compared with BTB for utilization in the PNC-based ELISA. SIC gave a slightly higher virus titre than BTB or the mixed pH indicator, but it often gave nonspecific reactions. Sodium or potassium salts of penicillin-G at 0.5-1.0 mg/ml and BTB at 0.2 mg/ml were found to be suitable as substrate-indicator mixture for PNC-based ELISA. The sensitivity of the PNC system was comparable to those of the alkaline phosphatase (ALP) and horseradish peroxidase (HRP) systems in detecting MMV, PMV, and TSWV. The PNC conjugate could be used at a greater dilution than those of the ALP and HRP conjugates and the BTB substrate mixture was stable for at least 3 weeks at 4 degrees C. Penicillin is readily available in developing countries, and at a substantially lower cost than p-nitrophenyl phosphate, the commonly used substrate for ALP in the plate ELISA. Thus the PNC-based ELISA provides a less expensive means for assaying plant viruses by ELISA.

  13. Kesesuaian Hasil Pemeriksaan Antibodi Virus Herpes Simpleks Metode Enzyme-Linked Immunofiltration Assay dengan Enzyme-Linked Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    Victor Immanuel

    2012-09-01

    Full Text Available Herpes simplex virus (HSV infections are very common and are caused by HSV type 1 (HSV-1 and HSV type 2 (HSV-2. HSV-1 being mostly associated with orofacial disease, whereas HSV-2 is usually associated with perigenital infection. Diagnosis of HSV infection is established based on history, physical and laboratory examination. Enzyme-linked immunosorbent assay (ELISA method to detect anti-HSV has a sensitivity 93–100% and specificity 95–100%, whereas enzyme-linked immunofiltration assay (ELIFA has a sensitivity 83.36–97% and specificity 83.93–98%. The aim of this study was to assess the agreement of anti-HSV between ELIFA and ELISA methods. This study was conducted in the clinical laboratory RSUP Dr. Hasan Sadikin Bandung since January to May 2011. The study design was cross sectional. Subjects of this study were serum of patients suspected HSV infection. Statistical analysis was performed to assess Kappa agreement. A total of 66 samples were examined anti-HSV using ELIFA and ELISA method. There was good agreement between test results of anti-HSV IgM ELIFA and ELISA method (p<0.001, κ=0.621, moderate agreement between test results of anti- HSV-1 IgG ELIFA and ELISA method (p<0.001, κ=0.533, and fair agreement between test results of anti-HSV-2 IgG ELIFA and ELISA method (p=0.006, κ= 0.260. In conclusions, only the anti-HSV IgM ELIFA method has good agreement with ELISA.

  14. Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

    Science.gov (United States)

    Funk, N D; Tabatabai, L B; Elzer, P H; Hagius, S D; Martin, B M; Hoffman, L J

    2005-02-01

    Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of melitensis-specific antibodies in goat milk.

  15. Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Klausen, Joan; Barfod, Kristen

    2002-01-01

    in samples of pig serum were detected by inhibition of the binding of polyclonal rabbit antibodies raised against Ap serotype 12. The assay was evaluated against sera from experimentally infected pigs, from pig herds naturally infected with Ap and from herds declared free of Ap serotypc 12 infection......The objective was to develop a blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Actinobacillus pleuropneumoniae (Ap) serotype 12 in pig serum. Lipopolysaccharide (LPS) from Ap serotype 12 was purified and used as antigen in the assay. Antibodies to the LPS antigen...

  16. Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of cashew nut in foods.

    Science.gov (United States)

    Gaskin, Ferdelie E; Taylor, Steve L

    2011-01-01

    The presence of undeclared cashew can pose a health risk to cashew-allergic consumers. The food industry has the responsibility to declare the presence of cashews on packaged foods even when trace residues are or might be present. The objective of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) for the detection of cashew residues. Raw and roasted cashews were defatted and used separately to immunize sheep, goats, and rabbits. The cashew ELISA was developed using sheep and rabbit polyclonal anti-roasted cashew sera as capture and detector reagents, respectively, with visualization through an alkaline phosphatase-mediated substrate reaction. The cashew ELISA was shown to have a limit of quantification of 1 ppm (1 μg cashew/g). The ELISA was highly specific except that substantial cross-reactivity was noted with pistachio and a lesser degree of cross-reactivity was noted with hazelnut. The performance of the ELISA was assessed by manufacturing cookies, ice cream, and milk chocolate with added known amounts (0 to 1000 ppm) of cashew. The mean percent recoveries for ice cream, cookies, and milk chocolate were 118%± 2.9%, 84.3%± 4.0%, and 104%± 3.0%, respectively. In a limited retail survey, 4/5 retail samples with cashew declared on ingredient labels tested positive for cashew compared to 5/36 samples of foods with precautionary labels indicating the possible presence of one or more tree nuts and 0/18 samples without cashew declared on the label in any manner. The cashew ELISA can be used to detect undeclared cashew residue in foods and as a potential tool for the food industry to assess the effectiveness of allergen control strategies and to guarantee compliance with food labeling regulatory requirements.

  17. Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period.From 6 to 12 h, the amount and enzyme activity ofprotease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5~6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 104 cell/mL in the indirect ELISA, while 105 cell/mL in the dot-ELISA.

  18. Application of dispersive liquid-liquid microextraction for estrogens' quantification by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Lima, Diana L D; Silva, Carla Patrícia; Schneider, Rudolf J; Otero, Marta; Esteves, Valdemar I

    2014-07-01

    Estrogens, such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), are the major responsible for endocrine-disrupting effects observed in aquatic environments due to their high estrogenic potency, even at concentrations ranging from pgL(-1) to ng L(-1). Thus, it is essential to develop analytical methodologies suitable for monitoring their presence in water samples. Dispersive liquid-liquid microextraction (DLLME) was used as a pre-concentration step prior to the quantification of E2 and EE2 by enzyme-linked immunosorbent assay (ELISA). First, an evaluation of the effect of DDLME on the E2 and EE2 ELISA calibration curves was performed. Since the extraction procedure itself had an influence on the ELISA optical density (OD), it became necessary to subject, not only the samples, but also all the standards to the DLLME process. Working ranges were determined, being between 1.2 and 8000 ng L(-1), for E2, and between 0.22 and 1500 ng L(-1), for EE2. The influence of organic matter, both in the extraction and quantification, was evaluated and it was observed that its presence in the solution did not affect considerably the calibration curve. Recovery rates were also determined, ranging from 77% to 106% for ultrapure water and from 104% to 115% for waste water samples, the most complex ones in what concerns matrix effects. Results obtained when applying the proposed method to real water samples can be considered quite satisfying. Moreover, the obtained working ranges encompass values generally reported in literature, confirming the practical use of the method for environmental samples.

  19. Enzyme-linked immunosorbent Assay for detecting of antibody to canine distemper virus

    Directory of Open Access Journals (Sweden)

    Sudarisman

    2006-03-01

    Full Text Available Serum neutralisation test (SNT has been established for evaluating canine distemper vaccination, but until now SNT was rarely used due to the need for continuous tissue culture facilities and requires 3 days to perform. For detecting antibody to canine distemper virus, an enzyme-linked immunosorbent assay (ELISA is relatively simple and rapid seroassay. ELISA for canine immunoglobulin (Ig G antibodies to canine distemper virus (CDV was developed by using Onderstepoort strain of canine distemper virus as coating antigen. Rabbit anti canine IgG labelled with horse radish peroxidase was used as the conjugate, while phenylenediamine dihydrochloride (OPD was used as the substrate. The ELISA results were then compared with the results of the SNT, using the sera of 312 random-source dogs from West Java. The two test-results had a high degree of correlation. Very few discrepancies occurred and most of these were at the lower limits of each test. When the sera were tested at 1 : 100 dilutions, there was a 95.5% agreement between the ELISA and SNT. Their sensitivity and spesificity were 83.9 and 98.4%. Titrated SNT and ELISA also were performed on sera from 7 dogs whose lifetime medical histories were known. The antibodies were inclining up after two months of post vaccination, where the titre was not in zero/lower position at the day of vaccination. However, antibody zero or low position were found at 28 days post vaccination. All of the results indicated that ELISA can be used for evaluating antibody to canine distemper virus response, replacing the SNT.

  20. Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis.

    Science.gov (United States)

    Tanyuksel, Mehmet; Yilmaz, Hasan; Ulukanligil, Mustafa; Araz, Engin; Cicek, Mutalip; Koru, Ozgur; Tas, Zeynep; Petri, William A

    2005-07-01

    Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (-) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey.

  1. Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis.

    Science.gov (United States)

    Riffelmann, M; Thiel, K; Schmetz, J; Wirsing von Koenig, C H

    2010-12-01

    Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.

  2. Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b

    Energy Technology Data Exchange (ETDEWEB)

    Kristensen, K. (Streptococcus Department, Statens Seruminstitut, Copenhagen (Denmark)); Weis Bentzon, M. (Department of Biostatistics, Statens Seruminstitut, Copenhagen (Denmark))

    1992-01-01

    The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was indepenedent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines. (au).

  3. Detection of Lassa virus antigens and Lassa virus-specific immunoglobulins G and M by enzyme-linked immunosorbent assay.

    OpenAIRE

    Niklasson, B S; Jahrling, P B; Peters, C. J.

    1984-01-01

    Rapid diagnosis of Lassa fever is desirable for the timely therapeutic intervention and implementation of strict quarantine procedures both in West Africa field hospitals where the disease is endemic and at international crossroads. An enzyme-linked immunosorbent assay (ELISA) to measure Lassa virus antigens in viremic sera was developed in which experimentally infected monkeys were used as a model for the human disease. In this test, Lassa virus antigens in test sera were captured in wells o...

  4. An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

    OpenAIRE

    Jensen, Trine Hammer; Ajjouri, Gitte; Handberg, Kurt; Slomka, Marek J.; Coward, Vivien J; Cherbonnel, Martine; Jestin, Véronique; Lind, Peter; Jørgensen, Poul Henrik

    2013-01-01

    BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the d...

  5. Seroprevalence of Japanese encephalitis virus using competitive enzyme linked immunosorbent assay (C-ELISA) in pigs in East Sumba, Indonesia

    OpenAIRE

    Annytha Detha; Diana A. Wuri; Ketut Santhia

    2015-01-01

    Japanese Encephalitis (JE), a vector-borne zoonotic viral disease, is mostly prevalent in Asian countries. The objective of this study was to investigate the occurence of JE virus (JEV) among pigs in East Sumba, Indonesia. Blood samples (n=52) were randomly collected from 52 apparantly healthy pigs where pig population was high in East Sumba. The samples were subjected for seroprevalence study for the presence of antibodies against JEV using competitive enzyme linked immunosorbent assay (C-EL...

  6. Technical and diagnostic performance of five commercial anti-diphtheria toxoid IgG enzyme-linked immunosorbent assay kits.

    Science.gov (United States)

    Faruq, A; Dadson, L; Cox, H; Alcock, F; Parker, A R

    2010-10-01

    The technical and diagnostic performances of five commercially available enzyme-linked immunosorbent assays for the measurement of anti-diphtheria toxoid IgG antibodies were evaluated. There was good agreement between the relative sensitivities of the five assays, but the relative specificity of one of the assays differed from that of the other four assays. Three of the five assays possessed recoveries of the international reference material NIBSC 00/496 within the range of 90% to 110% at antibody levels >0.1 IU/ml. The data suggest that there are manufacture-dependent differences in relative sensitivity, specificity, and accuracy for the determination of anti-diphtheria toxoid IgG antibodies that could result in different diagnostic interpretations.

  7. Diagnostic tests for amoebic liver abscess: comparison of enzyme - linked immunosorbent assay (Elisa and counterimmunoelectrophoresis (CIE

    Directory of Open Access Journals (Sweden)

    Marcos I. Restrepo

    1996-02-01

    Full Text Available The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoeletrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results iin both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100% than that of the CIE technique (66%.O abscesso hepático é a complicação mais freqüente da amebíase intestinal: o seu diagnótico sugere-se pelo quadro clínico, mas é confirmado pelos estudos paraclínicos. Para confirmar o diagnóstico precisa-se identificar a E. histolytica, o que é apenas possível em muito poucos casos. As provas sorolôgicas melhoram notadamente o diagnóstico das complicações severas da amebíase. Em nosso estudo comparamos o teste de ELISA e a Contraimunoeletroforese (CIE. Ambas as técnicas foram utilizadas para detectar anticorpos contra ameba em 50 pacientes sem amebíase, 30 pacientes com abscesso hepático e 30 com amebíase intestinal. Todos os soros dos pacientes sem amebíase foram negativos por ambas as técnicas. Quando analisamos os soros dos pacientes com amebíase intestinal, encontrou-se que 10% destes

  8. Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein

    DEFF Research Database (Denmark)

    Jensen, A T; Gaafar, A; Ismail, A

    1996-01-01

    An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from...... clinical history (GBPP ELISA; P = 0.038; amastigote ELISA; P = 0.004; and promastigote ELISA; P = 0.017). In the former group, the sensitivities of the five ELISAs were 100% (GBPP), 87% (amastigote), 93% (promastigote), 67% (L. donovani), and 53% (L. major), respectively....

  9. The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA using recombinant cathepsin L protease.

    Directory of Open Access Journals (Sweden)

    Bibiana Gonzales Santana

    Full Text Available BACKGROUND: Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. METHODOLOGY/PRINCIPAL FINDINGS: Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. CONCLUSIONS/SIGNIFICANCE: A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in

  10. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A., E-mail: amaquieira@qim.upv.es

    2014-02-06

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  11. Rapid enzyme-linked immunosorbent assay for the detection of hantavirus-specific antibodies in divergent small mammals.

    Science.gov (United States)

    Cautivo, Karla; Schountz, Tony; Acuña-Retamar, Mariana; Ferrés, Marcela; Torres-Pérez, Fernando

    2014-05-06

    We assessed the utility of an enzyme-linked immunosorbent assay (ELISA) for the detection of hantavirus-specific antibodies from sera of Oligoryzomys longicaudatus, the principal reservoir of Andes virus (ANDV), using an antigen previously developed for detection of antibodies to Sin Nombre virus (SNV) in sera from Peromyscus maniculatus. The assay uses a protein A/G horseradish peroxidase conjugate and can be performed in as little as 1.5 hours. Serum samples from Oligoryzomys longicaudatus collected in central-south Chile were used and the assay identified several that were antibody positive. This assay can be used for the rapid detection of antibodies to divergent hantaviruses from geographically and phylogenetically distant rodent species.

  12. Rapid Enzyme-Linked Immunosorbent Assay for the Detection of Hantavirus-Specific Antibodies in Divergent Small Mammals

    Directory of Open Access Journals (Sweden)

    Karla Cautivo

    2014-05-01

    Full Text Available We assessed the utility of an enzyme-linked immunosorbent assay (ELISA for the detection of hantavirus-specific antibodies from sera of Oligoryzomys longicaudatus, the principal reservoir of Andes virus (ANDV, using an antigen previously developed for detection of antibodies to Sin Nombre virus (SNV in sera from Peromyscus maniculatus. The assay uses a protein A/G horseradish peroxidase conjugate and can be performed in as little as 1.5 hours. Serum samples from Oligoryzomys longicaudatus collected in central-south Chile were used and the assay identified several that were antibody positive. This assay can be used for the rapid detection of antibodies to divergent hantaviruses from geographically and phylogenetically distant rodent species.

  13. Enzyme-linked immunosorbent assay for the detection and identification of plant pathogenic bacteria (in particular for Erwinia amylovora and Clavibacter michiganensis subsp. sepedonicus).

    Science.gov (United States)

    Kokoskova, Blanka; Janse, Jaap D

    2009-01-01

    Enzyme-linked immunosorbent assay (ELISA) is the most commonly used serological diagnostic technique. A number of different ELISA formats can be used for the detection of bacterial plant pathogens and in particular Erwinia amylovora and Clavibacter michiganensis subsp. sepedonicus.

  14. Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

    Science.gov (United States)

    This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

  15. Application of immunomagnetic particles to enzyme-linked immunosorbent assay (ELISA) for improvement of detection sensitivity of HCG.

    Science.gov (United States)

    Kuo, Hsiao-Ting; Yeh, Jay Z; Wu, Po-Hua; Jiang, Chii-Ming; Wu, Ming-Chang

    2012-01-01

    This investigation was aimed at using superparamagnetic particles to enzyme-linked immunosorbent assay (SPIO-ELISA) of human chorionic gonadotropin (hCG) to enhance detection sensitivity of hCG. We found that N-(3-dimethyl aminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was the best cross-linking reagent to link anti hCG α antibody to superparamagnetic particle (SPIO-anti hCG α antibody immunomagnetic particle). To improve the specificity of the assay, a horse radish peroxidase (HRP)-labeled anti-hCG beta monoclonal antibody was used to detect captured hCG using double antibody sandwich ELISA assay. SPIO-ELISA application to determine hCG increased the sensitivity to 1 mIU/mL, which is a level of sensitivity enabling the diagnosis of pregnancy during the early gestational period.

  16. A pH Indicator-linked Immunosorbent assay following direct amplification strategy for colorimetric detection of protein biomarkers.

    Science.gov (United States)

    Shao, Fengying; Jiao, Lei; Miao, Luyang; Wei, Qin; Li, He

    2017-04-15

    A new pH indicator-linked immunosorbent assay (PILISA) reached pg/mL sensitivity based on pH indicator molecules loaded carbon nitride nanosheets as signal enhancer has been developed for colorimetric detection of protein biomarkers. As the secondary antibody binds to the carbon nitride nanosheets, the carbon nitride nanosheets and pH indicator complex as the signal amplification platform for colour change by detecting absorbance of pH indicator. The colour change was resulted from the releasing of pH indicator molecules from carbon nitride nanosheets triggered by alkali solution (AS). In this novel PILISA, the intensity absorbance of pH indicator is proportional to the concentration of the disease marker. The outstanding detection performance of the PILISA can be attributed to the following reasons: (1) ultrathin carbon nitride nanosheets with a larger surface area could adsorb abundant phenolphthalein (PP) molecules through hydrophobic interactions as well as the resulted PP anions can be free easily released into aqueous solution, leading to an obvious allochroic response; (2) the signal intensity is precisely determined by the amount of PP molecules loading onto the carbon nitride nanosheets surface, which ensures simple, low-cost and stable colorimetric detection. As expected, this new PILISA method offered an enzyme-free approach followed enzyme-linked immunosorbent assay format, which showed great promising potential as an innovative robust assay method for practical clinical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. BOVINE LEUKEMIA VIRUS INFECTION IN TAIWAN : EVALUATION OF THE ENZYME-LINKED IMMUNOSORBENT ASSAY AND AGAR GEL IMMUNODIFFUSION TEST

    OpenAIRE

    WANG, Chun-Tshen

    1991-01-01

    I evaluated an enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test simultaneously for the detection of bovine leukemia virus (BLV) antibodies. Total 1,293 serum samples were tested for ELISA and AGID test and the results were compared. The results of ELISA and AGID agreed by 1,156 out of 1,293 (89.4%). All of AGID-positive 356 sera were positive by ELISA. However, of 451 ELISA-positive sera, 95 sera were either negative or equivocal by AGID test. Eleven animals ...

  18. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Brucella melitensis-Specific Antibodies in Goat Milk

    OpenAIRE

    Funk, N. D.; Tabatabai, L B; Elzer, P. H.; Hagius, S D; Martin, B. M.; Hoffman, L J

    2005-01-01

    Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection...

  19. Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus

    Science.gov (United States)

    Wilson, Anna; Goldberg, Tony; Marcquenski, Susan; Olson, Wendy; Goetz, Frederick; Hershberger, Paul; Hart, Lucas M.; Toohey-Kurth, Kathy

    2014-01-01

    Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

  20. Technical and Diagnostic Performance of Five Commercial Anti-Diphtheria Toxoid IgG Enzyme-Linked Immunosorbent Assay Kits ▿

    OpenAIRE

    Faruq, A.; Dadson, L.; H Cox; Alcock, F.; Parker, A R

    2010-01-01

    The technical and diagnostic performances of five commercially available enzyme-linked immunosorbent assays for the measurement of anti-diphtheria toxoid IgG antibodies were evaluated. There was good agreement between the relative sensitivities of the five assays, but the relative specificity of one of the assays differed from that of the other four assays. Three of the five assays possessed recoveries of the international reference material NIBSC 00/496 within the range of 90% to 110% at ant...

  1. Development of an enzyme-linked immunosorbent assay (ELISA) for identification of venoms from snakes in the Agkistrodon genus.

    Science.gov (United States)

    Li, Q; Ownby, C L

    1994-11-01

    An enzyme-linked immunosorbent assay (ELISA) using a purified myotoxin from the venom of Agkistrodon contortrix laticintus (broad-banded copperhead) as immunogen was developed for potential use in the identification of envenomation by snakes belonging to the genus Agkistrodon native to North America. The specificity of the assay was tested using a total of 43 venom samples from snakes of diverse geographic locations. Venom samples used for cross-reactivity determination represent eight snake families including 14 species from the genus Crotalus. The assay detected venom from all Agkistrodon species tested without significant cross-reactivity with other venoms except for samples from two species of Bothrops which do not occur naturally north of Southern Mexico. The detection limit of the assay was 2 ng/ml for homologous crude venom dissolved in normal human serum. The assay was highly accurate in correlating optical densities with venom concentrations (r = 0.997). The presence of the antigen in experimental envenomations was readily detected by the assay at an i.m. injection dosage of 0.1 microgram/g. This ELISA is a promising test for identification of envenomations by species of Agkistrodon found in most of North America. It can also be used to study the kinetics of the myotoxin in experimental envenomations.

  2. Detection of mutations by fill-in ligation reaction with enzyme-linked immunosorbent assay for rapid medical diagnosis.

    Science.gov (United States)

    Tang, Yi-Tong; Xiao, Na; Li, Zhi-Shan; Zou, Jiu-Ming; Cao, Rui; Zhao, Xue-Hong; Shao, Jin-Hui

    2014-01-01

    Several approaches for parallel genotyping have been developed with increasingly available information on DNA variation. However, these methods require either complex laboratory procedures or expensive instrumentation. None of these procedures is readily performed in local clinical laboratories. In this study, we developed a flexible genotyping method involving fill-in ligation reaction with enzyme-linked immunosorbent assay successfully applied to detect important single-nucleotide polymorphisms (SNPs) for EGFR c.2573T > G (L858R), EGFR c.2582T > A (L861Q), and EGFR c.2155G > T (G719C). This assay exhibited excellent specificity, with a sensitivity as low as 0.5%. Eight out of 62 clinical samples were identified as heterozygotes for the SNP site of L858R, whereas only two samples were identified as heterozygotes by direct sequencing. The developed method enabled accurate identification of SNP in a simple and cost-effective manner adapted to routine analysis.

  3. Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

    Directory of Open Access Journals (Sweden)

    Roldán William

    2006-01-01

    Full Text Available A dot enzyme-linked immunosorbent assay (dot-ELISA was standardized using excretory-secretory antigens of Toxocara canis for the rapid immunodiagnosis of human toxocariasis. Thirty patients with clinical signs of toxocariasis, 20 cases with other parasitic diseases, and 40 healthy subjects were tested. A total of 0.2 ng of antigen per dot, serum dilution of 1:160 and dilution conjugate of 1:1000 were found optimal. The sensitivity and specificity of the assay were 100 and 95%, respectively. Comparable sensitivity of dot-ELISA and the standard ELISA was obtained, but only 3 cross-reactions occurred in the dot-ELISA, compared with 6 in the standard ELISA. Dot-ELISA is simple to perform, rapid, and low cost. Large-scale screening studies should be done to evaluate its usefulness under field conditions.

  4. The development of an Enzyme Linked Immunosorbent Assay for detecting Injectious laryngotrachitis viral antibodies in chicken serum

    Directory of Open Access Journals (Sweden)

    Risa Indriani

    2002-06-01

    Full Text Available The aim of this study was to develop an Enzyme-linked immunosorbent assay (ELISA for detection of antibody against gallid herpes virus, the causal agent of infectious laryngotracheitis (ILT in chicken. Its application in experimental chicken under laboratory condition was also evaluated. Results showed that ELISA for ILT was developed successfully with sensitivity and specificity was 98% and 97,14% respectively. The ELISA was able to determine the dynamic of antibodies respond in experimental chickens following vaccination and artificial infection with ILT virus. It was concluded that this ELISA offers a simple, sensitive and specific antibody assay for detection of antibodies against ILT virus in chickens arising from vaccination or infection.

  5. Evaluation of a chemiluminescent enzyme-linked immunosorbent assay for the diagnosis of Trypanosoma cruzi infection in a nonendemic setting

    Directory of Open Access Journals (Sweden)

    Luis Izquierdo

    2013-11-01

    Full Text Available The disappearance of lytic, protective antibodies (Abs from the serum of patients with Chagas disease is accepted as a reliable indicator of parasitological cure. The efficiency of a chemiluminescent enzyme-linked immunosorbent assay based on a purified, trypomastigote-derived glycosylphosphatidylinositol-anchored mucin antigen for the serologic detection of lytic Abs against Trypanosoma cruzi was evaluated in a nonendemic setting using a panel of 92 positive and 58 negative human sera. The technique proved to be highly sensitive {100%; 95% confidence interval (CI = 96-100} and specific (98.3%; 95% CI = 90.7-99.7, with a kappa score of 0.99. Therefore, this assay can be used to detect active T. cruzi infection and to monitor trypanosomicidal treatment.

  6. A solid phase enzyme-linked immunosorbent assay for the antigenic detection of Legionella pneumophila (serogroup 1): A compliment for the space station diagnostic capability

    Science.gov (United States)

    Hejtmancik, Kelly E.

    1987-01-01

    It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events and environmental monitoring during long periods of space flight. The application of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be facilitated through employment of serological methods to aid in the identification of bacterial, fungal, and viral agents. A number of serological approaches are currently being considered, including the use of Enzyme Linked Immunosorbent Assay (ELISA) technology, which could be utilized during microgravity conditions. A solid phase, membrane supported ELISA for the detection of Legionella pneumophila, an expected disease agent, was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. These studies demonstrate the capability of membrane supported ELISA systems for identification of expected microbial disease agents as part of the HMF.

  7. Anti-Protective Antigen IgG Enzyme-Linked Immunosorbent Assay for Diagnosis of Cutaneous Anthrax in India

    Science.gov (United States)

    Ghosh, N.

    2012-01-01

    Anthrax caused by Bacillus anthracis is a public health problem in several developing countries whose main source of income is farming. Anthrax is a disease of herbivorous animals, and humans can be infected by handling infected animals or contaminated animal products. Specific diagnostic tests are unavailable in India for the detection and confirmation of cutaneous anthrax in humans. Here, we describe the development of an enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies against Bacillus anthracis protective antigen in the Indian population. A total of 405 serum samples collected from different groups were tested by the developed ELISA. The assay provided a specificity of 99.41% (95% confidence interval [CI], 97.89 to 99.93) and a sensitivity of 100% (CI, 94.4 to 100) using a cutoff value of 0.29 ELISA unit (EU). The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 97% and 100%, respectively. The efficiency and J index for the reliability of the assay were 99.5% and 0.994, respectively. The assay can be a very useful tool for surveillance as well as for diagnosis of cutaneous anthrax cases in India. PMID:22718130

  8. Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sails, A D; Bolton, F J; Fox, A J; Wareing, D R A; Greenway, D L A

    2002-03-01

    A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.

  9. Establishment of an Enzyme Linked Immunosorbent Assay for Total Triiodothyronine (T3)

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A sensitive and specific ELISA for total triiodothyronine (T3) is established. The anh-T3 antibody iscoated on the microtiter plate, the T4 antigen is conjugated to the biotin. The label is horseradishperoxidase(HRP) conjugate of streptavidin. TMB-H2O2 solution is used as the substrate of HRP.Thesensitivity of the assay is 0.2 ng/mL, the intra-assay CVs and the intre-assay CVs of 3 samples are lower

  10. An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle

    NARCIS (Netherlands)

    Oirschot, van J.T.; Kaashoek, M.J.; Maris-Veldhuis, M.A.; Weerdmeester, K.; Rijsewijk, F.A.M.

    1997-01-01

    A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and catt

  11. A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples.

    Science.gov (United States)

    Zhang, Xian; Wang, Xin; Sun, Mengjiao; Zhang, Xiaofeng; Song, Houhui; Yan, Yaxian; Sun, Jianhe; Li, Xiaoliang; Fang, Weihuan

    2015-10-20

    A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = -0.4287x + 0.3132 (R² = 0.9904). The working range was 0.07-2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%-111.9% and 91.7%-114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R² = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.

  12. A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples

    Directory of Open Access Journals (Sweden)

    Xian Zhang

    2015-10-01

    Full Text Available A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA was developed for rapid and sensitive detection of zearalenone (ZEN. The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R2 = 0.9904. The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R2 = 0.9283. We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.

  13. Analytical characterization and clinical evaluation of an enzyme-linked immunosorbent assay for measurement of afamin in human plasma☆

    Science.gov (United States)

    Dieplinger, Benjamin; Egger, Margot; Gabriel, Christian; Poelz, Werner; Morandell, Elisabeth; Seeber, Beata; Kronenberg, Florian; Haltmayer, Meinhard; Mueller, Thomas; Dieplinger, Hans

    2013-01-01

    Background Comparative proteomics has recently identified afamin, the newest member of the albumin gene family, as a potential biomarker for ovarian cancer. The aim of this study was the analytical and clinical evaluation of a sandwich enzyme-linked immunosorbent assay for the determination of afamin in human plasma. Methods We evaluated precision, linearity, and detection limit of the assay, analyte stability and biological variability, determined reference values and quantified afamin concentrations in various diseases. Results Within-run and total coefficients of variation were < 10%. The method was linear across the tested measurement range. Detection limit was 7 mg/L for the assay. The analyte was stable for 24 h at room temperature, for 48 h at 4 °C, and for at least one year at − 20 °C and − 80 °C. The reference change value for healthy individuals was 24%. Age- and sex-independent reference values in healthy blood donors were 45–99 mg/L (median 68 mg/L). In the clinical assay evaluation afamin plasma concentrations were modestly decreased in patients with heart failure. Patients with pneumonia or sepsis exhibited markedly decreased afamin plasma concentrations. However, patients with chronic renal disease or chronic obstructive pulmonary disease showed no difference in afamin plasma concentrations as compared to healthy individuals. Correlation analyses revealed an inverse association between afamin and inflammatory biomarkers. Conclusions The afamin assay meets quality specifications for laboratory medicine. The results of the clinical assay evaluation revealed novel insights with respect to afamin as a potential negative acute phase protein and should encourage further studies. PMID:23981841

  14. Development of atom transfer radical polymer-modified gold nanoparticle-based enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Chen, Feng; Hou, Shike; Li, Qingsheng; Fan, Haojun; Fan, Rong; Xu, Zhongwei; Zhala, Gahu; Mai, Xia; Chen, Xiaoyi; Chen, Xuyi; Liu, Yingfu

    2014-10-21

    In this work, a novel enzyme-linked immunosorbent assay (ELISA) with a low limit of detection and high sensitivity was developed using atom transfer radical polymer (ATRP)-modified gold nanoparticles (AuNPs). Clear signal amplification was achieved by introducing an abundance of horseradish peroxidase (HRP) to the AuNPs, because of the ATRP modification. This result suggested that the new ELISA was able to detect antigens in complex mixtures, and the limit of detection (LOD) was lower than that of conventional ELISA by a factor of 81. The new ELISA strategy greatly decreased the LOD during analysis and exhibited excellent reproducibility, stability, and feasibility. Therefore, it is a promising technique with many potential applications in biochemistry and medical science research.

  15. An enzyme-linked immunosorbent assay (ELISA) for the identification of Naegleria fowleri in environmental water samples.

    Science.gov (United States)

    Reveiller, Fabienne L; Varenne, Marie-Pierre; Pougnard, Claire; Cabanes, Pierre-Andre; Pringuez, Emmanuelle; Pourima, Benedicte; Legastelois, Stephane; Pernin, Pierre

    2003-01-01

    Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis, a fatal human disease of the central nervous system often contracted after swimming in fresh water. Identifying sites contaminated by N. fowleri is important in order to prevent the disease. An Enzyme-Linked ImmunoSorbent Assay (ELISA) has been developed for the specific identification of N. fawleri in primary cultures of environmental water samples. Of 939 samples isolated from artificially heated river water and screened by ELISA, 283 were positive. These results were subsequently confirmed by isoelectric focusing, the established reference method. A sensitivity of 97.4% and a specificity of 97% were obtained. These results indicate that this ELISA method is reliable and can be considered as a powerful tool for the detection of N. fowleri in environmental water samples.

  16. Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

    Directory of Open Access Journals (Sweden)

    Flábio R Araújo

    2005-11-01

    Full Text Available Indirect enzyme-linked immunosorbent assays (ELISAs based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests and specificities (100% for both tests were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

  17. Application of enzyme-linked immunosorbent assay for quantification of the insecticides imidacloprid and thiamethoxam in honey samples.

    Science.gov (United States)

    Ma, Huixin; Xu, Yanjun; Li, Qing X; Xu, Ting; Wang, Xintong; Li, Ji

    2009-05-01

    An enzyme-linked immunosorbent assay (ELISA) was used for the determination of residues of imidacloprid and thiamethoxam insecticides in honey after simple dilution of the samples without either extraction or cleanup. The ELISA enabled accurate determination of imidacloprid and thiamethoxam down to limits of 20 and 5 ng g(-1) in honey, respectively. Average recoveries of imidacloprid and thiamethoxam from the fortified honey samples were 90-120 and 96-122%, and coefficients of variation ranged 5-12 and 3-15%, respectively. The results from the ELISA agreed well with those by liquid chromatography-mass spectrometry (LC-MS) for the insecticide-spiked samples, with a correlation coefficient (r(2)) of 0.96 and a regression coefficient (slope) of 1.03. The results indicate that ELISA is a suitable tool for the quantification of imidacloprid and thiamethoxam in honey.

  18. An enzyme-linked immunosorbent assay, using an EDTA-extracted antigen for the serology of Haemophilus pleuropneumoniae.

    Science.gov (United States)

    Nicolet, J; Paroz, P; Krawinkler, M; Baumgartner, A

    1981-12-01

    An enzyme-linked immunosorbent assay (ELISA) was proposed as an alternative to the complement-fixation test (CF) for the detection of antibodies of Haemophilus pleuropneumoniae, agent of the pleuropneumonia in pigs. In tests done with different antigen-extraction procedures (including Tween 20, sodium dodecyl sulfate, aqueous phenol, sonification, and heat treatment at 120 C), ethylenediaminetetraacetic acid (EDTA) provided a satisfactorily reactive antigen. Chromatography purification on Sephacryl S200 improved the specificity of this antigen. Using hyperimmune rabbit sera, we investigated the specificity and the sensitivity of the ELISA with the EDTA-purified antigen of the different serotypes of H pleuropneumoniae on selected swine sera in herds with confirmed H pleuropneumoniae infection, from specific-pathogen-free animals showing doubtful CF reactions. The ELISA proved to be highly specific and more sensitive than the CF test. Furthermore, evidence of cross-reactions with H parasuis, a common bacteria isolated in swine populations, was not found.

  19. Evaluation of immunofluorescence microscopy and enzyme-linked immunosorbent assay in detection of Cryptosporidium and Giardia infections in asymptomatic dogs

    DEFF Research Database (Denmark)

    Rimhanen-Finne, R.; Enemark, Heidi L.; Kolehmainen, J.;

    2007-01-01

    The performance of immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA) in canine feces was evaluated. IF and Cryptosporidium ELISA detected 10(5) oocysts/g, while the detection limit for Giardia ELISA was 10(4) cysts/g. The Cryptosporidium ELISA showed 94% specificity...... but only 71% sensitivity. The Giardia ELISA correlated well with IF (sensitivity 100%, specificity 96%) and was capable of detecting animal specific Giardia duodenalis genotypes. Visual interpretation appeared appropriate for assessment of ELISA results. The proportion of positive samples and possible...... zoonotic character of Cryptosporidium and Giardia infections in 150 asymptomatic Finnish dogs from the Helsinki area were studied. The overall proportion of dogs positive for Cryptosporidium was 5% (7/150) and that for Giardia 5% (8/150). In dogs...

  20. Gelatin particle indirect agglutination and enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis using Strongyloides venezuelensis antigen.

    Science.gov (United States)

    Huaman, Maria Cecilia; Sato, Yoshiya; Aguilar, Jose Luis; Terashima, Angelica; Guerra, Humberto; Gotuzzo, Eduardo; Kanbara, Hiroji

    2003-01-01

    Routine microscopical examination of stool specimens for diagnosis of strongyloidiasis is insensitive and serological methods using Strongyloides stercoralis antigen are at present not available for field studies. We evaluated 2 techniques, enzyme-linked immunosorbent assay (ELISA) and gelatin particle indirect agglutination (GPIA), using an antigen obtained from the rodent parasite, S. venezuelensis. Fifty-four Peruvian patients with different clinical forms of strongyloidiasis were studied: 12 asymptomatic, 31 symptomatic, and 11 hyperinfection cases. Our results demonstrate that both ELISA and GPIA using S. venezuelensis antigen are useful for diagnosis of strongyloidiasis, with sensitivities of 74.1% and 98.2%, respectively and a specificity of 100% for both techniques. We found that GPIA is a highly sensitive test for patients with suspected chronic infection and/or hyperinfection. In the hyperinfection cases, significantly lower concentrations of specific immunoglobulin antibodies and eosinophils (P < 0.001) were found compared with the asymptomatic and symptomatic cases.

  1. Seroprevalence of Japanese encephalitis virus using competitive enzyme linked immunosorbent assay (C-ELISA in pigs in East Sumba, Indonesia

    Directory of Open Access Journals (Sweden)

    Annytha Detha

    2015-12-01

    Full Text Available Japanese Encephalitis (JE, a vector-borne zoonotic viral disease, is mostly prevalent in Asian countries. The objective of this study was to investigate the occurence of JE virus (JEV among pigs in East Sumba, Indonesia. Blood samples (n=52 were randomly collected from 52 apparantly healthy pigs where pig population was high in East Sumba. The samples were subjected for seroprevalence study for the presence of antibodies against JEV using competitive enzyme linked immunosorbent assay (C-ELISA. Results showed that 53% (n=28/52 blood samples from the pigs contained antibodies against JEV. This finding is suggestive that the JEV is circulating among pig population in East Sumba, Indonesia. The data may help in designing control strategies of the JEV in the East Sumba, Indonesia.

  2. The Establishment of an Enzyme Linked Immunosorbent Assay for Human Thyrotropin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A sensitive and specific ELISA for human thyrotropin(hTSH) is established with using two anti-hTSH monoclonal antibody. One of them is coated on the microtiter plate, the other is conjugate of biotin. The label is horseradish peroxidase(HRP) conjugate of streptavidin. TMB-H2O2 solution is used as the substrate of HRP. The sensitivity of the assay is 0.02 mIU/L. The intra-assay CVs and the intre-assay CVs of 3 samples are lower than 7.8% and 9.6%, respectively. The analytical recoveries are in the range from 93.3% to 107.8%. The assay is specific for hTSH and no cross reaction with other glyco-proteins, such as hLH , hHCG, hFSH. TSH concentrations range from 0.3 to 4,1 mlU/L in 142 normal subjects. As a reference IRMA(Multipact) method, the

  3. Quantitative determination of recombinant bovine somatotropin in commercial shrimp feed using a competitive enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Munro, James L; Boon, Virginia A

    2010-02-10

    Recombinant bovine somatotropin (rbST), also known as growth hormone, is used to enhance production and development of animals within the agriculture and aquaculture industries. Its use is controversial because of its potential effects on human and animal health. To screen for rbST in shrimp feed, a competitive enzyme-linked immunosorbent assay (ELISA) with an inhibition step was developed. Sample and rbST antibody (rabbit anti-rbST) were incubated at room temperature for 30 min. Subsequently, this competitive reaction was transferred to a microplate coated with rbST, using goat antirabbit IgG linked with horseradish peroxidise as the secondary antibody. Substrates for peroxidise were added, and the absorbance at 410 nm was determined. The applicability of the method was assessed using rbST extracted from "spiked" shrimp feed samples. The assay was reproducible and linear with R(2) values greater than 0.98 over the standard curve range of 20-500 microg/g. The intra- and interday precisions expressed as relative standard deviations were 3.4 and 5.3%, respectively. The mean recovery from 15 spiked feed samples was 105%. This assay will be a valuable tool for quantitative detection of rbST by both governments and commercial companies and can be modified for other types of feed.

  4. Screening of Dengue virus antiviral activity of marine seaweeds by an in situ enzyme-linked immunosorbent assay.

    Directory of Open Access Journals (Sweden)

    Andrea Cristine Koishi

    Full Text Available Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization.

  5. Enzyme-linked immunosorbent assay for serological diagnosis of Nocardia brasiliensis and clinical correlation with mycetoma infections.

    Science.gov (United States)

    Salinas-Carmona, M C; Welsh, O; Casillas, S M

    1993-11-01

    We previously identified three immunodominant antigens obtained from a Nocardia brasiliensis cell extract and recognized by sera from mycetoma patients (M. C. Salinas-Carmona, L. Vera, O. Welsh, and M. Rodríguez, Zentralbl. Bakteriol. 276:390-397, 1992). In the present work, we obtained a crude extract from a mass culture of N. brasiliensis HUJEG-1 and purified two immunodominant antigens, the 26- and 24-kDa proteins, by using simple physiochemical techniques. With these antigens, we developed a conventional solid-phase enzyme-linked immunosorbent assay and tested 30 serum samples from mycetoma patients, 29 from tuberculosis patients, 24 from a leprosy group, and 31 from healthy individuals. Our results show for the first time statistically significant differences in serology among these groups. All mycetoma patients with a positive culture for N. brasiliensis had absorbance values higher than 0.3. On the other hand, the mycobacterium-infected patients as well as the healthy individuals all had absorbance values below that level. Moreover, we found a close correlation between the clinical condition of the mycetoma patients and the anti-26- and anti-24-kDa protein antibody concentrations. We therefore propose the use of this assay in routine clinical laboratories to confirm the diagnosis of N. brasiliensis infection in human mycetoma cases. In addition, the possible application of this assay in the serodiagnosis of Nocardia asteroides infection is also discussed.

  6. Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

    Science.gov (United States)

    Rodak, L; Babiuk, L A; Acres, S D

    1982-07-01

    The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

  7. Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Fujita, Osamu; Uda, Akihiko; Morikawa, Shigeru; Yamada, Akio; Tanabayashi, Kiyoshi

    2013-01-01

    A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

  8. An indirect competitive enzyme-linked immunosorbent assay for determination of norfloxacin in waters using a specific polyclonal antibody.

    Science.gov (United States)

    Cui, Jianlan; Zhang, Kun; Huang, Qiuxin; Yu, Yiyi; Peng, Xianzhi

    2011-02-28

    A specific polyclonal anti-norfloxacin antibody was obtained, and a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for determining trace amounts of norfloxacin in various waters. Good linearity was achieved in the range from 0.1 to 10 μg L(-1). The average IC(50) value was determined to be 2.2 μg L(-1) and the limit of detection was 0.016 μg L(-1) at a signal-to-noise ratio of 3 in phosphate-buffered saline buffer. Recoveries of norfloxacin at various spiking levels ranged from 74 to 105% in groundwater, surface water, treated and untreated wastewater samples, with relative standard deviations of 3-5%. The assay was applied for determining norfloxacin in municipal wastewater, surface water, and groundwater collected in a metropolis of China. Raw wastewater samples were only submitted to filtration and pH adjustment while the other water samples were pre-concentrated by solid phase extraction prior to the icELISA assay. Good agreement of the results obtained by the icELISA and liquid chromatography tandem mass spectrometry further confirmed the reliability and accuracy of the icELISA for rapid detection of norfloxacin in waters. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Enzyme-linked immunosorbent assay for detection of streptolysin O antibodies.

    OpenAIRE

    Reitano, M; Pisano, M. A.; Eriquez, L A; D'Amato, R F

    1986-01-01

    An enzyme-linked immumosorbent assay (ELISA), based upon the detection of streptolysin O antibodies in human sera, was developed. Disposable polystyrene tubes, sensitized with streptolysin O antigen, were used as the test vehicles. Corresponding antibodies, present in test sera, were detected by binding of the antibodies to goat anti-human immunoglobulin G conjugated to horseradish peroxidase. Demonstration of bound conjugate was accomplished by monitoring peroxidase activity spectrophotometr...

  10. Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

    Indian Academy of Sciences (India)

    Harry E Grates; Richard M Mc Gowen; Smiti V Gupta; John R Falck; Thomas R Brown; Denis M Callewaert; Diane M Sasaki

    2003-02-01

    Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a proprietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3′,5,5,′-tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0.5 ng/ml for the AP assay, with 2 = 0.99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.

  11. Measuring immunoglobulin g antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay.

    Science.gov (United States)

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz

    2008-05-01

    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an "in-house" IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.

  12. Validation of a KHV antibody enzyme-linked immunosorbent assay (ELISA)

    DEFF Research Database (Denmark)

    Bergmann, S M; Wang, Q; Zeng, W

    2017-01-01

    Koi herpesvirus (KHV) causes KHV disease (KHVD). The virus is highly contagious in carp or koi and can induce a high mortality. Latency and, in some cases, a lack of signs presents a challenge for virus detection. Appropriate immunological detection methods for anti-KHV antibodies have not yet been...... and was particularly useful at the pond or farm levels. Considering the scale of the carp and koi industry worldwide, this assay represents an important practical tool for the indirect detection of KHV, also in the absence of clinical signs....

  13. Enzyme-Linked Immunosorbent Assay Using Vertical Micro Reactor Stack for the Detection of Biomolecules

    Science.gov (United States)

    Matsui, Katsuhiro; Morimoto, Syohei; Asano, Toshifumi; Ukita, Yoshiaki; Kato, Dai-Ichiro; Takeo, Masahiro; Utsumi, Yuichi; Negoro, Seiji

    Microreactors and micro total analysis system (μTAS) are recognized as powerful tools for genomics, proteomics, clinical diagnostics, and environmental testing. In this paper, we describe enzyme linked immunosorvent assay (ELISA) using a new microreactor with a vertical fluid flow operation. This microreactor is composed of two reaction vessels stacked on the vertical lines through PMMA fluid filters (φ3mm). The fluid filters constructed by deep X-ray lithography possess 2,100 pores (φ 40 μm), and have valve functions, which maintain liquid layer in each reaction vessel. In addition, the liquid can be selectively transferred by air pressure from upper vessel to lower, and vice versa. As a model of ELISA using the microreactor, we planed to detect mouse immunoglobulin (IgG). We bound the goat anti-IgG antibody to the surface of the PMMA filters, and assayed the IgG by ELISA using anti-IgG antibody/ peroxidase conjugate. We found that the mouse IgG (100 ng/ml) was quantitatively detected within 45 min of analytical period, which was ca. 1/3 of the period required for the conventional method using micro titer plate.

  14. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

    Science.gov (United States)

    Pickering, Jerry W; Martins, Thomas B; Schroder, M Carl; Hill, Harry R

    2002-07-01

    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

  15. Preparation of a specific monoclonal antibody to asiaticoside for the development of an enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Juengwatanatrakul, Thaweesak; Sritularak, Boonchoo; Amornnopparattanakul, Paveena; Tassanawat, Patcharin; Putalun, Waraporn; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-03-01

    Asiaticoside (AS), the major active component of Centella asiatica (L.) Urban, is used as a memory enhancer and for wound healing. We have successfully prepared monoclonal antibodies (MAbs) against AS, and developed an enzyme-linked immunosorbent assay (ELISA) system for its determination. AS was conjugated to the carrier protein bovine serum albumin (BSA), which acted as an immunogen. In order to confirm its immunogenicity, the ratio of hapten in the AS-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting MAbs against AS were produced by fusing splenocytes with the mouse myeloma cell line, SP2/0-Ag14. After the screening, anti-asiaticoside MAb 2B4 was obtained. Weak cross-reactivities occurred with madecassoside (7.08%), but no cross-reactivities were observed with other related triterpenoid glycosides (<0.01%). The assay was suitable for quantitating AS in the range of 0.78 to 50 µg mL(-1). A good correlation of AS concentrations in crude extracts of C. asiatica between ELISA and HPLC methods was obtained (r(2) = 0.999). The contents of AS in various cultivated C. asiatica samples were assayed by the newly established ELISA. The recovery rates of AS in the samples were in the range of 95-103% with coefficients of variation of <10%. The intra- and inter-assay variations were 3.9 and 4.5%, respectively. The ELISA method described should prove useful as an analytical tool for quality control and standardization of medicinal plants and pharmaceutical products containing AS.

  16. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Igoh, Akihisa; Tomotake, Sho; Doi, Yusuke

    2015-05-01

    Saliva is one of the most common body fluids found at a crime scene. Therefore, identifying saliva is important in forensic science. However, the current protein marker assays used to identify saliva are not sufficiently specific. Although proline-rich proteins (PRPs) are highly specific for saliva, their forensic potential has not yet been investigated. In this study, we developed enzyme-linked immunosorbent assays (ELISAs) to detect acidic salivary PRP HaeIII subfamily 1/2 (PRH1/2) and basic salivary PRP 2 (PRB2). The specificity, sensitivity, and efficiency of the ELISAs for PRH1/2 and PRB2 were compared with those of the ELISA for statherin (STATH), a known protein marker for saliva. The levels of PRH1/2 were significantly higher in saliva and saliva stains than in other body fluids (nasal secretions, urine, semen, vaginal fluid, blood, and sweat). PRB2 and STATH were detected in both nasal secretions and saliva. The PRH1/2 ELISA showed sensitivity similar to that of STATH ELISA. The detection rate of PRH1/2 ELISA was almost similar to that of STATH ELISA, followed by the ELISA for PRB2. The PRH1/2 ELISA had higher specificity for saliva than STATH ELISA. Therefore, the PRH1/2 ELISA has potential as a method to identify saliva for forensic investigation.

  17. Synthesis of novel haptens and development of an enzyme-linked immunosorbent assay for quantification of histamine in foods.

    Science.gov (United States)

    Luo, Lin; Xu, Zhen-Lin; Yang, Jin-Yi; Xiao, Zhi-Li; Li, Yong-Jun; Beier, Ross C; Sun, Yuan-Ming; Lei, Hong-Tao; Wang, Hong; Shen, Yu-Dong

    2014-12-24

    Novel haptens were designed and synthesized to prepare antibodies against free histamine, but none resulted in producing suitable antibodies for developing an enzyme-linked immunosorbent assay (ELISA). However, an antiserum was obtained having high specificity and affinity to p-nitrobenzoylated histamine (NPHA), which can be easily formed from reaction between histamine and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions. Based on rabbit polyclonal antibodies, a competitive indirect ELISA (ciELISA) for histamine determination in foods was developed. After ciELISA and derivatization optimization, the assay showed good sensitivity, with limits of detection of 1.8 mg/kg, 93.6 μg/L, and 93.6 μg/kg in fish, red wine, and yoghurt, respectively, with negligible cross-reactivity with related biogenic amines and amino acids. Average recovery of histamine in fortified food samples ranged from 80.9% to 110.1% with coefficients of variation below 16.3%. Good correlation between the ciELISA and liquid chromatography-tandem mass spectrometry was obtained for spiked food samples.

  18. Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

    Science.gov (United States)

    Quinn, Conrad P; Semenova, Vera A; Elie, Cheryl M; Romero-Steiner, Sandra; Greene, Carolyn; Li, Han; Stamey, Karen; Steward-Clark, Evelene; Schmidt, Daniel S; Mothershed, Elizabeth; Pruckler, Janet; Schwartz, Stephanie; Benson, Robert F; Helsel, Leta O; Holder, Patricia F; Johnson, Scott E; Kellum, Molly; Messmer, Trudy; Thacker, W Lanier; Besser, Lilah; Plikaytis, Brian D; Taylor, Thomas H; Freeman, Alison E; Wallace, Kelly J; Dull, Peter; Sejvar, Jim; Bruce, Erica; Moreno, Rosa; Schuchat, Anne; Lingappa, Jairam R; Martin, Sandra K; Walls, John; Bronsdon, Melinda; Carlone, George M; Bajani-Ari, Mary; Ashford, David A; Stephens, David S; Perkins, Bradley A

    2002-10-01

    The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

  19. Electrical signaling of enzyme-linked immunosorbent assays with an ion-sensitive field-effect transistor.

    Science.gov (United States)

    Jang, Hyun-June; Ahn, Junhyoung; Kim, Min-Gon; Shin, Yong-Beom; Jeun, Minhong; Cho, Won-Ju; Lee, Kwan Hyi

    2015-02-15

    Optical laboratory-based immunoassays, such as enzyme-linked immunosorbent assay (ELISA) give a high sensitivity and specificity of various fatal diseases. However, these assays are no longer efficient in on-spot diagnostics of wide-spreading and contagious infections. At this point in time, portable and handhold devices play a pivotal role in infectious diseases with quick diagnostics at or near the site of the disease propagation. In this paper, we demonstrated a novel electrical immunoassay of ELISA that was not based on optical signaling but on electrical signaling. This was done by combining an ion-sensitive field-effect transistor (ISFET) with ELISA. By harnessing the catalytic reaction of alkaline phosphatase that precipitated silver particles, we effectively overcame the chronic Debye screening length issue of the ISFET. Ultimately, small signal ranging from 1 pg/mL to 10 ng/mL was immensely amplified with the ALP label, regardless of buffer conditions. The sensor platform herein surpassed a sensing capability of conventional ELISA that is considered to have a LOD on the order of ~1 ng/mL. The results were compared with those of horseradish peroxidase label, which is generally used for optical analyses in ELISA. Our newly developed ISFET-based portable sensor holds a large potential for point-of-care tools in a variety of diseases, without being limited by the need for expensive equipment such as spectrophotometers.

  20. A highly rapid and simple competitive enzyme-linked immunosorbent assay for monitoring paralytic shellfish poisoning toxins in shellfish.

    Science.gov (United States)

    Kawatsu, Kentaro; Kanki, Masashi; Harada, Tetsuya; Kumeda, Yuko

    2014-11-01

    Using a streptavidin-coated well plate, a biotin-labelled anti-gonyautoxin 2/3 monoclonal antibody GT-13A, and a decarbamoyl saxitoxin-peroxidase conjugate, a direct competitive enzyme-linked immunosorbent assay (PSP-ELISA) was developed for monitoring paralytic shellfish poisoning (PSP) toxins in shellfish. This assay is simple to perform and can be completed in approximately 20 min. The PSP-ELISA was compared to the mouse bioassay (MBA) for the detection of PSP toxins in shellfish samples (n=83) collected from the coast of Osaka Prefecture, Japan. When positive and negative results were indicated based on the regulatory limit for PSP toxins (4 mouse unit(MU)/g of shellfish meat), the PSP-ELISA results showed a sensitivity of 100% (25 of 25) and a specificity of 89.7% (52 of 58 samples) compared to the MBA results. These results suggest that the PSP-ELISA could be used as a rapid and simple screening method prior to the MBA.

  1. Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Anaplasma phagocytophilum in sheep.

    Science.gov (United States)

    Woldehiwet, Z; Yavari, C

    2012-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Anaplasma phagocytophilum in ovine serum samples was evaluated. The assay used purified A. phagocytophilum grown in tick cell cultures as antigen. Serum samples were diluted 1 in 200 and binding was detected with anti-sheep IgG conjugated to horseradish peroxidase. All tests were carried out in the presence of positive and negative control samples. Optical density (OD) values obtained for each test sample at 490 nm were used to calculate percentage positivity (PP) of each sample based on the ratio of the OD of the test sample that of the positive reference sample. Known negative samples (n=69) obtained from uninfected sheep bred and maintained in a tick-free environment and subsequently shown to be susceptible to A. phagocytophilum were used to establish the cut-off point between negative and positive samples and to establish the specificity of the test. Serum samples obtained from 92 animals 14-21 days after infection were used to establish the sensitivity of the test. Using a cut-off point of 20PP (mean+2 standard deviations of the PP of 69 control samples) the test was shown to have a sensitivity of 84.8% and a specificity of 95.7%. Lowering the cut-off point to 15PP increased the sensitivity to 94.6%, but reduced the specificity to 92.8%.

  2. Development of monoclonal antibodies and quantitative sandwich enzyme linked immunosorbent assay for the characteristic sialoglycoprotein of edible bird's nest.

    Science.gov (United States)

    Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

    2013-01-01

    The article presents a sandwich enzyme linked immunosorbent assay (ELISA) for identification of edible bird's nest. The characteristic sialoglycoproteins were found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by liquid-phase isoelectric focusing (LIEF). According to the analysis, the molecular weight was 106-128 kDa and the isoelectric point was ≤pH 3.0. Two anti-characteristic sialoglycoprotein monoclonal antibodies were produced. The monoclonal antibodies were examined by western-blot assay. One of the monoclonal antibody was used as coating and the other as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). Based on the optimized ELISA condition, the method was established with IC(50) of 1.5 ng/mL, and low cross-reactivity with various fake materials (ELISA provided a suitable means for screening of a large number of samples. The coefficients of variation were between 2.9% and 5.8%.

  3. Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development.

    Science.gov (United States)

    Cho, Ki-hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-hee; Park, Seung-yong; Song, Chang-seon; Choi, In-soo; Lee, Joong-bok

    2014-12-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation- dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

  4. Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep.

    Science.gov (United States)

    García-Bocanegra, Ignacio; Allepuz, Alberto; Pérez, Julio José; Alba, Anna; Giovannini, Armando; Arenas, Antonio; Candeloro, Luca; Pacios, Alberto; Saez, José Luís; González, Miguel Ángel

    2014-03-01

    Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n=118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA). The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.

  5. Hapten synthesis and development of a competitive indirect enzyme-linked immunosorbent assay for acrylamide in food samples.

    Science.gov (United States)

    Wu, Jing; Shen, Yu-Dong; Lei, Hong-Tao; Sun, Yuan-Ming; Yang, Jin-Yi; Xiao, Zhi-Li; Wang, Hong; Xu, Zhen-Lin

    2014-07-23

    The high level of acrylamide in widely consumed processed foods poses a potentially significant risk to human health, which has led to an increasing demand for rapid, simple, and selective analytical methods. In the present work, several haptens for acrylamide were designed in an attempt to prepare antibodies with acrylamide affinity, but they failed their purpose. However, a polyclonal antibody was produced against 4-mercaptophenylacetic acid (4-MPA)-derivatized acrylamide, which showed high binding affinity to the derivative. As acrylamide easily reacted with 4-MPA at high derivation yield, a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for acrylamide via a preanalysis derivatization was developed. The derivatization and ELISA conditions were fully optimized to produce a method for acrylamide assay that exhibited an IC50 of 2.86 μg/kg, limit of detection at 0.036 μg/kg, and linear range of 0.25-24.15 μg/kg. The results of preanalysis recovery tests of acrylamide-spiked food samples and screening of blind food samples by both ciELISA and HPLC-MS/MS indicated the proposed ciELISA's good accuracy and reliability. This method was thus deemed suitable for routine acrylamide screening in food samples at low cost.

  6. Plastic enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor for botulinum neurotoxin A.

    Science.gov (United States)

    Han, Seung-Mok; Cho, Joung-Hwan; Cho, Il-Hoon; Paek, Eui-Hwan; Oh, Hee-Bok; Kim, Bong-Su; Ryu, Chunsun; Lee, Kyunghee; Kim, Young-Kee; Paek, Se-Hwan

    2007-03-21

    A plastic ELISA-on-a-chip (EOC) employing the concept of cross-flow immuno-chromatographic analysis was applied to the measurement of botulinum neurotoxin A (BoNT/A) as agent for bio-terrorism. Two monoclonal antibodies specific to the heavy chain of the toxin were raised and identified to form sandwich binding complexes as the pair with the analyte. For the construction of an immuno-strip, one was utilized as the capture antibody immobilized onto nitrocellulose membrane and the other as the detection coupled to an enzyme, horseradish peroxidase. The two plates of EOC used in this study were fabricated by injection molding of polycarbonate to improve the reproducibility of manufacture and, after inclusion of the immuno-strip, bonded using a UV-sensitive adhesive. Under optimal conditions of analysis, the chip produced a color signal in proportion to the analyte dose and the signal was quantified using a detector equipped with a digital camera. From the dose-response curve, the detection limit of BoNT/A was 2.0 ng mL(-1), approximately five times more sensitive than a commercial-version detection kit employing colloidal gold tracer.

  7. Enzyme-linked immunosorbent assay for group A Streptococcal anti-DNase B in human sera, using recombinant proteins - Comparison to the DNA methyl green micromethod.

    Science.gov (United States)

    Das, Sarita; Dileepan, T; Johnson, D R; Kaplan, E L; Patrick Cleary, P

    2017-09-19

    Among the four known Streptococcal nucleases comprising of DNase A, B, C and D; DNase B is the most common, and determination of the levels of antibody to DNase B (ADB) is often used to confirm a clinical diagnosis of Streptococcus pyogenes/group A Streptococcal (GAS) infection. The commonly used assays for antibodies that neutralize DNase B or streptolysin O activity use partially purified antigens that often fail to detect antibody changes subsequent to culture documented infections. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed employing his-tagged recombinant DNase B as plate antigen for comparison to the commonly used DNA methyl green micromethod (DMGM). DNAs from various Streptococcal species were screened for presence of dnaseB gene by PCR. Measurements of ADB in sera collected from subjects belonging to different ages, and ethnic groups were used to compare the two methods. dnaseB was not detected by PCR in DNA samples isolated from different strains of group B (GBS), C (GCS) and G (GGS) Streptococci. The ADB based ELISA proved to be highly sensitive and more responsive to changes in antibody concentration than DMGM. Use of recombinant DNase B eliminates the variability associated with the enzyme, partially purified from Streptococcal culture supernatants from various commercial sources and may provide a more reliable source of antigen to a wider group of laboratories concerned with GAS diagnosis. Copyright © 2017. Published by Elsevier B.V.

  8. Development of an enzyme-linked immuno-sorbent assay (ELISA) method for carbofuran residues.

    Science.gov (United States)

    Yang, Jinyi; Wang, Hong; Jiang, Yueming; Sun, Yuanming; Pan, Ke; Lei, Hongtao; Wu, Qing; Shen, Yudong; Xiao, Zhili; Xu, Zhenlin

    2008-04-17

    The haptens 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]butanoic acid (BFNB) and 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)-carbonylamino]hexanoic acid (BFNH) were synthesized and then used to develop a rapid,specific and sensitive ELISA method to determine residues of the pesticide carbofuran in a variety of matrices. A hybridoma cell line (5D3) producing anti-carbofuran monoclonal antibodies (MAbs) was also established. Based on the MAbs in combination with the heterologous hapten BFNH coupled to either horseradish peroxidase (HRP) or ovalbumin(OVA), four ELISAs (formats I-IV) for the quantification of carbofuran were developed and compared. Among them, the optimized format II (the conjugate-coated direct competitive ELISA) showed the best characteristics, with an IC50 value of 18.49 ng/mL, a limit of detection of 0.11 ng/mL and the shortest assay time (1 h). This ELISA method was then applied to the determinations of carbofuran in environmental water, soil and food samples. The relative standard deviations (R.S.D.s) ranged from 1.8% to 21.3% and the mean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for water, soil, lettuce and cabbage, respectively. Thus, the ELISA method of format II exhibited the potential to develop commercial ELISA kits for a rapid detection of carbofuran for human health and environmental safety.

  9. Development of an Enzyme-linked Immuno-Sorbent Assay (ELISA Method for Carbofuran Residues

    Directory of Open Access Journals (Sweden)

    Zhenlin Xu

    2008-04-01

    Full Text Available The haptens 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxycarbonyl]-amino]butanoic acid (BFNB and 6-[((2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy-carbonylamino]hexanoic acid (BFNH were synthesized and then used to develop a rapid,specific and sensitive ELISA method to determine residues of the pesticide carbofuran in avariety of matrices. A hybridoma cell line (5D3 producing anti-carbofuran monoclonalantibodies (MAbs was also established. Based on the MAbs in combination with theheterologous hapten BFNH coupled to either horseradish peroxidase (HRP or ovalbumin(OVA, four ELISAs (formats I-IV for the quantification of carbofuran were developedand compared. Among them, the optimized format II (the conjugate-coated directcompetitive ELISA showed the best characteristics, with an IC50 value of 18.49 ng/mL, alimit of detection of 0.11 ng/mL and the shortest assay time (1 h. This ELISA method wasthen applied to the determinations of carbofuran in environmental water, soil and foodsamples. The relative standard deviations (R.S.D.s ranged from 1.8% to 21.3% and themean recoveries were 104.6%, 108.3%, 106.3% and 100.1% for water, soil, lettuce andcabbage, respectively. Thus, the ELISA method of format II exhibited the potential todevelop commercial ELISA kits for a rapid detection of carbofuran for human health andenvironmental safety.

  10. Giant Magnetoresistive Sensors and Magnetic Labels for Chip-Scale Detection of Immunosorbent Assays

    Energy Technology Data Exchange (ETDEWEB)

    Millen, Rachel Lora [Iowa State Univ., Ames, IA (United States)

    2005-01-01

    The combination of giant magnetoresistive sensors, magnetic labeling strategies, and biomolecule detection is just beginning to be explored. New readout methods and assay formats are necessary for biomolecules detection to flourish. The work presented in this dissertation describes steps toward the creation of a novel detection method for bioassays utilizing giant magnetoresistive sensors as the readout method. The introduction section contains a brief review of some of the current methods of bioassay readout. The theoretical underpinnings of the giant magnetoresistive effect are also discussed. Finally, the more prominent types of giant magnetoresistive sensors are described, as well as their complicated fabrication. Four data chapters follow the introduction; each chapter is presented as a separate manuscript, either already published or soon to be submitted. Chapter 1 presents research efforts toward the production of a bioassay on the surface of a gold-modified GMR sensor. The testing of this methodology involved the capture of goat a-mouse-coated magnetic nanoparticles on the mouse IgG-modified gold surface. The second, third and fourth chapters describe the utilization of a self-referenced sample stick for scanning across the GMR sensor. The sample stick consisted of alternating magnetic reference and bioactive gold addresses. Chapter 2 is concerned with the characterization of both the scanning readout method and the binding and detection of streptavidin-coated magnetic particles to a biotinylated surface. Chapter 3 advances the sample stick readout with the use of the system for detection of a sandwich immunoassay with rabbit IgG proteins. Finally, simultaneous detection of three IgG proteins is demonstrated in Chapter 4. The dissertation is concluded with a brief summary of the research presented and a discussion of the possible future applications and direction of this work.

  11. Giant Magnetoresistive Sensors and Magnetic Labels for Chip-Scale Detection of Immunosorbent Assays

    Energy Technology Data Exchange (ETDEWEB)

    Rachel Lora Millen

    2005-12-17

    The combination of giant magnetoresistive sensors, magnetic labeling strategies, and biomolecule detection is just beginning to be explored. New readout methods and assay formats are necessary for biomolecules detection to flourish. The work presented in this dissertation describes steps toward the creation of a novel detection method for bioassays utilizing giant magnetoresistive sensors as the readout method. The introduction section contains a brief review of some of the current methods of bioassay readout. The theoretical underpinnings of the giant magnetoresistive effect are also discussed. Finally, the more prominent types of giant magnetoresistive sensors are described, as well as their complicated fabrication. Four data chapters follow the introduction; each chapter is presented as a separate manuscript, either already published or soon to be submitted. Chapter 1 presents research efforts toward the production of a bioassay on the surface of a gold-modified GMR sensor. The testing of this methodology involved the capture of goat a-mouse-coated magnetic nanoparticles on the mouse IgG-modified gold surface. The second, third and fourth chapters describe the utilization of a self-referenced sample stick for scanning across the GMR sensor. The sample stick consisted of alternating magnetic reference and bioactive gold addresses. Chapter 2 is concerned with the characterization of both the scanning readout method and the binding and detection of streptavidin-coated magnetic particles to a biotinylated surface. Chapter 3 advances the sample stick readout with the use of the system for detection of a sandwich immunoassay with rabbit IgG proteins. Finally, simultaneous detection of three IgG proteins is demonstrated in Chapter 4. The dissertation is concluded with a brief summary of the research presented and a discussion of the possible future applications and direction of this work.

  12. Study on the use of an enzyme-linked immunosorbent assay in determining human antibodies to diphtheria toxin as compared with a reference toxin neutralization assay.

    Science.gov (United States)

    Skoura, L; Efstratiou, A; Tsakris, A; Pournaras, S; George, R C; Douboyas, J

    1999-07-01

    Serum samples from 156 Greek persons were assessed by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a reference tissue culture toxin-neutralization (TN) assay for the quantitation of diphtheria toxin antibodies. By the reference method, 7.7% of the persons were susceptible to diphtheria (antitoxin or = 0.1 IU/ ml), while the corresponding figures were 17.9, 36.5 and 45.5% when they were tested by the immunoassay. None of the samples been susceptible by the TN assay were found to have some protection when tested by ELISA. However, three (6.7%) of the 45 samples showing a basic protection with TN, were fully protective when titrated by the immunoassay. In addition, 31 (31.3%) of the 99 samples been fully protective by the bioassay, were found to be either basically protective or susceptible by means of the ELISA. Overall, validity features of the immunoassay were: sensitivity 68.7%, specificity 94.7%, positive predictive value 95.8% and negative predictive value 63.5%. The ELISA tested in our study could be used to determine diphtheria antitoxin in individuals needed a booster immunization (susceptible or basic protective samples), although it might falsely include in the above categories samples that are within the fully protective levels of antibodies.

  13. Pengembangan Enzyme-Linked Immunosorbent Assay Paratuberkulosis dengan Antigen Protoplasmik Mycobacterium avium Subspecies Paratuberculosis Isolat Lapang (DEVELOPMENT OF PARATUBERCULOSIS ENZYME-LINKED IMMUNO-SORBENT ASSAY WITH PROTOPLASMIC ANTIGEN OF MYCO

    Directory of Open Access Journals (Sweden)

    Rahmat Setya Adji

    2015-08-01

    Full Text Available Enzyme-linked immunosorbent assay (ELISA is a serological test method most widely used for thediagnosis of paratuberculosis, because it has a better sensitivity compared to other serological test.Protoplasmic antigen (PPA or cellular extract is still the main choice for the diagnosis of paratuberkulosisdevelopment. The aim of research was to use the PPA Mycobacterium avium subspeciesparatuberculosis(MAP field isolates for the development of paratuberculosis ELISA (ELISA PPA-L. As many as 322cattle sera (300 negative and 22 positive were tested using this method and compared with IDEXXcommercial kit. The sensitivity and specificity of ELISA PPA-L test results were 68.18% and 97.0%,whereas for the IDEXX kit were 63.64% and 97.33%respectively. ELISA PPA-L had higher sensitivity andlower specificity compared to the IDEXX commercial kit. ELISA test using protoplasmic antigen of MAPfield isolates has good ability for paratuberculosis serological test and can be used for screening test of thedisease in Indonesia.

  14. A feasible enzyme-linked immunosorbent assay system using monoclonal and polyclonal antibodies against glucosyltransferase-B from Streptococcus mutans.

    Science.gov (United States)

    Shinozaki-Kuwahara, Noriko; Hashizume-Takizawa, Tomomi; Hirasawa, Masatomo; Takada, Kazuko

    2012-06-01

    Streptococcus mutans has been considered the principal etiological agent of dental caries in humans. S. mutans can secrete three kinds of glucosyltransferases (GTFs). One of these, GTF-B, which synthesizes water-insoluble glucans from sucrose, has been considered to be one of the most important factors of cariogenic dental plaque formation. Therefore, determination of whether GTF-B is present in plaque and saliva samples may contribute to the evaluation of individual virulence potential (caries risk). The aim of this study was to develop a feasible enzyme-linked immunosorbent assay (ELISA) for the routine quantification of GTF-B in plaque-derived cultures and clinical samples, and to apply this assay to an epidemiological study. To determine the presence of GTF-B in plaque samples, a sandwich-ELISA was devised, consisting of mouse monoclonal and rabbit polyclonal antibodies against GTF-B and a horseradish peroxidase-conjugated anti-rabbit antibody. The developed ELISA allowed for quantification of the amounts of purified GTF-B with satisfactory sensitivity and specificity; this method was not affected by other components such as plaque and saliva. Plaque samples from healthy volunteers were examined using this ELISA method and microbial analysis to apply the assay to an epidemiological study. A correlation was observed between the amount of extracted GTF-B and S. mutans levels as determined by ELISA and cultivated with Mitis Salivarius Bacitracin agar plates derived from plaque samples, although there were some exceptions. In this regard, this ELISA system has the advantage of estimating both the individual numbers of S. mutans and the productivity of GTF-B, namely, the cariogenic potential of S. mutans simultaneously. These results indicate that this ELISA method is a useful tool for the diagnosis of caries risk.

  15. Low sensitivity of type VII collagen enzyme-linked immunosorbent assay in epidermolysis bullosa acquisita : serration pattern analysis on skin biopsy is required for diagnosis

    NARCIS (Netherlands)

    Terra, J. B.; Jonkman, M. F.; Diercks, G. F. H.; Pas, H. H.

    BackgroundThe type VII collagen (coll VII) enzyme-linked immunosorbent assay (ELISA) has been reported to have high sensitivity (>93%) and specificity (>96%) for diagnosing epidermolysis bullosa acquisita (EBA) in patients who are seropositive on indirect immunofluorescence on salt-split skin (SSS).

  16. Simultaneous determination of 13 fluoroquinolone and 22 sulfonamide residues in milk by a dual-colorimetric enzyme-linked immunosorbent assay

    Science.gov (United States)

    Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor the two classes of antimicrobial residues in different food matrices. In th...

  17. Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders

    2015-01-01

    Background: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradica...

  18. Analysis of Mycobacterium avium subsp. paratuberculosis Antigens Used in an In-house Enzyme-linked Immunosorbent Assay for Johne's Disease

    Science.gov (United States)

    We developed a novel enzyme-linked immunosorbent assay (ELISA), called EVELISA, for the detection of MAP infection in cattle which showed a higher sensitivity than current ELISA test. We previously reported that the use of heat-killed M. flavascens for pre-absorption of cross-reactive antibodies im...

  19. Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Oviedo, J M; Valiño, F; Plasencia, I

    2001-01-01

    We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been...

  20. Evaluation of enzyme-linked immunosorbent assays based on monoclonal antibodies for the serology and antigen detection in canine parvovirus infections.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); N. Juntti; B. Klingeborn; J. Groen (Jan); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractAn enzyme-linked immunosorbent assay (ELISA) system was developed for the detection of canine parvovirus (CPV) or CPV antigen in dog faeces and two other ELISA systems were developed for the detection of CPV-specific antibodies in dog sera. The ELISA's were based on the use of CPV-specif

  1. Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Damgaard, Dres; Palarasah, Yaseelan; Skjødt, Karsten

    2014-01-01

    Abs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal...

  2. Enzyme-Linked Immunosorbent Assay To Differentiate the Antibody Responses of Animals Infected with Brucella Species from Those of Animals Infected with Yersinia enterocolitica O9

    OpenAIRE

    Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu

    2003-01-01

    Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

  3. Enzyme-linked immunosorbent assay to differentiate the antibody responses of animals infected with Brucella species from those of animals infected with Yersinia enterocolitica O9.

    Science.gov (United States)

    Erdenebaatar, Janchivdorj; Bayarsaikhan, Balgan; Watarai, Masahisa; Makino, Sou-ichi; Shirahata, Toshikazu

    2003-07-01

    Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

  4. Evaluation of a new antibody-based enzyme-linked immunosorbent assay for the detection of bovine leukemia virus infection in dairy cattle

    NARCIS (Netherlands)

    Monti, G.E.; Frankena, K.; Engel, B.; Buist, W.; Tarabla, H.D.; Jong, de M.C.M.

    2005-01-01

    The objective of this study was to validate a new blocking enzyme-linked immunosorbent assay (ELISA) (designated M108 for milk and S108 for serum samples) for detecting bovine leukemia virus (BLV) infection in dairy cattle. Milk, serum, and ethylenediaminetetraacetic acid-blood samples were collecte

  5. Development and validation of an indirect Enzyme-linked Immunosorbent Assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants

    NARCIS (Netherlands)

    Heijden, van der H.M.J.F.; Bouwstra, R.J.; Mars, M.H.; Poel, van der W.H.M.; Wellenberg, G.J.; Maanen, van C.

    2013-01-01

    To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood sample

  6. Low sensitivity of type VII collagen enzyme-linked immunosorbent assay in epidermolysis bullosa acquisita : serration pattern analysis on skin biopsy is required for diagnosis

    NARCIS (Netherlands)

    Terra, J. B.; Jonkman, M. F.; Diercks, G. F. H.; Pas, H. H.

    2013-01-01

    BackgroundThe type VII collagen (coll VII) enzyme-linked immunosorbent assay (ELISA) has been reported to have high sensitivity (>93%) and specificity (>96%) for diagnosing epidermolysis bullosa acquisita (EBA) in patients who are seropositive on indirect immunofluorescence on salt-split skin (SSS).

  7. Validation of an enzyme-linked immunosorbent assay that detects Histoplasma capsulatum antigenuria in Colombian patients with AIDS for diagnosis and follow-up during therapy.

    Science.gov (United States)

    Caceres, Diego H; Scheel, Christina M; Tobón, Angela M; Ahlquist Cleveland, Angela; Restrepo, Angela; Brandt, Mary E; Chiller, Tom; Gómez, Beatriz L

    2014-09-01

    We validated an antigen capture enzyme-linked immunosorbent assay (ELISA) in Colombian persons with AIDS and proven histoplasmosis and evaluated the correlation between antigenuria and clinical improvement during follow-up. The sensitivity of the Histoplasma capsulatum ELISA was 86%, and the overall specificity was 94%. The antigen test successfully monitored the response to therapy.

  8. The use of enzyme-linked immunosorbent assay systems for the serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. Groen (Jan); H.F. Egberink (Herman); G.H.A. Borst (Gerrit); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1991-01-01

    textabstractComplex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-,

  9. Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP) - Assessment of corresponding epitopes

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Larsen, D.V.; Zhang, C.

    2010-01-01

    Objectives: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. Methods: Monoclonal antibodies were raised against...

  10. Dual-color plasmonic enzyme-linked immunosorbent assay based on enzyme-mediated etching of Au nanoparticles

    Science.gov (United States)

    Guo, Longhua; Xu, Shaohua; Ma, Xiaoming; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2016-09-01

    Colorimetric enzyme-linked immunosorbent assay utilizing 3‧-3-5‧-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3‧-3-5‧-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.

  11. A simplified pretreatment method for the determination of polychlorinated biphenyls in transformer oil by enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Aranami, K.; Morita, M. [National Inst. for Environmental Studies, Tsukuba (Japan); Okuyama, A. [EnBio Tec Laboratories Co. Ltd., Tokyo (Japan)

    2005-07-01

    This paper presented a simplified pretreatment method for determining polychlorinated biphenyls (PCBs) in transformer oil using enzyme-linked immunosorbent assay (ELISA). The method used a combination of dimethylsulfoxide (DMSO) and hexane partition and solid phase extraction. Kanechlors (KC) was used along with a transformer oil comprised of paraffin, naphthene, and aromatic hydrocarbons. Waste oil samples contaminated with PCBs were prepared by adding KC to the oil. Capillary column gas chromatography electron capture detection (GC-ECD) was used to analyze the samples. Qualitative and quantitative measurements of KC and the oil were conducted. An ELISA kit was then used to analyze PCB samples in DMSO and horseradish peroxidase (HRP) PCB competitors in TBS. A sulfuric acid/silica gel cartridge was used to purify the samples with n-hexane solutions. The ratio of the DMSO/hexane partition was examined. Results showed that the ELISA system is subject to negative interference in the presence of hydrocarbon co-contaminations. It was suggested that the oil matrix must be eliminated in order to detect PCB using the system. A simplified pretreatment was then developed which included direct addition of waste oil samples to the silica cartridge followed by elution with hexane. ELISA values of the pretreatment samples were corrected by multiplying dilution factors. It was concluded that the method successfully pretreated samples in approximately 10 minutes. 10 refs., 3 figs.

  12. Influence of iodothyronine conjugates of bovine serum albumin and horseradish peroxidase on enzyme immunosorbent assay of thyroid hormones.

    Science.gov (United States)

    Kumari, G Lakshmi; Kumar, Sachin; Gupta, Satish; Saini, Anuradha; Sharma, Sudesh K; Kaur, Navneet

    2014-01-01

    Enzyme-linked immunosorbent assays (ELISA's) reported for thyroxine (T₄) and 3,5,3'-triiodothyronine (T₃), involved coupling of the haptens through (i) carboxylic group to carrier protein for producing antibodies and (ii) amino group to detection labels. To improve the titer and specificity of antibodies, immunogens were prepared by coupling of carboxyl group to bovine serum albumin (BSA) either directly or through adipic acid dihydrazide (ADH), after protecting amino group through acetylation of T₄ and T₃. Direct coupling resulted in the incorporation of 40-50 moles of T₄ and T₃ per BSA molecule and helped in improving immunogenic response and specificity, especially of T₄. High epitope density of immunogens evoked better antibody response, since attachement of ADH as spacer, introduced 18-27 moles of haptens into carrier protein and had less effect on antibody development, with T₃ being exception. Detection labels were prepared by coupling horseradish peroxidase (HRP) to amino group of thyroid hormones directly and after preparing their methyl esters, which provided sensitive displacement curves in combination with the antibodies developed against N-acetylated-T₄ and T₃. Unlike methyl esters, T₄-HRP and T₃-HRP showed higher sensitivity and seemed to be related to the affinity of the labels for binding the antibody.

  13. Advantages of soybean peroxidase over horseradish peroxidase as the enzyme label in chemiluminescent enzyme-linked immunosorbent assay of sulfamethoxypyridazine.

    Science.gov (United States)

    Sakharov, Ivan Yu; Berlina, Anna N; Zherdev, Anatoly V; Dzantiev, Boris B

    2010-03-24

    An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.

  14. Coupling solid-phase extraction and enzyme-linked immunosorbent assay for ultratrace determination of herbicides in pristine water

    Science.gov (United States)

    Aga, D.S.; Thurman, E.M.

    1993-01-01

    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were coupled for automated trace analysis of pristine water samples containing 2-chloro-4-ethylamino-6-isopropylamine-s-triazine (atrazine) and 2-chloro-2???,6???-diethyl-N-(methoxymethyl)acetanilide (alachlor). The isolation of the two herbicides on a C18-resin involved the selection of an elution solvent that both removes interfering substances and is compatible with ELISA. Ethyl acetate was selected as the elution solvent followed by a solvent exchange with methanol/water (20/80, % v/v). The SPE-ELISA method has a detection limit of 5.0 ng/L (5 ppt), >90% recovery, and a relative standard deviation of ??10%. The performance of a microtiter plate-based ELISA and a magnetic particle-based ELISA coupled to SPE was also evaluated. Although the sensitivity of the two ELISA methods was comparable, the precision using magnetic particles was improved considerably (??10% versus ??20%) because of the faster reaction kinetics provided by the magnetic particles. Finally, SPE-ELISA and isotope dilution gas chromatography/ mass spectrometry correlated well (correlation coefficient of 0.96) for lake-water samples. The SPE-ELISA method is simple and may have broader applications for the inexpensive automated analysis of other contaminants in water at trace levels.

  15. The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

    Directory of Open Access Journals (Sweden)

    Claudio R Madruga

    2006-11-01

    Full Text Available There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

  16. Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and PCR in diagnostics of enzootic bovine leukosis

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    Malovrh Tadej

    2005-01-01

    Full Text Available Bovine leukaemia virus (BLV is a retrovirus that induces a chronic infection in cattle. Once infected, cattle remain virus carriers for life and start to show an antibody response within a few weeks after infection. Eradication and control of the disease are based on early diagnostics and segregation of the carriers. The choice of a diagnostic method depends on the eradication programme, money resources and characteristics of the herd to be analysed. The agar gel immunodiffusion (AGID test has been the serological test of choice for routine diagnosis of serum samples. Nevertheless, in more recent years, the enzyme-linked immunosorbent assay (ELISA has replaced the AGID for large scale testing. For this purpose, commercially available BLV-ELISA kits were compared to the AGID and to the polymerase chain reaction (PCR method performed with two sets of primers, amplifying env region. The ELISA kit based on the p24 core protein was found to be less specific and served as a screening test. The ELISA kit based on the envelope glycoprotein (gpSI served as a verification test and gave a good correlation with the AGID test and PCR method. However, ELISA showed a higher sensitivity than AGID. The p24 based ELiSA was useful for screening a large number of samples, whereas gp51 based ELISA, AGID and PCR were more important for detecting the antibody response against the individual BLV-proteins and therefore for verification of the infection with BLV.

  17. Seroprevalence of contagious ecthyma in goats of Assam: An analysis by indirect enzyme-linked immunosorbent assay

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    Mousumi Bora

    2016-09-01

    Full Text Available Aim: The objective of this study was to screen the prevalence of contagious ecthyma (CE among the goat population of Assam owing to its high prevalence rate. Materials and Methods: In this study, a total of 231 serum samples were collected from 12 districts of Assam during September 2013 to July 2014. The serum samples were tested for the presence of antibodies against Orf virus (ORFV by indirect enzyme-linked immunosorbent assay (ELISA. Indirect ELISA was standardized using purified Orf reference virus produced in bulk in primary lamb testes cells. Results: Studies on seroprevalence showed 76.62% of goats were seropositive. The total number of animals were divided into different age groups starting from 0-2 months, 2-4 months, 4-6 months, and above 8 months and accordingly highest prevalence of antibodies against ORFV was recorded in the age-group above 8 months of age. Significantly, lower rates of infection were observed in goats of age group 2-4 months. This study recorded that seropositivity from naturally infected animals and in contact apparently healthy animals to be 53.67% and 46.32%, respectively. Conclusion: The results indicated that CE is a prevalent infection in goats of Assam, and the healthy population is at increased risk of infection.

  18. Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis-specific immunoglobulin G response in sera of pigs.

    Science.gov (United States)

    Holyoake, P K; Cutler, R S; Caple, I W; Monckton, R P

    1994-01-01

    Proliferative enteritis (PE) is a common intestinal disease on pig farms. The disease is caused by ileal symbiont (IS) intracellularis (Campylobacter-like organisms) bacteria. An enzyme-linked immunosorbent assay (ELISA) was developed to measure IS intracellularis-specific immunoglobulin G (IgG) response in the sera of pigs. The antigen used in the ELISA was filtered, percoll gradient-purified IS intracellularis extracted from the intestines of pigs affected with proliferative hemorrhagic enteropathy. The antibody responses of pigs challenged with intestinal homogenates from pigs affected with proliferative hemorrhagic enteropathy containing IS intracellularis or percoll-gradient purified IS intracellularis were low and variable. The low IgG titers measured in challenged pigs support previous findings that IgG plays a minor role in the immune response of pigs to IS intracellularis. On a farm in which infection was endemic, pigs seroconverted at between 7 and 24 weeks of age. High IgG titers, indicative of maternally acquired antibody, were present in 3-week-old pigs. The IgG titers in piglets were lowest at 6 weeks of age, which approximates the age of onset of clinical disease. These results suggest that IgG plays a role in determining the susceptibilities of pigs to natural infection. Measurements of seroconversion by the ELISA might aid in epidemiological investigations of PE in naturally infected herds. However, the variable antibody responses in experimentally challenged pigs would seem to limit its usefulness as an antemortem diagnostic test for PE. PMID:7989553

  19. Different strategies in the laboratory diagnosis of autoimmune disease: immunofluorescence, enzyme-linked immunosorbent assay or both?

    Science.gov (United States)

    Rondeel, J M; van Gelder, W; van der Leeden, H; Dinkelaar, R B

    1999-03-01

    We investigated the clinical utility of different strategies for antinuclear antibodies (ANA) and antibodies to extractable nuclear antigens (ENA) testing. All requests for ANA and ENA (n = 485) in a 20-week period were tested by immunofluorescence (FANA) and immunodiffusion (strategy 1), enzyme-linked immunosorbent assay (ELISA) techniques (strategy 2) or a combination of FANA and ELISA (strategy 3). Results of strategy 1 were positive by FANA in 8% (by immunodiffusion in 2%). By ELISA, 11% of the samples tested positive. In 12% (n = 60) of the cases the two strategies did not agree. The positive predictive value (PPV) for autoimmune disease of strategy 1 was significantly higher than that for strategy 2, but after exclusion of rheumatoid arthritis this difference was abolished. In strategy 2 reagent costs were high but working time comparably shorter. With strategy 3 PPV results were not better, whereas costs and working time were higher. The most frequently occurring reasons for ANA/ENA test requests were: joint symptoms (37%), follow up (30%) or abnormal laboratory result (7%). In a survey of the clinicians 66% replied that the test result did not have any consequences, irrespective of the result or the strategy used. We conclude that FANA and immunodiffusion are superior to ELISA techniques. However, the clinical value of ANA/ENA testing is low and more selective test ordering is strongly recommended.

  20. Development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses.

    Science.gov (United States)

    Kooijman, Lotte J; Mapes, Samantha M; Pusterla, Nicola

    2016-07-01

    Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations.

  1. Seroprevalence of contagious ecthyma in goats of Assam: An analysis by indirect enzyme-linked immunosorbent assay

    Science.gov (United States)

    Bora, Mousumi; Bora, Durlav Prasad; Barman, Nagendra Nath; Borah, Biswajyoti; Das, Sutopa

    2016-01-01

    Aim: The objective of this study was to screen the prevalence of contagious ecthyma (CE) among the goat population of Assam owing to its high prevalence rate. Materials and Methods: In this study, a total of 231 serum samples were collected from 12 districts of Assam during September 2013 to July 2014. The serum samples were tested for the presence of antibodies against Orf virus (ORFV) by indirect enzyme-linked immunosorbent assay (ELISA). Indirect ELISA was standardized using purified Orf reference virus produced in bulk in primary lamb testes cells. Results: Studies on seroprevalence showed 76.62% of goats were seropositive. The total number of animals were divided into different age groups starting from 0-2 months, 2-4 months, 4-6 months, and above 8 months and accordingly highest prevalence of antibodies against ORFV was recorded in the age-group above 8 months of age. Significantly, lower rates of infection were observed in goats of age group 2-4 months. This study recorded that seropositivity from naturally infected animals and in contact apparently healthy animals to be 53.67% and 46.32%, respectively. Conclusion: The results indicated that CE is a prevalent infection in goats of Assam, and the healthy population is at increased risk of infection.

  2. Validation of an enzyme-linked immunosorbent assay for qualitative screening of neomycin in muscle, liver, kidney, eggs and milk.

    Science.gov (United States)

    Solomun, B; Bilandzic, N; Varenina, I; Scortichini, G

    2011-01-01

    A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was used for the qualitative screening analysis of neomycin in food of animal origin (muscle, liver, kidney, eggs and milk) at levels corresponding to the European Union maximum residue limit (MRL) set for this substance. The method validation was performed according to the criteria of Commission Decision 2002/657/EC established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCβ), specificity, detection limit (LOD), quantification limit (LOQ), recovery, precision, linearity and ruggedness. LODs ranged from 5.7 microg kg(-1) in kidney to 29.3 microg kg(-1) in milk; LOQs ranged from 11.4 microg kg(-1) in kidney to 59.7 microkg(-1) in eggs. The recoveries from spiked samples at the MRL, half the MRL and double the MRL levels ranged from 65.8% to 122.8%, with a coefficient of variation (CV) between 5.9% and 28.6%. The CCβ value was less than the MRL for all examined matrices. Moderate variations of some critical factors in the sample pretreatment for muscle, milk and eggs were deliberately introduced for ruggedness evaluation and had a slight but not statistically significant effect on method performance. The proposed method is suitable for qualitative screening analysis of neomycin in the above-mentioned food in conformity with current European Union performance requirements.

  3. Comparison of enzyme-linked immunosorbent assay and radioimmunoassay for prostate-specific acid phosphatase in prostatic disease

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    Griffiths, J.; Rippe, D.F.; Panfili, P.R.

    1982-01-01

    Results of an enzyme-linked immunosorbent assay (ELISA) are compared with those of a standard radioimmunoassay (RIA) for detection and quantitation of prostate-specific acid phosphatase (EC 3.1.3.2) in serum. Control subjects, patients with benign prostatic hyperplasia, and patients in all four clinical stages of prostatic adenocarcinoma were tested. The upper limit of normal (95%of the population) by the ELISA was 2.0 ..mu..g/L, and by the RIA was 2.2 ..mu..g/L. In prostatic a denocarcinoma stage I (not detectable by digital rectal examination), ELISA was slightly more sensitive than RIA, but sensitivity was still relatively low (20%). As tumor mass increased (stages II through IV), the frequency of increased concentrations of prostatic acid phosphatase in serum also increased. We confirmed this increase in circulating enzyme in some cases of benign prostatic hyperplasia and suggest that this finding is related to either acinar cytolysis or an increase in acini size and number. Although prostate-specific acid phosphatase is not a cancer-specific enzyme, we conclude that its measurement may be of considerable value in monitoring prostatic disease.

  4. Development of a Specifically Enhanced Enzyme-Linked Immunosorbent Assay for the Detection of Melamine in Milk

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    Yuanming Sun

    2011-06-01

    Full Text Available An indirect competitive enzyme-linked immunosorbent assay (icELISA with enhanced specificity for melamine in milk was developed. Three haptens of melamine with different spacer-arms were used to prepare different plate coating antigens. It was found that the icELISA show best sensitivity and specificity to melamine when using the coating antigen prepared by coupling 3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthiopropanoic acid (Hapten C with ovalbumin (OVA. The 50% inhibitory concentration (IC50 value was 35.4 ng·mL−1, the limit of detection (LOD was 8.9 ng·mL−1 and the detectable working range (20–80% inhibitory concentration was from 14.9 to 108.5 ng·mL−1, respectively. Compared to the ELISA results previously reported, the developed icELISA in the present study showed a much lower cross-reactivity to cyromazine, a fly-killing insecticide widely used in vegetables and stables. Recoveries obtained from milk samples in this study were in agreement with those obtained using the HPLC-MS method, indicating the detection performance of the icELISA could meet the requirement of the residue limit set by the Codex Alimentarius Commission. Therefore, the developed immunoassay can be applied for the analysis of melamine presented in milk.

  5. Comparative analysis of enzyme-linked immunosorbent assay and direct microscopy for the diagnosis of Giardia intestinalis in fecal samples

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    Shipra Singhal

    2015-01-01

    Full Text Available Context: Giardiasis is one of the most common nonviral infections causing diarrheal illness worldwide. In this prospective cross-sectional study, we evaluated the RIDASCREEN ® Giardia kit for detection of Giardia intestinalis in stool samples and compared the results with direct microscopy. Materials and methods: A total of 360 fecal samples were collected. They were then processed by wet film, iodine preparation and an enzyme-linked immunosorbent assay (ELISA kit to determine the presence of Giardia trophozoites and cysts. Statistical analysis was performed by sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy. Results and Conclusion: Of the 360 cases, 17.2% samples were positive for Giardia by direct microscopy and 23.6% were found to be positive by ELISA (sensitivity ~97%, but specificity was ~92% only. Because of less specificity, we need to perform ELISA in congruence with direct microscopy, etc. Further studies need to be performed on a larger sample size using other molecular tests in order to get more accurate estimations.

  6. Large-scale purification of synaptophysin and quantification with a newly established enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Schlaf, G; Göddecke, M; Wolff, J R; Felgenhauer, K; Mäder, M

    1996-09-01

    Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

  7. Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in european wild boar

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    Gortázar Christian

    2008-11-01

    Full Text Available Abstract Background Bovine tuberculosis (bTB remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures. Results An enzyme-linked immunosorbent assay (ELISA to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off. Conclusion Although some negative group animals showed an ELISA positive reaction (

  8. Comparison of enzyme linked immunosorbent assay (ELISA with indirect immunofluorescence for detection of anti-nuclear antibody

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    G.L.S. Sumanth Kumar

    2014-10-01

    Full Text Available Background: Detection of antinuclear antibody (ANA is used as one of the diagnostic criteria for autoimmune rheumatic diseases (ARD. Both indirect immunofluorescence (IIF and enzyme linked immunosorbant assay (ELISA methods are used for this purpose. However, there are lack of data comparing these two tests from India. Methods: We prospectively studed 294 patients clinically suspected to be having ARD between April 2012 and September 2013. They were tested for ANA by IIF and ELISA methods. Representative samples positive by both the tests were processed again by a line immunoassay test to detect the specific antinuclear antibodies. Considering the IIF results as the ‘gold standard’, the utility of ELISA for ANA detection was analyzed. Results: Of the 294 samples processed, 181 (61.5% were from female patients. By IIF 30% of samples in males and 40.3% sample in females tested positive. We found ELISA to have a poor sensitivity (45.8% but good specificity (99.5%. The positive predictive value for ELISA were 98% and negative predictive value 76.2% respectively. Forty four samples positive by both IIF and ELISA were tested by Western blot to detect individual autoantibodies. Of these, only 24 samples showed the presence of one or more bands, while the remaining 20 (45.4% were negative by line immunoassay. In our study anti-nuclear ribonucleoprotein/Smith was the most common ANA detected. Conclusions: The poor sensitivity raises concerns regarding the practice of initial screening for ANA by ELISA

  9. Bluetongue in Bosnia: comparisons of competitive enzyme-linked immunosorbent assay and standard agar gel immunodiffusion tests.

    Science.gov (United States)

    Velić, L; Velić, R; Bajrović, T; Dukić, B; Camo, D

    2004-01-01

    At the end of August 2002, clinical symptoms of bluetongue (BT) (fever between 39 degrees C and 41 degrees C, muco-purulent or bloody nasal discharge, oedema of the lips and the intramandibular space, foot lesions including laminitis and coronitis in some cases, diarrhoea and dysentery) were recorded in Pramenka sheep flocks in north-east Bosnia in August 2002. A total of 9 599 serum samples (ovine: 8 967; bovine: 632) from 40 communities of Bosnia and Herzegovina were tested for the presence of anti-bluetongue virus (BTV) antibodies using competitive enzyme-linked immunosorbent assay (c-ELISA) and the standard agar gel immunodiffusion (AGID) test. The c-ELISA revealed BTV-seropositive reactions in 187 (1.94%) samples and the AGID test detected 141 (1.53%) cases. Complete agreement was recorded between the c-ELISA and AGID test results for bovine sera. These results indicate that the ability of c-ELISA to detect anti-bluetongue virus antibodies in ovine sera was superior to that of the AGID. All positive sera were collected from animals in the river areas of Bosnia and Herzegovina.

  10. Screening survey of deoxynivalenol in beer from the European market by an enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Papadopoulou-Bouraoui, A; Vrabcheva, T; Valzacchi, S; Stroka, J; Anklam, E

    2004-06-01

    Deoxynivalenol (DON) was analysed in 313 beer samples collected from the European retail market using a commercially available immunoassay kit (enzyme-linked immunosorbent assay, ELISA). The incidence rate was about 87%, while most samples (73%) had contamination levels lower than 20 ng m(-1). The contamination ranged between 4.0 and 56.7 ng ml(-1), with an average of 13.5 ng ml(-1). A statistically significant correlation between alcohol levels and DON contamination was found, as well as a significant difference between bottom, top and spontaneous fermenting beers. Twenty-seven beer samples were compared using a second ELISA kit and a good correlation was obtained between the two kits (r = 0.93). Although when compared with gas chromatography-mass spectrometry the ELISA tended to overestimate the results, a good correlation (r=0.94) between the two methods was observed. Monitoring of DON in beer is important considering that DON production is dependent on the weather and that it can contribute significantly to the tolerable daily intake of DON, especially for frequent beer consumers.

  11. Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of Salmonella serotype typhi antigens in urine.

    Science.gov (United States)

    Fadeel, Moustafa Abdel; Crump, John A; Mahoney, Frank J; Nakhla, Isabelle A; Mansour, Adel M; Reyad, Baheia; El Melegi, Dawlat; Sultan, Yehia; Mintz, Eric D; Bibb, William F

    2004-03-01

    We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies to capture somatic antigen 9 (O9), flagellar antigen d (Hd), and the Vi capsular polysaccharide antigen (Vi) from the urine of persons with and without typhoid fever. Sequential urine samples were collected from 44 patients with blood culture-confirmed typhoid fever and from two control groups. The first control group included patients with brucellosis (n = 12) and those with clinically diagnosed, non-typhoid, acute, febrile illness (n = 27). The second control group was a sample of healthy volunteer laboratory workers (n = 11). When assessed relative to date of fever onset, sensitivity was highest during the first week for all three antigens: Vi was detected in the urine of nine (100%) patients, O9 in 4 (44%) patients, and Hd in 4 (44%) patients. Sequential testing of two urine samples from the same patient improved test sensitivity. Combined testing for Vi with O9 and Hd produced a trend towards increased sensitivity without compromising specificity. The specificity for Vi exceeded 90% when assessed among both febrile and healthy control subjects, but was only 25% when assessed among patients with brucellosis. Detection of urinary Vi antigen with this ELISA shows promise for the diagnosis of typhoid fever, particularly when used within the first week after fever onset. However, positive reactions for Vi antigen in patients with brucellosis must be understood before urinary Vi antigen detection can be developed further as a useful rapid diagnostic test.

  12. Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus.

    Science.gov (United States)

    Alkahalaf, A N; Halvorson, D A; Saif, Y M

    2002-01-01

    Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.

  13. Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

    Science.gov (United States)

    Abdelwahab, Mohamed; Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long

    2015-06-01

    Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks.

  14. Sensitive and specific enzyme-linked immunosorbent assay for detecting serum antibodies against Mycobacterium avium subsp. paratuberculosis in fallow deer.

    Science.gov (United States)

    Prieto, José M; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2014-08-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations.

  15. Development and validation of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy.

    Science.gov (United States)

    Wattanaphansak, Suphot; Asawakarn, Tanong; Gebhart, Connie J; Deen, John

    2008-03-01

    The objective of this study was to develop an indirect enzyme-linked immunosorbent assay (ELISA) using a sonicated pure culture of Lawsonia intracellularis as the antigen (So-ELISA). A total of 332 serum samples, consisting of 232 experimentally infected animals and 100 animals naturally infected with L. intracellularis, were used to assess the diagnostic sensitivity. Three hundred and fifty-five sera from uninfected animals were used to determine the diagnostic specificity. The receiver operating characteristic and mean +3 standard deviation of optical density (OD) values from uninfected animals were used for selecting cut-off points. The diagnostic accuracy of So-ELISA was considered to be high as the area under the curve index was 0.991 with 0.0029 standard error. The optimal cut-off for So-ELISA was set at 0.45 OD with 89.8% sensitivity and 99.4% specificity based on a combination of good sensitivity and high specificity. No cross-reactivity was found in sera from pigs exposed to Brachyspira pilosicoli, B. hyodysenteriae, Campylobacter mucosalis, C. jejuni, or C. coli. Inter- and intracoefficient of variation of all control sera tested with So-ELISA was less than 10%. The observed agreements between So-ELISA and the immunoperoxidase monolayer assay tested with experimental challenge animals and field samples were 95.08% with 0.88 kappa and 90.65% with 0.74 kappa value, respectively. So-ELISA was able to detect the seroconversion of infected animals at 2 to 4 weeks after exposure to L. intracellularis. Based on the validation results, So-ELISA could be used as an alternative serology for proliferative enteropathy diagnosis.

  16. Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)

    Science.gov (United States)

    Ciaurriz, Paula; Fernández, Fátima; Tellechea, Edurne; Moran, Jose F

    2017-01-01

    The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased. PMID:28243563

  17. Development of a sandwich enzyme-linked immunosorbent assay for dTMP-GH fusion protein by rational immunogen selection.

    Science.gov (United States)

    Wang, Song; Shen, Mingqiang; Chen, Shilei; Wang, Cheng; Chen, Fang; Chen, Mo; Zhao, Gaomei; Ran, Xinze; Cheng, Tianmin; Su, Yongping; Xu, Yang; Wang, Junping

    2017-12-01

    dTMP-GH is a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide (dTMP) fused to human growth hormone (hGH) prepared previously by our team. It shows significant bioactivity in promoting thrombocytopoiesis, but detection of intact dTMP-GH in plasma is still a challenge due to the presence of endogenous hGH. In this study, a rabbit polyclonal antibody with high affinity to dTMP was obtained with a BSA-conjugated immunogen composed of 20 amino acids sequence spanning two TMP and the linker. A monoclonal antibody termed as 3B2 was screened out by using immunizing mice with whole dTMP-GH, which was proved to simultaneously interact with rhGH, TMP-GH, and dTMP-GH, respectively. In this study, we developed a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies (one polyclonal and one HRP-conjugated monoclonal) to quantify dTMP-GH. The polyclonal antibody and HRP-conjugated monoclonal antibody 3B2 were applied as the capture antibody and detection antibody, respectively. A good correlation between ELISA and bicinchoninic acid (BCA) assay in the quantification of diluted dTMP-GH was observed (r(2) = 0.996). Meanwhile, the standard curve of this ELISA method was found in a linear relationship between 0.2 and 10 ng/mL in the presence of rabbit plasma. In vivo experiments demonstrate that the newly developed method is effective to detect dTMP-GH in rabbits, which paves the way for further pharmacokinetic evaluation.

  18. Evaluation of five different antigens in enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

    Science.gov (United States)

    Maherchandani, Sunil; Patnayak, Devi P; Muñoz-Zanzi, Claudia A; Lauer, Dale; Goyal, Sagar M

    2005-01-01

    Five different antigens were evaluated in enzyme-linked immunosorbent assay (ELISA) tests for the detection of avian pneumovirus (APV) antibodies. Two of the 5 antigens were prepared from recent APV isolates from Minnesota. The 2 older isolates were passage 63 of a strain currently used as a live, attenuated vaccine and a Colorado strain isolated for the first time in the United States and currently used in an ELISA test. The fifth antigen is based on an APV recombinant N-protein. Basic parameters and positive-negative threshold of the assays were established for all 5 antigens on the basis of data obtained by testing 46 known negative and 46 known positive serum samples. Subsequently, 449 field samples were tested by all 5 ELISAs. The optical density difference (ODD) was calculated by subtracting optical density of the sample in the negative antigen well from that in the positive antigen well. In the current ELISA test based on the Colorado strain, an ODD of 0.2 is considered to be the cutoff value to classify samples as negative or positive. In this study, however, use of different cutoffs, based on ODD of negative control plus 3 SD or values estimated from Receiver operating characteristic analysis, was considered to be more appropriate for the various antigens used. Overall person-to-person and day-to-day variability was found to be large for all tests using either ODD or sample to positive ratio to report results. In addition, results suggest that antigenicity of the APV isolates in the United States has not changed between 1997 and 2000.

  19. Deoxynivalenol and its conjugates in beer: a critical assessment of data obtained by enzyme-linked immunosorbent assay and liquid chromatography coupled to tandem mass spectrometry.

    Science.gov (United States)

    Zachariasova, Milena; Hajslova, Jana; Kostelanska, Marta; Poustka, Jan; Krplova, Alexandra; Cuhra, Petr; Hochel, Igor

    2008-09-05

    Enzyme-linked immunosorbent assays (ELISAs) are often employed for the control of deoxynivalenol (DON) in barley and other intermediates involved in beer production chain. Because of the occurrence of high levels of DON-3-glucoside (DON-3-Glc) in malt and beer that have been reported for the first time in our earlier study, research focused on the accuracy of DON determination by immunoassays in cereal-based matrices has been initiated. DON-3-Glc was strongly cross-reacting in all examined commercial ELISA test kits (Ridascreen) DON (R-Biopharm), Veratox 5/5 DON) (Neogen Corporation), Deoxynivalenol EIA (Euro-Diagnostica), and AgraQuant) DON Assay 0.25/5.0 Test Kit (Romer Labs). The highest overestimation in beer analysis, up to 1000%, when taking the DON content determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as a reference method, was obtained by AgraQuant assay. Besides of DON-3-Glc and 3- and 15-acetyldeoxynivalenol (ADONs), also other, not known yet, matrix components contributed to false positive results. Similar phenomenon, although in a lesser extent due to lower content of these substances, was observed for using ELISA in the analysis of wheat. The relationship between a way of sample handling and DON overestimation was demonstrated; higher ELISA response was measured in an aqueous extract compared to that prepared by acetonitrile-water (84:16, v/v). Most of cross-reacting co-extracts were removed by MycoSep# 226 cartridge, what leads us to the hypothesis on the presence of currently unknown cross-reactive species.

  20. Anti-amebic antibody activity in patients, determined with antigens prepared from virulent parasites (indirect hemagglutination assay and enzyme-linked immunosorbent assay).

    Science.gov (United States)

    Israeli, Eitan; Talis, Batya; Peled, Nehama; Snier, Rachella; El-On, Joseph

    2007-09-01

    The serology of amebiasis is affected by low sensitivity and specificity. To evaluate the advantage of the indirect hemagglutination assay and enzyme-linked immunosorbent assay in the diagnosis of amebiasis, using Entamoeba histolytica soluble antigen (macerated amebic antigens) prepared from four different virulent isolates, continuously cultivated in the presence of the original enteric bacteria. Using IHA and ELISA with MAA antigen we examined 147 sera samples from patients with gastrointestinal symptoms, and 11 sera from amebiasis cases (confirmed by microscopy and copro-antigen ELISA). Of 104 of the 147 (70.7%) symptomatic cases that were amebiasis positive by IHA, 81 (55.1%) were positive by MAA-ELISA. In addition, of 11 amebiasis cases confirmed by microscopy and copro-antigen ELISA, 7 (64%) were amebiasis positive by both tests. Four species of bacteria were isolated from the ameba cultures: Escherichia coli, Morganella morganii, Proteus mirabilis, and Streptococcus lactis. Elimination of the bacteria from the cultures by an antibiotics cocktail containing gentamicin, imipenem, piperacillin-tazobactam and vancomycin was the preferred method. Absorption of patients' sera to bacterial antigen prior to serological analysis had only a marginal effect. These results indicate a correlation of 61% between the ELISA developed in this study and the IHA tests in the diagnosis of amebiasis.

  1. Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua.

    Science.gov (United States)

    Chikeka, Ijeuru; Matute, Armando J; Dumler, J Stephen; Woods, Christopher W; Mayorga, Orlando; Reller, Megan E

    2016-06-01

    Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies.

  2. Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

    Science.gov (United States)

    2013-07-12

    RESEARCH Open Access Direct comparison of the histidine -rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I...Walsh1, David L Saunders1 and Charlotte A Lanteri1* Abstract Background: Performance of the histidine -rich protein-2 enzyme-linked immunosorbent... histidine -rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in

  3. Development of Enzyme-linked Immunosorbent Assay for Neomycin%新霉素ELISA检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    刘沙洲; 桑小雪; 欧阳华学; 雷绍荣; 白林含

    2011-01-01

    In this study, we describe the advantages and disadvantages of direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and indirect competitive ELISA (idc-ELISA) and ELISA methods for the detection of neomycin. Antineomycin polyclonal antibodies were prepared and used to detect neomycin by dc-ELISA and idc-ELISA. The cross-reaction rates of prepared anti-neomycin polyclonal antibodies with gentamincin and kanamycin were 2.04% and 0.02%, respectively, and with ampicillin, erythromycin and tetracycline all less than 0.01%. The accuracy and recovery of idc-ELISA were tested with an intra-plate error of less than 4%, an inter-plate error of less than 11% and a recovery between 135.5% and 191.3%. The detection limits of dc-ELISA and idc-ELISA were 28.58 ng/mL and 51.74 ng/mL, respectively, both of which were below the national maximum residue limit (MRL) of 500 pg/kg. Therefore, a dc-ELISA method and an idc-ELISA method to detect neomycin have successfully established. Further, the idc-ELISA method where the working conditions were better optimized can be used for the development of neomycin test kit.%目的:比较直接和间接竞争酶联免疫法(enzyme linked immunosorbent assay,ELISA)的优缺点,建立新霉素残留ELISA检测方法。方法:利用自制的新霉素多克隆抗体,采用直接竞争和间接竞争ELISA方法检测新霉素残留,并比较两种方法的优缺点。结果:新霉素抗血清和庆大霉素的交叉反应率为2.04%,和卡那霉素的交叉反应率为0.02%,和氨苄青霉素、红霉素、四环素的交叉反应率均小于0.01%。初步测试新霉素间接竞争ELISA法的准确性和回收率。板内误差小于4%,板间误差小于11%,回

  4. Enzyme-linked immunosorbent assay characterization of basal variation and heritability of systemic microfibrillar-associated protein 4.

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    Susanne Gjørup Sækmose

    Full Text Available BACKGROUND: Microfibrillar-associated protein 4 (MFAP4 is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM, and variation in systemic MFAP4 (sMFAP4 has the potential to reflect diverse diseases with increased ECM turnover. Here, we aimed to validate an enzyme-linked immunosorbent assay (ELISA for the measurement of sMFAP4 with an emphasis on the robustness of the assay. Moreover, we aimed to determine confounders influencing the basal sMFAP4 variability and the genetic contribution to the basal variation. METHODS: The sandwich ELISA was based on two monoclonal anti-MFAP4 antibodies and was optimized and calibrated with a standard of recombinant MFAP4. The importance of pre-analytical sample handling was evaluated regarding sample tube type, time, and temperature conditions. The mean value structure and variance structure was determined in a twin cohort including 1,417 Danish twins (age 18-67 years by mixed-effect linear regression modeling. RESULTS: The practical working range of the sandwich ELISA was estimated to be 4-75 U/ml. The maximum intra- and inter-assay variation was estimated to be 8.7% and 6.6%, respectively. Sample handling and processing appeared to influence MFAP4 measurements only marginally. The average concentration of sMFAP4 in the serum was 18.9 ± 8.4 (SD U/ml in the twin cohort (95% CI: 18.5-19.4, median sMFAP4 17.3 U/ml. The mean structure model was demonstrated to include waist-hip ratio, age, and cigarette smoking status in interactions with gender. A relatively low heritability of h(2 = 0.24 was found after applying a model including additive genetic factors and shared and non-shared environmental factors. CONCLUSIONS: The described ELISA provides robust measures of the liver fibrosis marker sMFAP4. The low heritability and the relatively

  5. Quantification of urinary 5-hydroxyindoleacetic acid by in-house nitrosonaphthol reaction compared with nitrosonaphthol micro column chromatography and enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Joyce Matie Kinoshita da Silva

    2014-06-01

    Full Text Available The aim of this study was to compare the colorimetric "kit" and enzyme-linked immunosorbent assay (ELISA methods to quantify urinary 5-hydroxyindoleacetic acid through the Goldenberg's technique, exploring the potential of replacing it. 24-hour urine samples were tested by Goldenberg's assay and compared with kits. The agreement was almost perfect for the comparison of Goldenberg's assay with both colorimetric kit, and with ELISA kit, considering ≤ 7.5 mg/24h normal cutoff value. Therefore, both "kits" would be good alternatives to Goldenberg's technique due to practicality and agreement between values.

  6. Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation.

    Science.gov (United States)

    Ito, A; Honey, R D; Scanlon, T; Lightowlers, M W; Rickard, M D

    1988-05-01

    Serum antibody responses in two strains of mice infected with embryonated eggs of Hymenolepis nana were analysed by the enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) using sodium deoxycholate (DOC)-solubilized antigens prepared from embryonated eggs (eggs), mouse-derived cysticercoids (cysts) and adult tapeworms with immature segments only (adults). Highly susceptible dd mice, which harbour mature tapeworms for a long period (greater than 70 days), produced high levels of antibodies to all three different stages of H. nana. BALB/c mice, almost all of which expel adult tapeworms by 30 days after infection, produced high levels of antibody against egg antigens only. The high antibody titres to cyst and adult antigens in dd mice did not lead to expulsion of the worms. However, worms are rejected early in BALB/c mice when there is little or no detectable serum antibody. The antibody responses to eggs seen in BALB/c mice which had long since shed their adult worms were probably due to ingestion of eggs from faeces of other infected mice. Antibodies to eggs were not detected in BALB/c mice which were initially inoculated with eggs (day 0) and then treated with praziquantel on day 6 after the tissue phase of infection only. The different antibody responses to egg antigens and the other two antigens (cyst and adult) in BALB/c mice suggest a difference in antigen specificity between eggs and both cysts and adults. A major antigen component with Mr 32,000 appears to be specific to the egg (or oncosphere) stage of H. nana. Antibody to this major component of eggs was absorbed only with intact eggs, but not with intact cysts nor adults with immature segments only, so that the antigen appears to be on the surface of the oncosphere.

  7. Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP.

    Science.gov (United States)

    Goo, Youn-Kyoung; Jia, Honglin; Aboge, G Oluga; Terkawi, M Alaa; Kuriki, Ken; Nakamura, Chinatsu; Kumagai, Akiko; Zhou, Jinlin; Lee, Eung-goo; Nishikawa, Yoshifumi; Igarashi, Ikuo; Fujisaki, Kozo; Xuan, Xuenan

    2008-04-01

    The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.

  8. Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay or polymerase chain reaction.

    Science.gov (United States)

    Dhinakar Raj, G; Sivakumar, S; Matheswaran, K; Chandrasekhar, M; Thiagarajan, V; Nachimuthu, K

    2003-10-01

    Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS) virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein, presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDS virus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared with the other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum against EDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome. Following experimental infection of oestrogen-treated chickens with EDS virus, cloacal swabs, oviduct, uterus and spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly and after a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by AC-ELISA or PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommended due to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommend that PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged in embryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.

  9. Plasmon-enhanced enzyme-linked immunosorbent assay on large arrays of individual particles made by electron beam lithography.

    Science.gov (United States)

    Chen, Si; Svedendahl, Mikael; Antosiewicz, Tomasz J; Käll, Mikael

    2013-10-22

    Ultrasensitive biosensing is one of the main driving forces behind the dynamic research field of plasmonics. We have previously demonstrated that the sensitivity of single nanoparticle plasmon spectroscopy can be greatly enhanced by enzymatic amplification of the refractive index footprint of individual protein molecules, so-called plasmon-enhanced enzyme-linked immunosorbent assay (ELISA). The technique, which is based on generation of an optically dense precipitate catalyzed by horseradish peroxidase at the metal surface, allowed for colorimetric analysis of ultralow molecular surface coverages with a limit of detection approaching the single molecule limit. However, the plasmonic response induced by a single enzyme can be expected to vary for a number of reasons, including inhomogeneous broadening of the sensing properties of individual particles, variation in electric field enhancement over the surface of a single particle and variation in size and morphology of the enzymatic precipitate. In this report, we discuss how such inhomogeneities affect the possibility to quantify the number of molecules bound to a single nanoparticle. The discussion is based on simulations and measurements of large arrays of well-separated gold nanoparticles fabricated by electron beam lithography (EBL). The new data confirms the intrinsic single-molecule sensitivity of the technique but we were not able to clearly resolve the exact number of adsorbed molecules per single particle. The results indicate that the main sources of uncertainty come from variations in sensitivity across the surface of individual particles and between different particles. There is also a considerable uncertainty in the actual precipitate morphology produced by individual enzyme molecules. Possible routes toward further improvements of the methodology are discussed.

  10. Limitations of the BP26 protein-based indirect enzyme-linked immunosorbent assay for diagnosis of Brucellosis.

    Science.gov (United States)

    Xin, Ting; Yang, Hongjun; Wang, Nan; Wang, Fang; Zhao, Peng; Wang, Haiguang; Mao, Kairong; Zhu, Hongfei; Ding, Jiabo

    2013-09-01

    Brucellosis is a serious zoonosis that occurs worldwide, and its diagnosis is typically based on the detection of antibodies against Brucella lipopolysaccharide (LPS). However, the specificity of the LPS-based test is compromised by cross-reactivity with Escherichia coli O157:H7 and Yersinia enterocolitica O:9. Also, diagnosis based on the LPS test cannot differentiate between vaccinated and infected individuals. The detection of the 26-kDa cytosoluble protein (BP26) antibody is considered an alternative that circumvents these drawbacks because it is exclusively expressed by infectious Brucella. A BP26-based enzyme-linked immunosorbent assay (ELISA) has been tried for the diagnosis of Brucella-infected animals and humans, but a few results showed that BP26 couldn't react with all Brucella-positive sera. In order to explore whether different animals could produce antibodies against BP26 after being infected with various Brucella species, we infected sheep, goats, and beef cattle with common virulent reference Brucella species. All sera were collected from the experimental animals and tested using both LPS-based ELISAs and BP26-based ELISAs. The results showed that all Brucella-infected individuals could produce high levels of antibodies against LPS, but only B. melitensis 16M- and B. melitensis M28-infected sheep and B. melitensis 16M- and B. abortus 2308-infected goats could produce antibodies against BP26. Therefore, we concluded that the BP26-based indirect ELISA (i-ELISA) showed both Brucella species and host specificity, which obviously limits its reliability as a substitute for the traditional LPS-based ELISA for the detection of brucellosis.

  11. Development of an Enzyme-Linked Immunosorbent Assay Method Specific for the Detection of G-Group Aflatoxins.

    Science.gov (United States)

    Li, Peiwu; Zhou, Qian; Wang, Ting; Zhou, Haiyan; Zhang, Wen; Ding, Xiaoxia; Zhang, Zhaowei; Chang, Perng-Kuang; Zhang, Qi

    2015-12-28

    To detect and monitor G-group aflatoxins in agricultural products, we generated class-specific monoclonal antibodies that specifically recognized aflatoxins G₁ and G₂. Of the final three positive and stable hybridomas obtained, clone 2G6 produced a monoclonal antibody that had equal sensitivity to aflatoxins G₁ and G₂, and did not cross-react with aflatoxins B₁, B₂, or M₁. Its IC50 values for aflatoxins G₁ and G₂ were 17.18 ng·mL(-1) and 19.75 ng·mL(-1), respectively. Using this new monoclonal antibody, we developed a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA); the method had a limit of detection of 0.06 ng·mL(-1). To validate this CI-ELISA, we spiked uncontaminated peanut samples with various amounts of aflatoxins G₁ and G₂ and compared recovery rates with those determined by a standard HPLC method. The recovery rates of the CI-ELISA ranging from 94% to 103% were comparable to those of the HPLC (92% to 102%). We also used both methods to determine the amounts of G-group aflatoxins in five peanut samples contaminated by aflatoxin B₁-positive, and their relative standard deviations ranged from 8.4% to 17.7% (under 20%), which demonstrates a good correlation between the two methods. We further used this CI-ELISA to assess the ability of 126 fungal strains isolated from peanuts or field soils to produce G-group aflatoxins. Among these, seven stains producing different amounts of G-group aflatoxins were identified. Our results showed that the monoclonal antibody 2 G6-based CI-ELISA was suitable for the detection of G-group aflatoxins present in peanuts and also those produced by fungi.

  12. Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea).

    Science.gov (United States)

    Ghekiere, An; Fenske, Martina; Verslycke, Tim; Tyler, Charles; Janssen, Colin

    2005-09-01

    Mysid crustaceans have been put forward by several regulatory bodies as suitable test organisms to screen and test the potential effects of environmental endocrine disruptors. Despite the well-established use of mysid reproductive endpoints such as fecundity, egg development time, and time to first brood release in standard toxicity testing, little information exists on the hormonal regulation of these processes. Control of vitellogenesis is being studied intensively because yolk is an excellent model for studying mechanisms of hormonal control, and vitellogenesis can be chemically disrupted. Yolk protein or vitellin is a major source of nourishment during embryonic development of ovigorous egg-laying invertebrates. The accumulation of vitellin during oocyte development is vital for the production of viable offspring. In this context, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for vitellin of the estuarine mysid Neomysis integer. Mysid vitellin was isolated using gel filtration, and the purified vitellin was used to raise polyclonal antibodies. The ELISA was sensitive within a working range of 4 to 500 ng vitellin/mL. Serial dilutions of whole body homogenates from female N. integer and the vitellin standard showed parallel binding curves, validating the specificity of the ELISA. The intra- and interassay coefficients of variation were 8.2% and 13.8%, respectively. Mysid vitellin concentrations were determined from ovigorous females and eggs at different developmental stages. The availability of a quantitative mysid vitellin ELISA should stimulate further studies on the basic biology of this process in mysids. Furthermore, it could provide a means to better understand and predict chemically induced reproductive effects in mysids.

  13. The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex, varicella zoster and cytomegalovirus retinitis.

    Directory of Open Access Journals (Sweden)

    Madhavan Hajib

    2003-01-01

    Full Text Available Purpose: To evaluate the diagnostic usefulness of enzyme linked immuno-sorbent assay (ELISA in single serum samples to associate herpes simplex virus (HSV, varicella zoster virus (VZV or cytomegalovirus (CMV with viral retinitis as against polymerase chain reaction (PCR on intraocular specimens. It was also designed to study the seroprevalence in normal healthy individuals, and the genomic prevalence of HSV, VZV and CMV in patients without an active viral inflammatory process. Methods: PCR for the detection of HSV, VZV and CMV genomes was done on 33 and 90 intraocular fluids from viral retinal patients and non-viral controls respectively. ELISA was done on 30 and 100 serum samples from viral retinitis patients and normal healthy controls respectively. Results: PCR did not detect HSV, VZV and CMV genomes except one, in which VZV-DNA was detected. ELISA showed prevalence rates of 28%, 83% and 90% for antibodies against HSV, VZV and CMV respectively in the normal population. In the 30 viral retinitis patients, PCR detected HSV-DNA in 2 (6.7%, VZV-DNA in 7 (23.3% and CMV-DNA in 6 (20.0% patients, while ELISA detected antibodies against HSV, VZV and CMV in 13 (43.3%, 24 (80.0% and 23 (76.7% patients respectively. ELISA was of value in indirect diagnosis only in 6 (20.0% as compared to 15 (50.0% of 30 patients by PCR, this difference was statistically significant (McNemar test, P value = 0.005. Conclusion: Serology by ELISA is no longer a useful diagnostic tool to associate HSV, VZV and CMV viruses with viral retinitis.

  14. Cellphone-Based Hand-Held Microplate Reader for Point-of-Care Testing of Enzyme-Linked Immunosorbent Assays.

    Science.gov (United States)

    Berg, Brandon; Cortazar, Bingen; Tseng, Derek; Ozkan, Haydar; Feng, Steve; Wei, Qingshan; Chan, Raymond Yan-Lok; Burbano, Jordi; Farooqui, Qamar; Lewinski, Michael; Di Carlo, Dino; Garner, Omai B; Ozcan, Aydogan

    2015-08-25

    Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity.

  15. Salivary Desmoglein Enzyme-Linked Immunosorbent Assay for Diagnosis of Pemphigus Vulgaris: A Noninvasive Alternative Test to Serum Assessment

    Directory of Open Access Journals (Sweden)

    Hossein Mortazavi

    2015-01-01

    Full Text Available Background. Serum desmoglein enzyme-linked immunosorbent assay (ELISA is used for the diagnosis and monitoring of pemphigus diseases. Objectives. To compare the diagnostic accuracy of salivary antidesmoglein (Dsg 1 and 3 ELISA in the diagnosis of pemphigus vulgaris (PV patients with that of serum desmogleins ELISA. Methods. Eighty-six untreated PV patients and 180 age- and sex-matched PV-free controls were recruited in this case-control study. PV was diagnosed based on clinical, histopathological, and direct immunofluorescence findings. After processing, serum and salivary anti-Dsg 1 and 3 were measured by the ELISA method using Euroimmun kit (Lübeck, Germany. Results. Using the cut-off point of 20 relative units (RU/mL, the serum anti-Dsg 1 and 3 ELISA were positive in 62 (72.1% and 83 (96.5% patients, respectively, and the salivary anti-Dsg 1 and 3 ELISA were positive in 31 (36.1% and 63 (73.3% patients, respectively. The specificity of salivary anti-Dsg 1 and anti-Dsg 3 were both 98.9%. Optimal cut-off values of 7.7 and 13.4 RU/mL were determined for the salivary anti-Dsg 1 and anti-Dsg 3 ELISA, respectively. Conclusion. Salivary anti-Dsg 1 and 3 ELISA with high specificities (98.9% could be suggested as safe and noninvasive methods for the diagnosis of PV when obtaining a blood sample is difficult.

  16. Evaluation and optimization of a commercial enzyme linked immunosorbent assay for detection of Chlamydophila pneumoniae IgA antibodies

    Directory of Open Access Journals (Sweden)

    Gargouri Jalel

    2008-07-01

    Full Text Available Abstract Background Serologic diagnosis of Chlamydophila pneumoniae (Cpn infection routinely involves assays for the presence of IgG and IgM antibodies to Cpn. Although IgA antibodies to Cpn have been found to be of interest in the diagnosis of chronic infections, their significance in serological diagnosis remains unclear. The microimmunofluorescence (MIF test is the current method for the measurement of Cpn antibodies. While commercial enzyme linked immunosorbent assays (ELISA have been developed, they have not been fully validated. We therefore evaluated and optimized a commercial ELISA kit, the SeroCP IgA test, for the detection of Cpn IgA antibodies. Methods Serum samples from 94 patients with anti-Cpn IgG titers ≥ 256 (study group and from 100 healthy blood donors (control group were tested for the presence of IgA antibodies to Cpn, using our in-house MIF test and the SeroCP IgA test. Two graph receiver operating characteristic (TG-ROC curves were created to optimize the cut off given by the manufacturer. Results The MIF and SeroCP IgA tests detected Cpn IgA antibodies in 72% and 89%, respectively, of sera from the study group, and in 9% and 35%, respectively, of sera from the control group. Using the MIF test as the reference method and the cut-off value of the ELISA test specified by the manufacturer for seropositivity and negativity, the two tests correlated in 76% of the samples, with an agreement of Ƙ = 0.54. When we applied the optimized cut-off value using TG-ROC analysis, 1.65, we observed better concordance (86% and agreement (0.72 between the MIF and SeroCP IgA tests. Conclusion Use of TG-ROC analysis may help standardize and optimize ELISAs, which are simpler, more objective and less time consuming than the MIF test. Standardization and optimization of commercial ELISA kits may result in better performance.

  17. Mycoplasma agassizii strain variation and distinct host antibody responses explain differences between enzyme-linked immunosorbent assays and Western blot assays.

    Science.gov (United States)

    Wendland, Lori D; Klein, Paul A; Jacobson, Elliott R; Brown, Mary B

    2010-11-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A₄₀₅ values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

  18. Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus

    Institute of Scientific and Technical Information of China (English)

    MO Hong-ying; XU Jun; REN Xiao-lan; ZENG Guang-qiao; TAN Ya-xia; CHEN Rong-chang; Moira Chan-Yeung; ZHONG Nan-shan

    2005-01-01

    Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients' sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

  19. Evaluation of an egg yolk enzyme-linked immunosorbent assay antibody test and its use to assess the prevalence of Mycoplasma gallisepticum infection in laying hens in Italy

    Directory of Open Access Journals (Sweden)

    Marco Tamba

    2010-01-01

    Full Text Available The prevalence of Mycoplasma gallisepticum infection in commercial layers was established by the presence of antibodies in eggs. Saline-extracted yolks were used with a commercial enzyme-linked immunosorbent assay kit. For the prevalence study, yolks from 30 eggs were obtained from each of 66 flocks coming from 36 layer farms. The prevalence of egg antibodies to Mycoplasma gallisepticum was 33.3% in single-age farms and 77.8% in multi-age farms. In 27 flocks, antibody titers were compared with results obtained from blood samples taken in the same flock and in the same period and analyzed with the same kit. This study has confirmed that egg yolk enzyme-linked immunosorbent assay antibody test is a suitable and practical approach for assessing the flock prevalence of Mycoplasma gallisepticum infection in layer hens.

  20. Comparison of the latex agglutination test with the hemagglutination inhibition test, enzyme-linked immunosorbent assay, and neutralization test for detection of antibodies to rubella virus.

    OpenAIRE

    Meegan, J M; Evans, B. K.; Horstmann, D. M.

    1982-01-01

    The ability of a rapid, latex agglutination test to diagnose rubella infection and to measure immune status was evaluated by comparison with the hemagglutination-inhibition (HAI) test, enzyme-linked immunosorbent assay (ELISA), and the neutralization (NT) test. The latex agglutination test accurately detected serological conversions in 74 pairs of sera representing 21 natural infections and 53 immunizations. The antibody levels of 276 sera from the general population were determined by latex ...

  1. Rapid Detection of Campylobacter coli, C. jejuni, and Salmonella enterica on Poultry Carcasses by Using PCR-Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Hong, Yang; Mark E Berrang; Liu, Tongrui; Hofacre, Charles L.; Sanchez, Susan; Wang, Lihua; Maurer, John J.

    2003-01-01

    Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobac...

  2. Comparison of Three Enzyme-Linked Immunosorbent Assays for Detection of Immunoglobulin G Antibodies to Tetanus Toxoid with Reference Standards and the Impact on Clinical Practice▿

    OpenAIRE

    van Hoeven, Karen H.; Dale, Connie; Foster, Phil; Body, Barbara

    2008-01-01

    Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglo...

  3. Serology of Neisseria gonorrhoeae: W-antigen serogrouping by coagglutination and protein I serotyping by enzyme-linked immunosorbent assay both detect protein I antigens.

    OpenAIRE

    Sandstrom, E G; Knapp, J S; Buchanan, T B

    1982-01-01

    A total of 224 strains were serogrouped by coagglutination (COA) and serotyped by protein I enzyme-linked immunosorbent assay (ELISA). Of these strains, 61 were from patients with disseminated gonococcal infection, 21 were from patients with pelvic inflammatory disease, and 115 were from patients with uncomplicated gonococcal infection in Singapore, the Philippines, and Denmark. Twenty-seven were laboratory reference strains. Of the patient strains, 102 belonged to COA serogroup WI, and all o...

  4. Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus.

    Science.gov (United States)

    Devenish, J; Brooks, B; Perry, K; Milnes, D; Burke, T; McCabe, D; Duff, S; Lutze-Wallace, C L

    2005-11-01

    A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35 degrees C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus

  5. Multicountry Prospective Clinical Evaluation of Two Enzyme-Linked Immunosorbent Assays and Two Rapid Diagnostic Tests for Diagnosing Dengue Fever

    Science.gov (United States)

    Dauner, Allison L.; Valks, Andrea; Forshey, Brett M.; Long, Kanya C.; Thaisomboonsuk, Butsaya; Sierra, Gloria; Picos, Victor; Talmage, Sara; Morrison, Amy C.; Halsey, Eric S.; Comach, Guillermo; Yasuda, Chadwick; Loeffelholz, Michael; Jarman, Richard G.; Fernandez, Stefan; An, Ung Sam; Kochel, Tadeusz J.; Jasper, Louis E.; Wu, Shuenn-Jue L.

    2015-01-01

    We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 febrile individuals from Thailand were also used. Reference testing was performed on all samples using an algorithm involving virus isolation, in-house IgM and IgG capture ELISAs, and plaque reduction neutralization tests (PRNT) to determine the infection status of the individual. The primary endpoints were the clinical sensitivities and specificities of these devices. The SD Bioline Dengue Duo had an overall sensitivity of 87.3% (95% confidence interval [CI], 84.1 to 90.2%) and specificity of 86.8% (95% CI, 83.9 to 89.3%) during the first 14 days post-symptom onset (p.s.o.). The Panbio Dengue Duo Cassette demonstrated a sensitivity of 92.1% (87.8 to 95.2%) and specificity of 62.2% (54.5 to 69.5%) during days 4 to 14 p.s.o. The Panbio IgM capture ELISA had a sensitivity of 87.6% (82.7 to 91.4%) and specificity of 88.1% (82.2 to 92.6%) during days 4 to 14 p.s.o. Finally, the Panbio IgG capture ELISA had a sensitivity of 69.6% (62.1 to 76.4%) and a specificity of 88.4% (82.6 to 92.8%) during days 4 to 14 p.s.o. for identification of secondary dengue infections. This multicountry prospective study resulted in reliable real-world performance data that will facilitate data-driven laboratory test choices for managing patient care during dengue outbreaks. PMID:25588659

  6. Preparation of Monoclonal Antibody and Development of Enzyme-linked Immunosorbent Assay Specific for Escherichia coli O157 in Foods

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To prepare monoclonal antibodies (Mab) and antisera specific for Escherichia coli (E.coli) O157, and to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect E.coli O157 in foods. Methods Spleen cells from BALB/c mice immunized with the somatic antigen of E.coli O157:H7 were fused with murine Sp2/0 myeloma cells. The hybridoma cell line specific for E.coli O157 was established after having been subcloned. Antisera specific for E.coli O157 was prepared by intravenous injection into New Zealand rabbits with a stain of E.coli O157:H7. The sandwich ELISA was developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The inoculated ground poultry meat and pasteurized milk were tested to confirm efficiency of the method. Results Mab 3A5 specific for E.coli O157 and O113:H21 belonged to subtype IgM. The ascetic titers of the antibody was 1:1×106. No cross-reactivity of the Mab was observed with strains of Salmonella spp, Yersinia enterocolitica, Shigella dysenteriae, etc. The purified polyclonal antibody had a titer of 1:1×105 with E.coli O157. The detection limit of this sandwich ELISA was 103-104 cfu E.coli O157/mL in pure culture with a high specificity, which was characterized by every non-O157 strain with negative response. With 10h enrichment procedure, E.coli O157:H7 recovered well from inoculated ground poultry meat and pasteurized milk at levels of 0.1 cfu/g and 0.1 cfu/mL. Conclusion Mab 3A5 specific for E.coli O157 and O113:H21 can be produced by immunizing BALB/c mice with a strain of E.coli O157:H7. Then a sandwich ELISA can be developed with the polyclonal antibody as the capture antibody and the Mab 3A5 as the detection antibody. The method is proved to be a sensitive and specific technique to detect low number of E.coli O157 in food.

  7. Enzyme-linked immunosorbent assay for detection of antibodies to the venereal disease research laboratory (VDRL) antigen in syphilis.

    Science.gov (United States)

    Pedersen, N S; Orum, O; Mouritsen, S

    1987-09-01

    An enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin G (IgG) and IgM to cardiolipin, lecithin, and cholesterol (VDRL [Venereal Disease Research Laboratory] ELISA) is described. The specificity of the VDRL ELISA for IgG and IgM was 99.6 and 99.5%, respectively, with sera from 1,008 persons without syphilis. For a group of patients with false-positive results in traditional nontreponemal tests and for patients with autoimmune diseases, the VDRL ELISA for IgG had a higher specificity than the VDRL ELISA for IgM. The sensitivity for IgG and IgM with 118 sera from patients with untreated syphilis was 96.6 and 94.9%, respectively, which was equivalent to the sensitivities of the traditional nontreponemal tests. The performance of the VDRL ELISA was compared with that of an ELISA that uses cardiolipin as the antigen (cardiolipin ELISA). The VDRL ELISA was significantly more sensitive (P less than or equal to 0.01) than the cardiolipin ELISA with 25 sera from syphilis patients but was less sensitive (P less than or equal to 0.01) with 53 sera from patients with autoimmune diseases. The antibody reactivity in the VDRL ELISA could not be absorbed out by lecithin and cholesterol, and the sera from patients with syphilis did not react in an ELISA that uses cholesterol and lecithin as the antigen. This indicates that cholesterol and lecithin, although not antigenic by themselves, may change the structural form of the epitope on cardiolipin so that it becomes more recognizable for antibodies in syphilis and less recognizable for antibodies in autoimmune diseases. The results of the VDRL ELISA were expressed in percentages of the absorbance value of a positive control. The VDRL ELISA gave, without titration of sera, quantitative results that correlated with the quantitative results of the traditional nontreponemal tests obtained by titration. The VDRL ELISA will be well suited for large-scale testing for syphilis and may replace other nontreponemal tests.

  8. Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Sharon, Galit; Nath, Pulak R; Isakov, Noah; Zilberg, Dina

    2014-09-15

    Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse anti-guppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10 μL) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund's adjuvant (15,000 Tetrahymena/fish in total a volume of 20 μL). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from

  9. High seroprevalence of Mycoplasma pneumoniae IgM in acute Q fever by enzyme-linked immunosorbent assay (ELISA.

    Directory of Open Access Journals (Sweden)

    Chung-Hsu Lai

    Full Text Available Q fever is serologically cross-reactive with other intracellular microorganisms. However, studies of the serological status of Mycoplasma pneumoniae and Chlamydophila pneumoniae during Q fever are rare. We conducted a retrospective serological study of M. pneumoniae and C. pneumoniae by enzyme-linked immunosorbent assay (ELISA, a method widely used in clinical practice, in 102 cases of acute Q fever, 39 cases of scrub typhus, and 14 cases of murine typhus. The seropositive (57.8%, 7.7%, and 0%, p<0.001 and seroconversion rates (50.6%, 8.8%, and 0%, p<0.001 of M. pneumoniae IgM, but not M. pneumoniae IgG and C. pneumoniae IgG/IgM, in acute Q fever were significantly higher than in scrub typhus and murine typhus. Another ELISA kit also revealed a high seropositivity (49.5% and seroconversion rate (33.3% of M. pneumoniae IgM in acute Q fever. The temporal and age distributions of patients with positive M. pneumoniae IgM were not typical of M. pneumoniae pneumonia. Comparing acute Q fever patients who were positive for M. pneumoniae IgM (59 cases with those who were negative (43 cases, the demographic characteristics and underlying diseases were not different. In addition, the clinical manifestations associated with atypical pneumonia, including headache (71.2% vs. 81.4%, p=0.255, sore throat (8.5% vs. 16.3%, p=0.351, cough (35.6% vs. 23.3%, p=0.199, and chest x-ray suggesting pneumonia (19.3% vs. 9.5%, p=0.258, were unchanged between the two groups. Clinicians should be aware of the high seroprevalence of M. pneumoniae IgM in acute Q fever, particularly with ELISA kits, which can lead to misdiagnosis, overestimations of the prevalence of M. pneumoniae pneumonia, and underestimations of the true prevalence of Q fever pneumonia.

  10. Performance of four different indirect enzyme-linked immunosorbent assays (ELISAs) to detect specific IgG, IgA, and IgM in Legionnaires' disease

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Shand, G H; Hansen, K

    1994-01-01

    antigenic preparations of L. pneumophila serogroup 1 consisting of outer membrane protein (OMP), flagellin (FLA), and lipopolysaccharide (LPS) to a sonic extract (SON) in indirect immunosorbent assay (ELISA) measuring both IgG, IgA, and IgM. The reactivity of sera from 20 patients with culture......, closely followed by the corresponding SON tests. By combining two individual assays, a maximum nosographic sensitivity of 85% could be obtained. Whereas no benefit of using purified outer membrane protein or flagella instead of a sonic extract in the indirect ELISAs was found, the LPS antigen provided...... a sensitive and specific alternative to the sonic extract....

  11. Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1.

    Science.gov (United States)

    Karmali, M A; Petric, M; Winkler, M; Bielaszewska, M; Brunton, J; van de Kar, N; Morooka, T; Nair, G B; Richardson, S E; Arbus, G S

    1994-06-01

    The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group. A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA). The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test). With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6%. There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive. The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS. The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering. The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in

  12. Detection of flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA)--new prospects for diagnosis.

    Science.gov (United States)

    Monfort, J D; Bech-Nielsen, S; Stills, H F

    1994-01-01

    A new diagnostic procedure was developed to detect the flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected all C. jejuni or C. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative for C. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens of C. jejuni and C. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.

  13. Visual Detection of Canine Parvovirus Based on Loop-Mediated Isothermal Amplification Combined with Enzyme-Linked Immunosorbent Assay and with Lateral Flow Dipstick

    OpenAIRE

    Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

    2013-01-01

    ABSTRACT Loop-mediated isothermal amplification (LAMP) combined with enzyme-linked immunosorbent assay (LAMP–ELISA) and with lateral flow dipstick (LAMP–LFD) are rapid, sensitive and specific methods for the visual detection of clinical pathogens. In this study, LAMP–ELISA and LAMP–LFD were developed for the visual detection of canine parvovirus (CPV). For LAMP, a set of four primers (biotin-labeled forward inner primers) was designed to specifically amplify a region of the VP2 gene of CPV. T...

  14. Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus.

    Science.gov (United States)

    Chen, Xiaowei; Song, Cailing; Liu, Yun; Qu, Liandong; Liu, Dafei; Zhang, Yun; Liu, Ming

    2016-02-01

    For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV.

  15. Evaluation of a Commercial SD Dengue Virus NS1 Antigen Capture Enzyme-Linked Immunosorbent Assay Kit for Early Diagnosis of Dengue Virus Infection▿

    OpenAIRE

    Wang, Seok Mui; Sekaran, Shamala Devi

    2010-01-01

    Early definitive diagnosis of dengue virus infection may help in the timely management of dengue virus infection. We evaluated the Standard Diagnostics (SD, South Korea) dengue virus nonstructural protein NS1 antigen enzyme-linked immunosorbent assay (SD dengue NS1 Ag ELISA) for the detection of dengue virus NS1 antigen in patients' sera, using a total of 399 serum samples in a comparison with real-time reverse transcription (RT)-PCR, an in-house IgM capture (MAC)-ELISA, and a hemagglutinatio...

  16. Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using Au-Pt/SiO2 nanocomposites as labels.

    Science.gov (United States)

    Wu, Long; Yin, Wenmin; Tang, Kun; Shao, Kang; Li, Qin; Wang, Pan; Zuo, Yunpeng; Lei, Xiaomin; Lu, Zhicheng; Han, Heyou

    2016-08-15

    Improving the performance of conventional enzyme-linked immunosorbent assay (ELISA) is of great importance to meet the demand of early clinical diagnosis of various diseases. Herein, we report a feasible enzyme-free immunosorbent assay (EFISA) system using antibody conjugated Au-Pt/SiO2 nanocomposites (APS NCs) as labels. In this system, Au-Pt/SiO2 nanospheres (APS NPs) were first synthesized by wet chemical method and exhibited intrinsic peroxidase and catalase-like activity with excellent water-solubility. Then APS NCs were utilized as labels to replace HRP conjugated antibody, and Fe3O4 magnetic beads (MBs) to entrap the analyte. To discuss the performance of EFISA system, Human IgG was served as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples. The system boosted the detection limit of HIgG to 75pgmL(-1) with a RSD below 5%, a 264-fold improvement as compared with conventional ELISA. This is the first time that APS NCs have been used and successfully optimized for the sensitive dilution detection of PCV2 antibody (5:10(7)) in ELISA. Besides, APS NCs have advantages related to low cost, easy preparation, good stability and tunable catalytic activity, which make them a potent enzyme mimetic candidate and may find potential applications in bioassays and clinical diagnostics.

  17. Tetra-substituted Amino Aluminum Phthalocyanine as a New Red-region Fluorescent Substrate for Horseradish Peroxidase Based Enzyme-linked Immunosorbent Assay

    Institute of Scientific and Technical Information of China (English)

    YANG,Huang-Hao(杨黄浩); LI,Dong-Hui(李东辉); CHEN,Xiao-Lan(陈小兰); QU,Hui-Ying(曲会英); DING,Ma-Tai(丁马太); XU,Jin-Gou(许金钩)

    2002-01-01

    The use of tetra-substituted amino aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc displayed an excitation maximum at 610 mn and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H2O2 could rapidly and significantly react with TAAIPc, thus quenching the fluorescence of TAAIPc. The Michaelis- Menten parameters Km and Vmax were measured to be 2.82×10-6 mol/L-1 amt6.0×10-9 mol.L-1.s-1, respectively. In this paper, TAAlPc was used in an HRP-based ehzyme-linked immunosorbent assay (ELISA) of a-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5-200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.

  18. Enzyme-linked immunosorbent assay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies.

    Science.gov (United States)

    Pongkitwitoon, Benyakan; Sakamoto, Seiichi; Tanaka, Hiroyuki; Tsuchihashi, Ryota; Kinjo, Junei; Morimoto, Satoshi; Putalun, Waraporn

    2010-05-01

    Pueraria candollei (White Kwao Khuer) is a medicinal plant containing puerarin, daidzin, genistin, daidzein, and genistein as major isoflavonoids used for its rejuvenating and estrogenic effects. In order to analyze these compounds, a single enzyme-linked immunosorbent assay (ELISA) for total isoflavonoids was developed using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs). The range for calibration of isoflavonoids by ELISA was 0.05-6.25 microg/mL. Total isoflavonoid concentrations in P. candollei samples determined by the newly developed assay system showed good agreement with those analyzed by HPLC. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of total isoflavonoids in P. candollei.

  19. Enzyme-linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S)

    DEFF Research Database (Denmark)

    Leeming, Diana J; Nielsen, Mette J; Dai, Yueqin

    2012-01-01

    Aim:  The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100...... were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P collagen...... expression in BDL rats (r = 0.49, P collagen in CCL4 treated livers (P 

  20. A sensitive enzyme-linked immunosorbent assay amplified by biotin-streptavidin system for detecting non-steroidal anti-inflammatory drug ketoprofen.

    Science.gov (United States)

    Bu, Dan; Zhuang, Hui S; Yang, Guang X

    2014-01-01

    A sensitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed for detecting non-steroidal anti-inflammatory drug ketoprofen. Compared with traditional ELISA method, the sensitivity of proposed immunoassay was enhanced by the biotin-streptavidin system. Under the optimal condition, the median inhibitory concentration (IC50) was 0.25 ng mL(-1), with minor cross-reactivity to a number of structural analogs. This developed assay was successfully applied to detect the ketoprofen residues in different fish samples, and good recoveries (72.6-105.5%) were obtained. The results indicated that this immunoassay method could specifically detect trace ketoprofen residues and could be widely used for routine monitoring of food samples.

  1. Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    J.J.N. Ngeranwa

    2008-09-01

    Full Text Available The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra and domestic animal (cattle, sheep and goat populations was studied in wildlife / livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA, using a recombinant antigen (MSP-5 from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA, as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers.

  2. An undetectable source of technical error that could lead to false negative results in enzyme linked immunosorbent assay of antibodies to HIV-1.

    Science.gov (United States)

    Wiltbank, T B; McCarroll, D R; Wartick, M G

    1989-01-01

    Since the institution of routine testing for antibodies to Human Immunodeficiency Virus (HIV) using the enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of this assay system has received significant scrutiny. During previous use of this methodology, we have quantified rates of false biological positive results using commercial kit assays in a normal donor population. In this study, we have identified a potential source for false negative results. Using multiple lots of two different commercial ELISA kits, the absorbance readings at the test end point could not differentiate between normal non-reactive donor samples and blanks containing no sample. These results occur using normal donor samples, even though the assays could distinguish between blank wells and the manufacturers' "normal controls", provided with the assay. Our findings suggest that a technical pipetting error is presently undetectable, either visually or by statistical methods, and could permit an untested, potentially HIV-1 positive, unit to be released into the transfusable blood supply. A possible solution is suggested.

  3. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    Science.gov (United States)

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  4. Bovine viral diarrhea virus antigen detection across whole cattle hides using two antigen-capture enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Vander Ley, Brian L; Ridpath, Julia F; Sweiger, Shaun H

    2012-05-01

    Bovine viral diarrhea virus is a costly disease of cattle that can be controlled by vaccination, biosecurity, and removal of persistently infected cattle. Development and proficiency testing of assays to identify persistently infected cattle requires substantial quantities of known positive- and negative-sample material. The objective of this study was to determine what sections of bovine skin contained Bovine viral diarrhea virus antigen. Two commercially available antigen-capture enzyme-linked immunoassays were used to test subsamples representing the entire skin of 3 persistently infected calves. Both assays detected Bovine viral diarrhea virus antigen in the samples indicated for use by assay protocol. However, one assay identified all subsamples as positive, while the second assay identified 64.4% of subsamples as positive. These results show that use of samples other than those specified by the assay protocol must be validated for each individual assay. In this study, alternative sample sites and use of the entire hide for proficiency testing would be acceptable for only one of the assays tested.

  5. Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Sathe, Shridhar K

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

  6. Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein.

    Science.gov (United States)

    Fan, Jing-Hui; Zuo, Yu-Zhu; Shen, Xiao-Qiang; Gu, Wen-Yuan; Di, Jing-Mei

    2015-12-01

    The recent dramatic increase in reported cases of porcine epidemic diarrhea (PED) in pig farms is a potential threat to the global swine industry. Therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (PEDV) infection or vaccination would be essential in helping to control the spread of PED. We developed and validated an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant membrane (M) protein of PEDV. To detect PEDV antibodies in eight herds, 382 serum samples were collected from sows that had been immunized with a PED vaccine, and screened using the developed ELISA in parallel with a serum neutralization (SN) assay. Of the tested samples, 276 were positive for the presence of PEDV antibodies according to both assays, while 98 were negative. An excellent agreement between the ELISA and the SN assay was observed (kappa=0.947; 95% confidence interval=0.910-0.984; McNemar's test, P=0.727). No cross-reaction was detected for the developed ELISA with other coronaviruses or other common pig pathogens. The developed ELISA could be used for serological evaluation and indirect diagnosis of PED infection.

  7. A liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay for the detection of antibodies against Newcastle disease virus in serum of free-ranging pigeons.

    Science.gov (United States)

    de Oliveira, Elisabete Schirato; Silva, Ketherson Rodrigues; Fernando, Filipe Santos; Gonçalves, Mariana Costa Mello; Fernandes, Camila Cesário; Borzi, Mariana Monezi; dos Santos, Romeu Moreira; Tamanini, Maria de Lourdes Feres; Montassier, Maria de Fátima da Silva; Montassier, Helio José

    2013-11-01

    A competitive liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay (LPB-ConA-ELISA) was developed in the current study. The assay used ConA as a capture reagent, and the sera of specific pathogen-free chickens immunized with nonpurified Newcastle disease virus (NDV) suspension as detector antibodies, to detect and quantify specific antiviral antibodies in serum samples from free-ranging pigeons. The comparison between the LPB-ConA-ELISA and the hemagglutination inhibition (HI) test for the detection of antibodies in serum samples from 107 pigeons showed significant correlation between the assays (r = 0.875), a high sensitivity (100%), specificity (95.8%), accuracy (96.3%) for the ELISA, and good agreement (κ = 0.83) between the 2 assays. The results of this study suggest that the LPB-ConA-ELISA could be a useful alternative to HI test in the serodiagnosis of NDV in pigeons, or other species of birds.

  8. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley.

    Science.gov (United States)

    Rampitsch, Christof; Ames, Nancy; Storsley, Joanne; Marien, Lindsay

    2003-09-24

    A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.

  9. Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical.

  10. Detection of antibodies to Campylobacter in humans using enzyme-linked immunosorbent assays: a review of the literature.

    Science.gov (United States)

    Kuhn, Katrin Gaardbo; Falkenhorst, Gerhard; Ceper, Tina; Dalby, Tine; Ethelberg, Steen; Mølbak, Kåre; Krogfelt, Karen A

    2012-10-01

    Campylobacteriosis is the most common cause of bacterial foodborne illness in the European Union and the United States. Infection with Campylobacter spp. is frequently associated with different sequelae including neuropathies and reactive arthritis. Diagnosis is mainly by bacterial culturing which is time consuming, expensive, and not well suited for diagnosing sequelae or identifying infections from stool samples with nonviable bacteria. Serologic assays, in particular ELISAs, are well suited for this purpose, but, at present, there is no international consensus on antibody assays for human campylobacteriosis. In an extensive literature review, 19 studies validating such assays were identified of which 13 were more than 10 years old. We conclude that the best validated of these assays are developed and used in-house for research purposes rather than for routine diagnostics. Considering the burden of disease and potential long-term severity of Campylobacter infections, developing a standardized, commercially available antibody assay could be of great benefit for diagnostic and surveillance purposes worldwide. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Diagnostic criteria for an enzyme-linked immunosorbent assay for occult heartworm disease: standardization of the test system in naturally exposed dogs.

    Science.gov (United States)

    Gillis, J M; Smith, R D; Todd, K S

    1984-11-01

    The development of criteria for interpreting and reporting the results of an occult heartworm enzyme-linked immunosorbent assay to practitioners is described. The antigen is a saline extract of adult female Dirofilaria immitis. The cutoff absorbance A400 nm values were estimated, using 106 dogs free of infection. Any A400 nm value less than 0.526 is considered negative and values greater than or equal to 0.784 are positive. Intermediate A400 nm values are interpreted as suspect. Absorbance values for serum samples from 13 client-owned amicrofilaremic dogs revealed a bimodal distribution consistent with presumptive diagnosis based on clinical signs, which indicates that the test may be used to support a diagnosis of occult heartworm disease. The present serotest, however, is unable to distinguish microfilaremic dogs from noninfected dogs. Serum from dogs infected with other common helminths failed to crossreact with the D immitis antigen, with the exception of Dipetalonema reconditum.

  12. Simultaneous determination of soy isoflavone glycosides, daidzin and genistin by monoclonal antibody-based highly sensitive indirect competitive enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sakamoto, Seiichi; Yusakul, Gorawit; Pongkitwitoon, Benyakan; Paudel, Madan Kumar; Tanaka, Hiroyuki; Morimoto, Satoshi

    2015-02-15

    Soy isoflavones are known as major bioactive compounds in soybean (Glycine max), which is an indispensable food. Despite their utility, the consumption of isoflavones has recently been limited because they exhibit oestrogenic and topoisomerase II inhibitory effects. To assess their intake limitation, accurate, sensitive, and effective quantitative analyses are necessary. In this study, we produced the monoclonal antibody (MAb) against daidzin (DZ) and applied it to an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the simultaneous determination of DZ and genistin (GEN), which are known as two major soy isoflavone glycosides in soy products. Using the DZ-MAb, we developed a sensitive icELISA method, where the limit of detection for DZ and GEN was 1.95ng/ml. Several validation analyses revealed that the icELISA is sufficiently accurate and sensitive to be used to assess the overconsumption of soy isoflavones, which would lead to the safe dietary intake of soy products.

  13. Tips and step-by-step protocol for the optimization of important factors affecting cellular enzyme-linked immunosorbent assay (CELISA).

    Science.gov (United States)

    Morandini, R; Boeynaems, J M; Wérenne, J; Ghanem, G

    2001-01-01

    CELISA, or cellular enzyme-linked immunosorbent assay, is a powerful and easy to use technique to study cell surface antigens under different stimulations. Nevertheless, some factors must be discussed and optimized prior to reaching a reproducible CELISA. These include the choice of cell density, fixative agent, blocking agent, culture medium, optimal antibody dilutions, and incubation time. In this paper, we first present a short review of some references devoted to CELISA by means of a comparison of these parameters, followed by their description. Then, we describe and study these different parameters using practical examples comparing TNF-induced ICAM-1 expression as an end point, on HBL melanoma and HUVEC. These cell lines were also chosen because they differ in their ability to grow as discontinuous and continuous layers, respectively. Furthermore, we designed a comprehensive flow chart, as well as a complete step-by-step protocol for CELISA optimization.

  14. Simultaneous determination of 13 fluoroquinolone and 22 sulfonamide residues in milk by a dual-colorimetric enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Jiang, Wenxiao; Wang, Zhanhui; Beier, Ross C; Jiang, Haiyang; Wu, Yongning; Shen, Jianzhong

    2013-02-19

    Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor two classes of antimicrobial residues in different food matrixes. In this paper, we describe a dual-colorimetric ELISA for the simultaneous detection of 13 fluoroquinolone and 22 sulfonamide residues. The limit of detection for fluoroquinolones and sulfonamides was 2.4 and 5.8 ng/mL, respectively. The developed immunoassay is suitable for high-throughput screening of these low-molecular weight contaminants. This is the first report where two different enzymes (alkaline phosphatase and horseradish peroxidase) were used in one immunoassay and together in a single well for simultaneous detection of multiple low-molecular weight chemical residues.

  15. The use of enzyme-linked immunosorbent assay and immunoblotting for the detection of Campylobacter fetus immunoglobulins in the cervico-vaginal mucus of female cattle

    Directory of Open Access Journals (Sweden)

    A.O. Pellegrin

    2011-03-01

    Full Text Available An indirect enzyme-linked immunosorbent assay was developed to detect antigen-specific secretory IgA antibodies to Campylobacter fetus subsp. venerealis in bovine vaginal mucus with a protein extract of the Campylobacter fetus subsp. venerealis by the acid glycine extraction method. Mean optical density measurement (λ=450 nm was 0.143±0.9. The most immunoreactive protein bands of the Campylobacter fetus subsp. venerealis or Campylobacter fetus subsp. fetus recognized by IgA in immunoblotting, using bovine vaginal mucus samples, migrate at 42.6 kDa. The protein that migrates at 93 kDa was recognized exclusively for C. fetus subsp. venerealis. A positive vaginal mucus sample of a cow from negative herd recognized antigens of C. jejuni subsp. jejuni e C. fetus subsp. fetus.

  16. Evaluation of an O antigen enzyme-linked immunosorbent assay for screening of milk samples for Salmonella dublin infection in dairy herds

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Lind, Peter; Bitsch, V.

    1995-01-01

    Levels of antibodies to the O antigens (0:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum......-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, r(s) = 0.69, P milk samples to screening and, eventually, regular certification of herds....... of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA...

  17. Detection of plum pox virus by enzyme-linked immunosorbent assay in some apricot and peach varieties and hybrids in Romania.

    Science.gov (United States)

    Toma, S; Isac, M; Balan, V; Ivascu, A

    1998-09-01

    Plum pox virus (PPV) is a potyvirus widely spread in many species of the Prunus genus such as plum, apricot, peach, sweet cherry and others. This potyvirus causes great damage to stone fruit trees in Romania and other European countries as Hungary, Italy, Czech Republic, France, Spain, Greece, Turkey, and Slovak Republic. The Research Station for Fruit Tree Growing Baneasa in Bucharest has realized many studies on the epidemiology and spread of PPV and also on the disease symptomatology and detection possibilities. The control of sharka disease by sanitary selection measures requires corresponding detection techniques. The aim of this study was to determine the presence or absence of PPV in some apricot and peach varieties and hybrids in 1995-1997 by the enzyme-linked immunosorbent assay (ELISA) and to verify if some of our biological materials evaluated as symptom-free under field conditions for many years are also virus-free and can be considered healthy.

  18. The enzyme-linked immunosorbent assay (ELISA) as a serological test for detecting antibodies against Leptospira interrogans serovar hardjo in sheep.

    Science.gov (United States)

    Adler, B; Faine, S; Gordon, L M

    1981-09-01

    The enzyme-liked immunosorbent assay (ELISA) was compared with the standard microscopic agglutination test (MAT) as a method for detecting antibodies against Leptospira interrogans serovar hardjo in sheep. Peak antibody levels detected by the 2 tests occurred at different times following experimental infection of sheep. In serums from flocks of sheep with naturally acquired infection there was a 95% correlation between MAT and ELISA with respect to the presence or absence of antibody to serovar hardjo, although the levels of correlation of the titres of the 2 tests was low. The 2 tests appeared to measure different antigen-antibody systems. The ELISA would be a useful test for screening large numbers of serums for antibodies to L. interrogans serovar hardjo.

  19. Development and application of an enzyme-linked immunosorbent assay (ELISA) for the quantification of amygdalin, a cyanogenic glycoside, in food.

    Science.gov (United States)

    Bolarinwa, Islamiyat F; Orfila, Caroline; Morgan, Michael R A

    2014-07-09

    Amygdalin is a member of the cyanogenic glycoside group of plant secondary metabolites capable of generating hydrogen cyanide under certain conditions. As a consequence, the cyanogenic glycosides have been associated with incidents of acute and subacute food poisoning. Specific antibodies were raised against an amygdalin-bovine serum albumin immunogen synthesized using a novel approach. The antibodies were used in a microtitration plate enzyme-linked immunosorbent assay (ELISA) for the quantification, for the first time, of amygdalin in commercially available foods. Correlation of results with high-performance liquid chromatography was very high (r = 0.983). The limit of detection of the immunoassay was 200 ± 0.05 pg mL(-1), and the 50% inhibitory concentration of amygdalin was 50 ± 0.02 ng mL(-1), making the ELISA particularly sensitive.

  20. Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals.

    Science.gov (United States)

    Beaugrand, Johnny; Gebruers, Kurt; Ververken, Cedric; Fierens, Ellen; Dornez, Emmie; Goddeeris, Bruno M; Delcour, Jan A; Courtin, Christophe M

    2007-09-19

    To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals.

  1. [Evaluation of usefulness of commercial recomwell Campylobacter enzyme--linked immunosorbent assays for routine serodiagnosis of Campylobacter jejuni and Campylobacter coli infections].

    Science.gov (United States)

    Rokosz, Natalia; Rastawicki, Waldemar; Jagielski, Marek

    2008-01-01

    The commercially available enzyme-linked immunosorbent assays (ELISA recomWell Campylobacter) from Mikrogen was evaluated for the diagnosis of Campylobacter jejuni and Campylobacter coli infections. Serum samples from 20 healthy controls, 44 persons with symptoms of primary Campylobacter infection and 24 serum samples from patients with Yersinia enterocolitica or Salmonella infections were tested. This ELISA assay detects IgA and IgG antibodies against three recombinant antigens of the Campylobacter jejuni and Campylobacter coli: OMP 18 (18 kDa), PEB4 (31 kDa) and P39 (39 kDa). The healthy controls showed significantly lower antibody titers in all two immunoglobulin classes. The IgA antibodies were diagnosed only in 2 (18.2%) serum samples obtained from patients with bacteriologically confirmed campylobacteriosis. The presence of IgG antibodies was confirmed in 82% of serum samples. Furthermore, we showed that 66.7% of the 33 serum samples obtained from the patients suspected for campylobacteriosis not confirmed by isolation, were positive for IgG and 15.2% for IgA antibodies. We observed also not specific reactions in ELISA recom Well Campylobacter with sera obtained form patients with yersiniosis and salmonelosis. This study demonstrates the usefulness of commercially available assay for the routine diagnosis of Campylobacter infection but with some limitations.

  2. Development and application of a double-antigen sandwich enzyme-linked immunosorbent assay for detection of antibodies to porcine circovirus 2.

    Science.gov (United States)

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong; Yu, Xinglong

    2012-09-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.

  3. An electric detection of immunoglobulin G in the enzyme-linked immunosorbent assay using an indium oxide nanoparticle ion-sensitive field-effect transistor

    Science.gov (United States)

    Lee, Dongjin; Cui, Tianhong

    2012-01-01

    Semiconducting nanoparticle ion-sensitive field-effect transistors (ISFETs) are used to detect immunoglobulin G (IgG) in the conventional enzyme-linked immunosorbent assay (ELISA). Indium oxide and silica nanoparticles were layer-by-layer self-assembled with the oppositely charged polyelectrolyte as the electrochemical transducer and antibody immobilization site, respectively. The assay was conducted on a novel platform of indium oxide nanoparticle ISFETs, where the electric signals are generated in response to the concentration of target IgG using the labeled detecting antibody. The sandwiched ELISA structure catalyzed the conversion of the acidic substrate into neutral substance with the aid of horseradish peroxidase. The pH change in the substrate solution was detected by nanoparticle ISFETs. Normal rabbit IgG was used as a model antigen whose detection limit of 0.04 ng ml-1 was found. The facile electric detection in the conventional assay through the semiconducting nanoparticle ISFET has potential applications as a point-of-care detection or a sensing element in a lab-on-a-chip system.

  4. An evaluation study of enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 for detection of antibody against Bartonella bacilliformis infection among the Peruvian population.

    Science.gov (United States)

    Angkasekwinai, Nasikarn; Atkins, Erin H; Romero, Sofia; Grieco, John; Chao, Chien Chung; Ching, Wei Mei

    2014-04-01

    Reliable laboratory testing is of great importance to detect Bartonella bacilliformis infection. We evaluated the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 (rPap31) for the detection of antibodies against B. bacilliformis as compared with immunofluorescent assay (IFA). Of the 302 sera collected between 1997 and 2000 among an at-risk Peruvian population, 103 and 34 samples tested positive for IFA-immunoglobulin G (IgG) and IFA-IgM, respectively. By using Youden's index, the cutoff values of ELISA-IgG at 0.915 gave a sensitivity of 84.5% and specificity of 94%. The cutoff values of ELISA-IgM at 0.634 gave a sensitivity of 88.2% and specificity of 85.1%. Using latent class analysis, estimates of sensitivity and specificity of almost all the assays were slightly higher than those of a conventional method of calculation. The test is proved beneficial for discriminating between infected and non-infected individuals with the advantage of low-cost and high-throughput capability.

  5. Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

    Science.gov (United States)

    Heller, Martin; Gicheru, Nimmo; Tjipura-Zaire, Georgina; Muriuki, Cecilia; Yu, Mingyan; Botelho, Ana; Naessens, Jan; Jores, Joerg; Liljander, Anne

    2016-06-01

    Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

  6. Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

    Science.gov (United States)

    Liu, Changqi; Chhabra, Guneet S; Zhao, Jing; Zaffran, Valerie D; Gupta, Sahil; Roux, Kenneth H; Gradziel, Thomas M; Sathe, Shridhar K

    2017-09-04

    A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. © 2017 Institute of Food Technologists®.

  7. Serodiagnosis of Human Cutaneous Anthrax in India Using an Indirect Anti-Lethal Factor IgG Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Ghosh, N.; Tomar, I.; Lukka, H.

    2013-01-01

    Anthrax, caused by Bacillus anthracis, is primarily a zoonotic disease. Being a public health problem also in several developing countries, its early diagnosis is very important in human cases. In this study, we describe the use of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of anti-lethal factor (anti-LF) IgG in human serum samples. A panel of 203 human serum samples consisting of 50 samples from patients with confirmed cutaneous anthrax, 93 samples from healthy controls from areas of India where anthrax is nonendemic, 44 samples from controls from an area of India where anthrax is endemic, and 16 patients with a disease confirmed not to be anthrax were evaluated with an anti-LF ELISA. The combined mean anti-LF ELISA titer for the three control groups was 0.136 ELISA unit (EU), with a 95% confidence interval (CI) of 0.120 to 0.151 EU. The observed sensitivity and specificity of the ELISA were 100% (95% CI, 92.89 to 100%) and 97.39% (95% CI, 93.44 to 99.28%), respectively, at a cutoff value of 0.375 EU, as decided by receiver operating characteristic (ROC) curve analysis. The likelihood ratio was found to be 49.98. The positive predictive value (PPV), negative predictive value (NPV), efficiency, and Youden's index (J) for reliability of the assay were 92.5%, 100%, 98.02%, and 0.97, respectively. The false-positive predictive rate and false-negative predictive rate of the assay were 2.61% and 0%. The assay could be a very useful tool for early diagnosis of cutaneous anthrax cases, as antibodies against LF appear much earlier than those against other anthrax toxins in human serum samples. PMID:23269414

  8. Antibodies with specificity for native and denatured forms of ovalbumin differ in reactivity between enzyme-linked immunosorbent assays

    DEFF Research Database (Denmark)

    Holm, B.E.; Bergmann, A.C.; Hansen, Paul Robert

    2015-01-01

    In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme...... to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially...... with three-dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94-06 and 94-07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures...

  9. Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

    Science.gov (United States)

    Yokomizo, Y; Kishima, M; Mori, Y; Nishimori, K

    1991-08-01

    An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.

  10. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases

    Science.gov (United States)

    Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines. PMID:26184194

  11. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

    Science.gov (United States)

    Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih

    2014-02-12

    Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples.

  12. Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Tyramine as an Index of Freshness in Meat and Seafood.

    Science.gov (United States)

    Sheng, Wei; Sun, Congcong; Fang, Guozhen; Wu, Xuening; Hu, Gaoshuang; Zhang, Yan; Wang, Shuo

    2016-11-23

    A competitive indirect enzyme-linked immunosorbent assay (ciELISA) using a polyclonal antibody was developed to detect tyramine in meat and seafood. This ciELISA had a 50% inhibition concentration (IC50) of 0.20 mg/L and a limit of detection (LOD) of 0.02 mg/L and showed no cross-reactivity with tyrosine or other biogenic amines. The average recoveries of tyramine from spiked samples for this ciELISA ranged from 85.6 to 102.6%, and the results exhibited good correlation with high-performance liquid chromatography (HPLC) results. The LOD of this assay for tyramine in meat and seafood samples was 1.20 mg/kg. The ciELISA was successfully applied to detect tyramine in positive fish samples, and the results were validated by HPLC to be reliable. The developed ciELISA allows for the rapid, specific, and accurate detection of tyramine in meat and seafood samples, and it could be a potentially useful tool for the evaluation of the freshness of protein-rich foods.

  13. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of Campylobacter jejuni antibodies, and comparison with a complement fixation test (CFT).

    Science.gov (United States)

    Oosterom, J; den Uyl, C H; Bänffer, J R; Lauwers, S; Huisman, J; Busschbach, A E; Poelma, F G; Bellemans, R

    1985-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of total anti-Campylobacter immunoglobulins in human sera. In this assay disintegrated Campylobacter bacteria were used as the antigen. Absorption tests including other possibly enteropathogenic bacterial species showed that the ELISA system displayed a high immunological specificity for Campylobacter. Using this ELISA it was found that in about 80% of Campylobacter patients these Campylobacter antibodies are produced to almost maximal levels within 8 days after onset of disease, and that they may persist for at least 4 months. Indeed, Campylobacter antibodies were demonstrated at low levels in a large number of control sera. However, accepting an antibody titre of 1:640 as indicative of Campylobacter infection, the statistical sensitivity of the ELISA system was 77% and the specificity 95%. In an epidemiological survey a high association was demonstrated between the severity of Campylobacter-related symptoms and antibody titre values. Assessment of Campylobacter antibody titres by means of this ELISA and by a complement fixation test in 92 sera from index patients and contacts with and without symptoms showed a high association of results.

  14. Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle, chicken muscle, egg, fish, milk and kidney.

    Science.gov (United States)

    Wang, S; Xu, B; Zhang, Y; He, J X

    2009-05-01

    A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10μg/kg in kidney and 20μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015μg/kg, and the detection limits were 1.5μg/kg in pig muscle and 6μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r(2)) which was greater than 0.9.

  15. Monoclonal antibody production and indirect competitive enzyme-linked immunosorbent assay development of 3-methyl-quinoxaline-2-carboxylic acid based on novel haptens.

    Science.gov (United States)

    Li, Guopeng; Zhao, Liang; Zhou, Feng; Li, Jiaying; Xing, Yuan; Wang, Tiangang; Zhou, Xilong; Ji, Baoping; Ren, Wanpeng

    2016-10-15

    Two novel immunizing haptens of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) were synthesized and conjugated with cationized bovine serum albumin. Female BALB/c mice were immunized with above conjugates, splenocytes were fused with Sp2/0 cells to produce monoclonal antibody. Compared with previous studies, antibodies raised in this work showed higher sensitivity. Meantime, a novel heterologous coating hapten was also prepared. The indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the optimum condition showed an IC50 of 3.1μg/kg (ppb), and the linear range of 0.46-10.5ppb for MQCA. The limit of detect (LOD) of MQCA in swine muscle, swine liver and chicken was 0.32, 0.54, and 0.28ppb, respectively. The LOD of this assay can satisfy the minimum required performance levels (4ppb) for MQCA. These results indicated that the proposed ELISA, with high sensitivity and specificity, as well as good reproducibility and accuracy, is suitable for determination of MQCA residues in food samples.

  16. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA) for Infectious Diseases.

    Science.gov (United States)

    Singh, Harpal; Shimojima, Masayuki; Shiratori, Tomomi; An, Le Van; Sugamata, Masami; Yang, Ming

    2015-07-08

    Enzyme-linked Immunosorbent Assay (ELISA)-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a '3D well' was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

  17. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA for Infectious Diseases

    Directory of Open Access Journals (Sweden)

    Harpal Singh

    2015-07-01

    Full Text Available Enzyme-linked Immunosorbent Assay (ELISA-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

  18. Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an identical marker, in White Kwao Krua using a monoclonal antibody.

    Science.gov (United States)

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Tanaka, Hiroyuki; Putalun, Waraporn

    2017-04-15

    Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen-rich plant widely used among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have been added as an active ingredient for skin rejuvenation and breast enlargement effects in various functional foods. However, most of the products on the market containing WKK have not been sufficiently standardized with respect to the active compound or identical marker. To control the quality of these plant materials, an enzyme-linked immunosorbent assay (ELISA) using anti-isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and was thus selected to develop the ELISA. Based on the validation analysis and the tested performance of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant.

  19. An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection.

    Science.gov (United States)

    Nakajima, Hizuru; Okuma, Yukiko; Morioka, Kazuhiro; Miyake, Mayo; Hemmi, Akihide; Tobita, Tatsuya; Yahiro, Masayuki; Yokoyama, Daisuke; Adachi, Chihaya; Soh, Nobuaki; Nakano, Koji; Xue, Shuhua; Zeng, Hulie; Uchiyama, Katsumi; Imato, Toshihiko

    2011-10-01

    A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 μW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.

  20. Development of a Fibrinogen-Specific Sandwich Enzyme-Linked Immunosorbent Assay Microarray Assay for Distinguishing Between Blood Plasma and Serum Samples

    Energy Technology Data Exchange (ETDEWEB)

    Gonzales, Rachel M.; Zhang, Qibin; Zangar, Richard C.; Smith, Richard D.; Metz, Thomas O.

    2011-07-01

    We have developed a fibrinogen-specific sandwich ELISA microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies, 49D2, HPA001900, and F8512, were evaluated in conjunction with 1D6 as detection antibody, and the data show that 49D2 and, to a lesser extent, F8512 successfully identify previously unknown plasma and serum samples based upon a ~28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high throughput manner prior to proteomics analyses.

  1. Development and evaluation of a Sarcocystis neurona-specific IgM capture enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Murphy, J E; Marsh, A E; Reed, S M; Meadows, C; Bolten, K; Saville, W J A

    2006-01-01

    Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. Currently available antemortem diagnostic testing has low specificity. The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S neurona would have an increased S neurona-specific IgM (Sn-IgM) concentration after infection, as determined by an IgM capture enzyme linked immunoassay (ELISA). The ELISA was based on the S neurona low molecular weight protein SNUCD-1 antigen and the monoclonal antibody 2G5 labeled with horseradish peroxidase. The test was evaluated using serum and CSF from 12 horses experimentally infected with 1.5 million S neurona sporocysts and 16 horses experimentally infected with varying doses (100 to 100,000) of S neurona sporocysts, for which results of histopathologic examination of the central nervous system were available. For horses challenged with 1.5 million sporocysts, there was a significant increase in serum Sn-IgM concentrations compared with values before infection at weeks 2-6 after inoculation (P neurona, there were significant increases in serum Sn-IgM concentration at various points in time after inoculation, depending on the challenge dose (P < .01). In addition, there was a significant increase between the CSF Sn-IgM concentrations before and after inoculation (P < .0001). These results support further evaluation of the assay as a diagnostic test during the acute phase of EPM.

  2. Direct Agglutination Test and Enzyme Linked Immunosorbent Assay with Urine Samples for the Diagnosis of Visceral Leishma-niasis

    Directory of Open Access Journals (Sweden)

    Sarkari B

    2007-07-01

    Full Text Available Background: Visceral leishmaniasis (VL or Kala azar is an infectious disease caused by various species of Leishmania parasites. The aim of this study was to detect and compare the presence of anti-Leishmania antibodies in the urine of vis-ceral leishmaniasis patients using ELISA and DAT methods."nMethods: A total of 30 urine samples were collected from VL patients referred to Shiraz (southeast of Iran hospitals. Moreover 31 urine samples were collected from healthy individuals and patients with other diseases such as malaria, brucellosis, hydatidosis and cutaneous leishmaniasis. Collected samples were examined to detect anti-Leishmania antibod-ies in urine, using ELISA and DAT."nResults: Anti-Leishmania antibody was detected in urine of 18 out of 30 (60% VL patients by DAT while ELISA detected anti-Leishmania antibodies in urine of 28 out of 30 (93.3% of VL cases. Sensitivity and specificity of urine-based DAT was 60% and 83.9%, respectively while sensitivity and specificity of urine-based ELISA were 93.3% and 93.5%, corre-spondingly. "nConclusion: Urine-based DAT and ELISA have a reasonable specificity and sensitivity in diagnosis of VL. Accordingly, urine-based ELISA might be a suitable alternative for serum based assays for diagnosis of VL.

  3. Development and validation of an enzyme-linked immunosorbent assay for detection of cortisol in human saliva.

    Science.gov (United States)

    Ozgocer, Tuba; Yildiz, Sedat; Uçar, Cihat

    2017-01-01

    Non-invasive measurement of cortisol in saliva is of prime importance as it represents a bioavailable neuroendocrine marker for stress. Therefore, in this study, we developed an enzyme immune assay that was suitable for salivary cortisol measurements. For that purpose, rabbit polyclonal antibody was raised against cortisol-3-CMO:BSA conjugate. The test was based on competition of liquid phase cortisol with conjugated cortisol on the solid phase. Primary antibody was used to bind available sites on the conjugate, which was proportional to numbers of cortisol in liquid phase. Biotinylated secondary anti-rabbit antibody was used to detect primary antibodies by addition of streptavidin peroxidase and substrate, respectively. Color formation was stopped and yellow color was read by a plate-reader spectrophotometer. Additionally, validated test was used to met all validation criteria including. Test developed was used to establish cortisol awakening response (CAR) in saliva samples collected in the morning after awakening (0, 15, 30, and 60(th) min) from women (n = 4) and men (n = 4) at 8 or 4 different days, respectively. Diurnal cortisol levels were assessed (n = 8) at after awaking 60 min at morning, 12:00, 19:00, and 22:00 hr. In conclusion, an enzyme immunoassay test was successfully produced, validated and used for cortisol measurement in saliva samples.

  4. Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

    NARCIS (Netherlands)

    J. Groen (Jan); G. van Dijk (Grietje); H.G.M. Niesters (Bert); W.I. van der Meijden (Willem); A.D.M.E. Osterhaus (Albert)

    1998-01-01

    textabstractThe sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme

  5. An indirect enzyme-linked immunosorbent assay for the identification of antibodies to Senecavirus A in swine.

    Science.gov (United States)

    Dvorak, Cheryl M T; Akkutay-Yoldar, Zeynep; Stone, Suzanne R; Tousignant, Steven J P; Vannucci, Fabio A; Murtaugh, Michael P

    2017-02-15

    Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal diseases. We have developed and characterized an indirect ELISA assay to specifically identify serum antibodies to SVA. Viral protein 1, 2 and 3 (VP1, VP2, VP3) were expressed, isolated, and purified from E. coli and used to coat plates for an indirect ELISA. Sera from pigs with and without IVD symptoms as well as a time course following animals from an infected farm, were analyzed to determine the antibody responses to VP1, VP2, and VP3. Antibody responses to VP2 were higher than VP1 and VP3 and showed high affinity binding on an avidity ELISA. ROC analysis of the SVA VP2 ELISA showed a sensitivity of 94.2% and a specificity of 89.7%. Compared to IFA, the quantitative ELISA showed an 89% agreement in negative samples and positive samples from 4-60 days after appearance of clinical signs. Immune sera positive for FMDV, encephalomyocarditis virus, and porcine epidemic diarrhea virus antibodies did not cross-react. A simple ELISA based on detection of antibodies to SVA VP2 will help to differentially diagnose IVD due to SVA and rule out the presence of economically devastating foreign animal diseases.

  6. Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies.

    Science.gov (United States)

    Zasada, A A; Rastawicki, W; Śmietańska, K; Rokosz, N; Jagielski, M

    2013-07-01

    Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, Virion\\Serion, IBL International and Euroimmun. All assays were performed according to the manufacturers' instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material-International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7%); Immunolab, 11/72 samples (15.3%); Sekisui Virotech, 0/72 samples (0%); NovaTec 18/72 samples (25.0%); Serion 12/72 samples (16.7%); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6%). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.

  7. Establishment of mouse Mac-2 binding protein enzyme-linked immunosorbent assay and its application for mouse chronic liver disease models.

    Science.gov (United States)

    Iwata, Ayumi; Kamada, Yoshihiro; Ebisutani, Yusuke; Yamamoto, Akiko; Ueda, Yui; Arai, Hitomi; Fujii, Hironobu; Takamatsu, Shinji; Maruyama, Nobuhiro; Maeda, Masahiro; Takehara, Tetsuo; Miyoshi, Eiji

    2017-08-01

    We identified Mac-2 (galectin-3) binding protein (Mac-2bp) as a novel diagnostic and liver fibrosis predicting biomarker for nonalcoholic steatohepatitis in humans. In mouse models, there are no serum biomarkers predicting liver disease severity. In this study, we developed a mouse Mac-2bp enzyme-linked immunosorbent assay (ELISA) system and determined its efficacy for predicting the severity of liver disease in mouse models, especially in non-alcoholic fatty liver disease (NAFLD) models. We established several rat monoclonal antibodies against mouse Mac-2bp, selected two clones for the ELISA, and checked the accuracy and reproducibility of the ELISA, especially for NAFLD models and liver fibrosis models. We also investigated the relationships between serum levels and hepatic gene expression of Mac-2bp in mouse models. Our ELISA system had high accuracy and reproducibility (R(2)  = 0.999). The intra-assay and inter-assay results for the coefficient of variation were 2.0-3.7% and 1.7-6.9%, respectively. The levels of bilirubin, hemoglobin, and chyle did not affect the Mac-2bp serum levels detected by our ELISA kit. In the mouse models, serum Mac-2bp levels increased with liver disease progression (F0/F1/F2/F3, 239.1 ± 36.7 / 259.1 ± 43.0 / 457.5 ± 162.0 / 643.7 ± 116.0 ng/mL; P Mac-2bp (R = 0.42, P Mac-2bp ELISA system effectively predicts severity of NAFLD and liver fibrosis in mouse models. © 2016 The Japan Society of Hepatology.

  8. A comparison of intradermal testing and detection of allergen-specific immunoglobulin E in serum by enzyme-linked immunosorbent assay in horses affected with skin hypersensitivity.

    Science.gov (United States)

    Morgan, Erin E; Miller, William H; Wagner, Bettina

    2007-12-15

    Skin hypersensitivities (allergies) in horses are often diagnosed using clinical signs only. Intradermal testing or serological assays are diagnostic options to confirm the allergic nature of the disease and to identify the allergen(s). Our objective was to develop an allergen-specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for horse IgE and to examine its potential for allergen detection in serum in comparison to intradermal testing. Intradermal testing with 61 allergen extracts was performed on 10 horses affected with skin hypersensitivity. Their sera were analyzed by ELISA for IgE antibodies to the same allergens. The kappa test of concordance was used for comparison of the results of both tests. Out of 61 allergen extracts, only two (Timothy and Quack) had kappa values greater than 0.60, suggesting a substantial agreement between skin testing and IgE ELISA. The statistical comparison of the remaining 59 allergens showed little or no concordance between the tests beyond chance. To identify parameters that may influence the sensitivity of the ELISA, the assay was modified to detect allergen-specific IgGb and IgG(T) in serum, and the protein content in all allergen extracts was determined by SDS-PAGE. The commercial allergen extracts revealed a high variation in detectable protein. High concentrations of allergen-specific IgG in horse serum were found to compete with IgE for binding to the plates. In conclusion, an ELISA using whole serum and crude allergen preparations provides limited diagnostic information in horses. The reliable diagnosis of allergens in equine skin hypersensitivity is essential to improve allergen-specific treatments, such as hyposensitization, or the development of allergy vaccines.

  9. Application of the Ceditest FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against Foot-and-mouth disease virus in selected livestock and wildlife species in Uganda

    DEFF Research Database (Denmark)

    Ayebazibwe, Chrisostom; Mwiine, Frank Norbert; Balinda, Sheila Nina

    2012-01-01

    Diagnosis and control of Foot-and-mouth disease virus (FMDV) requires rapid and sensitive diagnostic tests. Two antibody enzyme-linked immunosorbent assay (ELISA) kits, Ceditest FMDV-NS for the detection of antibodies against the nonstructural proteins of all FMDV serotypes and Ceditest FMDV type...

  10. Enzyme-linked immunosorbent assay using a virus type-specific peptide based on a subdomain of envelope protein e(rns) for serologic diagnosis of pestivirus infections in swine

    NARCIS (Netherlands)

    Langedijk, J.P.; Middel, W.G.; Meloen, R.H.; Kramps, J.A.; Smit, de J.A.

    2001-01-01

    Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein Erns were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure

  11. Absorption of p,p'-dichlorodiphenyldichloroethylene and dieldrin in largemouth bass from a 60-D slow-release pellet and detection using a novel enzyme-linked immunosorbent assay method for blood plasma

    Science.gov (United States)

    Muller, Jennifer K.; Sepulveda, Maria S.; Borgert, Christopher J.; Gross, Timothy S.

    2005-01-01

    This work describes the uptake of two organochlorine pesticides from slow-release pellets by largemouth bass and the utility of a blood plasma enzyme-linked immunosorbent assay (ELISA) method for exposure verification. We measured blood and tissue levels by gas chromatography/mass spectrometry and by a novel ELISA method, and present a critical comparison of the results.

  12. Use of Baculovirus-Expressed Glycoprotein H in an Enzyme-Linked Immunosorbent Assay Developed To Assess Exposure to Chelonid Fibropapillomatosis-Associated Herpesvirus and Its Relationship to the Prevalence of Fibropapillomatosis in Sea Turtles▿

    OpenAIRE

    Herbst, Lawrence H.; Lemaire, Shefali; Ene, Ada R.; Heslin, David J.; Ehrhart, Llewellyn M.; Dean A Bagley; Klein, Paul A.; Lenz, Jack

    2008-01-01

    Chelonid fibropapillomatosis-associated herpesvirus (CFPHV) is an alphaherpesvirus believed to cause marine turtle fibropapillomatosis (FP). A serodiagnostic assay was developed for monitoring sea turtle populations for CFPHV exposure. CFPHV glycoprotein H (gH) expressed in recombinant baculovirus was used in an enzyme-linked immunosorbent assay (ELISA) to detect virus-specific 7S turtle antibodies. Using captive-reared green turtles (Chelonia mydas) with no history of virus exposure as “know...

  13. A Novel Method of Safely Measuring Influenza Virus Aerosol Using Antigen-Capture Enzyme-Linked Immunosorbent Assay for the Performance Evaluation of Protective Clothing Materials.

    Science.gov (United States)

    Shimasaki, Noriko; Nojima, Yasuhiro; Okaue, Akira; Takahashi, Hitoshi; Kageyama, Tsutomu; Hamamoto, Itsuki; Shinohara, Katsuaki

    2016-01-01

    Currently, threats caused by pathogens are serious public health problems worldwide. Protective clothing is essential when one is treating infected patients or dealing with unknown pathogens. Therefore, it is necessary to evaluate the performance of protective clothing against pathogens. In Japan, some methods for evaluating the performance of protective clothing have been established in the Japanese Industrial Standards (JIS). However, a test method against virus aerosols has not been established. Because there is a risk of infection from a live virus during the test, it is necessary to devise a safe method for the virus-aerosol-based test. Here, we propose a new method of safely measuring virus aerosols for the performance evaluation of protective clothing materials. To ensure safety, an inactivated virus was used. As a model virus, the influenza virus was selected owing to the proper small diameter of the virus particles. To quantitatively measure the particle-amount of the inactivated influenza virus, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting the M1 protein. Furthermore, we evaluated two materials using our method. Significant differences in the protection performance against the virus aerosol were observed between different sample materials, thereby confirming the applicability of our new method for performance evaluation.

  14. Plasmonic Enzyme-Linked Immunosorbent Assay Using Nanospherical Brushes as a Catalase Container for Colorimetric Detection of Ultralow Concentrations of Listeria monocytogenes.

    Science.gov (United States)

    Chen, Rui; Huang, Xiaolin; Xu, Hengyi; Xiong, Yonghua; Li, Yanbin

    2015-12-30

    Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth exhibits ultrahigh sensitivity for detecting disease-related biomarkers using sandwich formats. However, the limit of detection (LOD) of this strategy for Listeria monocytogenes is only around 10(3) CFU/mL, which considerably exceeds the amount of L. monocytogenes commonly present in food products (container" to increase enzyme loading for enhancing the detection signal. Under optimal conditions, the proposed pELISA exhibits good specificity and excellent sensitivity for L. monocytogenes with a LOD of 8 × 10(1) CFU/mL in 0.01 M phosphate-buffered saline, via a reaction that can be discriminated by the naked eye. The LOD obtained by this method was 2 and 5 orders of magnitude lower than that of conventional CAT-based pELISA and horseradish peroxidase (HRP)-based conventional ELISA, respectively. Coupled with large-volume immunomagnetic separation, the LOD for L. monocytogenes-spiked lettuce samples reached 8 × 10(1) CFU/g. The improved pELISA also exhibited a great potential in detecting a single cell of L. monocytogenes in 100 μL of solution.

  15. A novel enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 using immunomagnetic and beacon gold nanoparticles.

    Science.gov (United States)

    Shen, Zhiqiang; Hou, Nannan; Jin, Min; Qiu, Zhigang; Wang, Jingfeng; Zhang, Bin; Wang, Xinwei; Wang, Jie; Zhou, Dongsheng; Li, Junwen

    2014-01-01

    This paper presents a functional nanoparticle-enhanced enzyme-linked immunosorbent assay (FNP-ELISA) for detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Immunomagnetic nanoparticles (IMMPs) conjugated with monoclonal anti-O157:H7 antibody were used to capture E. coli O157:H7. Beacon gold nanoparticles (B-GNPs) coated with polyclonal anti-O157:H7 and biotin single-stranded DNA (B-DNA) were then subjective to immunoreaction with E. coli O157:H7, which was followed by streptavidin-horseradish peroxidase (Strep-HRP) conjugated with B-GNPs based on a biotin-avidin system. The solutions containing E. coli O157:H7, IMMPs, B-GNPs, and Strep-HRP were collected for detecting color change. The signal was significantly amplified with detection limits of 68 CFU mL(-1) in PBS and 6.8 × 10(2) to 6.8 × 10(3) CFU mL(-1) in the food samples. The FNP-ELISA method developed in this study was two orders of magnitude more sensitive than immunomagnetic separation ELISA (IMS-ELISA) and four orders of magnitude more sensitive than C-ELISA. The entire detection process of E. coli O157:H7 lasted only 3 h, and thus FNP-ELISA is considered as a time-saving method.

  16. The effect on kaolin adsorption of serum on the virus neutralization and enzyme-linked immunosorbent assays of antibody to bovine herpesvirus 1.

    Science.gov (United States)

    Darcel, C L; Kozub, G C

    1984-01-01

    Two procedures have been used for measuring antibody titres to bovine herpes virus 1 (BHV1): the serum neutralization (SN) test and enzyme-linked immunosorbent assay (ELISA). One hundred and thirty-two sera selected for their low SN titres were tested both unadsorbed and after adsorption with kaolin to determine the effect of kaolin on the titres. With ELISA, the titres of unadsorbed and kaolin adsorbed were not significantly different but with the SN test many treated sera, originally with weak positive titres, became negative after kaolin adsorption. Thus, if the ELISA results are specific for BHV1 antibody then the SN test findings suggest that treatment of sera with kaolin, rather than removing a viral inhibitor, removes a substance from the serum which potentiates SN antibody. This in turn indicates that low SN titres (reciprocal of titre less than or equal to 4, for instance) are probably specific for BHV1 SN antibody whether or not they are abolished by kaolin treatment of the serum.

  17. Rheumatoid arthritis and its association with HLA-DR antigens. II. Antibodies to native connective tissue antigens detected by enzyme linked immunosorbent assay.

    Science.gov (United States)

    Pesoa, S A; Vullo, C M; Onetti, C M; Riera, C M

    1989-01-01

    The distribution of frequencies of HLA-DR alloantigens in HLA-DR4 negative subjects was determined in patients with Rheumatoid arthritis (RA) and normal individuals. An increased incidence of HLA-DR1 alloantigen in DR4 negative RA patients (45.9%) compared with DR4 negative healthy controls (23.6%) was found. The difference became significant when the incidence of DR1 was compared between patients with severe disease stages (III-IV) (75%) in contrast to 32% of incidence in patients of the milder stages (I-II) (p less than 0.05). Using Enzyme Linked Immunosorbent Assay we have determined the incidence of serum antibodies to native bovine type I and type II collagens and proteoglycans in patients with RA. Presence of serum antibodies to native type I collagen was detected in 59% of patients with RA, 60% of sera exhibited reactivity to type II collagen and 12% had antibodies to proteoglycans. There was no correlation between the presence of antibodies to type I and II collagens and disease stages, however, the incidence of serum antibodies to proteoglycans was increased in severe disease stages. On the other hand, the presence of high levels of antibodies to type I collagen was associated to HLA-DR1 antigen, (p less than 0.05).

  18. Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

    Science.gov (United States)

    Singh, Harpal; Shimojima, Masayuki; Fukushi, Shuetsu; Le Van, An; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

  19. Use of chemiluminescence for the serological diagnosis of bovine and ovine brucellosis with indirect and competitive enzyme-linked immunosorbent assays

    Directory of Open Access Journals (Sweden)

    Manuela Tittarelli

    2008-06-01

    Full Text Available The official methods specified in the national bovine and ovine/caprine brucellosis eradication plan are the Rose Bengal and complement fixation tests. In the current phase of the eradication plan, it is often difficult to interpret the results obtained with the official tests. Consequently, additional tests that offer greater sensitivity and specificity are thus required. For this reason, two methods, the indirect chemiluminescence enzyme-linked immunosorbent assay (i-ELISA CL and the competitive chemiluminescence ELISA (c-ELISA CL that use a chemiluminescent substrate to determine anti-Brucella antibodies in bovine and ovine serum were validated. The methods are based on the detection of anti-Brucella antibodies in serum by catalysis of a chemiluminescent enzyme substrate (luminol/ H2O2/enhancer system by peroxidase conjugated to secondary anti IgG antibodies in i-ELISA CL and to monoclonal anti-lipopolysaccharide (LPS antibodies in c-ELISA CL. From the results obtained, a cut-off of 60% for bovine serum and 37.5% for ovine serum, expressed as positivity rate (PR, were established Using these cut-off values, for the i-ELISA CL, 100% sensitivity and specificity was obtained for bovine serum and 100% sensitivity and 99.8% specificity for ovine serum. Cut-off values of 30% for bovine serum and 40% for ovine serum, expressed as inhibition rate, were selected for c-ELISA CL, which ensured 100% sensitivity and specificity in both cases.

  20. Analysis of Benzo[a]pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISPE Coupled with Enzyme-Linked Immunosorbent Assay (ELISA

    Directory of Open Access Journals (Sweden)

    Michael Pschenitza

    2014-05-01

    Full Text Available This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE method coupled with enzyme-linked immunosorbent assay (ELISA for determination of the PAH benzo[a]pyrene (B[a]P in vegetable oils. Different molecularly imprinted polymers (MIPs were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q of ~32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values.

  1. Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

    Institute of Scientific and Technical Information of China (English)

    Yi; Huang; Youjie; Zhu; Mengshi; Yang; Zhenqing; Zhang; Donglin; Song; Zhiming; Yuan

    2014-01-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  2. Validation of the World Health Organization Enzyme-Linked Immunosorbent Assay for the Quantitation of Immunoglobulin G Serotype-Specific Anti-Pneumococcal Antibodies in Human Serum.

    Science.gov (United States)

    Lee, Hyunju; Lim, Soo Young; Kim, Kyung Hyo

    2017-10-01

    The World Health Organization (WHO) enzyme-linked immunosorbent assay (ELISA) guideline is currently accepted as the gold standard for the evaluation of immunoglobulin G (IgG) antibodies specific to pneumococcal capsular polysaccharide. We conducted validation of the WHO ELISA for 7 pneumococcal serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by evaluating its specificity, precision (reproducibility and intermediate precision), accuracy, spiking recovery test, lower limit of quantification (LLOQ), and stability at the Ewha Center for Vaccine Evaluation and Study, Seoul, Korea. We found that the specificity, reproducibility, and intermediate precision were within acceptance ranges (reproducibility, coefficient of variability [CV] ≤ 15%; intermediate precision, CV ≤ 20%) for all serotypes. Comparisons between the provisional assignments of calibration sera and the results from this laboratory showed a high correlation > 94% for all 7 serotypes, supporting the accuracy of the ELISA. The spiking recovery test also fell within an acceptable range. The quantification limit, calculated using the LLOQ, for each of the serotypes was 0.05-0.093 μg/mL. The freeze-thaw stability and the short-term temperature stability were also within an acceptable range. In conclusion, we showed good performance using the standardized WHO ELISA for the evaluation of serotype-specific anti-pneumococcal IgG antibodies; the WHO ELISA can evaluate the immune response against pneumococcal vaccines with consistency and accuracy. © 2017 The Korean Academy of Medical Sciences.

  3. Performance of Helicobacter pylori acid extract and urease enzyme-linked immunosorbent assays in relation to 14C-urea breath test.

    Science.gov (United States)

    von Wulffen, H; Gatermann, S; Windler, E; Gabbe, E; Heinrich, H C

    1993-09-01

    The 14C-urea breath test has been shown to be a reliable non-invasive method to detect the presence or absence of H. pylori infection. Alternatively, a number of techniques have been devised to detect circulating antibodies against H. pylori in serum, the most commonly used being enzyme-linked immunosorbent assays (ELISA). In the present study we compared the value of two ELISA antigen preparations, an acid glycine extract and a urease preparation, in relation to the results achieved in a 14C-urea breath test. Seventy-five gastroenterology outpatients were screened for the presence of H. pylori infection using the urea breath test. At the same time serum specimens were obtained. Thirty-seven patients had a positive breath test, i.e. they expired more than 2% of the oral 14C test dose within 60 min. Using the breath test as reference, sensitivity and specificity for the acid extract were 89.2% and 84.2% respectively, and for the urease ELISA 81.1% and 89.5%. Agreement between the two ELISAs was found in 82.7%, overall agreement between all three tests was observed in 77.3%. All three tests were found to be useful for monitoring therapy directed against H. pylori.

  4. Development of an antigen-capture enzyme-linked immunosorbent assay using monoclonal antibodies for detecting H6 avian influenza viruses.

    Science.gov (United States)

    Chen, Yi-Tung; Tsao, Zak; Chang, Shu-Ting; Juang, Ron-Huay; Wang, Lih-Chiann; Chang, Chung-Ming; Wang, Ching-Ho

    2012-06-01

    The H6 subtype of avian influenza virus (AIV) infection occurs frequently in wild and domestic birds. AIV antigen detection is preferred for controlling AIV as birds are infected before they produce antibodies. The purpose of this study was to develop an early diagnostic method for AIV detection. Six monoclonal antibodies (mAbs) developed from a field H6N1 AIV strain were tested for their ability to bind to viruses. The two that showed the greatest binding ability to AIVs were used for antigen detection. An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect H6 AIVs was developed using these mAbs. One mAb was coated onto an ELISA plate as the capture antibody. The other mAb was used as the detector antibody after labeling with horseradish peroxidase. The antigen-capture ELISA detected H6N1 AIVs but not H5 AIVs, human H1N1, H3N2 influenza or other viruses. This antigen-capture ELISA could be used to specifically detect H6N1 AIV.

  5. Development of an enzyme-linked immunosorbent assay system for detecting β'-component (Onk k 5), a major IgE-binding protein in salmon roe.

    Science.gov (United States)

    Shimizu, Yutaka; Oda, Hiroshi; Seiki, Kohsuke; Saeki, Hiroki

    2015-08-15

    A novel enzyme-linked immunosorbent assay (ELISA) system has been established for selective detection of chum salmon (Oncorhynchus keta) yolk protein (SYP). Rabbit and rat polyclonal Immunoglobulin G antibodies to β'-component (the major allergic protein in fish roe; anti-β) were applied for designing the ELISA system. The sandwich ELISA using rabbit anti-β for the capture antibody and horseradish peroxidase-labeled F(ab')2 fragment of rat anti-β for the detection antibody obtained high sensitivity and narrow specificity for SYP. Protein extraction using sodium dodecyl sulfate and 2-mercaptoethanol ensured strict specificity of the ELISA, and components of three popular processed foods had no effect on the ELISA response. The limits of determination and quantification of SYP were estimated to be 0.78 μg/g and 2.60 μg/g of food sample, respectively. In conclusion, the developed ELISA system has a probability to be applied for the detection of contaminated chum salmon roe in processed food.

  6. Development of polyclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of Alicyclobacillus strains in apple juice.

    Science.gov (United States)

    Wang, Zhouli; Yue, Tianli; Yuan, Yahong; Cai, Rui; Guo, Caixia; Wang, Xin; Niu, Chen

    2012-11-01

    A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10(5) colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.

  7. Comparison of Enzyme-Linked Immunosorbent Assay, Surface Plasmon Resonance and Biolayer Interferometry for Screening of Deoxynivalenol in Wheat and Wheat Dust

    Directory of Open Access Journals (Sweden)

    Melanie Sanders

    2016-04-01

    Full Text Available A sample preparation method was developed for the screening of deoxynivalenol (DON in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA was compared to the sensor-based techniques of surface plasmon resonance (SPR and biolayer interferometry (BLI in terms of sensitivity, affinity and matrix effect. The matrix effects from wheat and wheat dust using SPR were too high to further use this screenings method. The preferred ELISA and BLI methods were validated according to the criteria established in Commission Regulation 519/2014/EC and Commission Decision 2002/657/EC. A small survey was executed on 16 wheat lots and their corresponding dust samples using the validated ELISA method. A linear correlation (r = 0.889 was found for the DON concentration in dust versus the DON concentration in wheat (LOD wheat: 233 μg/kg, LOD wheat dust: 458 μg/kg.

  8. Determination of alachlor and its sulfonic acid metabolite in water by solid-phase extraction and enzyme-linked immunosorbent assay

    Science.gov (United States)

    Aga, D.S.; Thurman, E.M.; Pomes, M.L.

    1994-01-01

    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were combined for the trace analysis of the herbicide alachlor and its major soil metabolite, ethanesulfonic acid (ESA). The anti-alachlor antibody cross-reacted with ESA, which produced false-positive detections of alachlor in water samples by immunoassay screens. Alachlor and ESA were isolated from water by SPE on a C18 resin and eluted sequentially with ethyl acetate and methanol. Alachlor is soluble in ethyl acetate while the anionic ESA is not. Thus ESA remained adsorbed on the C18 resin and was eluted later with methanol. The combination of SPE with ELISA effectivety separated and quantified both alachlor and ESA using the same antibody for two ELISA methods. The general method may have applicability for the separation of other herbicides and their ionic metabolites. The SPE-ELISA method has a, detection limit of 0.01 ??g/L for alachlor and 0.05 ??g/L for ESA, with a precision of ?? 10%. Analyses of surface and ground water samples were confirmed by gas chromatography/mass spectrometry and high-performance liquid chromatography with photodiode-array detection. Results showed widespread occurrence of ESA in surface and ground water of the midwestern United States, with concentrations ranging from 10 ??g/L.

  9. A polymerase chain reaction and enzyme linked immunosorbent assay based approach for diagnosis and differentiation between vaccinated and infected cattle with Mycobacterium bovis

    Directory of Open Access Journals (Sweden)

    Mohamed Sabry

    2014-01-01

    Full Text Available Background: In most African and Arabic countries tuberculosis (TB causes great economic losses in bovine species and constitutes serious zoonotic problem. As the traditional diagnostic method delay the research because of low sensitivity and specificity, a rapid method of diagnosis is of outmost importance. Aim: The study was designed to evaluate the two rapid diagnostic methods of TB in cattle, further to differentiate between infected and bacillus Calmette-Guerin (BCG vaccinated animals. Materials and Methods: Intradermal tuberculin test was applied to 300 cattle. Of these cattle, 15 cattle were vaccinated from cattle negative to tuberculin test with BCG. Blood samples were taken for lymphocyte separation to apply polymerase chain reaction (PCR upon and for serum preparation for the enzyme-linked immunosorbent assay (ELISA application, this blood collected from 65 cattle classified into three groups, viz. positive tuberculin test (35 animals, negative tuberculin test (15 animals, and vaccinated cow with BCG (15 animals. From blood samples lymphocytes were separated and the isolated lymphocytes were subjected to PCR and serum for ELISA application. Blood samples, specimens from lymph nodes and specific tissues were taken for PCR and for cultivation and isolation of Mycobacterium bovis. Results and Conclusions: The results of this study revealed that PCR can be used as rapid efficient and accurate diagnostic test in detection of ruminant TB. Moreover, cattle′s ELISA reading showed higher sensitivity in positive tuberculin animals. However, the differentiations between vaccinated and infected animals not clear by using a single antigen only.

  10. Immunodiagnosis of bovine trypanosomiasis in Anambra and Imo states, Nigeria, using enzyme-linked immunosorbent assay: zoonotic implications to human health

    Directory of Open Access Journals (Sweden)

    M.C. Ezeani

    2008-11-01

    Full Text Available Background & objectives: The prevalence of trypanosomiasis was studied in cattle, being a major source of animal protein in Nigeria, thus, a very likely means of spread of Human African Trypano-somosis (HAT. Methods: Enzyme-linked immunosorbent assay (ELISA was used to diagnose bovine trypanosomiasis in 264 samples collected from adult cattle of mixed breeds, age and sex, in Anambra and Imo states, Nigeria. Results: Out of 264 samples analysed, 21 (7.96% were seropositive for Trypanosoma congolense while 20 (7.58% were seropositive for T. vivax and 8 (3.03% were seropositive for T. brucei infections in both the states. Interpretation & conclusion: The predominant species was found to be T. congolense. Mixed infection of three species, T. vivax, T. congolense and T. brucei was found to dominate other mixed infections in both the states. ELISA detected the infection of the three species of trypanosomes in the same group of animals. The usefulness of antigen capture ELISA in the diagnosis of human or animal trypanosomiasis was established, and the possibility of the spread of HAT caused by T. brucei gambiense and T.b. rhodesiense through cattle was expressed.

  11. Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

    Directory of Open Access Journals (Sweden)

    Virgínia MG Silva

    2006-08-01

    Full Text Available Indirect enzyme-linked immunosorbent assays (ELISAs based on recombinant major surface protein 5 (rMSP5 and initial body (IB antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2% and specificities (100% for rMSP5 and 93.8% for IB ELISA which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA to 15% (IB ELISA of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

  12. Biotin-streptavidin enzyme-linked immunosorbent assay for the detection of antibodies to Campylobacter jejuni and C. coli in chickens.

    Science.gov (United States)

    Haas, B; Hinz, K H; Glünder, G

    1999-04-01

    An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.

  13. Utility of Schistosoma bovis Adult Worm Antigens for Diagnosis of Human Schistosomiasis by Enzyme-Linked Immunosorbent Assay and Electroimmunotransfer Blot Techniques

    Science.gov (United States)

    Pardo, J.; Carranza, C.; Turrientes, M. C.; Arellano, J. L. Pérez; Vélez, R. López; Ramajo, V.; Muro, A.

    2004-01-01

    Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive. PMID:15539523

  14. Synthesis of Reabsorption-Suppressed Type-II/Type-I ZnSe/CdS/ZnS Core/Shell Quantum Dots and Their Application for Immunosorbent Assay

    Science.gov (United States)

    Wang, Sheng; Li, Jin Jie; Lv, Yanbing; Wu, Ruili; Xing, Ming; Shen, Huaibin; Wang, Hongzhe; Li, Lin Song; Chen, Xia

    2017-06-01

    We report a phosphine-free one-pot method to synthesize ZnSe/CdS/ZnS core-shell quantum dots (QDs) with composite type-II/type-I structures and consequent reabsorption suppression properties. The as-synthesized QDs possess high efficient red emission (with quantum yield of 82%) and high optical stability. Compared to type-I QDs, the ZnSe/CdS/ZnS QDs show larger Stokes shift and lower reabsorption which can reduce the emission loss and improve the level of fluorescence output. The ZnSe/CdS/ZnS QDs are used as fluorescent labels to exploit their application in fluorescence-linked immunosorbent assay (FLISA) for the first time in the detection of C-reactive protein (CRP) with a limit of detection (LOD) of 0.85 ng/mL, which is more sensitive than that of CdSe/ZnS type-I QDs based FLISA (1.00 ng/mL). The results indicate that the ZnSe/CdS/ZnS type-II/type-I QDs may be good candidates for applications in biomedical information detection.

  15. Development and field application of a competitive enzyme-linked immunosorbent assay for detection of Newcastle disease virus antibodies in chickens and ducks.

    Science.gov (United States)

    Phan, L V; Park, M-J; Kye, S-J; Kim, J-Y; Lee, H-S; Choi, K-S

    2013-08-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) using a baculovirus-expressed recombinant nucleocapsid protein antigen (rNDV-N) and an rNDV-N-specific monoclonal antibody (5B3) was developed for the detection of Newcastle disease virus (NDV) antibodies, and its diagnostic performance was evaluated. The specificity and sensitivity of the C-ELISA was found to be 98.4 and 98.9%, respectively, for chickens, and 98.2 and 97.9% for ducks. However, the C-ELISA showed weak cross-reaction with hyperimmune antisera to some other avian paramyxovirus serotypes. In all experimentally vaccinated chickens, seroconversion rates at 7 d postinoculation were 100 and 40% when measured by C-ELISA and hemagglutination inhibition (HI), respectively. In field trials, the C-ELISA showed positive results in 98.9% of HI-positive sera and 40.8% of HI-negative sera from NDV-vaccinated chickens (n = 705). In domestic ducks (n = 158) from NDV-positive duck farms (n = 8), the positive rates according to C-ELISA were significantly higher than those according to the HI test. At the same time, 98.1% of ducks (n = 209) from NDV-negative duck farms (n = 11) were also negative by C-ELISA. Our results indicate that C-ELISA could be a useful alternative to HI testing for detecting NDV antibodies in different avian species such as chickens and ducks.

  16. Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of serum antibodies against Akabane virus in cattle.

    Science.gov (United States)

    Kittelberger, Reinhold; McFadden, Andrew M J; Kirkland, Peter D; Hannah, Michaela J; Orr, Della; Bueno, Rudolfo; Swainsbury, Richard; Keen, Denise; Jenner, Judy; French, Jennifer; Pigott, Clive J

    2013-09-01

    In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods' characteristics.

  17. Commercial enzyme-linked immunosorbent assay versuspolymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis.

    Science.gov (United States)

    Brasil, Pedro Emmanuel Alvarenga Americano do; Castro, Rodolfo; Castro, Liane de

    2016-01-01

    Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects.

  18. Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis.

    Science.gov (United States)

    Vanasco, N B; Jacob, P; Landolt, N; Chiani, Y; Schmeling, M F; Cudos, C; Tarabla, H; Lottersberger, J

    2016-04-01

    Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations.

  19. Evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of Theiler's murine encephalomyelitis virus antibodies in mice colonies.

    Science.gov (United States)

    Laborde, Juan M; Carbone, Cecilia; Corva, Santiago G; Galosi, Cecilia M

    2008-11-01

    The current study demonstrates the ability of an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against Theiler's murine encephalomyelitis virus in mice colonies. The antigen was produced from infected baby hamster kidney (BHK)-21 cells and treated with 1% Nonidet P40 in saline buffer. Control antigen was prepared following the same procedure using uninfected BHK-21 cells. The optimal antigen and serum dilutions were established. The reaction was revealed using an anti-mouse-horseradish peroxidase conjugate and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Optimized iELISA was validated by detection of antibodies in known positive and negative serum samples before testing the samples of unknown status. Performance of the iELISA was compared with the indirect fluorescent antibody test, and the cutoff value was determined by receiver operating curve. Indirect ELISA showed 100% sensitivity, 99.38% specificity, and 97.78% predictive positive value. The antigen used is easy to produce, and no special equipment is required. The iELISA developed is simple and provides a rapid and less costly tool for diagnosis and research.

  20. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows.

    Science.gov (United States)

    Walsh, Robert B; Kelton, David F; Hietala, Sharon K; Duffield, Todd F

    2013-04-01

    Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.

  1. Detecting quinoxaline-2-carboxylic acid in animal tissues by using sensitive rapid enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay.

    Science.gov (United States)

    Le, Tao; Yu, Huan; Niu, Xiaodong

    2015-05-15

    An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluoroimmunoassay (TR-FIA) based on an anti-N-butylquinoxaline-2-carboxamide (BQCA) monoclonal antibody were standardized and validated for quinoxaline-2-carboxylic acid (QCA) screening in animal tissues and its performance were compared to HPLC. The sensitivities obtained for edible tissue extracts were 1.62 and 1.12 ng ml(-1) for ic-ELISA and TR-FIA detection, respectively. Two samples were spiked with QCA and analyzed by both methods. The recovery values ranged from 92.6% to 112.2% and the coefficients of variation were less than 15% for QCA spiking into swine tissue samples at concentrations of 2.5-50.0 μg kg(-1). Excellent correlations (r(2)=0.987-0.996) of the ic-ELISA/HPLC and TR-FIA/HPLC data were observed for processed samples. The results demonstrated that the ic-ELISA and TR-FIA methods were rapid and accurate for the residue detection of QCA in animal tissues.

  2. Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

    Science.gov (United States)

    Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

    2014-12-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  3. Preparation of monoclonal antibody of anti-feline calicivirus and establishment of double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

    Science.gov (United States)

    Yuan, B; Ai, C-X; Yuan, L; Gao, W; Hu, J-P; Chen, J; Ren, W-Z

    2014-09-12

    This study aimed to prepare monoclonal antibody of feline calicivirus (FCV) and identify its basic biological characteristics. Saturated ammonium sulfate precipitation, combined differential centrifugation, and cesium chloride density gradient centrifugation were used for the purification of FCV. The purified FCV was used as antigen to immunize BALB/c mice. The hybridoma lines of anti-FCV monoclonal antibodies were established using cell fusion and hybridoma screening techniques. The subtypes of the monoclonal antibody were identified. The results showed that 3 strains of hybridoma cell lines stably secreted anti-FCV monoclonal antibody; they were named as D8, E5, and H4. The D8 and E5 were IgM subtype antibodies, and H4 was IgG2b subtype antibody. The monoclonal antibody obtained shared no cross-reactivity with feline parvovirus, canine parvovirus, and canine distemper virus. According to the different recognition sites of 2 monoclonal antibodies E5 and H4 to the FCV, they were used to coat microtiter plates and prepare 2 enzyme-labeled secondary antibodies to establish double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

  4. A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc.

    Science.gov (United States)

    Thiha, Aung; Ibrahim, Fatimah

    2015-05-18

    The enzyme-linked Immunosorbent Assay (ELISA) is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD) platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density) of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT) in resource-limited settings.

  5. A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc

    Directory of Open Access Journals (Sweden)

    Aung Thiha

    2015-05-01

    Full Text Available The enzyme-linked Immunosorbent Assay (ELISA is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT in resource-limited settings.

  6. Commercial enzyme-linked immunosorbent assay versus polymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

    Science.gov (United States)

    do Brasil, Pedro Emmanuel Alvarenga Americano; Castro, Rodolfo; de Castro, Liane

    2016-01-01

    Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects. PMID:26814640

  7. Differential enzyme-linked immunosorbent assay and ligand-binding mass spectrometry for analysis of biotransformation of protein therapeutics: application to various FGF21 modalities.

    Science.gov (United States)

    Hager, Todd; Spahr, Chris; Xu, Jing; Salimi-Moosavi, Hossein; Hall, Michael

    2013-03-05

    Novel protein therapeutics have become increasingly important modalities for treating diseases. Such therapeutics include recombinant fusions of pharmacoactive polypeptides to half-life extenders such as monoclonal antibodies, fragments of antibodies, and albumin. Half-life extension can also be achieved via chemical attachment to polymers such as polyethylene glycol. Any of these therapeutics may be susceptible to biotransformation, most notably in vivo proteolytic truncation, and it is vital to understand this phenomenon during early drug development to ensure correct pharmacokinetic profiling and optimize the in vivo stability through re-engineering. In this paper, we describe an integrated approach that combines differential enzyme-linked immunosorbent assay (ELISA) with ligand-binding-mass spectrometry (LB-MS) to provide a thorough understanding of the biotransformation of novel protein therapeutics. Differential ELISA allows for a fast, high-throughput means to reveal gross in vivo proteolytic liabilities. Ensuing LB-MS analysis provides higher resolution details such as specific vulnerable loci to allow design refinement of the molecule. In this work, the power of the approach is elucidated by application to the optimization of a promising drug candidate, FGF21.

  8. Significance of enzyme linked immunosorbent assay (ELISA) for antibodies to double stranded and single stranded DNA in patients with lupus nephritis: correlation with severity of renal histology.

    Science.gov (United States)

    Okamura, M; Kanayama, Y; Amastu, K; Negoro, N; Kohda, S; Takeda, T; Inoue, T

    1993-01-01

    The correlation between renal histology and class specific (IgG and IgM) antibodies to double stranded DNA (dsDNA) and single stranded DNA (ssDNA) was studied by enzyme linked immunosorbent assay (ELISA) in 40 untreated patients with systemic lupus erythematosus (SLE). The levels of IgG antibodies to dsDNA were significantly higher in patients with World Health Organisation class IV nephritis than in those with class I, class II, or class III nephritis. IgG antibodies to ssDNA were higher in patients with class IV than in those with class II nephritis. IgG antibodies to dsDNA showed a close correlation with the histological activity score and the amount of electron dense deposit. IgG antibodies to ssDNA showed only a weak correlation with the renal histological activity score. IgM antibodies to dsDNA and IgM antibodies to ssDNA were not correlated with renal histological features. Patients with moderate to severe nephritis had a lower ratio of IgM antibodies to dsDNA to IgG antibodies to dsDNA than those with mild nephritis. These results indicate that the measurement of IgG antibodies to dsDNA is predictive in evaluating renal histological activity in patients with SLE.

  9. Comparison of indirect immunofluorescence (IF) test with enzyme-linked immunosorbent assay (ELISA) in screening of hybridomas to very virulent Marek's disease virus.

    Science.gov (United States)

    Nakajima, K; Shibayama, T; Naito, M; Kurimura, T; Hirai, K

    1992-01-01

    For identifying virus-specific antigens of Marek's disease virus (MDV), monoclonal antibodies (MAbs) against strain Md5 of serotype 1, which is known to be a very virulent MDV (vvMDV), were isolated. Fifty-eight hybridoma clones that secreted MAbs against vvMDV were obtained. Of these MAbs, 36 gave positive reactions in an immunofluorescence (IF) test, and 22 gave positive reactions on enzyme-linked immunosorbent assay (ELISA). None of these MAbs gave positive reactions in both the IF test and ELISA. Of the MAbs that gave positive reactions in the IF test, 33 clones reacted with MDV1-specific epitopes, the other three reacting with MDV1-HVT intertypic epitopes. None of the clones reacted with MDV1-MDV2 intertypic epitopes. Three virus-specific polypeptides were identified by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or immunoblotting. These polypeptides were recognized by 12 MAbs giving positive reactions by IF, but by none of those giving positive reactions by ELISA. In addition, size heterogeneity of the MDV1-specific phosphorylated polypeptides in the MDV1 strains was shown using the MAbs against Md5.

  10. Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial enzyme-linked immunosorbent assay kits.

    Science.gov (United States)

    Lee, Hyun Jung; Meldrum, Alexander D; Rivera, Nicholas; Ryu, Dojin

    2014-10-01

    Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests.

  11. Development and validation of an indirect competitive enzyme linked-immunosorbent assay for the determination of potentially allergenic sesame (Sesamum indicum) in food.

    Science.gov (United States)

    Husain, Fatima Tazeen; Bretbacher, Ines Elisabeth; Nemes, Albert; Cichna-Markl, Margit

    2010-02-10

    This study was designed to develop an indirect competitive enzyme linked-immunosorbent assay (ELISA) to detect traces of sesame in food. Antibodies against sesame were prepared by immunizing a hen with a protein extract of white, peeled sesame. The ELISA did not show any cross-reactivity with 12 of 13 food ingredients tested, only for chocolate was a low cross-reactivity of 0.7% observed. To eliminate matrix effects, sesame protein standard solutions were prepared by diluting the sesame extract with blank food matrix (1:20 diluted with PBS). Recovery of sesame protein in food samples (crisp toasts, snacks, and rolls) spiked with different sesame protein concentrations ranged from 85% to 120%, with the exception of multigrain crisp toast, resulting in too high recoveries (117%-160%) and whole grain bread, yielding too low recoveries (70%-85%). In crisp bread, cracker, cereals, and snacks the limit of detection (LOD) was found to be 5 microg of sesame protein/g of food, in fresh breads and rolls, the LOD was 11 microg of sesame protein/g of food.

  12. Relative efficiency of polymerase chain reaction and enzyme-linked immunosorbant assay in determination of viral etiology in congenital cataract in infants

    Directory of Open Access Journals (Sweden)

    Shyamala G

    2008-01-01

    Full Text Available Background: Perinatal viral infections of fetus are among the leading causes of congenital cataract and identifying the viral etiology is important. Objectives: To detect the presence of Rubella virus (RV, herpes simplex virus (HSV and cytomegalovirus (CMV in lens aspirate specimens obtained from patients with congenital cataract and relate the results with serology. Setting and Design: Prospective study carried out in tertiary care hospital. Materials and Methods: Fifty lens aspirates from 50 infants with congenital cataract were subjected to HSV, RV isolation and polymerase chain reaction (PCR for detection of HSV and CMV. Reverse transcription polymerase chain reaction (RT-PCR was applied for RV detection. Peripheral blood specimens were screened for anti-HSV, RV and CMV antibodies by enzyme-linked immunosorbant assay (ELISA. Results: Rubella virus was detected in nine (18% lens aspirates, by nRT-PCR which includes six positive by culture. HSV-2 DNA was detected in nine other lens aspirates, while CMV was not detected by PCR. Serological results did not correlate with the presence of viruses in the lens aspirates. This is the first report of detection of HSV-2 DNA in cases of congenital cataract. Conclusions: Cytomegalovirus may not be playing a significant role in causation of congenital cataract. The role of serology in identifying causative viral infection for congenital cataract needs to be re-evaluated.

  13. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    2009-01-01

    Full Text Available To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver, we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2>0.95, which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

  14. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Adungo, Ferdinard; Yu, Fuxun; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu; Morita, Kouichi

    2016-08-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units.

  15. Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.

    Science.gov (United States)

    de Medina, Philippe; Paillasse, Michael R; Segala, Gregory; Al Saati, Talal; Boyes, Jeannine; Delsol, Georges; Allal, Cuider; Marsili, Sabrina; Silvente-Poirot, Sandrine; Poirot, Marc

    2013-03-01

    We have recently discovered the existence of 5α-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-o-hemisuccinate with a carboxylic spacer arm attached to the 3β-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

  16. hYKL-40 cancer biomarker electroanalysis in serum samples and model cell lysates: capacitive immunosensing compared with enzyme label immunosorbent assays (ELISA).

    Science.gov (United States)

    Chaocharoen, W; Schulte, A; Suginta, W

    2017-01-26

    Human chitinase 3-like protein 1 (CHI3L1 or hYKL-40), a potential molecular marker for several cancers, was measured in clinical human serum samples and model cell lysates by indirect and competitive enzyme-linked immunosorbent assay (ELISA) and by capacitive immunosensing, so as to evaluate a recently introduced electrochemical method for routine use in cancer-monitoring studies. The clinical samples tested included serum from four healthy individuals, five breast cancer and four glioblastoma patients; cultures of the human monocytic cell line THP-1, known to secrete hYKL-40, and of the human embryonic kidney 293t cell line, which does not express hYKL-40, provided cell lysates and cell culture media for positive and negative bio- and electrochemical control trials. A good agreement was observed between the results of the three tested methods during hYKL-40 quantifications in human serum and cell lysates. Measurements of 'spiked' samples from healthy volunteers, cancer patients and hYKL-40-free 293t cell lysates revealed that capacitive immunosensing and the two types of ELISA all recovered the analyte with an efficiency close to 100%. On this basis, capacitive hYKL-40 immunosensor screening is a promising stand-alone or complementary analytical tool for the analysis of hYKL-40 in serum, and would be useful for the validation of standard ELISA data and also, because of the significantly lower hYKL-40 detection limit of the electroanalytical procedure, would permit assay of the marker and cancer observation at earlier stages than is currently possible using ELISA.

  17. Improved diagnostic performance of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5-glutathione S-transferase fusion protein as antigen.

    Science.gov (United States)

    Chung, Chungwon; Wilson, Carey; Bandaranayaka-Mudiyanselage, Chandima-Bandara; Kang, Eunah; Adams, D Scott; Kappmeyer, Lowell S; Knowles, Donald P; McElwain, Terry F; Evermann, James F; Ueti, Massaro W; Scoles, Glen A; Lee, Stephen S; McGuire, Travis C

    2014-01-01

    The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (Anaplasma-negative sera, which included the 139 sera with significant (≥30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested polymerase chain reaction. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera. In summary, the rMSP5-GST cELISA was a faster and simpler assay with higher specificity, comparable sensitivity, and improved resolution in comparison with the rMSP5-MBP cELISA with MBP adsorption.

  18. Development and validation of an indirect Enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants.

    Science.gov (United States)

    van der Heijden, H M J F; Bouwstra, R J; Mars, M H; van der Poel, W H M; Wellenberg, G J; van Maanen, C

    2013-10-01

    To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

  19. Comparison of Three Enzyme-Linked Immunosorbent Assays for Detection of Immunoglobulin G Antibodies to Tetanus Toxoid with Reference Standards and the Impact on Clinical Practice▿

    Science.gov (United States)

    van Hoeven, Karen H.; Dale, Connie; Foster, Phil; Body, Barbara

    2008-01-01

    Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y = 1.09x − 0.08), whereas the Scimedx ELISA gave results that were consistently lower (y = 0.21x − 0.07) and the Euroimmun ELISA gave results that were consistently higher (y = 1.5x + 0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results. PMID:18845832

  20. Comparison of three enzyme-linked immunosorbent assays for detection of immunoglobulin g antibodies to tetanus toxoid with reference standards and the impact on clinical practice.

    Science.gov (United States)

    van Hoeven, Karen H; Dale, Connie; Foster, Phil; Body, Barbara

    2008-12-01

    Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y=1.09x-0.08), whereas the Scimedx ELISA gave results that were consistently lower (y=0.21x-0.07) and the Euroimmun ELISA gave results that were consistently higher (y=1.5x+0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results.

  1. Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

    Directory of Open Access Journals (Sweden)

    Zoheiry Mona K

    2011-09-01

    Full Text Available Abstract Background This research was carried out to develop a reliable monoclonal antibody (MoAb-based sandwich enzyme linked immunosorbent assay (ELISA for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags, a pair (12B/11D/3F and 10A/9D/10G was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

  2. Parallel enzyme-linked immunosorbent assay screening for human immunodeficiency virus among blood donors in five Chinese blood centres: a retrospective analysis.

    Science.gov (United States)

    Zeng, P; Liu, J; Wang, J; Dong, X; Li, J; Bi, X; Ma, H; Wen, X; He, M; Liu, Y; Ness, P; Shan, H

    2015-08-01

    To evaluate the strategy of parallel screening with different enzyme-linked immunosorbent assays (ELISA) among Chinese blood donors. Parallel screening with ELISA has been the main strategy to detect human immunodeficiency virus (HIV) in blood donations in China for more than a decade. The performance of the strategy should be analysed. A total of 821,927 donations collected from five Chinese blood centres in 2008-2010 were tested using two third-generation ELISAs by different manufacturers licenced and confirmed by the Western blot (WB) in this study. The confirmatory positive predictive values (PPV), false positive rates (FPR), false negative rates (FNR) and potential risks for transfusion resulting from single or sequential ELISA screening were evaluated. A total of 5318 (0·647%) of donations screened HIV reactive and were discarded. WB confirmatory results on 1668 available samples suggested that PPVs for dual ELISA, one round ELISA reactive and grey zone samples were 75·1, 0·7 and 0·5%, respectively. Eight out of 1124 one round ELISA reactive and 1 out of 195 grey zone samples were WB confirmed positive. All but one ELISA assay displayed comparable PPVs but variable FPRs and FNRs that differed by blood centre. In the absence of nucleic acid testing (NAT), parallel ELISA screening prevented a substantial number of HIV infected donations from entering the Chinese blood supply. However, the loss of false positive donors should be re-evaluated especially given the frequently reported blood supply shortage in China. © 2015 British Blood Transfusion Society.

  3. Identification and expression of Babesia ovis secreted antigen 1 and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sevinc, Ferda; Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, Onur

    2015-05-01

    In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis.

  4. Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

    Directory of Open Access Journals (Sweden)

    Raba Julio

    2011-10-01

    Full Text Available Abstract Background Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA for quantification of B. cinerea in apple (Red Delicious, table grape (pink Moscatel, and pear (William's tissues. Results The method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation. Conclusions The developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide.

  5. Prediction of estrus cyclicity in Asian elephants (Elephas maximus) through estimation of fecal progesterone metabolite: development of an enzyme-linked immuno-sorbent assay.

    Science.gov (United States)

    Ghosal, R; Sukumar, R; Seshagiri, P B

    2010-05-01

    Asian elephants (Elephas maximus), prominent "flagship species", are listed under the category of endangered species (EN - A2c, ver. 3.1; IUCN Red List 2009) and there is a need for their conservation. This requires understanding demographic and reproductive dynamics of the species. Monitoring reproductive status of any species is traditionally being carried out through invasive blood sampling and this is restrictive for large animals such as wild or semi-captive elephants due to legal, ethical, and practical reasons. Hence, there is a need for a non-invasive technique to assess reproductive cyclicity profiles of elephants, which will help in the species' conservation strategies. In this study, we developed an indirect competitive enzyme linked immuno-sorbent assay (ELISA) to estimate the concentration of one of the progesterone-metabolites i.e., allopregnanolone (5 alpha-P-3OH) in fecal samples of Asian elephants. We validated the assay which had a sensitivity of 0.25 microM at 90% binding with an EC(50) value of 1.37 microM. Using female elephants, kept under semi-captive conditions in the forest camps of Mudumalai Wildlife Sanctuary, Tamil Nadu and Bandipur National Park, Karnataka, India, we measured fecal progesterone-metabolite (5 alpha-P-3OH) concentrations in six animals and showed their clear correlation with those of serum progesterone, measured by a standard radio-immuno assay. Statistical analyses using a Linear Mixed Effect model showed a positive correlation (P<0.1) between the profiles of fecal 5 alpha-P-3OH (range: 0.5-10 microg/g) and serum progesterone (range: 0.1-1.8 ng/mL). Therefore, our studies show, for the first time, that the fecal progesterone-metabolite assay could be exploited to predict estrus cyclicity and to potentially assess the reproductive status of captive and free-ranging female Asian elephants, thereby helping to plan their breeding strategy. (c) 2010 Elsevier Inc. All rights reserved.

  6. Evaluation of a fecal pancreatic elastase-1 enzyme-linked immunosorbent assay: Assessment versus an established assay and implication in classifying pancreatic function.

    Science.gov (United States)

    Erickson, J Alan; Aldeen, William E; Grenache, David G; Ashwood, Edward R

    2008-11-01

    Disagreement continues regarding 2 fecal pancreatic elastase-1 (PE-1) ELISAs and their respective capabilities to assess pancreatic function. The BioServ Diagnostics polyclonal PE-1 ELISA was validated and its performance characteristics compared to the previously validated ScheBo Biotech monoclonal PE-1 ELISA. Split sample study results were analyzed by Deming regression and Bland-Altman plot analysis. Data mining was utilized to explore PE-1 distribution and evaluate PE-1 and fecal fat correlation. Analysis demonstrates limited quantitative agreement; slope=0.9640, intercept=10.787, R(2)=0.633. Means were 228.8 and 226.2 microg PE-1/g stool for the polyclonal and monoclonal assays respectively. Bland-Altman analysis showed 91% of paired values within 2 SD of their means. There was good qualitative agreement when interpreted against established intervals with 91% of results equivalent in pancreatic function classification. The remaining 9% varied by one classification level with no bias evident. The distribution of PE-1 concentrations (n=400, 0-25 years) classified 78% of subjects with normal pancreatic function, 7% with moderate pancreatic insufficiency and 15% with severe insufficiency. There was little agreement between PE-1 and fecal fat results. The polyclonal PE-1 ELISA is an acceptable alternative to the monoclonal PE-1 ELISA. PE-1 is a potential substitute for fecal fat for evaluating pancreatic function.

  7. Validation according to OIE criteria of a monoclonal, recombinant p26-based, serologic competitive enzyme-linked immunosorbent assay as screening method in surveillance programs for the detection of Equine infectious anemia virus antibodies.

    Science.gov (United States)

    Nardini, Roberto; Autorino, Gian Luca; Ricci, Ida; Frontoso, Raffaele; Rosone, Francesca; Simula, Massimiliano; Scicluna, Maria Teresa

    2016-03-01

    The Italian National Reference Center for equine infectious anemia (CRAIE; Rome, Italy) developed and validated a monoclonal, recombinant p26-based competitive enzyme-linked immunosorbent assay (cELISA) for the detection of EIA virus antibodies employing the 2010 criteria of the World Organization for Animal Health (OIE). The following parameters were evaluated: cutoff values, repeatability, reproducibility, concordance, analytical sensitivity (Se), absolute analytical specificity (Sp), and diagnostic Se and Sp. Positive and negative predictive values were also defined in relation to the estimated prevalence. When the cELISA was used as a screening test for 96,468 samples in the Italian EIA surveillance program, 17% more EIA cases were detected than by the agar gel immunodiffusion test, and the apparent diagnostic Sp estimated from these samples was 99.8%, which was more than the diagnostic Sp (80.2%) estimated from validation. The high Se and Sp of the cELISA confirm its fit for purpose as a screening test. © 2015 The Author(s).

  8. Differentiation of foot-and-mouth disease virus-infected from vaccinated pigs by enzyme-linked immunosorbent assay using nonstructural protein 3AB as the antigen and application to an eradication program

    DEFF Research Database (Denmark)

    Chung, Wen Bin; Sørensen, Karl Johan; Liao, Pei Chih

    2002-01-01

    Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field...... correlation coefficient (r = 0.93) was found between the test results from sow sera and those from their offspring. Therefore, piglet serum was a good substitute for sow serum to monitor the infection status of the dam. The application of this assay to serological surveillance in an FMD eradication program...

  9. A comparative study of Toxoplasma gondii seroprevalence in mink using a modified agglutination test, a Western blot, and enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Gu, Yi; Wang, Zedong; Cai, Yufeng; Li, Xiaoxing; Wei, Feng; Shang, Limin; Li, Jiping; Liu, Quan

    2015-09-01

    Toxoplasma gondii can infect almost all warm-blooded animals, and many serological methods have been developed to detect T. gondii infection in a variety of animal species. In the present study, the seroprevalence of T. gondii infection in farmed mink in northeast China was determined using the modified agglutination test (MAT), a Western blot (WB), and 3 enzyme-linked immunosorbent assays (ELISAs) with protein A/G conjugate, using either of 2 recombinant dense granule antigens, GRA1 and GRA7, or Toxoplasma soluble antigens (TSA). There was no significant difference between the detection results of the GRA1-, GRA7-, and TSA-ELISAs and WB (McNemar chi-square, P > 0.05), but a significant difference was observed between MAT and WB (P < 0.05). A near perfect agreement (97.0%) was found between the GRA7-ELISA and WB (κ = 0.83), and a substantial agreement (92.4-93.1%) was observed in the TSA- and GRA1-ELISAs (κ = 0.68-0.73). The GRA7-ELISA showed the highest sensitivity and specificity, and the lowest false-positive and negative rates, while the MAT gave both a low sensitivity and frequent false positives in comparison to the WB. Receiver operating characteristic analysis revealed the largest area under curve of 0.85 (95% confidence interval: 0.74-0.96), and the highest relative sensitivity (72.7%) and specificity (99.0%) for a cutoff value of 0.19 in the GRA7-ELISA. These results indicate that the GRA7-ELISA is suitable for detection of T. gondii infection in mink and that MAT should be used with caution.

  10. Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

    Science.gov (United States)

    Peng, Dapeng; Ye, Shengqiang; Wang, Yulian; Chen, Dongmei; Tao, Yanfei; Huang, Lingli; Liu, Zhenli; Dai, Menghong; Wang, Xiaoqing; Yuan, Zonghui

    2012-01-11

    Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.

  11. Assessment of urine solute and matrix effects on the performance of an enzyme-linked immunosorbent assay for measurement of interleukin-6 in dog urine.

    Science.gov (United States)

    Wood, Michael W; Nordone, Sushila K; Vaden, Shelly L; Breitschwerdt, Edward B

    2011-03-01

    Measurement of cytokine concentrations within body fluids is a means of recognizing subclinical and/or unresolved, infectious and inflammatory states in patients. In the urinary tract, such information may be useful for identifying patients with pyelonephritis, asymptomatic bacteriuria, recurrent infections, and cystitis. One such cytokine, interleukin-6 (IL-6), is recognized as a primary cytokine that is produced following exposure of the urothelium to bacterial virulence factors. Complicating reliable testing for this and other cytokines is the nature of urine itself. Urine varies widely in its composition as indicated by the range of pH and urine specific gravity (USG) observed in healthy patients. An additional variable is the protein and carbohydrate matrix capable of hindering immunologic testing modalities, such as enzyme-linked immunosorbent assays (ELISAs). The purpose of the current study was to examine the role of urine pH, USG, and matrix while optimizing a canine-specific chemiluminescent ELISA for the measurement of IL-6 in the urine of dogs. Urine spiked with IL-6 obtained maximal IL-6 quantitative recoveries of only 55 ± 10% (mean ± 1 standard deviation) when an ELISA optimized for cell culture supernatants was used. The urine matrix and variations in USG were determined to by contributing to this poor IL-6 recovery. Using specific matrix inhibitors and optimal dilutions improved the IL-6 quantitative recovery to 91 ± 5%. Urine pH (5.5-9.5) had no effect. The current work underscores the importance of critically optimizing testing modalities for biomarkers, particularly if they are immunologic in origin.

  12. Facile preparation of glycoprotein-imprinted 96-well microplates for enzyme-linked immunosorbent assay by boronate affinity-based oriented surface imprinting.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-01-01

    Molecularly imprinted polymers (MIPs), as inexpensive and stable substitutes of antibodies, have shown great promise in immunoassays. Glycoproteins are of significant diagnostic value. To facilitate the application of MIPs in clinical diagnostics, a general and facile imprinting method toward glycoproteins oriented for an enzyme-linked immunosorbent assay (ELISA) in the form of a 96-well microplate is essential but has not been fully explored yet. In this study, a new method called boronate affinity-based oriented surface imprinting was proposed for facile preparation of glycoprotein-imprinted microplates. A template glycoprotein was first immobilized by a boronic acid-modified microplate through boronate affinity binding, and then, a thin layer of polyaniline was formed to cover the microplate surface via in-water self-copolymerization. After the template was removed by an acidic solution, 3D cavities that can rebind the template were fabricated on the microplate surface. Using horseradish peroxidase (HRP) as a model target, the effects of imprinting conditions as well as the properties and performance of the prepared MIPs were investigated. α-Fetoprotein (AFP)-imprinted microplate was then prepared, and thereby, a MIP-based ELISA method was established. The prepared MIPs exhibited several highly favorable features, including excellent specificity, widely applicable binding pH, superb tolerance for interference, high binding strength, fast equilibrium kinetics, and reusability. The MIP-based ELISA method was finally applied to the analysis of AFP in human serum. The result was in good agreement with that by radioimmunoassay, showing a promising prospect of the proposed method in clinical diagnostics.

  13. Development of an antigen-capture enzyme-linked immunosorbent assay for Clostridium perfringens beta2-toxin in porcine feces and the neonatal piglet intestine.

    Science.gov (United States)

    Kircanski, Jasmina; Hodgins, Douglas; Soltes, Glenn; Pei, Yanlong; Parreira, Valeria R; Songer, J Glenn; Prescott, John F

    2012-09-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantitation of beta2-toxin in neonatal piglet intestinal contents. Polystyrene plates were coated with polyclonal capture antibodies prepared against consensus recombinant beta2-toxin. The ELISA was developed using consensus recombinant beta2-toxin, atypical recombinant beta2-toxin, purified consensus native beta2-toxin, and field samples of neonatal porcine intestinal contents. Captured antigen was detected using a horseradish peroxidase-labeled monoclonal antibody against consensus recombinant beta2-toxin. The limit of detection of the ELISA for consensus beta2-toxin was between 2.0 and 3.5 ng/ml. The ELISA detected atypical recombinant beta2-toxin only weakly. Optical density was protein concentration dependent. The test confirmed differences between consensus and atypical recombinant beta2-toxin, but similar results obtained when testing pure consensus recombinant beta2-toxin and native beta2-toxin. Results obtained from intestinal content samples, particularly from the small intestine, were highly inconsistent and suggested variable protease activity. Addition of protease inhibitors partially prevented degradation of the toxin; however, sample processing at low temperature, at a lower pH (citrate buffer with 5% of bovine serum albumin, pH 6.1), and "cold incubation" of applied antigens abolished protease activity. The recombinant toxin was preserved in spiked intestinal samples by freezing at -70°C, suggesting that necropsy samples can be stored frozen for periodic testing. With appropriate sample preparation, antigen-capture ELISA can detect beta2-toxin in the intestinal content and feces of neonatal piglets.

  14. An Improved Ultrasensitive Enzyme-Linked Immunosorbent Assay Using Hydrangea-Like Antibody-Enzyme-Inorganic Three-in-One Nanocomposites.

    Science.gov (United States)

    Wei, Tianxiang; Du, Dan; Zhu, Mei-Jun; Lin, Yuehe; Dai, Zhihui

    2016-03-01

    Protein-inorganic nanoflowers, composed of protein and copper(II) phosphate (Cu3(PO4)2), have recently grabbed people's attention. Because the synthetic method requires no organic solvent and because of the distinct hierarchical nanostructure, protein-inorganic nanoflowers display enhanced catalytic activity and stability and would be a promising tool in biocatalytical processes and biological and biomedical fields. In this work, we first coimmobilized the enzyme, antibody, and Cu3(PO4)2 into a three-in-one hybrid protein-inorganic nanoflower to enable it to possess dual functions: (1) the antibody portion retains the ability to specifically capture the corresponding antigen; (2) the nanoflower has enhanced enzymatic activity and stability to produce an amplified signal. The prepared antibody-enzyme-inorganic nanoflower was first applied in an enzyme-linked immunosorbent assay to serve as a novel enzyme-labeled antibody for Escherichia coli O157:H7 (E. coli O157:H7) determination. The detection limit is 60 CFU L(-1), which is far superior to commercial ELISA systems. The three-in-one antibody (anti-E. coli O157:H7 antibody)-enzyme (horseradish peroxidase)-inorganic (Cu3(PO4)2) nanoflower has some advantages over commercial enzyme-antibody conjugates. First, it is much easier to prepare and does not need any complex covalent modification. Second, it has fairly high capture capability and catalytic activity because it is presented as aggregates of abundant antibodies and enzymes. Third, it has enhanced enzymatic stability compared to the free form of enzyme due to the unique hierarchical nanostructure.

  15. Enzyme-linked immunosorbent assay by enhanced chemiluminescence detection for the standardization of estrogenic miroestrol in Pueraria candollei Graham ex Benth.

    Science.gov (United States)

    Yusakul, Gorawit; Udomsin, Orapin; Tanaka, Hiroyuki; Morimoto, Satoshi; Juengwatanatrakul, Thaweesak; Putalun, Waraporn

    2015-08-01

    Miroestrol (ME) is a potent phytoestrogen from the P. candollei tuberous root. It has been approved for use in clinical trials due to its beneficial effect on disorders associated with estrogen deficiency. To ensure medical efficacy and safety, high performance analytical methods for ME analysis are required to standardize products from the P. candollei root. An enhanced chemiluminescence enzyme-linked immunosorbent assay (ECL-ELISA) was developed and validated using a polyclonal antibody against ME and a chemiluminescent system of luminol-H2 O2 -horseradish peroxidase-4-(1-imidazolyl) phenol. The ECL-ELISA system exhibited linearity over a concentration range of 0.31-10.00 ng mL(-1) , for which the relative standard variation (%RSD) was less than 10% for both intra- and interplate determinations. The ECL-ELISA is reliable for the determination of ME as reflected by the high recovery percentage (101.22-103.06%). As a comparative analysis, the ME content in each sample determined by ECL-ELISA was correlated with high coefficients of determination with colorimetric ELISA (R(2)  = 0.998) and high performance liquid chromatography (HPLC) (R(2)  = 0.998) methods. The ECL-ELISA method could be applied to all of the commercial products containing P. candollei root, when the products contain between 0.706 ± 0.046 and 13.123 ± 0.794 µg g(-1) dry wt. of ME. This method is useful as a high performance analytical method for the quantity control of ME in raw materials and end products at both the research and industrial levels.

  16. Development of phosphonate modified Fe 1-x MnxFe2O4 mixed ferrite nanoparticles: novel peroxidase mimetics in enzyme linked immunosorbent assay.

    Science.gov (United States)

    Bhattacharya, Dipsikha; Baksi, Ananya; Banerjee, Indranil; Ananthakrishnan, Rajakumar; Maiti, Tapas K; Pramanik, Panchanan

    2011-10-30

    A highly facile and feasible strategy on the fabrication of advanced intrinsic peroxidase mimetics based on Mn(2+) doped mixed ferrite (Mn(II)(x)Fe(II)(1-x)Fe(III)(2)O(4)) nanoparticles was demonstrated for the quantitative and sensitive detection of mouse IgG (as a model analyte). Mn(2+) doped Fe(1-x)Mn(x)Fe(2)O(4) nanoparticles were synthesized using varying ratios of Mn(2+):Fe(2+) ions and characterized by the well known complementary techniques. The increase of Mn(2+) proportion had remarkably enhanced the peroxidase activity and magnetism. The catalytic activity of mixed ferrites was found to follow Michaelis-Menten kinetics and was noticeably higher than native Fe(3)O(4). The calculated K(m) and K(cat) exhibited strong affinity with substrates which were remarkably higher than similar sized native magnetite nanoparticles and horseradish peroxidase (HRP). These findings stimulated us to develop carboxyl modified Fe(1-x)Mn(x)Fe(2)O(4) nanoparticles using phosphonomethyl immunodiacetic acid (PMIDA) to engineer PMIDA-Fe(1-x)Mn(x)Fe(2)O(4) fabricated enzyme linked immunosorbent assay (ELISA). Results of both PMIDA-Fe(1-x)Mn(x)Fe(2)O(4) linked ELISA revealed that the enhancements in absorbance during the catalysis of enzyme substrate were linearly proportional to the concentration of mouse IgG within the range between 0.1 μg/ml and 2.5 μg/ml. Further, this detection was ten times lower than previous reports and the detection limit of mouse IgG was 0.1 μg/ml. The advantages of our fabricated artificial peroxidase mimetics are combined of low cost, easy to prepare, better stability and tunable catalytic activity. Moreover, this method provides a new horizon for the development of promising analytical tools in the application of biocatalysis, bioassays, and bioseparation.

  17. Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

    Science.gov (United States)

    Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

    2014-03-01

    Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

  18. Enzyme-linked immunosorbent assay of aflatoxins B1, B2, and G1 in corn, cottonseed, peanuts, peanut butter, and poultry feed: collaborative study.

    Science.gov (United States)

    Trucksess, M W; Stack, M E; Nesheim, S; Park, D L; Pohland, A E

    1989-01-01

    A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of less than 30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain greater than or equal to 20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain less than 20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing less than 2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and greater than or equal to 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 10:1:3) were 52, 86, and 96%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Studies to assess the biological relevance of anti-Tamm-Horsfall protein antibodies detected by direct-binding enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Hunt, J S; Groufsky, A; Lynn, K L

    1987-11-01

    1. A role has been suggested for anti-Tamm-Horsfall protein (THP) antibodies in renal disease based on the results of immunoassays of pathological sera. The putative autoantibodies have not been isolated from such sera nor have definitive inhibition studies of their binding been carried out. We have carried out such studies using rabbit anti-THP antibodies as control reagents. 2. Urinary THP prepared by salt precipitation was used to prepare four immunoabsorbent columns by covalent coupling to CNBr-activated Sepharose 4B. After washing with a variety of dissociating agents to remove any non-covalently bound subunit THP, each column was incubated with normal and immune rabbit serum. Fractions washed and eluted from columns were tested for anti-THP antibodies by enzyme-linked immunosorbent assay (ELISA) and THP antigen by radioimmunoassay, and showed NH4SCN (3 mol/l) and guanidine hydrochloride (GuHCl) (6 mol/l) equivalent and sodium dodecyl sulphate (20 g/l) to be inferior in their capacity to produce immunoabsorbent THP capable of isolating specific antibodies from immune rabbit serum. 3. The column treated with GuHCl (6 mol/l) was used further in attempts to isolate putative anti-THP antibodies from five patients, who had a history of urinary tract infections and whose sera showed strong binding by ELISA. 4. Results from direct and inhibition ELISA experiments on fractions collected after washing and elution with all sera suggested that the putative human anti-THP antibodies were of very low affinity and/or directed against non-subunit THP. 5. The pathological relevance of human anti-THP antibodies measured by ELISA remains to be established.

  20. Enzyme-linked immunosorbent assay for S100A9 in the stool of rats with dextran sulfate sodium-induced colitis.

    Science.gov (United States)

    Sekiya, Shunsuke; Murata, Makoto; Arai, Satoshi; Murayama, Hiroshi; Kawasaki, Atushi; Ashida, Noriyuki; Okada, Kohki; Ikemoto, Masaki

    2016-12-01

    Calprotectin, a heterodimer of S100A8 and S100A9, has been reported to be a useful biomarker in inflammatory bowel disease (IBD); however, the relationship between the fecal level of S100A9 and the extent of inflammation in IBD remains unclear. Our aim was to develop a new enzyme-linked immunosorbent assay (ELISA) for rat S100A9, and to investigate whether changes in fecal S100A9 levels reflect the inflammatory conditions in the intestinal tracts of rats with dextran sulfate sodium (DSS)-induced colitis. Anti-rat S100A9 monoclonal antibodies were raised in mice and used for the development of a novel ELISA for rat S100A9. The performance of our ELISA was assessed by dilution and recovery tests, and the detection range was 3.75-240ng/mL. The dilution test showed good linearity. The recovery of fecal S100A9 was 95.1% (mean), with a range of 86.1%-108.8%. Colitis was induced in rats by oral administration of 3% DSS/drinking water (DW) for 11days (D group), while DW alone was provided to rats of the control group (C group) during the same period. The extent of inflammation was evaluated with the disease activity index (DAI), and the concentration of fecal S100A9 was determined by ELISA. Both the DAI scores and the fecal S100A9 levels were significantly higher in the D group than in the C group. Microscopic observation revealed that S100A9 was dominantly produced in many immune cells of myeloid origin in rat rectal tissues. These results indicate that the novel ELISA may be applied to clinically evaluate IBD in rats with high sensitivity. In conclusion, our ELISA is useful in toxicological and pharmacological evaluations. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Antibody responses to Campylobacter infections determined by an enzyme-linked immunosorbent assay: 2-year follow-up study of 210 patients.

    Science.gov (United States)

    Strid, M A; Engberg, J; Larsen, L B; Begtrup, K; Mølbak, K; Krogfelt, K A

    2001-03-01

    An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody to Campylobacter jejuni and Campylobacter coli. Heat-stable antigen, a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verified Campylobacter enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter species or C. jejuni serotype, with the important exception of response to C. jejuni O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (P = 0.006) and IgA (P = 0.06) compared with other C. jejuni and C. coli serotypes.

  2. Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle.

    Science.gov (United States)

    Zhao, Hailing; Liu, Huifang; Du, Yanfen; Liu, Siguo; Ni, Hongbo; Wang, Yong; Wang, Chunlai; Si, Wei; Yang, Jinguo; Ling, Jingkai

    2010-06-01

    Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398bp) and SapA-C (1422bp) fragments were amplified from the C. fetusSapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (PELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P0.45: positive; S/PS/P0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications. Copyright 2009 Elsevier Ltd. All rights reserved.

  3. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples.

    Science.gov (United States)

    Brooks, B W; Devenish, J; Lutze-Wallace, C L; Milnes, D; Robertson, R H; Berlie-Surujballi, G

    2004-10-05

    A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.

  4. Rapid detection of Campylobacter coli, C. jejuni, and Salmonella enterica on poultry carcasses by using PCR-enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Hong, Yang; Berrang, Mark E; Liu, Tongrui; Hofacre, Charles L; Sanchez, Susan; Wang, Lihua; Maurer, John J

    2003-06-01

    Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 x 10(2) and 4 x 10(1) CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P ELISA was used to screen 60 tetrathionate enrichment cultures for Salmonella. With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.

  5. Development and comparative evaluation of a plate enzyme-linked immunosorbent assay based on recombinant outer membrane antigens Omp28 and Omp31 for diagnosis of human brucellosis.

    Science.gov (United States)

    Tiwari, Sapana; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-08-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  6. Evaluation of the sensitivity and specificity of an enzyme-linked immunosorbent assay for diagnosing brucellosis in African buffalo (Syncerus caffer).

    Science.gov (United States)

    Gorsich, Erin E; Bengis, Roy G; Ezenwa, Vanessa O; Jolles, Anna E

    2015-01-01

    Brucellosis is a disease of veterinary and public health importance worldwide. In sub-Saharan Africa, where the bacterium Brucella abortus has been identified in several free-ranging wildlife species, successful disease control may be dependent on accurate detection in wildlife reservoirs, including African buffalo (Syncerus caffer). We estimated the sensitivity and specificity of a commercial enzyme-linked immunosorbent assay (ELISA) (IDEXX Brucellosis Serum Ab test, IDEXX Laboratories, Westbrook, Maine, USA) for B. abortus based on a data set of 571 serum samples from 258 buffalo in the Kruger National Park, South Africa. We defined a pseudogold standard test result as those buffalo that were consistently positive or negative on two additional serologic tests, namely, the rose bengal test (RBT) and the complement fixation test (CFT). The ELISA's cutoff value was selected using receiver operating characteristics analysis, the pseudogold standard, and a threshold criterion that maximizes the total sensitivity and specificity. Then, we estimated the sensitivity and specificity of all three tests using Bayesian inference and latent class analysis. The ELISA had an estimated sensitivity of 0.928 (95% Bayesian posterior credibility interval [95% BCI] = 0.869-0.974) and specificity of 0.870 (95% BCI = 0.836-0.900). Compared with the ELISA, the RBT had a higher estimated sensitivity of 0.986 (95% BCI = 0.928-0.999), and both the RBT and CFT had higher specificities, estimated to be 0.992 (95% BCI = 0.971-0.996) and 0.998 (95% BCI = 0.992-0.999), respectively. Therefore, no single serologic test perfectly detected the antibody. However, after adjustment of cutoff values for South African conditions, the IDEXX Brucellosis Serum Ab Test may be a valuable additional screening test for brucellosis in Kruger National Park's African buffalo.

  7. Use of Heavy Water (D2O in Developing Thermostable Recombinant p26 Protein Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Equine Infectious Anemia Virus Infection

    Directory of Open Access Journals (Sweden)

    Harisankar Singha

    2014-01-01

    Full Text Available Thermostabilizing effect of heavy water (D2O or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA for serodiagnosis of equine infectious anemia virus (EIAV infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4°C, 37°C, 42°C, and 45°C over a storage time from 2 weeks to 10 months. A set of positive serum (n=12 consisting of strong, medium, and weak titer strength (4 samples in each category and negative serum (n=30 were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37°C < 42°C < 45°C was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45°C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.

  8. Competitive binding inhibition enzyme-linked immunosorbent assay that uses the secreted aspartyl proteinase of Candida albicans as an antigenic marker for diagnosis of disseminated candidiasis.

    Science.gov (United States)

    Morrison, Christine J; Hurst, Steven F; Reiss, Errol

    2003-09-01

    The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 10(7) C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition +/- standard error [SE] compared with preinfection control urine, 15.7% +/- 2.7% [P ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition +/- SE by day 3 after infection, 32.9% +/- 2.7% [P test sensitivity was 83%, the specificity was 92%, the positive predictive value was 84%, the negative predictive value was 91%, and the efficiency was 89% (166 urine samples from 33 rabbits tested). The specificity, positive predictive value, and efficiency could be increased to 97, 95, and 92%, respectively, if at least two positive test results were required for a true positive designation. The ELISA was sensitive and specific for the detection of Sap in urine specimens from rabbits with disseminated C. albicans infection, discriminated between colonization and invasive disease, reflected disease progression and severity, and has the potential to be a noninvasive means to diagnose disseminated candidiasis.

  9. Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis from milk samples from dairy cows.

    Science.gov (United States)

    Buddle, Bryce M; Wilson, Tania; Luo, Dongwen; Voges, Hinrich; Linscott, Richard; Martel, Edmond; Lawrence, John C; Neill, Mark A

    2013-12-01

    Milk samples from dairy cows provide a ready source of material for measuring antibody responses to Mycobacterium bovis antigens. In this study, we evaluated the IDEXX enzyme-linked immunosorbent assay (ELISA) for the measurement of antibody responses to M. bovis antigens MPB70 and MPB83 in milk samples from New Zealand cattle. Test sensitivities for individual milk and serum samples were assessed in samples collected from 44 M. bovis-infected cows, and test specificities were assessed in milk samples collected from 356 cows from tuberculosis (TB)-free herds. Milk vat samples were collected from 505 herds from regions with relatively high or low prevalences of infection. The ELISA had a sensitivity of 50% and a specificity of 97.5% for milk samples, and the test sensitivities for milk and serum samples were the same. Dilution of the positive test milk samples in milk from noninfected cows at 1/10, 1/20, and 1/50 dilutions reduced the proportions of positive responses to 13/21, 9/21, and 4/21, respectively. Small differences were observed in the ELISA responses of milk samples from individual TB-free cows collected at different times during lactation. No significant differences were detected in the ELISA responses of milk vat samples collected from infected and noninfected herds. This study shows that milk samples can be substituted for serum samples for screening individual cows for M. bovis infection, and pooling of milk samples from 10 to 20 animals can result in a reduction in the sensitivity by approximately 50%. However, screening of milk vat samples is unlikely to be useful in countries with low prevalences of M. bovis in cattle and large herd sizes.

  10. Evaluation of a new antibody-based enzyme-linked immunosorbent assay for the detection of bovine leukemia virus infection in dairy cattle.

    Science.gov (United States)

    Monti, Gustavo E; Frankena, Klaas; Engel, Bas; Buist, Willem; Tarabla, Héctor D; de Jong, Mart C M

    2005-09-01

    The objective of this study was to validate a new blocking enzyme-linked immunosorbent assay (ELISA) (designated M108 for milk and S108 for serum samples) for detecting bovine leukemia virus (BLV) infection in dairy cattle. Milk, serum, and ethylenediaminetetraacetic acid-blood samples were collected from 524 adult Holstein cows originating from 6 dairy herds in Central Argentina. The M108 and S108 were compared with agar gel immunodiffusion (AGID), polymerase chain reaction and a commercial ELISA. Because there is currently no reference test capable of serving as a gold standard, the test sensitivity (SE) and specificity (SP) were evaluated by the use of a latent class model. Statistical inference was performed by classical maximum likelihood and by Bayesian techniques. The maximum-likelihood analysis was performed assuming conditional independence of tests, whereas the Bayesian approach allowed for conditional dependence. No clear conclusion could be drawn about conditional dependence of tests. Results with maximum likelihood (under conditional independence) and posterior Bayes (under conditional dependence) were practically the same. Conservative estimates of SE and SP (with 95% confidence intervals) for M108 were 98.6 (96.7; 99.6) and 96.7 (92.9; 98.8) and for S108 99.5 (98.2; 99.9) and 95.4 (90.9; 98.1), respectively. The ELISA 108 using either milk or serum to detect BLV-infected animals had comparable SE and SP with the official AGID and a commercial ELISA test, which are currently the most widely accepted tests for the serological diagnosis of BLV infection. Therefore, ELISA 108 can be used as an alternative test in monitoring and control programs.

  11. Use of heavy water (D2O) in developing thermostable recombinant p26 protein based enzyme-linked immunosorbent assay for serodiagnosis of equine infectious anemia virus infection.

    Science.gov (United States)

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Singh, Raj K

    2014-01-01

    Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4 °C, 37 °C, 42 °C, and 45 °C over a storage time from 2 weeks to 10 months. A set of positive serum (n = 12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n = 30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37 °C < 42 °C < 45 °C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45 °C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.

  12. Enzyme-linked immunosorbent assay gliadin assessment in processed food products available for persons with celiac disease: a feasibility study for developing a gluten-free food database.

    Science.gov (United States)

    Agakidis, Charalampos; Karagiozoglou-Lampoudi, Thomais; Kalaitsidou, Marina; Papadopoulos, Theodoros; Savvidou, Afroditi; Daskalou, Efstratia; Dimitrios, Triantafyllou

    2011-12-01

    Inappropriate food labeling and unwillingness of food companies to officially register their own gluten-free products in the Greek National Food Intolerance Database (NFID) result in a limited range of processed food products available for persons with celiac disease (CDP). The objective of the study was to evaluate the feasibility of developing a gluten-free food product database based on the assessment of the gluten content in processed foods available for CDP. Gluten was assessed in 41 processed food products available for CDP. Group A consisted of 26 products for CDP included in the NFID, and group B contained 15 food products for CDP not registered in the NFID but listed in the safe lists of the local Celiac Association (CA). High-sensitivity ω-gliadin enzyme-linked immunosorbent assay (ELISA) was used for analysis. Gluten was lower than 20 ppm in 37 of 41 analyzed products (90.2%): in 24 of 26 (92.3%) products in group A and in 13 of 15 (86.7%) products in group B (P = .61). No significant difference was found between the 2 groups regarding gluten content. No product in either group contained gluten in excess of 100 ppm. Most of the analyzed products included in the Greek NFID or listed in the lists of the local CA, even those not officially labeled "gluten free," can be safely consumed by CDP. The use of commercially available ω-gliadin ELISA is able to identify those products that contain inappropriate levels of gluten, making feasible it to develop an integrated gluten-free processed food database.

  13. [Detection of Autoantibodies Against the 1-Adrenergic Receptor in the Sera of Patients via the Competitive cell-Based Enzyme Linked Immunosorbent Assay].

    Science.gov (United States)

    Shevelev, A Y; Kostiukevich, M V; Efremov, E E; Vlasik, T N; Mironova, N A; Zykov, K A; Kashirina, N M; Kuznetsova, I B; Sharf, T V; Mamochkina, E N; Lipatova, L N; Peklo, M M; Rutkevich, P N; Yanushevskaya, E V; Rybalkin, I N; Stukalova, O V; Malkina, T A; Belyaeva, M M; Kuznetsova, T V; Tkachev, G A; Zinchenko, L V; Gupalo, E M; Agapova, O Y; Yureneva-Tkhorzhevskaya, T V; Rvacheva, A V; Sidorova, M V; Sadgyan, A S; Tereshchenko, S N; Golitsyn, S P

    2016-12-01

    This study aimed to assess the level of anti-1-adrenergic receptor autoantibodies in patients with ventricular arrhythmias with no signs of organic heart disease and with presence of cardiovascular pathology in comparison with a group of healthy volunteers. The study included 44 patients with ventricular arrhythmias with no signs of organic heart disease ("idiopathic"), 34 patients with diagnosed dilated cardiomyopathy (DCM) of inflammatory origin, 35 patients with coronary heart disease and ventricular arrhythmias, 12patients with coronary heart disease with no ventricular arrhythmias, and 19 healthy volunteers (control group). The level of autoantibodies against the 1-adrenergic receptor was determined by the developed competitive cell-based enzyme-linked immunosorbent assay (ELISA) and by the standard ELISA using peptides corresponding to the second extracellular loop of the 1-adrenergic receptor. Elevated level of autoantibodies detected by a competitive cell-based ELISA was observed in 62% of patients with DCM compared to 21% of healthy volunteers (p=0.0006). In patients with "idiopathic" ventricular arrhythmias, the level of 1-adrenergic receptor autoantibodies was lower than in healthy subjects (p=0.003). Coronary heart disease patients with or without ventricular arrhythmias exhibited no differences from the control group. The number of significantly positive signals in peptide-based ELISA did not exceed 10% in any of the groups. No correlation between the data from competitive cell-based ELISA and peptide-based ELISA was found. This study demonstrated that competitive cell-based ELISA technique can be applied for detection of 1-adrenergic receptor autoantibodies. The results in DCM patients generally correspond to the expected. Decreased level of autoantibodies in patients with "idiopathic" ventricular arrhythmias indicates that this disease is related to changes in the immune system. Such relation is not observed in the case of coronary heart disease

  14. Comparison of enzyme-linked immunosorbent assay and gas chromatography procedures for the detection of cyanazine and metolachlor in surface water samples

    Science.gov (United States)

    Schraer, S.M.; Shaw, D.R.; Boyette, M.; Coupe, R.H.; Thurman, E.M.

    2000-01-01

    Enzyme-linked immunosorbent assay (ELISA) data from surface water reconnaissance were compared to data from samples analyzed by gas chromatography for the pesticide residues cyanazine (2-[[4-chloro-6-(ethylamino)-l,3,5-triazin-2-yl]amino]-2-methylpropanenitrile ) and metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide). When ELISA analyses were duplicated, cyanazine and metolachlor detection was found to have highly reproducible results; adjusted R2s were 0.97 and 0.94, respectively. When ELISA results for cyanazine were regressed against gas chromatography results, the models effectively predicted cyanazine concentrations from ELISA analyses (adjusted R2s ranging from 0.76 to 0.81). The intercepts and slopes for these models were not different from 0 and 1, respectively. This indicates that cyanazine analysis by ELISA is expected to give the same results as analysis by gas chromatography. However, regressing ELISA analyses for metolachlor against gas chromatography data provided more variable results (adjusted R2s ranged from 0.67 to 0.94). Regression models for metolachlor analyses had two of three intercepts that were not different from 0. Slopes for all metolachlor regression models were significantly different from 1. This indicates that as metolachlor concentrations increase, ELISA will over- or under-estimate metolachlor concentration, depending on the method of comparison. ELISA can be effectively used to detect cyanazine and metolachlor in surface water samples. However, when detections of metolachlor have significant consequences or implications it may be necessary to use other analytical methods.

  15. Milk progesterone enzyme-linked immunosorbent assay as a tool to investigate ovarian cyclicity of water buffaloes in relation to body condition score and milk production

    Directory of Open Access Journals (Sweden)

    Banu Turgish A

    2012-05-01

    Full Text Available Abstract Background Application of assisted reproductive technologies in buffaloes is limited to some extent by farmers’ inability to detect oestrus because of its poor expression. The present study aimed at investigating reliability of a milk progesterone enzyme-linked immunosorbent assay (ELISA to assess the ovarian cyclicity during post partum, oestrus and post-breeding periods in water buffaloes. Methods Progesterone concentrations were measured by an ELISA in milk of 23 postpartum buffaloes in relation to oestrus, pregnancy, body condition score (BCS and milk production. Two milk samples were taken at 10 days intervals, every month starting from day 30 and continued to day 150 post partum. BCS and milk production were recorded during sample collection. Milk samples from bred buffaloes were collected at Day 0 (day of breeding, Days 10–12 and Days 22–24. Defatted milk was preserved at −80°C until analysis. Pregnancy was confirmed by palpation per rectum on Days 70–90. Results Seventeen buffaloes had 47 ovulatory cycles, one to four in each, 13 were detected in oestrus once (28 % oestrus detection rate. Progesterone concentration ≥1 ng/ml in one of the two 10-day-interval milk samples reflected ovulation and corpus luteum formation. The intervals between calving to first luteal activity and to first detected oestrus varied from 41 to 123 days (n = 17 and 83 to 135 (n = 13 days, respectively. Eight buffaloes were bred in the course of the study and seven were found pregnant. These buffaloes had a progesterone profile of low (P P  Conclusions Milk progesterone ELISA is a reliable tool for monitoring ovarian cyclicity and good BCS may be an indicator of resuming cyclicity in water buffalo.

  16. Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

  17. An indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay for the determination of dimethyl phthalate (DMP) in milk and milk products.

    Science.gov (United States)

    Sun, Rui Y; Zhuang, Hui S

    2015-01-01

    After the "plasticizer event" in Taiwan, phthalic acid esters (PAEs) have been listed in "Inedible materials possibly added into food illegally" and "Commonly abused food additives." As one of the PAEs family, DMP has long been a problem of great concern due to its potential impacts on human health. In order to detect DMP with high sensitivity and specificity, a sensitive indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established in this study. A high-titer rabbit polyclonal antibody (pAb-DMP) targeting DMP was obtained, and the procedures of BA-ELISA were optimized for the determination of DMP in milk and milk products. Under optimal conditions, good linearity was achieved within a range of 0.024 to 6.027 μg L(-1), with low cross-reactivity values for DMP structural analogues (lower than 10%). The median inhibitory concentration (IC50) was 0.356 μg L(-1) and the limit of detection (LOD) was 0.0082 μg L(-1). Finally, the concentrations of DMP in milk and milk products ranged from 1.03 μg kg(-1) to 7.23 μg kg(-1) by BA-ELISA. Satisfactory recoveries (90.26-112.38%) and coefficient of variation (CV) values (5.08-8.46%) were obtained. These results were consistent with those using gas chromatography-mass spectrometry (GC-MS), which further confirmed that the proposed BA-ELISA was accurate, specific, reliable and rapid for routine monitoring trace DMP residues in foodstuff, especially milk and milk products.

  18. Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE.

    Science.gov (United States)

    Liang, F T; Steere, A C; Marques, A R; Johnson, B J; Miller, J N; Philipp, M T

    1999-12-01

    VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR(6). In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C(6)) with the IR(6) sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C(6) peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.

  19. Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum.

    Science.gov (United States)

    Muleme, Michael; Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M; Nguyen, Chelsea; Stevenson, Mark A; Wilks, Colin R; Firestone, Simon M

    2016-06-01

    Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants.

  20. A multilaboratory evaluation of a commercial enzyme-linked immunosorbent assay test for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in cattle.

    Science.gov (United States)

    Dargatz, David A; Byrum, Beverly A; Collins, Michael T; Goyal, Sagar M; Hietala, Sharon K; Jacobson, Richard H; Kopral, Christine A; Martin, Barbara M; McCluskey, Brian J; Tewari, Deepanker

    2004-11-01

    Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA

  1. Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus.

    Science.gov (United States)

    Seepiban, Channarong; Charoenvilaisiri, Saengsoon; Warin, Nuchnard; Bhunchoth, Anjana; Phironrit, Namthip; Phuangrat, Bencharong; Chatchawankanphanich, Orawan; Attathom, Supat; Gajanandana, Oraprapai

    2017-05-30

    Tomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars. The recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene. Two MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72-81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78-96% identity with each other and 72-91% identity with those that were not detected by MAb D2. TAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of

  2. Evaluation of Chicken IgY Generated Against Canine Parvovirus Viral-Like Particles and Development of Enzyme-Linked Immunosorbent Assay and Immunochromatographic Assay for Canine Parvovirus Detection.

    Science.gov (United States)

    He, Jinxin; Wang, Yuan; Sun, Shiqi; Zhang, Xiaoying

    2015-11-01

    Immunoglobulin Y (IgY) antibodies were generated against canine parvovirus virus-like particles (CPV-VLPs) antigen using chickens. Anti-CPV-VLPs-IgY was extracted from hen egg yolk and used for developing enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) for the detection of CPV in dog feces. The cutoff negative values for anti-CPV-VLPs-IgY were determined using negative fecal samples (already confirmed by polymerase chain reaction [PCR]). In both ELISA and ICA, there was no cross-reaction with other diarrheal pathogens. Thirty-four fecal samples were collected from dogs with diarrhea, of which 26.47% were confirmed as CPV-positive samples by PCR, while 29.41% and 32.35% of the samples were found to be positive by ELISA and ICA, respectively. The developed ELISA and ICA exhibited 97.06% and 94.12% conformity with PCR. Higher sensitivity and specificity were observed for IgY-based ELISA and ICA. Thus, they could be suitable for routine use in the diagnosis of CPV in dogs.

  3. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay

    Science.gov (United States)

    Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L.; Day, Nicholas P. J.

    2016-01-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus. PMID:27008880

  4. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay.

    Science.gov (United States)

    Blacksell, Stuart D; Lim, Cherry; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L; Paris, Daniel H; Limmathurotsakul, Direk; Day, Nicholas P J

    2016-06-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.

  5. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Science.gov (United States)

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  6. Evaluation of enzyme-linked immunosorbent assays and a haemagglutination inhibition tests for the detection of antibodies to Newcastle disease virus in village chickens using a Bayesian approach.

    Science.gov (United States)

    Chaka, H; Thompson, P N; Goutard, F; Grosbois, V

    2015-04-01

    Newcastle disease (ND) is an endemic disease in village chickens in Ethiopia with substantial economic importance. The sensitivity (Se) and specificity (Sp) of a blocking enzyme-linked immunosorbent assay (bELISA, Svanova Biotech), indirect ELISA (iELISA, Laboratoire Service International) and haemagglutination inhibition (HI) test for ND virus (NDV) antibody detection were evaluated in a Bayesian framework in the absence of a gold standard test, on sera collected from unvaccinated chickens kept under the village production system in household flocks and at markets in two woredas (i.e. districts) of the Eastern Shewa zone, Ethiopia. The outcomes of the iELISA test differed dramatically from those of the two other tests with 92% of the samples testing positive as compared with less than 15% for bELISA and HI. iELISA results were also inconsistent with previous estimations of Newcastle serological prevalence. The information provided by the iELISA test was thus considered as highly unreliable, probably due to an extremely low specificity, and thus not considered in the Bayesian models aiming at estimating serological prevalence and test performance parameters. Bayesian modelling of HI and bELISA test results suggested that bELISA had both the highest Se (86.6%; 95% posterior credible interval (PCI): 61.8%; 98.5%), and the highest Sp (98.3%; 95% PCI: 97.2%; 99.5%), while HI had a Se of 80.2% (95% PCI: 59.1%; 94.3%), and a Sp of 96.1% (95% PCI: 95.1%; 97.4%). Model selection and the range of the posterior distribution of the correlation between bELISA and HI test outcomes for truly seropositive animals (median at 0.461; PCI: -0.055; 0.894) suggested a tendency for bELISA and HI to detect the same truly positive animals and to fail to detect the same truly positive animals. The use of bELISA in screening and surveillance for NDV antibodies is indicated given its high Se and Sp, in addition to its ease of automation to handle large numbers of samples compared to HI. The

  7. Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

    Institute of Scientific and Technical Information of China (English)

    XIN Jiu-qing; GAO Yun-long; LI Yuan; WANG Yan-fan; QIAN Ai-dong

    2007-01-01

    Mycoplasma mycoides subsp mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA (Trp) codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI Ⅹ strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL-1, and coated to microtiter enzyme-linked immunosorbent assay (ELISA) plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 (0.934/0.193), the sensitivity to be 95.8% (46/48), and the specificity to be 98.9% (161/163). 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

  8. Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens.

    Science.gov (United States)

    Ferra, Bartłomiej; Holec-Gąsior, Lucyna; Kur, Józef

    2015-10-01

    Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric antigens in place of the TLA, which was confirmed by studies in the serodiagnosis of toxoplasmosis in humans. In this paper, we assess, for the first time, the diagnostic utility of five T. gondii recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and GRA1-GRA2-GRA6) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs) with sera from three different groups of livestock animals (horses, pigs, and sheep). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISAs based on a mixture of three antigens (M1: rSAG1+rMIC1+rMAG1, M2: rSAG2+rGRA1+rROP1, and M3: rGRA1+rGRA2+rGRA6) and referenced to TLA. All chimeric antigens were characterized by high specificity (100%), and the sensitivity of the IgG ELISAs based on chimeric antigens was variable (between 28.4% and 100%) and mainly dependent on the animal species. The chimeric antigens were generally more reactive than mixtures of three antigens. The most effective for the diagnosis of toxoplasmosis was SAG2-GRA1-ROP1L, which can detect specific anti-T. gondii antibodies in 100%, 93.8%, and 100% of positive serum samples from horses, pigs, and sheep, respectively. The present study shows that recombinant chimeric antigens can be successfully used to diagnose T. gondii infection in farm animals, and can replace the commonly

  9. Use of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian Altiplano.

    Science.gov (United States)

    Hillyer, G V; Soler de Galanes, M; Rodriguez-Perez, J; Bjorland, J; Silva de Lagrava, M; Ramirez Guzman, S; Bryan, R T

    1992-05-01

    A collaborative study between the University of Puerto Rico School of Medicine, the Centers for Disease Control, the Bolivian Ministry of Health, and private voluntary organizations (Foster Parents Plan International and Danchurchaid) working in Bolivia has identified a region in the northwestern Altiplano of Bolivia near Lake Titicaca as harboring the highest prevalence of human fascioliasis in the world reported to date. Two serologic techniques (the Falcon assay screening test-enzyme-linked immunosorbent assay [FAST-ELISA] and the enzyme-linked immunoelectrotransfer blot [EITB]) were used in the determination of its prevalence. One hundred serum samples and 73 stool samples were obtained from Aymara Indians from Corapata, Bolivia. Antibody absorbance levels to Fasciola hepatica excretion-secretion antigens were compared with EITB banding patterns using the same antigen preparation. A positive FAST-ELISA result was defined as an absorbance value greater than the mean plus three standard deviations of two sets of normal negative controls (Puerto Rican and Bolivian). Using this criterion, 53 of 100 sera tested were found positive by this technique. Within this group, 19 (95%) of 20 individuals who were parasite positive were also positive by FAST-ELISA. An additional 24 individuals who were negative for F. hepatica eggs and 10 individuals for whom no specimens were received were also positive by FAST-ELISA. Among the 53 individuals negative for F. hepatica eggs, 29 were also negative by FAST-ELISA. The EITB analysis of the sera from confirmed infected individuals revealed at least three F. hepatica (Fh) bands with molecular weights of 12, 17, and 63 kD, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Anju Mohan

    2016-07-01

    Full Text Available Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT, microtiter plate agglutination test (MAT, indirect hemagglutination assay (IHA, and indirect enzyme-linked immunosorbent assay (iELISA as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001. The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005. The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002. However, the difference in mean iELISA titers of infected cattle (1.3678±0.014 and healthy vaccinated cattle (1.367±0.014 was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.

  11. Enzyme-linked immunosorbent assay and colloidal gold immunoassay for sulphamethazine residues in edible animal foods: investigation of the effects of the analytical conditions and the sample matrix on assay performance.

    Science.gov (United States)

    Wang, Lei; Wang, Shuo; Zhang, Jiayi; Liu, Junwei; Zhang, Yan

    2008-03-01

    To determine sulphamethazine (SMZ) residues in edible animal foods (pig muscle, chicken muscle, egg, fish, milk and liver), a competitive direct enzyme-linked immunosorbent assay (ELISA) and a colloidal gold immunoassay were established. The limits of detection of the ELISA and the colloidal gold immunoassay were 0.02 and 0.5 microg kg(-1), respectively. The specificity of the ELISA developed to the SMZ was high according to the results of cross-reactivity testing with 14 kinds of sulphonamides. To obtain a more sensitive immunoassay, buffer solution (30 mmol L(-1) phosphate-buffered saline with 0.05% Tween 20, pH 8.5) was optimized through the whole test procedure. A simple and efficient extraction method for the rapid detection of SMZ residues in foods was developed, with recoveries between 74 and 117.5%. Matrix effects can be avoided by 1:10 dilution of pig muscle, chicken muscle, egg, fish, milk and liver with optimal buffer. The detection limit of SMZ was 5 microg kg(-1) in liver and 2 microg kg(-1) in the other five samples. For the validation of the ELISA tests, sample extracts were analysed by ELISA and high-performance liquid chromatography. The results obtained by these two methods showed a good correlation (r(2)) which was greater than 0.9. The colloidal gold immunoassay presented in this assay was successfully applied to determine SMZ in pig muscle, milk and fish below or equal to the maximum residue level (20 microg kg(-1)).

  12. Rapid identification and susceptibility testing of uropathogenic microbes via immunosorbent ATP-bioluminescence assay on a microfluidic simulator for antibiotic therapy.

    Science.gov (United States)

    Dong, Tao; Zhao, Xinyan

    2015-02-17

    The incorporation of pathogen identification with antimicrobial susceptibility testing (AST) was implemented on a concept microfluidic simulator, which is well suited for personalizing antibiotic treatment of urinary tract infections (UTIs). The microfluidic device employs a fiberglass membrane sandwiched between two polypropylene components, with capture antibodies immobilized on the membrane. The chambers in the microfluidic device share the same geometric distribution as the wells in a standard 384-well microplate, resulting in compatibility with common microplate readers. Thirteen types of common uropathogenic microbes were selected as the analytes in this study. The microbes can be specifically captured by various capture antibodies and then quantified via an ATP bioluminescence assay (ATP-BLA) either directly or after a variety of follow-up tests, including urine culture, antibiotic treatment, and personalized antibiotic therapy simulation. Owing to the design of the microfluidic device, as well as the antibody specificity and the ATP-BLA sensitivity, the simulator was proven to be able to identify UTI pathogen species in artificial urine samples within 20 min and to reliably and simultaneously verify the antiseptic effects of eight antibiotic drugs within 3-6 h. The measurement range of the device spreads from 1 × 10(3) to 1 × 10(5) cells/mL in urine samples. We envision that the medical simulator might be broadly employed in UTI treatment and could serve as a model for the diagnosis and treatment of other diseases.

  13. A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; He, Y.; Veidal, S. S.

    2011-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) an......-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.......) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung-and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically...

  14. Clinical Research of Enzyme Linked Immunosorbent Assay for The Detection of Hepatitis B Virus Surface Antigen False Positive%酶联免疫法检测HBsAg假阳性临床研究

    Institute of Scientific and Technical Information of China (English)

    张海军

    2013-01-01

    目的:研究分析酶联免疫法(ELISA)检测乙肝病毒表面抗原(HBsAg)的假阳性情况.方法:对酶联免疫法检测HBsAg结果为阳性的1500例血清标本用金标法验证,并用化学发光仪微粒子捕捉免疫发光法(MEIA)定量检测HBsAg的含量,以确认酶联免疫法检测结果的假阳性.结果:1500例酶联免疫法检测HBsAg阳性的血清标本,其中1479例为真阳性,真阳性率为98.6%(1479/1500);21例为假阳性,假阳性率为1.4%(21/1500).结论:酶联免疫法检测HBsAg有一定的假阳性,临床检测时应高度注意,避免错报误诊.%Objective: To study enzyme linked immunosorbent assay ( ELISA ) in detecting hepatitis B virus surface antigen ( HBsAg ) of the false positive cases. Method: ELISA for detection of HBsAg results for 1500 cases with positive serum samples using the colloidal gold method validation, and using chemical luminous instrument microparticle enzyme lmmunoassay ( MEIA ) for quantitative detection of HBsAg content, to confirm the enzyme-linked immunosorbent assay for detection of false positive results. Result: 1500 cases of enzyme-linked immunosorbent assay for detection of HBsAg positive serum samples, including 1479 cases of true positive, true positive rate was 98. 6% ( 1479/1500 ); 21 were false positive, false positive rate was 1. 4% ( 21/1500 ). Conclusion: Enzyme-linked immunosorbent assay for detection of HBsAg have false positive, clinical test should be highly attention, avoid the error diagnosis.

  15. The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens, followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period

    DEFF Research Database (Denmark)

    Skov, M.N.; Feld, Niels Christian; Carstensen, B.

    2002-01-01

    uninfected as controls. The groups were monitored bacteriologically by examination of cloacal swabs and organs and serologically by examination of serum and egg yolk by a lipopolysaccharide enzyme-linked immunosorbent assay throughout the period. Within the first week, 100% of birds in both infected groups...... were excreting salmonella bacteria in the feces. However, the number of fecal excretors declined rapidly with time, down to 6% in 16 wk for S. typhimurium and down to a similar level within the first 3 wk for S. enteritidis. For the latter, relapses with up to 40% positive birds were observed...

  16. Development of an enzyme-linked immunosorbent assay for detection of chicken osteocalcin and its use in evaluation of perch effects on bone remodeling in caged White Leghorns

    Science.gov (United States)

    Osteocalcin (OC) is a sensitive biochemical marker for evaluating bone turnover in mammals. The role of avian OC is less clear because of a need for a chicken assay. Our objectives were to develop an assay using indirect competitive ELISA for detecting chicken serum OC and use the assay to examine t...

  17. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Moller, T

    1999-01-01

    The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L....... donovani GBP (GBPP) in the diagnosis of L. donovani infections in Sudan. The sensitivity of the rGBP ELISA in diagnosing visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) was 92% and 93%, respectively. In contrast, the sensitivity of the GBPP ELISA was 55% for VL and 63% for PKDL...... for malaria but free of leishmaniasis was negative in both assays....

  18. Dot enzyme-linked immunosorbent assay (dot-ELISA for schistosomiasis diagnosis using dacron as solid-phase Dot-ELISA (dot enzyme-linked immunosorbent assay, utilizando o dacron como suporte sólido para o diagnóstico da esquistossomose

    Directory of Open Access Journals (Sweden)

    Silvia Maria Lucena Montenegro

    1999-04-01

    Full Text Available Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA for schistosomiasis and compared to indirect immunofluorescence (IMF. Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05. The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05. In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.O dacron e a nitrocelulose foram utilizados como matrizes para realização do dot-ELISA na esquistossomose e comparadas com a imunofluorescência indireta (IMF. A titulação dos soros de 18 pacientes esquistossomóticos foi feita, utilizando o antígeno solúvel de verme adulto (SWAP e soro de pessoas normais não endêmicas foram usadas como controle. A IMF foi menos sensível do que os dot-ELISAs, apesar da diferença não ter sido estatisticamente significativa (p > 0,05. O dot-ELISA, utilizando a nitrocelulose foi tão sensível do que aquele utilizando o dacron como suporte. Não houve diferenças significativas entre os suportes em relação à estabilidade do antígeno. Entretanto, a especificidade, utilizando o dacron como suporte foi menor do que a nitrocelulose, apesar da diferença não ter sido estatisticamente significativa (p > 0,05. Em resumo, este trabalho mostrou que os resultados dos suportes utilizados em dot-ELISA para o diagnóstico da esquistossomose mansônica foram

  19. Teste imunoenzimático (enzyme-linked immunosorbent assay para diagnóstico da cisitcercose bovina e estudo da cinética de produção de anticorpos contra-Cysticercus bovis Enzyme-linked immunosorbent assay (ELISA for imunodiagnostic of bovine cysticercosis and kinetics of antibodies production against-Cysticercus bovis

    Directory of Open Access Journals (Sweden)

    João Carlos Minozzo

    2004-06-01

    Full Text Available Um teste de ELISA indireto (ENZYME-LINKED IMMUNOSORBENT ASSAY foi desenvolvido para pesquisa de anticorpos contra-C. bovis em bovinos experimental e naturalmente infectados. Foram estudados três antígenos: antígeno parcial de C. cellulosae, antígeno total de C. bovis e antígeno total de C. longicollis. Na padronização do ELISA foram analisadas as seguintes combinações: antígeno 250 e 500ng de proteína/cavidade, diluição dos soros 50, 100, 200 e 400 vezes, diluição do conjugado (IgG de cabra anti-IgG bovina conjugada com peroxidase 400 e 800 vezes. Do cruzamento das condições acima resultou a seguinte padronização: antígeno 250ng/cavidade, soro e conjugado diluídos 100 e 400 vezes, respectivamente. O nível de corte (cut-off da reação entre animais reagentes e não reagentes foi determinado pela média das densidades óticas de 54 soros negativos acrescidas de três desvio-padrão, resultando no valor de 0,303. Através da prova ELISA foram comparadas as reatividades dos antígenos parcial de C. cellulosae, total de C. bovis e total de C. longicollis com soros de bovinos portadores de cisticercose, empregando as diluições de soros e de conjugados padronizados anteriormente. O antígeno de C. bovis mostrou alta correlação com o teste padronizado com C. cellulosae. Entretanto, os valores de absorbância foram sensivelmente menores. Com C. longicollis observou-se reatividade bastante baixa, porém aumentando-se a quantidade de antígeno, até 3000ng/cavidade, houve um aumento proporcional da resposta. Após a padronização do teste foi analisado o comportamento imunológico de bezerros infectados experimentalmente com ovos de Taenia saginata. Dez bezerros foram infetados oralmente com 2 x 104 ovos de T. saginata. Seis bezerros não infetados foram usados como controle. Treze amostras de soro de cada animal foram analisadas. A primeira foi colhida no dia da infecção e o restante, quinzenalmente até o abate. A produ

  20. Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax

    Directory of Open Access Journals (Sweden)

    N Ghosh

    2015-01-01

    Full Text Available Background & objectives: Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA in human cutaneous anthrax cases. Methods: Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. Results: The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] =0.9982; slope=0.9186; intercept = 0.1108. Interpretation & conclusions: The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

  1. Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax.

    Science.gov (United States)

    Ghosh, N; Gunti, D; Lukka, H; Reddy, B R; Padmaja, Jyothi; Goel, A K

    2015-08-01

    Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA) in human cutaneous anthrax cases. Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] = 0.9982; slope = 0.9186; intercept = 0.1108). The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

  2. Serine transhydroxymethylase: a simplified radioactive assay; purification and stabilization of enzyme activity employing Affi-Gel Blue.

    Science.gov (United States)

    Braman, J C; Black, M J; Mangum, J H

    1981-01-01

    An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the [14C]HCHO which is generated when [3- 14C]-serine is employed as the substrate. The new assay eliminates the need for a solvent extraction of a [14C]HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach. The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4 degrees indefinitely.

  3. Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

    Science.gov (United States)

    Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

    2016-02-01

    To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

  4. Detection and identification of platelet-associated alloantibodies by a solid-phase modified antigen capture enzyme-linked immunosorbent assay method and its correlation to platelet refractoriness in multiplatelet concentrate-transfused patients.

    Science.gov (United States)

    Jain, Neelesh; Sarkar, Shankar; Philip, Joseph

    2014-01-01

    Platelets express a variety of polymorphic glycoproteins (GPs), such as GPIIb/IIIa, GPib/IX, GPla/Ila, GPIV, and class I human leukocyte antigen. In the platelet transfusion setting, alloimmunization involves the production of antibodies against these glycoproteins. Patients transfused with multiple units of platelet concentrates for longer periods are the main individuals with platelet alloimmunization. This study was performed to detect the development of platelet antibodies in patients who are transfused with multiple units of leukodepleted platelet concentrates, such as those with hemato-oncologic diseases and bone marrow failure syndromes. The method used was solid phase modified antigen capture enzyme-linked immunosorbent assay. Platelet refractoriness was assessed by measuring the corrected count increment at 1 and 24 hours after transfusion.

  5. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.; Marks, James D.; Varnum, Susan M.

    2012-11-15

    Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.

  6. Development of an enzyme-linked immunosorbent assay for detection of chicken osteocalcin and its use in evaluation of perch effects on bone remodeling in caged White Leghorns.

    Science.gov (United States)

    Jiang, S; Cheng, H W; Hester, P Y; Hou, J-F

    2013-08-01

    Osteocalcin (OC) is a sensitive biochemical marker for evaluating bone turnover in mammals. The role of avian OC is less clear because of the need for a chicken assay. Our objectives were to develop an assay using indirect competitive ELISA for detecting chicken serum OC and use the assay to examine the effects of perches on bone remodeling in caged hens. Anti-chicken OC polyclonal antibody was produced by immunization of rabbits with a recombinant OC from Escherichia coli. Chicken OC extracted from bone was used as a coated protein, and purified chicken OC was used for calibration. The limit of detection of the developed OC ELISA was 0.13 ng/mL. The intra- and interassay CV were chickens that never had access to perches during their life cycle. Treatment 2 chickens had perches during the pullet phase (0 to 16.9 wk of age), whereas treatment 3 chickens had perches only during the egg-laying phase of the life cycle (17 to 71 wk of age). Treatment 4 chickens always had access to perches (0 to 71 wk of age). Correlation between the 2 assays was 0.62 (P chicken ELISA were higher than that detected using the Rat-Mid ELISA (P chicken ELISA assay showed that hens with perch access had higher concentrations of serum OC than hens without perches during egg laying (P = 0.04). Pullet access to perches did not affect serum OC levels in 71-wk-old hens (P = 0.15). In conclusion, a chicken OC ELISA has been validated that is sensitive and accurate with adequate discriminatory power for measuring bone remodeling in chickens.

  7. Sensitivity and specificity enhanced enzyme-linked immunosorbent assay by rational hapten modification and heterogeneous antibody/coating antigen combinations for the detection of melamine in milk, milk powder and feed samples.

    Science.gov (United States)

    Cao, Biyun; Yang, Hong; Song, Juan; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-11-15

    The adulteration of food products with melamine has led to an urgent requirement for sensitive, specific, rapid and reliable quantitative/screening methods. To enhance the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the detection of melamine in milk, milk powder and feed samples, rational hapten modification and heterogeneous antibody/coating antigen combinations were adopted. Three melamine derivatives with different length of carboxylic spacer at the end were synthesized and linked to carrier proteins for the production of immunogens and coating antigens. Monoclonal antibody against melamine was produced by hybridoma technology. Under optimal experimental conditions, the standard curves of the ELISAs for melamine were constructed in range of 0.1-100 ng mL(-1). The sensitivity was 10-300 times enhanced compared to those in the published literatures. The cross-reactivity values of the ELISAs also demonstrated the assays exhibited high specificity. Five samples were spiked with melamine at different concentrations and detected by the ELISA. The recovery rates of 72.8-123.0% and intra-assay coefficients of variation of 0.8-18.9% (n=3) were obtained. The ELISA for milk sample was confirmed by high-performance liquid chromatography with a high correlation coefficient of 0.9902 (n=6). The proposed ELISA was proven to be a feasible quantitative/screening method for melamine analysis.

  8. Comparison of Microcystis aeruginosa (PCC7820 and PCC7806) growth and intracellular microcystins content determined by liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay anti-Adda and phosphatase bioassay.

    Science.gov (United States)

    Ríos, V; Moreno, I; Prieto, A I; Soria-Díaz, M E; Frías, J E; Cameán, A M

    2014-03-01

    Cyanobacteria are able to produce several metabolites that have toxic effects on humans and animals. Among these cyanotoxins, the hepatotoxic microcystins (MC) occur frequently. The intracellular MC content produced by two strains of Microcystis aeruginosa, PCC7806 and PCC7820, and its production kinetics during the culture time were studied in order to elucidate the conditions that favour the growth and proliferation of these toxic strains. Intracellular MC concentrations measured by liquid chromatography (LC) coupled to electrospray ionization mass spectrometer (MS) were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) anti-Adda and protein phosphatase 2A (PP2A) inhibition assays. It has been demonstrated there are discrepancies in the quantification of MC content when comparing ELISA and LC-MS results. However, a good correlation has been obtained between PP2A inhibition assay and LC-MS. Three MC were identified using LC-MS in the PCC7806 strain: MC-LR, demethylated MC-LR and a new variant detected for the first time in this strain, [L-MeSer(7)] MC-LR. In PCC7820, MC-LR, D-Asp(3)-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW and MC-LF were identificated. The major one was MC-LR in both strains, representing 81 and 79% of total MC, respectively. The total MC content in M. aeruginosa PCC7820 was almost three-fold higher than in PCC7806 extracts.

  9. Measuring Immunoglobulin G Antibodies to Tetanus Toxin, Diphtheria Toxin, and Pertussis Toxin with Single-Antigen Enzyme-Linked Immunosorbent Assays and a Bead-Based Multiplex Assay▿

    OpenAIRE

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz

    2008-01-01

    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assa...

  10. Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP.

    Science.gov (United States)

    Passos, Angela Noronha; Kohara, Valdelene Sayuri; de Freitas, Roseli Santos; Vicentini, Adriana Pardini

    2014-01-01

    Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information.

  11. Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP

    Science.gov (United States)

    Passos, Angela Noronha; Kohara, Valdelene Sayuri; de Freitas, Roseli Santos; Vicentini, Adriana Pardini

    2014-01-01

    Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information. PMID:25763041

  12. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    Directory of Open Access Journals (Sweden)

    Yanil R Parma

    2012-06-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC, a subset of Shiga toxin producing E. coli (STEC is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic uremic syndrome (HUS. Regardless of serotype, Shiga toxins (Stx1 and/or Stx2 are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx was developed using anti-Stx2 B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933 and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 400 ng /ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for 2 strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli.

  13. Performance of a Redesigned HIV Selectest Enzyme-Linked Immunosorbent Assay Optimized To Minimize Vaccine-Induced Seropositivity in HIV Vaccine Trial Participants

    Science.gov (United States)

    Penezina, Oksana; Krueger, Neil X.; Rodriguez-Chavez, Isaac R.; Busch, Michael P.; Hural, John; Kim, Jerome H.; O'Connell, Robert J.; Hunter, Eric; Aboud, Said; Higgins, Keith; Kovalenko, Victor; Clapham, David; Crane, David

    2014-01-01

    Vaccine-induced seropositivity (VISP) or seroreactivity (VISR), defined as the reaction of antibodies elicited by HIV vaccines with antigens used in HIV diagnostic immunoassays, can result in reactive assay results for vaccinated but uninfected individuals, with subsequent misclassification of their infection status. The eventual licensure of a vaccine will magnify this issue and calls for the development of mitigating solutions in advance. An immunoassay that discriminates between antibodies elicited by vaccine antigens and those elicited by infection has been developed to address this laboratory testing need. The HIV Selectest is based on consensus and clade-specific HIV peptides that are omitted in many HIV vaccine constructs. The assay was redesigned to enhance performance across worldwide clades and to simplify routine use via a standard kit format. The redesigned assay was evaluated with sera from vaccine trial participants, HIV-infected and uninfected individuals, and healthy controls. The HIV Selectest exhibited specificities of 99.5% with sera from uninfected recipients of 6 different HIV vaccines and 100% with sera from normal donors, while detecting HIV-1 infections, including intercurrent infections, with 95 to 100% sensitivity depending on the clade, with the highest sensitivities for clades A and C. HIV Selectest sensitivity decreased in very early seroconversion specimens, which possibly explains the slightly lower sensitivity observed for asymptomatic blood donors than for clinical HIV cases. Thus, the HIV Selectest provides a new laboratory tool for use in vaccine settings to distinguish the immune response to HIV vaccine antigens from that due to true infection. PMID:24403525

  14. Performance and diagnostic usefulness of commercially available enzyme linked immunosorbent assay and rapid kits for detection of HIV, HBV and HCV in India

    Directory of Open Access Journals (Sweden)

    Maity Susmita

    2012-11-01

    Full Text Available Abstract Background HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. Results A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.; ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd. gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd., Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd. did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100% except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.. The kit efficiency didn’t vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p  Conclusions ELISA is a good screening assay for markers of HIV, HBV and HCV infections. Rapid tests are useful for

  15. Glycidyl methacrylate-co-N-vinyl-2-pyrrolidone coated polypropylene strips: Synthesis, characterization and standardization for dot-enzyme linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi, Charu; Tomar, Lomas [Centre for Biomedical Engineering, Indian Institute of Technology, Delhi 110016 (India); Singh, Harpal [Centre for Biomedical Engineering, Indian Institute of Technology, Delhi 110016 (India)], E-mail: tyagicharu11@rediffmail.com

    2009-01-26

    Glycidyl methacrylate and N-vinyl-2-pyrrolidone (GMA-co-NVP) copolymers with various GMA:NVP ratios were synthesized by solution polymerization technique in toluene using 2,2'-azobisisobutyronitrile (AIBN) as free radical initiator and dip coated onto polypropylene strips. The copolymer composition in polymeric coatings was confirmed by proton NMR spectroscopy. Various techniques like FTIR, SEM and contact angle were used for surface characterization of the polymer coatings. These polymer coated strips were evaluated and standardized for their application in dot-ELISA in two steps. In first step, specificity, sensitivity and reproducibility of the assay on developed polymer coated strips was evaluated through a model system using rabbit anti-goat IgG, goat anti-rabbit IgG and goat anti-rabbit IgG HRP (horseradish peroxidase)-conjugate. Polymer coating with GMA-NVP mol% ratio of 78:22 was able to detect rabbit anti-goat IgG antibody at a concentration as low as 2 ng mL{sup -1} with 1% BSA as blocking agent using antispecies IgG peroxidase conjugate diluted 1500 times. In the second step, the sensitivity and specificity of the developed system was established with human blood and finally used to identify the source of mosquito blood meal which is an important parameter in epidemiological studies, particularly in determining the role of mosquito in malaria transmission. The time duration of standardized assay with developed polymer coated strips was cut down to one hour compared to the 3-4 h required in usual dot-ELISA.

  16. Development and validation of an enzyme-linked immunosorbent assay for the quantification of cytochrome 3A4 in human liver microsomes.

    Science.gov (United States)

    De Bock, Lies; Colin, Pieter; Boussery, Koen; Van Bocxlaer, Jan

    2012-09-15

    Little is known about the influence of hepatic pathologies on cytochrome P450 (CYP) mediated drug metabolism in children. The determination of the abundance of the different isoforms in pediatric microsomes may provide valuable information on the mechanisms of possible changes in activity. Until now, western blotting was mostly used for abundance measurements, but this technique only provides semi-quantitative data. Therefore, this study aimed to develop and validate an indirect ELISA for the quantification of the most important CYP isoform, CYP3A4, in human liver microsomes, using commercially available reagents. Samples, calibrators and validation samples were diluted to a final concentration of 10 μg microsomal protein/ml. A polyclonal antibody raised against the full length human protein was used as primary antibody; horseradish peroxidase conjugated secondary antibodies for detection. The assay was validated for sensitivity, working range and calibration, accuracy and precision. Amounts of CYP3A4 between 2 and 300 pmol/mg microsomal protein could be quantified with a 5-parameter logistics function with 1/x weighting factor. Coefficients of variation of intra and inter assay variability were between 9.54 and 13.98% (16.34% at LLOQ), and between 10.51 and 14.55% (19.44% at LLOQ), respectively. The relative error (%RE) varied between -5.96 and 6.68% (11.53% at LLOQ), and the total error between 11.93 and 21.23% (30.97% at LLOQ). The cross-reactivity of the method with human CYP2E1 showed to have no significant effect on the accuracy of the results. Successful analysis of five samples from an ongoing study demonstrated the usefulness of the method.

  17. Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis.

    Science.gov (United States)

    Preston, Andrew; Fodey, Terence; Elliott, Christopher

    2008-02-11

    The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered. These foods include bread and other bakery products, crisps, chips, breakfast cereals, and coffee. To date, the diminutive size of acrylamide (71.08 Da) has prevented the development of screening immunoassays for this chemical. In this study, a polyclonal antibody capable of binding the carcinogen was produced by the synthesis of an immunogen comprising acrylamide derivatised with 3-mercaptobenzoic acid (3-MBA), and its conjugation to the carrier protein bovine thyroglobulin. Antiserum from the immunised rabbit was harvested and fully characterised. It displayed no binding affinity for acrylamide or 3-MBA but had a high affinity for 3-MBA-derivitised acrylamide. The antisera produced was utilised in the development of an ELISA based detection system for acrylamide. Spiked water samples were assayed for acrylamide content using a previously published extraction method validated for coffee, crispbread, potato, milk chocolate and potato crisp matrices. Extracted acrylamide was then subjected to a rapid 1-h derivatisation with 3-MBA, pre-analysis. The ELISA was shown to have a high specificity for acrylamide, with a limit of detection in water samples of 65.7 microgkg(-1), i.e. potentially suitable for acrylamide detection in a wide range of food commodities. Future development of this assay will increase sensitivity further. This is the first report of an immunoassay capable of detecting the carcinogen, as its small size has necessitated current analytical detection via expensive, slower, physico-chemical techniques such as Gas or Liquid Chromatography coupled to Mass Spectrometry.

  18. Evaluation of two Taenia solium cysticercal antigenic preparations (vesicular fluid and a glycoprotein fraction with affinity for lentil lectin for the immunodiagnosis of neurocysticercosis by enzyme-linked immunosorbent assay (ELISA

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

    2011-06-01

    Full Text Available OBJECTIVE: To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. METHOD: Fifty-six cerebrospinal fluid (CSF samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA. RESULTS: The VF ELISA showed 100% sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9% and 97.1%, with the CSF samples and 95.5% and 100% with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. CONCLUSION: Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.

  19. Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Malgor, R; Nonaka, N; Basmadjian, I; Sakai, H; Carámbula, B; Oku, Y; Carmona, C; Kamiya, M

    1997-12-01

    A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotinylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67,700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, showing a sensitivity of 100% and a specificity of 96%.

  20. [Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Salmonella Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in persons from outbreak of typhoid fever].

    Science.gov (United States)

    Rastawicki, Waldemar; Kałużewski, Stanisław

    2015-01-01

    The laboratory diagnosis of typhoid fever is dependent upon either isolation of S. Typhi from a clinical sample or the detection of raised titers of serum antibodies in the Widal test or the passive hemagglutination assay (PHA). In this study we evaluated the usefulness of ELISA for detection of antibodies to S. Typhi lipopolysaccharide O and capsular polysaccharide Vi antigens in the sera of persons from outbreak of typhoid fever. Fifteen serum samples from patients with laboratory confirmed typhoid fever and 140 sera from persons suspected for contact with typhoid fever patients from outbreak in 1974/75 in Poland were tested by ELISA. Additionally, as the control group, we tested 115 sera from blood donors for the presence of S. Typhi anti-LPS and anti-Vi antibodies. Anti-LPS and anti-Vi antibodies were detected in 80% and 53.3% of sera obtained from patients with laboratory confirmed typhoid fever, respectively. The high percentages of positive results in ELISA were also noted in the group of persons suspected for contact with typhoid fever patients (51.4% and 45%) but not in the group of blood donors (7.8% and 6.1%, respectively). The ELISA could be a useful tool for the serological diagnosis of typhoid fever in patients who have clinical symptoms but are culture negative, especially during massive outbreaks of typhoid fever.

  1. Detection of Campylobacter jejuni and Campylobacter coli in foods by enrichment culture and polymerase chain reaction enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Bolton, F J; Sails, A D; Fox, A J; Wareing, D R A; Greenway, D L A

    2002-05-01

    A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.

  2. Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population.

    Science.gov (United States)

    Kumar, Sanjay; Kumar, Rajender; Gupta, Ashok K; Yadav, Suresh C; Goyal, Sachin K; Khurana, Sandip K; Singh, Raj K

    2013-11-15

    Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.

  3. Evaluation of three commercial enzyme-linked immunosorbent assays for the detection of antibodies against Salmonella spp. in meat juice from finishing pigs in Spain.

    Science.gov (United States)

    Vico, J P; Engel, B; Buist, W G; Mainar-Jaime, R C

    2010-11-01

    The control of animal salmonellosis is considered as a major objective in Europe and indirect ELISAs will be important tools for the implementation of control programs for this infection in pigs. We analyse the results yielded by three commercial ELISAs (Herdcheck Swine Salmonella, SALMOTYPE Pig Screen, and PrioCHECK Salmonella) on meat juice samples from a population of slaughter pigs of Aragon, NW Spain, to assess their efficacy using traditional and latent-class approaches. Overall, the Herdcheck Swine Salmonella detected more Salmonella-infected pigs than the other two tests, but its relative sensitivity was low (65.9%). A similar result was observed when only serotypes detectable by this test were considered (69.1%). When a Bayesian approach was used the Herdcheck Swine Salmonella showed also the highest overall accuracy (sensitivity = 88% and specificity = 74%). Our results suggest that a relatively small proportion of the observed prevalence in herds would be explained by using these ELISAs. Also, this study points out that when different ELISA tests are used within the same herd, results may differ substantially. Thus, caution is advised if it is decided to use these assays for herd health classification in Spanish Salmonella control programs.

  4. Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Zhang, Yan; Wang, Zhanfeng; Zhan, Yang; Gong, Qian; Yu, Wanting; Deng, Zhibang; Wang, Aibing; Yang, Yi; Wang, Naidong

    2016-06-01

    Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.

  5. Use of baculovirus-expressed glycoprotein H in an enzyme-linked immunosorbent assay developed to assess exposure to chelonid fibropapillomatosis-associated herpesvirus and its relationship to the prevalence of fibropapillomatosis in sea turtles.

    Science.gov (United States)

    Herbst, Lawrence H; Lemaire, Shefali; Ene, Ada R; Heslin, David J; Ehrhart, Llewellyn M; Bagley, Dean A; Klein, Paul A; Lenz, Jack

    2008-05-01

    Chelonid fibropapillomatosis-associated herpesvirus (CFPHV) is an alphaherpesvirus believed to cause marine turtle fibropapillomatosis (FP). A serodiagnostic assay was developed for monitoring sea turtle populations for CFPHV exposure. CFPHV glycoprotein H (gH) expressed in recombinant baculovirus was used in an enzyme-linked immunosorbent assay (ELISA) to detect virus-specific 7S turtle antibodies. Using captive-reared green turtles (Chelonia mydas) with no history of virus exposure as "known negatives" and others with experimentally induced FP as "known positives," the assay had 100% specificity but low sensitivity, as seroconversion was detected in only half of the turtles bearing experimentally induced tumors. Antibodies were detected only in samples collected after cutaneous fibropapillomas appeared, consistent with observations that tumors are significant sites of virion production and antigen expression and the possibility that prolonged/repeated virus shedding may be required for adequate stimulation of 7S antibody responses to gH. Natural routes of infection, however, may produce higher seroconversion rates. High gH antibody seroprevalences ( approximately 80%) were found among wild green turtles in three Florida localities with different FP prevalences, including one site with no history of FP. In addition, all eight loggerhead turtles (Caretta caretta) tested were seropositive despite FP being uncommon in this species. The possibility that CFPHV infection may be common relative to disease suggests roles for environmental and host factors as modulators of disease expression. Alternatively, the possibility of other antigenically similar herpesviruses present in wild populations cannot be excluded, although antibody cross-reactivity with the lung/eye/trachea disease-associated herpesvirus was ruled out in this study.

  6. 动物性食品中氨苯砜dcELISA的检测%Development of a direct competitive enzyme-linked immunosorbent assay for detection of dapsone in animal foods

    Institute of Scientific and Technical Information of China (English)

    郑小娇; 张晶; 张亚卓; 史晨杉; 王向红

    2015-01-01

    A direct competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect Dapsone in animal foods.Hapten dapsone was conjugated with Bovine Sera Albumin (BSA) by diazotization to produce immunogen BSA-DDS used for immunizing rabbit.The rabbit antiserum was purified with ProteinA-Sephros 4B to prepare polyclonal antibody against dapsone.A direct enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantitate dapsone.The bewt synthesis of BSA-DDS and the conjugated-ratio was 1 ∶ 10.The IC50 was 4.78 ng/mL,visual detection limit was 0.03 ng/mL,and recoveries of dapsone in spiked samples ranged from 75.45% ~ 91.75%.The artificial antigens can be used for preparing specificity anti-dapsone antibody.The method is sensitive and the procedure of sample pretreatment is simple and quick.It is suitable to use in the detection of DDS residues in animal foods on site.%建立了动物性食品中氨苯砜残留快速检测技术.将氨苯砜重氮化后连接到牛血清蛋白(BSA)上制得免疫原BSA-DDS,免疫新西兰大耳白兔获得多克隆抗体,经ProteinA-Sephros 4B对抗体进行纯化,在此基础上建立直接竞争ELISA方法.结果显示:该方法可制备目标抗原BSA-DDS和抗体,氨苯砜与载体蛋白偶联比可达到1:10;建立的直接竞争ELISA方法的IC50为4.78 ng/mL,最低检测限达0.03 ng/mL,加标回收率为75.45% ~91.75%.

  7. Is a Plasmodium lactate dehydrogenase (pLDH) enzyme-linked immunosorbent (ELISA)-based assay a valid tool for detecting risky malaria blood donations in Africa?

    Science.gov (United States)

    Atchade, Pascal S; Doderer-Lang, Cécile; Chabi, Nicodème; Perrotey, Sylvie; Abdelrahman, Tamer; Akpovi, Casimir D; Anani, Ludovic; Bigot, André; Sanni, Ambaliou; Candolfi, Ermanno

    2013-08-08

    Malaria is a leading cause of mortality in southern Benin. The main causative agent, Plasmodium falciparum, poses a threat on critical transfusions in pregnant women and children. This study's objective was to compare the performance of different malaria screening methods in blood donors in southern Benin, a malaria-endemic country. Blood from 2,515 voluntary blood donors in Benin was collected over a period of 10 months in ethylenediaminetetraacetic acid (EDTA) tubes, which were then classified according to extraction time: long rainy season, short dry season, short rainy season, and long dry season. Microscopic examination was used to count parasites. Parasite density (PD) was expressed as the number of parasites per μL of blood. Pan Plasmodium pLDH detection was assessed by an ELISA-malaria antigen test. Using crude soluble P. falciparum antigens, an ELISA-malaria antibody test detected anti-Plasmodium antibodies. Among the 2,515 blood donors (2,025 males and 488 females) screened, the rate of asymptomatic Plasmodium carriage was 295/2,515 (11.72%, 95% CI: 10.5-13.1%). Males had a higher infection rate (12.4%) than did females (8.8%). Parasite density was very low: between seven and100 parasites per μL of blood was reported in 80% of donors with parasitaemia. Three Plasmodium species were diagnosed: P. falciparum in 280/295 patients (95.0%), Plasmodium malariae in 14/295 (5.0%), and Plasmodium ovale in 1/295 (0.34%). Malaria prevalence in donors was higher during the rainy seasons (13.7%) compared with the dry seasons (9.9%). The use of a highly sensitive assay enabled pan Plasmodium pLDH detection in 966/2,515 (38.4%, 95% CI: 36.5%-40.3%). Malaria antibody prevalence was 1,859/2,515 (73.9%, 95% CI: 72.16-75.6%). Donors' antigenaemia and antibody levels varied significantly (P Plasmodium can transmit malaria to donation recipients. Malaria diagnostic methods are currently available, but the feasibility criteria for mass screening in endemic areas become

  8. Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary.

    Science.gov (United States)

    Zhu, Qingfu; El-Mergawy, Rabab G; Heinemann, Stefan H; Schönherr, Roland; Jáč, Pavel; Scriba, Gerhard K E

    2013-09-01

    A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.

  9. Employing carbon dots modified with vancomycin for assaying Gram-positive bacteria like Staphylococcus aureus.

    Science.gov (United States)

    Zhong, Dan; Zhuo, Yan; Feng, Yuanjiao; Yang, Xiaoming

    2015-12-15

    By employing attractive performance of fluorescent carbon dots, we herein successfully established an assay for analyzing bacteria firstly. Specifically, carbon dots with blue fluorescence were initially synthesized according to a previous report, and modified with vancomycin on their surfaces. Subsequently, the prepared carbon dots were applied to detect Staphylococcus aureus accompanied with a linear range of 3.18×10(5)-1.59×10(8) cfu/mL as well as a detection limit of 9.40×10(4) cfu/mL. Compared with other regular methods, our method is more rapid and convenient in term of methodology. Meanwhile, the current strategy was applied for detection of other bacteria including Bacillus subtilis, Listeria monocytogenes, Salmonella, Pseudomonas aeruginosa and Escherichia coli, and the modified carbon dots showed obvious affinity with Gram-positive bacteria owing to the ligand-receptor interactions between vancomycin and the cell walls, suggesting its value for detecting Gram-positive bacteria. Additionally, the practicability of this sensing approach was validated by recovery experiments conducted in orange juice, confirming its potential to broaden avenues for detection of Gram-positive bacteria.

  10. Design of indirect solid-phase immunosorbent methods for detecting arenavirus antigens and antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, A.P.; Rezapkin, G.V.; Dzagurova, T.K.; Tkachenko, E.A.

    1984-05-01

    Specifications have been elaborated for formulating indirect solid-phase enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (SPRIA) methods that employ anti-human and anti-mice G class immunoglobulin (IgG), conjugated with horseradish peroxidase and /sup 125/I for detecting the arenaviruses Junin, Machupo, Tacaribe, Amalpari, Tamiami, Lassa, and LCM (lymphocytic choriomeningitis). These methods make it possible to identify with a high degree of sensitivity arenavirus antigens and antibodies in various kinds of material.

  11. Comparison of bacterial culture, polymerase chain reaction, and a mix-enzyme-linked immunosorbent assay for the detection of Salmonella status in grow-to-finish pigs in western Canada with a Bayesian approach

    Science.gov (United States)

    Wilkins, Wendy; Waldner, Cheryl; Rajić, Andrijana; McFall, Margaret; Chow, Eva; Muckle, Anne

    2011-01-01

    Among grow-to-finish pigs from 10 herds in Alberta and Saskatchewan, 23 (16%) of 144 fecal samples were culture-positive and 40 (28%) of 144 pigs were seropositive for Salmonella. With a Bayesian model specifying dependence between the 2 tests, the sensitivity (Se) of culture and real-time polymerase chain reaction (RT-PCR) was 79% to 86%, depending on the cut-off value for the enzyme-linked immunosorbent assay (ELISA). Culture specificity (Sp) was assumed to be 100%; RT-PCR Sp was found to be 94%. The ELISA Se was 76% and 51% at optical density cut-off values ≥ 20% and ≥ 40%, respectively; the Sp was 94% at each cut-off value. The model showed some sensitivity to ELISA prior information, the ELISA Se being approximately 8% lower when informative prior information was specified in the model. When there was no adjustment for dependence between culture and RT-PCR, the posterior estimates for both culture and RT-PCR Se were 11% higher than with the conditional-dependence model and had considerably narrower probability intervals, which suggests that correlation between culture and PCR is important and should be adjusted for in future studies. PMID:22468029

  12. Enzyme linked immunosorbent assay (ELISA for the detection of IgG, IgM, IgE and IgA against Cysticercus cellulosae in cerebrospinal fluid of patients with neurocysticercosis

    Directory of Open Access Journals (Sweden)

    Odashima Newton Satoru

    2002-01-01

    Full Text Available The objective of this study was to analyze different immunoglobulins classes (IgG, IgM, IgE and IgA against Cysticercus cellulosae in the cerebrospinal fluid (CSF, through enzyme linked immunosorbent assay (ELISA, correlating them to clinical and tomographic profiles in patients with neurocysticercosis (NCC. Eighty-five specimens of CSF were obtained from 43 cases with NCC (26 with the active form and 17 with the inactive form and from 42 patients with other neurological diseases. The inactive form of NCC presented a profile in CSF similar to the group without NCC. The active form of NCC presented elevation of specific immunoglobulins (IgG, IgM, IgE, and IgA in decreasing order, with the highest values being detected among the cases with intraventricular cysts, or with inflammation signs in CSF or in those with multiple clinical manifestations. The highest sensitivity and specificity were obtained with ELISA-IgG (88.5% and 93.2%, respectively. This study confirmed the importance of ELISA in the immunologic diagnosis of NCC.

  13. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran

    Directory of Open Access Journals (Sweden)

    Mehrangiz Rajaii

    2015-01-01

    Full Text Available Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%, 102 (26%, 74 (18.92% in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF, enzyme-linked immunosorbent assay (ELISA and IgG Avidity techniques. Results: We have found 111 (28.38% seronegative and 280 (71.61% seropositive cases by IIF and 124 (31.70% seronegative, 267 (68.28% seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23% and 7 (10.76% doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.

  14. Expression of Leptin and Visfatin in Gingival Tissues of Chronic Periodontitis With and Without Type 2 Diabetes Mellitus: A Study Using Enzyme-Linked Immunosorbent Assay and Real-Time Polymerase Chain Reaction.

    Science.gov (United States)

    Ghallab, Noha A; Amr, Eman M; Shaker, Olfat G

    2015-07-01

    The aim of this study is to investigate the protein and gene expression of leptin and visfatin in gingival tissue from patients with chronic periodontitis (CP), patients with CP and type 2 diabetes mellitus (T2DM), and healthy individuals. The study includes 50 individuals: 10 healthy individuals, 20 patients with CP, and 20 patients with CP and T2DM. Plaque index, gingival index, probing depth, and clinical attachment loss were measured, and gingival biopsies were obtained. Leptin and visfatin protein expression in gingival tissues was determined using enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was measured via real-time polymerase chain reaction. The highest leptin mRNA and protein expression was observed in the control group and was significantly (P ≤0.05) different from the CP and CP+T2DM groups. Gingival tissues from patients with CP and T2DM had a significant increase in visfatin and a decrease in leptin gene and protein expression (P <0.05) compared with both controls and patients with CP. Expression of leptin and visfatin in the gingival tissues suggests a possible role for these adipokines in the pathogenesis of CP and T2DM.

  15. Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus-infected pigs from pigs vaccinated with CP7_E2alf.

    Science.gov (United States)

    Pannhorst, Katrin; Fröhlich, Andreas; Staubach, Christoph; Meyer, Denise; Blome, Sandra; Becher, Paul

    2015-07-01

    Infections with Classical swine fever virus (CSFV) are a major economic threat to pig production. To combat CSF outbreaks and to maintain trade, new marker vaccines were developed that allow differentiation of infected from vaccinated animals (DIVA principle). The chimeric pestivirus CP7_E2alf was shown to be safe and efficacious. Its DIVA strategy is based on the detection of CSFV E(rns)-specific antibodies that are only developed on infection. However, for the new marker vaccine to be considered a valuable control tool, a validated discriminatory assay is needed. One promising candidate is the already commercially available enzyme-linked immunosorbent assay, PrioCHECK CSFV E(rns) ELISA (Prionics BV, Lelystad, The Netherlands). Four laboratories of different European Union member states tested 530 serum samples and country-specific field sera from domestic pigs and wild boar. The ELISA displayed a good robustness. However, based on its reproducibility and repeatability, ranges rather than single values for diagnostic sensitivity and specificity were defined. The ELISA displayed a sensitivity of 90-98% with sera from CSFV-infected domestic pigs. A specificity of 89-96% was calculated with sera from domestic pigs vaccinated once with CP7_E2alf. The ELISA detected CSFV infections in vaccinated domestic pigs with a sensitivity of 82-94%. The sensitivity was lower with sera taken ≤21 days post-challenge indicating that the stage of CSFV infection had a considerable influence on testing. Taken together, the PrioCHECK CSFV E(rns) ELISA can be used for detection of CSFV infections in CP7_E2alf-vaccinated and nonvaccinated domestic pig populations, but should only be applied on a herd basis by testing a defined number of animals.

  16. Enumeration of Gut-Homing β7-Positive, Pathogen-Specific Antibody-Secreting Cells in Whole Blood from Enterotoxigenic Escherichia coli- and Vibrio cholerae-Infected Patients, Determined Using an Enzyme-Linked Immunosorbent Spot Assay Technique.

    Science.gov (United States)

    Bhuiyan, Taufiqur Rahman; Hoq, Mohammad Rubel; Nishat, Naoshin Sharmin; Al Mahbuba, Deena; Rashu, Rasheduzzaman; Islam, Kamrul; Hossain, Lazina; Dey, Ayan; Harris, Jason B; Ryan, Edward T; Calderwood, Stephen B; Svennerholm, Ann-Mari; Qadri, Firdausi

    2015-10-28

    Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are noninvasive mucosal pathogens that cause acute watery diarrhea in people in developing countries. Direct assessment of the mucosal immune responses to these pathogens is problematic. Surrogate markers of local mucosal responses in blood are increasingly being studied to determine the mucosal immune responses after infection. However, the volume of blood available in children and infants has limited this approach. We assessed whether an approach that first isolates β7-positive cells from a small volume of blood would allow measurement of the antigen-specific immune responses in patients with cholera and ETEC infection. β7 is a cell surface marker associated with mucosal homing. We isolated β7-expressing cells from blood on days 2, 7, and 30 and used an enzyme-linked immunosorbent spot (ELISPOT) assay to assess the gut-homing antibody-secreting cells (ASCs) specific to pathogen antigens. Patients with ETEC diarrhea showed a significant increase in toxin-specific gut-homing ASCs at day 7 compared to the levels at days 2 and 30 after onset of illness and to the levels in healthy controls. Similar elevations of responses to the ETEC colonization factors (CFs) CS6 and CFA/I were observed in patients infected with CS6- and CFA/I-positive ETEC strains. Antigen-specific gut-homing ASCs to the B subunit of cholera toxin and cholera-specific lipopolysaccharides (LPS) were also observed on day 7 after the onset of cholera using this approach. This study demonstrates that a simple ELISPOT assay can be used to study the mucosal immunity to specific antigens using a cell-sorting protocol to isolate mucosal homing cells, facilitating measurement of mucosal responses in children following infection or vaccination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans.

    Science.gov (United States)

    Galula, Jedhan U; Chang, Gwong-Jen J; Chuang, Shih-Te; Chao, Day-Yu

    2016-02-01

    The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great

  18. Validation and evaluation of VapA-specific IgG and IgG subclass enzyme-linked immunosorbent assays (ELISAs) to identify foals with Rhodococcus equi pneumonia.

    Science.gov (United States)

    Sanz, M G; Oliveira, A F; Loynachan, A; Page, A; Svansson, V; Giguère, S; Horohov, D W

    2016-01-01

    Rhodococcus equi (Rhodococcus hoagii/Prescottella equi) is a common cause of foal pneumonia, but its diagnosis remains a challenge for equine veterinarians. While the VapA-specific (virulence-associated protein A) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) has low sensitivity and specificity for detecting pneumonic foals, little is known about VapA-specific IgG subclasses. To evaluate the performance of VapA-specific ELISA for IgG and its subclasses IgGa, IgGb and IgG(T) in the early diagnosis of pneumonia caused by R. equi. Assay validation followed by assessment of diagnostic performance using archived samples from animals of known status. Serum samples from exposed (n = 125) and nonexposed adult horses (n = 10) and from experimentally challenged and naturally infected foals were used for ELISA validation. Post mortem and tissue culture records of the last 24 years from the Institute for Experimental Pathology at the University of Iceland in Keldur, Iceland laboratory were evaluated to confirm the absence of R. equi cases in Iceland. The diagnostic performance of VapA-specific IgG and its subclasses was evaluated using banked serum samples from pneumonic (n = 21) and healthy foals (n = 80). To evaluate each IgG assay, a cut-off value was selected based on receiver operating characteristic curve analysis and used to calculate sensitivity and specificity. The intra- and interassay coefficients of variation were calculated for each ELISA. Using sera from Iceland, where R. equi infection has not been reported, the VapA-specific IgG ELISA differentiated exposed from nonexposed horses. When used to identify infected foals, VapA-specific IgG, IgGa and IgGb had no diagnostic value. In contrast, IgG(T) had high sensitivity and specificity. Horses from Iceland are not exposed to VapA(+) R. equi and can serve as negative controls. VapA-specific IgG subclasses, with the exception of IgG(T), are poor predictors of disease. Further

  19. Erysipelothrix rhusiopathiae and Mycoplasma hyopneumoniae: the sensitivities of enzyme-linked immunosorbent assays for detecting vaccinated sows of unknown disease status using serum and colostrum, and the correlation of the results for sow serum, colostrum, and piglet serum.

    Science.gov (United States)

    Jenvey, Caitlin J; Reichel, Michael P; Cockcroft, Peter D

    2015-03-01

    Due to relatively high concentrations of immunoglobulins, colostrum has the potential to improve the sensitivity of diagnostic tests for diseases in pigs when compared with serum. It is possible that colostrum could improve the sensitivity of the antibody enzyme-linked immunosorbent assay (ELISA) compared with serum. Colostrum is also essential for piglets, providing protection against infections in the first few weeks and months of life. The sensitivity of 2 commercially available ELISAs, one for the detection of Erysipelothrix rhusiopathiae and the second for Mycoplasma hyopneumoniae antibodies, when used with sow colostrum in comparison with serum was investigated. The correlation of maternal E. rhusiopathiae- and M. hyopneumoniae-specific antibody levels with specific-antibody serum levels in the piglet was also determined. The sensitivity was defined as the proportion of vaccinated sows that were correctly identified as vaccinated at a given cutoff point. The true disease status of the sows with regard to the 2 infections was unknown. Blood and colostrum samples were collected from 20 sows, 10 primiparous and 10 multiparous, and blood samples were also collected from the piglets of each sow, 48-72 hr post-farrowing. The sensitivities of both ELISAs were significantly improved when using colostrum compared with serum. Sow serum and colostrum optical density (OD) values were significantly correlated. The mean sow OD values for serum for E. rhusiopathiae and M. hyopneumoniae and colostrum for E. rhusiopathiae were significantly correlated with piglet serum OD levels. If the improved sensitivity of colostrum can be demonstrated in infected animals, this will increase the ability of the test to identify infected animals using both individual and pooled colostrum. Testing serum and/or colostrum using ELISA can be useful predictors of piglet disease-specific OD values.

  20. Serum antibodies from a subset of horses positive for Babesia caballi by competitive enzyme-linked immunosorbent assay demonstrate a protein recognition pattern that is not consistent with infection.

    Science.gov (United States)

    Awinda, Peter O; Mealey, Robert H; Williams, Laura B A; Conrad, Patricia A; Packham, Andrea E; Reif, Kathryn E; Grause, Juanita F; Pelzel-McCluskey, Angela M; Chung, Chungwon; Bastos, Reginaldo G; Kappmeyer, Lowell S; Howe, Daniel K; Ness, SallyAnne L; Knowles, Donald P; Ueti, Massaro W

    2013-11-01

    Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)-competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1-cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi.

  1. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail: hyhan@mail.hzau.edu.cn

    2015-08-05

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL{sup −1} with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

  2. Improved methods for urinary atrazine mercapturate analysis-Assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

    Energy Technology Data Exchange (ETDEWEB)

    Koivunen, Marja E. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Dettmer, Katja [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Vermeulen, Roel [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); Bakke, Berit [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); National Institute of Occupational Health, Oslo (Norway); Gee, Shirley J. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Hammock, Bruce D. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States)]. E-mail: bdhammock@ucdavis.edu

    2006-07-21

    Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis[reg] HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL{sup -1}), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis[reg] HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL{sup -1}) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R {sup 2} values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R {sup 2} = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine.

  3. Development and validation of a competitive enzyme-linked immunosorbent assay for the measurement of total plasma immunoglobulins in healthy loggerhead sea (Caretta caretta) and green turtles (Chelonia mydas).

    Science.gov (United States)

    Kaplan, Amy J; Stacy, Nicole I; Jacobson, Elliott; Le-Bert, Carolina R; Nollens, Hendrik H; Origgi, Francesco C; Green, Linda G; Bootorabi, Shadi; Bolten, Alan; Hernandez, Jorge A

    2016-01-01

    The quantification of circulating plasma immunoglobulins represents a valuable diagnostic tool in human and veterinary immunology, although its application is very limited in reptile medicine to date. The objectives of our study were the development and standardization of a competitive enzyme-linked immunosorbent assay (cELISA) for the measurement of total plasma immunoglobulins (Igs; both IgM and IgY) in loggerhead sea turtles (LST; Caretta caretta; n = 254) and green turtles (GT; Chelonia mydas; n = 111), the establishment of reference intervals for Ig for both species, and the examination of associations between Ig and total protein (TP), condition index, and water temperature. The cELISA for Ig was successfully developed and optimized. Reference intervals for Ig were 0.38-0.94 g/dL in LST (median: 0.59 g/dL; range: 0.16-2.15 g/dL) and 0.40-0.85 g/dL in GT (median: 0.58 g/dL; range: 0.18-1.80 g/dL). In LST, there were positive linear relationships of Ig with TP, and TP with Ig and condition index, and a negative relationship of Ig with condition index. The positive linear relationships of Ig with TP, and TP with Ig were also identified in GT. These positive associations of Ig and TP were expected, as Ig represents fractions of TP, and TP reportedly increases with straight carapace length and weight. The negative association of Ig with condition index may indicate potential biological variations. The cELISA and reference intervals for total Ig of LST and GT presented herein have the potential to be useful as a diagnostic and research tool for sea turtle immunology.

  4. Application of a type-specific enzyme-linked immunosorbent assay for equine herpesvirus types 1 and 4 (EHV-1 and -4) to horse populations inoculated with inactivated EHV-1 vaccine.

    Science.gov (United States)

    Yasunaga, S; Maeda, K; Matsumura, T; Kondo, T; Kai, K

    2000-07-01

    A type-specific enzyme-linked immunosorbent assay (ELISA) using equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) glycoprotein G was applied for sero-epizootiology of EHV infections in Japan. Recently, an inactivated EHV-1 vaccine has been administered to racehorses for prevention of upper respiratory disease. To examine the effect of the vaccination on the result of the ELISA, 6 horses were experimentally inoculated three times intramuscularly or intranasally with inactivated EHV-1 vaccine. Sera collected from these horses were used to the type-specific ELISA and complement-fixation (CF) test. Although the CF test detected a significant increase of antibody elicited by vaccination, the ELISA did not detect any antibody response. Next, sera collected from thirty-eight horses, which were intramuscularly inoculated with inactivated EHV-1 twice at an interval of four weeks, were used in the ELISA and CF test. The results also indicated that CF titers increased by vaccine inoculation, but ELISA titers did not. To examine epizootiology of EHVs serologically in racehorse populations at two Training Centers of the Japan Racing Association, the type-specific ELISA and CF test were carried out using paired sera collected from racehorses before and after the winter season. The results showed that the ELISA could distinguish EHV-1 and EHV-4 infections in vaccinated horses serologically. In conclusion, the type-specific ELISA is considered to be useful for sero-diagnosis and sero-epizootiological research on EHV-1 and EHV-4 infections not only in unvaccinated horses, but also in vaccinated horses in Japan.

  5. 抗恩诺沙星抗血清的制备及其ELISA检测%Preparation of enrofloxacin antiserum and its determination by enzyme - linked immunosorbent assay (ELISA)

    Institute of Scientific and Technical Information of China (English)

    刘兵; 曾华金; 杨冉; 雷利芳; 屈凌波

    2011-01-01

    Objective;To develop a rapid and sensitive enzyme - linked immunosorbent assay(ELISA) for determination of enrofloxacin( ENR). Method; Immunogen enrofloxacin - bovine serum albumin( Enrofloxacin - BSA) and coating antigen enrofloxcin -ovum albumin (Enrofloxacin - OVA) were prepared by a succinic anhydride method. By subcutaneous injection, the polyclonal antiserum was produced from big ear rabbits immunized with conjugates ENR -BSA. Result; After the assay procedure was optimized,the standard curve of ENR was established and the practical measuring range of the ELISA extended from 5 ng ? mL-1 to 2. 5 |xg ? mL '' ( R2 =0. 9935 ) . In a comparison of the assay results obtained by ELISA and HPLC, there is a good correlation between these two methods (R2 = 0. 9892, n = 9 ) . The proposed method has been satisfactorily applied for the determination of ENR in rat plasma and Pharmaceuticals with recoveries in the range of 98. 4% to 105. 8% for milk sample,91. 7% to 101. 2% for rat plasma and 97.0% to 110.3% for rat urine. Conclusion; The experimental data indicated that in some extent the ELISA method was more suitable for high throughput and real - time ENR analysis in biological samples and animal food with lower detection limit,low background and no requirement of sample pre -treatment.%目的:建立快速、灵敏的恩诺沙星残留酶联免疫检测方法(ELISA).方法:采用混合酸酐法,将恩诺沙星与牛血清白蛋白和卵清蛋白分别合成免疫抗原(Enrofloxacin-BSA)和包被抗原(Enrofloxacin-OVA).通过背部多点免疫法免疫日本大白耳兔,制备抗恩诺沙星的特异性抗血清.结果:利用所获得的特异性抗血清,建立的恩诺沙星ELISA检测方法,其线性范围为5 ng·mL.~2.5μg·mL(R=0.9935),且与HPLC方法具有良好的相关性(R=0.9892,n=9).牛奶、大鼠血浆和尿液中的加样回收率分别为98.4%~105.8%,91.7%~101.2%,97.0%~110.3%.运用所建立的方法,对大鼠体内血浆及药品

  6. Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis.

    Directory of Open Access Journals (Sweden)

    Priscila Zacarias de Azevedo

    Full Text Available The diagnosis of chronic pulmonary aspergillosis (CPA depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA and four chronic fibrosing pulmonary aspergillosis (CFPA; G2: 28 patients with pulmonary tuberculosis (TB; G3: 23 patients with histoplasmosis (HST; G4: 50 patients with paracoccidioidomycosis (PCM; G5: 20 patients with cryptococcosis (CRC; and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations--Aspergillus fumigatus (DID1, ELISA1 and a pool of A. fumigatus, A. flavus and A. niger antigens (DID2, ELISA2. The Platélia Aspergillus Enzyme Immunoassay (EIA kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio-especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA1 and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA1 and ELISA2. The detection of serum galactomannan showed high sensitivity, comparable to ELISA2. The immunodiffusion test showed an excellent relationship with the progression after

  7. Test of Syphilis by Treponema Pallidum Enzyme-linked Immunosorbent Assay and Treponema Pallidum Particle Assay%梅毒酶联免疫吸附试验与梅毒螺旋体明胶凝集试验检测梅毒的效果比较

    Institute of Scientific and Technical Information of China (English)

    吴险

    2014-01-01

    目的:探讨梅毒酶联免疫吸附试验与梅毒螺旋体明胶凝集试验检测梅毒的效果。方法对100例梅毒患者,分别运用梅毒酶联免疫吸附试验与梅毒螺旋体明胶凝集试验进行检测,对比检测结果。结果两种方法的阳性检出率分别为97.0%和92.0%,特异性分别为94.0%和100%。结论在梅毒阳性检出率上梅毒酶联免疫吸附试验较高,而特异性梅毒螺旋体明胶凝集试验较高。%Objective To compare the different test results of syphilis by treponema pallidum enzyme linked immunosorbent assay(TP-ELISE) and treponema pallidum particle assay(TPPA). Methods 100 patients with syphilis were tested respectively by TP-ELISE and TPPA, and the results were compared. Results The positive rates of the two methods were 97%and 92%, and the speciifcity were 94%and 100%. Conclusions The positive rate of TP-ELISE was higher, and the speciifcity of TPPA was the highest.

  8. A novel enzyme-linked immunosorbent assay for quantitative detection of anti-thyroid peroxidase antibodies in serum%血清抗甲状腺过氧化物酶抗体ELISA定量方法的建立

    Institute of Scientific and Technical Information of China (English)