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Sample records for anti-filamin-a antibody detects

  1. Photonic crystal fiber based antibody detection

    DEFF Research Database (Denmark)

    Duval, A; Lhoutellier, M; Jensen, J B; Hoiby, P E; Missier, V; Pedersen, L H; Hansen, Theis Peter; Bjarklev, Anders Overgaard; Bang, Ole

    An original approach for detecting labeled antibodies based on strong penetration photonic crystal fibers is introduced. The target antibody is immobilized inside the air-holes of a photonic crystal fiber and the detection is realized by the means of evanescent-wave fluorescence spectroscopy and...

  2. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  3. Antibody conjugated graphene nanocomposites for pathogen detection

    Science.gov (United States)

    Sign, Chandan; Sumana, Gajjala

    2016-04-01

    Graphene oxide (GO), due to its excellent electrochemical properties and large surface area, known to be highly suitable material for biosensing application. Here, we report in situ synthesis of silver nanopaticles (AgNPs) onto the GO sheets for the electrochemical detection of Salmonella typhimurium (S.typhimurium). The GO-AgNPs composites have been deposited onto the indium tin oxide (ITO) coated glass substrate by the electrophoretic deposition technique. Carbodiimide coupling (EDC-NHS) has been used for the immobilization of antibodies of Salmonella typhimurium (anti-S.typhimurium) for detection of S.typhimurium. The electron microscopy and UV-visible studies reveal successful synthesis GO-AgNPs composites while FT-IR studies suggest the proper immobilization of anti-S.typhi. The cyclic voltammetry (CV) has been utilized for detection using anti-S.typhi/GOAgNPs/ITO based immunoelectrode as a function of S.typhimurium concentration. The fabricated immunosensor shows improved sensitivity of 33.04 μACFU-1mLcm-2 in a wide detection range of 101 to 106 CFUmL-1. This immunosensor may be utilized for the detection of other food borne pathogens like aflatoxin and E.coli also.

  4. Detection of Campylobacter species using monoclonal antibodies

    Science.gov (United States)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  5. Evaluation of Salivary Antibodies to Detect Infection with Helicobacter pylori

    OpenAIRE

    1997-01-01

    Helicobacter pylori infection is an important cause of peptic ulcer disease and chronic gastritis. Infection with this bacterium stimulates the production of immunoglobulin (Ig) G antibody. Salivary IgG antibody tests to detect H pylori infection offer a convenient and noninvasive method of diagnosis. To evaluate an IgG salivary antibody kit, saliva was collected from 157 out-patients with dyspepsia referred for endoscopy to a tertiary centre. A salivary IgG ELISA antibody assay was performed...

  6. Occult choriocarcinoma: Detection using radiolabeled monoclonal antibodies

    International Nuclear Information System (INIS)

    Occult choriocarcinoma, manifested only by an elevated B-hCG level, can be a difficult management problem. The authors evaluated the ability of I-131-labeled 5F9.3, a murine monoclonal antibody reactive with choriocarcinomas but not hCG, to detect foci of choriocarcinoma in five patients referred with elevated B-hCG levels but in whom the location of residual disease was uncertain. I-131 5F9.3, 0.5-1.0 mCi, was injected intravenously in each patient and images with dynamic background subtraction of TcHSA were obtained at later time points. In four patients chest studies were true positive (confirmed surgically in all), the chest CT scans in these patients had been interpreted as not definitely showing active disease. In the fifth patient no abnormal focus of uptake was seen and subsequent B-hCG levels normalized. In two of the patients with chest lesions, foci of abdominal uptake were seen that were not due to tumor. One of these patients had a partial small bowel obstruction; the other appeared to have a false-positive study. I-131 5F9.3 is a promising agent for the detection of occult choriocarcinomas

  7. 42 CFR 493.861 - Standard; Unexpected antibody detection.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 5 2010-10-01 2010-10-01 false Standard; Unexpected antibody detection. 493.861 Section 493.861 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN..., Or Any Combination of These Tests § 493.861 Standard; Unexpected antibody detection. (a) Failure...

  8. Method for detecting pathogens attached to specific antibodies

    Science.gov (United States)

    Miles, Robin R.; Venkateswaran, Kodumudi S.; Fuller, Christopher K.

    2005-01-25

    The use of impedance measurements to detect the presence of pathogens attached to antibody-coated beads. In a fluidic device antibodies are immobilized on a surface of a patterned interdigitated electrode. Pathogens in a sample fluid streaming past the electrode attach to the immobilized antibodies, which produces a change in impedance between two adjacent electrodes, which impedance change is measured and used to detect the presence of a pathogen. To amplify the signal, beads coated with antibodies are introduced and the beads would stick to the pathogen causing a greater change in impedance between the two adjacent electrodes.

  9. Development of Monoclonal Antibodies Suitable for Rabies Virus Antibody and Antigen Detection

    OpenAIRE

    Chander, Vishal; Singh, R.P.; Verma, P. C.

    2012-01-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the pro...

  10. Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

    Science.gov (United States)

    Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

    Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

  11. [Detection of antiadenoviral antibodies by surface plasmon resonance].

    Science.gov (United States)

    Nosach, L M; Boltovets', P M; Zahorodnia, S D; Povnytsia, O Iu; Holovan', A V; Netreba, N I; Dobrochyns'ka, L I

    2009-01-01

    A possibility to detect antiadenoviral antibodies by surface plasmon resonance (SPR) was demonstrated. Immobilization on the surface of a sensor of the hexone or degraded purified adenovirus of one of the types (Ad 2) allows finding antibodies to the hexone antigenic determinants of wide specificity common for human adenoviruses of different types. Optimum conditions for immobilization of the antigen and formation of the complex antigen-antibody are determined. The comparative assays of the levels of antibodies in rabbit antisera obtained to the hexone and adenoviruses of different types (1, 2 and 6) by SPR and ELISA was analyzed. The biosensor was used to detect antiadenoviral antibodies in the blood sera of children with aggravation of chronic nonspecific broncho-pulmonary diseases. The sensitivity of SPR in comparison with ELISA was 86.9%, in comparison with the method of fluorescing antibodies (MFA)--89.5%. PMID:20387633

  12. Selective detection of antibodies in microstructured polymer optical fibers

    DEFF Research Database (Denmark)

    Jensen, Jesper Bo Damm; Hoiby, P.E.; Emiliyanov, Grigoriy Andreev;

    2005-01-01

    We demonstrate selective detection of fluorophore labeled antibodies from minute samples probed by a sensor layer of complementary biomolecules immobilized inside the air holes of microstructured Polymer Optical Fiber (mPOF). The fiber core is defined by a ring of 6 air holes and a simple procedure...... was applied to selectively capture either α-streptavidin or α-CRP antibodies inside these air holes. A sensitive and easy-to-use fluorescence method was used for the optical detection. Our results show that mPOF based biosensors can provide reliable and selective antibody detection in ultra small...

  13. Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

    Science.gov (United States)

    Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

    2015-08-01

    Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

  14. Antibodies for detecting and quantifying anticoagulant agents

    OpenAIRE

    Salvador, Juan Pablo; Marco, María Pilar

    2012-01-01

    [EN] The present invention relates to the design of haptens that are structurally related to coumarin oral anticoagulant compounds (COAC), to be used for the production of specific antibodies against said type of substances and the subsequent use thereof for the development of diagnosis tools for use in laboratories or in point-of-care (PoC) devices. In particular, the produced antibodies have been used to develop a diagnosis tool that enables the plasma levels of COAC to be quantified in pat...

  15. T-cell detection with monoclonal antibody T101 kits.

    OpenAIRE

    Pollack, S M; Cimino, E F; Robbins, D S; Hoffman, P M

    1986-01-01

    A solid-phase immunoadsorption procedure (Quantigen T&B cell kit; Bio-Rad Laboratories, Richmond, Calif.) employing monoclonal antibody T101 detected mean percentages of peripheral blood T cells comparable to those obtained by rosetting with sheep erythrocytes, while lower values were obtained with an indirect immunofluorescence procedure (Cytotag T&B cell kit; Hybritech, Inc., San Diego, Calif.) employing the same antibody. Therefore, T101 binding appears to be more easily detected by solid-...

  16. Detection of thrombocyte antibodies by 125I labeled protein A

    International Nuclear Information System (INIS)

    Protein A from Staphylococcus aureus interacts in a specific manner with most subclasses of human IgG. In the present study a method is described which utilizes Protein A labeled with 125I for the detection of antibody sensitization of platelets. The clinical applicability of the test for detection of in vivo or in vitro sensitization is demonstrated in three patients with platelet antibodies. (author)

  17. Developing recombinant antibodies for biomarker detection

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    2010-10-01

    Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.

  18. Detection of enterovirus 70 with monoclonal antibodies.

    OpenAIRE

    Anderson, L J; Hatch, M. H.; Flemister, M R; Marchetti, G E

    1984-01-01

    To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave ...

  19. Clinical experience in humans with radiolabeled antibody for tumor detection

    International Nuclear Information System (INIS)

    I-131 and Tc-99m labeled polyclonal or monoclonal antibody and fragments of antibody, specific to human chorionic gonadotropin (hCG) or to a melanoma cell surface antigen (MCSA) were injected into proven cancer patients. Using standard homeostasis parameters, and scanning techniques, the safety and efficacy of each antibody was evaluated. Antibody fragments were expected to clear faster from the circulation allowing for earlier imaging and a better target-to-non-target ratio. The technetium label may perturb the antiboby's kinetics so that clearance is more rapid for both whole antibody and fragments. After a statistical evaluation of all parameters measured pre and post injection it was concluded that no acute toxicity reactions were present in any patient studied. Scan results were not acceptable for a tumor detecting procedure used in routine practice. Tumor upake was seen in less than 10% of scans

  20. Detecting Lyme disease using antibody-functionalized carbon nanotubes

    Science.gov (United States)

    Dailey, Jennifer; Lerner, Mitchell; Goldsmith, Brett; Brisson, Dustin; Johnson, A. T. Charlie

    2011-03-01

    We combine antibodies for Lyme flagellar protein with carbon nanotube transistors to create an electronic sensor capable of definitive detection of Lyme disease. Over 35,000 cases of Lyme disease are reported in the United States each year, of which more than 23 percent are originally misdiagnosed. Rational design of the coupling of the biological system to the electronic system gives us a flexible sensor platform which we can apply to several biological systems. By coupling these antibodies to carbon nanotubes in particular, we allow for fast, sensitive, highly selective, electronic detection. Unlike antibody or biomarker detection, bacterial protein detection leads to positive identification of both early and late stage bacterial infections, and is easily expandable to environmental monitoring.

  1. Development and characterization of antibody reagents for detecting nanoparticles

    OpenAIRE

    Ravichandran, Supriya; Sullivan, Mark A.; Callahan, Linda M.; Bentley, Karen L.; DeLouise, Lisa A.

    2015-01-01

    The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in sol...

  2. Development of monoclonal antibodies suitable for rabies virus antibody and antigen detection.

    Science.gov (United States)

    Chander, Vishal; Singh, R P; Verma, P C

    2012-12-01

    The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects. PMID:24293819

  3. Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

    1982-10-01

    An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

  4. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Luis Mario Rodríguez-Martínez

    Full Text Available Current Ebola virus (EBOV detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV proteins. In particular, several monoclonal antibodies (mAbs have been described that bind the capsid glycoprotein (GP of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude and they are easily and economically produced in bacterial cultures.Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.

  5. Detection of serum antitrichomonal antibodies in urogenital trichomoniasis by immunofluorescence.

    Directory of Open Access Journals (Sweden)

    Bhatt R

    1992-04-01

    Full Text Available Trichomonas vaginalis is a frequently encountered genital pathogen in both males and females. In females, vaginitis due to this parasite is one of the most common manifestation. The indirect fluorescent technique (IFA test was carried out to detect antitrichomonal antibodies in 370 female patients using whole cell antigen. Seventy one (19.18% gave positive reaction for either of the class IgG, IgM and IgA antibodies. The level of the IgG class antibodies was found to be higher i.e. 58 (81.69% than IgM 11 (15.27% antibodies, which may be suggestive of past infection or a prolonged manifestation by the organisms.

  6. Detection of IgE insulin antibody with radioallergosorbent test

    International Nuclear Information System (INIS)

    An in vitro method for detecting IgE insulin antibody using the principle of the radioallergosorbent test (RAST) is described. In six patients with insulin allergy, the RAST values were higher than in normal persons or insulin-treated diabetics without insulin allergy. No differences were observed between normal persons and insulin-treated diabetics without insulin allergy. Moreover, it was observed that in one patient treated with highly purified insulin, there was a gradual decrease of RAST value parallel to the radioinsulin binding activity and clinical allergic symptoms. The RAST value of insulin is slightly inhibited by non-IgE antibodies and is, therefore, a semiquantitative value. However, the RAST is simple to perform and reproducible; it is therefore very useful in the detection of IgE insulin antibodies. (orig.)

  7. [Antibody detection after antepartal rhesus prophylaxis: normal values or sensitization].

    Science.gov (United States)

    Behrens, O; Bader, W; Holle, W; Maas, D H; Schneider, J

    1993-05-01

    Antibody screening tests were performed in 29 unsensitized pregnant women after antepartum Rh immune prophylaxis, using the indirect Coombs test (ICT) and a more sensitive ID-microtyping-system (IDM). With the ICT, anti-D antibodies were detected in 85% for at least 4 weeks and at most 8 weeks after immunisation. The maximum titer was 1:8. With the IDM, 97% showed antibodies against 'D' for at least 4 weeks and at most 11 weeks with a maximum of 1:16. The IDM titer was always 1 to 3 steps more sensitive than the ICT. After postpartum Rh immune prophylaxis, anti-D titers were again positive in many of the patients (ICT: 42%; IDM: 60%). In conclusion, it is nearly always possible to measure antibodies against 'D' after antepartum Rh immune prophylaxis and IDM was superior in comparison to ICT. However, maternal isoimmunisation to the rhesus antigen cannot be excluded for sure and patients have then to be controlled. As isoimmunisation could not be confirmed in any of our patients, postpartum Rh immune prophylaxis has to be administered even after detection of an antibody titer against 'D' after antepartum Rh prophylaxis. PMID:8514107

  8. Development and characterization of antibody reagents for detecting nanoparticles

    Science.gov (United States)

    Ravichandran, Supriya; Sullivan, Mark A.; Callahan, Linda M.; Bentley, Karen L.; Delouise, Lisa A.

    2015-11-01

    The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields.The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields. Electronic supplementary information (ESI) available: Figures and detailed methods of various techniques

  9. Detection of specific immunoglobulin M antibodies to cytomegalovirus by using monoclonal antibody to immunoglobulin M in an indirect immunofluorescence assay.

    OpenAIRE

    M. Zerbini; Musiani, M; G. Gentilomi; La Placa, M.

    1986-01-01

    The detection of immunoglobulin M (IgM) antibodies to cytomegalovirus-induced late antigens by an indirect immunofluorescence assay was improved by the use of monoclonal antibodies to human IgM. Nonspecific background fluorescence was absent, facilitating the reading of the slides and the detection of a specific fluorescence reaction in sera with low levels of specific IgM. Moreover, the indirect immunofluorescence assay with monoclonal antibodies to IgM proved more sensitive than the indirec...

  10. Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms

    Directory of Open Access Journals (Sweden)

    Michael R. Kierny

    2012-07-01

    Full Text Available The utility of biomarker detection in tomorrow's personalized health care field will mean early and accurate diagnosis of many types of human physiological conditions and diseases. In the search for biomarkers, recombinant affinity reagents can be generated to candidate proteins or post-translational modifications that differ qualitatively or quantitatively between normal and diseased tissues. The use of display technologies, such as phage-display, allows for manageable selection and optimization of affinity reagents for use in biomarker detection. Here we review the use of recombinant antibody fragments, such as scFvs and Fabs, which can be affinity-selected from phage-display libraries, to bind with both high specificity and affinity to biomarkers of cancer, such as Human Epidermal growth factor Receptor 2 (HER2 and Carcinoembryonic antigen (CEA. We discuss how these recombinant antibodies can be fabricated into nanostructures, such as carbon nanotubes, nanowires, and quantum dots, for the purpose of enhancing detection of biomarkers at low concentrations (pg/mL within complex mixtures such as serum or tissue extracts. Other sensing technologies, which take advantage of ‘Surface Enhanced Raman Scattering’ (gold nanoshells, frequency changes in piezoelectric crystals (quartz crystal microbalance, or electrical current generation and sensing during electrochemical reactions (electrochemical detection, can effectively provide multiplexed platforms for detection of cancer and injury biomarkers. Such devices may soon replace the traditional time consuming ELISAs and Western blots, and deliver rapid, point-of-care diagnostics to market.

  11. Evaluation of a quantitative fluorescence immunoassay (FIAX) for detection of serum antibody to Borrelia burgdorferi.

    OpenAIRE

    Pennell, D R; Wand, P J; Schell, R F

    1987-01-01

    A quantitative, indirect, fluorescence immunoassay (FIAX; Whittaker Bioproducts, Inc.) was compared with the conventional indirect fluorescent-antibody test for detection of serum antibody to Borrelia burgdorferi. FIAX correlated well with the indirect fluorescent-antibody test (r = 0.72). FIAX is a convenient and dependable means of measuring serum antibody to B. burgdorferi.

  12. Detection of polysaccharide antigens of Candida albicans interfering with specific antibodies in human sera

    International Nuclear Information System (INIS)

    A double antibody sandwich radioimmunoassay was developed for the detection of circulating polysaccharide antigens of Candida albicans. The sensitivity of the assay for polysaccharides was 1 ng/mL. The in-vitro interference of specific polysaccharide antibodies, even from sera with low antibody levels, could be demonstrated. The sensitivity of the antigen detection decreased proportionally to the amount of polysaccharide antibodies in the sera. The sensitivity of the assay was almost completely restored by heating the sera. This procedure destroyed antibodies and the released polysaccharide antigens were detectable in the test system by using radiolabelled anti-polysaccharide antibodies. (author)

  13. Cantilever with immobilized antibody for liver cancer biomarker detection

    International Nuclear Information System (INIS)

    A novel cantilever array-based bio-sensor was batch-fabricated with IC compatible MEMS technology for precise liver cancer bio-marker detection. A micro-cavity was designed in the free end of the cantilever for local antibody-immobilization, thus the adsorption of the cancer biomarker takes place only in the local region of the cantilever instead of the whole lever, and the effect of adsorption-induced k variation can be dramatically reduced. These structural features offer several advantages: high sensitivity, high throughput, high mass detection accuracy, and a portable system. In addition, an analytical model has been established to eliminate the effect of the adsorption-induced lever stiffness change and has been applied to the precise mass detection of the cancer biomarker AFP; the experimentally detected AFP antigen mass by the sensor (7.6 pg/mL) is quite close to the calculated one (5.5 pg/mL), two orders of magnitude better than those of the fully antibody-immobilized cantilever sensor. These approaches can promote real applications of the cantilever sensors in cancer diagnosis. (semiconductor devices)

  14. Cantilever with immobilized antibody for liver cancer biomarker detection

    Science.gov (United States)

    Shuaipeng, Wang; Jingjing, Wang; Yinfang, Zhu; Jinling, Yang; Fuhua, Yang

    2014-10-01

    A novel cantilever array-based bio-sensor was batch-fabricated with IC compatible MEMS technology for precise liver cancer bio-marker detection. A micro-cavity was designed in the free end of the cantilever for local antibody-immobilization, thus the adsorption of the cancer biomarker takes place only in the local region of the cantilever instead of the whole lever, and the effect of adsorption-induced k variation can be dramatically reduced. These structural features offer several advantages: high sensitivity, high throughput, high mass detection accuracy, and a portable system. In addition, an analytical model has been established to eliminate the effect of the adsorption-induced lever stiffness change and has been applied to the precise mass detection of the cancer biomarker AFP; the experimentally detected AFP antigen mass by the sensor (7.6 pg/mL) is quite close to the calculated one (5.5 pg/mL), two orders of magnitude better than those of the fully antibody-immobilized cantilever sensor. These approaches can promote real applications of the cantilever sensors in cancer diagnosis.

  15. A new sensitive immunosorbent radioassay for the detection of circulating antibodies to polypeptide hormones and proteins

    International Nuclear Information System (INIS)

    A solid-phase immunosorbent radioassay for the detection of circulating antibodies to protein hormones is described. The assay is based on the binding of the homologous 125I-labelled antigen to the antibodies which are then bound to anti-IgG antibodies covalently coupled to Sepharose. It can easily be applied as a complement to any radioimmunoassay for the detection of circulating antibodies to the ligand measured. The assay system avoids falsely elevated values due to interference of high serum concentrations of the antigen. The assay was applied to measure antibodies to FSH, LH, TSH, GH, prolactin, insulin and thyroglobulin (Tg). Among patients with chronic thyroiditis Tg antibodies were found in 100% of the sera. In diffuse toxic goitre 73% of the patients had detectable Tg antibodies. Insulin antibodies were present in 82% of the sera from patients with insulin treated diabetes. No antibodies were found against the other protein hormones tested. (author)

  16. [Detection of anti-granulocyte antibodies using flow cytometry].

    Science.gov (United States)

    Kumagai, T; Furihata, K; Misawa, K; Aoki, M; Kameko, M; Ichikawa, T; Okubo, Y; Ito, S; Ohto, H

    1994-03-01

    To detect anti-granulocyte antibodies (AGAs) in the sera of granulocytopenic patients is important to study the mechanism of the disease. In this report, we studied neutrophil associated immunoglobulin (NAIg) and neutrophil binding immunoglobulin (NBIg) in patients' sera using flow cytometry (FCM). We investigated the interference of circulating immune complexes (CIC) on measuring the NAIg and NBIg. No apparent effect of CIC was observed at concentrations up to 140 micrograms/ml. NAIg and NBIg were semiquantitated by determining the relative fluorescence intensity (RFI) on a flow cytometer and the normal ranges of NAIg and NBIg were less than 15 RFI and less than 10 RFI, respectively. Of 100 sera from patients with neutropenia, 5 were positive for NBIg and 3 of them were positive for granulocyte-specific antibodies. One serum of a patient with benign chronic neutropenia showed clear specificity for NA1 alloantigen but the other 4 AGAs were not specific for NA alloantigen system. Our NAIg, NBIg screening procedure, including NA specificity testing of NBIg and detection of reactivity with normal lymphocytes using FCM, is simple and useful for measuring and studying serological and immunological characteristics of AGAs. PMID:8152165

  17. Detection of grass carp reovirus (GCRV) with monoclonal antibodies.

    Science.gov (United States)

    Hongli, Jing; Lifeng, Zhang; Zhenzhen, Fang; Lipu, Xu; Min, Zhang; Na, Wang; Yulin, Jiang; Xiangmei, Lin

    2014-04-01

    Grass carp reovirus (GCRV) is a pathogen that causes hemorrhagic disease of grass carp. It is the most serious infectious disease of carp and causes serious losses of fingerlings of grass carp and black carp. In this study, a recombinant VP4, one of the viral core proteins, was constructed with a histidine tag and expressed at a high level in E. coli, and the expressed protein was mainly found in the form of inclusion bodies. The expressed VP4 protein was recognized by an anti-His-tag monoclonal antibody and goat anti-GCRV serum. Four monoclonal antibodies (16B7, 39E12, 13C3 and 14D1) against the recombinant VP4 protein were produced. These MAbs did not react with any of the tested viruses or fish cells lines in the ELISA tests except GCRV. In western blotting analysis, a protein band was observed when the recombinant VP4 protein of GCRV was used as an antigen, but a 68-kDa band was observed when natural capsid proteins of GCRV were used as antigens. Furthermore, a sandwich ELISA was developed for detection of GCRV. The detection limit of the test was 105 TCID50 of GCRV per mL. PMID:24122108

  18. Antibody immobilized cysteamine functionalized-gold nanoparticles for aflatoxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Aditya; Matharu, Zimple; Sumana, G.; Solanki, Pratima R. [Department of Science and Technology Centre on Biomolecular Electronics, Biomedical Instrumentation Section, Materials Physics and Engineering Division, National Physical Laboratory (Council of Scientific and Industrial Research), Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Kim, C.G. [Centre for NanoBioEngineering and Spintronics, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Malhotra, B.D., E-mail: bansi.malhotra@gmail.co [Department of Science and Technology Centre on Biomolecular Electronics, Biomedical Instrumentation Section, Materials Physics and Engineering Division, National Physical Laboratory (Council of Scientific and Industrial Research), Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Centre for NanoBioEngineering and Spintronics, Chungnam National University, Daejeon, 305-764 (Korea, Republic of)

    2010-11-30

    Aflatoxin B{sub 1} antibody (aAFB{sub 1}) covalently attached to cysteamine functionalized-gold nanoparticles (C-AuNP) has been immobilized onto 4-mercaptobenzoic acid (MBA) based self assembled monolayer (SAM) on gold electrode (MBA/Au), for the fabrication of BSA/aAFB{sub 1}-C-AuNP/MBA/Au immunoelectrode. This immunoelectrode has been characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM) and electrochemical characterization techniques. The electrochemical response studies reveal that the BSA/aAFB{sub 1}-C-AuNP/MBA/Au immunoelectrode can be used to detect AFB{sub 1} in the range of 10-100 ng dL{sup -1} and has sensitivity as 0.45 {mu}A ng{sup -1} dL, limit of detection as 17.90 ng dL{sup -1} and a response time of 60 s.

  19. Antibody immobilized cysteamine functionalized-gold nanoparticles for aflatoxin detection

    International Nuclear Information System (INIS)

    Aflatoxin B1 antibody (aAFB1) covalently attached to cysteamine functionalized-gold nanoparticles (C-AuNP) has been immobilized onto 4-mercaptobenzoic acid (MBA) based self assembled monolayer (SAM) on gold electrode (MBA/Au), for the fabrication of BSA/aAFB1-C-AuNP/MBA/Au immunoelectrode. This immunoelectrode has been characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM) and electrochemical characterization techniques. The electrochemical response studies reveal that the BSA/aAFB1-C-AuNP/MBA/Au immunoelectrode can be used to detect AFB1 in the range of 10-100 ng dL-1 and has sensitivity as 0.45 μA ng-1 dL, limit of detection as 17.90 ng dL-1 and a response time of 60 s.

  20. Detection of HBs antigen in routine paraffin embedded liver tissue by enzyme-labelled antibody technique

    Directory of Open Access Journals (Sweden)

    Tsuji,Takao

    1976-02-01

    Full Text Available HB surface antigen (HBs Ag was detected using the enzyme-labelled antibody technique on routinely processed liver biopsy material fixed in Bouin's fixative and embedded in paraffin. Of 85 examined specimens, 45 cases were HBs Ag positive by both the immunofluorescent test and the enzyme labelled antibody technique. The remaining 40 cases were negative by both techniques. The specificity of HBs Ag detected by the enzyme-labelled antibody technique was confirmed by the blocking test using guinea pig specific HBs antibody. The results indicate that the enzyme-labelled antibody technique may be useful for detecting HBs Ag on routine paraffin sections.

  1. Immunoradiometric assay for the detection of circulating antibodies to murine monoclonal antibodies in humans (HAMA)

    International Nuclear Information System (INIS)

    The increasing clinical use of monoclonal antibodies (MAb) has focussed attention on the importance of the generation of human anti-mouse antibodies (HAMA). HAMA can not only be life threatening but can also be associated with reduced image quality and accelerated MAb clearance in vivo as well as interfere with in vitro MAb based assays. The development of a two step immunoradiometric assay (IRMA) for detecting circulating HAMA is reported. Preliminary results have been generated with specific MAb coated polystyrene wells. The appropriately diluted serum sample is first incubated with the MAb coated wells followed by a wash step and a second incubation with 125I labeled goat anti-human IgG. After a final wash, the wells are assayed for 125I and the results expressed as percent of the input bound. The prototype assay is compared with an existing commercially available ELISA kit using patient sera obtained at various time periods up to 7 months after IV MAb injection for radioimmunoscintigraphy. 18 refs., 2 tabs., 1 fig

  2. Indirect micro-immunofluorescence test for detecting type-specific antibodies to herpes simplex virus.

    OpenAIRE

    Forsey, T; Darougar, S

    1980-01-01

    A rapid indirect micro-immunofluorescence test capable of detecting and differentiating type-specific antibodies to herpes simplex virus is described. The test proved highly sensitive and, in 80 patients with active herpes ocular infection, antibody was detected in 94%. No anti-herpes antibody was detected in a control group of 20 patients with adenovirus infections. Testing of animal sera prepared against herpes simplex virus types 1 and 2 and of human sera from cases of ocular and genital h...

  3. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter; Frøkiær, Hanne; Hearty, Stephen; O'Kennedy, Richard; Hejgaard, Jørn; Nicolaisen, Mogens; Justesen, Annemarie Fejer

    2007-01-01

    The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine m......Ab-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard...... curves for both assays had good intra- and interday reproducibility. The subtractive inhibition ELISA had greater sensitivity with a detection limit of 1.5 105 spores ml1. Cross-reactivity studies of Pst mAb8 in the subtractive inhibition ELISA, showed reaction with other Puccinia spores only, suggesting...

  4. Chemo-enzymatic production of O-glycopeptides for the detection of serum glycopeptide antibodies

    DEFF Research Database (Denmark)

    Nøstdal, Alexander; Wandall, Hans H

    Protein microarray is a highly sensitive tool for antibody detection in serum. Monitoring of patients' antibody titers to specific antigens is increasingly employed in the diagnosis of several conditions, ranging from infectious diseases, allergies, autoimmune diseases, and cancer. In this protoc...... we present a detailed method for enzymatic generation of disease-specific O-glycopeptides and how to monitor the antibody response to these in serum using microarray technology.......Protein microarray is a highly sensitive tool for antibody detection in serum. Monitoring of patients' antibody titers to specific antigens is increasingly employed in the diagnosis of several conditions, ranging from infectious diseases, allergies, autoimmune diseases, and cancer. In this protocol...

  5. Detection of Breda virus antigen and antibody in humans and animals by enzyme immunoassay.

    OpenAIRE

    Brown, D W; Beards, G M; Flewett, T. H.

    1987-01-01

    Enzyme immunoassays were developed for the detection of Breda virus antibody and antigen. Cattle sera collected in the United Kingdom were found to have a high prevalence of antibody (55%) to Breda virus when examined in a competitive enzyme-linked immunosorbent assay. A low prevalence of antibody was found in pigs (2.2%), and no antibody was found in sheep or goat sera. No antibody to either Breda virus or Berne virus was detected in human sera collected from veterinarians and farm workers. ...

  6. [Detection of antigen-antibody interaction of human adenovirus by the method of surface plasmon resonance].

    Science.gov (United States)

    Nosach, L M; Boltovets', P M; Povnytsia, O Iu; Zhovnovata, V L; Zakharenko, O M; Snopok, B A; Shyrshov, Iu M; Diachenko, N S

    2005-01-01

    A possibility to detect adenoviral protein--hexon, using specific antibodies by surface plasmon resonance (SPR) was demonstrated. The hexon of the human adenovirus 2 (Ad2) binds to antibodies immobilized on the sensor surface treated by KNCS and protein A Staphylococcus aureus. The specificity of antihexon antibodies was demonstrated by indirect method of fluorescent antibodies (MFA) and cellular variant of the immunoassay (cELISA). PMID:16250237

  7. Antibody Detection and Kinetics of Antibody Production during Early Stages of Immunization with Hepatitis B Virus Vaccine▿

    OpenAIRE

    Odinsen, Odd; Owusu-Ofori, Shirley; Dompreh, Albert; Sarkodie, Francis; Opare-Sem, Ohene; Parker, David; Allain, Jean-Pierre

    2007-01-01

    Antibodies to influenza virus and human immunodeficiency virus are detectable in B cells during the early stages of the immune response, prior to their occurrence in plasma. To investigate similar phenomena in a model of immunization against hepatitis B virus (HBV) infection, medical students in Ghana were screened for HBV markers, HBV surface (HBs) antigen (HBsAg), and HBV core antibodies (anti-HBc). Consenting volunteers, 24 of whom were seronegative (susceptible) and 2 of whom were positiv...

  8. A new approach to detection of incomplete antibodies using hydrogel chromatography medium.

    Science.gov (United States)

    Wang, HongMei; Chen, YeZhou; Ding, ShaoHua; Duan, ShengBao; Tian, JingJing; Meng, QingLin; Wei, ShuangShi; Li, Yong

    2015-12-01

    In assays for incomplete antibody detection, several washing steps are required to remove unbound globulins which may cause false negatives. Here, we present an improved approach employing hydrogel chromatography medium (HCM) in the detection of incomplete antibodies. After a rapid single-step centrifugation, incomplete antibodies, attached to red blood cells (RBCs), were separated from the reaction mixture using HCM and sedimentation. This method obviates the need for multiple centrifugation steps found in conventional Tube-Coombs tests. The HCM-Coombs tests may have a wide range of applications for incomplete antibody detection. PMID:26099667

  9. The production of recombinant single chain antibody fragments for the detection of illicit drug residues

    OpenAIRE

    Brennan, Joanne

    2005-01-01

    Recombinant antibodies represent a more sensitive and specific detection tool for immunoanalysis. The research carried out for this thesis describes the production of genetically-derived single chain antibody fragments to detect illicit drugs. A variety of novel recombinant antibody fragments against morphine-3-glucuronide, a metabolite of heroin has been produced. A monomeric, dimeric and enzyme-labelled scFv were characterised with respect to their binding abilities and cross reactiviti...

  10. Detection of IgG antibodies against Bordetella pertussis with 125I-protein A

    International Nuclear Information System (INIS)

    A method for the detection of IgG antibodies against Bordetella pertussis is described, based on the principle of 'sandwich' radioimmunoassay. 125I protein A is used as radioactive tracer. The influence of amounts of antigen, antibody, radioactive tracer, incubation time and temperature were tested and the optimal conditions for the assay are described. The procedure offers a simple, quick, and sensitive method for detecting antibodies against B. pertussis. Application and limitation of the test are discussed. (orig.)

  11. Blood collection on filter paper for HIV antibodies detection: experience of SampaCentro Project

    OpenAIRE

    Marcia J. Castejon; Rosemeire Yamashiro; Carmem A. F. Oliveira; Carmen Lúcia Soares; Maria Amélia de S M Veras

    2015-01-01

    Introduction: Blood samples collected on filter paper (dried blood spot [DBS]) is an immunoassay that has been used for antibodies screening. Objective: To evaluate the strategy of DBS blood collection for detection of HIV antibodies, evaluation of Q-Preven HIV 1 + 2 - DBS kit lot, and to analyze the stability of DBS samples. Methods: Blood collection on DBS was performed according to World Health Organization (WHO) recommendations. The evaluation of the kit lot for HIV antibodies detection w...

  12. Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis

    OpenAIRE

    Pramanik, J; Lodam, A. N.; Badole, C.M.; Reddy, M. V. R.; Patond, K. R.; Harinath, B. C.

    2000-01-01

    Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity ...

  13. Technetium-99m pertechnetate - a tracer for radiolabelling antibody for inflammation detection

    International Nuclear Information System (INIS)

    The polyclonal antibody, Human Immunoglobulin G (HlgG) was reduced by using 2-mercaptoethanol with molar ratio of 1000:1 (i.e. mercaptoethanol:antibody). The reduction of the antibody, was carried out for 30 minutes at room temperature. The reduced antibody was purified by using Sephadex G-25 fine column. The antibody kit for the detection of inflammation was prepared aseptically in Class 1 Laminar Flow cabinet. The kit passed the sterility test. Upon reconstitution of the antibody kit with sodium pertechnetate-99m (99mTc) solution, the labelling efficiency obtained was more than 95%. This preparation was stable up to 24-hour stored at room temperature. Gamma camera scans showed the accumulation of technetium-99m labelled antibody (99mTc-HIgG) at the turpentine-induced inflammation of female Sprague-Dawley rats. This indicated the possibility of using 99mTc-HIgG for inflammation detection. (author)

  14. 9 CFR 113.47 - Detection of extraneous viruses by the fluorescent antibody technique.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Detection of extraneous viruses by the fluorescent antibody technique. 113.47 Section 113.47 Animals and Animal Products ANIMAL AND PLANT HEALTH... fluorescent antibody technique. The test for detection of extraneous viruses by the fluorescent...

  15. Stochastic simulation modeling to determine time to detect Bovine Viral Diarrhea antibodies in bulk tank milk

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Enøe, Claes; Krogh, Kaspar;

    2014-01-01

    A stochastic simulation model was developed to estimate the time from introduction ofBovine Viral Diarrhea Virus (BVDV) in a herd to detection of antibodies in bulk tank milk(BTM) samples using three ELISAs. We assumed that antibodies could be detected, after afixed threshold prevalence of...

  16. Performance of three microimmunofluorescence assays for detection of Chlamydia pneumoniae immunoglobulin M, G, and A antibodies

    DEFF Research Database (Denmark)

    Bennedsen, Mette; Berthelsen, Lene; Lind, Inga

    2002-01-01

    , 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from...

  17. A solid-phase radioimmunoassay for detection of tetanus antibodies

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay has been developed as a screening technique for tetanus antibodies in blood plasma. It is based on the principle of a commercial test for Hepatitis B antibody. Compared to previous screening techniques, the radioimmunoassay showed better stability with no apparent loss of sensitivity over a 2 month period. This technique has proved useful in determining tetanus immunity and in monitoring free antibody level in treated cases of clinical tetanus. (U.K.)

  18. Sensitive detection of platelet-specific antibodies with a modified MAIPA using biotinylated antibodies and streptavidin-coated beads.

    Science.gov (United States)

    Mörtberg, Anette; Meinke, Stephan; Berg, Petra; Killie, Mette Kjær; Kjeldsen-Kragh, Jens; Järås, Kerstin; Refsum, Erle; Höglund, Petter; Wikman, Agneta

    2016-07-01

    We have developed a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) with enhanced sensitivity in detecting antibodies against human platelet antigens (HPA), using biotinylated monoclonal antibodies, streptavidin-coated beads and detection by flow cytometry. The beads-MAIPA gave superior signal-to-noise resolution (>10-fold higher) for detection of anti-HPA-1a and anti-HPA-5b compared with the in-house standard MAIPA. Also, efficient and reproducible detection of anti-HPA-15 (CD109) was shown. The enhanced sensitivity was confirmed using WHO International Reference Reagents for anti-HPA-1a, anti-HPA-3a and anti-HPA-5b, which allowed comparison of detection endpoints with other laboratories. Finally, the beads-MAIPA was validated for quantification of anti-HPA-1a. The lower limit of quantification was 0.4IU/mL for beads-MAIPA, compared to 1IU/mL previously reported for standard MAIPA. Based on improved performance against all HPA-antibodies tested, the beads-MAIPA has replaced the standard MAIPA in our laboratory in diagnostics of conditions due to HPA-immunization, such as fetal and neonatal alloimmune thrombocytopenia (FNAIT). PMID:27059653

  19. Detection of Rh antibodies using two low ionic diluents: Extension of the incubation time and the number of Rh antibodies detected

    Directory of Open Access Journals (Sweden)

    Skaik Y

    2008-01-01

    Full Text Available Background: Low ionic strength solution (LISS is used to increase the rate of association of antibody to the corresponding antigen during antibody detection tests. A number of LISSs are available on the market. Aims: The efficiency of two commercial low ionic diluents, DiaMed ID-CellStab and Inverclyde LISS were assessed using the DiaMed-ID LISS Coombs microtube column system and an incubation time varying from 15 to 35 min. Materials and Methods: One-hundred samples containing five Rh antibodies (anti-D, anti-C, anti-E, anti-c and anti-e were tested against commercial red cells using the two low ionic diluents after 15, 25 and 35 min. Results: The Inverclyde LISS detected 91, 95 and 96% of the Rh antibodies compared to 78, 79 and 83% for ID-CellStab after 15, 25 and 35 min incubation time, respectively, for both methods. Conclusions: The Inverclyde LISS is a more suitable and efficient diluent than ID-CellStab for the detection of Rh antibodies. The sensitivity of Rh antibody detection increased for the both methods as the incubation time increased.

  20. Detection of novel diagnostic antibodies in ankylosing spondylitis: An overview.

    Science.gov (United States)

    Quaden, Dana H F; De Winter, Liesbeth M; Somers, Veerle

    2016-08-01

    Ankylosing spondylitis (AS) is a debilitating, chronic, rheumatic disease characterized by inflammation and new bone formation resulting in fusion of the spine and sacroiliac joints. Since early treatment is impeded by a delayed diagnosis, it is highly important to find new biomarkers that improve early diagnosis and may also contribute to a better assessment of disease activity, prognosis and therapy response in AS. Because of the absence of rheumatoid factor, AS was long assumed to have a seronegative character and antibodies are thus not considered a hallmark of the disease. However, emerging evidence suggests plasma cells and autoantibodies to be involved in the disease course. In this review, the role of B cells and antibodies in AS is discussed. Furthermore, an overview is provided of antibodies identified in AS up till now, and their diagnostic potential. Many of these antibody responses were based on small study populations and further validation is lacking. Moreover, most were identified by a hypothesis-driven approach and thus limited to antibodies against targets that are already known to be involved in AS pathogenesis. Hence, we propose an unbiased approach to identify novel diagnostic antibodies. The already successfully applied techniques cDNA phage display and serological antigen selection will be used to identify antibodies against both known and new antigen targets in AS plasma. These newly identified antibodies will enhance early diagnosis of AS and provide more insight into the underlying disease pathology, resulting in a more effective treatment strategy and eventually an improved disease outcome. PMID:27288842

  1. Detection of antibodies to the 20s proteasome by ELISA

    DEFF Research Database (Denmark)

    Jørgensen, Karin Meinike; Frederiksen, Jette Lautrup; Nielsen, Christoffer Tandrup;

    2013-01-01

    The presence of antibodies against the 20S proteasome has been correlated with diseases like multiple sclerosis (MS) and systemic lupus erythematosus (SLE) but no definite association has been established. In order to investigate this further, we optimized an ELISA for proteasome antibodies and...

  2. A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

    Directory of Open Access Journals (Sweden)

    Yuan-Cheng Cao

    2015-01-01

    Full Text Available This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen; goat anti-human IgG (Cy3 or FITC was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody; finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were applied to the concomitant assay to analyze the feasibility. The first approach applied FITC and Cy3 when both targets were present at the same time, resulting in 50 ng/mL of the antibody detection limit and 10 ng/mL of antigen detection limit in the quantitative measurements of target concentration, taking the consideration of FRET efficiency of 68% between donor and acceptor. The sequential approach tended to lower the signal/noise (S/N ratio and the detection of the model antigen (lower than 1 ng/mL had better sensitivity than the model antibody (lower than 50 ng/mL. This combined antigen/antibody method might be useful for combined detection of antigens and antibodies. It will be helpful to screen for both antigen and antibody particularly in the situations of the multiserotype and high-frequency mutant virus infections.

  3. DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

    Science.gov (United States)

    Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

    2016-09-15

    We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. PMID:27111124

  4. Detection of antitrophoblast antibodies in the sera of patients with anticardiolipin antibodies and fetal loss.

    Science.gov (United States)

    McCrae, K R; DeMichele, A M; Pandhi, P; Balsai, M J; Samuels, P; Graham, C; Lala, P K; Cines, D B

    1993-11-01

    Women with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with ACLA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P trophoblast cells. In most cases, sera from which ACLA were adsorbed by cardiolipin-containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an approximately 62-kD protein from the trophoblast cell surface, stimulated the release of arachidonic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with ACLA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise. PMID:7693045

  5. Radiolabeled antibody in the detection of infection using endocarditis as a model

    International Nuclear Information System (INIS)

    The authors have examined a method to detect infections using radiolabeled antibodies. Staphylococcal endocarditis was chosen as a model because it poses a common clinical diagnostic problem. The experiments demonstrate that biologically active antibodies may be extracted and efficiently labeled by a relatively simple process. This has the potential to make the specificity of the in vivo antigen-antibody reaction available through the use of autologously extracted, labeled γ-globulin

  6. Anticomplement immunofluorescence test that uses isolated fibroblast nuclei for detection of antibodies to human cytomegalovirus.

    OpenAIRE

    Mintz, L; Miner, R. C.; Yeager, A S

    1980-01-01

    Cytomegalovirus antibodies were measured in human sera by a nuclear anticomplement immunofluorescence test that used as antigen the isolated nucleic of virus-infected fibroblasts cells lysed in distilled water. The method exhibited less nonspecific fluorescence than either a conventional whole-cell anticomplement immunofluorescence test or an indirect fluorescent antibody test applied to the same isolated nuclear substrate. The assay detected 97.5% of 40 antibody-positive sera, compared with ...

  7. Radioimmunoassay for detection of VP1 specific neutralizing antibodies of foot and mouse disease virus

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay was developed for the detection of antibodies against a specific region of the VP1 protein of the A24 and O1 serotypes of foot and mouth disease virus. The antibody titers from the radioimmunoassay showed a positive correlation with neutralizing antibody titers determined by a mouse protection assay. The specificity of the assay resides in the peptide used as antigen. The assay is rapid, reproducible and does not require the use of whole virions. (orig.)

  8. Evaluation of a competitive enzyme immunoassay for detection of Coxiella burnetii antibody in animal sera.

    OpenAIRE

    Soliman, A.K.; Botros, B A; Watts, D M

    1992-01-01

    A competitive enzyme immunoassay (CEIA) was established and compared with other serological techniques for detecting Coxiella burnetii antibody in camels, goats, and sheep. This technique was evaluated because a conjugated anti-camel immunoglobulin was not available to serve as a direct signal for the demonstration of antigen-antibody reaction. A C. burnetii antibody-positive human serum and a peroxidase-conjugated anti-human immunoglobulin G were used as an indicator system competing against...

  9. Referencing cross-reactivity of detection antibodies for protein array experiments.

    Science.gov (United States)

    Lemass, Darragh; O'Kennedy, Richard; Kijanka, Gregor S

    2016-01-01

    Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays. PMID:27335636

  10. Enhanced antigen detection in immunohistochemical staining using a 'digitized' chimeric antibody.

    Science.gov (United States)

    Eng, Hui-Yan; Wang, Cheng-I; Xue, Yuezhen; Lee, Chia-Yin; Zulkifli, Sarah Binte; Chiam, Poh-Cheang; Ghadessy, Farid J; Lane, David P

    2016-01-01

    The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality. PMID:26508747

  11. Characterization of Antibodies for Grain-Specific Gluten Detection.

    Science.gov (United States)

    Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa

    2016-03-01

    Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation. PMID:26878584

  12. Cell adhesion molecules: detection with univalent second antibody

    OpenAIRE

    1980-01-01

    Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against c...

  13. Solid phase radioimmunoassay for detection of antibodies to extractable nuclear antigens

    Energy Technology Data Exchange (ETDEWEB)

    Whittingham, S. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1983-06-24

    A solid phase radioimmunoassay is described for the detection of autoantibodies to the saline-soluble extractable nuclear antigens, ribonucleoprotein (RNP) and SS-B (or La). This assay depends on enrichment of antigens from a crude, commercially available (Pel Freez, USA) extract of rabbit thymus by absorption to the F(ab)/sub 2/ fraction of specific high titre antibody attached to a microtitre plate. Serum antibody reactive with this antigen is then detected by /sup 125/I-labelled Protein A. The assay is simple and is more sensitive than the gel diffusion assays in general use for detecting such antibodies.

  14. A solid phase radioimmunoassay for detection of antibodies to extractable nuclear antigens

    International Nuclear Information System (INIS)

    A solid phase radioimmunoassay is described for the detection of autoantibodies to the saline-soluble extractable nuclear antigens, ribonucleoprotein (RNP) and SS-B (or La). This assay depends on enrichment of antigens from a crude, commercially available (Pel Freez, USA) extract of rabbit thymus by absorption to the F(ab)2 fraction of specific high titre antibody attached to a microtitre plate. Serum antibody reactive with this antigen is then detected by 125I-labelled Protein A. The assay is simple and is more sensitive than the gel diffusion assays in general use for detecting such antibodies. (Auth.)

  15. SPR Biosensor for the Detection of L. monocytogenes using Phage Displayed Antibody

    Science.gov (United States)

    Whole cells of Listeria monocytogenes were detected with a compact, surface plasmon resonance (SPR) sensor using a phage-displayed scFv antibody to the virulence factor ActA for biorecognition. Phage Lm P4:A8, expressing the scFv antibody fused to the pIII surface protein was immobilized to the se...

  16. Towards a peptide-based suspension array for the detection of pestivirus antibodies in swine.

    Science.gov (United States)

    van der Wal, Fimme J; Jelsma, Tinka; Fijten, Helmi; Achterberg, René P; Loeffen, Willie L A

    2016-09-01

    Classical swine fever (CSF) is a highly contagious and lethal disease in swine. Serological tests for the diagnosis of CSF need not only to detect antibodies against CSFV, but also need to differentiate these from antibodies against other pestiviruses. To investigate the possibilities of specific peptide-based serology, various synthetic peptides that represent a well-described linear epitope of the CSFV E2 protein (TAVSPTTLR) were used to test the viability of a peptide-based suspension array for the detection of antibodies against pestiviruses in swine. The results show that N-terminally biotinylated peptides can bind to avidin conjugated beads, and function in detection of the corresponding monoclonal antibody WH303. There are indications that the length of the spacer between epitope and biotin affect the efficiency of the peptide-antibody interaction. A protocol was established that enables probing for antibodies in porcine sera, where neutravidin-blocking of serum and the use of empty control beads for normalization was crucial. With a set of porcine sera with antibodies against various pestiviruses, the proof of concept of a peptide-based suspension array for specific detection of antibodies against pestiviruses in porcine sera was demonstrated. PMID:27166561

  17. Antigens of Trypanosoma cruzi detected by different classes and subclasses of antibodies.

    Science.gov (United States)

    Araujo, F G; Heilman, B; Tighe, L

    1984-01-01

    The kinetics of the appearance of specific IgM and of subclasses of IgG antibodies following infection of mice with Trypanosoma cruzi and the antigens of amastigotes and epimastigotes recognized by these antibodies were investigated by using the indirect immunofluorescent antibody test and the protein transfer technique. IgM and IgG2 antibodies were detected almost at the same time and peaked on day 30 and 40 of infection respectively. On day 150 of infection IgM antibodies were barely detectable whereas IgG2 antibodies were still at a high titre. IgG3 and IgG1 antibodies were first detected on days 20 and 30, peaked on days 30 and 50 respectively, and were still detected at low titres on day 150 of infection. The immunofluorescent test with each antibody revealed differences in the patterns of the fluorescent staining of the organisms, particularly with amastigotes. These differences were most striking with IgG3 antibodies. Fluorescent staining with IgM or IgG1 was localized mostly on one or two poles of the amastigotes; with IgG2 it was over the entire body of either amastigotes or epimastigotes; and with IgG3 it was in the form of very small spots over the entire body of organisms of both stages. The Western blots revealed that each antibody apparently recognized the same antigens in both the epimastigote and amastigote antigen preparations. The 90 Kd MW antigen of epimastigotes as well as two antigens of MW 92 Kd and 90 Kd of amastigotes were recognized by each of the antibodies examined. PMID:6438837

  18. Towards On-site Pathogen Detection Using Antibody-based Sensors

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Nicolaisen, Mogens; Justesen, Annemarie Fejer

    2008-01-01

    In this paper the recent progress within biosensors for plant pathogen detection will be reviewed. Bio-recognition layers on sensors can be designed in various ways, however the most popular approach is to immobilise antibodies for specific capture of analytes. Focus will be put on antibody surface......-immobilisation strategies as well as the use of antibodies in the widely used sensors, quartz crystal microbalance, surface plasmon resonance and cantilevers. We will describe the available data on antibody-based plant pathogen detection and furthermore use examples from detection of the pathogens Salmonella, Listeria...... monocytogenes, Streptococcus mutans, Bacillus cereus, Bacillus anthracis, Campylobacter and Escherichia coli. We will touch upon optimal assay design and further discuss the strengths and limitations of current sensor technologies for detection of viruses, bacteria and fungi....

  19. Detection of antinuclear antibodies by solid-phase immunoassays and immunofluorescence analysis

    DEFF Research Database (Denmark)

    Fenger, Mogens; Wiik, Allan; Høier-Madsen, Mimi; Lykkegaard, Jens Jørgen; Rozenfeld, Teresa; Hansen, Michael S; Samsoe, Bente Danneskjold; Jacobsen, Søren

    2004-01-01

    BACKGROUND: Antinuclear antibodies (ANAs) are associated with several inflammatory rheumatic diseases. The aim of the present work was to evaluate enzyme immunoassays (EIAs) and compare them with classic immunofluorescent analysis (IFA) for the detection of ANA. METHODS: Seven enzyme immunoassays...

  20. Towards on-site pathogen detection using antibody-based sensors.

    Science.gov (United States)

    Skottrup, Peter Durand; Nicolaisen, Mogens; Justesen, Annemarie Fejer

    2008-11-15

    In this paper, the recent progress within biosensors for plant pathogen detection will be reviewed. Bio-recognition layers on sensors can be designed in various ways, however the most popular approach is to immobilise antibodies for specific capture of analytes. Focus will be put on antibody surface-immobilisation strategies as well as the use of antibodies in the widely used sensors, quartz crystal microbalance, surface plasmon resonance and cantilevers. We will describe the available data on antibody-based plant pathogen detection and furthermore use examples from detection of the pathogens Salmonella, Listeria monocytogenes, Streptococcus mutans, Bacillus cereus, Bacillus anthracis, Campylobacter and Escherichia coli. We will touch upon optimal assay design and further discuss the strengths and limitations of current sensor technologies for detection of viruses, bacteria and fungi. PMID:18675543

  1. Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

    OpenAIRE

    Anderson, L J; Tsou, C; Parker, R. A.; Chorba, T L; Wulff, H; Tattersall, P; Mortimer, P P

    1986-01-01

    Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscop...

  2. Detection of thrombi using a Tc-99m labelled antifibrin monoclonal antibody (MoAb)

    International Nuclear Information System (INIS)

    This thesis presents an investigation into the possibility of immunoscintigraphic detection of thrombi using an antifibrin monoclonal antibody, and fragments of the latter. The antifibrin antibody and tis fragments were labelled with Ec-99m, which has excellent characteristics for imaging with a gamma camera. The characterization of the antifibrin antibody and its fragments, the assessment of quality of labelling with Tc-99m, and results of experiments in vitro and in animals, which show the potential of immunoscintigraphic detection, are described. (author). 142 refs.; 44 figs.; 5 tabs

  3. Detection of antibodies to single-stranded DNA in naturally acquired and experimentally induced viral hepatitis

    International Nuclear Information System (INIS)

    A sensitive ''Farr'' assay, utilizing 125I-labelled DNA was developed for detecting antibody to single-stranded DNA (anti-ssDNA). The test was shown to be specific and as sensitive as assays using 14C-labelled DNA, for the detection of antibody in patients with connective tissue diseases. Groups of sera from patients with naturally acquired viral hepatitis and experimentally infected chimpanzees were tested for anti-ssDNA by the 125I assay and by counterimmunoelectrophoresis (CIEP). No consistent pattern was observed with either technique, indicating the elevated levels of this antibody are not as reliable markers of parenchymal liver damage as had been previously suggested

  4. A Model System for Concurrent Detection of Antigen and Antibody Based on Immunological Fluorescent Method

    OpenAIRE

    2015-01-01

    This paper describes a combined antigen/antibody immunoassay implemented in a 96-well plate using fluorescent spectroscopic method. First, goat anti-human IgG was used to capture human IgG (model antigen); goat anti-human IgG (Cy3 or FITC) was used to detect the model antigen; a saturating level of model antigen was then added followed by unlabelled goat anti-human IgG (model antibody); finally, Cy3 labelled rabbit anti-goat IgG was used to detect the model antibody. Two approaches were appli...

  5. Anti-cysticercus antibody detection in saliva as a potential diagnostic tool for neurocysticercosis

    Science.gov (United States)

    Saha, Rumpa; Roy, Priyamvada; Das, Shukla; Shah, Dheeraj; Agarwal, Sunil; Kaur, Iqbal Rajinder

    2016-01-01

    Objectives: This study was planned to determine the usefulness of anti-cysticercus IgG antibody detection in saliva for neurocysticercosis (NCC) diagnosis, along with serum C-reactive protein (CRP) level to serve as a surrogate marker. Materials and Methods: In this prospective study of 14 months duration, blood and saliva samples were collected from 40 patients suspected to be suffering from NCC and were subjected to anti-cysticercus IgG antibody detection by ELISA. Serum CRP levels were estimated as acute-phase reactant by high sensitivity CRP ELISA. Results: Anti-cysticercus IgG was detected in serum and saliva of 34 and 30 patients, respectively. Cases positive for salivary antibody were positive for serum antibody and their serum CRP level was higher than normal. Cases negative for salivary antibody had low serum CRP levels. Anti-cysticercus IgG detection in saliva was 88.24% sensitive, 100% specific, and had a positive predictive value of 100% and negative predictive value of 60%. Positive salivary anti-cysticercus IgG and high serum CRP level showed a significant association. Difference between CRP levels of patients positive for anti-cysticercus antibody in both serum and saliva, and patients positive for antibody in serum but not saliva was highly significant. Conclusions: Saliva, being painless and noninvasive, can be used as alternative to serum for NCC diagnosis. PMID:27570404

  6. In vivo detection of prostatic carcinoma with antibodies against prostatic acid phosphatase

    International Nuclear Information System (INIS)

    Serum prostatic acid phosphates (PAP) immunoassay is used to evaluate patients with prostatic carcinoma; however, as with other tumor markers, the enzyme levels do not necessarily reflect the presence or extent of tumor. The authors investigated the use of radiolabeled PAP antibodies for the in vivo detection of prostatic carcinoma by external scintillation imaging. Nine patients with prostatic carcinoma were entered into the study. Each received from 2.0 to 2.5 mCi of I-131 labeled antibody to PAP, administered i.v. The immunogen (PAP) was purified from normal human seminal fluid. Antiserum was prepared in rabbits by injecting the purified PAP. The antibodies were labeled with I-131 by chloramine-T method (10 to 20 Ci/g of IgG). Total body images were obtained at 24 and 48 hrs following administration of the labeled antibody. Nontarget I-131 activity was diminished by computer processing. Tumor sites detected by I-131 antibodies were correlated with other diagnostic procedures. In 7 of 9 patients primary and metastatic sites of cancer were detected by antibody imaging, however, no bone lesions were detected (6 cases). In 3 patients with concomitant pulmonary tumors, one was identified as of prostate origin. The serum PAP was normal in 4 patients; however, the primary tumor was identified in 3 of these. These findings suggest that the localization of prostatic carcinoma by means of in-vivo imaging of labeled antibodies to PAP is feasible and offers diagnostic opportunities based upon the functional characteristics

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

    International Nuclear Information System (INIS)

    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 D3 and should be useful for the detection of antibodies to ligand-binding proteins in general

  8. 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.

  9. Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

    Science.gov (United States)

    Arts, Remco; den Hartog, Ilona; Zijlema, Stefan E; Thijssen, Vito; van der Beelen, Stan H E; Merkx, Maarten

    2016-04-19

    Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays. PMID:27018236

  10. Detection of B-lymphocytes secreting antibodies to Dermatophagoides antigens

    DEFF Research Database (Denmark)

    Sparholt, S H; Barington, T; Schou, C

    1991-01-01

    An enzyme-linked immunospot assay (ELI-spot assay) has been established to count individual cells secreting antibodies to Dermatophagoides spp. allergens. Initial optimization of the assay was performed using Der p I-specific murine hybridoma cell lines. Inhibition with soluble purified allergen ...

  11. DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens.

    Science.gov (United States)

    Seymour, Elif; Daaboul, George G; Zhang, Xirui; Scherr, Steven M; Ünlü, Nese Lortlar; Connor, John H; Ünlü, M Selim

    2015-10-20

    Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications. PMID:26378807

  12. Antikörpernachweis bei der Notfalltransfusion [Antibody detection in emergency transfusions. A comparison of 3 different methods

    OpenAIRE

    Heim, M. U.; Alraun, K.; Hansen, Ernil; Pachmann, U; Böck, M.; Mempel, W.

    1988-01-01

    We compared the manual Polybrene technique to three standard methods (albumin/Coombs, LISS/Coombs, and enzyme/papain) on 113 red cell antibodies. Polybrene identified 8 antibodies of the Rhesus system missed by standard methods, whereas 3 antibodies could only be detected by the standard techniques. Four antibodies were identified only in saline solution at room temperature; 6 were found by use of Polybrene exclusively in the additional Coombs phase. In addition, 61 antibodies were tested by ...

  13. Referencing cross-reactivity of detection antibodies for protein array experiments [version 1; referees: 1 approved, 2 approved with reservations

    OpenAIRE

    Darragh Lemass; Richard O'Kennedy; Kijanka, Gregor S.

    2016-01-01

    Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human protei...

  14. A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

    Directory of Open Access Journals (Sweden)

    Larry H. Stanker

    2013-11-01

    Full Text Available Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT, produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A–H have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s for individual antibodies ranging from 10 to 48 × 10−11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D., ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule and readily detects toxin in those food samples tested.

  15. Double-antigen sandwich ELISA for the detection of anti-hepatitis C virus antibodies.

    Science.gov (United States)

    He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Ling, Shigan; Zhang, Heqiu

    2011-01-01

    A double-antigen sandwich ELISA was developed a detection of HCV antibodies by a recombinant multi-epitope HCV antigen and a biotin-streptavidin amplification system. Three plasma specimens from 1708 individuals who were suspected previously to be HCV-positive using an HCV antibody diagnostic kit (Chuangxin, Xiamen, China) displayed negative results when using the ELISA. These results were validated by a recombinant immunoblotting assay (two were negative, and one was indeterminate). Among 889 blood specimens donated for clinical evaluation, 246 were positive and 630 were negative using the ELISA. The sensitivity and specificity of the ELISA were 98.7% and 100%, respectively. In 43 donors and 14 patients with chronic hepatitis C, the detectable rates for HCV IgM by both ELISA and the HCV anti-IgM detection reagents (Huimin, Shenyang, China) were 100%, and the detectable rate for HCV IgG using an indirect HCV-antibody detection kit (GWK, Beijing, China) was 98.3%. Thus, the double-antigen sandwich ELISA exhibits strong specificity and sensitivity and has been approved by the China State Food and Drug Administration (SFDA). The performance of the double-antigen sandwich ELISA was similar to the Ortho ELISA 3.0. It did not give false-negative results otherwise IgM was undetectable using an indirect HCV-antibody detection kit. This ELISA provides another method for the detection of HCV antibodies. PMID:21029749

  16. Detection of Root Mucilage Using an Anti‐fucose Antibody

    OpenAIRE

    Sinha Roy, S; Mittra, B.; Sharma, S.; Das, T K; C.R. Babu

    2002-01-01

    Plant root mucilage is known to enhance soil quality by contributing towards the soil carbon pool, soil aggregation, detoxification of heavy metal ions and interactions with rhizospheric microflora. Mucilage consists of many monosaccharide units, including fucose which can be used as an indicator for plant root based polysaccharides. This is the first report of an immunological technique developed to use anti‐fucose antibodies as markers for probing and localizing fucosyl residues in mucilage...

  17. Detection of Rh antibodies using two low ionic diluents: Extension of the incubation time and the number of Rh antibodies detected

    OpenAIRE

    Skaik Y

    2008-01-01

    Background: Low ionic strength solution (LISS) is used to increase the rate of association of antibody to the corresponding antigen during antibody detection tests. A number of LISSs are available on the market. Aims: The efficiency of two commercial low ionic diluents, DiaMed ID-CellStab and Inverclyde LISS were assessed using the DiaMed-ID LISS Coombs microtube column system and an incubation time varying from 15 to 35 min. Materials and Methods: One-hundred samples containing five Rh anti...

  18. Early detection of antibody to human immunodeficiency virus type 1 by using an antigen conjugate immunoassay correlates with the presence of immunoglobulin M antibody.

    OpenAIRE

    Gallarda, J L; Henrard, D R; Liu, D.; Harrington, S.; Stramer, S L; Valinsky, J E; Wu, P

    1992-01-01

    Sequential plasma samples obtained from 16 individuals who seroconverted were tested for the presence of antibody to human immunodeficiency virus type 1 (HIV-1) by an antigen conjugate enzyme immunoassay (EIA) and a conventional antibody conjugate assay. In 11 of these individuals, the antigen conjugate assay detected antibody to HIV-1 2 to 11 days (mean, 5.5 days) earlier than the antibody conjugate assay. In 11 individuals, HIV-1 p24 antigen was detected a median of 6.5 days (range, 3 to 14...

  19. Evaluation of a monoclonal antibody able to detect live Listeria monocytogenes and Listeria innocua

    DEFF Research Database (Denmark)

    Sølve, Marianne; Boel, Jeppe; Nørrung, Birgit

    2000-01-01

    A monoclonal Listeria antibody, designated B4, was evaluated. The ability of the antibody to bind to viable bacteria belonging to Listeria spp, compared to bacteria of the same species killed by beat treatment, acid or base treatment, sanitizers, and irradiation was examined. The antibody was found...... to react with viable L. monocytogenes and L. innocua, but not with heat-killed (72 degrees C, 5 min) strains of these organisms. When L. monocytogenes and L. innocua were killed by methods other than heat treatment, it was ambiguous whether the antibody detected the organism or not. It was concluded...... that the B4 antibody has potential to be used in an immune capture step to capture live L, monocytogenes and L. innocua from foods prior to identification of L. monocytogenes by polymerase chain reaction (PCR)....

  20. Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues

    DEFF Research Database (Denmark)

    Couchman, J R; Caterson, B; Christner, J E;

    1984-01-01

    glycosaminoglycan (GAG), particularly with respect to self-association and interactions with other extracellular matrix components. Interactions with specific molecules from different connective tissue types, such as the collagens and their associated glycoproteins, could be favoured by particular charge...... organizations on the GAG molecule endowed by the sulphate groups. So far, it has not been possible to identify and map chondroitins of differing sulphation in tissues, but we have now raised three monoclonal antibodies which specifically recognize unsulphated, 4-sulphated and 6-sulphated chondroitin and...

  1. Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Frøkiær, Hanne; Hearty, Stephen;

    2007-01-01

    The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine m......Ab-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard...

  2. Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

    Science.gov (United States)

    Di Cristofaro, Julie; Frassati, Coralie; Montagnie, Rolande; Basire, Agnes; Merieux, Yves; Picard, Christophe

    2015-01-01

    Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future. PMID:25101933

  3. Experimental results of antigliadin antibodies detection using long period fiber grating

    Science.gov (United States)

    Corres, J. M.; Matias, I. R.; Goicoechea, J.; Arregui, F. J.; Viegas, D.; Araújo, F. M.; Santos, J. L.

    2008-04-01

    In this work a new nano-biofilm is proposed for the detection of celiac disease (CD). A long-period fiber grating (LPFG) is used as a transducer and the surface of the fiber is coated with a precursor layer of SiO2-nanospheres using the electrostatic self-assembly technique (ESA). This layer has been designed in order to create a substrate of high porosity where the gliadins could be deposited. Under the presence of specific antibodies antigliadin antibodies (AGA) the refractive index of the overlay changes giving a detectable shift in the resonance wavelength of the LPFG. Concentrations as low as 5 ppm were detected.

  4. Comparison between indirect immunofluorescence and microneutralization for detection of antibodies to polioviruses.

    OpenAIRE

    Pettit, C.; Minnich, L L; Shehab, Z M; Ray, C G

    1987-01-01

    Indirect immunofluorescence and microneutralization methods for the detection of antibodies to poliovirus serotypes 1, 2, and 3 were compared. Of the 41 sera tested for poliovirus type 1 antibody, 40% were in complete agreement, 55% differed by one dilution, and 5% differed by two dilutions. For poliovirus type 2, 37 sera were tested; 56% completely agreed, and 44% differed by only one dilution. For poliovirus type 3, complete agreement occurred in 59% of 33 sera, while 41% differed by one di...

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

    OpenAIRE

    Sudarisman

    2006-01-01

    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 dis...

  6. Modified indirect hemagglutination test for detection of treponemal antibodies in finger-prick blood.

    OpenAIRE

    Backhouse, J L; Lee, M. H.; Nesteroff, S I; Hudson, B J; Hamilton, P. A.

    1992-01-01

    A modified indirect hemagglutination test for the detection of treponemal antibodies was developed for use with finger-prick blood. By using paired serum and absorbed finger-prick blood from 58 patients from an area previously endemic for yaws and 12 patients without yaws, the modified hemagglutination test was compared with a hemagglutination test for Treponema pallidum and the fluorescent treponemal antibody absorption test. The modified hemagglutination test showed 100% specificity and an ...

  7. Comparison of three commercially available assays for detection of varicella-zoster virus antibody.

    OpenAIRE

    Martins, T B; Jaskowski, T D; Schroder, C; Streeter, B; Hill, H R

    1994-01-01

    Three commercially available diagnostic assays for the detection of antibodies to varicella-zoster virus were evaluated to determine which would be the most suitable for our clinical laboratory. Three different methods were examined: an enzyme-linked immunosorbent assay (ELISA), latex agglutination (LA), and an indirect fluorescent-antibody technique. For the 141 serum specimens tested, the ELISA had an agreement of 90.1% and LA had an agreement of 92.2% with the indirect immunofluorescent-an...

  8. Detection of Antibody against Helicobacter pylori in the Saliva of Patients with Dyspepsia

    OpenAIRE

    Clancy, Robert L.; Cripps, Allan W.; Taylor, Diana C; McShane, Lois A; Webster, Victor J

    1994-01-01

    There is a need to develop noninvasive assays to detect Helicobacter pylori infection in the gastric mucosa, Current dogma predicts that the presence of antibody within saliva should accurately reflect contemporary colonization of the gut mucosa. This study examined the clinical value of a saliva enzyme-linked immunoadsorbent assay (ELISA) for anti-H pylori antibody, compared with the serum ELISA assay, and found the sensitivity of the saliva assay was 89%, specificity 94%, accuracy 93%, posi...

  9. 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.

  10. Potential application of labeled antibodies for thrombus detection

    International Nuclear Information System (INIS)

    Labeling platelets with monoclonal antibodies in whole blood for imaging thrombi is less cumbersome than the established 111In-oxine method. 7E3, a murine monoclonal antibody directed against glycoprotein IIb and/or IIIa on both human and dog platelets was used to label canine platelets. Thrombi were induced by transcatheter placement of a copper coil followed by electrocoagulation. 7E3 was iodinated with 131I and labelled with 111In using 7E3-DTPA conjugate. Whole blood was incubated with 0.5 - 1.0 μg labeled 7E3/mL blood. In 4/4 dogs, experimental deep vein thrombi were identified using both 131I-and 111In-labeled 7E3 within 5-30 min after injection. For both isotopes, 1 h blood clearance was 54 + - 9%. In 1/3 dogs, experimental coronary thrombus could be identified ex vivo at 4 h. Clot to blood ratios ranged between 7 to 13:1. Using the 111In-oxine method, 0/3 coronary thrombi were seen. Thus, 131I-and 111In-labeled 7E3 may be used to readily identify peripheral venous thrombi. For reliable and prompt identification of coronary thrombi, more rapid clearance of the labeled platelets is required. (author)

  11. Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.

    Directory of Open Access Journals (Sweden)

    Marisa J Fortunato

    Full Text Available Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5-2, 29-5, and 45-3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

  12. Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus.

    Science.gov (United States)

    Nielsen, K; Smith, P; Yu, W L; Elmgren, C; Nicoletti, P; Perez, B; Bermudez, R; Renteria, T

    2007-09-20

    A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection thus allowing for true standardization. The assay utilized a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3 but which did not react with protein A/G. This monoclonal antibody was used to compete with antibody in the bovine test serum. Binding of bovine antibody to the smooth lipopolysaccharide antigen was then measured directly with the protein A/G enzyme conjugate. In this case, development of colour in the reaction was indicative of the presence of bovine antibody. The performance characteristics, sensitivity, specificity and exclusion of B. abortus S19 vaccinated animals, of the assay were very similar to those of the classical CELISA. PMID:17467200

  13. Red Blood Cell Antibodies in Hematology/Oncology Patients: Interpretation of Immunohematologic Tests and Clinical Significance of Detected Antibodies.

    Science.gov (United States)

    Hendrickson, Jeanne E; Tormey, Christopher A

    2016-06-01

    Red blood cell (RBC) transfusion is a cornerstone of the management of patients with hematology/oncology disorders. However, a potentially deleterious consequence of transfusion is the development of alloantibodies against blood group antigens present on RBCs. Such alloantibodies can be an obstacle in providing compatible units for transfusion. Providers in this arena must fully understand the testing performed by blood banks, as well as the consequences of detected antibodies. This article reviews immunohematologic tests, describes how autoimmune hemolytic anemia is classified by autoantibodies; outlines RBC alloimmunization rates, and presents strategies to prevent/mitigate the impact of RBC alloimmunization. PMID:27113001

  14. The use of monoclonal antibodies in competitive ELISA for the detection of antibodies to rinderpest and peste des petits ruminants viruses

    International Nuclear Information System (INIS)

    A monoclonal antibody against the haemagglutinin of rinderpest virus has been used in a competitive ELISA (C-ELISA) for the detection of antibodies to rinderpest virus in cattle, sheep, goat and game sera. Unlike the indirect ELISA and the virus neutralisation test (VNT), the C-ELISA detects only antibodies to rinderpest virus and gives no cross-reactivity with antibodies to peste des petits ruminants (PPR) virus. Antibodies to a wide range of strains of rinderpest virus have been detected using this assay, suggesting its suitability for both sero-monitoring and sero-surveillance. Analysis of C-ELISA results from the examination of field sera shows a much greater separation of negative and positive populations as compared to the indirect ELISA. A further monoclonal antibody against the H protein of PPR has also been found suitable for use in a C-ELISA for the detection of antibodies to PPR virus. The use of these two C-ELISA's has made possible rapid differential sero-diagnosis without recourse to cross-VNT testing. The use of monoclonal antibody-based assays will allow much greater standardisation of rinderpest and PPR diagnosis, and following field-trials the C-ELISA will replace the indirect ELISA for sero-monitoring throughout the Pan African Rinderpest Campaign. (author). 3 refs, 6 figs, 1 tab

  15. Development of recombinant antigen array for simultaneous detection of viral antibodies.

    Directory of Open Access Journals (Sweden)

    Yi Liu

    Full Text Available Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV, Rubella virus (RV core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs. The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

  16. Comparative efficacy of antigen and antibody detection tests for human trichinellosis

    International Nuclear Information System (INIS)

    Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation

  17. Rapid detection of antibodies against cytomegalovirus induced immediate early and early antigens by an enzyme linked immunosorbent assay.

    OpenAIRE

    M. Musiani; Carpi, C; Zerbini, M

    1984-01-01

    An enzyme linked immunosorbent assay (ELISA) for detecting antibodies against cytomegalovirus induced immediate early antigens and early antigens was developed using purified nuclear antigens and was compared with the indirect immunofluorescence test. The tests were comparable in their ability to detect positive and negative sera, and antibody titres determined by both assays were similar. The use of ELISA for the detection of antibodies against cytomegalovirus induced immediate early and ear...

  18. Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using horseradish peroxidase/detection antibody-coated gold nanoparticles as nanoprobes.

    Science.gov (United States)

    Zhou, Yuan; Zhou, Tao; Zhou, Rui; Hu, Yonggang

    2014-06-01

    A rapid, simple, facile, sensitive and enzyme-amplified chemiluminescence immunoassay (CLIA) method to detect antibodies against porcine parvovirus has been developed. Horseradish peroxidase (HRP) and the detection antibody were simultaneously co-immobilized on the surface of gold nanoparticles using the electrostatic method to form gold nanoparticle-based nanoprobes. This nanoprobe was employed in a sandwich-type CLIA, which enables CL signal readout from enzymatic catalysis and results in signal amplification. The presence of porcine parvovirus infection was determined in porcine parvovirus antibodies by measuring the CL intensity caused by the reaction of HRP-luminol with H2 O2 . Under optimal conditions, the obtained calibration plot for the standard positive serum was approximately linear within the dilution range of 1:80 to 1:5120. The limit of detection for the assay was 1:10,240 (S/N = 3), which is much lower than that typically achieved with an enzyme-linked immunosorbent assay (1:160; S/N = 3). A series of repeatability measurements using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation of 4.9% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor had an efficiency of 90%, a sensitivity of 93.3%, and a specificity of 87.5% relative to the enzyme-linked immunosorbent assay results. PMID:23832716

  19. Initial study with Tc99m antigranulocyte antibody (MAK-47) in detection sources of infection

    International Nuclear Information System (INIS)

    Antigranulocytes antibodies (AGAB) are antibodies directed against glycoprotein on the surface of granulocytes and as such provides in vivo cell labelling. They are easily labelled with Tc99m and comes a one step labelling technique. 20 patients have been studied 1 h and 4-6 hours after administration of 200 MBq of Tc99m AGAB (MAK-47). Less then 0.5 mg of antibody was given to each patients. Sites of uptake and outside of the reticular-endothelial system were reported as showing positive accumulation. Clinical results were confirmed by microbiological, pathological examinations, clinical follow-up and autopsy. There were 8 patients who had sites of infection confirmed by additional examination. All patients were visualized by Tc99m AGAB (MAK-47). There were 4 cases of osteomyelitis and septic arthritis and 4 cases of focal intra-abdominal infection. Two patients had uptake in non-infected/inflammatory arthritis, both in the knee. The remaining patients had true negative studies. The diagnostic accuracy of this study was as follows: sensitivity 100%, specificity 83%, positive predictive value (PPV) was 80% and negative predictive value (NPV) was 100%. Antigranulocyte antibody (MAK-47) seems to by promising tool in detecting focal infection in bone and soft tissue, except physiological accumulation in some parts of the body. It should be considered that antibodies can have non-specific uptake in non-infected, inflammation sites. It is easy to use and no had allergic reaction and HAMA antibody (human antimouse antibody). (author)

  20. Aerosol delivered radiolabeled antibodies to ectopic lung carcinoma antigens in scintigraphic tumour detection

    International Nuclear Information System (INIS)

    Biologically specific antibodies offer increased specificity of inhalation scintigraphy in pulmonary malignancy at present limited to detection of airways obstruction. Polypeptide ectopic antigens produced by epithelial lung tumours provide a targeting focus for radiolabeled antibodies. Image resolution using the intravenous route is poor due to the small proportion of the dose targeting to the tumor site and ineffective clearance of the background. The inhalation route minimizes non-specific distribution and utilizes mucociliary clearance as a contrast enhancement mechanism. Mucociliary deposition is cleared more effectively than alveolar deposition. Submicronic aerosol droplets produced by airlet nebulisers ensure peripheral airways penetration and deposition. Affinity purified polyclonal goat antibodies against calcitonin labeled with 150 MBq of Tc-99m are delivered to the lungs to produce dynamic images over 24 hours. Pure synthetic human calcitonin ensures production of monospecific antibodies that can be affinity purified. Labeling with Tc-99m by stannous chloride reduction is effected on the solid phase Sepharose beads. An antibody-antigen concentration gradient over a small distance can be provided with radiolabeled antibodies from aerosol deposition. Histologically neoplastic cells are typically separated from the mucous layer by bronchial mucosa and a thin uninvolved lamina. A localised high concentration of labile antigen is present in extra-cellular spaces at the tumour site. A study is progressing to determine if specific antibody-antigen agglutination contributes towards localised impairment of mucociliary clearance at tumour sites creating contrast

  1. Improved detection of Pneumocystis carinii by an immunofluorescence technique using monoclonal antibodies

    DEFF Research Database (Denmark)

    Orholm, M; Holten-Andersen, W; Lundgren, Jens Dilling

    1990-01-01

    To assess whether a recently developed indirect immunofluorescent stain using monoclonal antibodies was more sensitive in detecting Pneumocystis carinii than the combination of Giemsa and methenamine silver nitrate stains which has routinely been used in the laboratory, 88 lavage fluid specimens...... and 34 induced sputum specimens were examined. All specimens were stained by five techniques: immunofluorescence using a combination of three monoclonal antibodies (from the National Institutes of Health, USA), immunofluorescence using a single monoclonal antibody (from Dakopatts), Giemsa, methenamine...... silver nitrate and toluidine blue O. Immunofluorescence using the monoclonal antibodies from the NIH was significantly more sensitive than any other single staining method and than the combination of Giemsa and methenamine silver nitrate staining. The study also showed that the cytospin centrifuge was...

  2. Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

    International Nuclear Information System (INIS)

    The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using 125I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodies in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women

  3. [Dynamics of the antismallpox antibodies detectable in the passive hemagglutination reaction with various animal immunization schemes].

    Science.gov (United States)

    Matsevich, G R; Shelukhina, E M; Konikova, R E; Marennikova, S S

    1975-10-01

    Dynamics of accumulation and preservation of antibodies detectable in the PHAT (PHAT-AT) was studied on rabbits and guinea pigs with the use of various doses of the living inactivated virus and their combination in comparison with the virus-neutralizing antibodies, antihemagglutinins and precipitins. Accumulation of the virus-neutralizing antibodies did not coincide in time with the curve of the PHAT-AT accumulation; the titres of the virus-neutralizing antibodies were higher than the PHAT-AT titres. At the same time the percentage of seroconversions determined by PHAT was equal to 100 and the PHAT-AT level directly depended on the immunizing dose, the time of administration and the type of the antigen. On the basis of the data obtained PHAT could be recommended as a test for the assessment of the immunological efficacy of the smallpox vaccinations. PMID:1082220

  4. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    Science.gov (United States)

    Elango, Chinnasamy

    2016-01-01

    Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline. PMID:27556048

  5. Development of QCM Biosensor with Specific Cow Milk Protein Antibody for Candidate Milk Adulteration Detection

    Directory of Open Access Journals (Sweden)

    Setyawan P. Sakti

    2016-01-01

    Full Text Available Adulteration of goat milk is usually done using cow’s milk product. Cow milk is used as it is widely available and its price is cheaper compared to goat milk. This paper shows a development of candidate tools for milk adulteration using cow milk. A quartz crystal microbalance immunosensor was developed using commercial crystal resonator and polyclonal antibody specific to cow milk protein. A specific protein at 208 KDa is found only in cow milk and does not exist in goat milk. The existence of this protein can be used as an indicator of cow milk content in a target solution. To detect the PSS 208 kDa protein, antibody specific to the PSS 208 was developed. The purified antibody was immobilized on top of the sensor surface on a polystyrene layer. The fraction of the immobilized antibody on the sensor was found at 1.5% of the given antibody. Using a static reaction cell, the developed immunosensor could detect the specific cow milk protein in buffer solution. The detection limit is 1 ppm. A linear relationship between frequency change and specific protein of cow milk concentration is found from a concentration of 1 ppm to 120 ppm.

  6. DEVELOPMENT OF ANTIBODY TO RALSTONIA SOLANACEARUM AND ITS APPLICATION FOR DETECTION OF BACTERIAL WILT

    Directory of Open Access Journals (Sweden)

    YADI SURYADI

    2009-01-01

    Full Text Available The serological assay for the detection of bacterial wilt caused by Ralstonia solanacearum (RSwas able to provide information regarding the presence of the pathogen in plant materials. Theresearch is was aimed to develop polyclonal antibody (PAb for RS detection. Bacterial wholecells of RS isolates mixed with glutaraldehyde were used to immunize New Zealand femalewhite rabbit. The titre of antibody in culture supernatant was 1: 1024. The PAb developed froma ground nut RS isolates reacted with infected plant samples from various locations. It was ableto detect RS antigen of crude extract and pure cultures from tomato and potato plant samples4-5using dot blot ELISA; however, the minimum detectable concentration of RS antigen was 10cells/ml. The PAb obtained in this study is sensitive enough to detect RS isolates in routineserological assay

  7. A liquid phase blocking ELISA for the detection of antibodies against infectious bronchitis virus

    Directory of Open Access Journals (Sweden)

    Cardoso T.C.

    1999-01-01

    Full Text Available A liquid phase blocking ELISA (LPB-ELISA was developed for the detection and measurement of antibodies against infectious bronchitis virus (IBV. The purified and nonpurified virus used as antigen, the capture and detector antibodies, and the chicken hyperimmune sera were prepared and standardized for this purpose. A total of 156 sera from vaccinated and 100 from specific pathogen-free chickens with no recorded contact with the virus were tested. The respective serum titers obtained in the serum neutralization test (SNT were compared with those obtained in the LPB-ELISA. There was a high correlation (r2 = 0.8926 between the two tests. The LPB-ELISA represents a single test suitable for the rapid detection of antibodies against bronchitis virus in chicken sera, with good sensitivity (88%, specificity (100% and agreement (95.31%.

  8. Development of a double-antibody radioimmunoassay for the detection of methotrexate in the serum

    International Nuclear Information System (INIS)

    This double-antibody radioimmunoassay (RIA) designed to detect methotrexate (MTX) in the serum is a precise, rapid and cost-saving quantitative procedure, which meets all the requirements regarding accuracy and reproducibility. Its main components are anti-methotrexate as antiserum, 3H-MTX as tracer substance and a second antibody as separating agent. The measuring technique using tritium is based on a method involving removal of the supernate by suction after separation of the antibody, dissolution of the precipitate in NaCl and addition of the scintillator. As opposed to separation techniques based on carbon the radioactivity to be determined is contained in the precipitate, not in the supernate. The procedure permits the required amount of scintillator and the associated radioactive waste to be reduced by 90%. A further decisive advantage is its low limit of detection. (TRV)

  9. Detection of antibodies to single-stranded DNA in naturally acquired and experimentally induced viral hepatitis

    Energy Technology Data Exchange (ETDEWEB)

    Gust, I.D.; Feinstone, S.M.; Purcell, R.H.; Alter, H.J.

    1980-01-01

    A sensitive ''Farr'' assay, utilizing /sup 125/I-labelled DNA was developed for detecting antibody to single-stranded DNA (anti-ssDNA). The test was shown to be specific and as sensitive as assays using /sup 14/C-labelled DNA, for the detection of antibody in patients with connective tissue diseases. Groups of sera from patients with naturally acquired viral hepatitis and experimentally infected chimpanzees were tested for anti-ssDNA by the /sup 125/I assay and by counterimmunoelectrophoresis (CIEP). No consistent pattern was observed with either technique, indicating the elevated levels of this antibody are not as reliable markers of parenchymal liver damage as had been previously suggested.

  10. [Immunochemical Detection of Azospirilla in Soil with Genus-Specific Antibodies].

    Science.gov (United States)

    Shirokov, A A; Krasov, A I; Selivanov, N Yu; Burygin, G L; Shchegolev, S Yu; Matora, L Yu

    2015-01-01

    Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil. PMID:26263631

  11. Surface plasmon resonance detection of biological warfare agent Staphylococcal enterotoxin B using high affinity monoclonal antibody

    International Nuclear Information System (INIS)

    A novel sensitive method was developed for the detection as well as quantification of Staphylococcal enterotoxin B (SEB) using surface plasmon resonance (SPR). It is well known that the amount of SEB needed to cause the intoxication to human beings is very less and this concentration (0.02 μg/kg) is highly dangerous, hence, it is used as biological warfare agent. Thus, the need to develop a reliable and potential detection system against SEB is warranted. In the present work, SEB antibody was immobilized on carboxymethyldextran modified gold chip. The immobilization of SEB antibody and interaction of antigen with immobilized antibody were in-situ characterized by SPR and electrochemical impedance spectroscopy. A sample solution containing SEB antigen was injected in a working channel and the results revealed linearity in the concentration from 2.0 to 32.0 pM with a detection limit of 1.0 pM. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) values were calculated and found to be 13 pM and 424.23, respectively. Moreover, the thermodynamic parameter, change in Gibb's free energy was deduced and found to be -62.08 kJ/mol and this value shows the spontaneous interaction between SEB antigen and SEB antibody. In order to optimize the detection method, temperature and pH variation studies were also performed. Interference study was conducted to know the selectivity for the antigen-antibody interaction of SEB. The selectivity efficiency of SEB, SEC, SEA and SED were 100, 27.15, 20.01 and 12.05%, respectively towards SEB antibody.

  12. Comparison of an enzyme-linked immunoassay and a quantitative indirect fluorescent-antibody test with the conventional indirect fluorescent-antibody test for detecting antibodies to Toxoplasma gondii.

    OpenAIRE

    Violand, S A; Mitchell, T G; Kleeman, K T

    1982-01-01

    Two new methods for the detection of antibodies to Toxoplasma gondii, an enzyme-linked immunosorbent assay and a quantitative immunofluorescence assay, were evaluated and compared with the conventional indirect fluorescent-antibody slide test. Each of 100 human sera was assayed twice by the three procedures. Both the enzyme-linked immunosorbent assay and the quantitative immunofluorescence assay correlated well with serologically positive (indirect fluorescent-antibody titer greater than or e...

  13. Polyclonal antibodies to tropoelastin and the specific detection and measurement of tropoelastin in vitro.

    Science.gov (United States)

    Prosser, I W; Whitehouse, L A; Parks, W C; Stahle-Bäckdahl, M; Hinek, A; Park, P W; Mecham, R P

    1991-01-01

    Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays. PMID:2060302

  14. Comparison of antibody detection by RIA and PCR in the diagnosis of tuberculous meningitis

    International Nuclear Information System (INIS)

    Diagnosis of tuberculous meningitis (TBM) by conventional microbiological techniques such as microscopy and culture is very difficult, as the sensitivity of either test is not good. Radioimmunoassays for the detection of M tuberculosis antigen and anti M tuberculosis antibody have been reported. Polymerase chain reaction based on amplification of DNA sequences is one of the recent techniques being evaluated for diagnosis

  15. Field evaluation of a fast anti-Leishmania antibody detection assay in Ethiopia

    NARCIS (Netherlands)

    A. Hailu; G.J. Schoone; E. Diro; A. Tesfaye; Y. Techane; T. Tefera; Y. Assefa; A. Genetu; Y. Kebede; T. Kebede; H.D.F.H. Schallig

    2006-01-01

    A fast agglutination screening test (FAST) for the detection of Leishmania antibodies in human serum samples was evaluated under harsh field conditions in northern Ethiopia. Test performance was compared with a standard serological test, namely the direct agglutination test (DAT), and with parasitol

  16. Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies

    NARCIS (Netherlands)

    J. Groen (Jan); P. Koraka (Penelopie); J. Velzing (Jans); C. Copra (Cederick); A.D.M.E. Osterhaus (Ab)

    2000-01-01

    textabstractThe performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromato

  17. Laboratory evaluation of three rapid diagnostic tests for dual detection of HIV and Treponema pallidum antibodies.

    Science.gov (United States)

    Humphries, Romney M; Woo, Jennifer S; Chung, Jun Ho; Sokovic, Anita; Bristow, Claire C; Klausner, Jeffrey D

    2014-12-01

    The performance of three research-use-only, dual HIV and syphilis rapid diagnostic tests (RDTs) was evaluated for 150 patient serum samples and compared to reference HIV and Treponema pallidum antibody detection methods. The RDTs performed comparably, with sensitivities of 93 to 99% and specificities of 97 to 100%. The kappa statistic between the RDTs was 0.95. PMID:25297332

  18. An ELISA for the detection of antibodies against Newcastle disease virus in African village poultry

    International Nuclear Information System (INIS)

    A standardised and robust ELISA was developed for detecting antibodies against Newcastle disease in poultry. Results from 28 field samples were compared with the results from a haemagglutination test. It will be necessary to validate the ELISA in the field by assessing the results of serum samples collected from village chickens following vaccination or field infection. (author)

  19. A comparison of indirect immunofluorescence and radioimmunoassay for detecting antibody to varicella-zoster virus

    International Nuclear Information System (INIS)

    Immunofluorescence (IF) and radioimmunoassay (RIA) were found to be more sensitive methods than complement fixation (CF) for detecting antibody to varicella-zoster (V-Z) virus. RIA yielded titres about 30 times greater than those obtained by IF, but for screening purposes RIA was only about six times more sensitive since the minimum serum dilutions that could be tested were 1/100 and 1/16 respectively. When 539 sera from subjects of different ages were screened for V-Z antibody, IF and RIA gave concordant results with 527 specimens (98%). When 19 patients were tested who had not previously had varicella but were experiencing primary infection with herpes simplex (HS) virus, crossreacting antibodies to V-Z antigens were detected in six patients by IF but in only two of these by RIA. IF and RIA are preferable to CF as tests for immune status because of their greater sensitivity, but weak positive reactions caused by presumptive low titres of homologous antibody or by higher titres of heterologous antibody can occur in one or both tests. Such reactions could cause difficulty in assessing the need for vaccine or for specific immune globulin, and in interpreting the response to vaccination. (Auth.)

  20. Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus.

    Science.gov (United States)

    Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi

    2015-09-01

    A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology. PMID:25952731

  1. An agar gel enzyme assay (AGEA) for simple detection of Salmonella enteritidis antibodies in chicken sera.

    Science.gov (United States)

    Kim, C J; Nagaraja, K V

    1991-01-01

    An agar gel enzyme assay (AGEA) was developed for the detection of antibodies to Salmonella enteritidis (SE). The assay was based on the ability of antibodies to diffuse through an agar gel and react with antigen coated on a polystyrene surface. The antigen-antibody reaction was then made visible by applying an enzyme-conjugated anti-immunoglobulin and the addition, subsequently, of a substrate-containing gel. The color change in circular zones was taken as the indication for the presence of antibodies. The present investigation reports identification of an antigen specific for SE and its use in the development of a relatively simple AGEA procedure. The results of AGEA were compared with those of conventional microagglutination (MA) test and serum plate (SP) test. The percentage agreement between MA and AGEA in positive serum sample was found to be 94.4%, and in negative serum samples it was found to be 88.8%. The present results suggest that the AGEA could be a very useful screening test for the detection of SE antibodies because the assay is inexpensive, specific and simple to perform without much equipment, and give results within a 3-hr period. PMID:1832368

  2. Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

    OpenAIRE

    Baldi, Pablo C.; Araj, George F.; Racaro, Graciela C.; Wallach, Jorge C.; Fossati, Carlos A.

    1999-01-01

    The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to...

  3. Diagnosis of Paracoccidioidomycosis by Detection of Antigen and Antibody in Bronchoalveolar Lavage Fluids▿

    OpenAIRE

    Marques-da-Silva, Silvia Helena; Colombo, Arnaldo Lopes; Blotta, Maria Heloisa Souza Lima; Queiroz-Telles, Flávio; Balthazar, Alípio Barbosa; Lopes, José Daniel; Camargo, Zoilo Pires

    2006-01-01

    Paracoccidioidomycosis (PCM) is a systemic infection caused by the fungus Paracoccidioides brasiliensis and is believed to be the leading cause of fungal pulmonary infection. In this study, we used an inhibition enzyme-linked immunosorbent assay to diagnose pulmonary PCM based on the detection of 43-kDa and 70-kDa molecules in bronchoalveolar lavage fluids. The results were compared with results obtained by classical methods for antibody detection.

  4. Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody.

    OpenAIRE

    Bezeaud, A; Rosenswajg, M; Guillin, M C

    1989-01-01

    Five commercially available assays for hepatitis delta (HD) virus markers were compared for sensitivity, specificity, and reproducibility: three assays for antibody (anti-HD), provided by Diagnostics Pasteur, Organon Teknika, and Abbott Laboratories, and two assays for antigen (HD Ag), from Pasteur and Organon Teknika. The assay from Organon Teknika is the less sensitive assay for anti-HD detection. Although the sensitivities of the Pasteur and Abbott assays for anti-HD detection are similar,...

  5. Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture.

    OpenAIRE

    Stephens, R S; Kuo, C C; Tam, M R

    1982-01-01

    Monoclonal antibodies which recognize the species-specific major outer membrane protein antigen of Chlamydia trachomatis were used for immunofluorescence staining of chlamydial inclusions in cell culture. A total of 115 clinical specimens were inoculated onto replicate HeLa 229 cell monolayers and assayed for chlamydial inclusions by immunofluorescence staining and Giemsa staining. Of the isolates, 38 were detected by immunofluorescence staining on passage 1 and 1 was detected on passage 2; 2...

  6. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

    Science.gov (United States)

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-01-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  7. Detection of antibodies against Chlamydophila abortus in Costa Rican sheep flocks

    OpenAIRE

    R. Villagra-Blanco; Dolz, G.; Montero-Caballero, D.; Romero-Zúñiga, J.J.

    2015-01-01

    A total of 359 sheep samples from 15 flocks were analyzed for the presence of antibodies against Chlamydophila abortus using a commercial Enzyme linked Immunosorbent Assay (ELISA). Antibodies were detected in 19 (5.29%) sheep from 12 (80%) flocks. Seropositive animals were found in all analyzed regions (Central, Chorotega, Atlantic Huetar, North Huetar and Central Pacific) determining prevalence between 0.28% and 4.4%, and intra-flock positivity between 3.7% and 25.0%. The survey revealed two...

  8. Oral Fluid Antibody Detection in the Diagnosis of Gastric Helicobacter pylori Infection

    OpenAIRE

    Behzad Hooshm; Alireza Monsef; Mohammad Amirmadglesi; Mani Kashani

    2004-01-01

    The aim of this study was to evaluate an enzyme - linked immunosorbent assay (ELISA) for the detection of anti - helicobacter pylori (H. pylori) specific IgG antibodies in specimens of oral fluid.All subjects over the age 18 years undergoing endoscopy for any reason were asked to participate in the study. Two groups of 44 patients in each were selected as HP+ and HP-. At the same time, 5 milliliters of unstimulated saliva was collected from these patients, and the antibody titration against H...

  9. [Significance of human leptospirosis in Mexico. Detection of Leptospira antibodies in a blood donor population].

    Science.gov (United States)

    Gavaldón, D G; Cisneros, M A; Rojas, N; Moles-Cervantes, L P

    1995-01-01

    The presence of specific serum antibodies has been used as a diagnostic test for human leptospirosis. The presence of these antibodies in humans is indicative of an active natural infection. Its detection after exposure denotes the presence of immunity. Serum samples from 206 adult blood donors were analyzed with a microscopic agglutination assay against 7 serovars of Leptospira interrogans. A total of 7% were positive with the following serovar distribution; shermani 53%, canicola 33%, pyrogens 20%, pomona 13% and icterohaemorrhagiae 6%. The highest frequency of seropositivity was found in the 20 year to 39 age group. These results in asymptomatic individuals show that leptospirosis is a frequent zoonosis in Mexico. PMID:8582567

  10. Comparison of Two Rapid Diagnostic Assays for Detection of Immunoglobulin M Antibodies to Dengue Virus

    OpenAIRE

    Wu, Shuenn-Jue L.; Paxton, Helene; Hanson, Barbara; Kung, Cheryl G.; Chen, Timothy B.; Rossi, Cindy; David W Vaughn; Murphy, Gerald S.; Hayes, Curtis G.

    2000-01-01

    Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-hu...

  11. Multiplexed immunoassay for the rapid detection of anti-tumor-associated antigens antibodies.

    Science.gov (United States)

    Desmet, C; Le Goff, G C; Brès, J-C; Rigal, D; Blum, L J; Marquette, C A

    2011-07-21

    TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors). PMID:21666912

  12. A nanofluidic bioarray chip for fast and high-throughput detection of antibodies in biological fluids

    Science.gov (United States)

    Lee, Jonathan; Gulzar, Naveed; Scott, Jamie K.; Li, Paul C. H.

    2012-10-01

    Immunoassays have become a standard in secretome analysis in clinical and research analysis. In this field there is a need for a high throughput method that uses low sample volumes. Microfluidics and nanofluidics have been developed for this purpose. Our lab has developed a nanofluidic bioarray (NBA) chip with the goal being a high throughput system that assays low sample volumes against multiple probes. A combination of horizontal and vertical channels are produced to create an array antigens on the surface of the NBA chip in one dimension that is probed by flowing in the other dimension antibodies from biological fluids. We have tested the NBA chip by immobilizing streptavidin and then biotinylated peptide to detect the presence of a mouse monoclonal antibody (MAb) that is specific for the peptide. Bound antibody is detected by an AlexaFluor 647 labeled goat (anti-mouse IgG) polyclonal antibody. Using the NBA chip, we have successfully detected peptide binding by small-volume (0.5 μl) samples containing 50 attomoles (100 pM) MAb.

  13. Development of Robust and Standardized Cantilever Sensors Based on Biotin/Neutravidin Coupling for Antibody Detection

    Directory of Open Access Journals (Sweden)

    Christoph Gerber

    2013-04-01

    Full Text Available A cantilever-based protein biosensor has been developed providing a customizable multilayer platform for the detection of antibodies. It consists of a biotin-terminated PEG layer pre-functionalized on the gold-coated cantilever surface, onto which NeutrAvidin is adsorbed through biotin/NeutrAvidin specific binding. NeutrAvidin is used as a bridge layer between the biotin-coated surface and the biotinylated biomolecules, such as biotinylated bovine serum albumin (biotinylated BSA, forming a multilayer sensor for direct antibody capture. The cantilever biosensor has been successfully applied to the detection of mouse anti-BSA (m-IgG and sheep anti-BSA(s-IgG antibodies. As expected, the average differential surface stress signals of about 5.7 ± 0.8 ´ 10−3 N/m are very similar for BSA/m-IgG and BSA/s-IgG binding, i.e., they are independent of the origin of the antibody. A statistic evaluation of 112 response curves confirms that the multilayer protein cantilever biosensor shows high reproducibility. As a control test, a biotinylated maltose binding protein was used for detecting specificity of IgG, the result shows a signal of bBSA layer in response to antibody is 5.8 ´ 10−3 N/m compared to bMBP. The pre-functionalized biotin/PEG cantilever surface is found to show a long shelf-life of at least 40 days and retains its responsivity of above 70% of the signal when stored in PBS buffer at 4 °C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay.

  14. Development of Robust and Standardized Cantilever Sensors Based on Biotin/Neutravidin Coupling for Antibody Detection

    Science.gov (United States)

    Zhang, Jiayun; Lang, Hans Peter; Battiston, Felice; Backmann, Natalija; Huber, Francois; Gerber, Christoph

    2013-01-01

    A cantilever-based protein biosensor has been developed providing a customizable multilayer platform for the detection of antibodies. It consists of a biotin-terminated PEG layer pre-functionalized on the gold-coated cantilever surface, onto which NeutrAvidin is adsorbed through biotin/NeutrAvidin specific binding. NeutrAvidin is used as a bridge layer between the biotin-coated surface and the biotinylated biomolecules, such as biotinylated bovine serum albumin (biotinylated BSA), forming a multilayer sensor for direct antibody capture. The cantilever biosensor has been successfully applied to the detection of mouse anti-BSA (m-IgG) and sheep anti-BSA(s-IgG) antibodies. As expected, the average differential surface stress signals of about 5.7 ± 0.8 × 10−3 N/m are very similar for BSA/m-IgG and BSA/s-IgG binding, i.e., they are independent of the origin of the antibody. A statistic evaluation of 112 response curves confirms that the multilayer protein cantilever biosensor shows high reproducibility. As a control test, a biotinylated maltose binding protein was used for detecting specificity of IgG, the result shows a signal of bBSA layer in response to antibody is 5.8 × 10−3 N/m compared to bMBP. The pre-functionalized biotin/PEG cantilever surface is found to show a long shelf-life of at least 40 days and retains its responsivity of above 70% of the signal when stored in PBS buffer at 4 °C. The protein cantilever biosensor represents a rapid, label-free, sensitive and reliable detection technique for a real-time protein assay. PMID:23604028

  15. Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis

    Directory of Open Access Journals (Sweden)

    Lin Qiu

    2014-01-01

    Full Text Available In this report, fluorescence detection coupled capillary electrophoresis (CE-FL was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

  16. Dye Labelled Monoclonal Antibody Assay for Detection of Toxic Shock Syndrome Toxin -1 from Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    V Javid Khojasteh

    2011-12-01

    Full Text Available Objective: The aim of study was to develop a rapid assay, dye labelled monoclonal antibody assay (DLMAA, using non-radioactive organic synthetic dyes for identification of Toxic Shock Syndrome Toxin-1 (TSST-1 producing strains of Staphylococcus aureus.Materials and Methods: The assay protocol required only two simple steps; addition of TSST-1 antigen to a nitrocellulose membrane and then adding a colloidal dye labelled antibody (D/A suspension detection reagent.Results: The sensitivity and specificity of the assay was determined relative to positive and negative strains compared to an ELISA assay. Overall 100% agreement was found between both assays. The sensitivity for detection of TSST-1 was 30 ng.Conclusion: The DLMAA did not require handling and disposal of radioactive materials. It is a rapid qualitative technique for detection of TSST-1 toxin at room temperature within a short time.

  17. Oral Fluid Antibody Detection in the Diagnosis of Gastric Helicobacter pylori Infection

    Directory of Open Access Journals (Sweden)

    Behzad Hooshm

    2004-09-01

    Full Text Available The aim of this study was to evaluate an enzyme - linked immunosorbent assay (ELISA for the detection of anti - helicobacter pylori (H. pylori specific IgG antibodies in specimens of oral fluid.All subjects over the age 18 years undergoing endoscopy for any reason were asked to participate in the study. Two groups of 44 patients in each were selected as HP+ and HP-. At the same time, 5 milliliters of unstimulated saliva was collected from these patients, and the antibody titration against H.P. was evaluated by “ELISA” method.In overall, the level of salivary antibody in H.P+ group was significantly more than those in H.P- group (POral fluid ELISA is relatively a comfortable, fast and noninvasive test for diagnosis of H. pylori infection.

  18. Detecting Lyme Disease Using Antibody-Functionalized Single-Walled Carbon Nanotube Transistors

    CERN Document Server

    Lerner, Mitchell B; Goldsmith, Brett R; Brisson, Dustin; Johnson, A T Charlie

    2013-01-01

    We examined the potential of antibody-functionalized single-walled carbon nanotube (SWNT) field-effect transistors (FETs) for use as a fast and accurate sensor for a Lyme disease antigen. Biosensors were fabricated on oxidized silicon wafers using chemical vapor deposition grown carbon nanotubes that were functionalized using diazonium salts. Attachment of Borrelia burgdorferi (Lyme) flagellar antibodies to the nanotubes was verified by Atomic Force Microscopy and electronic measurements. A reproducible shift in the turn-off voltage of the semiconducting SWNT FETs was seen upon incubation with Borrelia burgdorferi flagellar antigen, indicative of the nanotube FET being locally gated by the residues of flagellar protein bound to the antibody. This sensor effectively detected antigen in buffer at concentrations as low as 1 ng/ml, and the response varied strongly over a concentration range coinciding with levels of clinical interest. Generalizable binding chemistry gives this biosensing platform the potential to...

  19. Detection of Serum Antibodies to Borna Disease Virus in Patients with Psychiatric Disorders

    Science.gov (United States)

    Rott, R.; Herzog, S.; Fleischer, B.; Winokur, A.; Amsterdam, J.; Dyson, W.; Koprowski, H.

    1985-05-01

    Borna disease virus causes a rare meningoencephalitis in horses and sheep and has been shown to produce behavioral effects in some species. The possibility that the Borna virus is associated with mental disorders in humans was evaluated by examining serum samples from 979 psychiatric patients and 200 normal volunteers for the presence of Borna virus-specific antibodies. Antibodies were detected by the indirect immunofluorescence focus assay. Antibodies to the virus were demonstrated in 16 of the patients but none of the normal volunteers. The patients with the positive serum samples were characterized by having histories of affective disorders, particularly of a cyclic nature. Further studies are needed to define the possible involvement of Borna virus in human psychiatric disturbances.

  20. Detection of antibodies against Fasciola hepatica in cirrhotic patients from Peru.

    Science.gov (United States)

    Marcos, L A; Bussalleu, A; Terashima, A; Espinoza, J R

    2009-03-01

    The prevalence of Fasciola hepatica infection, in endemic countries, in patients with established cirrhosis is unknown. We hypothesized that, in endemic countries, the presence of fascioliasis may be detected in a serum pool of cirrhotic patients. Forty-four previously stored serum samples of patients with established liver cirrhosis, in the Hospital Nacional Cayetano Heredia in Lima, Peru, were collected from 1998 to 2003 and assessed for hepatitis B, C and fascioliasis antibodies (Fas2 ELISA). Hepatitis B surface antigen (HBsAg) was positive in 8.8% (n = 34), hepatitis B core antibody (anti-HBc) in 32.5% (n = 34), hepatitis C antibodies (anti-HCV) in 9.1% (n = 33), and 9.1% (n = 44) were Fas2 ELISA positive. This disease is an example of an emerging tropical infection which can be present in chronic liver diseases, requiring greater clinician awareness especially in endemic rural areas. Further clinical studies are warranted. PMID:18817587

  1. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

    Directory of Open Access Journals (Sweden)

    Luisa W. Cheng

    2015-11-01

    Full Text Available Botulinum neurotoxins (BoNT are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL immunoassay for BoNT/B, using monoclonal antibodies (mAbs MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

  2. Development of a double-antibody sandwich ELISA for rapid detection of Bacillus Cereus in food.

    Science.gov (United States)

    Zhu, Longjiao; He, Jing; Cao, Xiaohan; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-01-01

    Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 10(4)-2.8 × 10(6) cells/mL with a detection limit (LOD) of 0.9 × 10(3) cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens. PMID:26976753

  3. Clinical significance of detection of antibodies to fetal and adult acetylcholine receptors in myasthenia gravis

    Institute of Scientific and Technical Information of China (English)

    Qi-Guang Shi; Zhi-Hong Wang; Xiao-Wei Ma; Da-Qi Zhang; Chun-Sheng Yang; Fu-Dong Shi; Li Yang

    2012-01-01

    Objective To evaluate the frequency,distribution and clinical significance of the antibodies to the fetal and/or adult acetylcholine receptor (AChR) in patients with myasthenia gravis (MG).Methods AChR antibodies were detected by cell-based assay in the serum of ocular MG (OMG) (n =90) and generalized MG (GMG) patients (n =110).The fetaltype (2α∶ β∶ γ∶ δ) and adult-type (2α∶ β∶ ε∶ δ) AChR were used as antigens,and their relevance to disease presentation was assessed.Results The overall frequencies of anti-adult and anti-fetal AChR antibodies were similar in all 200 patients examined,with 14 having serum specific to the AChR-γ subunit,and 22 to the AChR-ε subunit.The overall sensitivity when using the fetal and adult AChR antibodies was higher than that when using the fetal AChR antibody only (P =0.015).Compared with OMG patients,the mean age at disease onset and the positive ratio of antibodies to both isoforms of the AChR were significantly higher in patients who subsequently progressed to GMG.Older patients and patients with both anti-fetal and anti-adult AChR antibodies had a greater risk for developing generalized disease [odds ratio (OR),1.03;95% confidence interval (CI),1.01-1.06 and OR,5.09;95% CI,2.23-11.62].Conclusion Using both fetal-and adulttype AChRs as the antigens may be more sensitive than using either subtype.Patients with serum specific to both isoforms are at a greater risk of progressing to GMG.Patients with disease onset at an advanced age appear to have a higher frequency of GMG conversion.

  4. Detection of osteomyelitis using a Tc-99m labeled antigranulocyte antibody immunoscintigraphy

    International Nuclear Information System (INIS)

    The purpose of this study was to evaluate the diagnostic accuracy of Tc-99m labeled antigranulocyte antibody immunoscintigraphy in the diagnosis of osteomyelitis and compare with the results of triphasic bone scan. The study population was 39 patients (22 male, 17 female) who had uncertain diagnoses of osteomyelitis. Fifteen patients had history of orthopedic surgery, and 5 had previous fracture. One milligram of monoclonal antibody against NCA-95 was labeled with 370 MBq of Tc-99m, injected intravenously, and 4 hour images were obtained. Triphasic bone scan images were obtained in 30 patients. The final diagnosis was confirmed by bacteriologic culture, biopsy or long term clinical follow up. Twenty one patients were confirmed to have osteomyelitis (1 acute, 20 chronic). Eighteen patients were without osteomyelitis. Antigranulocyte antibody immunoscintigraphy had a sensitivity of 71% (15/21), and a specificity of 89% (16/18), while the sensitivity and specificity of triphasic bone scan was 93% (13/14) and 38% (6/16), respectively. Antigranulocyte antibody scan showed higher specificity of 100% (11/11) in comparison with 33% (3/9) of triphasic bone scan in patients with history of orthopedic surgery or fracture. Antigranulocyte antibody immunoscintigraphy is more specific than that of triphasic bone scan and may be helpful in patients with history of surgery or fracture. However, sensitivity is lower than triphasic bone scan in the detection of chronic osteomyelitis

  5. Comparison of antibody capture radio- and enzyme immunoassays with immunofluorescence for detecting IgM antibody in infants with congenital rubella

    Energy Technology Data Exchange (ETDEWEB)

    Chantler, S.; Evans, C.J. (Wellcome Research Lab., Beckenham (UK)); Mortimer, P.P. (Virus Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London, (UK)); Cradock-Watson, J.E.; Ridehalgh, M.K.S. (Withington Hospital, Manchester (UK))

    1982-08-01

    IgM antibody capture radioimmunoassay (MACRIA) and enzyme immunoassay (MACEIA) were compared with immunofluorescence (IF) for detecting specific IgM antibody in 99 sera from 76 infants with confirmed congenital rubella, and 61 sera from a comparative group of 59 infants who had miscellaneous abnormalities but in whom congenital rubella was not confirmed. All of 35 specimens collected from confirmed cases within 12 weeks of birth were positive by all three methods and all but one of 17 specimens collected after the age of 18 months were uniformly negative. At intermediate ages discrepancies occurred in 18 specimens, of which eight were positive and 10 negative by IF. Three of these 18 specimens were negative by both antibody capture procedures but showed weak fluorescence; the other 15 were negative by MACEIA, but positive by MACRIA which appears to be the more sensitive of the antibody capture methods. Sera from five infants in the comparative group were clearly positive by all three methods. These five infants were probably congenitally infected with rubella. Sera from the other 54 infants were negative, except for one that gave a weakly positive result by MACRIA alone. Antibody capture procedures offer several advantages over previous methods for detecting IgM antibody. Although MACRIA was found to be slightly more sensitive than MACEIA, the greater stability of the enzyme label, together with the possibility of both visual and quantitative assessment, could make MACEIA the method of choice for detecting rubella-specific IgM.

  6. A comparison of antibody capture radio- and enzyme immunoassays with immunofluorescence for detecting IgM antibody in infants with congenital rubella

    International Nuclear Information System (INIS)

    IgM antibody capture radioimmunoassay (MACRIA) and enzyme immunoassay (MACEIA) were compared with immunofluorescence (IF) for detecting specific IgM antibody in 99 sera from 76 infants with confirmed congenital rubella, and 61 sera from a comparative group of 59 infants who had miscellaneous abnormalities but in whom congenital rubella was not confirmed. All of 35 specimens collected from confirmed cases within 12 weeks of birth were positive by all three methods and all but one of 17 specimens collected after the age of 18 months were uniformly negative. At intermediate ages discrepancies occurred in 18 specimens, of which eight were positive and 10 negative by IF. Three of these 18 specimens were negative by both antibody capture procedures but showed weak fluorescence; the other 15 were negative by MACEIA, but positive by MACRIA which appears to be the more sensitive of the antibody capture methods. Sera from five infants in the comparative group were clearly positive by all three methods. These five infants were probably congenitally infected with rubella. Sera from the other 54 infants were negative, except for one that gave a weakly positive result by MACRIA alone. Antibody capture procedures offer several advantages over previous methods for detecting IgM antibody. Although MACRIA was found to be slightly more sensitive than MACEIA, the greater stability of the enzyme label, together with the possibility of both visual and quantitative assessment, could make MACEIA the method of choice for detecting rubella-specific IgM. (Auth.)

  7. A rapid method for the detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes

    International Nuclear Information System (INIS)

    Cell membranes isolated from murine lymphocytes or ascites tumors bind tightly to the surface of flexible plastic microtiter plates in the absence of additional proteins. This allows the detection of membrane associated molecules by specific antibodies and thus forms the basis for a rapid and sensitive radioimmunoassay for antibodies to membrane-bound components. The assay compares favourably with a variety of methods currently used to detect antibodies to cell surface antigens. The assay detects a variety of well characterized murine cell surface antigens (H-2, I-A, T-200, Thy-1.2, Ig). The level of antibody binding to membranes on plates correlates well with antigen density on intact cells. A modification of the assay involving competition between cross-reacting antibodies allows detection and resolution of closely spaced antigenic determinants. (Auth.)

  8. Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine.

    Science.gov (United States)

    Goodell, C K; Prickett, J; Kittawornrat, A; Johnson, J; Zhang, J; Wang, C; Zimmerman, J J

    2016-02-01

    Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti-IAV antibodies using homologous and heterologous haemagglutination-inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)-blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut-off of S/N ≤ 0.60, the sensitivity and specificity of the NP-blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post-inoculation and the interactions between those factors (P cost-effective approach for the detection and surveillance of IAV infections in swine populations. PMID:24571447

  9. Radioimmunoassay for the detection of hepatitis e antigen (HBeAG) and antibody (ANTI-HBe)

    International Nuclear Information System (INIS)

    A solid phase micro-immunoradiometric assay (micro-SPIRA) for the detection of hepatitis e antigen (HBeAg) and antibody has been developed. Chimpanzee anti-HBe/2 was developed by repeated immunizations with purified antigen containing HBeAg/1 and HBeAg/2. An anti-HBe/2 titer of 1:4 was determined by immunodiffusion (ID) analysis. Anti-HBe/1 was not detected. The anti-HBe IgG used in the assay was purified from plasma by a combination of DEAE-cellulose and affinity chromatography. The sensitivity of the micro-SPIRA for antigen and antibody was 193 ng/ml and 65 ng/ml, respectively. By comparing relative endpoint titers obtained by ID to micro-SPIRA, it was determined that micro-SPIRA for antigen and antibody is 320 and greater than 1300 times more sensitive, respectively, than ID. The specificity of the assay was ascertained by the examination of various non-B specimens. The application of the assay to a panel of 50 hepatitis B surface antigen (HBsAg)-positive specimens resulted in an increase in positivity of 18% for antigen and 22% for antibody

  10. Blood collection on filter paper for HIV antibodies detection: experience of SampaCentro Project

    Directory of Open Access Journals (Sweden)

    Marcia J. Castejon

    2015-04-01

    Full Text Available Introduction: Blood samples collected on filter paper (dried blood spot [DBS] is an immunoassay that has been used for antibodies screening. Objective: To evaluate the strategy of DBS blood collection for detection of HIV antibodies, evaluation of Q-Preven HIV 1 + 2 - DBS kit lot, and to analyze the stability of DBS samples. Methods: Blood collection on DBS was performed according to World Health Organization (WHO recommendations. The evaluation of the kit lot for HIV antibodies detection was performed using delta (d values from the results of 774 DBS samples from volunteers men who have sex with men (MSM recruited in the central region of São Paulo city, Brazil. Results: DBS blood collection was performed without complications. The positive (5.26 and negative (5.23 delta values allowed to clearly differentiate HIV antibodies reactive and non-reactive samples. We observed good performance of the kit lot and samples were stable on DBS form. Conclusion: Blood collection on DBS is feasible for the study of MSM population and is suitable for laboratory routine. The overall performance of Q Preven HIV-1 + 2 - DBS kit was satisfactory, having reached the quality levels required for the development of this study.

  11. Ochratoxin A Detection on Antibody- Immobilized on BSA-Functionalized Gold Electrodes

    Science.gov (United States)

    2016-01-01

    Ochratoxin A (OTA)—a toxin produced by Aspergillus carbonarius, Aspergillus ochraceus, and Penicillium verrucosum—is one of the most-abundant food-contaminating mycotoxins. To avoid the risk of OTA consumption for humans and animals, the rapid detection and quantitation of OTA level in different commodities are of great importance. In this work, an impedimetric immunosensor for ochratoxin A (OTA) detection, a common toxic botanical contaminant, was developed via the immobilization of anti-OTA antibody on bovine serum albumin modified gold electrodes. A four-step reaction protocol was tested to modify the gold electrode and obtain the sensing substrate. All the steps of the immunosensor elaboration and also the immunochemical reaction between surface-bound antibody and ochratoxin A were analyzed using cyclic voltammetry and electrochemical impedance spectroscopy. Modification of the impedance due to the specific antigen-antibody reaction at immunosensor surface, was used in order to detect ochratoxin A. Linear proportionality of the charge transfer resistance to the concentration of OTA allows ochratoxin A detection in the range of 2.5–100 ng/mL. PMID:27467684

  12. Combined antibody and DNA detection for early diagnosis of leptospirosis after a disaster.

    Science.gov (United States)

    Iwasaki, Hiroko; Chagan-Yasutan, Haorile; Leano, Prisca Susan A; Koizumi, Nobuo; Nakajima, Chie; Taurustiati, Delsi; Hanan, Firmanto; Lacuesta, Talitha Lea; Ashino, Yugo; Suzuki, Yasuhiko; Gloriani, Nina G; Telan, Elizabeth Freda O; Hattori, Toshio

    2016-04-01

    Early diagnosis based on laboratory confirmation is essential for managing leptospirosis. This study investigated the effectiveness of a novel method of detecting leptospirosis that combines measurement of anti-Leptospira antibodies by the microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic test (ICT) and leptospiral DNA by loop-mediated isothermal amplification (LAMP) and real-time PCR in plasma and 2 types of urine pellets. Of 113 suspected cases, 68.1%, 76.1%, and 60.2% were positive by MAT, ELISA, and ICT, respectively. Real-time PCR using DNA purified from urine pellets collected by low-speed centrifugation yielded positive signals for patients in late acute as well as early phase who were positive by LAMP using plasma DNA or urine pellets. Among antibody-negative patients, 9.5% were positive by DNA detection. These findings indicate that the leptospirosis detection rate is increased by combining antibody and DNA detection, providing a new tool for timely diagnosis of infection. PMID:26860351

  13. Ochratoxin A Detection on Antibody- Immobilized on BSA-Functionalized Gold Electrodes.

    Science.gov (United States)

    Badea, Mihaela; Floroian, Laura; Restani, Patrizia; Cobzac, Simona Codruta Aurora; Moga, Marius

    2016-01-01

    Ochratoxin A (OTA)-a toxin produced by Aspergillus carbonarius, Aspergillus ochraceus, and Penicillium verrucosum-is one of the most-abundant food-contaminating mycotoxins. To avoid the risk of OTA consumption for humans and animals, the rapid detection and quantitation of OTA level in different commodities are of great importance. In this work, an impedimetric immunosensor for ochratoxin A (OTA) detection, a common toxic botanical contaminant, was developed via the immobilization of anti-OTA antibody on bovine serum albumin modified gold electrodes. A four-step reaction protocol was tested to modify the gold electrode and obtain the sensing substrate. All the steps of the immunosensor elaboration and also the immunochemical reaction between surface-bound antibody and ochratoxin A were analyzed using cyclic voltammetry and electrochemical impedance spectroscopy. Modification of the impedance due to the specific antigen-antibody reaction at immunosensor surface, was used in order to detect ochratoxin A. Linear proportionality of the charge transfer resistance to the concentration of OTA allows ochratoxin A detection in the range of 2.5-100 ng/mL. PMID:27467684

  14. Clinical significance of combined detection of multiple serum antibodies (AsAb, EmAb, AcAb, AoAb, ToxAb) in infertile women

    International Nuclear Information System (INIS)

    Objective: To determine the clinical significance of combined detection of multiple serum antibodies in infertile women. Methods; Serum multiple antibodies were examined in 120 infertile women, including 88 failed to get pregnancy and 32 with repeated spontaneous abortion. The antibodies tested were: (1) anti-sperm antibody (AsAb) (2) endometrial antibody (EmAb) (3) anti-cardiophospholipid antibody (AcAb) (4) Anti-ovarian antibody (AoAb) and Toxoplasmosis antibody (ToxAb). Results: In 48 of the infertile women, none of the five antibodies were positive (40% of 120). The rest were: one antibody positive--38/120 or 31.6%; two antibodies positive--31/120 or 25.83%, three and four antibodies positive--4/120 or 3.33%. None of the women were positive with all five antibodies. Conclusion: Immune factor was the chief cause of infertility in women. (authors)

  15. Evaluation of an In-House-Developed Radioassay Kit for Antibody Detection in Cases of Pulmonary Tuberculosis and Tuberculous Meningitis

    OpenAIRE

    Kameswaran, M.; Shetty, K.; Ray, M. K.; Jaleel, M. A.; Kadival, G. V.

    2002-01-01

    A radioassay for the detection of antitubercular antibody has been developed. The technique involves the addition of 125I-labeled Mycobacterium tuberculosis antigen as a tracer, diluted clinical sample (serum or cerebrospinal fluid [CSF]), and heat-inactivated Staphylococcus aureus to capture the antibody, incubation for 4 h, and quantitation of the amount of antibody present in the sample. A total of 330 serum samples from patients with pulmonary tuberculosis and 138 control serum samples fr...

  16. Interpretation of the Detection of Antibodies to Sarcocystis neurona in the serum and CSF of young horses

    OpenAIRE

    Cook, Anne Grimsley

    2001-01-01

    Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24 hours of age (post...

  17. Comparison of immunodiffusion and enzyme linked immunosorbent assay in the detection of abnormal antibodies in pigeon breeder's disease.

    OpenAIRE

    Simpson, C.; Shirodaria, P V; J. P. Evans; Simpson, D I; Stanford, C F

    1992-01-01

    AIMS: To compare the sensitivity of two methods for the detection of serum antibodies to pigeon faecal antigens in patients with pigeon breeder's disease. METHODS: Serum samples stored at -20 degrees C from 50 patients with pigeon breeder's disease, 50 control samples from patients with other respiratory diseases, and 50 healthy blood donors were examined for the precipitating antibodies and IgG antibodies to antigens present in extract of pigeon droppings by immunodiffusion and enzyme linked...

  18. A novel polymorphism of human complement component C3 detected by means of a monoclonal antibody

    DEFF Research Database (Denmark)

    Koch, C; Behrendt, N

    1986-01-01

    A mouse monoclonal antibody, HAV 4-1, obtained after immunization of a BALB/c mouse with purified C3F, detected a novel genetic polymorphism of human complement component C3 in a simple immunoblotting system. The frequency of HAV 4-1-positive genes was 20.1%. Reactivity of HAV 4-1 was closely...... related to C3F, but certain individuals with the C3F allele did not react with HAV 4-1. Conversely, certain C3S homozygous individuals did react with HAV 4-1. The polymorphism detected by this monoclonal antibody is therefore different from the previously described polymorphism based on charge differences....

  19. Detection of anti-Giardia lamblia serum antibody among children of day care centers

    Directory of Open Access Journals (Sweden)

    Guimarães Semíramis

    2002-01-01

    Full Text Available OBJETIVES: To detect anti-Giardia lamblia serum antibodies in healthy children attending public day care centers and to assess serological tests as tools for estimating the prevalence of G. lamblia in endemic areas. METHODS: Three separate stool specimens and filter paper blood samples were collected from 147 children ranging from 0 to 6 years old. Each stool sample was processed using spontaneous sedimentation and zinc sulfate flotation methods. Blood samples were tested by indirect immunofluorescence (IIF and enzyme-linked immunosorbent assay (ELISA for Giardia IgG. RESULTS AND CONCLUSIONS: Of 147 individuals tested, 93 (63.3% showed Giardia cysts in their feces. Using IIF and ELISA, serum antibodies were detected in 93 (63.3% and 100 (68% samples , respectively. Sensitivity of IIF and ELISA was 82% and 72%, respectively. However, ELISA revealed to be less specific (39% than IIF (70%. IIF also showed a higher concordance with microscopic examination than ELISA.

  20. Detection of anti-Giardia lamblia serum antibody among children of day care centers

    Directory of Open Access Journals (Sweden)

    Semíramis Guimarães

    2002-02-01

    Full Text Available OBJETIVES: To detect anti-Giardia lamblia serum antibodies in healthy children attending public day care centers and to assess serological tests as tools for estimating the prevalence of G. lamblia in endemic areas. METHODS: Three separate stool specimens and filter paper blood samples were collected from 147 children ranging from 0 to 6 years old. Each stool sample was processed using spontaneous sedimentation and zinc sulfate flotation methods. Blood samples were tested by indirect immunofluorescence (IIF and enzyme-linked immunosorbent assay (ELISA for Giardia IgG. RESULTS AND CONCLUSIONS: Of 147 individuals tested, 93 (63.3% showed Giardia cysts in their feces. Using IIF and ELISA, serum antibodies were detected in 93 (63.3% and 100 (68% samples , respectively. Sensitivity of IIF and ELISA was 82% and 72%, respectively. However, ELISA revealed to be less specific (39% than IIF (70%. IIF also showed a higher concordance with microscopic examination than ELISA.

  1. Detection of anti neutrophil antibodies by radio-iodinated protein A binding test

    International Nuclear Information System (INIS)

    The granulocyte associated IgG in normal and neutropenic subjects has been determined by a direct quantitative assay using radiolabeled staphylococcal protein A. This assay allows to postulate an immunological mechanism to explain the neutropenia in 19 cases of neutropenias associated with malfunctions of the immune system and in 4 cases of idiopathic neutropenias. Discussed in this report is the possible interaction of immune complexes bound in vivo to the granulocytes. By an immunofluorescence test, it has been possible to detect IgG or IgM antibodies in only 52% of patients with a positive direct assay. The determination of granulocyte-associated IgG is therefore a better indicator for defining an auto-immune neutropenia than the detection of free antibodies in the sera

  2. Agar gel immunodiffusion assay to detect antibodies to Type A influenza virus.

    Science.gov (United States)

    Jenson, Terra A

    2014-01-01

    The agar gel immunodiffusion (AGID) test is used to detect antibodies to Type A influenza group-specific antigens, i.e., the ribonucleoprotein (RNP) and matrix (M) proteins. Therefore, this test will detect antibodies to all influenza A virus subtypes. AGID is commonly used to screen poultry flocks for avian influenza virus infection. The AGID is a simple and economical serological test. All serological testing has its advantages and disadvantages which should be considered before choosing the optimal test for the laboratory needs. Each laboratory must evaluate the laboratory's resources, the volume of testing, the goal of testing, how the test results are used and what types of samples are being tested in order to select the optimal test. PMID:24899427

  3. Referencing cross-reactivity of detection antibodies for protein array experiments [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Darragh Lemass

    2016-01-01

    Full Text Available Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires requires a platform-dependent, lot-to-lot validation of secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. The cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Secondary antibody characterisation using protein arrays enables generation of reference lists of cross-reactive proteins, which can be then excluded from analysis in follow-up experiments. Furthermore, making such cross-reactivity lists accessible to the wider research community may help to interpret data generated by the same antibodies in applications not related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

  4. New method of antibody detection by indirect immunoperoxidase plaque staining for serodiagnosis of African swine fever.

    OpenAIRE

    Pan, I. C.; Huang, T. S.; Hess, W. R.

    1982-01-01

    An indirect immunoperoxidase plaque-staining method was developed for detecting antibody to African swine fever virus infection. In both sensitivity and specificity, the test was comparable to indirect immunofluorescence. Because it has all of the desirable features of the indirect immunofluorescence test and may also be readily used for testing large numbers of sera, the indirect immunoperoxidase plaque-staining method can be used as a single and final serodiagnostic test in a large-scale su...

  5. A latex slide agglutination test for rapid detection of antimyeloperoxidase antibody.

    OpenAIRE

    Ko, K.H.; S. S. Lee; Lawton, J W

    1999-01-01

    AIM: To develop and test a new latex slide agglutination test (MPO-LSAT) to detect antimyeloperoxidase (anti-MPO) antibody in serum. METHODS: Latex bead coating was adjusted to give maximum sensitivity by attending to latex size, MPO to latex ratio for coupling, ratio of diluted serum to MPO-latex, reaction time and temperature for coupling, and reaction time for agglutination. Inhibition studies were performed using MPO, proteinase 3, bactericidal/permeability increasing protein, and lactofe...

  6. Detection of Foot-and-mouth Disease Serotype O by ELISA Using a Monoclonal Antibody

    OpenAIRE

    Chen, Hao-tai; Peng, Yun-hua; ZHANG, YONG-GUANG; Liu, Xiang-tao

    2012-01-01

    An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, and A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Furthermore, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suita...

  7. Immunological characterization of proteins detected by phosphotyrosine antibodies in cells transformed by Rous sarcoma virus.

    OpenAIRE

    Linder, M E; Burr, J G

    1988-01-01

    Phosphotyrosine antibodies were used to identify tyrosine-phosphorylated proteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. A large number of tyrosine phosphoproteins were detected. A similar set of proteins was observed in RSV-transformed murine cells. An 85,000-dalton protein, however, was present in transformed avian cells but missing in transformed murine cells. Neither the 85,000-dalton protein nor any of the other tyrosine phosphoproteins appeared to be viral s...

  8. PCR and antibody methods: Research compares two cattle feed tests that detect bovine byproduct contaminants

    OpenAIRE

    Sawyer, Mary M.; Smith, Wayne L.; Rensen, Gabriel J.; Osburn, Bennie I.; Cullor, James S.

    2005-01-01

    Preventing the spread of mad cow disease through contaminated cattle feed is a major concern of beef and dairy producers, regulators and consumers around the world. Routine testing of cattle feeds for the presence of banned substances is a critical control point in assuring animal health and food safety. We compared the results of two test procedures (a real-time polymerase chain reaction [PCR] assay and a commercially available ruminant antibody detection kit) on five cattle rations spiked w...

  9. Evaluation of Four Methods for Detection of Immunoglobulin M Antibodies to Dengue Virus

    OpenAIRE

    Branch, Songee L.; Levett, Paul N

    1999-01-01

    Dengue has become hyperendemic in many islands of the Caribbean region. The performance in a diagnostic laboratory of four commercial assays for detection of immunoglobulin M (IgM) antibodies was evaluated. Sera from 62 patients with dengue virus infection were studied. These included 18 patients from whom dengue virus type 2 was isolated in a 1997 outbreak (specimens collected a mean of 14 days after onset of symptoms), 8 patients with dengue hemorrhagic fever (me...

  10. Comparative Evaluation of Seven Commercial Tests for Detection of Heterophile Antibody in Infectious Mononucleosis

    OpenAIRE

    Skulnick, Martin; Low, Donald E.; Simor, Andrew E.; Patel, Mohan; George, Pauline; Chua, Robert

    1992-01-01

    Detection of heterophile antibodies in infectious mononucleosis is the most rapid and cost-effective method for confirming the clinical diagnosis of the disease. This study compared seven commercial test kits (the Oxoid Infectious Mononucleosis Kit [Oxoid Ltd], Immunoscan Im-Latex [Baxter Travenol], Mono-Latex [Wampole Laboratories], Monospot and Im Screen Test [Ortho Diagnostics], Immunoscan Im-RBC Test [Baxter Travenol], and Infectious Mononucleosis Test [NCS Diagnostics]) to the Davidsohn ...

  11. 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.

  12. Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

    OpenAIRE

    Weare, J A; Robertson, E F; Madsen, G; Hu, R; Decker, R H

    1991-01-01

    Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a...

  13. Enzyme immunoassay for detection of human immunodeficiency virus-specific immunoglobulin A antibodies.

    OpenAIRE

    Connor, E; Z. Wang; STEPHENS, R; Holland, B.; Palumbo, P.; McSherry, G; Oleske, J; Denny, T

    1993-01-01

    Early diagnosis of human immunodeficiency virus (HIV) infection may be difficult in adults with acute or recent HIV infection and in infants with perinatally acquired HIV. Detection of HIV-specific immunoglobulin A (IgA) antibodies in infant serum by Western blot (immunoblot) has been suggested as a reliable method to identify HIV-infected infants, especially those over the age of 6 months, and as an adjunct to diagnosis of acute HIV infection in adults. We developed a simple enzyme immunoass...

  14. Monoclonal antibodies targeted to alpha-oligonucleotides. Characterisation and application in nucleic acid detection.

    OpenAIRE

    Cros, P.; Kurfürst, R; Allibert, P; Battail, N; Piga, N; Roig, V; Thuong, N T; Mandrand, B; Hélène, C

    1994-01-01

    The aim of the present study was to test the antigenicity of alpha-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize alpha-deoxyribonucleotides. Two were raised against a poly(alpha-dT) sequence and specifically recognized the alpha-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as alpha-nucleotides and, surpr...

  15. Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies.

    OpenAIRE

    Stamm, W. E.; Tam, M; Koester, M; Cles, L

    1983-01-01

    We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C. trachomatis. Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 ver...

  16. Specific antibodies to detect Tamarillo leaf malformation virus (TALMV) in Tamarillo

    International Nuclear Information System (INIS)

    In Colombia, yields of Tamarillo are seriously affected by a complex viral disease known as virosis. This pathology was first reported in 1991 in the north of Antioquia and currently affects all Tamarillo growing regions in the country. Recent works have demonstrated the association of two potyviruses (potyviridae) with this disease: potato virus y (PVY) and Tamarillo leaf malformation virus (TALMV, proposed species). Specific diagnostic tools are required for early asymptomatic detection of these viruses and Tamarillo certification programs. In this study, we report the obtention of TALMV specific antibodies using a 15 residues peptide mimicking the n-terminal coat protein. Specificity and sensitivity of the anti-TALMV antibodies was determined by Elisa and dot-blot using recombinant protein and synthetic peptides as controls. The usefulness of these antibodies was validated from a preliminary trial of TALMV detection in plant samples obtained from Tamarillo crops in eastern Antioquia and results were compared with a TALMV specific coat RT-PCR detection protocol.

  17. Fluorescent microbead-based immunoassay for anti-Erysipelothrix rhusiopathiae antibody detection in cetaceans.

    Science.gov (United States)

    Melero, Mar; Giménez-Lirola, Luis G; Rubio-Guerri, Consuelo; Crespo-Picazo, José Luis; Sierra, Eva; García-Párraga, Daniel; García-Peña, Francisco Javier; Arbelo, Manuel; Álvaro, Teresa; Valls, Mónica; Sánchez-Vizcaíno, José Manuel

    2016-01-13

    A fluorescent microbead-based immunoassay (FMIA) for detection of anti-Erysipelothrix rhusiopathiae antibodies in pigs was adapted for use in cetaceans. The FMIA was validated and adjusted using serum samples from 10 vaccinated captive bottlenose dolphins Tursiops truncatus collected between 1 and 13 mo after immunization. The technique was then used to analyze specimens from 15 free-ranging cetaceans stranded alive on the Valencian Mediterranean coast between 2006 and 2014: 11 striped dolphins Stenella coeruleoalba, 3 Risso's dolphins Grampus griseus and 1 bottlenose dolphin Tursiops truncatus. One of these wild animals was confirmed to have died from E. rhusiopathiae septicemia, but no anti-E. rhusiopathiae antibodies were detected in its serum, pericardial fluid or milk samples. Another free-ranging individual, which lacked any signs or lesions that might be indicative of E. rhusiopathiae infection, showed high fluorescence intensity similar to that measured in captive dolphins at 6-13 mo after vaccination. These results suggest that this animal underwent an E. rhusiopathiae infection several months before stranding. The findings in the present study suggest that FMIA can be useful for detecting anti-E. rhusiopathiae antibodies in cetaceans, and its application to free-ranging animals is particularly interesting because of the great value of these specimens. Furthermore, the FMIA can be multiplexed to allow the determination of up to 100 analytes per sample in a single well, thereby reducing the cost, time and sample volume needed. PMID:26758657

  18. Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

    Science.gov (United States)

    Apel, W A; Dugan, P R; Filppi, J A; Rheins, M S

    1976-07-01

    An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique. PMID:61736

  19. Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

    Science.gov (United States)

    Fukuma, Aiko; Fukushi, Shuetsu; Yoshikawa, Tomoki; Tani, Hideki; Taniguchi, Satoshi; Kurosu, Takeshi; Egawa, Kazutaka; Suda, Yuto; Singh, Harpal; Nomachi, Taro; Gokuden, Mutsuyo; Ando, Katsuyuki; Kida, Kouji; Kan, Miki; Kato, Nobuyuki; Yoshikawa, Akira; Kitamoto, Hiroaki; Sato, Yuko; Suzuki, Tadaki; Hasegawa, Hideki; Morikawa, Shigeru; Shimojima, Masayuki; Saijo, Masayuki

    2016-01-01

    Background Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. Methodology/Principal Findings We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID50/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>105 copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV. Conclusions The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia. PMID:27045364

  20. Technetium-99m labeled monoclonal antibodies in the detection of metastatic melanoma

    International Nuclear Information System (INIS)

    Twenty-six stage II/III malignant melanoma patients with 321 measurable metastatic lesions were imaged using Fab fragments of an IgG murine monoclonal antibody labeled specifically with 10-30 mCi Tc-99m with a bi-functional chelating method (NeoRx, Seattle, WA). There were no side effects or adverse reactions. Immunoscintigraphy demonstrated 66.6% of lesions larger than 1 cm and 92.5% of lesions larger than 3 cm. Most frequently detected metastases were in lymph nodes, subcutaneous areas, and bone. Of lesions less than 1 cm, 23.6% were detected if superficial cutaneous lesions were excluded. The smallest detectable lesion was 4 mm. Twenty-one additional clinically unsuspected sites were visualized in 12 of the 26 patients studied. Of these, 56% were confirmed as metastasis by other tests. There were apparent nonspecific localizations owing to other causes, including fracture, varicosities, skin abscess and pneumonitis. Increased experience in image analysis facilitates correct interpretation of these localizations. This study demonstrates that imaging with Tc-99m labeled antibody fragments detects melanoma lesions in organs routinely surveyed and in other areas not routinely assessed by other imaging techniques. The procedure is readily performed and safe. The principal advantage of the test is its ability to survey the entire body and all organs with a single test. Its principal limitation, in common with other diagnostic imaging procedures, is its poor sensitivity for detecting lesions less than 1 cm

  1. Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

    Science.gov (United States)

    Gong, Haibiao; Holcomb, Ilona; Ooi, Aik; Wang, Xiaohui; Majonis, Daniel; Unger, Marc A; Ramakrishnan, Ramesh

    2016-01-20

    The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method's application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and

  2. Characterization of a Monoclonal Antibody against Syringate Derivatives: Application of Immunochemical Detection of Methyl Syringate in Honey.

    Science.gov (United States)

    Kato, Yoji; Fujinaka, Rie; Juri, Maki; Yoshiki, Yui; Ishisaka, Akari; Kitamoto, Noritoshi; Nitta, Yoko; Ishikawa, Hirohito

    2016-08-24

    Syringic acid is one of the key skeletal structures of plant-derived chemicals. The derivatives of syringic acid have certain biological functions. In this study, a monoclonal antibody to syringic acid-based phytochemicals was prepared and characterized. The obtained antibody reacted with methyl syringate, syringic acid, and leonurine. Methyl syringate is a characteristic compound found in manuka honey, other honey varieties, and plants. Manuka honey was fractionated using HPLC, and the reactivity of the fractions with the antibody was examined. The antibody reacted with the fraction in which methyl syringate was eluted. The amount of methyl syringate in honeys as estimated by ELISA using the antibody had a good linearity compared with that estimated by HPLC. These results suggest that the antibody is applicable for the immunochemical detection of syringic acid derivatives in plants and foods. PMID:27477590

  3. Identification of five serum protein markers for detection of ovarian cancer by antibody arrays.

    Directory of Open Access Journals (Sweden)

    Weidong Jiang

    Full Text Available BACKGROUND: Protein and antibody arrays have emerged as a promising technology to study protein expression and protein function in a high-throughput manner. These arrays also represent a new opportunity to profile protein expression levels in cancer patients' samples and to identify useful biosignatures for clinical diagnosis, disease classification, prediction, drug development and patient care. We applied antibody arrays to discover a panel of proteins which may serve as biomarkers to distinguish between patients with ovarian cancer and normal controls. METHODOLOGY/PRINCIPAL FINDINGS: Using a case-control study design of 34 ovarian cancer patients and 53 age-matched healthy controls, we profiled the expression levels of 174 proteins using antibody array technology and determined the CA125 level using ELISA. The expression levels of those proteins were analyzed using 3 discriminant methods, including artificial neural network, classification tree and split-point score analysis. A panel of 5 serum protein markers (MSP-alpha, TIMP-4, PDGF-R alpha, and OPG and CA125 was identified, which could effectively detect ovarian cancer with high specificity (95% and high sensitivity (100%, with AUC =0.98, while CA125 alone had an AUC of 0.87. CONCLUSIONS/SIGNIFICANCE: Our pilot study has shown the promising set of 5 serum markers for ovarian cancer detection.

  4. Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

    Science.gov (United States)

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2014-01-01

    Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin. PMID:24830148

  5. Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases

    DEFF Research Database (Denmark)

    Samuelsen, Simone V; Maity, Arindam; Nybo, Mads;

    2016-01-01

    Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take ...... correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens' composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.......Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take...... structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid...

  6. 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.

  7. Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

    International Nuclear Information System (INIS)

    Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL-1 with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 μg mL-1 and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 μL of human blood was sufficient to perform the assay with the modified fibers.

  8. Surface modification of polyacrylonitrile fiber for immobilization of antibodies and detection of analyte

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Swati, E-mail: swatijain.iitd@gmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Chattopadhyay, Sruti, E-mail: srutic@hotmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Jackeray, Richa, E-mail: richajackeray.iitd@gmail.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India); Singh, Harpal, E-mail: harpal2000@yahoo.com [Center for Biomedical Engineering, Indian Institute of Technology, New Delhi, 110016 (India)

    2009-11-10

    Pendent nitrile groups of multifilamentous polyacrylonitrile (PAN) fibers were reduced to amino groups using lithium aluminum hydride for different time of reduction and amine content was estimated by performing acid-base titrations. Attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) and Differential Scanning Calorimetry (DSC) were used for the characterization of the generated amino groups and thermal properties of the reduced fibers, respectively. The surface morphology of the fibers after reduction and immobilization was characterized using Scanning Electron Microscope (SEM). The newly formed amino groups of the fibers were activated by using glutaraldehyde for the covalent linking of Goat anti-Rabbit IgG-HRP (GAR-HRP) antibody enzyme conjugate. Modified PAN fibers were evaluated as a matrix for sandwich ELISA by using Goat anti-Rabbit antibody (GAR-IgG), Rabbit anti-Goat (RAG-IgG) as analyte and enzyme conjugate GAR-HRP. The fibers reduced for 24 h were able to detect the analyte RAG-IgG at a concentration as low as 3.75 ng mL{sup -1} with 12% skimmed milk as blocking reagent for the optimized concentration of primary antibody GAR-IgG 3 {mu}g mL{sup -1} and peroxidase conjugate GAR-HRP dilution of 8000 fold. The sensitivity, specificity and reproducibility of the developed immunoassay was further established with antibodies present in human blood using Rabbit anti-Human (RAH-IgG) antibody and the corresponding HRP enzyme conjugate. As low as 0.1 {mu}L of human blood was sufficient to perform the assay with the modified fibers.

  9. Application of a human osteogenic sarcoma cell culture for detection of human cytomegalovirus antibody by immunofluorescence tests.

    OpenAIRE

    Furukawa, T; Herold, R; Zolnick, P; Plotkin, S A

    1981-01-01

    A human osteogenic sarcoma cell culture is useful in the immunofluorescence serological test for detecting human cytomegalovirus antibody. These sarcoma cells are chronically infected with human cytomegalovirus and provide a constant number of immunofluorescence-positive cells.

  10. Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

    Directory of Open Access Journals (Sweden)

    Qian Yajuan

    2011-05-01

    Full Text Available Abstract Background Cucumber green mottle mosaic virus (CGMMV, a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA, triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA, Dot-immunobinding assay (DBIA, direct tissue blot immunoassay (DTBIA and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR were described for detection and diagnosis of CGMMV. Results Using the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA. Conclusions The established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.

  11. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies.

    Science.gov (United States)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-11-15

    A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use. PMID:25076184

  12. 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.

  13. Direct radioimmunoassay for the detection of IgM antibodies against Mycoplasma pneumoniae

    International Nuclear Information System (INIS)

    A direct solid-phase radioimmunoassay is described for detecting IgM-class antibodies against Mycoplasma pneumoniae. The assay achieves a preliminary separation of IgM from other serum proteins by immunoadsorption to anti-IgM-coated wells in microtitre plates. The IgM is then tested for antigen specificity by measuring its ability to bind radiolabelled M. pneumoniae. Positive results are confirmed by retesting sera after treatment with 2-mercaptoethanol. The assay is specific for IgM-class antibodies and specific for M. pneumoniae. It is not affected by competitive inhibition from IgG and avoids the use of density gradient centrifugation or gel filtration to separate IgM from other immunoglobulins. It is sensitive, reproducible, rapid, simple and requires very little serum. (Auth.)

  14. A multiplexed immunoassay for detection of antibodies to Actinobacillus pleuropneumoniae (App) in pigs

    DEFF Research Database (Denmark)

    Berger, Sanne Schou; Boas, Ulrik; Andresen, Lars Ole;

    2014-01-01

    The bacterium Actinobacillus pleuropneumoniae (App) is the causative agent of porcine pleuropneumoniae, a contagious and severe respiratory disease in pigs. Based on capsular antigens, 15 App serovars have been described, and the prevalence and morbidity of these serovars vary with geographic...... regions (1). In Denmark, the most important serovars are considered to be App 1, 2, 5, 6, 7, 10 and 12. As part of the Danish surveillance program for App, the Danish Veterinary Institute uses ELISAs and complement fixation tests (CFT) to test for porcine anti-App antibodies (2-7). In an effort to improve...... our diagnostic tools, we are currently developing a novel indirect fluorescent microsphere immunoassay that can facilitate simultaneous detection of antibodies towards multiple App serovars within a single serum sample volume. The multiplex immunoassay is based on Luminex technology (8) and has...

  15. DETECTION OF ANTIBODIES AGAINST AVIAN PNEUMOVIRUS IN BROILER BREEDER FLOCKS IN PAKISTAN

    Directory of Open Access Journals (Sweden)

    M. D. Ahmad, M. Chaudhry and H. B. R. Chaudhry1

    2005-04-01

    Full Text Available A total of 282 serum samples were collected from 10 flocks, representing eight poultry breeding companies. These birds were reared in Punjab and North West Frontier Province of Pakistan. The antibodies against Turkey Rhinotracheitis virus/Swollen Head Syndrome (TRT/SHS were detected by using a commercial kit based on Blocking Enzyme Immuno-Assay. Out of 282 samples tested for TRT/SHS antibodies, 53 were positive, 28 were in grey-zone and the remaining 201 samples were declared non-reactive. On the basis of this study it is evident that the poultry industry of Pakistan is at risk of avian pneumovirus (TRT/SHS infection. It would be advisable to protect the valuable poultry flocks using TRT/SHS vaccine.

  16. Bluetongue virus: comparative evaluation of enzyme-linked immunosorbent assay, immunodiffusion, and serum neutralization for detection of viral antibodies.

    OpenAIRE

    Poli, G.; Stott, J.; Liu, Y. S.; Manning, J S

    1982-01-01

    Comparative studies on the detection of bovine serum immunoglobulin G antibodies to bluetongue virus with an enzyme-linked immunosorbent assay, an immunodiffusion method, and a serum neutralization assay demonstrated complete concordance between the enzyme-linked immunosorbent assay and the serum neutralization assay results. However, the immunodiffusion method failed to detect bluetongue virus antibody in a substantial number of sera found to possess bluetongue virus immunoglobulin G with th...

  17. Development of a polyclonal competitive enzyme-linked immunosorbent assay for detection of antibodies to Ehrlichia ruminantium

    OpenAIRE

    Sumption, Keith J.; Paxton, Edith; Bell-Sakyi, Lesley

    2003-01-01

    A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnosti...

  18. Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immunosorbent assay.

    OpenAIRE

    Anusz, K Z; Mason, P H; Riggs, M W; Perryman, L E

    1990-01-01

    A monoclonal antibody enzyme-linked immunosorbent assay (ELISA) was developed to detect Cryptosporidium parvum oocysts in bovine feces. Fecal oocysts were concentrated by centrifugation through Formalin-ethyl acetate solution and captured with monoclonal antibody 18.280.2 reactive with C. parvum oocysts. Captured oocysts were detected with goat anti-oocyst serum, following the addition of a peroxidase conjugate of rabbit anti-goat immunoglobulin and O-phenylenediamine substrate. The assay was...

  19. Colorimetric detection of influenza A virus using antibody-functionalized gold nanoparticles.

    Science.gov (United States)

    Liu, Yuanjian; Zhang, Linqun; Wei, Wei; Zhao, Hongyu; Zhou, Zhenxian; Zhang, Yuanjian; Liu, Songqin

    2015-06-21

    Early and accurate diagnosis is considered the key issue to prevent the further spread of viruses and facilitate influenza therapy. Herein, we report a colorimetric immunosensor for influenza A virus (IAV) based on gold nanoparticles (AuNPs) modified with monoclonal anti-hemagglutinin antibody (mAb). The immunosensor allows for a fast, simple, and selective detection of IAV. In this assay, influenza-specific antibodies are conjugated to AuNPs to create mAb-AuNP probes. Since IAV has multiple recognition sites for probes on the surface, the mAb-AuNP probes can be specifically arranged on the virus surface due to their very specific antigen recognition. In this case, this aggregation of the mAb-AuNP probes produces a red shift in the absorption spectrum due to plasmon coupling between adjacent AuNPs, and it can be detected with the naked eye as a color change from red to purple and quantified with the absorption spectral measurements. The aggregate formation is also confirmed with transmission electron microscopy (TEM) imaging and dynamic light scattering (DLS). Under the optimal conditions, the present immunoassay can sensitively measure H3N2 IAV (A/Brisbane/10/2007) with a detection limit of 7.8 hemagglutination units (HAU). This proposed immunosensor revealed high specificity, accuracy, and good stability. Notably, it is a single-step detection using AuNP probes and UV-vis spectrophotometer for readout, and no additional amplification, e.g., enzymatic, is needed to read the result. This assay depends on an ordered AuNP structure covering the virus surface and can be applied to any virus pathogen by incorporating the appropriate pathogen-specific antibody. PMID:25899840

  20. Detection of infectious laryngotracheitis virus antibodies by glycoprotein-specific ELISAs in chickens vaccinated with viral vector vaccines.

    Science.gov (United States)

    Godoy, Alecia; Icard, Alan; Martinez, Mellisa; Mashchenko, Anna; García, Maricarmen; El-Attrachea, John

    2013-06-01

    Two glycoproteins of infectious laryngotracheitis virus (ILTV), gI and gB, were expressed in baculovirus and purified for the development of ILTV recombinant protein-based ELISAs. The ability of gB and gI ELISAs to detect ILTV antibodies in chickens vaccinated with viral vector vaccines carrying the ILTV gB gene, Vectormune FP-LT (the commercial fowlpox vector laryngotracheitis vaccine) and Vectormune HVT-LT (commercial turkey herpesvirus vector laryngotracheitis vaccine), was evaluated using serum samples from experimentally vaccinated and challenge chickens. The detection of gB antibodies in the absence of gI antibodies in serum from chickens vaccinated with FP-LT indicated that the gB ELISA was specific for the detection of antibodies elicited by vaccination with this viral vector vaccine. The gB ELISA was more sensitive than the commercial ILTV ELISA to detect seroconversion after vaccination with the FP-LT vaccine. Both gI and gB antibodies were detected in the serum samples collected from chickens at different times postchallenge, indicating that the combination of these ELISAs was suitable to screen serum samples from chickens vaccinated with either recombinant viral vector FP-LT or HVT-LT vaccines. The agreement between the gI ELISA and the commercial ELISA to detect antibodies in serum samples collected after challenge was robust. However, further validation of these ELISAs needs to be performed with field samples. PMID:23901757

  1. Detection of newly antibody-defined epitopes on HLA class I alleles reacting with antibodies induced during pregnancy.

    Science.gov (United States)

    Duquesnoy, R J; Hönger, G; Hösli, I; Marrari, M; Schaub, S

    2016-08-01

    The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry. PMID:27312793

  2. A three-layer immunoradiometric assay for antibodies in different immunoglobulin classes and its application to the detection of chicken thyroglobulin autoantibodies and of antibodies to sheep erythrocytes

    International Nuclear Information System (INIS)

    A versatile solid-phase assay for detection of antibodies in different immunoglobulin classes is described. The assay has been applied to: (1) the detection of IgG and IgM antithyroglobulin autoantibodies in chickens with spontaneous autoimmune thyroiditis, and (2) the detection of anti-sheep cell antibodies in normal chickens. Thyroglobulin-coated plastic tubes or formaldehyde-fixed sheep erythrocytes were used as the solid phase. Antisera were added in succession to the solid-phase antigen so as to form 3 antibody layers: (1) chicken antibody against the solid-phase antigen; (2) heavy-chain-specific rabbit anti-chicken immunoglobulin; and (3) 125I-labelled goat anti-rabbit immunoglobulin. The assay is suitable for routine determinations on large numbers of samples; its sensitivity enables small volumes of serum to be tested and allows considerable economy in the use of valuable class-specific antisera. The radiolabelled reagent can be readily applied to other assays employing rabbit antisera. (Auth.)

  3. Evaluation of Nobuto filter paper strips for the detection of avian influenza virus antibody in waterfowl

    Science.gov (United States)

    Dusek, Robert J.; Hall, Jeffrey S.; Nashold, Sean W.; TeSlaa, Joshua L.; Ip, Hon S.

    2011-01-01

    The utility of using Nobuto paper strips for the detection of avian influenza antibodies was examined in mallards (Anas platyrhynchos) experimentally infected with low pathogenic avian influenza viruses. Blood was collected 2 wk after infection and was preserved either as serum or whole blood absorbed onto Nobuto strips. Analysis of samples using a commercially available blocking enzyme-linked immunosorbent assay revealed comparable results (≥96% sensitivity for all methods) between sera stored at -30 C and the Nobuto strip preservation method even when the Nobuto strips were stored up to 3 mo at room temperature (RT). Significant differences were detected in the ratio of sample absorbance to negative control absorbance for Nobuto strips stored at RT compared with sera stored at -30 C, although these differences did not affect the ability of the test to reliably detect positive and negative samples. Nobuto strips are a convenient and sensitive alternative to the collection of serum samples when maintaining appropriate storage temperatures is difficult.

  4. 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.

  5. Monoclonal and polyclonal antibodies for ante- and post-mortem detection of PrPSc in sheep

    Directory of Open Access Journals (Sweden)

    Michele Dietrich Moura Costa

    2015-04-01

    Full Text Available Scrapie is a disease that affects sheep and goats and is characterized by the accumulation of an abnormal isoform (PrPSc of the cellular prion protein, PrPC, in the central nervous system (CNS and in lymphoid tissues. Detection of PrPSc in these tissues can be attempted by a variety of techniques, including immunohistochemistry (IHC and western blotting (WB, for which a wide range of monoclonal and polyclonal antibodies are commercially available. The objective of this study was to test and compare the efficacy of monoclonal antibodiesF89/160.1.5, F99/97.6.1, and P4 and polyclonal antibodies M52 and R486 in the detection of PrPSc in lymphoid and CNS tissue samples by using IHC. Positive and negative control samples of sheep brain and tonsils were provided by the Animal Health and Veterinary Laboratories Agency (AHVLA, UK. The IHC examination of CNS samples with both monoclonal and polyclonal antibodies confirmed the granular deposition of PrPSc in the neurons of the positive control tissues. However, while the monoclonal antibodies did not produce positive reactions in the negative controls, the polyclonal antibodies showed some non-specific staining. The testing of positive control tonsil samples with polyclonal and monoclonal antibodies identified positive control-specific reactions, whereas the negative control tissues were IHC-negative with all antibodies, although P4 and the polyclonal antibodies produced some background staining. In summary, although the polyclonal antibodies may be more accessible, their use is not advisable because of possible false positive reactions. The polyclonal antibody M52 was able to identify PrPC in brain and spleen samples by WB but other lymphoid tissues were negative.

  6. Validation of serological tests for the detection of antibodies against Treponema pallidum in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Sascha Knauf

    2015-03-01

    Full Text Available There is evidence to suggest that the yaws bacterium (Treponema pallidum ssp. pertenue may exist in non-human primate populations residing in regions where yaws is endemic in humans. Especially in light of the fact that the World Health Organizaiton (WHO recently launched its second yaws eradication campaign, there is a considerable need for reliable tools to identify treponemal infection in our closest relatives, African monkeys and great apes. It was hypothesized that commercially available serological tests detect simian anti-T. pallidum antibody in serum samples of baboons, with comparable sensitivity and specificity to their results on human sera. Test performances of five different treponemal tests (TTs and two non-treponemal tests (NTTs were evaluated using serum samples of 57 naturally T. pallidum-infected olive baboons (Papio anubis from Lake Manyara National Park in Tanzania. The T. pallidum particle agglutination assay (TP-PA was used as a gold standard for comparison. In addition, the overall infection status of the animals was used to further validate test performances. For most accurate results, only samples that originated from baboons of known infection status, as verified in a previous study by clinical inspection, PCR and immunohistochemistry, were included. All tests, TTs and NTTs, used in this study were able to reliably detect antibodies against T. pallidum in serum samples of infected baboons. The sensitivity of TTs ranged from 97.7-100%, while specificity was between 88.0-100.0%. The two NTTs detected anti-lipoidal antibodies in serum samples of infected baboons with a sensitivity of 83.3% whereas specificity was 100%. For screening purposes, the TT Espline TP provided the highest sensitivity and specificity and at the same time provided the most suitable format for use in the field. The enzyme immune assay Mastblot TP (IgG, however, could be considered as a confirmatory test.

  7. Validation of serological tests for the detection of antibodies against Treponema pallidum in nonhuman primates.

    Science.gov (United States)

    Knauf, Sascha; Dahlmann, Franziska; Batamuzi, Emmanuel K; Frischmann, Sieghard; Liu, Hsi

    2015-03-01

    There is evidence to suggest that the yaws bacterium (Treponema pallidum ssp. pertenue) may exist in non-human primate populations residing in regions where yaws is endemic in humans. Especially in light of the fact that the World Health Organizaiton (WHO) recently launched its second yaws eradication campaign, there is a considerable need for reliable tools to identify treponemal infection in our closest relatives, African monkeys and great apes. It was hypothesized that commercially available serological tests detect simian anti-T. pallidum antibody in serum samples of baboons, with comparable sensitivity and specificity to their results on human sera. Test performances of five different treponemal tests (TTs) and two non-treponemal tests (NTTs) were evaluated using serum samples of 57 naturally T. pallidum-infected olive baboons (Papio anubis) from Lake Manyara National Park in Tanzania. The T. pallidum particle agglutination assay (TP-PA) was used as a gold standard for comparison. In addition, the overall infection status of the animals was used to further validate test performances. For most accurate results, only samples that originated from baboons of known infection status, as verified in a previous study by clinical inspection, PCR and immunohistochemistry, were included. All tests, TTs and NTTs, used in this study were able to reliably detect antibodies against T. pallidum in serum samples of infected baboons. The sensitivity of TTs ranged from 97.7-100%, while specificity was between 88.0-100.0%. The two NTTs detected anti-lipoidal antibodies in serum samples of infected baboons with a sensitivity of 83.3% whereas specificity was 100%. For screening purposes, the TT Espline TP provided the highest sensitivity and specificity and at the same time provided the most suitable format for use in the field. The enzyme immune assay Mastblot TP (IgG), however, could be considered as a confirmatory test. PMID:25803295

  8. Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases

    Science.gov (United States)

    Nybo, Mads; Macaubas, Claudia; Lønstrup, Lars; Balboni, Imelda M.; Mellins, Elizabeth D.; Astakhova, Kira

    2016-01-01

    Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid cardiolipin, and as the last step, coupling of the product with azide-containing linker and copper-catalyzed click chemistry with β2-glycoprotein I and prothrombin. To prove utility of the product antigens, we used enzyme-linked immunosorbent assay and three cohorts of samples obtained from patients in Denmark (n = 34) and the USA (n = 27 and n = 14). Afterwards we analyzed correlation of the obtained autoantibody titers with clinical parameters for each patient. Our results prove that using novel antigens clinically relevant autoantibodies can be detected with high repeatability, sensitivity and specificity. Unlike previously used antigens the obtained autoantibody titers strongly correlate with high disease activity and in particular, with arthritis, renal involvement, anti-Smith antibodies and high lymphocyte count. Importantly, chemical composition of antigens has a strong influence on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens’ composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity. PMID:27257889

  9. Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

    Science.gov (United States)

    Baldi, Pablo C.; Araj, George F.; Racaro, Graciela C.; Wallach, Jorge C.; Fossati, Carlos A.

    1999-01-01

    The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis. PMID:10473531

  10. Solid phase radioimmunoassay for detection of malaria antigen. Comparison of monoclonal and polyclonal antibodies

    International Nuclear Information System (INIS)

    A solid phase competitive binding radioimmunoassay (RIA) was developed for the detection of Plasmodium falciparum in infected blood. A suspension of NP40 treated red blood cells was mixed with labelled antimalarial IgG, incubated and then added to malarial antigen coated microtitre plate. Antimalarial IgGs were purified either from high titre sera from individuals living in a malaria endemic area in Thailand or from a locally produced monoclonal antibody (MAB) which showed a bright generalized immunofluorescent staining pattern against all blood stages of P. falciparum, including gametocytes. This MAB reacted with 27 of 31 P. falciparum isolates from Thailand. Using dilution of red blood cells from in vitro cultures of P. falciparum, the test was found to detect parasites at levels equivalent to 13 and 2.2 parasites/106 red blood cells with labelled polyclonal IgG (PIgG) and labelled monoclonal IgG (MIgG), respectively. No false positive results were obtained among samples from non-malarial subjects. Of the samples that gave negative results upon microscopic examination, 50 and 35% were still positive with RIA using MIgG and PIgG, respectively. There was a correlation between RIA and the number of parasites, especially when MIgG was used. The results indicate that the IgG fraction of sera from individuals with natural acquired immunity to malaria showed a lower degree of sensitivity in parasite detection than the IgG from monoclonal antibody. (author)

  11. Development and evaluation of the rVP-ELISA for detection of antibodies against porcine parvovirus.

    Science.gov (United States)

    Kong, Miaomiao; Peng, Yonggang; Cui, Yuchao; Chang, Tiecheng; Wang, Xiaoling; Liu, Zhaoxia; Liu, Yonggang; Zhu, Yu; Luo, Yakun; Tang, Qinghai; Feng, Li; Cui, Shangjin

    2014-09-01

    The gene encoding the VP2 protein of porcine parvovirus (PPV) was expressed in an insect-baculovirus system. The recombinant (r) VP2 was similar antigenically/functionally to the native capsid protein as demonstrated by hemagglutination (HA), Western blotting using PPV positive sera. The purified rVP2 proteins were used as coating antigen to establish a rVP-ELISA method for detection of PPV positive and negative sera from pigs. The optimal operating conditions of the rVP-ELISA were: the concentration of rVP2 proteins coated on the wells was 2 μg/mL; the diluted concentration of serum was 1: 150 and that of the enzyme-labeled antibody was 1: 6000. A total of 596 sera were detected by this assay, and the average positive rate was 87%. Compared with France LSI kit, the result showed that the coincidence rate was 96.7%. In conclusion, the rVP2-ELISA is a sensitive and specific method for detecting antibodies against PPV. PMID:24945904

  12. Detection of leptospiral antibodies by microscopic agglutination test in north-east of Iran

    Institute of Scientific and Technical Information of China (English)

    Ehsanollah Sakhaee; Gholam Reza Abdollah pour

    2011-01-01

    Objective: To detect leptospiral antibodies by microscopic agglutination test (MAT) in north-east of Iran. Methods: This study was conducted to evaluate prevalence of human leptospiral infections by MAT, using six current reference strains of Leptospira interrogans in north-east of Iran. A total of 285 serum samples were collected from three north-east provinces of Iran, from December, 2009 to June, 2010. Results: Antibodies were detected at least against one serovar of Leptospira interrogans in 45 sera (15.79 %) among 285 samples at a dilution 1:100 or greater. Positive titers against more than one serovar were detected in 24 sera of the positive samples. Therefore, there were 75 positive reactions against different serovar of Leptospira interrogans. Positive titers were recorded against serovar icterohaemorrhagiae (31 samples), hardjo (26 samples), grippotyphosa (7 samples), pomona (5 samples), canicola (4 samples) and ballum (2 sample).Conclusions:In present study the most prevalent (Leptospira icterohaemorrhagiae) and the least prevalent (Leptospira ballum) serovar are different from previous studies. Maybe, species and prevalence of serovars change during the time in one area and between regions.

  13. Detection of plasma granule membrane protein (GMP-140) using radiolabelled monoclonal antibodies in thrombotic diseases

    International Nuclear Information System (INIS)

    Radioimmunoassay with two monoclonal antibodies to granule membrane protein (GMP-140) is used to determine whether plasma GMP-140 including its soluble and microparticle forms can be detected in patients with acute myocardial infarction (AMI) or during cardio-pulmonary bypass (CPB) and in platelet concentrates during storage. Monoclonal antibody (McAb) SZ-51 was used as a solid phase and 125I-labelled McAb S12 was used as a fluid phase. The assay showed sufficient sensitivity to detect as few as 1 ng/mL of purified GMP-140. There was only 10.0 ± 4.5 ng/mL in normal plasma (n 20) with no significant different between the male and the female controls. Ten patients undergoing CPB demonstrated a transient increase in the concentration of plasma GMP-140, especially 2 h after CPB, and its plasma level was inversely correlated with the platelet counting during bypass (r -0.81, P < 0.01). Patients with AMI (n = 16) were found to have significantly increased concentration of plasma GMP-140 after AMI, reaching the peak within 3 days and changing with the progression of AMI. The concentration of plasma GMP-140 increased progressively in platelet concentrates during storage, particularly 5 days after storage. In short, these data suggest that plasma GMP-140 can be reliably detected by radioimmunoassay with two McAbs to GMP-140 and its plasma level may serve as a useful marker of thrombosis and thrombotic diseases

  14. Laser-induced breakdown detection of temperature-ramp generated aggregates of therapeutic monoclonal antibody.

    Science.gov (United States)

    Menzen, Tim; Friess, Wolfgang; Niessner, Reinhard; Haisch, Christoph

    2015-08-01

    The detection and characterization of protein aggregation is essential during development and quality control of therapeutic proteins, as aggregates are typically inactive and may trigger anti-drug-antibody formation in patients. Especially large multi-domain molecules, such as the important class of therapeutic monoclonal antibodies (mAbs), can form various aggregates that differ in size and morphology. Although particle analysis advanced over the recent years, new techniques and orthogonal methods are highly valued. To our knowledge, the physical principle of laser-induced breakdown detection (LIBD) was not yet applied to sense aggregates in therapeutic protein formulations. We established a LIBD setup to monitor the temperature-induced aggregation of a mAb. The obtained temperature of aggregation was in good agreement with the results from previously published temperature-ramped turbidity and dynamic light scattering measurements. This study demonstrates the promising applicability of LIBD to investigate aggregates from therapeutic proteins. The technique is also adaptive to online detection and size determination, and offers interesting opportunities for morphologic characterization of protein particles and impurities, which will be part of future studies. PMID:26158409

  15. Direct Detection of Protein Biomarkers in Human Fluids Using Site-Specific Antibody Immobilization Strategies

    Directory of Open Access Journals (Sweden)

    Maria Soler

    2014-01-01

    Full Text Available Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

  16. Detection of Babesia divergens in southern Norway by using an immunofluorescence antibody test in cow sera

    Directory of Open Access Journals (Sweden)

    Røed Knut H

    2010-10-01

    Full Text Available Abstract Background The incidence of bovine babesiosis, caused by Babesia divergens (Apicomplexa: Piroplasmida has decreased markedly since the 1930 s, but may re-emerge as a consequence of climate change and changes in legislation and pasturing practices. This is a potentially serious disease, with both economical and animal welfare consequences. Therefore, there is a need to survey the distribution of B. divergens. Methods We tested sera from 306 healthy pastured cows from 24 farms along the southern Norwegian coast by using an indirect immunofluorescence IgG antibody test (IFAT. Fractions of seropositive cows were compared by calculating 95% CI. Results The results of this test showed that 27% of the sera were positive for B. divergens antibodies. The fraction of antibody-positive sera that we detected showed a two-humped distribution, with a high fraction of positives being found in municipalities in the western and eastern parts of the study area, while the municipalities between these areas had few or no positive serum samples. Conclusions Neither the farmers' observations nor the Norwegian Dairy Herd Recording System give an adequate picture of the distribution of bovine babesiosis. Serological testing of cows by using IFAT is a convenient way of screening for the presence of B. divergens in an area.

  17. Antibody-based fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine.

    Science.gov (United States)

    Huynh Nhat, Kim Phuong; Watanabe, Takayoshi; Yoshikoshi, Kensuke; Hohsaka, Takahiro

    2016-08-01

    Fluorescent indicators for protein phosphorylation are very important in not only fundamental biology but also biomedical applications. In this study, we developed novel fluorescent and fluorescent ratiometric indicators for detection of phosphotyrosine (pTyr) derivatives. A single-chain antibody variable fragment (scFv) against phosphotyrosine was fluorescent-labeled by incorporation of tetramethylrhodamine (TAMRA)-linked nonnatural amino acid at the N- or C-terminus. The TAMRA-labeled scFv showed fluorescence enhancement upon addition of pTyr-containing peptides based on antigen-dependent fluorescence quenching effect on TAMRA. The TAMRA-labeled scFv was further fused with enhanced green fluorescent protein (EGFP) to generate a double-labeled scFv for pTyr. In the absence of antigen, fluorescence resonance energy transfer (FRET) occurred from EGFP to TAMRA but TAMRA was quenched. The antigen-binding removed the quenching of TAMRA while FRET occurred without altering its efficiency. As a result of the FRET and antigen-dependent fluorescence quenching effect, the double-labeled scFv exhibited fluorescence ratio enhancement upon the antigen-binding. The fluorescent and fluorescent ratiometric indicators obtained in this study will become a novel tool for analysis of protein phosphorylation. Moreover, this strategy utilizes antibody derivatives, and therefore, can be easily applied to other antigen-antibody pairs to generate fluorescent ratiometric indicators for various target molecules. PMID:26896314

  18. Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection

    Directory of Open Access Journals (Sweden)

    Ingeborg Hospach

    2014-12-01

    Full Text Available Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα and Interleukin 6 (IL-6 were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D. The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established.

  19. Evaluation of five commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19.

    OpenAIRE

    Bruu, A L; Nordbø, S A

    1995-01-01

    The following commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19 were evaluated: Ideia Parvovirus B19-IgM, MRL Diagnostics Human Parvovirus B19 IgM ELISA, Parvoscan-B19, and Biotrin Parvo B19 IgM EIA and IF. A total of 203 serum specimens from patients who probably have current B19 infections or have other viral infections and sera with rheumatoid factor were investigated. Between 75 and 79 of 102 serum samples from patients thought to have current B19 infec...

  20. Detection of foot-and-mouth disease serotype O by ELISA using a monoclonal antibody.

    Science.gov (United States)

    Chen, Hao-Tai; Peng, Yun-Hua; Zhang, Yong-Guang; Liu, Xiang-Tao

    2013-02-01

    An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, and A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Furthermore, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suitable for diagnosis of FMDV serotype O infection in field stations. PMID:23600506

  1. Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus

    OpenAIRE

    Goutama Michael; Murtini Sri; Soejoedono Retno D; He Fang; Kwang Jimmy

    2010-01-01

    Abstract Background Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection. Results In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189A...

  2. Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody to hepatitis B core antigen.

    OpenAIRE

    Kryger, P; Mathiesen, L R; Møller, A M; Aldershvile, J; Hansson, B G; Nielsen, J O

    1981-01-01

    An enzyme-linked immunosorbent assay for detection of specific immunoglobulin M (IgM) antibodies against the core antigen of the hepatitis B virus (anti-HBc IgM) is described. The interference of IgM rheumatoid factor was evaluated quantitatively. In the anti-HBc IgM test, the rheumatoid factor gave false-positive results when the concentration exceeded 20 IU/ml. The rheumatoid-positive sera were disclosed by a control and retested for anti-HBc IgM after absorption of rheumatoid factor with l...

  3. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

    DEFF Research Database (Denmark)

    Boas, Ulrik; Lind, Peter; Riber, Ulla

    2014-01-01

    A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene...... against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the...... polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use....

  4. Evaluation of an Enzyme Immunoassay Technique for Detection of Antibodies against Treponema pallidum

    OpenAIRE

    Castro, Rita; Prieto, Emília S.; Santo, Irene; Azevedo, Jacinta; Exposto, Filomena da L.

    2003-01-01

    In the present study, the performance of an enzyme-linked immunosorbent assay (ELISA) technique (Eti-syphilis-G and Eti-syphilis-M; DiaSorin) for detection of Treponema pallidum immunoglobulin M (IgM) and IgG antibodies for the laboratory diagnosis of syphilis was evaluated. Four hundred forty-one samples were studied. The sensitivity and specificity of the ELISA were 100 and 93%, respectively, compared with the results of a microhemagglutination assay for Treponema pallidum (MHA-TP) and 99.4...

  5. Study of FAO/IAEA/PANAFTOSA ELISA kit for the detection of antibodies against FMD

    International Nuclear Information System (INIS)

    Two groups of sera were used to evaluate a liquid phase blocking ELISA (LPBE) for the detection of antibody against foot-and-mouth disease virus. One hundred and twenty sera, from animals with no previous history of FMD infection or vaccination, were analyzed by screening assay at a final dilution of 1:32. A second group of 120 sera, from animals vaccinated with an oil trivalent vaccine (O, A, C) were tested by titration in the LPBE. All the sera were tested against virus of three FMD serotypes, using O1 Campos, A24 Cruzeiro, C3 Indaial virus strains. (author)

  6. Solid-phase radioimmunoassay for the detection of antibodies to collagens

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay using 125I-protein A is described for the detection of antibodies to collagens of different types. The optimal conditions for the adsorption of collagen onto polystyrene microplates, then the incubations with the antiserum and finally with the 125I-protein A have been evaluated. The technique was applied successfully to antisera raised in rabbit, goat, guinea pig and mouse against human type I, II, III, IV, V and bovine type I, II, 1α2α3α, X1-X7 collagens

  7. Development and utilization of camelid VHH antibodies from alpaca for 2,2',4,4'-tetrabrominated diphenyl ether detection.

    Science.gov (United States)

    Bever, Candace R S; Majkova, Zuzana; Radhakrishnan, Rajeswaran; Suni, Ian; McCoy, Mark; Wang, Yanru; Dechant, Julie; Gee, Shirley; Hammock, Bruce D

    2014-08-01

    An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies. PMID:25005746

  8. A Generic Method for Fungal Spore Detection: The use of a monoclonal antibody and surface plasmon resonance

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Hearty, Stephen; Frøkiær, Hanne; Leonard, Paul; Hejgaard, Jørn; O'Kennedy, Richard; Nicolaisen, Mogens; Justesen, Annemarie Fejer

    This study describes a biosensing principle for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a monoclonal antibody (mab) and a SPR sensor for label-free detection of the model organism Puccinia striiformis f.sp. tritici (Pst) a biotrophic fungus...... causing wheat yellow rust. We have developed mabs towards intact whole spores and used a subtractive inhibition format for detection of spores in solution. The antibody was incubated with different spore concentrations and the remaining free antibody was quantified using a BIAcore® 3000 sensor....... Decreasing binding of mab to the sensor surface was observed as the Pst urediniospore concentration was increased. The detection range for the assay was 1.7 x 106 – 5.3 x 104 spores/ml. This study describes the first use of SPR for detection of fungal spores and the generic principle has the potential to...

  9. A Generic Method for Fungal Spore Detection: The use of a monoclonal antibody and surface plasmon resonance

    DEFF Research Database (Denmark)

    Skottrup, Peter; Hearty, Stephen; Frøkiær, Hanne;

    This study describes a biosensing principle for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a monoclonal antibody (mab) and a SPR sensor for label-free detection of the model organism Puccinia striiformis f.sp. tritici (Pst) a biotrophic fungus...... causing wheat yellow rust. We have developed mabs towards intact whole spores and used a subtractive inhibition format for detection of spores in solution. The antibody was incubated with different spore concentrations and the remaining free antibody was quantified using a BIAcore® 3000 sensor. Decreasing...... binding of mab to the sensor surface was observed as the Pst urediniospore concentration was increased. The detection range for the assay was 1.7 x 106 – 5.3 x 104 spores/ml. This study describes the first use of SPR for detection of fungal spores and the generic principle has the potential to be used in...

  10. A Generic Method for Fungal Spore Detection: The use of a monoclonal antibody and surface plasmon resonance

    DEFF Research Database (Denmark)

    Skottrup, Peter; Hearty, Stephen; Frøkiær, Hanne;

    2005-01-01

    This study describes a biosensing principle for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a monoclonal antibody (mab) and a SPR sensor for label-free detection of the model organism Puccinia striiformis f.sp. tritici (Pst) a biotrophic fungus...... causing wheat yellow rust. We have developed mabs towards intact whole spores and used a subtractive inhibition format for detection of spores in solution. The antibody was incubated with different spore concentrations and the remaining free antibody was quantified using a BIAcore® 3000 sensor. Decreasing...... binding of mab to the sensor surface was observed as the Pst urediniospore concentration was increased. The detection range for the assay was 1.7 x 106 – 5.3 x 104 spores/ml. This study describes the first use of SPR for detection of fungal spores and the generic principle has the potential to be used in...

  11. Surface plasmon resonance biosensor for direct detection of antibodies against human growth hormone.

    Science.gov (United States)

    Kausaite-Minkstimiene, Asta; Ramanaviciene, Almira; Ramanavicius, Arunas

    2009-10-01

    A direct, label-free detection method of antibodies against the human growth hormone (anti-HGH) using a surface plasmon resonance (SPR) biosensor is reported. The sensing surface of the surface plasmon resonance biosensor chip (SPR-chip) was modified by covalent coupling of the human growth hormone (HGH) to the self-assembled monolayer of 11-mercaptoundecanoic acid (MUA). HGH was immobilized via primary amine groups after activation of the MUA carboxyl groups with a mixture of N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The specific binding of monoclonal anti-HGH antibody on the HGH-modified surface was examined in the concentration range from 0.25 nM to 10 microM. The experimentally observed detection minimum for anti-HGH was 2.47 nM. A single immunoassay cycle could be done within 30 min including the HGH and anti-HGH association, HGH/anti-HGH complex dissociation and surface regeneration steps. The SPR biosensor response for repeatable detections of anti-HGH was highly reproducible and very stable. On the SPR-chip the formed HGH and anti-HGH complex (HGH/anti-HGH) could be gently dissociated and the sensing surface might be regenerated by 50 mM NaOH and 0.5% sodium dodecylsulfate (SDS) solution. Any changes in the original baseline level were detected during the 40 detection-regeneration cycles. This means that damage of the immobilized HGH-based sensitive layer during regeneration was minimal. It was demonstrated that the developed SPR-chip could be stored for at least 21 days before use without considerable loss of sensitivity towards anti-HGH. PMID:19768212

  12. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

    Directory of Open Access Journals (Sweden)

    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  13. Antibody-Based Detection of ERG Rearrangement-Positive Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Kyung Park

    2010-07-01

    Full Text Available TMPRSS2-ERG gene fusions occur in 50% of prostate cancers and result in the overexpression of a chimeric fusion transcript that encodes a truncated ERG product. Previous attempts to detect truncated ERG products have been hindered by a lack of specific antibodies. Here, we characterize a rabbit anti-ERG monoclonal antibody (clone EPR 3864; Epitomics, Burlingame, CA using immunoblot analysis on prostate cancer cell lines, synthetic TMPRSS2-ERG constructs, chromatin immunoprecipitation, and immunofluorescence. We correlated ERG protein expression with the presence of ERG gene rearrangements in prostate cancertissues using a combined immunohistochemistry(IHC and fluorescence in situ hybridization (FISH analysis. We independently evaluated two patient cohorts and observed ERG expression confined to prostate cancer cells and high-grade prostatic intraepithelial reoplasia associated with ERG-positive cancer, as well as vessels and lymphocytes (where ERG has a known biologic role. Image analysis of 131 cases demonstrated nearly 100% sensitivity for detecting ERG rearrangement prostate cancer, with only 2 (1.5% of 131 cases demonstrating strong ERG protein expression without any known ERG gene fusion. The combired pathology evaluation of 207 patient tumors for ERG protein expression had 95.7% sensitivity and 96.5% specificity for determining ERG rearrangement prostate cancer. Ir conclusion, this study qualifies a specific anti-ERG antibody and demonstrates exquisite association between ERG gene rearrangement and truncated ERG protein product expression. Giver the ease of performing IHC versus FISH, ERG protein expression may be useful for molecularly subtypirg prostate cancer based or ERG rearrangement status and suggests clinical utility it prostate needle biopsy evaluation.

  14. Detection of antibodies against Toxoplasma gondii in Mirounga leonina Linnaeus, 1758 (Pinnipedia, Phocidae from Elephant Island

    Directory of Open Access Journals (Sweden)

    Tony Silveira

    2016-03-01

    Full Text Available Objective: To investigate the presence of antibodies against Toxoplasma gondii (T. gondii in Mirounga leonina (M. leonina Linnaeus from Elephant Island, Antarctica. Methods: The animals were anesthetized, restrained, measured, weighed and had their blood collected by venipuncture of intervertebral lumbar epidural vein. Blood samples were collected from 102 individuals. Indirect hemagglutination and serum dilution at a proportion of 1:25 was used for specific immunoglobulin G anti-T. gondii detection. Results: Only one (0.98% specimen, a newly weaned calf, was seropositive. Conclusions: This study is the highest serological survey for antibody detection against T. gondii in M. leonina. The results suggest a low level of exposure to T. gondii, probably due to the absence of felids in the study area. The seropositivity presented by the elephant seal may be related to the presence of oocysts in water or cysts in their preys. Despite being reported the presence of the parasite in fish and molluscs, there are no records of tissue cysts or oocysts in squid or fish of the diet of M. leonina. Thus, further parasitological studies focused on preys of elephant seals are needed for a better understanding of infection of M. leonina by T. gondii.

  15. A 125I-protein A-binding assay detecting antibodies to cell surface antigens

    International Nuclear Information System (INIS)

    A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were than investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells. (orig.)

  16. Precise detection of L. monocytogenes hitting its highly conserved region possessing several specific antibody binding sites.

    Science.gov (United States)

    Jahangiri, Abolfazl; Rasooli, Iraj; Reza Rahbar, Mohammad; Khalili, Saeed; Amani, Jafar; Ahmadi Zanoos, Kobra

    2012-07-21

    Listeria monocytogenes, a facultative intracellular fast-growing Gram-positive food-borne pathogen, can infect immunocompromised individuals leading to meningitis, meningoencephalitis and septicaemias. From the pool of virulence factors of the organism, ActA, a membrane protein, has a critical role in the life cycle of L. monocytogenes. High mortality rate of listeriosis necessitates a sensitive and rapid diagnostic test for precise identification of L. monocytogenes. We used bioinformatic tools to locate a specific conserved region of ActA for designing and developing an antibody-antigen based diagnostic test for the detection of L. monocytogenes. A number of databases were looked for ActA related sequences. Sequences were analyzed with several online software to find an appropriate region for our purpose. ActA protein was found specific to Listeria species with no homologs in other organisms. We finally introduced a highly conserved region within ActA sequence that possess several antibody binding sites specific to L. monocytogenes. This protein sequence can serve as an antigen for designing a relatively cheap, sensitive, and specific diagnostic test for detection of L. monocytogenes. PMID:22575546

  17. Supramolecular biosensors based on electropolymerised pyrrole-cyclodextrin modified surfaces for antibody detection.

    Science.gov (United States)

    Wajs, Ewelina; Fernández, Núria; Fragoso, Alex

    2016-06-01

    The self-assembly of an adamantane-appended polymer bearing an antigen fragment on a polypyrrole-cyclodextrin modified surface provides a highly sensitive immunosensor with low limits of detection for celiac disease related targets. The pyrrole-carboxylic acid films were formed on the surface of gold electrodes by electropolymerisation and followed by covalent attachment of cyclodextrin units. Surface plasmon resonance measurements confirmed the role of the host/guest interactions between adamantane moieties and β-cyclodextrin hosts in the formation of the supramolecular sensor interface. Furthermore, this novel electrochemical supramolecular platform was effective in the amperometric detection of anti-gliadin antibodies in spiked serum samples with very good signal recovery. PMID:27097527

  18. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    Science.gov (United States)

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  19. Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Yan Chen; Hong Tian; Jian-Hui He; Jin-Yin Wu; You-jun Shang; Xiang-tao Liu

    2011-01-01

    Porcine reproductive and respiratory syndrome is caused by the PRRS virus(PRRSV), which has six structural proteins(GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay(ELISA)and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7%(266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.

  20. Porous silicon biosensor for detection of variable domain of heavy-chain of HCAb antibody

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong-yan; Lü Xiao-yi; JIA Zhen-hong; LI Jiang-wei; ZHANG Fu-chun

    2012-01-01

    In this paper,we produce porous silicon (PSi) by electrochemical etching,and it is the first time to evaluate the performance of label-free porous silicon biosensor for detection of variable domain of heavy chain of heavy-chain antibody (VHH).The binding of hen egg white lysozyme (HEWL) and VHH causes a red shift in the reflection spectrum of the biosensor.The red shift is proportional to the VHH concentration in the range from 14 μg · ml-1 to 30μg·ml-1 with a detection limit of 0.648 ng ·ml-1.The research is useful for the development of label-free biosensor applied in the rapid and sensitive determination of small molecules.

  1. 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.

  2. Immunodot blot assay to detect Helicobacter pylori using monoclonal antibodies against the 26 kDa protein.

    Science.gov (United States)

    Amini Najafabadi, Hossein; Paknejad, Maliheh; Farshad, Shohreh; Mohammadian, Taher; Seyyed Ebrahimi, Shadi Sadat; Amini Najafabadi, Azadeh

    2012-12-01

    Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The monoclonal antibodies were produced using the hybridoma technique. Reactivity of monoclonal antibodies was tested with the purified 26 kDa antigen and cell surface proteins from cultured H. pylori by ELISA. Furthermore reactivity of monoclonal antibodies was tested on negative and positive stool samples for H. pylori and suspensions of several major bacteria in stool by immunodot blot assay. Five stable hybridoma monoclones were obtained. The concordant reactivity of the monoclonal antibodies with H. pylori present in the stool samples, which had been tested previously using an ACON ELISA kit for H. pylori stool antigen testing, and unreactivity with several different major fecal bacteria in immunodot blotting indicates high specificity of the immunodot blot based on the reaction of produced monoclonal antibodies with the H. pylori antigen in stools. The findings indicate that the novel immunodot blot developed based on new monoclonal antibodies for stool antigens would be useful as a noninvasive method of diagnosing H. pylori infection. PMID:23244318

  3. A rapid assay for Hendra virus IgG antibody detection and its titre estimation using magnetic nanoparticles and phycoerythrin.

    Science.gov (United States)

    Gao, Yuan; Pallister, Jackie; Lapierre, Florian; Crameri, Gary; Wang, Lin-Fa; Zhu, Yonggang

    2015-09-15

    Detection of Hendra viral IgG antibody in animal sera is useful for surveillance following a virus outbreak. The commonly used enzyme-linked immunosorbent assay and fluorescence-based Luminex assay typically consist of three steps and take at least several hours to complete. We have simplified the procedure to two steps in an effort to develop a rapid procedure for IgG antibody, but not IgM antibody, detection. This is achieved by conjugating the fluorescence label R-phycoerythrin directly onto the IgG binding protein Protein G. The use of magnetic nanoparticles, due to their large specific surface area, has helped reduce each of the binding steps to 20 min. As a result, the whole assay can be completed in 60 min. We also demonstrate a method to quickly estimate IgG antibody titres by assaying the sera at only two dilutions (i.e. 1:20 and 1:1000) and using the fluorescence ratio at these dilutions as an indicator of antibody titre. The results of this approach correlated well with the well-regarded serum neutralization test in virus antibody assays. This protocol reported here can be adopted in Luminex assays, fluorescence-linked immunosorbent assays and assays on microfluidics platforms for rapid antibody surveillance of Hendra and other viruses. PMID:26141730

  4. Detection of vibrio cholerae O1 by using cerium oxide nanowires - based immunosensor with different antibody immobilization methods

    Science.gov (United States)

    Tam, Phuong Dinh; Hoang, Nguyen Luong; Lan, Hoang; Vuong, Pham Hung; Anh, Ta Thi Nhat; Huy, Tran Quang; Thuy, Nguyen Thanh

    2016-05-01

    In this work, we evaluated the effects of different antibody immobilization strategies on the response of a CeO2-nanowires (NWs)-based immunosensor for Vibrio cholerae O1 detection. Accordingly, the changes in the electron-transfer resistance ( R et ) from before to after cells bind to an antibody-modified electrode prepared by using three different methods of antibody immobilization were determined. The values were 16.2%, 8.3%, and 6.65% for the method that utilized protein A, antibodies activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS), and absorption, respectively. Cyclic voltammetry confirmed that the change in the current was highest for the immunosensors prepared using protein A (11%), followed by those prepared with EDC/NHS-activated antibodies (9%), and finally, those prepared through absorption (7.5%). The order of the antibody immobilization strategies in terms of resulting immunosensor detection limit and sensitivity was as follows order: absorption (3.2 × 103 CFU/mL; 45.1 Ω/CFU·mL-1) < EDC/NHS-activated antibody (1.0 × 103 CFU/mL; 50.6 Ω/CFU·mL-1) < protein A (1.0 × 102 CFU/mL; 65.8 Ω/CFU·mL-1). Thus, we confirmed that the protein A - mediated method showed significantly high cell binding efficiencies compared to the random immobilization method.

  5. Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization

    International Nuclear Information System (INIS)

    Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody. (orig.)

  6. Use of immunoblotting to detect antibodies to Mycoplasma crocodyli infection in the sera of crocodiles (Crocodylus niloticus).

    Science.gov (United States)

    Dawo, Fufa; Mohan, Krishna

    2008-02-01

    An immunoblotting protocol for the detection of antibodies to Mycoplasma crocodyli was developed using sonicated antigen of the reference strain 266/93. Immunoblotting detected nine reacting antigens, of which the 33 and 40kDa antigens were immunodominant. There was no difference in reactivity of the antigens against sera obtained from vaccinated and infected crocodiles. Both antigens are candidates for other serological and molecular studies. This is the first report to develop and apply an immunoblotting test for detection of antibody to M. crocodyli infection in crocodiles. PMID:17360201

  7. Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease.

    OpenAIRE

    Schumacher, I M; Brown, M B; Jacobson, E R; Collins, B R; Klein, P A

    1993-01-01

    Mycoplasma agassizii (proposed species novum) is the etiologic agent of an upper respiratory tract disease in the desert tortoise (Gopherus agassizii), which is threatened in most of its range. An enzyme-linked immunosorbent assay (ELISA) for the detection of M. agassizii-specific antibodies in desert tortoises was developed with a monoclonal antibody with specificity for desert tortoise immunoglobulin light chain. Plasma samples from one group of tortoises were tested immediately before and ...

  8. Microenzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G and Immunoglobulin M Antibodies to Legionella pneumophila

    OpenAIRE

    Elder, E M; Brown, A.; Remington, J S; Shonnard, J.; Naot, Y

    1983-01-01

    The microenzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin M and G (IgM, IgG) antibodies to Legionella pneumophila serogroup 1 antigens was evaluated. IgM antibodies were measured by both double-sandwich and single-sandwich techniques. These assays were compared with the previously standardized indirect immunofluorescence test in four groups of subjects: (i) pneumonia patients with culture-proven Legionnaires disease with serogroup 1 isolates, (ii) pneumonia patien...

  9. Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

    OpenAIRE

    Luciane A Amado; Livia M. Villar; Vanessa S de Paula; de Almeida, Adilson J; Ana Maria C Gaspar

    2006-01-01

    In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oral fluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One h...

  10. Comparison of four enzyme immunoassays for detection of human T-cell lymphotropic virus type 2 antibodies.

    OpenAIRE

    Gallo, D; Yeh, E T; Moore, E S; Hanson, C V

    1996-01-01

    Four licensed enzyme immunoassay (EIA) kits for the measurement of antibody to human T-cell lymphotropic virus (HTLV) type 1, one from Organon Teknika Corp. (OTC), one from Cambridge Biotech Corp. (CBC), and two from Abbott Laboratories (the 1993 modification [Abb 93] and the 2.0 version licensed in 1995 [Abb 95]), were evaluated for sensitivity and specificity in the detection of HTLV type 2 antibody, and the results were compared with those previously obtained with earlier kit versions. The...

  11. Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

    OpenAIRE

    Sofía Duque-Beltrán; Rubén Santiago Nicholls-Orejuela; Adriana Arévalo-Jamaica; Rafael Guerrero-Lozano; Sonia Montenegro; James, Mark A

    2002-01-01

    The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were pu...

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

    OpenAIRE

    2002-01-01

    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 experiment...

  13. Micro-indirect hemagglutination test for detection of antibodies to the Ibc protein of group B Streptococcus.

    OpenAIRE

    Thangavelu, C P; Koshi, G

    1980-01-01

    A micro-indirect hemagglutination test was developed for detecting antibody against the Ibc protein of group B Streptococcus. Formalin-preserved, tanned sheep erythrocytes were sensitized with a partially purified preparation of Ibc protein from a type Ic strain of group B streptococci. A total of 76% of 103 sera from pregnant and nonpregnant women had demonstrable antibody against this protein, with titers ranging from 10 to 320. Examination of five pairs of mother and cord sera revealed pas...

  14. Detection of Ebola Viral Antigen by Enzyme-Linked Immunosorbent Assay Using a Novel Monoclonal Antibody to Nucleoprotein

    OpenAIRE

    Niikura, Masahiro; Ikegami, Tetsuro; Saijo, Masayuki; Kurane, Ichiro; Miranda, Mary E.; Morikawa, Shigeru

    2001-01-01

    With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope ...

  15. The UW 21/123 monoclonal antibody in the radioimmunological detection of tumour tissue in the head and neck region

    International Nuclear Information System (INIS)

    When incorporated in a radioimmunoassay the UW 21/123 the monoclonal antibody permits the diagnosis of certain squamous cell carcinoma of the cephalocervical region and is particularly reliable in the detection laryngeal carcinomas. It even permits the assessment of tumour cells that are not yet fully developed and thus still escape histological detection. (MBC)

  16. E4 antibodies facilitate detection and type-assignment of active HPV infection in cervical disease.

    Directory of Open Access Journals (Sweden)

    Heather Griffin

    Full Text Available High-risk human papillomavirus (HPV infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins (HPV-16, -18, and -58 were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias (CIN, (n = 247 and normal controls (n = 28. All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76% and 5/9 (55.6% expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination.

  17. Detection and Quantification of Gluten during the Brewing and Fermentation of Beer Using Antibody-Based Technologies.

    Science.gov (United States)

    Panda, Rakhi; Zoerb, Hans F; Cho, Chung Y; Jackson, Lauren S; Garber, Eric A E

    2015-06-01

    In 2013 the U.S. Food and Drug Administration (FDA) defined the term ''gluten-free'' and identified a gap in the analytical methodology for detection and quantification of gluten in foods subjected to fermentation and hydrolysis. To ascertain the ability of current enzyme-linked immunosorbent assays (ELISAs) to detect and quantify gluten in fermented and hydrolyzed products, sorghum beer was spiked in the initial phases of production with 0, 20, and 200 μg/ml wheat gluten, and samples were collected throughout the beer production process. The samples were analyzed using five sandwich ELISAs and two competitive ELISAs and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western analysis employing four antibodies (MIoBS, R5, G12, and Skerritt). The sensitivity of the MIoBS ELISA (0.25 ppm) enabled the reliable detection of gluten throughout the manufacturing process, including fermentation, when the initial concentration of 20 μg/ml dropped to 2 μg/ml. The R5 antibody-based and G12 antibody-based sandwich ELISAs were unable to reliably detect gluten, initially at 20 μg/ml, after the onset of production. The Skerritt antibody-based sandwich ELISA overestimated the gluten concentration in all samples. The R5 antibody-based and G12 antibody-based competitive ELISAs were less sensitive than the sandwich ELISAs and did not provide accurate results for quantifying gluten concentration. The Western analyses were able to detect gluten at less than 5 μg/ml in the samples and confirmed the results of the ELISAs. Although further research is necessary before all problems associated with detection and quantification of hydrolyzed and fermented gluten are resolved, the analytical methods recommended by the FDA for regulatory samples can detect ≥ 20 μg/ml gluten that has undergone brewing and fermentation processes associated with the manufacture of beer. PMID:26038908

  18. Evaluation of a blocking ELISA for the detection of antibodies against Lawsonia intracellularis in pig sera

    Directory of Open Access Journals (Sweden)

    Merza Malik

    2011-04-01

    Full Text Available Abstract Background Lawsonia intracellularis is a common cause of chronic diarrhoea and poor performance in young growing pigs. Diagnosis of this obligate intracellular bacterium is based on the demonstration of the microbe or microbial DNA in tissue specimens or faecal samples, or the demonstration of L. intracellularis-specific antibodies in sera. The aim of the present study was to evaluate a blocking ELISA in the detection of serum antibodies to L. intracellularis, by comparison to the previously widely used immunofluorescent antibody test (IFAT. Methods Sera were collected from 176 pigs aged 8-12 weeks originating from 24 herds with or without problems with diarrhoea and poor performance in young growing pigs. Sera were analyzed by the blocking ELISA and by IFAT. Bayesian modelling techniques were used to account for the absence of a gold standard test and the results of the blocking ELISA was modelled against the IFAT test with a "2 dependent tests, 2 populations, no gold standard" model. Results At the finally selected cut-off value of percent inhibition (PI 35, the diagnostic sensitivity of the blocking ELISA was 72% and the diagnostic specificity was 93%. The positive predictive value was 0.82 and the negative predictive value was 0.89, at the observed prevalence of 33.5%. Conclusion The sensitivity and specificity as evaluated by Bayesian statistic techniques differed from that previously reported. Properties of diagnostic tests may well vary between countries, laboratories and among populations of animals. In the absence of a true gold standard, the importance of validating new methods by appropriate statistical methods and with respect to the target population must be emphasized.

  19. Further evaluation of an ELISA kit for detection of antibodies to a nonstructural protein of foot-and-mouth disease virus.

    Science.gov (United States)

    Fukai, Katsuhiko; Nishi, Tatsuya; Morioka, Kazuki; Yamada, Manabu; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

    2016-04-01

    An ELISA kit for detection of antibodies to a nonstructural protein of foot-and-mouth disease (FMDV) was further evaluated using sequentially collected serum samples of experimentally infected animals, because the sensitivity of the kit used in a previous study was significantly low in field animals. The kit fully detected antibodies in infected animals without vaccination; however, the first detections of antibodies by the kit were later than those by the liquid-phase blocking ELISA that is used for serological surveillance in the aftermath of outbreaks in Japan, for detection of antibodies to structural proteins of FMDV. Additionally, although the kit effectively detected antibodies in infected cattle with vaccination, there were several infected pigs with vaccination for which the kit did not detect antibodies during the experimental period. Taken together, the kit may not be suitable for serological surveillance after an FMD outbreak either with or without emergency vaccination in FMD-free countries. PMID:26498533

  20. Detection of Staphylococci aureus cells with single domain antibody functionalized Raman nanoparobes

    Science.gov (United States)

    Tay, Li-Lin; Tanha, Jamshid; Ryan, Shannon; Veres, Teodor

    2007-06-01

    Raman spectroscopy has demonstrated to be an effective tool in the detection and classification of pathogenic microorganisms. The technique is, however, limited by the inherently low cross-section of the Raman scattering process. Among the many enhanced Raman processes, surface enhanced Raman scattering (SERS) technique provides the highest sensitivity and can be easily adapted in the bio-sensing applications such as DNA hybridization and protein binding events. In this study, we report the targeted detection of the pathogenic bacteria, Staphylococcus aureus, with novel single domain antibody (sdAb) conjugated SERS nanoprobes. A sdAb specific to protein A of S. aureus cells was conjugated to silver nanoparticles (Ag-NP). Bacteria recognition was achieved through specific binding of the sdAb (conjugated to SERS nanoprobe) to protein A. Binding rendered the nanoparticle-labeled S. aureus cells SERS active. As a result, S. aureus cells could be detected rapidly and with excellent sensitivity by monitoring the SERS vibrational signatures. This work demonstrates that the SERS imaging technique offers excellent sensitivity with a detection limit of a single bacterium.

  1. Detection of antibodies specific to Campylobacter fetus subsp. venerealis in the vaginal mucus of Nigerian breeding cows

    Directory of Open Access Journals (Sweden)

    Zerai Woldehiwet

    2010-09-01

    Full Text Available The presence of bovine venereal campylobacteriosis in the Lake Chad Basin of Nigeria was investigated using an enzyme-linked immunosorbent assay (ELISA for the detection of IgA antibodies specific to Campylobacter fetus subsp. venerealis in vaginal mucus (n = 66. IgA antibodies specific to C. fetus subsp. venerealis were detected in 7 (11% vaginal mucus samples. All but one of the IgA-positive samples originated from cows belonging to herds with a history of abortion and infertility which suggested an association between antibody detection and poor herd fertility. It was concluded that bovine venereal campylobacteriosis is prevalent in the Lake Chad Basin of Nigeria and its contribution to reduced reproductive performance in cattle herds may be grossly underestimated in this part of the world.

  2. Electrochemical immunosensor for detection of antibodies against influenza A virus H5N1 in hen serum.

    Science.gov (United States)

    Jarocka, Urszula; Sawicka, Róża; Góra-Sochacka, Anna; Sirko, Agnieszka; Zagórski-Ostoja, Włodzimierz; Radecki, Jerzy; Radecka, Hanna

    2014-05-15

    This paper describes the development of an immunosensor for detection of anti-hemagglutinin antibodies. Its preparation consists of successive modification steps of glassy carbon electrodes: (i) creation of COOH groups, (ii) covalent immobilization of protein A with EDC/NHS coupling reaction, (iii) covering with anti-His IgG monoclonal antibody, (iv) immobilization of the recombinant His-tagged hemagglutinin (His6-H5 HA), (v) filling free space with BSA. The interactions between two variants of recombinant HA (short and long) from highly pathogenic avian influenza virus H5N1 and the anti-H5 HA monoclonal antibody (Mab 6-9-1) have been explored with electrochemical impedance spectroscopy (EIS). The impedimetric immunosensor displayed a very good detection limit (LOD) of 2.1 pg/mL, the quantification limit (LOQ) of 6.3 pg/mL and a dynamic range from 4 pg/mL to 20 pg/mL. In addition, this analytical device was applied for detection of antibodies against His6-H5 HA in serum of vaccinated hen using serial 10-fold dilutions of serum. The immunosensor proposed was able to detect antibody in hen serum diluted up to 7 × 10(7)-fold. The sensitivity of immunosensor was about four orders of magnitude much better than ELISA. PMID:24412426

  3. Detection of viral hemorrhagic septicemia virus-IVb antibodies in sera of muskellunge Esox masquinongy using competitive ELISA.

    Science.gov (United States)

    Millard, Elena V; Brenden, Travis O; LaPatra, Scott E; Marcquenski, Susan; Faisal, Mohamed

    2014-04-01

    A competitive enzyme-linked immunosorbent assay (cELISA) was developed for the detection of antibodies to viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb) in fish sera. Assay conditions were standardized using known negative and positive muskellunge Esox masquinongy. A positive-negative threshold of 14.6% inhibition was established based on analysis of sera of 60 muskellunge with no previous exposure to VHSV-IVb. The cELISA was then used to investigate immune responses of wild muskellunge sampled from 5 water bodies in Michigan and Wisconsin, USA, between 2005 and 2012. Antibodies were detected in fish from Lake St. Clair, Michigan, and Lower Fox River/Green Bay, Wisconsin. Both water systems were considered enzootic for VHSV-IVb. Additionally, antibodies were detected in muskellunge from Thornapple Lake, a Michigan inland lake previously considered negative for VHSV-IVb based on virus isolation methods. Muskellunge populations from Lake Hudson, Michigan, and Butternut Lake, Wisconsin, lacked evidence of an immune response to VHSV-IVb. When results of the cELISA were compared to the 50% plaque neutralization test for several groups of fish, there was 78.4% agreement between the tests for antibody presence. The cELISA is a rapid and efficient test for the detection of binding antibodies to VHSV-IVb and will be a useful non-lethal tool for monitoring the spread of this serious pathogen. PMID:24695232

  4. Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

    Science.gov (United States)

    Tran, Quang Huy; Hanh Nguyen, Thi Hong; Mai, Anh Tuan; Thuy Nguyen, Thi; Khue Vu, Quang; Nga Phan, Thi

    2012-03-01

    This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml‑1 to 1 μg ml‑1, and the limit of detection was about 10 ng ml‑1. This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks.

  5. Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml−1 to 1 μg ml−1, and the limit of detection was about 10 ng ml−1. This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks

  6. Detection of antibodies against Chlamydophila abortus in Costa Rican sheep flocks

    Directory of Open Access Journals (Sweden)

    R. Villagra-Blanco

    2015-09-01

    Full Text Available A total of 359 sheep samples from 15 flocks were analyzed for the presence of antibodies against Chlamydophila abortus using a commercial Enzyme linked Immunosorbent Assay (ELISA. Antibodies were detected in 19 (5.29% sheep from 12 (80% flocks. Seropositive animals were found in all analyzed regions (Central, Chorotega, Atlantic Huetar, North Huetar and Central Pacific determining prevalence between 0.28% and 4.4%, and intra-flock positivity between 3.7% and 25.0%. The survey revealed two risk factors associated with seropositivity; introducing animals (males and females, embryos, or semen from other farms or from abroad without any sanitary certification, and flocks not having quarantine areas or separated boxes for diseased animals. No clinical signs of disease were observed in positive seroreactors. C. abortus seems to be present in Costa Rica in a very low prevalence in sheep flocks. Further studies, to isolate the bacteria are required. Finally, implementation of control measures to prevent the spread of C. abortus is recommended.

  7. Detection of antibodies against Chlamydophila abortus in Costa Rican sheep flocks.

    Science.gov (United States)

    Villagra-Blanco, R; Dolz, G; Montero-Caballero, D; Romero-Zúñiga, J J

    2015-01-01

    A total of 359 sheep samples from 15 flocks were analyzed for the presence of antibodies against Chlamydophila abortus using a commercial Enzyme linked Immunosorbent Assay (ELISA). Antibodies were detected in 19 (5.29%) sheep from 12 (80%) flocks. Seropositive animals were found in all analyzed regions (Central, Chorotega, Atlantic Huetar, North Huetar and Central Pacific) determining prevalence between 0.28% and 4.4%, and intra-flock positivity between 3.7% and 25.0%. The survey revealed two risk factors associated with seropositivity; introducing animals (males and females), embryos, or semen from other farms or from abroad without any sanitary certification, and flocks not having quarantine areas or separated boxes for diseased animals. No clinical signs of disease were observed in positive seroreactors. C. abortus seems to be present in Costa Rica in a very low prevalence in sheep flocks. Further studies, to isolate the bacteria are required. Finally, implementation of control measures to prevent the spread of C. abortus is recommended. PMID:26623377

  8. Detection of antibody-antigen reaction by silicon nitride slot-ring biosensors using protein G

    Science.gov (United States)

    Taniguchi, Tomoya; Hirowatari, Anna; Ikeda, Takeshi; Fukuyama, Masataka; Amemiya, Yoshiteru; Kuroda, Akio; Yokoyama, Shin

    2016-04-01

    Biosensors using ring resonators with silicon nitride (SiN) slot waveguides have been fabricated. The temperature coefficient of the resonance wavelength of the SiN resonator is 0.006 nm/°C, which is one order of magnitude smaller than that of Si. The sensitivity of the biosensor has been improved by using slot waveguide together with Si-binding protein (designated as Si-tag), which bonds to SiN or SiO2 surface, as an anchoring molecule to immobilize bioreceptors on the SiN rings in an oriented manner. Furthermore, the protein G, which strongly bonds to many kinds of mammalian antibodies only by mixing the antibody solution, is used to efficiently immobilize the antigen on the sensor surface. By means of these devises the sensitivity of the biosensor has been improved by factor of 10-100 compared with that of normal Si ring resonator sensors without slot. Then the detection of prostate specific antigen (PSA) with the sensitivity of ~1×10-8 g/ml, which is the concentration of strongly suspicious for the prostate cancer, has been achieved.

  9. Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

    Science.gov (United States)

    Masiri, Jongkit; Benoit, Lora; Katepalli, Madhu; Meshgi, Mahzad; Cox, David; Nadala, Cesar; Sung, Shao-Lei; Samadpour, Mansour

    2016-05-11

    Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing. PMID:27087556

  10. Detection of serum antibodies against Bartonella species in cats with sporotrichosis from Rio de Janeiro, Brazil.

    Science.gov (United States)

    Kitada, Amanda A B; Favacho, Alexsandra R M; Oliveira, Raquel V C; Pessoa, Adonai A; Gomes, Raphael; Honse, Carla O; Gremião, Isabella D F; Lemos, Elba R S; Pereira, Sandro A

    2014-04-01

    Cat scratch disease is a zoonosis caused by Bartonella species, transmitted to humans through scratches or bites from infected cats and via direct contact with infected feces. Sporotrichosis, caused by the fungal complex Sporothrix, is transmitted by traumatic inoculation of the fungus. Cats are important in zoonotic transmission. Serum samples from 112 domestic cats with sporotrichosis and 77 samples from healthy cats were analyzed by indirect immunofluorescence assay (IFA), using the commercial kit Bartonella henselae IFA IgG (Bion). The presence of antibodies against feline leukemia virus (FeLV) and of feline immunodeficiency virus (FIV) core antigens was detected using the commercial kit Snap Combo FIV-FeLV (Idexx). The group of animals with sporotrichosis contained 93 males with a median age of 22 months, eight (7.1%) of which were positive for FIV and 15 (13.4%) for FeLV. The group of animals without sporotrichosis contained 36 males with a median age 48 months, 10 (13.0%) of which were positive for FIV and eight (10.4%) for FeLV. Of the 112 cats with sporotrichosis and 77 cats without mycosis, 72 (64.3%) and 35 (45.5%), respectively, were IFA reactive. No association was found between age, sex, FIV/FeLV and the presence of antibodies to Bartonella species. The results suggest that the study population can be considered a potential source of zoonotic infection for both diseases. PMID:24127458

  11. Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

    Directory of Open Access Journals (Sweden)

    Endimiani Andrea

    2009-03-01

    Full Text Available Abstract Background β-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed. Results E. coli DH10B cells producing SHV-1 β-lactamase and a clinical isolate of K. pneumoniae producing SHV-5 β-lactamase were rendered membrane permeable, fixed and adhered to poly-L-lysine coated slides, and stained with purified polyclonal anti-SHV antibodies that were fluorescein labeled. E. coli DH10B cells without a blaSHV gene were used as a negative control. The procedure generated a fluorescence signal from those slides containing cells expressing SHV β-lactamase that was sufficient for direct imaging. Conclusion We developed a rapid and accurate method of visualizing the SHV family of enzymes in clinical samples containing Gram-negative bacilli using a fluorescein-labeled polyclonal antibody.

  12. Developments of sensitive immunoassays for detection of antibodies against hepatitis B surface antigen

    International Nuclear Information System (INIS)

    Three micro solid phase immunoassays (a micro-SPRIA and two ELISA techniques) were developed and tested for the detection of anti-HBs antibodies. Two different crosslinkers (glutaraldehyde and N-succinimidyl 3-(2-pyridyldithio) propionate) were used to couple a goat anti-mouse IgG reagent to alkaline phosphatase for use as enzyme-labeled probes in the two ELISA tests. With the latter cross-linker, a defined conjugate with a 1 : 1 antibody-enzyme molar ratio was obtained. The sensitivities of micro-SPRIA and the two types of ELISA were compared to that of the commercial solid phase radioimmunoassay AUSAB test. All three microtests were significantly more sensitive than the AUSAB test. The ELISA using the glutaraldehyde cross-linked conjugate was 3-5 times less sensitive than micro-SPRIA, while the ELISA using the disulfide-linked conjugate was 2.6-4.0 times more sensitive than micro-SPRIA. (Auth.)

  13. A Rapid, Self-confirming Assay for HIV: Simultaneous Detection of Anti-HIV Antibodies and Viral RNA

    Science.gov (United States)

    Chen, Zongyuan; Zhu, Hui; Malamud, Daniel; Barber, Cheryl; Ongagna, Yhombi Yvon Serge; Yasmin, Rubina; Modak, Sayli; Janal, Malvin N.; Abrams, William R.; Montagna, Richard A.

    2016-01-01

    Objective We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. Methods We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention. Results The finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample. Conclusion The ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for

  14. Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays

    Science.gov (United States)

    Dias, J. T.; Lama, L.; Gantelius, J.; Andersson-Svahn, H.

    2016-04-01

    Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 103-fold (5 pg mL-1) lower than when no ultrasound was applied (50 ng mL-1) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09207h

  15. A solid-phase radioimmunoassay to detect antibodies produced by hybridomas to antigens derived from human melanoma cells

    International Nuclear Information System (INIS)

    A solid-phase radioimmunoassay to detect antibodies that react with antigens derived from human melanoma cells is described. A soluble preparation derived from Nonidet P-40 lysates of tissue-cultured melanoma cells was dried on the surfaces of wells of polyvinyl chloride microtiter plates and fixed with 0.02% glutaraldehyde. Antibody preparations were added and incubated for 18 h at 40C. The wells were washed and bound antibodies were detected using radioactive Staphyloccoccal protein A (125I-SpA). Optimal conditions are described for all the steps employed. Concentrations of antigen selected, the amount of 125I-SpA employed and the duration of incubation of antibodies with antigen were found to be critical. The assay was sensitive and reproducible, and lent itself to the simultaneous evaluation of many individual antibody samples in a short period of time. The assay was particularly valuable for rapid screening of hybridoma supernatants for antibodies to antigens derived from melanoma cells and from a panel of other tumor and normal cells. (orig.)

  16. Detection of serum anti-sperm antibody in infertile couples with dot-immunogold filtration assay (DIGFA)

    International Nuclear Information System (INIS)

    Objective: To develop a new method for rapid detection of serum anti-sperm antibody in infertile couples. Methods: Human sperm antigen was prepared from pooled semen specimens of fertile males. Nitro-cellulose membrane was used as solid-phase carrier of the antigen. Colloidal gold pellet combined goat anti-human IgG was taken as labelled antibody. A dot-immunogold filtration assay system was established for test of serum anti-human sperm antibody. Serum specimens from 137 infertile couples were tested and the result compared with flat from ELISA. Results: The human sperm antigen would react with the anti-sperm antibody in the tested serum over the cellulose membrane through filtration and the result could be read with naked eye within 6 minutes. In this study of 137 infertile coupled, the anti-sperm antibody was positive in 21.9% of the female serum specimens and 13.19% of the males. Compared with the result from ELISA, the consistency rate was 96.1%. The sensitivity of the assay was 90.2% and specificity was 95.4%. The p reparation was stable after 6 months refrigerator storage. Conclusion: This newly developed DIGFA is very adequate for rap id detection of anti-sperm antibody and deserves popularization. (authors)

  17. Detecting Proteins in Highly Autofluorescent Cells Using Quantum Dot Antibody Conjugates

    Directory of Open Access Journals (Sweden)

    Karen M. Orcutt

    2009-09-01

    Full Text Available We have applied quantum dot (Qdot antibody conjugates as a biomolecular probe for cellular proteins important in biogeochemical cycling in the sea. Conventional immunological methods have been hampered by the strong autofluorescence found in cyanobacteria cells. Qdot conjugates provide an ideal alternative for studies that require long-term imaging of cells such as detection of low abundance cellular antigens by fluorescence microscopy. The advantage of Qdot labeled probes over conventional immunological methods is the photostability of the probe. Phycoerythrin bleaches in cyanobacterial cells under prolonged UV or blue light excitation, which means that the semiconducting nanocrystal probe, the Qdot, can yield a strong fluorescent signal without interference from cellular pigments.

  18. The use of ELISA to detect antibodies to Brucella abortus in naturally infected cattle

    International Nuclear Information System (INIS)

    An indirect ELISA using the immunodominant antigen, smooth lipopolysaccharide (SLPS) of Brucella abortus, was used to detect antibodies in a statistically selected dairy cattle population from the Sabana of Bogota (Colombia). Based on the FAO/IAEA Brucella ELISA kit, the test was standardized in terms of plates, reagents and reference sera. Quality control for routine testing was also established. Calculation of the appropriate cut-off optical density (O.D) value for negativity was made after discriminant analysis and examination of the frequency of the sera distributed as positive or negative. The sensitivity and specificity of the ELISA for diagnosis of Brucella infection were compared to the complement fixation (CF) test results and were 94.8% and 96.0% respectively. The use of ELISA for monitoring bovine populations undergoing an eradication of brucellosis is recommended. The application of an ELISA using B. bovis outer membrane proteins (OMP) as antigen is discussed. (author). 38 refs, 3 figs, 4 tabs

  19. Detection of antibodies against Leishmania infantum in cats (Felis catus from the State of Pernambuco, Brazil

    Directory of Open Access Journals (Sweden)

    Rita de Cássia Nascimento Silva

    2014-01-01

    Full Text Available Introduction: Little information is available concerning infection by Leishmania infantum in cats. Therefore, the aim of this study was to perform a serological study in domestic cats. Methods: Serum samples (n=153 obtained from animals living in the Cities of Recife and Petrolina, State of Pernambuco, Brazil, were tested by ELISA/S7® (Biogene. Results: Anti-L. infantum antibodies were detected in 3.9% (6/153 of the cats. All seroreagent animals were from Petrolina. Conclusions: These results serve as an important alert, and future studies are needed to better understand the possible role of cats in the epidemiology of visceral leishmaniasis (VL in this area.

  20. Studies on the detection of erythrocytic antibodies with a radioimmuno-antiglobulin assay

    International Nuclear Information System (INIS)

    The RIA with I-125-anti-IgG and I-125-anti-C3b was used. Various factors in RIA were tested for the purpose of optimisation. As a rule, incomplete anti-D (test serum of one charge) was used. Anti-Kell was used for specific problems. After optimisation of the method, patient sera were studied which contained auto- or allo-antibodies in the fresh state. More significant findings were obtained with the direct RIAGT. Indirect methods did not show a clearly enhanced detection sensitivity of erythrocyte autoantibodies, but they may serve for a closer investigation of diffuse reactions of patient sera in the enzyme test. The direct RIA is clearly of advantage in cases in which the complement Coombs test alone is positive. (orig./MG)

  1. Evaluation of classical swine fever virus antibody detection assays with an emphasis on the differentiation of infected from vaccinated animals

    DEFF Research Database (Denmark)

    Schroeder, S.; von Rosen, Tanya; Blome, S.;

    2012-01-01

    The aim of this study was to evaluate the general characteristics of commercially available enzyme-linked immunosorbent assays (ELISAs) to detect antibody against classical swine fever (CSF), as well as to assess their potential use as accompanying marker tests able to differentiate infected from...... vaccinated animals (DIVA). The Chekit* CSF-Sero and the HerdChek* CSFV Ab, both of which detect antibodies against the E2 protein of classical swine fever virus (CSFV), had the highest sensitivity. Both tests were practicable and showed good reproducibility. Comparable sensitivity was shown by the Chekit...

  2. Use of Piezoelectric Immunosensors for Detection of Interferon-Gamma Interaction with Specific Antibodies in the Presence of Released-Active Forms of Antibodies to Interferon-Gamma

    Directory of Open Access Journals (Sweden)

    Elena Don

    2016-01-01

    Full Text Available In preliminary ELISA studies where released-active forms (RAF of antibodies (Abs to interferon-gamma (IFNg were added to the antigen-antibody system, a statistically significant difference in absorbance signals obtained in their presence in comparison to placebo was observed. A piezoelectric immunosensor assay was developed to support these data and investigate the effects of RAF Abs to IFNg on the specific interaction between Abs to IFNg and IFNg. The experimental conditions were designed and optimal electrode coating, detection circumstances and suitable chaotropic agents for electrode regeneration were selected. The developed technique was found to provide high repeatability, intermediate precision and specificity. The difference between the analytical signals of RAF Ab samples and those of the placebo was up to 50.8%, whereas the difference between non-specific controls and the placebo was within 5%–6%. Thus, the piezoelectric immunosensor as well as ELISA has the potential to be used for detecting the effects of RAF Abs to IFNg on the antigen-antibody interaction, which might be the result of RAF’s ability to modify the affinity of IFNg to specific/related Abs.

  3. Use of Piezoelectric Immunosensors for Detection of Interferon-Gamma Interaction with Specific Antibodies in the Presence of Released-Active Forms of Antibodies to Interferon-Gamma.

    Science.gov (United States)

    Don, Elena; Farafonova, Olga; Pokhil, Suzanna; Barykina, Darya; Nikiforova, Marina; Shulga, Darya; Borshcheva, Alena; Tarasov, Sergey; Ermolaeva, Tatyana; Epstein, Oleg

    2016-01-01

    In preliminary ELISA studies where released-active forms (RAF) of antibodies (Abs) to interferon-gamma (IFNg) were added to the antigen-antibody system, a statistically significant difference in absorbance signals obtained in their presence in comparison to placebo was observed. A piezoelectric immunosensor assay was developed to support these data and investigate the effects of RAF Abs to IFNg on the specific interaction between Abs to IFNg and IFNg. The experimental conditions were designed and optimal electrode coating, detection circumstances and suitable chaotropic agents for electrode regeneration were selected. The developed technique was found to provide high repeatability, intermediate precision and specificity. The difference between the analytical signals of RAF Ab samples and those of the placebo was up to 50.8%, whereas the difference between non-specific controls and the placebo was within 5%-6%. Thus, the piezoelectric immunosensor as well as ELISA has the potential to be used for detecting the effects of RAF Abs to IFNg on the antigen-antibody interaction, which might be the result of RAF's ability to modify the affinity of IFNg to specific/related Abs. PMID:26791304

  4. Advantages of detecting monoclonal antibody binding to tissue sections with biotin and avidin reagents in Coplin jars.

    Science.gov (United States)

    Bindl, J M; Warnke, R A

    1986-04-01

    We describe a method of biotin/avidin-peroxidase detection using second and third stage reagents in Coplin jars. This method allows a large quantity of sections to be stained simultaneously with a minimal amount of technical time involved. A wide range of mouse monoclonal antibodies of varying specificities and isotypes were used to stain both frozen and paraffin-embedded sections of various normal and neoplastic tissues. Three different biotinylated anti-mouse antibodies were tested, including F(ab')2 antibody fragments of one, followed by horseradish peroxidase conjugated avidin. All monoclonal antibodies employed gave good staining, using incubation times of 30-50 minutes. The staining was done during a mean period of 25 to 27 days with an average staining load of 500 sections per Coplin jar. PMID:2420169

  5. Comparative evaluation of antibody detection tests to facilitate the diagnosis of multibacillary leprosy.

    Science.gov (United States)

    Duthie, Malcolm S; Orcullo, Florenda M; Abbelana, Junie; Maghanoy, Armi; Balagon, Marivic F

    2016-04-01

    Despite control efforts, leprosy persists as a significant health concern in many regions. Diagnosis is achieved by a combination of clinical, histopathological, and bacteriological examinations, each of which presents a barrier to expeditious diagnosis, particularly by non-experts. Immunological investigations in research laboratories have clearly indicated that antibody detection tests could aid the diagnosis of leprosy. In this study, we detected circulating antibodies with two rapid diagnostic tests (RDT) involving immunochromatographic lateral flow platforms and one rapid ELISA system. Leprosy patients were identified with a high degree of sensitivity in each assay (over 80 % in all; over 90 % among cases with bacterial indices >1+), although critical differences were observed in specificity. While the specificity of CTK OnSite Leprosy Ab Rapid Test and InBios Leprosy Detect™ fast ELISA were high (96.4 and 93.7 % in the general population, respectively), there was a marked reduction in OrangeLife NDO-LID® RDT (only 25.0 %). As anticipated, seropositivity rates were marginally higher in contacts of leprosy patients than in endemic controls. Although we observed a slight drop in test band intensity when blood, rather than serum, was used to develop OnSite Leprosy Ab Rapid Tests, the sensitivity and specificity of these tests was unaffected. When we contrasted test performance with clinical and bacteriological information, we found that RDT and ELISA results positively correlated with the bacteriological index. These data indicate that these assays could be a ready replacement of invasive, insensitive, and time consuming skin slit smear procedures that additionally require expert microscopic examinations. We propose that, due to their speed and point of care applicability, the RDT could be used as an initial entry point to the diagnostic protocols, with confirmation of results attained in a highly quantitative manner following serum transfer to a reference

  6. Detection of anti-HAY antibody with dot immunogold filtration assay

    Institute of Scientific and Technical Information of China (English)

    Zhong-Jun Shao; De-Zhong Xu; Yong-Ping Yan; Jing-Hua Li; Jing-Xia Zhang; Zhi-Ying Zhang; Bo-Rong Pan

    2003-01-01

    AIM: To establish a rapid, sensitive and specific immunogold assay for detection of hepatitis A virus infection.METHODS: Rabbit monoclonal antibodies to anti-human IgM and IgG (Dako) were dotted on a nitrocellulose membrane (NCM) respectively to capture the human sera IgM and IgG. Then the captured antibodies would conjugate to HAV antigen, which was revealed by mouse anti-HAV IgG conjugated to gold particles. Final results were assessed by blind method.RESULTS: Sera from 96 patients with acute hepatitis were used for our study. Compared with well-recognized standard (Abbott Laboratory, USA), the sensitivity and specificity of IgM-DIGFA (self-made) were 91.3 % (42/46) and 96.0 %(48/50), and those of IgM-ELISA (Kehua, Shanghai) were 97.8 % (45/46) and 100.0 % (50/50). The identical results were produced from the study with reagents at different conditions, and the study was repeated in 15 negative sera and 10 positive sera. The serum anti-HAV IgG was tested with DIGFA at the same time. In comparison with ELISA,the sensitivity and specificity of DIGFA for IgG anti-HAV were 87.2 % (41/47) and 91.8 % (45/49), respectively.CONCLUSION: This assay can detect anti-HAV IgM and IgG simultaneously, and be done within 3 minutes. The simplicity, rapidity and specificity of the assay were useful for screening and epidemiological study.

  7. A comprehensive immunoassay for the detection of microcystins in waters based on polyclonal antibodies

    International Nuclear Information System (INIS)

    Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae), and microcystin-leucine-arginine (MC-LR) is among the most frequent and most toxic microcystin congeners. In this study, a free amino group was introduced to MC-LR at its seventh amino acid residue with 2-mercaptoethylamine, and the product aminoethyl-MC-LR was coupled to bovine serum albumin (BSA) and horseradish peroxidise (HRP) by glutaraldehyde to be complete antigen (MC-LR-BSA) and labelled hapten (MC-LR-HRP), respectively. Polyclonal antibodies against MC-LR were generated by immunization with MC-LR-BSA. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was established to detect the MCs in waters, which showed a good cross-reactivity with MC-LR, MC-RR, MC-YR, MC-LF, MC-LW and nodularin, and have a detection limit for MC-LR 0.12 μg L-1, the 50% inhibition concentration (IC50) for MC-LR was 0.63 ± 0.06 μg L-1 and the quantitative detection range was from 0.17 to 2.32 μg L-1, the analysis result of water samples showed good recovery and reliability. So the comprehensive and reliable dc-ELISA will well potentially suit for sensitive analysis for total MCs in drinking as well as resource water samples

  8. Detection of lymphocystis disease virus infection to flounder gill cells in vitro by monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Flounder gill (FG) cells were used to isolate lymphocystis disease virus (LCDV) and two monoclonal antibodies (Mabs) (1A8 and 3G3) against LCDV were used to trace LCDV infection to FG cells. FG monolayer cells was inoculated with LCDV supernatant, obtained from lymphocystis cells of diseased flounder, Paralichthys olivaceus. LCDV infection was detected with Mabs employing immunocytochemical assay (ICA) and indirect immunofluorescence assay test (ⅡFAT) technique. Detected by ⅡFAT, they were specific for LCDV. The results of experimental infection illustrated that FG cells was sensitive to LCDV, and showed virus-infection positive detected by ICA. Cytopathic effect (CPE) occurred 1-2 days post inoculation (PI), and half tissue culture infection dosage (TCID50) of virus supernatant was 22.57 per 40μl. Tracing by ⅡFAT showed that LCDV positive signal first appeared at the cell membrane immediately PI, and then in cytoplasm at 24h PI, it reached the strongest positive at 48-72 h PI, and began to decrease at 96h PI.

  9. Detection and specificity of antibodies secreted by spleen cells in mice immunized with Streptococcus mutans.

    OpenAIRE

    Russell, M. W.; Czerkinsky, C; Moldoveanu, Z

    1986-01-01

    Immune responses of mice to Streptococcus mutans serotype c were analyzed by means of the enzyme-linked immunospot assay to determine the predominant specificities of the antibodies developed. In general, the numbers of splenic antibody-secreting cells correlated with serum antibody levels. A low dose (10(8) CFU) of killed whole cells injected twice intraperitoneally induced antibodies mainly against surface protein antigen I/II. A higher dose (10(9) CFU) given two to six times also resulted ...

  10. Detection of Neutralising Antibody Titration in Vaccinated Owned and Stray Dogs against Rabies Virus

    OpenAIRE

    GAZİ, Aliye Ebru; AK, Seyyal

    2011-01-01

    The aim of this study was to determine the titers of neutralising antibodies against rabies virus in five hundred vaccinated owned and stray dogs within the Istanbul province. The levels of the neutralising antibody titration in the blood serum were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA) method. Sixty five (13%) of the dogs examined had adequate level (0.5 IU/ml and over) of antibody titration. Also, the effects on the formation of the neutralising antibody levels following th...

  11. Detection of hepatitis B virus infection in HBsAg-negative patients by monoclonal antibodies against HBsAg

    International Nuclear Information System (INIS)

    The technique of producing antibody secreting hybridomas has made available high-affinity antibodies of predefined specificity for use as diagnostic reagents. Recently, high-affinity monoclonal antibodies to hepatitis B surface antigens (HBsAg) were produced and characterized. Immunoassay was developed using these antibodies for the detection of HBsAg-associated determinants. The present study indicated the significance of the enhanced detection by monoclonal radioimmunoassay (M-RIA) of HBsAg in sera of patients with hepatitis B virus infection. The M-RIA detected HBsAg in sera of hemodialysis patients and blood donor defined as HBsAg-negative by polyclonal RIA (2.2 %, 0.14 %, respectively). Furthermore, individuals with chronic liver diseases were reactive only in the M-RIA (chronic hepatitis 4.8 %, liver cirrhosis 10.0 %, hepatocellular carcinoma 22.2 %). It is noteworthy that some of these patients were diagnosesed as so-called non-A non-B hepatitis because of no serological markers of hepatitis B virus infection such as HBsAb and HBcAb. The enhanced performance of the monoclonal RIA compared to conventional RIA was due to the increased sensitivity of the assay (55 pg vs 230 pg/ml). In immunohistochemical study, one of the monoclonal antibody named 5C3 was applied for detection of HBsAg in the formalin-fixed paraffin-embedded liver. HBsAg was detected in 6 out of 41 HBsAg-seronegative liver specimen. Thus, the studies showed the importance of the clinical application of monoclonal antibodies such as immunoassay and immunohistochemical study in the diagnosis of hepatitis B virus infection. (author)

  12. The diagnosis of proventricular dilatation disease: use of a Western blot assay to detect antibodies against avian Borna virus.

    Science.gov (United States)

    Villanueva, Itamar; Gray, Patricia; Mirhosseini, Negin; Payne, Susan; Hoppes, Sharman; Honkavuori, Kirsi S; Briese, Thomas; Turner, Debra; Tizard, Ian

    2010-07-14

    Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently "healthy" birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleoprotein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces. PMID:20036080

  13. Detection of anti-Chlamydia trachomatis Antibodies in Patients with Acquired Immune Deficiency Syndrome in Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Izebe, K. S.

    2008-01-01

    Full Text Available Chlamydia trachomatis (CT infections are among the sexually transmitted diseases known to increase the risk for human immunodeficiency virus infection. Serum samples from 34 consenting AIDS patients which attended the Government-approved Antiretroviral Treatment (ART Facility at the National Institute for Pharmaceutical Research and Development (NIPRD, Abuja between April 2005 and March 2006 were screened by enzyme immunoassay (EIA for the presence of anti-CT antibodies using ImmunoComb® Chlamydia Bivalent IgG Test kit (Orgenics, Israel. Anti-CT antibodies were detected in ten (29.4% of the thirty-four patients tested. The detection rate was higher among the females (33.3% than the males (23.1%. Patients of the age group 31-45 years had the highest detection of anti-C. trachomatis antibodies, followed by those of age group 16-30 years. The result of the present study suggests the presence of anti-CT antibodies in AIDS patients, and reinforces the need for routine screening for anti-CT antibodies as a necessary intervention to reduce the burden of chlamydial diseases and to reduce the risk of HIV and its spread in Nigeria. The outcome of this study also provides justification for the possible inclusion of anti-chlamydial agents in the National AIDS Management Plan to treat associated C. trachomatis infections.

  14. Detection of ruminant meat and bone meal in feeds by sandwich ELISA with monoclonal antibodies.

    Science.gov (United States)

    Yamamoto, Takayuki; Kato, Masatoshi; Endo, Kiwamu; Kotoura, Satoshi; Takeda, Zenya

    2016-01-01

    A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against a synthetic peptide with an amino-acid sequence related to the C-terminus of bovine myoglobin and the whole molecule of sodium dodecyl sulphate (SDS)-denatured bovine myoglobin was adapted for detecting bovine myoglobin in contaminated feeds. The ELISA employed bovine meat extract of a known myoglobin concentration as a calibration standard and had an limit of detection (LOD) of 3.54 ng/ml and an limit of quantification (LOQ) of 11.0 ng/ml corresponding to 0.022% and 0.067% (wt/wt) bovine meat-and-bone-meal (MBM) mixed in 20-fold-diluted feed extracts, respectively. A cut-off threshold of 20.6 ng/ml bovine myoglobin was set to simplify ELISA and facilitate quick assessment of test results without a tedious calibration process. The ELISA was able to detect bovine MBM in artificially prepared model feeds, mixed botanical feeds, mixed botanical feeds with skimmed milk, fish meal, pork meal and pork/chicken meal at 0.1% (wt/wt). It was also able to detect sheep MBM in test feeds, but showed no reactivity to swine MBM, chicken MBM, skimmed milk or gelatine of bovine origin. The advantages of this method are the quick and easy extraction protocol of proteins from test feeds, using 100 mM sodium sulphide and 0.6% sodium dodecyl sulphate in the extraction solution and the effective detection of bovine and sheep MBM at 0.1% (wt/wt). PMID:26166832

  15. Variability in detection and quantification of interferon β-1b–induced neutralizing antibodies

    Directory of Open Access Journals (Sweden)

    Hartung Hans-Peter

    2012-06-01

    Full Text Available Abstract Background Interferon-beta (IFNB therapy for multiple sclerosis can lead to the induction of neutralizing antibodies (NAbs against IFNB. Various methods are used for detection and quantification of NAbs. Methods Blood samples from 125 IFNB-1b–treated patients, which were tested NAb negative or NAb positive after conclusion of a clinical study, were retested three years after first being assessed in four different laboratories that offer routine NAb testing to practicing neurologists. The myxovirus protein A (MxA induction assay, the cytopathic effect (CPE assay (two laboratories, or the luciferase assay were used. Intra- and inter-laboratory agreement between assays with respect to NAb detection and NAb titer quantification were evaluated. Results High agreement for NAb detection (kappa coefficient, 0.86 and for titer levels was observed for the intra-laboratory comparison in the laboratory using the MxA induction assay performed three years ago and now. A similarly high agreement for NAb detection (kappa coefficient, 0.87 and for titer quantification was noted for the MxA assay of this laboratory with one of two laboratories using the CPE assay. All other inter-laboratory comparisons showed kappa values between 0.57 and 0.68 and remarkable differences in individual titer levels. Conclusions There are considerable differences in the detection and quantification of IFNB-induced NAbs among laboratories offering NAb testing for clinical practice using different assay methods. It is important that these differences are considered when interpreting NAb results for clinical decision-making and when developing general recommendations for potentially clinically meaningful NAb titer levels.

  16. Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Mark Schütte

    Full Text Available BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16 which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.

  17. Validation of an indirect ELISA for the detection of Trypanosoma congolense antibodies in Ethiopian cattle

    International Nuclear Information System (INIS)

    Control and eradication of African Animal Trypanosomosis can be achieved if the reliability of the methods to diagnose the disease could be improved. The techniques currently used in the diagnosis of trypanosomosis in Ethiopia are not sufficiently sensitive to detect all infected animals. In order to improve disease diagnosis the indirect antibody detection ELISA, developed by FAO/IAEA, was evaluated under field conditions. Accordingly, reference serum samples were collected from trypanosomosis free and endemic areas. Serum samples negative for trypanosomes were collected from the Central highlands of Ethiopia where there is no previous record of trypanosomosis. The samples were used to establish the threshold and the specificity of the test. Trypanosoma congolense positive sera (based on thin and thick smears and BCT) were collected from endemic areas in the Southwestern part of the country to estimate the sensitivity of the test. Out of 701 negative serum samples, 690 were identified as negative with the indirect antibody ELISA, whereas the remaining 11 were detected as positive. Moreover, of the 282 infected samples the ELISA detected 155 sera as positive, but the remaining 127 cases fell in the negative range. The positive/negative threshold established from negative reference sera was found to be 81.38%. Based on this threshold the specificity of the test was 98.43%, whilst the sensitivity was calculated as 54.96%. Thus, the complementary use of both the ELISA and parasitological methods is encouraged. Since the internal quality controls (IQC) did not fall in the ranges prescribed in the protocol provided by FAO/IAEA precision was achieved by comparing the plate to plate variation of the IQC based on the means plot. Accordingly the assay process indicated that there was no significant difference between individual mean of each plate regarding the strong positive (C++) and moderate positive (C+) controls. Nevertheless, considerable discrepancies were

  18. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

    DEFF Research Database (Denmark)

    Staalsoe, T; Giha, H A; Dodoo, D;

    1999-01-01

    BACKGROUND: Naturally induced antibodies binding to surface antigens of Plasmodium falciparum-infected erythrocytes can be detected by direct agglutination of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously...... been shown to recognise Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This protein is inserted by the parasite into the host cell membrane and mediates the adhesion to the venular endothelium of the host organism in vivo. METHODS: Erythrocytes infected at high parasitaemias with...... subsequently detected by two-colour flow cytometry. RESULTS: Binding of human antibodies to the surface of erythrocytes infected with adhesive strains of Plasmodium falciparum can be measured by the two-colour flow cytometry (FCM) assay described. In addition, we demonstrate that the adhesive capacity of a...

  19. Comparison of radiolabeled monoclonal antibody and magnetic resonance imaging in the detection of metastatic neuroblastoma in bone marrow: Preliminary results

    International Nuclear Information System (INIS)

    Magnetic resonance imaging (MRI) was compared with iodine-131-labeled monoclonal antibody scanning for ability to detect bone marrow metastases in the spine, pelvis, and femurs of children with disseminated neuroblastoma. The five patients in this study had received high-dose chemotherapy and radiation, either with (N=2) or without (N=3) bone marrow transplants. MRI disclosed marrow abnormalities at all sites detected with the radiolabeled antibody, which is highly specific for neuroblastoma. However, several diffuse and multifocal marrow changes apparent on MR scans were not present on scintigrams, indicating that MRI is probably less specific than monoclonal antibody imaging. Both methods were more useful than conventional radiography, computed tomography, and 99mTc-MDP bone scans for identifying sites of marrow involvement by neuroblastoma. (orig.)

  20. Evaluation of enzyme-linked immunosorbent assays for detection of Mycoplasma bovis-Specific antibody in bison sera

    Science.gov (United States)

    Mycoplasma bovis has recently emerged as a significant and costly infectious disease problem in bison. This report demonstrates that ELISAs for detection of M. bovis-specific antibody in cattle are not optimal for identification of seropositive bison. An ELISA optimized for use with bison sera is ...

  1. Dot immunobinding assay for simultaneous detection of specific immunoglobulin G antibodies to measles virus, mumps virus, and rubella virus.

    OpenAIRE

    F. Condorelli; Ziegler, T.

    1993-01-01

    A dot immunobinding assay was used to detect antibodies to measles virus, mumps virus, and rubella virus antigens. Filter paper soaked with serum or whole blood was directly applied to the antigen-coated nitrocellulose sheets. The test was easy to perform, and its results agreed very well with those obtained by standard enzyme immunoassay.

  2. Detection of thrombocytic antibodies with the direct and indirect haemolysis inhibition test and the radioimmuno-Coombs test

    International Nuclear Information System (INIS)

    Methods of application of the direct and indirect haemolysis inhibition test were studied in order to optimise the test parameters: The ultimate aim was to standardize the test method and compare its sensitivity in detecting various platelet antibodies with platelet indirect radioactive Coombs-test and the platelet immunofluorescence test. (orig.)

  3. Monoclonal antibodies against troponin I for the detection of rendered muscle tissues in animal feedstuffs.

    Science.gov (United States)

    Chen, Fur-Chi; Hsieh, Y-H Peggy; Bridgman, Roger C

    2002-12-01

    Regulatory controls to prevent the spread of BSE have prohibited the use of certain animal proteins in feed in several countries. Accurate analytical methods for detecting prohibited material in feedstuffs are needed to ensure compliance with the new regulations. Six IgG class monoclonal antibodies (MAbs) against troponin I (TnI), a thermostable marker protein, have been developed for the detection and differentiation of rendered muscle tissue in animal feed. MAbs 1F9, 2G3 and 7F7 reacted to TnI of all species, including mammalian, poultry and fish, while MAbs 7A12 and 8A12 recognized only mammalian TnI (porcine, bovine, ovine, equine, and deer). MAb 2A8 was able to differentiate TnI of ruminant origin (bovine, ovine and deer) from other species. Three indirect enzyme-linked immunosorbent assays (ELISAs) employing these MAbs were developed for the determination of animal muscle, mammalian muscle or ruminant muscle in animal feeds. PMID:22061747

  4. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.

  5. Glycopeptide-Based Antibody Detection in Multiple Sclerosis by Surface Plasmon Resonance

    Directory of Open Access Journals (Sweden)

    Elisa Peroni

    2012-05-01

    Full Text Available In multiple sclerosis (MS the gold standard for the diagnosis and prognosis is, up to now, the use of magnetic resonance imaging markers. No alternative simpler assays proven of use, except for cerebrospinal fluid analysis, have been provided in MS diagnosis. Therefore, there is a need to develop non-invasive, sensitive, simple new techniques for the clinical routine. Herein we present the evaluation of the feasibility of a glycopeptide-based biosensor to detect MS specific antibodies in sera using the surface plasmon resonance technology. The previously described glycopeptide antigen CSF114(Glc has been immobilized on a gold sensor chip and the method has been optimized for real-time specific autoantibody detection directly in sera. A population of 60 healthy blood donors and 61 multiple sclerosis patients has been screened. The receiver operating characteristic (ROC-based analysis has established the optimal diagnostic cut-off value for the method obtaining a sensitivity of 36% and a specificity of 95%. Sample sera have been also screened with a previously validated ELISA.

  6. Serological detection of circulating Angiostrongylus vasorum antigen and specific antibodies in dogs from central and northern Italy.

    Science.gov (United States)

    Guardone, L; Schnyder, M; Macchioni, F; Deplazes, P; Magi, M

    2013-02-18

    The most frequently employed method for the diagnosis of Angiostrongylus vasorum in dogs is the detection of first stage larvae (L1) in faeces. The sensitivity of coproscopy, however, is limited in case of low parasite load, intermittent larval excretion, and during pre-patency. An epidemiological survey on dogs was conducted applying serological methods in two Italian regions where angiostrongylosis is endemic in foxes. 265 dog serum samples from Tuscany (central Italy - site A) and 447 from Liguria (north-western Italy - site B) were tested with a sandwich-ELISA for detection of circulating antigen, and with an ELISA using A. vasorum adult somatic antigen purified by monoclonal antibodies for specific antibody detection. During previous examinations dogs naturally infected with Leishmania infantum (n=149), Dirofilaria immitis (n=40), Dirofilaria repens (n=30), Acanthocheilonema reconditum (n=27), Crenosoma vulpis (n=1), A. vasorum (n=2), Capillaria aerophila (n=35), Capillaria boehmi (n=3), Toxocara canis (n=68), Toxascaris leonina (n=5), hookworms (n=37) and Trichuris vulpis (n=39) were detected. Sera of these dogs were used to evaluate cross reactions. In site A, 2 dogs (0.8%) were seropositive for antibody and antigen detection and 4 (1.5%) for antibody detection only. From site B, 4 dogs (0.9%) were seropositive for both tests, while other 4 dogs (0.9%) for antigen detection only and 9 dogs (2%) for antibody detection only. Considering a subgroup of 347 dogs from site B which had also been tested with the Baermann technique, 2 (0.6%) were positive for both tests, 4 (1.2%) for antigen detection only and 9 (2.6%) for antibody detection only. The two dogs which were positive for both serological tests were also positive for A. vasorum L1 in the faeces. No significant difference in seropositivities was observed in the group of dogs with other proven parasitic infections. A. vasorum serology presents significant advantages (diagnosis before patency, single serum

  7. Evaluation of Indirect Fluorescence Antibody Assay for Detection of Bartonella clarridgeiae and Seroprevalence of B. clarridgeiae among Patients with Suspected Cat Scratch Disease

    OpenAIRE

    Tsuneoka, Hidehiro; Umeda, Akiko; Tsukahara, Masato; Sasaki, Kohsuke

    2004-01-01

    The possibility of Bartonella clarridgeiae being a causative agent of cat scratch disease (CSD) was investigated by using indirect fluorescence antibody assays with 288 suspected CSD patients. Immunoglobulin G antibody to noncocultivated B. clarridgeiae was suitable only for detection of B. clarridgeiae antibody. Significant cross-reactivity between Bartonella henselae and B. clarridgeiae was noted, and no CSD case caused by B. clarridgeiae was detected.

  8. A new rapid diagnostic test for detection of anti-Schistosoma mansoni and anti-Schistosoma haematobium antibodies

    Directory of Open Access Journals (Sweden)

    Coulibaly Jean T

    2013-01-01

    Full Text Available Abstract Background Parasitological methods are widely used for the diagnosis of schistosomiasis. However, they are insensitive, particularly in areas of low endemicity, and labour-intensive. Immunoassays based on detection of anti-schistosome antibodies have the merit of high sensitivity and recently a rapid diagnostic test (RDT, incorporating Schistosoma mansoni cercarial transformation fluid (SmCTF for detection of anti-schistosome antibodies in blood has been developed. Here, we assessed the diagnostic performance of the SmCTF-RDT for S. mansoni and S. haematobium infections by comparing it with microscopy for egg detection. Methods A cross-sectional survey was carried out in Azaguié, south Côte d’Ivoire. 118 pre-school-aged children submitted two stool and two urine samples, which were subjected to the Kato-Katz and urine filtration methods for the detection of S. mansoni and S. haematobium eggs, respectively. Urine was also subjected to a commercially available cassette test for S. mansoni, which detects circulating cathodic antigen. A finger-prick blood sample was used for the SmCTF-RDT for detection of anti-S. mansoni and anti-S. haematobium antibodies. Results The prevalence of both anti-S. mansoni and anti-S. haematobium antibodies was more than three times higher than the prevalence of infection estimated by egg detection under a microscope. Using quadruplicate Kato-Katz as the reference standard for the diagnosis of S. mansoni infection, the sensitivity, negative predictive value (NPV, and positive predictive value (PPV of the SmCTF-RDT was 75.0%, 84.2% and 22.5%, respectively. When two urine filtrations were considered as the reference standard for the diagnosis of S. haematobium infection, the sensitivity, NPV and PPV of SmCTF-RDT was 66.7%, 94.9% and 5.1%, respectively. The specificity of SmCTF-RDT, when using egg-detection as the reference standard, was estimated to be 34.4%. This low specificity may be a reflection of the

  9. A Generic Method for Fungal Spore Detection: The use of a monoclonal antibody and surface plasmon resonance

    DEFF Research Database (Denmark)

    Skottrup, Peter; Hearty, Stephen; Frøkiær, Hanne; Leonard, Paul; Hejgaard, Jørn; O’Kennedy, Richard; Nicolaisen, Mogens; Justesen, Annemarie Fejer

    This study describes a biosensing principle for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a monoclonal antibody (mab) and a SPR sensor for label-free detection of the model organism Puccinia striiformis f.sp. tritici (Pst) a biotrophic fungus...... binding of mab to the sensor surface was observed as the Pst urediniospore concentration was increased. The detection range for the assay was 1.7 x 106 – 5.3 x 104 spores/ml. This study describes the first use of SPR for detection of fungal spores and the generic principle has the potential to be used in...

  10. Production and characterization of a biotinylated single-chain variable fragment antibody for detection of parathion-methyl.

    Science.gov (United States)

    Wang, Huimin; Zhao, Fengchun; Han, Xiao; Yang, Zhengyou

    2016-10-01

    In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM. The selected scFv antibody was fused with a biotin acceptor domain (BAD) and inserted into pET-28a(+) vector for high-level expression in Escherichia coli BL2 (DE3). After optimizing expression conditions, the scFv-BAD antibody was expressed as a soluble protein and biotinylated in vitro by the E. coli biotin ligase (BirA). Subsequently, the biotinylated scFv-BAD antibody was purified with a high yield of 59.2 ± 3.7 mg/L of culture, and was characterized by SDS-PAGE and western blotting. Finally, based on the biotinylated scFv-BAD, a sensitive IC-ELISA for detection of PM was developed, and the 50% inhibition value (IC50) of PM was determined as 14.5 ng/mL, with a limit of detection (LOD, IC10) of 0.9 ng/mL. Cross-reactivity (CR) studies revealed that the scFv antibody showed desirable specificity for PM. PMID:27181246

  11. Role of SPECT immunoscintigraphy with radiolabelled monoclonal antibodies for the early detection of malignancy and infection

    International Nuclear Information System (INIS)

    The study was undertaken to determine to what extent it is possible to improve immunoscintigraphic diagnosis by using single photon emission computed tomography (SPECT) imaging, in addition to the conventional planar method. Seventy-two patients (40 with infection, 17 with colorectal carcinoma and 15 with malignant melanoma) with 135 known lesions were studied using radiolabelled antibodies. Twenty-eight patients with 53 known lesions underwent additional SPECT imaging of the relevant body region. The overall sensitivity of the study was 86%. Sixteen sites (12%) were detected only by SPECT. Twenty occult lesions were diagnosed, 16 (80%) of them by SPECT. False negative results were mainly related to localization in organs with physiologically high count rates, such as kidneys, the urinary bladder, liver, stomach and the cardiac blood pool. False positive results were probably caused by normal tissue expressing the antigen. The results reveal that an 11% improvement in overall sensitivity can be attained by the utilization of the SPECT procedure with no false negative results. However, higher rates of false positive results were obtained by using SPECT, with an overall increase of the false positive studies by 11%. The planar method offers the advantage of an easier procedure, and is better suited for whole body studies. The SPECT method was of value in differentiating between overlapping physiological and pathological concentrations and in equivocal planar studies. In general, much better demonstration of the tumoral lesions was obtained by SPECT and it also enables sizing of the suspected lesion. However, owing to the prolonged imaging time and the higher rate of false positive studies, restriction of SPECT to selected body areas, after initial screening by planar whole body scintigraphy, is suggested. In conclusion, SPECT represents a useful supplement to the planar study. The combination of both studies seems to be promising for the early detection of tumoral

  12. Commercial serological antibody detection tests for the diagnosis of pulmonary tuberculosis: a systematic review.

    Directory of Open Access Journals (Sweden)

    Karen R Steingart

    2007-06-01

    Full Text Available BACKGROUND: The global tuberculosis epidemic results in nearly 2 million deaths and 9 million new cases of the disease a year. The vast majority of tuberculosis patients live in developing countries, where the diagnosis of tuberculosis relies on the identification of acid-fast bacilli on unprocessed sputum smears using conventional light microscopy. Microscopy has high specificity in tuberculosis-endemic countries, but modest sensitivity which varies among laboratories (range 20% to 80%. Moreover, the sensitivity is poor for paucibacillary disease (e.g., pediatric and HIV-associated tuberculosis. Thus, the development of rapid and accurate new diagnostic tools is imperative. Immune-based tests are potentially suitable for use in low-income countries as some test formats can be performed at the point of care without laboratory equipment. Currently, dozens of distinct commercial antibody detection tests are sold in developing countries. The question is "do they work?" METHODS AND FINDINGS: We conducted a systematic review to assess the accuracy of commercial antibody detection tests for the diagnosis of pulmonary tuberculosis. Studies from all countries using culture and/or microscopy smear for confirmation of pulmonary tuberculosis were eligible. Studies with fewer than 50 participants (25 patients and 25 control participants were excluded. In a comprehensive search, we identified 68 studies. The results demonstrate that (1 overall, commercial tests vary widely in performance; (2 sensitivity is higher in smear-positive than smear-negative samples; (3 in studies of smear-positive patients, Anda-TB IgG by enzyme-linked immunosorbent assay shows limited sensitivity (range 63% to 85% and inconsistent specificity (range 73% to 100%; (4 specificity is higher in healthy volunteers than in patients in whom tuberculosis disease is initially suspected and subsequently ruled out; and (5 there are insufficient data to determine the accuracy of most

  13. Rapid Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Peste des Petits Ruminants Virus

    OpenAIRE

    Choi, Kang-Seuk; Nah, Jin-Ju; Ko, Young-Joon; Kang, Shien-Young; Jo, Nam-In

    2005-01-01

    Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants that is of economic importance in Africa, the Middle East, and Asia. We developed a rapid competitive enzyme-linked immunosorbent assay (rapid c-ELISA) for the diagnosis and surveillance of PPR. This assay detects PPR virus (PPRV) antibodies in serum samples by quantifying the amount of monoclonal antibody (MAb) P-3H12 after 30 min of incubation of a serum-MAb conjugate mixture on plates coated with a PPRV recom...

  14. Antibody-based biosensor assays for the detection of zilpaterol and markers for prostate cancer

    OpenAIRE

    Dillon, Mary

    2008-01-01

    The research presented in this thesis describes the production and application of antibodies against the drug of abuse zilpaterol, and the application of antibodies against prostate-specific antigen (PSA), a cancer marker. Polyclonal antibodies were used in the development of immunoassays in a competitive ELISA format and on the Biacore (a surface plasmon resonance-based optical biosensor capable of monitoring biomolecular interactions in 'real-time'). A zilpaterol-HSA conjugate was u...

  15. Detection of aquaporin-4 antibody using aquaporin-4 extracellular loop-based carbon nanotube biosensor for the diagnosis of neuromyelitis optica.

    Science.gov (United States)

    Son, Manki; Kim, Daesan; Park, Kyung Seok; Hong, Seunghun; Park, Tai Hyun

    2016-04-15

    Here we propose a carbon nanotube (CNT) field-effect transistor (FET) functionalized with aquaporin-4 (AQP4) extracellular loop peptides for the rapid detection of AQP4 antibody without pretreatment. Neuromyelitis optica (NMO) is a rare disease of the central nerve system that affects the optic nerves and the spinal cord. NMO-IgG, a serum antibody in patients, is highly specific for NMO and targets AQP4. We synthesized AQP4 extracellular loop peptides, known as primary autoimmune target in NMO, and immobilized them onto CNT-FET. The sensor showed p-type FET characteristics after the functionalization of peptides. The sensor was able to detect antibody with a detection limit of 1 ng l(-1). Moreover, AQP4 antibody in human serum was detected without any pretreatment. These results indicate that the biosensor can be used for rapid and simple detection of NMO antibody. PMID:26594890

  16. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.

    Science.gov (United States)

    Tang, Yu Qian; Han, Shuang Yan; Zheng, Hong; Wu, Lin; Ueda, Mitsuyoshi; Wang, Xiao Ning; Lin, Ying

    2008-07-01

    In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen. PMID:18542951

  17. Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

    Science.gov (United States)

    Paredes, Adriana; Sáenz, Patricia; Marzal, Miguel W; Orrego, Miguel A; Castillo, Yesenia; Rivera, Andrea; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E

    2016-07-01

    Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections. PMID:27018063

  18. Rapid Detection and Identification of Streptococcus Iniae Using a Monoclonal Antibody-Based Indirect Fluorescent Antibody Technique

    Science.gov (United States)

    Streptococcus iniae is among the major pathogens of a large number of fish species cultured in fresh and marine recirculating and net pen production systems . The traditional plate culture technique to detect and identify S. iniae is time consuming and may be problematic due to phenotypic variations...

  19. Validation of Serological Antibody Profiles Against Human Papillomavirus Type 16 Antigens as Markers for Early Detection of Cervical Cancer.

    Science.gov (United States)

    Salazar-Piña, Dolores Azucena; Pedroza-Saavedra, Adolfo; Cruz-Valdez, Aurelio; Ortiz-Panozo, Eduardo; Maldonado-Gama, Minerva; Chihu-Amparan, Lilia; Rodriguez-Ocampo, Angelica Nallelhy; Orozco-Fararoni, Emilia; Esquivel-Guadarrama, Fernando; Gutierrez-Xicotencatl, Lourdes

    2016-02-01

    Cervical cancer (CC) is the second most frequent neoplasia among women worldwide. Cancer prevention programs around the world have used the Papanicolaou (Pap) smear as the primary diagnostic test to reduce the burden of CC. Nevertheless, such programs have not been effective in developing countries, thus leading to research on alternative tests for CC screening. During the virus life cycle and in the process toward malignancy, different human papillomavirus (HPV) proteins are expressed, and they induce a host humoral immune response that can be used as a potential marker for different stages of the disease. We present a new Slot blot assay to detect serum antibodies against HPV16 E4, E7, and VLPs-L1 antigens. The system was validated with sera from a female population (n = 485) aged 18 to 64 years referred to the dysplasia clinic at the General Hospital in Cuautla, Morelos, Mexico. To evaluate the clinical performance of the serological markers, the sensitivity, specificity, positive, and negative predictive values and receiver-operating characteristic curves (for antibodies alone or in combination) were calculated in groups of lesions of increasing severity. The results showed high prevalence of anti-E4 (73%) and anti-E7 (80%) antibodies in the CC group. Seropositivity to 1, 2, or 3 antigens showed associations of increasing magnitude with CC (odds ratio [OR] = 12.6, 19.9, and 58.5, respectively). The highest association with CC was observed when the analysis was restricted to only anti-E4+E7 antibodies (OR = 187.7). The best clinical performance to discriminate CC from cervical intraepithelial neoplasia 2 to 3 was the one for the combination of anti-E4 and/or anti-E7 antibodies, which displayed high sensitivity (93.3%) and moderate specificity (64.1%), followed by anti-E4 and anti-E7 antibodies (73.3% and 80%; 89.6% and 66%, respectively). In addition, the sensitivity of anti-E4 and/or anti-E7 antibodies is high at any time of sexual activity (TSA

  20. Comparison of the performance of five different immunoassays to detect specific antibodies against emerging atypical bovine pestivirus

    DEFF Research Database (Denmark)

    Larska, Magdalena; Polak, Mirosław P.; Liu, Lihong;

    2013-01-01

    Bovine pestiviruses represent a considerably variable group. In addition to the two accepted species BVDV-1 and BVDV-2, a number of atypical bovine pestiviruses have been detected both in foetal calf sera and in field samples. The sera collected during the initial six weeks of experimental...... BVDV-1 or atypical pestivirus, whereas the animals inoculated with a mixture of these two viruses had a reaction pattern very similar to the homologous virus alone. It was found that immunoassays using whole virus and polyclonal antibodies are the most robust, but all tests examined were able to detect...... antibodies also from cattle infected with atypical pestivirus a few weeks after infection. The detection, however, was at a lower level and slightly delayed. Statistical validation of the threshold suggested by the manufacturer showed that in some cases the reduction of the cut-off values would improve the...

  1. Experiments toward the development of a radioimmunoassay for the detection of serum antibodies for the respiratory syncytial virus

    International Nuclear Information System (INIS)

    In order to detect an infection by the respiratory syncytial virus (RSV) quickly and safely, a radioimmunassay (RIA) should be developed. Various antigen preparations were compared to one another. The immune serums used in the RIA came from guinea pigs with a RSV antibody titer of up to 320 in the complement binding reaction. A number of observations lead to the discussion of the possibility of the formation (incomplete) of cross-reactive antibodies between virus and host cell. This hypothesis could be well supported through references in the literature. Under the assumption of the existence of cross-reactive antibodies, a further model of the pathogenesis of the RSV illness allows itself to be developed, which could be preceived as an illness with autoimmune components. With this model the varying courses of this disease in different age groups can be easily explained. (orig.)

  2. Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo.

    Science.gov (United States)

    Dai, Hua; Xu, Zheng-Zhong; Wang, Meiling; Chen, Jun-Hua; Chen, Xiang; Pan, Zhi-Ming; Jiao, Xin-An

    2016-04-01

    The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ. PMID:27127340

  3. Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies.

    Science.gov (United States)

    Combs, Benjamin; Hamel, Chelsey; Kanaan, Nicholas M

    2016-10-01

    changes in tau indicating that detection of conformational alterations involving PAD exposure is not achieved by all N-terminal tau antibodies and that a relatively discrete region of the N-terminus (i.e., amino acids 7-12, the TNT1 and TNT2 epitope) is central to the differences between normal and pathological tau. The appearance of PAD in early tau pathology and its disappearance in late-stage tangles suggest that toxic forms of tau are associated with the earliest forms of tau deposits. Collectively, these findings demonstrate that the TNT antibodies are useful markers for early conformational display of PAD and provide information regarding conformational changes that have potential implications in the toxic mechanisms of tau pathology. PMID:27260838

  4. Monoclonal antibody based immunodot for specific detection of proteins of the shrimp Penaeus species.

    Science.gov (United States)

    Abhiman, P B; Shankar, K M; Patil, Rajreddy; Suresh Babu, P P; Sahoo, A K; Shamasundar, B A

    2014-05-01

    Frozen shrimp continued to be the single largest item of export from India in terms of value accounting for about 44% of the total marine export earnings. Headless, peeled frozen shrimp is a common and dominant item in the market and there is need for differentiating peeled Penaeus sp from Metapenaeus, Parapenopsis and Macrobrachium sp as consumer preference and price vary. Furthermore, there is need to find out original species used in value addition of shrimp products. Hence, it is essential for development of simple and consumer friendly technique for the identification of shrimp and their products in the market. Two monoclonal antibodies (MAbs) C-15 (IgG3) and C-52 (IgG2a) reacting with 65 and 47 kD proteins of Penaeus monodon respectively in the Western blot were selected. In epitope analysis by immunodot, the two MAbs reacted and recognized specific proteins of P. monodon, Fenneropenaeus indicus and Littopenaeus vannamei and not that of Metapenaeus, Parapenopsis, Macrobrachium rosenbergii, crabs and fishes. The immunodot required 120 min for completion. The sensitivity of the immunodot to detect proteins of P. monodon was 0.225 mg with MAb C-15 and 0.028 mg with MAb C-52. The MAb based immunodot developed, could be used for identifying and differentiating meat of P. monodon, F. indicus, and L. vannamei from that of Metapenaeus, Parapenopsis, M. rosenbergii, crabs and fishes. PMID:24803705

  5. Validation of pre-coated ELISA tests to detect antibodies against T. congolense and T. vivax

    International Nuclear Information System (INIS)

    The anti-trypanosomal antibody detecting enzyme linked immunosorbent assay (ELISA) was first described in 1977 and was further developed for use in large scale surveys in Zimbabwe. More recently, the IAEA initiated a programme to improve the robustness and standardisation of the assay. The IAEA supplied plates pre-coated with either a crude T. congolense or T. vivax antigen and the reagents necessary for analysing samples. Parasitologically positive and negative sera were used to validate and determine the cut-off values of the two tests. The samples were tested and results analysed using a variety of cut-off values. The tests provided similar information although the T. congolense pre-coated plates gave significantly higher optical density values than the plates coated with T. vivax. Sensitivity and specificity values were calculated using the different cut-off points. Results indicate that the test using T. congolense antigen had the highest specificity and sensitivity for a given cut-off value. Although the test could distinguish positive from negative sera, it was quite difficult to provide a suitable cut-off value, but the value should be dictated by the use of the test. (author)

  6. Detecting antibody-antigen reaction using nano ripple gold LSPR based biosensor

    Science.gov (United States)

    Saleem, Iram; Wijesundera, Dharshana; Tilakaratne, Buddhi; Widger, William; Chu, Wei-Kan

    We introduce a simple and cost-effective scheme for bio-sensing using nano-ripple structures. One-dimension metallic nano-ripple structures formed by gas cluster ion beam irradiation have shown polarization of light as well as the localized surface plasmon resonance. These localized surface plasmon resonance (LSPR) based bio sensors not only are capable of label free real time analytical detection but also show high sensitivity. The nano surface morphology determines the changes in the plasmonic properties of nanostructures hence the plasmonic response is tunable. By immobilizing a stable and sterically accessible monolayer of antibody on the surface of these substrates and loading different concentrations of the specific antigen we identified the shift in the LSPR peaks triggered by the change of dielectric function in the neighborhood of the structures. These plasmonic nano-metallic structures can be utilized to observe the shift in the LSPR resonance frequency due to the cycle of adsorption, re-adsorption and reactions taking place on the surface that can potentially be mapped in to reaction mechanics. The bio-sensor has monolayer molecule-coating sensitivity and specific selectivity.

  7. Standardization of serological tests for detecting anti-Trypanosoma cruzi antibodies in dogs

    Directory of Open Access Journals (Sweden)

    M. A. Lauricella

    1993-09-01

    Full Text Available This paper reports on the standardization of four serological reactions currently used in human serodiagnosis for the detection of anti-Trypanosoma cruzi antibodies in naturally and experimentally infected dogs. Indirect immunofluorescence test (IFAT and hemagglutination test (IHAT were standardized, and complement fixation test (CFT and direct agglutination test (DAT were used for diagnostic confirmation. Four hundred and eighty one mongrel dogs that were studied by xenodiagnosis were used: (1 parasitemic dogs of two localities of endemic area (EA of Santiago del Estero province in Argentina (n = 134; (2 non-parasitemic dogs of the same area (n = 285; (3 dogs experimentally infected with T. cruzi in the patent period (n = 6; (4 non-infected dogs (n = 56 which were born in the city of Buenos Aires (BA, one non-EA for Chagas' disease. For IFAT, parasitemic dogs EA showed 95% of reactive sera. Non parasitemic dogs EA showed 77% of non reactive sera. None sera from BA were reactive for dilutions higher than four. For IHAT, 84% of sera of parasitemic dogs EA showed serological reactivity and among non parasitemic dogs BA, 61% were non reactive, while the remainder showed at most titres of 1/16. The cut-off titres for IFAT and IHAT were 1/16 and 1/32 respectively, and for CFT and DAT 1/1 and 1/128 respectively. Sensitivity for IFAT, IHAT, CF and DAT were 95%, 84%, 97% and 95% respectively.

  8. Detection of antibodies against the orthopox virus cameli in sera of East African dromedaries using two different ELISAs

    International Nuclear Information System (INIS)

    The suitability of using two different enzyme-linked immunosorbent assays (ELISAs) for the detection of immunoglobulin G antibodies against the camel pox virus has been tested by examining 297 sera of dromedaries from Kenya, Somalia and Sudan. The ELISAs were based on an indirect method of antibody detection. One technique used direct conjugation of enzymes to antispecies-antibodies (C-EL), the other a biotin-avidin amplification system (BA-EL). Using the conventional technique (C-EL) on 196 sera of dromedaries from different ranches in Kenya during the period 1981 to 1984 we determined a high prevalence of antibodies, with titres of up to 1:4096. A comparison made for the years 1981 to 1984 showed a slight decrease in antibody titres. Five animals suffering from acute camel pox showed titres between 1:8192 and 1:32768; eight serum samples from Somalia, collected during an outbreak of camel pox, showed titres of 1:2048 to 1:8192. A high prevalence of antibodies was also found in 93 samples from Sudan; 95% of the animals showed titres of between 1:128 and 1:4096. Differences due to sex or age could not be determined. According to these results, camel pox appears to be endemic in the areas investigated. To test the applicability of the new BA-EL method, 60 camel sera were investigated. It was found that the sensitivity and specificity of this technique seem to be superior to those of the standard ELISA. The advantage of BA-EL is that it avoids direct conjugation of enzymes to antispecies-globulins which, in the case of exotic animals, are not commercially available. Both ELISAs can be regarded as suitable for serological screening tests and for rapid serological differentiation of orthopox and parapox virus infections, also in camels. (author)

  9. Development of two murine antibodies against Neospora caninum using phage display technology and application on the detection of N. caninum.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1 and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

  10. Mountain Yellow-legged Frogs (Rana muscosa) did not Produce Detectable Antibodies in Immunization Experiments with Batrachochytrium dendrobatidis.

    Science.gov (United States)

    Poorten, Thomas J; Stice-Kishiyama, Mary J; Briggs, Cheryl J; Rosenblum, Erica Bree

    2016-01-01

    Chytridiomycosis is a devastating infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). A growing number of studies have examined the role of amphibian adaptive immunity in response to this pathogen, with varying degrees of immune activation reported. Here we present immunologic data for the mountain yellow-legged frog, Rana muscosa, and the Sierra Nevada yellow-legged frog, Rana sierrae, which are two endangered and ecologically important species experiencing Bd-inflicted declines. Previous studies on these species that examined transcriptional response during Bd infection, and the effective of immunization, provided little evidence of immune activation to Bd. However, the studies did not directly assay immune effectors in the frog hosts. We performed experiments to examine antibody production, which is a hallmark of systemic adaptive immune activation. We used controlled laboratory experiments and enzyme-linked immunosorbent assays to examine the antibody response to Bd immunization and live Bd exposure. Rana muscosa and R. sierrae individuals did not produce detectable antibodies with the capacity to bind to denatured Bd antigens under our experimental conditions. While we cannot rule out antibody response to Bd in these species, our results suggest weak, poor, or inefficient production of antibodies to denatured Bd antigens. Our findings are consistent with susceptibility to chytridiomycosis in these species and suggest additional work is needed to characterize the potential for adaptive immunity. PMID:26540180

  11. Multiplex Antibody Detection for Noninvasive Genus-Level Diagnosis of Prosthetic Joint Infection

    Science.gov (United States)

    Marmor, Simon; Bauer, Thomas; Desplaces, Nicole; Heym, Beate; Roux, Anne-Laure; Sol, Olivier; Rogé, Julie; Mahé, Florence; Désiré, Laurent; Aegerter, Philippe; Ghout, Idir; Ropers, Jacques; Gaillard, Jean-Louis

    2016-01-01

    We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcus species (Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens), Streptococcus agalactiae (4 antigens), and Propionibacterium acnes (4 antigens) were selected by comparative immunoproteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed. We conducted a prospective study to evaluate the performance of the assay. A PJI was defined by the presence of a sinus tract and/or positive intraoperative sample cultures (at least one sample yielding a virulent organism or at least two samples yielding the same organism). A total of 455 consecutive patients undergoing revision or resection arthroplasty (hip, 66.3%; knee, 29.7%; shoulder, 4%) at two French reference centers for the management of PJI were included: 176 patients (38.7%) were infected and 279 (61.3%) were not. About 60% of the infections involved at least one of the species targeted by the assay. The sensitivity/specificity values were 72.3%/80.7% for targeted staphylococci, 75%/92.6% for S. agalactiae, and 38.5%/84.8% for P. acnes. The assay was more sensitive for infections occurring >3 months after arthroplasty and for patients with an elevated C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, it detected 64.3% and 58.3% of targeted staphylococcal infections associated with normal CRP and ESR values, respectively. This new multiplex immunoassay approach is a novel noninvasive tool to evaluate patients suspected of having PJIs and provides information complementary to that from inflammatory marker values. PMID:26865683

  12. Direct relationship between radiobiological hypoxia in tumors and monoclonal antibody detection of EF5 cellular adducts.

    Science.gov (United States)

    Lee, J; Siemann, D W; Koch, C J; Lord, E M

    1996-07-29

    While the potential importance of hypoxia in limiting the sensitivity of tumor cells to ionizing radiation has long been appreciated, methods for accurately quantifying the number of radiation-resistant hypoxic cells within tumors have been lacking. We have used the pentafluorinated derivative [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acet amide] of etanidazole (EF5), which binds selectively to hypoxic cells. The adducts formed between EF5 and cellular proteins in the hypoxic cells were detected using the specific monoclonal antibody (MAb), ELK3-51 conjugated to the flurochrome Cy3, and the number of hypoxic cells was quantified by flow cytometry. To verify the validity of this technique for the detection of hypoxic cells, mice bearing KHT sarcomas were treated with various agents to alter tumor oxygenation and hence vary the fraction of radiobiologically hypoxic tumor cells. The percentage of EF5 binding cells was then compared directly with the clonogenic survival of the tumor cells following radiation treatment under the various pretreatment conditions. The results showed that allowing the mice to breathe carbogen (5% CO2/95% O2) prior to irradiation reduced clonogenic cell survival approx. 6-fold and led to an absence of cells binding high levels of EF5. In contrast, pretreating the tumor-bearing animals with either hydralazine, which decreased tumor blood flow, or phenylhydrazine hydrochloride, which made the mice anemic, increased tumor cell survival following irradiation 2- to 4-fold, indicative of an increase in the fraction of hypoxic tumor cells. EF5 measurements made under identical conditions illustrated a shift in the cells in the tumor to high EF5 binding. Our results demonstrate that flow cytometric measurement by fluorescent MAb binding to EF5 adducts may relate directly to radiobiological hypoxia in KHT tumors measured by conventional methods. PMID:8707411

  13. Multiplex Antibody Detection for Noninvasive Genus-Level Diagnosis of Prosthetic Joint Infection.

    Science.gov (United States)

    Marmor, Simon; Bauer, Thomas; Desplaces, Nicole; Heym, Beate; Roux, Anne-Laure; Sol, Olivier; Rogé, Julie; Mahé, Florence; Désiré, Laurent; Aegerter, Philippe; Ghout, Idir; Ropers, Jacques; Gaillard, Jean-Louis; Rottman, Martin

    2016-04-01

    We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcusspecies (Staphylococcus aureus,Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens),Streptococcus agalactiae(4 antigens), and Propionibacterium acnes(4 antigens) were selected by comparative immune proteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed. We conducted a prospective study to evaluate the performance of the assay. A PJI was defined by the presence of a sinus tract and/or positive intraoperative sample cultures (at least one sample yielding a virulent organism or at least two samples yielding the same organism). A total of 455 consecutive patients undergoing revision or resection arthroplasty (hip, 66.3%; knee, 29.7%; shoulder, 4%) at two French reference centers for the management of PJI were included: 176 patients (38.7%) were infected and 279 (61.3%) were not. About 60% of the infections involved at least one of the species targeted by the assay. The sensitivity/specificity values were 72.3%/80.7% for targeted staphylococci, 75%/92.6% forS. agalactiae, and 38.5%/84.8% forP. acnes The assay was more sensitive for infections occurring >3 months after arthroplasty and for patients with an elevated C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, it detected 64.3% and 58.3% of targeted staphylococcal infections associated with normal CRP and ESR values, respectively. This new multiplex immunoassay approach is a novel noninvasive tool to evaluate patients suspected of having PJIs and provides information complementary to that from inflammatory marker values. PMID:26865683

  14. Evaluation of four methods for cytomegalovirus antibody detection for use by a bone marrow transplantation service.

    OpenAIRE

    Leland, D S; Barth, K A; Cunningham, E B; Jansen, J; Tricot, G J; French, M L

    1989-01-01

    Four methods, latex agglutination, indirect fluorescent antibody, enzyme immunoassay, and complement fixation, were compared for cytomegalovirus antibody screening and for pre- and posttransplant determinations on bone marrow transplant recipients. Latex agglutination was most sensitive (98%) and specific (97%) for screening and pretransplant determinations and was quickest and easiest to perform. In posttransplant sera from allogeneic bone marrow transplant recipients, all methods except com...

  15. Monoclonal antibody-based Surface Plasmon Resonance sensors for pathogen detection

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand

    2007-01-01

    striiformis f.sp. tritici, the cause of wheat yellow rust and Phytophthora infestans, the cause of late blight disease in potato. As no antibody existed against urediniospores from P. striiformis, mouse monoclonal antibodies (mAbs) were produced and characterised. IgM-isotype mAbs from nine hybridoma cell...

  16. Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    Sofía Duque-Beltrán

    2002-12-01

    Full Text Available The present study developed and standardized an enzime-linked immunosorbent assay (ELISA to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate. One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%; specificity, 95% (95% CI: 88.6-97.6%; positive predictive value, 91% (95% CI: 81.4-95.9%; and negative predictive value, 100% (95% CI: 96.1-100%. This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

  17. 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. PMID:23114700

  18. Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

    Directory of Open Access Journals (Sweden)

    Luciane A Amado

    2006-03-01

    Full Text Available In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oral fluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One hundred and ten paired serum and saliva specimens from suspect patients of having acute hepatitis were collected to detect antibodies to hepatitis A (total and IgM, hepatitis B (anti-HBs, total anti-HBc and IgM anti-HBc, and hepatitis C (anti-HCV using commercially available enzyme-linked immunossorbent assay (EIA. In relation to serum samples, oral fluid assay sensitivity and specificity were as follows: 87 and 100% for total anti-HAV, 79 and 100% for anti-HAV IgM, 6 and 95% for anti-HBs, 13 and 100% for total anti-HBc, 100 and 100% for anti-HBc IgM, and 75 and 100% for anti-HCV. The consistency observed between antibodies tests in saliva and expected risk factors for hepatitis A and C suggests that the saliva method could replace serum in epidemiological studies for hepatitis A and C.

  19. Development of immunochromatographic strip test using fluorescent, micellar silica nanosensors for rapid detection of B. abortus antibodies in milk samples.

    Science.gov (United States)

    Vyas, Swati S; Jadhav, Sushma V; Majee, Sharmila B; Shastri, Jayanthi S; Patravale, Vandana B

    2015-08-15

    Presence of bacteria such as Brucella spp. in dairy products is an immense risk to public health. Point of care immunoassays are rapid in that they can quickly screen various samples in a relatively short amount of time, are sensitive, specific and offer a great advantage in accurate and fast diagnosis of infectious diseases. We have fabricated a point of care rapid diagnostic assay that employs fluorescent, micellar silica nanosensors capable of specifically detecting Brucella IgG antibodies in milk samples of afflicted animals. Currently, point of care detection assays are not commercially available for field testing of farm animals using milk samples. The nanosensing allows precise detection of antibodies with low sample volumes (50 μl). We demonstrate recognition of B. abortus antibodies through capture by fluorescent silica nanosensors using spiked and raw milk samples validated by ELISA and PCR. The test results are accurate and repeatable with high sensitivity and specificity, and a short assay time of 10 min for antigenic recognition and do not require any sample processing procedures such as isolation and separation. Additionally, well defined antigenic components and surface biomarkers of various disease causing microbes can be broadly incorporated within the purview of this technology for accurate and rapid detection of suspected bovine pathological conditions, and can largely enable rapid field testing that can be implemented in farms and food industry. PMID:25829223

  20. Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples

    Directory of Open Access Journals (Sweden)

    M. F. Kearney

    2011-01-01

    Full Text Available The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick, we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples ( or prostate specimens ( from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.

  1. Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

    Science.gov (United States)

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Brasil, Tatiana de Arruda Campos; Foti, Leonardo; Souza, Wayner Vieira de; Silva, Edmilson Domingos; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2016-01-01

    The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies. PMID:27517281

  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. The SOLID (Signs Of LIfe Detector) instrument concept: an antibody microarray-based biosensor for life detection in astrobiology

    Science.gov (United States)

    Parro, V.; Rivas, L. A.; Rodríguez-Manfredi, J. A.; Blanco, Y.; de Diego-Castilla, G.; Cruz-Gil, P.; Moreno-Paz, M.; García-Villadangos, M.; Compostizo, C.; Herrero, P. L.

    2009-04-01

    Immunosensors have been extensively used since many years for environmental monitoring. Different technological platforms allow new biosensor designs and implementations. We have reported (Rivas et al., 2008) a shotgun approach for antibody production for biomarker detection in astrobiology and environmental monitoring, the production of 150 new polyclonal antibodies against microbial strains and environmental extracts, and the construction and validation of an antibody microarray (LDCHIP200, for "Life Detector Chip") containing 200 different antibodies. We have successfully used the LDCHIP200 for the detection of biological polymers in extreme environments in different parts of the world (e.g., a deep South African mine, Antarctica's Dry valleys, Yellowstone, Iceland, and Rio Tinto). Clustering analysis associated similar immunopatterns to samples from apparently very different environments, indicating that they indeed share similar universal biomarkers. A redundancy in the number of antibodies against different target biomarkers apart of revealing the presence of certain biomolecules, it renders a sample-specific immuno-profile, an "immnuno-fingerprint", which may constitute by itself an indirect biosignature. We will present a case study of immunoprofiling different iron-sulfur as well as phylosilicates rich samples along the Rio Tinto river banks. Based on protein microarray technology, we designed and built the concept instrument called SOLID (for "Signs Of LIfe Detector"; Parro et al., 2005; 2008a, b; http://cab.inta.es/solid) for automatic in situ analysis of soil samples and molecular biomarkers detection. A field prototype, SOLID2, was successfully tested for the analysis of grinded core samples during the 2005 "MARTE" campaign of a Mars drilling simulation experiment by a sandwich microarray immunoassay (Parro et al., 2008b). We will show the new version of the instrument (SOLID3) which is able to perform both sandwich and competitive immunoassays. SOLID3

  4. Generation of monoclonal antibodies against prostate specific antigen (PSA) for the detection of PSA and its purification

    International Nuclear Information System (INIS)

    The prostate cancer in Cuba is a problem of health (2672 diagnosed cases and 2769 deaths in 2007). Various diagnostic methods have been implemented for the detection and management of this disease, emphasizing among them (PSA) prostate-specific antigen serological determination. At this work was generated and characterized a panel of 11 antibodies (AcMs) monoclonal IgG1 detected with high affinity described major epitopes of the PSA, both in solution and attached to the test plate. From the panel obtained AcMs was the standardization of an essay type ELISA for the detection of serum total PSA (associated and free) equimolar, based on antibody monoclonal CB-PSA.4 in the coating and the CB-PSA.9 coupled with biotin as liner, with a detection limit of 0.15 ng/mL. Similarly, standardized system for detection in serum free PSA, based on the AcMs CB-PSA.4 (coating) and CB-PSA.2 coupled with biotin (liner), with a detection limit of 0.5 ng/mL. Finally, with the purpose of using PSA as standard in trials type ELISA, developed a simple method of inmunopurificación based on the AcM, CB-PSA.2, which was obtained the PSA with a purity exceeding 90%. Immunoassay Centre on the basis of the AcMs panel and the results of this study, developed and recorded two diagnostic systems for the detection of PSA in human serum. (author)

  5. Advantage of highly immunoreactive monoclonal antibodies in radioimmunoscintigraphy for tumor detection, (2)

    International Nuclear Information System (INIS)

    There is theoretically a potential benefit in using a highly immunoreactive monoclonal antibody. The effect of immunoreactivity (IR) on the antibody biodistribution, however, has not yet been described in detail. Thus, this study was designed to investigate the effect of IR on the biodistribution in an animal model. The hydroxylapatite high performance liquid chromatography (HA-HPLC) system has been tested and confirmed to separate the F ab 96.5, an anti melanoma p97 antigen, into high and low IR fractions. 125I-F ab 96.5 preparations with a different IR were administered to groups of nude mice bearing FEM-XII human skin melanoma xenografts for biodistribution and imaging studies. The biodistribution data showed that the high IR antibody improved tumor targeting by increasing activity ratios of tumor to non tumor tissue; the mechanism for the increased tumor to non tumor ratios was increased tumor activity uptake and prolonged tumor activity retention with associated rapid clearance from the blood and non tumor sites. The imaging study visually supported the results obtained in the biodistribution study; the high IR antibody demonstrated better and earlier tumor delineation and the tumor to non tumor contrast continued to improve with time. In this model system, where the whole body clearance rate was the same for the high IR and low IR preparations, the overall antibody metabolism and excretion were not significantly dependent on IR. Therefore, the effect of IR is to alter the distribution of antibody between tumor and blood, with high IR having increased tumor activity and reduced blood activity (consequently reduced non tumor organ activity). This would also be beneficial for therapeutic use of radiolabeled antibodies, since high IR antibodies can minimize undesirable radiation exposure to normal organs. In conclusion, high IR antibodies are essential for optimal tumor targeting. (author)

  6. In vivo germinal mutation detection with "monospecific" antibody against lactate dehydrogenase-X.

    OpenAIRE

    Ansari, A. A.; Baig, M A; Malling, H. V.

    1980-01-01

    This report describes developments toward a cell specific-locus test for measuring point mutations directly in sperm based upon the use of a monospecific antibody against sperm-specific lactate dehydrogenase-X (LDH-X). The antibody recognizes amino acid differences between mouse and rat LDH-X molecules. In general, mouse sperm do not bind the monospecific antibody against rat LDH-X, but a few exceptional mouse sperm do. Such mouse sperm are believed to contain LDH-X molecules in which an amin...

  7. Detection of serum anti-B/B’ UsnRNP antibodies in patients with connective tissue diseases by immunoblotting

    Directory of Open Access Journals (Sweden)

    L. Iaccarino

    2011-09-01

    Full Text Available Objective: To investigate the reliability of the immunoblot method in the detection of serum immunoreactivity towards the B/B’ polypeptides of U small nuclear ribonucleoproteins (UsnRNP and to assess the significance of these antibodies in connective tissue disease (CTD patients. Methods: We tested the sera of 348 patients with CTD (101 SLE, 51 systemic sclerosis, 53 primary Sjogren’s syndrome, 27 poly/dermatomyositis, 15 rheumatoid arthritis and 101 overlap CTD, of 31 matched healthy subjects and 13 patients with primary Epstein-Barr virus (EBV infection with high titre IgG anti-EBV antibodies. IgG anti-UsnRNP antibodies were determined by immunoblotting on nuclear extract from Raji cells (an EBV-immortalised human B lymphoid cell line and Jurkat cells (a human T lymphoid cell line. Anti-dsDNA antibodies were detected by indirect immunofluorescence on Crithidia luciliae and anti-ENA by counterimmunoelectrophoresis. Anti-dsDNA activity and avidity were measured in SLE sera by ELISA with Scatchard analysis. Results were statistically analysed by chi-square and Mann-Whitney tests. Results: A high frequency of anti-B/B’ antibodies was found in the sera of CTD patients, confined to SLE (54.4% and overlap CTD with SLE features (55,2%. Anti-B/B’ immune reactivity was closely associated with other anti-UsnRNP specificities, gel precipitating anti-nRNP and anti-P antibodies. Nine out of 15 (60% anti-B/B’ positive/anti-ENA negative lupus sera on Raji blots were confirmed to be positive also on Jurkat blots. The sera from patients with EBV infection provided, on Raji blots, completely different band patterns from those obtained with auto-immune sera. Conclusions. The Sm B/B’ proteins are the predominant or, at least, the most frequently targeted antigens of the UsnRNP auto-immune response in SLE and “lupus-like” overlap CTD. Moreover, anti-B/B’ is diagnostically specific for CTD with SLE features. Immunoblotting on human B lymphoid cells

  8. [Detection of bactericidal antibody in the breast milk of a mother infected with enterohemorrhagic Escherichia coli O157:H7].

    Science.gov (United States)

    Adachi, E; Tanaka, H; Toyoda, N; Takeda, T

    1999-05-01

    A 21 years-old pregnant woman developed diarrhea, fresh bloody stools and abdominal pain on April 6th 1997 at 32 weeks of gestation, and was admitted to the hospital on April 11th. The stool culture on admission was positive for enterohemorrhagic Escherichia coli (EHEC) O157:H7 (Stx1 and 2). Clinical laboratory data during admission showed only slight elevation of beta-microglobulin and N-acetyl glucosaminidase in the urine, and no neurological or hemolytic symptoms were seen. After the antibiotic and lactobacillus administration, all her symptoms were relieved and no abnormal findings in pregnancy were observed. She delivered a baby girl normally on May 30th. Serum (between 41 and 120 days from the onset) and milk (between 4 and 64 days post partum) samples from the mother, and serum (64 days of age) from a baby and cord blood were obtained to monitor the immune status against EHEC O157:H7 and against Shiga toxins (Stx). Anti-E. coli O157 LPS antibodies (IgA, G and M) were assayed by the ELISA method. Neutralizing anti-Stx antibodies were measured by using ACHN cell cytotoxicity assay. In the colostrum and mature milk, high levels of IgA and IgM, and no IgG antibodies against EHEC O157 LPS were detected. In one of the control colostrum samples obtained from 4 healthy mothers IgA antibody against EHEC O157 LPS was detected. To assess the potency of protection against EHEC O157:H7 by the breast milk, we monitored it by the bactericidal activity for the organism under complement-coincubation experiment, and by the neutralization test for the Stx cytotoxicity. As a result, breast milk samples (both colostrum and mature milk) from a patient were demonstrated to kill the organisms. One of 4 healthy milk samples, showed bactericidal activity though it was negative in O157-LPS antibody. This bactericidal activity seen in one healthy colostrum is possibly due to a nonspecific reaction caused by non-O157 E. coli infection. From these observations, it was suggested that the

  9. Frequencies and Specificities of “Enzyme-Only” Detected Erythrocyte Alloantibodies in Patients Hospitalized in Austria: Is an Enzyme Test Required for Routine Red Blood Cell Antibody Screening?

    OpenAIRE

    Dietmar Enko; Claudia Habres; Franz Wallner; Barbara Mayr; Gabriele Halwachs-Baumann

    2014-01-01

    The aim of this study was to determine the frequencies and specificities of “enzyme-only” detected red blood cell (RBC) alloantibodies in the routine antibody screening and antibody identification in patients hospitalized in Austria. Routine blood samples of 2420 patients were investigated. The antibody screening was performed with a 3-cell panel in the low-ionic strength saline- (LISS-) indirect antiglobulin test (IAT) and with an enzyme-pretreated (papain) 3-cell panel fully automated on th...

  10. Synthesis of a peptide-universal nucleotide antigen: towards next-generation antibodies to detect topoisomerase I-DNA covalent complexes.

    Science.gov (United States)

    Perkins, Angela L; Peterson, Kevin L; Beito, Thomas G; Flatten, Karen S; Kaufmann, Scott H; Harki, Daniel A

    2016-04-26

    The topoisomerase (topo) I-DNA covalent complex represents an attractive target for developing diagnostic antibodies to measure responsiveness to drugs. We report a new antigen, peptide , and four murine monoclonal antibodies raised against that exhibit excellent specificity for recognition of in comparison to structurally similar peptides by enzyme-linked immunosorbent assays. Although topo I-DNA complex detection was not achieved in cellular samples by these new antibodies, a new strategy for antigen design is reported. PMID:27113574

  11. Development of Multiple ELISAs for the Detection of Antibodies against Classical Swine Fever Virus in Pig Sera

    Institute of Scientific and Technical Information of China (English)

    Zhen-hua Yang; Ling Li; Zi-shu Pan

    2012-01-01

    The major immunogenic proteins (Ems,E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E.coli and purified by affinity chromatography.The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies in pig sera.Optimum cut-off values were determined by receiver operating characteristic (ROC) analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets.The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results.The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination.

  12. Enzyme-Linked Immunosorbent Assay Employing a Recombinant Antigen for Detection of Protective Antibody against Swine Erysipelas

    OpenAIRE

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-01-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher y...

  13. Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Min Long; Jun Luo; Yan Li; Fang-Yin Zeng; Ming Li

    2009-01-01

    AIM: To detect and evaluate the antibodies against Helicobacter pylori ( H pylori) neutrophil-activating protein (HP-NAP) in patients with gastric cancer and other gastroduodenal diseases.METHODS: Recombinant HP-NAP was prepared from a prokaryotic expression system in Escherichia coli. Serum positivity and level of HP-NAP-specific antibodies in sera from 43 patients with gastric cancer, 28 with chronic gastritis, 28 with peptic ulcer, and 89 healthy controls were measured by rHP-NAP-based ELISA. rHP-NAP-stimulated production of interleukin-8 (IL-8) and growth-related oncogene (GROα) cytokines in the culture supernatant of SGC7901 gastric epithelial cells was also detected. RESULTS: The serum positivity and mean absorbance value of HP-NAP-specific antibodies in the gastric cancer group (97.7% and 1.01 ± 0.24) were significantly higher than those in the chronic gastritis group (85.7% and 0.89 ± 0.14, P < 0.005) and healthy control group (27.7% and 0.65 ± 0.18, P < 0.001). The sensitivity and specificity of ELISA for the detection of HP-NAP-specific antibodies were 95.5% and 91.5%, respectively. HP-NAP could slightly upregulate IL-8 production in gastric epithelial cell lines but had no effect on GROα production. CONCLUSION: Infection with virulent H pylori strains secreting HP-NAP is associated with severe gastroduodenal diseases, and HP-NAP may play a role in the development of gastric carcinoma. rHP-NAPbased ELISA can be used as a new method to detect H pylori infection. The direct effect of HP-NAP on gastric epithelial cells may be limited, but HP-NAP may contribute to inflammatory response or carcinogenesis by activating neutrophils.

  14. Generation of monoclonal antibodies against MGA and comparison of their application in breast cancer detection by immunohistochemistry

    OpenAIRE

    Cuimi Duan; Xiqin Yang; Xuhui Zhang; Jiannan Feng; Zhiqiang Liu; Haiping Que; Heather Johnson; Yanfeng Zhao; Yawen Fan; Yinglin Lu; Heqiu Zhang; Yan Huang; Bingshui Xiu; Xiaoyan Feng

    2015-01-01

    Mammaglobin A (MGA) is an organ specific molecular biomarker for metastatic breast cancer diagnosis. However, there are still needs to develop optimal monoclonal antibodies (mAbs) to detect MGA expression in breast carcinoma by immunohistochemistry. In this study, we first generated mAbs against MGA. Then, we used epitope prediction and computer-assisted structural analysis to screen five dominant epitopes and identified mAbs against five epitopes. Further immunohistochemical analysis on 42 b...

  15. Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems.

    OpenAIRE

    Faude, U C; Höfle, M.G.

    1997-01-01

    Strain-specific monoclonal antibodies (MAbs) were developed for three different bacterial isolates obtained from a freshwater environment (Lake Plusssee) in the spring of 1990. The three isolates, which were identified by molecular methods, were as follows: Cytophaga johnsonae PX62, Comamonas acidovorans PX54, and Aeromonas hydrophila PU7718. These strains represented three species that were detected in high abundance during a set of mesocosm experiments in Lake Plusssee by the direct analysi...

  16. Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay

    OpenAIRE

    Kohlgraf, Karl G.; Ackermann, Abbey R.; Burnell, Kindra K.; Srikantha, Rupasree N.; Joly, Sophie A.; Bartlett, Jennifer; Gakhar, Lokesh; Johnson, Georgia K.; McCray, Paul B.; Guthmiller, Janet M.; Brogden, Kim A

    2011-01-01

    The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1...

  17. Suitability of a Rapid Immunochromatographic Test for Detection of Antibodies to Human Immunodeficiency Virus in Ghana, West Africa

    OpenAIRE

    Aidoo, S.; Ampofo, W. K.; Brandful, J. A. M.; Nuvor, S. V.; Ansah, J. K.; Nii-Trebi, N.; Barnor, J. S.; Apeagyei, F; Sata, T.; Ofori-Adjei, D.; Ishikawa, K.

    2001-01-01

    In West African countries such as Ghana, efficient human immunodeficiency virus (HIV) testing is a priority in the fight against AIDS. A new immunochromatographic rapid test, Determine HIV-1/2 (Abbott Diagnostics, North Chicago, Ill.), that detects antibodies against HIV type 1 (HIV-1) and/or HIV-2 was evaluated using Ghanaian blood samples. Two hundred four serum and/or plasma specimens were tested. HIV screening was done by a particle agglutination test and confirmed by a Western blot (WB) ...

  18. Graphical control and evaluation of the operational performance of ELISA method for detection of trypanosomal antibodies

    International Nuclear Information System (INIS)

    The performance of four indirect trypanosomal antibody detection enzyme-linked immunosorbent assays (I-TAB ELISA's), exploiting native and denatured antigens of Trypanosoma congolense (T.c.AGn, T.c.AGd) and T. vivax (T.v.AGn, T.v.AGd), has been evaluated in fifteen laboratories from Africa and Europe. Standardised internal quality control samples (IQCs) were used as indicators and data plotted on charts to monitor and control the ELISA's at individual laboratories. Based on the overall data, dispersion of true values from the population data range was estimated plotting the location and deviation of raw and normalised absorbance values of the IQCs. Binding ratios were calculated to estimate the assay proficiency with respect to the accuracy of assessing that the QC samples tested positive or negative in the test proper. The frequency distribution of coefficients of variation <10% of IQCs was monitored. The use of this standardised and transparent IQC data charting provides a useful quality control tool for the evaluation of the performance of the ELISA's used for trypanosomosis serology. The method provides a measure of confidence in estimating the proficiency of national laboratories with respect to reported ELISA results on disease occurrence. Moreover, data were compiled of the inter-laboratory ELISA performance by means of summary data charts with reference to the performance criteria described. The data were also analysed using modified Youden plots. The analysis demonstrated similar laboratory proficiency for the I-TAB ELISA (T.c.AGn) (four of five laboratories), I-TAB ELISA (T.c.AGd) (eleven of fourteen laboratories), I-TAB ELISA (T.v.AGn) (three of five laboratories), and I-TAB ELISA (T.v.AGd) (eleven of fifteen laboratories). The impact of data variation between test and specified values as a sole decision criterion for acceptance or rejection of test plates is discussed. (author)

  19. The use of monoclonal antibodies to detect Bacteroides gingivalis in biological samples.

    OpenAIRE

    Chen, P; Bochacki, V; Reynolds, H S; Beanan, J; Tatakis, D.N.; Zambon, J J; Genco, R J

    1986-01-01

    Hybridomas were established which produce monoclonal antibodies specific for Bacteroides gingivalis, a pathogen associated with human periodontal disease. Spleen cells from BALB/c mice immunized with formalinized B. gingivalis were fused with Sp2/0-Ag14 myeloma cells. Of 1,050 wells with positive growth, 60 contained antibody reactive with the immunizing strain of B. gingivalis by enzyme-linked immunosorbent assay. Expansion of these cultures and cloning by limited dilution resulted in 28 clo...

  20. Comparison of three ELISA methods with CLIF test for detection of Anti-dsDNA antibody

    OpenAIRE

    Feyza Çetin; Alparslan Toyran; Özlem Aytaç; Feride Alaca Coşkun; İpek Mumcuoğlu; Feyza Alp; Altan Aksoy

    2015-01-01

    INTRODUCTION[|]Systemic lupus erythematosus (SLE) is an autoimmune disease caused by antibodies which produced against antigens commonly on cell nuclei. According to the criteria of American College of Rheumatology (ACR), the immunological parameters which are used for diagnosis of SLE, are anti- nuclear antibodies (ANA) and anti- dsDNA autoantibodies. In this study it is aimed to investigate the specifity of three different ELISA tests by comparing with CLIF test, as ...

  1. Detection of anti-Neospora caninum antibodies in Iranian native cattle

    OpenAIRE

    Jamal Gharekhani; Heidar Heidari

    2014-01-01

    Neospora caninum is an Apicomplexan parasite which may cause abortion in cattle. This study investigated occurrences of antibodies against N. caninum in Iranian native cattle. From September 2010 to September 2011, blood samples (n=768) of native cows were collected randomly from different rural regions of Hamedan (n=400) and Kurdistan provinces (n=368) located to the western part of Iran. All the samples were evaluated for IgG antibodies against N. caninum using Enzyme Linked Immunosorbent A...

  2. Preparation of the monoclonal antibodies against tumor M2-type pyruvate kinase and establishment of immunoradiometric analysis for detection

    International Nuclear Information System (INIS)

    Objective: To produce monoclonal antibodies against tumor M2-type pyruvate kinase (M2-PK), and to establish immunoradiometric analysis for detection. Methods: The BALB/c mice were immunized with M2-PK antigen. Monoclonal antibodies were generated after cell fusion and screening. Established detection method of immunoradiometric analysis and then evaluated its sensitivity, specificity and coefficient of variation. Results: Two monoclonal antibodies (1A6 and 3F8) with high-titer and high-specificity were generated and detection method of immunoradiometric analysis was established. The analytical sensitivity of immunoradiometric analysis was 2 U/ml, while its clinical sensitivity and specificity was 70.5% and 89.0% respectively. The reference value of M2-PK level was less than 14.8 U/nd according to immunoradiometric analysis. Intra-assay coefficient of variation was less than 5% , while inter-assay coefficient of variation was less than 10% . Conclusion: This sensitive immunoradiometric analysis can be used in early diagnosis of tumor and physical examination. (authors)

  3. Detection of antibodies against Brucella abortus, Leptospira spp., and Apicomplexa protozoa in water buffaloes in the Northeast of Argentina.

    Science.gov (United States)

    Konrad, José L; Campero, Lucía M; Caspe, Gastón S; Brihuega, Bibiana; Draghi, Graciela; Moore, Dadin P; Crudeli, Gustavo A; Venturini, María C; Campero, Carlos M

    2013-11-01

    Water buffalo industry has become a profitable activity worldwide, including the Northeast of Argentina (NEA). However, research on diseases affecting this species is scarce. The aim of the present study was to detect antibodies against Brucella abortus, Leptospira spp., Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in 500 water buffalo cows from five ranches (100 animals each) in the NEA. Serum samples were tested for B. abortus by fluorescence polarization assay, Leptospira spp. by microagglutination test, and N. caninum, T. gondii, and Sarcocystis spp. by indirect fluorescent antibody tests. Overall, the proportion of seropositive animals was 6.4, 22.2, 42.2, 25.4, and 50.8 % for brucellosis, leptospirosis, neosporosis, toxoplasmosis, and sarcocystosis, respectively. The proportion of seropositive animals for all diseases was statistically different among herds (p < 0.05). Statistical differences were also detected among age groups for brucellosis and neosporosis (p < 0.05). The detection of specific antibodies to B. abortus, Leptospira spp., and several Apicomplexa protozoans in water buffaloes in the NEA is reported in this study. PMID:23765549

  4. Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

    Directory of Open Access Journals (Sweden)

    Ruiz Patrícia M Gonçalves

    2001-01-01

    Full Text Available A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina, or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days. The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

  5. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    Science.gov (United States)

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  6. Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.

    Science.gov (United States)

    Stoddard, Robyn A; Quinn, Conrad P; Schiffer, Jarad M; Boyer, Anne E; Goldstein, Jason; Bagarozzi, Dennis A; Soroka, Stephen D; Dauphin, Leslie A; Hoffmaster, Alex R

    2014-06-01

    Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

  7. Use of liquid phase ELISA kit for detection of antibodies against food-and-mouth disease virus (FMDV) in Colombia

    International Nuclear Information System (INIS)

    Colombia has FMDV type O and type A in some regions with endemic characteristics. At present, the country is involved in the hemispheric plan for eradication of FMDV. To attain this objective, it is necessary to use modern techniques with high sensitivity and specificity as opposed to traditional serological tests. The use of liquid ELISA is of great benefit in places where prevention, control and eradication programmes for FMD are running. This technique gives more security because it is very sensitive and specific and the results are obtained on the same day. In addition, it does not need any cell culture nor a CO2 environment. The test is based on specific blocking of the liquid phase of FMDV antigen by antibodies in the test sample. After the test serum has been allowed to react with the plate coated with FMDV serotype specific trapping antibodies. The presence of antibodies to FMDV in the serum sample will result in the formation of immune complexes and, consequently, reduce the amount of free antigen trapped by the immobilized rabbit antisera. In turn, fewer guinea pig anti-FMDV detecting antibodies will react in the next incubation step. After the addition of enzyme labelled horseradish peroxidase, anti-guinea-pig Ig and substrate/chromogen solution, a reduction in colour development will be observed when compared to controls containing free antigens only

  8. In vitro molecular magnetic resonance imaging detection and measurement of apoptosis using superparamagnetic iron oxide + antibody as ligands for nucleosomes

    International Nuclear Information System (INIS)

    Recent research in cell biology as well as oncology research has focused on apoptosis or programmed cell death as a means of quantifying the induced effects of treatment. A hallmark of late-stage apoptosis is nuclear fragmentation in which DNA is degraded to release nucleosomes with their associated histones. In this work, a method was developed for detecting and measuring nucleosome concentration in vitro with magnetic resonance imaging (MRI). The indirect procedure used a commercially available secondary antibody-superparamagnetic iron oxide (SPIO) particle complex as a contrast agent that bound to primary antibodies against nucleosomal histones H4, H2A and H2B. Using a multiple-echo spin-echo sequence on a 1.5 T clinical MRI scanner, significant T2 relaxation enhancement as a function of in vitro nucleosomal concentration was measured. In addition, clustering or aggregation of the contrast agent was demonstrated with its associated enhancement in T2 effects. The T2 clustering enhancement showed a complex dependence on relative concentrations of nucleosomes, primary antibody and secondary antibody + SPIO. The technique supports the feasibility of using MRI measurements of nucleosome concentration in blood as a diagnostic, prognostic and predictive tool in the management of cancer. (paper)

  9. Sensitive solid-phase detection of donor-specific antibodies as an aid highly relevant to improving allograft outcomes.

    Science.gov (United States)

    Schlaf, Gerald; Pollok-Kopp, Beatrix; Altermann, Wolfgang W

    2014-04-01

    Transplant recipients who have had sensitizing events such as pregnancies, blood transfusions and previous transplants often develop antibodies directed against human leukocyte antigen (HLA)-molecules of the donor tissue. These pre-formed donor-specific antibodies (DSA) represent a high risk of organ failure as a consequence of antibody-mediated hyper-acute or acute allograft rejection. As a first assay to detect DSA, the complement-dependent lymphocytotoxicity assay (CDC) was established more than 40 years ago. However, this assay is characterized by several drawbacks such as a low sensitivity and a high susceptibility to various artificial factors generally not leading to valid and reliable outcomes under several circumstances that are reviewed in this article. Furthermore, only those antibodies that exert complement-fixing activity are detected. As a consequence, novel procedures that act independently of the complement system and that do not represent functional assays were generated in the format of solid phase assays (SPAs) (bead- or ELISA-based). In this article, we review the pros and cons of these sensitive SPA in comparison with the detection of DSA through the use of the traditional methods such as CDC and flow cytometric analyses. Potential drawbacks of the alternative methodological approaches comprising high background reactivity, susceptibility to environmental factors and the possible influence of subjective operators' errors concerning the interpretation of the results are summarized and critically discussed for each method. We provide a forecast on the future role of SPAs reliably excluding highly deleterious DSA, thus leading to an improved graft survival. PMID:24170304

  10. A novel monoclonal antibody for detection of galectin-9 in tissue sections: application to human tissues infected by oncogenic viruses

    Directory of Open Access Journals (Sweden)

    Barjon Clément

    2012-07-01

    Full Text Available Abstract Background Galectin-9 is a mammalian lectin which possesses immunosuppressive properties. Excessive production of galectin-9 has been reported in two types of human virus-associated diseases chronic hepatitis C and nasopharyngeal carcinoma associated to the Epstein-Barr virus. The objective of this study was to produce new monoclonal antibodies targeting galectin-9 in order to improve its detection in clinical samples, especially on tissue sections analysed by immunohistochemistry. Methods Hybridomas were produced through immunization of mice with the recombinant c-terminus part of galectin-9 (residues 191 to 355 of the long isoform and semi-solid fusion of spleen cells with Sp2/0 cells. Monoclonal antibodies were characterized using ELISA, epitope mapping, western blot and immunohistochemistry. Results We selected seven hybridomas producing antibodies reacting with our recombinant c-terminus galectin-9 in ELISA. Five of them reacted with the epitope “TPAIPPMMYPHPA” (common to all isoforms, residues 210 to 222 of the long isoform and stained all three isoforms of galectin-9 analysed by western blot. One of them, 1G3,demonstrated very good sensitivity and specificity when used for immunohistochemistry. Using 1G3, we could confirm the intense and constant expression of galectin-9 by Epstein-Barr virus positive malignant cells from nasopharyngeal carcinomas. In most samples, specific staining was detected in both cytoplasm and nuclei. Galectin-9 was also detected in liver biopsies from patients infected by the human hepatitis C or B viruses with expression not only in inflammatory leucocytes and Kupffer cells, but also in hepatocytes. In contrast, galectin-9 was virtually absent in non-infected liver specimens. Conclusion The 1G3 monoclonal antibody will be a powerful tool to assess galectin-9 expression and distribution especially in diseases related to oncogenic viruses.

  11. Detection of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii in horses from Costa Rica.

    Science.gov (United States)

    Dangoudoubiyam, S; Oliveira, J B; Víquez, C; Gómez-García, A; González, O; Romero, J J; Kwok, O C H; Dubey, J P; Howe, D K

    2011-06-01

    Serum samples from 315 horses from Costa Rica, Central America, were examined for the presence of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii by using the surface antigen (SAG) SnSAG2 enzyme-linked immunosorbent assay (ELISA), the NhSAG1 ELISA, and the modified agglutination test, respectively. Anti- S. neurona antibodies were found in 42.2% of the horses by using the SnSAG2 ELISA. Anti- Neospora spp. antibodies were found in only 3.5% of the horses by using the NhSAG1 ELISA, and only 1 of these horses was confirmed seropositive by Western blot. Antibodies to T. gondii were found in 34.0% of the horses tested, which is higher than in previous reports from North and South America. The finding of anti- S. neurona antibodies in horses from geographical areas where Didelphis marsupialis has wide distribution suggests that D. marsupialis is a potential definitive host for this parasite and a source of infection for these horses. PMID:21506839

  12. Detection of immunocomplex formation by enhanced photoluminescence of antibody-functionalized diatom biosilica

    Science.gov (United States)

    Gale, Debra K.; Gutu, Timothy; Jiao, Jun; Chang, Chih-Hung; Rorrer, Gregory L.

    2009-05-01

    Diatoms are single-celled photosynthetic algae that make silica shells or "frustules" with intricate features patterned at the nano and microscales. In this study, antibody-functionalized diatom biosilica frustules serve as a biosensor platform for selective and label free antibody-antigen immunocomplex formation by enhanced photoluminescence. Biosilica frustules of 10 micron diameter were isolated from cells of the centric marine diatom Cyclotella sp. They were then mounted on glass and covalently functionalized with the model antibody Rabbit Immunoglobulin G (IgG) to yield a uniform nanostructured surface that selectively binds to its complimentary antigen, Goat anti-Rabbit IgG. Diatom frustules possess an intrinsic capacity to emit blue light when excited with a UV laser light source, a property called photoluminescence. Binding the antibody-functionalized diatom frustule with its complimentary antigen selectively enhanced the intrinsic photoluminescence intensity of the diatom frustule by a factor of three, whereas challenging the antibody-functionalized diatom frustule with a non-complimentary antigen, Goat anti-human IgG did not change the intrinsic photoluminescence intensity. The nucleophilic immunocomplex increases the photoluminescence by donating electrons to non-radiative sites on the photoluminescent diatom biosilica, thereby decreasing non-radiative electron decay and increasing radiative emission. The intensified photoluminescence intensity is correlated to the antigen, goat anti-rabbit IgG concentration, with a binding constant of 2.8 +/- 0.7x10-7 M.

  13. The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses.

    Science.gov (United States)

    Morley, P S; Hanson, L K; Bogdan, J R; Townsend, H G; Appleton, J A; Haines, D M

    1995-06-01

    Antibodies specific for equine influenza viruses are usually quantified using single radial hemolysis (SRH), hemagglutination inhibition (HI) or virus neutralization (VN). Neutralizing antibodies are thought to provide optimum protection to challenged animals. The purpose of this study was to determine the extent to which SRH and HI assays detect antibodies which neutralize equine influenza viruses. Acute and convalescent sera from 41 horses were analyzed using VN, SRH, and HI assays. These horses were present in a population of Thoroughbred racehorses during an epidemic of upper respiratory tract disease associated with influenza A/equine/Saskatoon/1/91 (H3N8), infections. Concentrations of antibodies binding to influenza A/equine/Kentucky/1/81 (H3N8), A/equine/Miami/1/63 (H3N8), and A/equine/Prague/1/56 (H7N7) were determined. Results of the VN assay were compared with results from the SRH and HI assays for acute antibody levels, changes in antibody concentrations between acute and convalescent sampling, and the occurrence of seroconversion. The correlation between assays for pre-exposure antibody levels ranged from 88% to 96%. The correlation between assays for change in antibody concentration ranged from 83% to 90% for the H3N8 viruses. This study shows that antibody concentrations specific for equine influenza virus, measured using SRH and HI assays, are highly correlated with concentrations detected using a VN assay. PMID:7653031

  14. Use of Monoclonal Antibodies in an ELISA for Detecting an Invasive Pest Insect, Liriomyza trifolii (Diptera: Agromyzidae).

    Science.gov (United States)

    Zhao, W C; Shang, H W; Guo, W; Xu, D; Huang, T Y; Zhu, L X

    2015-04-01

    A monoclonal antibody was prepared by the hybridoma technology. It reacted only with the protein of Liriomyza trifolii (Burgess) and not with that of Chromatomyia horticola Goureau or Liriomyza sativae Blanchard in indirect enzyme-linked immunosorbent assay. It was effective even after being diluted more than 8.192×10(6)-fold. The detection sensitivity of the antibody was 31.3 µg/ml under controlled conditions. Positive reaction was achieved with all laboratory-reared L. trifolii samples, including larvae, pupae, and adults. An indirect enzyme-linked immunosorbent assay system was successfully established to detect L. trifolii in the field. This antibody was successfully used to determine the L. trifolii collected in different locations, from different host plants, and in different seasons. More than 50% of leafminers collected on Brassica chinensis var chinensis, Apium graveolens (Miller) Persoon, Vigna unguiculata (L.) Walpers, Phaseolus vulgaris L., Lactuca sativa L., and Chrysanthemum coronarium (L.) Cassini ex Spach were L. trifolii, indicating that those six plant species might be the preference host plants of L. trifolii. Population of L. trifolii peaked in September, October, or November in Hangzhou, Zhejiang. These results suggest a great potential of using this McAb for precisely identifying L. trifolii and monitoring the population dynamics of L. trifolii in the field. PMID:26470159

  15. Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

    Science.gov (United States)

    Takakura, T.; Yanagi, I.; Goto, Y.; Ishige, Y.; Kohara, Y.

    2016-03-01

    We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

  16. Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp

    Institute of Scientific and Technical Information of China (English)

    Tossapon WONGTANGPRASERT; Wirongrong NATAKUATHUNG; Umaporn PIMPITAK; Anumart BUAKEAW; Tanapat PALAGA; Kittinan KOMOLPIS; Nanthika KHONGCHAREONPORN

    2014-01-01

    A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cellfusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50%inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118%for an intra-assay and 96%-113%for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9%and 5.5%-14.9%, respectively.%本文题目:抗土霉素单克隆抗体的制备及其在虾中土霉素残留的检测应用Production of a monoclonal antibody against oxytetracycline and its application for oxy-tetracycline residue detection in shrimp研究目的:抗土霉素单克隆抗体的制备和特性分析,并用于间接竞争酶联免疫吸附测定法(icELISA)分析虾样品中土霉素的残留。创新要点:本研究建立分泌抗土霉素单克隆抗体的杂交瘤细胞株,筛选出对虾中土霉素残留检测具有较高灵敏度的单克隆抗体2-4F。研究方法:用细胞杂交技术使土霉素-牛血清白蛋白偶联物免疫的BALC/c雌性小鼠的脾细胞与骨髓瘤细胞融合,建立三株分泌抗土霉素单克隆抗体的杂交瘤细胞株(2-4F、7-3G和11-11A),并制备它们的单克隆抗体。通过 icELISA 法分析单克隆抗体对土霉素的半抑制质量浓度(IC50)和交叉反

  17. Optical detection of antibody using silica-silver core-shell particles

    Science.gov (United States)

    Kalele, S. A.; Ashtaputre, S. S.; Hebalkar, N. Y.; Gosavi, S. W.; Deobagkar, D. N.; Deobagkar, D. D.; Kulkarni, S. K.

    2005-03-01

    Nearly monodispersed spherical particles of silica were synthesized and coated with thin layer of silver nanoparticles. Silver coated silica particles, forming core-shell particles exhibited a strong surface plasmon resonance peak at 453 nm. A very small amount (20 μg) of rabbit immunoglobulin in core-shell particle solution results in to a marked shift in surface plasmon resonance. Addition of 20 μg quantity of goat anti rabbit antibodies results in to a red shift of surface plasmon resonance to 494 nm. This demonstrates that silver coated silica particles are sensitive probes for rapid antibody-anti antibody kind of interaction investigations. Fourier transform infra red spectroscopy and scanning electron microscopy have been used to interpret the optical extinction spectroscopy results.

  18. Coombs Antiglobulin Test Using Brucella abortus 99 as Antigen To Detect Incomplete Antibodies Induced by B. abortus RB51 Vaccine in Cattle

    OpenAIRE

    Ciuchini, Franco; Adone, Rosanna; Pasquali, Paolo

    2002-01-01

    This study showed that vaccination of cattle with Brucella abortus rough strain RB51 induces incomplete antibodies that can be detectable by a Coombs antiglobulin test using the B. abortus 99 smooth strain.

  19. Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti.

    Science.gov (United States)

    Schares, G; Basso, W; Majzoub, M; Rostaher, A; Scharr, J C; Langenmayer, M C; Selmair, J; Dubey, J P; Cortes, H C; Conraths, F J; Haupt, T; Pürro, M; Raeber, A; Buholzer, P; Gollnick, N S

    2011-01-10

    Bovine besnoitiosis is an economically important disease in cattle caused by the protozoan parasite Besnoitia besnoiti, which occurs endemically in many countries of Africa and Asia and is spreading in Europe. Serological identification of subclinically infected cattle is important to avoid the introduction of infected animals into naive herds. Here we determine the sensitivity and specificity of the PrioCHECK(®) Besnoitia Ab, a serological test recently introduced into the European market. Analytical specificity was examined using sera from animals experimentally infected with parasites related to B. besnoiti (n=27). Three animals experimentally infected with Neospora caninum or Toxoplasma gondii showed inconclusive reactions in the ELISA (percent positivity relative to the positive control [PP] 10% ≤ 20%) while all other sera reacted negative (PPbovine herds without besnoitiosis but with abortion problems associated to N. caninum (n=403). The analysis revealed a specificity of 94.3% or 96.8% depending on the applied cut-off (PP 10% or 20%, respectively). Sensitivity was assessed with sera from 110 animals of a herd in Germany where clinical bovine besnoitiosis was first diagnosed in September 2008. A positive serological reference standard was defined regarding sera from animals as reference positive, if these animals had tested positive in at least two of a panel of three other serological tests (two different B. besnoiti immunoblots and one immunofluorescence antibody test) on both of two sampling dates, November 2008 and April 2009. A diagnostic sensitivity of 91.8% or 75.5% was determined for sera collected in November 2008 and a sensitivity of 82.7% or 50% for sera collected in April 2009 (cut-off PP 10% or PP 20%, respectively). The marked drop in sensitivity from November 2008 to April 2009 was predominantly observed in reference-positive cattle without clinical signs. We conclude that PrioCHECK(®) Besnoitia Ab is a valuable diagnostic tool to detect

  20. Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies.

    Science.gov (United States)

    Tsoukas, C D; Lambris, J D

    1988-08-01

    The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage. PMID:2970972

  1. [Preparation and detection of anti-influenza A virus polymerase basic protein 1 polyclonal antibody].

    Science.gov (United States)

    Qin, Yujie; Zhang, Tinghong; Ye, Xin

    2016-01-01

    Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1. PMID:27363203

  2. Labelled monoclonal antibodies used for the detection of inflammatory processes and bone marrow metastases

    International Nuclear Information System (INIS)

    This review gives a short history of the developments of immunoscintigraphy using radiolabelled monoclonal antibodies and will look at some clinical indications studied in close cooperation with international groups. Examples are presented of infected orthopaedic prostheses and the diagnosis of osteomyelitis in diabetic foot in which the method is considered diagnostically helpful. Furthermore, the accuracy of the bone marrow immunoscintigraphy is discussed in evaluating results of a multi-centre study, clearly demonstrating its diagnostic superiority over bone scanning in metastatic cancer. It is concluded that the future diagnostic trend is going towards more specific agents (antibodies, peptides) and a speedier availability of the diagnostic results in cases of supposed infection. (author)

  3. A radioimmunoassay method for the rapid detection of Candida antibodies is experimental systematic candidiasis

    International Nuclear Information System (INIS)

    Rabbits were employed as experimental models to evaluate a solid-phase radioimmunoassay (RIA) method for the diagnosis of systematic candidiasis. Ten rabbits were incubated subcutaneously to mimic superficial candidiasis and were found to produce no antibodies to Candida as determined by both immunodiffusion and RIA procedures. However, 94 per cent of 18 rabbits systematically infected by intravenous injection of Candida cells were observed to produce antibody as assessed by the RIA technique. These data encourage further tests with human sera and the continued development of this RIA procedure as a useful tool in the early serodiagnosis of systematic candidiasis. (Auth.)

  4. Early detection of neutralizing antibodies to interferon-beta in multiple sclerosis patients

    DEFF Research Database (Denmark)

    Hegen, H; Millonig, A; Bertolotto, A;

    2014-01-01

    BACKGROUND: Neutralizing antibodies (NAb) affect efficacy of interferon-beta (IFN-b) treatment in multiple sclerosis (MS) patients. NAbs evolve in up to 44% of treated patients, usually between 6-18 months on therapy. OBJECTIVES: To investigate whether early binding antibody (BAb) titers or...... after 3, 12 and 24 months on that therapy; for determination of NAbs, BAbs, gene expression of MxA and protein concentrations of MMP-9, TIMP-1, sTRAIL, CXCL-10 and CCL-2. RESULTS: We found that 22 of 164 (13.4%) patients developed NAbs during a median time of 23.8 months on IFN-b treatment. Of these...

  5. Detection and purification of rat and goat immunoglobulin G antibodies using protein G-based solid phase radioimmunoassays

    International Nuclear Information System (INIS)

    Using the newly described streptococcal surface protein, protein G, which has powerful immunoglobulin G binding properties, solid-phase radioimmunoassays were developed for the quantitation of goat and rat immunoglobulin G bound to the plastic surface of microtiter plates. The binding of goat immunoglobulin G to the surface via a specific antigen (guinea pig alpha1-microglobulin) permitted the determination of antigen-specific antibodies with a detection limit of 50-100 ng. Optimum assay conditions were established and the whole assay procedure could be brought to completion at room temperature in less than a working day. The value of the assays was illustrated by monitoring rat and goat immunoglobulin G antibodies during their purification from whole sera by classical chromatographic procedures. (Auth.)

  6. Use of radiolabeled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning

    International Nuclear Information System (INIS)

    To determine whether tumors containing carcinoembryonic antigen could be detected by administration of a radiolabeled, affinity-purified, goat IgG having 70% immunoreactivity against carcinoembryonic antigen, 18 patients with a history of cancer of diverse histopathology received an average total dose of 1.0 mCi of 131I-labeled IgG. Total-body photoscans were performed with a gamma scintillation camera at various intervals after administration of the radioactive antibody. Ordinary photoscans proved difficult to interpret because of blood-pool background radioactivity, thus necessitating the computer subtraction of radioactive blood-pool agents from the antibody's 131I activity. Tumor location could be demonstrated at 48 hours after injection in almost all cases studied. The scans were negative in patients without demonstrable tumors or with tumors apparently devoid of carcinoembryonic antigen. Circulating antigen levels of up to 350 ng per milliliter did not prevent successful tumor imaging after injection of the radioantibody

  7. Comparison of serological assays for detecting antibodies in ducks exposed to H5 subtype avian influenza virus

    Directory of Open Access Journals (Sweden)

    Wibawa Hendra

    2012-07-01

    Full Text Available Abstract Background Chicken red blood cells (RBCs are commonly used in hemagglutination inhibition (HI tests to measure hemagglutinating antibodies against influenza viruses. The use of horse RBCs in the HI test can reportedly increase its sensitivity when testing human sera for avian influenza antibodies. This study aims to compare the proportion of positives detected and the agreement between two HI tests using either chicken or horse red blood cells for antibody detection in sera of ducks experimentally infected or naturally exposed to Indonesian H5 subtype avian influenza virus. In addition, comparison with a virus neutralisation (VN test was conducted with the experimental sera. Results In the experimental study, the proportion of HI antibody-positive ducks increased slightly, from 0.57 when using chicken RBCs to 0.60 when using horse RBCs. The HI tests indicated almost perfect agreement (kappa = 0.86 when results were dichotomised (titre ≥ 4 log2, and substantial agreement (weighted kappa = 0.80 for log titres. Overall agreements between the two HI tests were greater than between either of the HI tests and the VN test. The use of horse RBCs also identified a higher proportion of antibody positives in field duck sera (0.08, compared to chicken RBCs 0.02, with also almost perfect agreements for dichotomized results (Prevalence and bias adjusted Kappa (PABAK = 0.88 and for log titres (weighted PABAK = 0.93, respectively. Factors that might explain observed differences in the proportion of antibody-positive ducks and in the agreements between HI tests are discussed. Conclusion In conclusion, we identified a good agreement between HI tests. However, when horse RBCs were used, a higher proportion of sera was positive (titre ≥ 4 log2 than using chicken RBCs, especially during the early response against H5N1 virus. The HRBC-HI might be more responsive in identifying early H5N1 HPAI serological response and could be a

  8. Preparation of Monoclonal Antibody Against HPT and Its Application to Detecting Marker Protein in Genetically Modified Rice

    Institute of Scientific and Technical Information of China (English)

    LI-CHEN YANG; SU-XIANG ZHANG; GUO-HUA PI; YING-HUA LI; ZHEN ZHU; XIAO-GUANG YANG

    2005-01-01

    Objective To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1, D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 1×10-4 to 1×10-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay. Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.

  9. Detection of IgM antibrucella antibody in the absence of IgGs: a challenge for the clinical interpretation of brucella serology.

    OpenAIRE

    Julián Solís García Del Pozo; Santiago Lorente Ortuño; Elena Navarro; Javier Solera

    2014-01-01

    The use of enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies antibrucella has become widespread in the diagnosis of human brucellosis. IgM anti-Brucella antibodies are indicative of acute infection. Between 2009-2013, 5307 patients were evaluated for serologic diagnosis at the Microbiology Laboratory of the Albacete General Hospital. A ELISA IgM-positive, IgG-negative anti-Brucella antibody serology pattern was detected in 17 of those patients. Epidemiology...

  10. Elaboration of optical immunosensors based on the surface plasmon resonance for detecting specific antibodies and antigens of Epstein-Barr virus and human adenovirus.

    Science.gov (United States)

    Nesterova, N V; Nosach, L M; Zagorodnya, S D; Povnitsa, O Y; Boltovets, P M; Baranova, G V; Golovan, A V

    2008-01-01

    The study of antigen-antibody interaction on the model of Epstein-Barr virus (EBV) and second type adenovirus (Ad2) based on the surface plasmon resonance (SPR) was carried out. Kinetic and concentration dependences between virus antigens and specific antisera to them at different pH were determined. Experimental samples of biosensors for the detection by SPR method of virus (EBV and Ad2) antigens using monospecific antibodies, immobilized on the surface of gold, and also for detection of specific antibodies in the blood sera of patients with EBV or adenovirus infection were elaborated PMID:19351051

  11. Enhanced Signal and Quantitative Detection of Anti-Interferon-Gamma Antibody by Using a Nanometer Biolinker

    Science.gov (United States)

    Tsai, Pei-I; Lee, Adam Shih-Yuan; Lee, Shu-Sheng; Chung, Ming-Han; Liu, Meng-Wei; Lee, Chih-Kung

    2016-01-01

    For rapid screening and quantification of an antisera antibody, a nanometer bithiophene-based conductive biolinker can enhanced signal performance and can be used to verify the interaction of an anti-IFN-γ antibody with an IFN-γ protein. The experimental measurements take a generic approach which takes advantage of the functionality of thiophene-based linkers for biosensors. Effects associated with using bithiophene as a biolinker for surface plasmon resonance (SPR) spectroscopy are examined in this paper. By using an atomic force microscope (AFM), it was observed that the morphology of the bithiophene modified gold sensor surface became smoother than the original gold surface. We compared the response and concentration of the anti-IFN-γ antibody on a bithiophene-coated and dextran-coated biochip as well as on different thickness-modified surfaces under SPR relevant conditions. The results indicate that a response to IFN-γ molecules immobilized on a sensor using a bithiophene biolinker improved more than 8-fold when compared to that of a sensor using a dextran biolinker. Furthermore, the regeneration ability of the sensor surface shows good repeatability as only less than a 1% decrease was found after repeating the experimental work over 6 cycles. The characteristics provided us with a good platform for rapid screening, real-time monitoring and quantitative concentration of the autoimmune antibody activities. PMID:27459633

  12. Detection of Mycoplasma pneumoniae in cerebrospinal fluid of children: Quantification of CSF-IgG antibody

    Directory of Open Access Journals (Sweden)

    Noorbakhsh S

    2010-08-01

    Full Text Available "nBackground: M. pneumoniae infection in children is usual and diagnosis of its neurologic complications for rapid treatment is very important. To compare the CSF- M. pneumoniae antibody level between febrile children with acute neurologic signs (Menigoencephalitis, Guillan Barre Syndrome (GBS, Transverse myelitis, Ataxia and so on with unaffected ones. "n"nMethods: A cross sectional/ case control study in pediatric wards of Rasoul-e-Akram & Mofid hospitals (2007-2009 was done. The amount of Specific M. pneumoniae IgG (ELISA antibody level determined in CSF of 55 cases and in 10 controls. Chi square values (CI 95%, p< 0.05 calculated for all categorical variables. Sensitivity; specificity; Positive Predictive Value (PPV; Negative Predictive Value (NPV of CSF antibody level determined by using the Area under the ROC Curve. "n"nResults: Cases (n= 55 aged between five month to 13 years with mean age of 3.84±3.43 years. Area Under Curve (AUC in ROC was 0.876 (%95 CI, 0.78- 0.96; p< 0.0001. Cut off level for antibody was 0.0025 with 73% sensitivity; 90% specificity; 100% PPV; 28.8% NPV. CSF antibody level had significant difference between cases and controls [0.08± 0.26 Versus 0.001± 0.001; p: 0.02]; It had poor agreement between cases and controls (Kappa= 0.27. Lowest amount seen in cases with aseptic meningitis; highest amount observed in cases with GBS and cases with focal neurologic signs. "n"nConclusion: The presence of very low amount (0.0025 of M. pneumoniae antibody in CSF of febrile children with acute neurologic signs had 70% sensitivity and 90% specificity; 100% PPV; but had low (28.8% NPV. M. pneumoniae would be a rare cause in cases with aseptic meningitis. Finding the M. pneumoniae-DNAs in CSF are not so frequent (2% but in high suspicious cases adding this test to determining the CSF antibody level might be helpful.

  13. Antinuclear antibody detection by automated multiplex immunoassay in untreated patients at the time of diagnosis.

    Science.gov (United States)

    Op De Beéck, Katrijn; Vermeersch, Pieter; Verschueren, Patrick; Westhovens, René; Mariën, Godelieve; Blockmans, Daniel; Bossuyt, Xavier

    2012-12-01

    Fully automated multiplex immunoassays are increasingly used as first line screening for antinuclear antibodies. The diagnostic performance of such multiplex assays in untreated patients at the time of diagnosis has not been reported. Antinuclear antibodies were measured by indirect immunofluorescence (IIF) (dilution 1:160) and by BioPlex 2200 ANA screen (antibodies to dsDNA, chromatin, ribosomal protein, SSA-52, SSA-60, SSB, Sm, SmRNP, RNP-A, RNP-68, Scl-70, Jo-1, and centromere B) in 236 patients with a systemic rheumatic disease at the time of diagnosis, 149 blood donors, 139 patients with chronic fatigue syndrome (CFS), and 134 diseased controls. BioPlex ANA screen and IIF were positive in, respectively, 79% and 90% of patients with systemic lupus erythematosus (SLE), 60% and 60% with cutaneous lupus, 72% and 93% with systemic sclerosis (SSc), 100% and 100% with mixed connective tissue disease (MCTD), 89% and 56% with primary Sjögren's (SS) syndrome, 36% and 36% with polymyositis/dermatomyositis, 5.4% and 6% of blood donors, 7.2% and 3.6% of patients with CFS, and 11% and 18% of diseased controls. BioPlex test result interval specific likelihood ratios increased with increasing antibody concentration. The simultaneous presence of at least three antibodies by BioPlex was found in 35% of patients with SLE, 4% with SSc, 100% with MCTD, 64% with SS, 7% with inflammatory myopathy, 0.7% of CFS and diseased controls, and none of the blood donors. In conclusion, test result specific likelihood ratios and the presence of multiple autoantibodies help with the interpretation of data generated by multiplex immunoassays. PMID:22387973

  14. Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay.

    Science.gov (United States)

    Petersen, J S; Hejnaes, K R; Moody, A; Karlsen, A E; Marshall, M O; Høier-Madsen, M; Boel, E; Michelsen, B K; Dyrberg, T

    1994-03-01

    Autoantibodies to glutamic acid decarboxylase (GAD) are frequent at or before the onset of insulin-dependent diabetes mellitus (IDDM). We have developed a simple, reproducible, and quantitative immunoprecipitation radioligand assay using as antigen in vitro transcribed and translated [35S]methionine-labeled human islet GAD65. By using this assay, 77% (77 of 100) of serum samples from recent-onset IDDM patients were positive for GAD65 antibodies compared with 4% (4 of 100) of serum samples from healthy control subjects. In competition analysis with unlabeled purified recombinant human islet GAD65, binding to tracer was inhibited in 74% (74 of 100) of the GAD65-positive IDDM serum samples compared with 2% of the control samples. The levels of GAD antibodies expressed as an index value relative to a standard serum, analyzed with or without competition, were almost identical (r = 0.991). The intra- and interassay variations of a positive control serum sample were 2.9 and 7.6%, respectively (n = 4). The frequency of GAD antibodies was significantly higher with IDDM onset before the age of 30 (80%, 59 of 74) than after the age of 30 (48%, 10 of 21) (P DNA autoantibodies (8% [2 of 25] and 4% [1 of 25] in competition analysis) or rheuma factor autoantibodies [12% (4 of 35) and 3% (1 of 35) in competition analysis] was not different from that in control samples. In contrast, in sera positive for ribonucleoprotein antibodies the frequency of GAD antibodies was significantly increased (73% [51 of 70] and 10% [7 of 70] in competition analysis [P history of IDDM for the presence of this marker. PMID:8314020

  15. A Halotyrosine Antibody that Detects Increased Protein Modifications in Asthma Patients

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Hallstrand, Teal S.; Daly, Don S.; Matzke, Melissa M.; Nair, Parameswaran; Bigelow, Diana J.; Pounds, Joel G.; Zangar, Richard C.

    2014-01-31

    Background-Airway inflammation plays an important pathophysiological role in asthma. Eosinophils produce hypobromite and bromotyrosine while neutrophils produce hypochlorite and chlorotyrosine. Objective-To evaluate halotyrosine modifications of individual airway proteins as a marker of inflammation in asthma using an antibody-based assay. Methods-We developed a novel monoclonal antibody (BTK-94C) that binds halogenated tyrosine residues, and used this antibody in a custom enzyme-linked immunosorbent assay (ELISA) microarray platform to examine halotyrosine levels in 23 proteins in three independent sets of sputum samples (52 samples total). Results-In 15 subjects with either no asthma, or with asthma characterized by high or low sputum eosinophil counts, we found associations between increased halotyrosine levels of at least three proteins and severity of airway hyperresponsiveness (AHR). Treatment with mepolizumab in 17 patients with sputum eosinophilia markedly reduced the sputum eosinophilia and significantly reduced halotyrosine levels in one sputum protein. Further analysis of 10 subjects with neutrophilic asthma and 10 health controls demonstrated a broad increase in halotyrosine in the patients with airway neutrophilia. Conclusions-Significantly higher levels of halotyrosine are associated with asthma in the asthma phenotypes we examined. The halotyrosine levels correlated with indirect AHR in the form of exercise-induced bronchoconstriction. Clinical Implication-An antibody-based assay for tyrosine halogenation in specific proteins may prove useful for assessing airway inflammation in asthma. Capsule Summary-An antibody to measure protein monobrominated tyrosine and other halotyrosine modifications was developed and used to evaluate halogenation in specific proteins in the airways for the first time. Associations were found between levels of halotyrosine and exercise-induced bronchoconstriction, and eosinophil and neutrophil inflammation in sputum from

  16. A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals

    DEFF Research Database (Denmark)

    Ajua, Anthony; Engleitner, Thomas; Esen, Meral; Theisen, Michael; Issifou, Saadou; Mordmüller, Benjamin

    2012-01-01

    ABSTRACT: BACKGROUND: Antibodies play a central role in naturally acquired immunity against Plasmodium falciparum. Current assays to detect anti-plasmodial antibodies against native antigens within their cellular context are prone to bias and cannot be automated, although they provide important...... information about natural exposure and vaccine immunogenicity. A novel, cytometry-based workflow for the quantitative detection of anti-plasmodial antibodies in human serum is presented. METHODS: Fixed red blood cells (RBCs), infected with late stages of P. falciparum were utilized to detect malaria......-specific antibodies by flow cytometry with subsequent automated data analysis. Available methods for data-driven analysis of cytometry data were assessed and a new overlap subtraction algorithm (OSA) based on open source software was developed. The complete workflow was evaluated using sera from two GMZ2 malaria...

  17. Combined Serological Detection of Circulating Angiostrongylus vasorum Antigen and Parasite-specific Antibodies in Dogs from Hungary.

    Science.gov (United States)

    Schnyder, Manuela; Schaper, Roland; Lukács, Zoltán; Hornok, Sándor; Farkas, Róbert

    2015-08-01

    The occurrence of the nematode Angiostrongylus vasorum, also known as the French heartworm, is increasingly being reported from various European countries. The adults of this parasite species live in the pulmonary arteries and right cardiac ventricle of wild canids and domestic dogs. Larval stages and eggs in the lungs induce inflammatory verminous pneumonia, causing severe respiratory disease in dogs. Furthermore, haematological and neurological signs and even death may occur. In Hungary, A. vasorum has been identified in red foxes, golden jackals and in two dogs and some slugs. In this first large-scale survey, 1247 sera from pet dogs were collected and tested by an ELISA for the detection of circulating antigen of A. vasorum and by a separate ELISA to detect specific antibodies against the parasite. A total of 1.36% (n = 17, 95 % confidence intervals, CI: 0.80 - 2.17 %) of the animals were positive in both ELISAs, while 1.76 % (n = 22, CI: 1.11 - 2.66 %) of the tested dogs were antigen-positive only and 2.73 % (n = 34, CI: 1.90 - 3.79 %) were positive for specific antibodies only. Regions with antigen- and antibody-positive animals overlapped and were distributed over nearly the whole sampled areas of the country. A considerable number of cases was observed in Budapest and also in the southern part of the country bordering Croatia, while in the most eastern part bordering Ukraine no positive samples were detected. These results confirm the endemic occurrence of A. vasorum in dogs originating from different parts of Hungary and the significant advantages of A. vasorum serology in epidemiological studies. PMID:26152415

  18. Diagnostic potential of monoclonal antibodies against the capsid protein of chikungunya virus for detection of recent infection.

    Science.gov (United States)

    Damle, R G; Jayaram, N; Kulkarni, S M; Nigade, K; Khutwad, K; Gosavi, S; Parashar, D

    2016-06-01

    Chikungunya fever is self-limiting. However, neurological and hemorrhagic complications have been seen in recent outbreaks. The clinical manifestations of this disease are similar to those of dengue virus infection, indicating the need for differential diagnosis in areas such as India, which are endemic for both viruses. The aim of the present study was to develop monoclonal antibodies (MAbs) against Chikungunya virus (CHIKV) and assess their use in MAb-based IgM capture ELISA (MAC ELISA). The ELISA detects CHIKV-specific IgM antibodies, a marker of recent infection, in a patient's serum. One IgG1 and two IgM isotype hybrids were obtained. All of the subclones derived from the IgG1 hybrid recognized the C protein of CHIKV. The anti-C MAb ClVE4/D9 was the most promising as a detector antibody in MAC ELISA (C-MAb ELISA) yielding higher positive-to-negative (P/N) ratios. When compared with the CHIKV MAC ELISA kit developed by the National Institute of Virology (NIV), Pune (NIV MAC ELISA), the sensitivity of the test was 87.01 % with 100 % specificity. The positive and negative predictive values (PPV and NPV) were 100 % and 94.47 %, respectively. In precision testing, standard deviation (SD) and coefficient of variation (% CV) values of the C-MAb ELISA were within acceptable limits. The C-MAb ELISA detected anti-CHIKV IgM in serum of patients up to five months after the onset of infection, indicating that anti-C MAbs have strong potential for use in MAC ELISA to detect recent CHIKV infection. PMID:27016930

  19. Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits

    Science.gov (United States)

    Mattera, Lucia; Bhuckory, Shashi; Wegner, K. David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J.; Hildebrandt, Niko; Reiss, Peter

    2016-05-01

    A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter 26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL-1 obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL-1) and demonstrate their direct applicability in clinical diagnostics.A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter 26 nm without biomolecules). The LODs

  20. Detection of anti-U3-RNP/fibrillarin IgG antibodies by line immunoblot assay has comparable clinical significance to immunoprecipitation testing in systemic sclerosis.

    Science.gov (United States)

    Peterson, Lisa K; Jaskowski, Troy D; Mayes, Maureen D; Tebo, Anne E

    2016-04-01

    The aim of this study was to evaluate the performance and clinical relevance of a commercially available line immunoblot assay (LIA) for detecting anti-U3-RNP/fibrillarin (anti-U3-RNP), against immunoprecipitation (gold standard). This study involved a multi-ethnic cohort of 1000 American systemic sclerosis (SSc) patients and 50 healthy controls. Antinuclear antibodies and centromere antibodies were detected by indirect immunofluorescent antibody test, anti-topo I by immunodiffusion and anti-RNAP III by ELISA. The presence of anti-U3-RNP in select serum samples was detected by immunoprecipitation (IP) and LIA. By IP, U3-RNP antibody was detected in 75 (7.5 %) patients with SSc. Overall agreement between LIA and IP was very good (κ = 0.966). Analytic sensitivity and specificity of the U3-RNP LIA was 100 and 94.7 %, respectively. Clinical features associated with positivity for the anti-U3-RNP antibody include diffuse cutaneous SSc and increased prevalence of renal crisis, consistent with previous studies that used IP. Testing for U3-RNP antibodies is only performed by a small number of laboratories due to the complexity of both performance and interpretation of the IP. LIA is faster and less complex than IP. Excellent agreement between IP and LIA demonstrates that LIA is an acceptable and attractive alternative to IP for anti-U3-RNP detection. PMID:26467972

  1. Comparative sensitivity of 125I-protein A and enzyme-conjugated antibodies for detection of immunoblotted proteins

    International Nuclear Information System (INIS)

    Immunoblotting is a powerful technique for the detection of small amounts of immunologically interesting proteins in unpurified preparations. Iodinated protein A (PA) has been widely used as a second antibody for detection of proteins; however, it does not bind equally well to immunoglobulins from different species nor does it bind to all subclasses of immunoglobulin G (IgG). We compared the sensitivity of [125I]PA with those of both horseradish peroxidase-conjugated second antibodies (HRP) and glucose oxidase-anti-glucose oxidase (GAG) soluble complexes for visualizing bovine serum albumin, human IgG, or human C3 which was either dot blotted or electroblotted to nitrocellulose. [125I]PA was uniformly 10- to 100-fold less sensitive than either HRP or GAG. GAG was more sensitive than HRP except for C3 (electroblotting) and bovine serum albumin and IgG (dot blotting), in which they were equivalent. In general, dot blotting was 10- to 1000-fold more sensitive than electroblotting. Although relative sensitivities varied depending on the proteins analyzed and the antisera used, GAG appeared to be superior to [125I]PA and HRP for detection of immunoblotted proteins

  2. Evaluation of an enzyme linked immunosorbent assay kit for the detection of Babesia bovis antibodies in cattle in Argentina

    International Nuclear Information System (INIS)

    An enzyme linked immunosorbent assay (ELISA) for the detection of antibodies to Babesia bovis was evaluated by using sera of 874 cattle carrying B. bovis antibodies, 700 sera of uninfected cattle, and 357 sera from calves from 16 herds subjected to different B. bovis inoculation rates. The seropositive/ seronegative cut-off point set as double the mean percent positivity of negative cattle sera (= 16%). The sensitivity of the ELISA (four trials) ranged from 97.1% to 100% and the specificity (three trials) varied from 92.0% to 97.0%. The agreement between ELISA and immunofluorescent antibody test was ≥ 90.0% in 18 of 23 evaluations and it ranged from 86.0% to 88.0% in the remainder. The correlation coefficient between percentage of sera positive to ELISA and IFA test in 16 herds was 0.9958 (P<0.001). The ELISA has the advantages of a high sensitivity, objectivity and capacity to test large number of samples in short period of time and could replace the IFA test specially for epidemiological studies. (author)

  3. Efficacy of serum samples stored on filter paper for the detection of antibody to Leptospira spp. by microagglutination test (MAT).

    Science.gov (United States)

    Blanco, R M; Romero, E C

    2012-12-14

    The aim of this study was to investigate the microagglutination test (MAT) results in serum samples dried on filter paper and stored at different temperatures during 1day, 7days, 30days and 1year to determine the stability of sera antibody against leptospires. Serum samples collected onto filter paper for the detection of leptospires antibody was compared with MAT in a study of 300 serum samples from patients with suspected leptospirosis. Among 300 fresh serum samples analyzed by MAT 156 (52%) were positive and 144 (48%) negative. All the negative fresh serum samples were negative when dried on filter paper (specificity 100%). The sensitivity of MAT performed on dried serum samples was 100%. Storage on filter paper at room temperature and at 4°C for 1 and 7days did not affect the MAT titers. For up to 7days, 98.72% of dried serum samples had titers identical to those of the corresponding serum samples, and 1.18% of dried serum samples showed 1 dilution of difference. After a storage period of one month a prozone phenomenon was observed. After a storage period of one year all serum samples were negative. Serum samples collected onto filter paper are a convenient source of antibodies for serological diagnosis and epidemiological surveys. PMID:22960422

  4. The Utilization of a New Immunochromatographic Test in Detection of Helicobacter pylori Antibody from Maternal and Umbilical Cord Serum

    Directory of Open Access Journals (Sweden)

    Fu-Chen Kuo

    2014-01-01

    Full Text Available Background. Helicobacter pylori (H. pylori was linked with several extragastrointestinal diseases, including preeclampsia and intrauterine growth restriction of fetus. One of the signals which can be transferred from mother to fetus is the H. pylori IgG antibody. Aims. We utilized a commercial immunochromatographic kit to detect the antibody in maternal and cord serum. Methods. Three hundred and forty-six females were enrolled and the blood samples were collected on antenatal examination and on delivery. The maternal H. pylori infection was determined by stool H. pylori antigen test. Results. One hundred and five females (30.3% were H. pylori-infected, and the prevalence was higher in immigrants (43.5% than in Taiwanese (28.7%, P=0.058. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the kit were 77.1%, 88.0%, 73.6%, 89.8%, and 84.7%, respectively. This kit also had similar performance in cord serum. Comparing to the maternal result on delivery, this kit offered a consistent performance in antenatal maternal serum (kappa coefficient 0.92 and in cord serum (kappa coefficient 0.88. Conclusions. H. pylori IgG antibody can be transferred through the placenta into the fetal circulation. However, accuracy of the test kit needs to be evaluated before utilization in screening.

  5. Development of an Indirect Competitive ELISA Based on Polyclonal Antibody for the Detection of Diethylstilbestrol in Water Samples

    Institute of Scientific and Technical Information of China (English)

    WANG,Wen-Jun; LING,Yun; XU,Ting; GAO,Hong-Bin; SHENG,Wei; LI,Ji

    2007-01-01

    An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on polyclonal antibody for the estrogen diethylstilbestrol (DES) was developed. With this aim, two different haptens mono-O-3-carboxypropyldiethylstilbestrol (DES-CP) and mono-O-carboxymethyldiethylstilbestrol (DES-CM) with carboxylic group that preserve the molecular structure character of diethylstilbestrol were synthesized. The haptens were conjugated with the carrier proteins bovine serum albumin (BSA) by mixed-anhydride method for immunogen and conjugated with ovalbumin (OVA) by active ester method for coating antigen. Polyclonal antibodies for diethylstilbestrol were raised by immunizing mice with immune antigen DES-CP-BSA. Under optimized system, the lowest limit of detection (LLD) of diethylstilbestrol was 0.01 ng/mL, and IC50= 1.02 ng/mL. Its analogs were tested and no obvious cross-reactivity was found to anti-diethylstilbestrol antibody. DES-fortified water samples were determined by simple dilution to diminish the matrix effect. The comparison between the amount of DES estimated by ELISA and the amount added indicates good agreement for all water samples tested, with mean recovery values ranging from 86% to 120.2%.

  6. Comparison of commercially available enzyme immunoassay with traditional serological tests for detection of antibodies to Coccidioides immitis.

    OpenAIRE

    Martins, T B; Jaskowski, T D; Mouritsen, C L; Hill, H R

    1995-01-01

    A newly released commercially available enzyme-linked immunosorbent assay (ELISA) was evaluated for its ability to detect immunoglobulin M (IgM) and IgG antibodies against the tube precipitin and complement fixation (CF) antigens of Coccidioides immitis. The ELISA was compared with more traditional diagnostic assays, CF, latex agglutination (LA), and immunodiffusion (ID). When the IgM-specific portion of the ELISA was compared with LA, there was an agreement of 81.8%, a specificity of 75.0%, ...

  7. Serological detection of H-Y antigen in humans with a cellular radioimmunobinding assay and monoclonal antibody

    International Nuclear Information System (INIS)

    A sensitive and reliable serological assay for the detection of H-Y antigen is described which uses monoclonal H-Y antibody and a cellular radioimmunobinding assay on cultured human fibroblasts. Cell lines from 2 normal human females, 2 normal human males and one XX human male were tested in 3 separate assays. Cells from XY and XX males were found to contain H-Y antigen; however, the reaction against the XX male cells was found to be intermediate between the XY male and normal XX female cells. (Auth.)

  8. Direct fluorescent-antibody confirmation of chlamydial antigen below the detection threshold of the chlamydiazyme enzyme-linked immunosorbent assay.

    OpenAIRE

    Kellogg, J A; Seiple, J W; Stroll, E S

    1993-01-01

    Of 4,000 endocervical specimens tested with the Chlamydiazyme enzyme-linked immunosorbent assay (Abbott Laboratories), 233 (5.8%) gave positive results (A492 above the cutoff), which were confirmed with a blocking reagent (Abbott). An additional 34 specimens (14.6%) with chlamydial antigen were detected and confirmed with the direct fluorescent-antibody test (Syva) from among those 66 Chlamydiazyme-negative specimens which had A492s that ranged from 0.030 to the cutoff and that could be block...

  9. Cloning and expression of a truncated pigeon circovirus capsid protein suitable for antibody detection in infected pigeons.

    OpenAIRE

    Daum, Iris; Tim, Finsterbusch; Stefan, Härtle; Thomas, Göbel; Mankertz, Anette; Korbel, Rüdiger; Grund, Christian

    2009-01-01

    Abstract Infections with pigeon circovirus (PiCV, also termed CoCV) occur in meat and racing pigeons (Columba livia) of all ages and have been reported worldwide. A PiCV infection is associated with immunosuppression and the development of young pigeon disease syndrom (YPDS). An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of virus-specific serum antibody was developed for research purposes. In the absence of a method to propagate PiCV in cell culture, the a...

  10. Novel Point-of-Care Test for Simultaneous Detection of Nontreponemal and Treponemal Antibodies in Patients with Syphilis ▿

    OpenAIRE

    Castro, Arnold R.; Esfandiari, Javan; Kumar, Shailendra; Ashton, Matthew (British painter, active from 1728); Kikkert, Susan E.; Park, Mahin M.; Ballard, Ronald C.

    2010-01-01

    We describe a point-of-care immunochromatographic test for the simultaneous detection of both nontreponemal and treponemal antibodies in the sera of patients with syphilis that acts as both a screening and a confirmatory test. A total of 1,601 banked serum samples were examined by the dual test, and the results were compared to those obtained using a quantitative rapid plasma reagin (RPR) test and the Treponema pallidum passive particle agglutination (TP-PA) assay. Compared to the RPR test, t...

  11. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

    OpenAIRE

    Su, Mingjun; Li, Chunqiu; Guo, Donghua; Wei, Shan; Wang, Xinyu; GENG, Yufei; Yao, Shuang; Gao, Jing; WANG, Enyu; ZHAO, Xiwen; Wang, Zhihui; Wang, Jianfa; Wu, Rui; Feng, Li; SUN, Dongbo

    2015-01-01

    Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-re...

  12. [Detection of the level of antibodies against bovine leucosis virus in the cow milk by immune sensor].

    Science.gov (United States)

    Pyrohova, L V; Starodub, M F; Nahaeva, L I

    2005-01-01

    An immune sensor based on the surface plasmon resonance (SPR) was developed for express diagnostics of bovine leucosis. Sensor used for detection of the level of antibodies against bovine leukaemia virus (BLV) in the milk serum. It was shown that immune sensor analysis is more sensitive, rapid and simple in comparison with the traditional AGID test. It was stated that the developed immune sensor may be used for performance of screening of bovine leucosis at the farms and the minimal dilution of the milk serum should be 1:20. PMID:16335252

  13. Detection of specific IgE antibodies to major and minor antigenic determinants in sera of penicillin allergic patients

    Institute of Scientific and Technical Information of China (English)

    赵永星; 乔海灵

    2003-01-01

    Objective To investigate the mechanism (s) of penicillins allergic reaction.Methods The radioallergosorbent test (RAST) was used to detect 9 specific IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO), phenoxomethylpenicilloyl (PVO) and flucloxacilloyl (FLUO), and minor antigenic determinants: benzylpenicillanyl (BPA), amoxicillanyl (AXA), 6-aminopenicillanic (APA) and phenoxomethylpenicillany (PVA), in the sera of 32 penicillin allergic patients. The relationship between specific IgE antibodies and penicillins chemical structures was studied by radioallergosorbent inhibition test.Results Nineteen of 32 patients (59.4%) were RAST positive, among whom, five cases were positive only to one or two antigenic minor determinants, and three cases were positive only to one or three major antigenic determinants. The remaining 11 patients were positive not only to major antigenic determinants but also minor antigenic determinants. In 9 specific IgE antibodies, the positive rate of PVA-IgE was the highest (34.38%), followed by BPO-IgE (31.25%). The positive rate of FLUO-IgE was the lowest (15.63%). Of the total patient group, 53.13% were positive to one or more minor antigenic determinants, while 37.5% (12/32) were positive to one or more major antigenic determinants. The percentage of patients with urticarial reactions who were positive to minor antigenic determinants (63.16%) was significantly higher than observed in the anaphylactic shock group (38.5%, P<0.05).Conclusions The minor antigenic determinant was important in allergic reaction. The combining sites of the specific IgE antibodies were likely to be the side-chain of drug or the overwhelming drug molecule.

  14. Heterophile antibody interference in a solid phase sandwich immunoassay for detection of equine growth hormone in plasma.

    Science.gov (United States)

    Borromeo, V; Berrini, A; Gaggioli, D; Secchi, C

    2007-01-15

    Heterophile antibodies (HAs) present in serum recognize animal immunoglobulins and are one of the most unpredictable causes of false results in immunoassays. However, no study has yet reported their interference on the diagnostic reliability of immunochemical analyses on horse plasma. Recently, we developed a sandwich ELISA for detection of equine growth hormone (eGH) in plasma. In a pilot study to measure basal eGH levels (blood samples were drawn from 13 horses every 10 min for 1h), we noted one horse with abnormally high eGH (>100 ng/mL). We demonstrate here that this plasma eGH level was falsely elevated due to interference from HAs. The interfering antibodies were polyspecific immunoglobulins, with fairly broad species-specificity, which affected the eGH immunoassay by bridging the mouse IgG capture antibody and the rabbit IgG conjugate. This produced artificial sandwiches which led to overestimation of the eGH plasma concentration. Spiking horse plasma with pure mouse and rabbit immunoglobulins or whole plasma of several species significantly reduced but did not totally eliminate the HAs interference. Immunoglobulins and whole plasma differed in their ability to block the interference, suggesting that HAs may recognize other proteins beside immunoglobulins in animal sera. To investigate whether HAs have any implications in equine clinical practice, we decided to seek information on the incidence of HAs interference in normal animals. We collected single plasma samples from another 114 horses and we found that 5 of these had plasma HAs. Therefore, in total 6 out of the 127 horses examined (4.7%) had plasma HAs generating falsely elevated eGH measures. In conclusion, this study provides the first evidence of HAs in horse plasma interfering with an immunoassay and indicates that veterinary surgeons and diagnostic laboratory staff should be aware of this potential for interference in tests on horse plasma using monoclonal or polyclonal antibody reagents. PMID

  15. A diagnostic algorithm for detection of antibodies to influenza A viruses in dogs in Italy (2006-2008).

    Science.gov (United States)

    De Benedictis, Paola; Anderson, Tara C; Perez, Andrés; Viale, Elisabetta; Veggiato, Crispina; Tiozzo Caenazzo, Silvia; Crawford, P Cynda; Capua, Ilaria

    2010-11-01

    Since 2004, there have been several reports of Influenza A virus (FLUAV) infection in dogs. Dogs have been infected with equine influenza H3N8, avian influenza H3N2 and H5N1, and the pandemic H1N1 virus. Because of recent avian and equine influenza outbreaks in Italy, the objectives of the present study were to estimate the level of exposure of Italian dogs to influenza A viruses and to assess a diagnostic algorithm for detection of FLUAV exposure in dogs. Sera collected from 6,858 dogs from 2006 to 2008 were screened in a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the highly conserved influenza A nucleoprotein. Samples positive in the cELISA were confirmed by testing in hemagglutination inhibition (HI) and fluorescent antibody test (FAT). Two seropositive dogs had antibodies to H3 hemagglutinin proteins, consistent with exposure to recent canine and equine subtype H3N8 viruses. Using a Bayesian model, the sensitivity and specificity of the cELISA were estimated as 93.98% (probability intervals [PI]: 81.67-99.08%) and 98.71% (PI: 98.43-98.96%), respectively. After accounting for the imperfect sensitivity and specificity of the cELISA, the Bayesian posterior prevalence of FLUAV exposure among tested Italian dogs was 0.5% (PI: 0.1-1.4%). The study results indicate that screening with a cELISA for influenza A nucleoprotein antibody, followed by confirmatory testing with HI and/or FAT, is a highly sensitive and highly specific approach for diagnosing FLUAV exposure in dogs. PMID:21088175

  16. Evaluation of a solid-phase immunofluorescence assay for detection of antibodies to Legionella pneumophila.

    OpenAIRE

    Thompson, T. A.; Wilkinson, H W

    1982-01-01

    A semiautomated solid-phase immunofluorescence technique (FIAX) was compared with the standard indirect immunofluorescence assay (IFA) for the determination of antibody levels to Legionella pneumophila serogroup 1 in paired human serum samples. The FIAX method was in agreement with the IFA test for 91.8% of the serum pairs but gave evidence of a recent Legionella infection significantly fewer times than did the IFA. These results suggest that the FIAX technique may eventually be a useful alte...

  17. Detection of specific antibodies to an antigenic mannoprotein for diagnosis of Penicillium marneffei penicilliosis

    OpenAIRE

    Cao, Liang; Chen, Da-Liang; Lee, Cindy; Chan, Che-Man; Chan, King-Man; Vanittanakom, Nongnuch; Tsang, Dominic N.C.; Yuen, Kwok-Yung

    1998-01-01

    The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marne...

  18. Multiplex Assay Detection of Immunoglobulin G Antibodies That Recognize Giardia intestinalis and Cryptosporidium parvum Antigens ▿

    OpenAIRE

    Priest, Jeffrey W.; Moss, Delynn M.; Visvesvara, Govinda S.; Jones, Cara C.; Li, Anna; Isaac-Renton, Judith L

    2010-01-01

    Giardiasis and cryptosporidiosis are common enteric parasitic diseases that have similar routes of transmission. In this work, we have identified epitopes within the Giardia variant-specific surface protein (VSP) sequences that are recognized by IgG antibodies from 13 of 14 (93%) sera from patients with stool-confirmed giardiasis. The conserved epitopes are shared among VSPs from both of the assemblages that commonly infect humans, and they are likely to be structural, as both sodium dodecyl ...

  19. Radiolabeled anti granulocyte antibodies in the detection of osteo-articular prosthesis infection

    International Nuclear Information System (INIS)

    Radiolabeled anti-granulocyte antibodies (immunoscintigraphy) are of demonstrated effectiveness in the diagnosis of infection. The osteo-articular prosthesis infection, instead of its low prevalence, needs an accurate and fast diagnosis. The immunoscintigraphy in this group of patients is a debated subject as the results seem to be related to the experience, interpretation criteria and patients clinical features. In general, immunoscintigraphy is an useful technique with an easy, safe and fast preparation. (author)

  20. Evaluation of enzyme immunoassay for detection of salivary antibody to Helicobacter pylori.

    OpenAIRE

    Simor, A E; Lin, E; Saibil, F; Cohen, L; Louie, M; Pearen, S; Donhoffer, H A

    1996-01-01

    The Helisal test is a quantitative enzyme immunoassay for the measurement of Helicobacter pylori-specific immunoglobulin G antibodies in saliva. This test was evaluated in comparison with culture and histopathologic examination of gastric biopsy specimens obtained from 195 patients who underwent 200 endoscopic procedures for the investigation of gastrointestinal symptoms. Forty-one (21%) patients were found to have peptic ulcer disease, and one other patient had a gastric carcinoma. H. pylori...

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

    OpenAIRE

    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. neuron...

  2. Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

    OpenAIRE

    Qian Yajuan; Zhou Xueping; Xie Yan; Shang Haili; Wu Jianxiang

    2011-01-01

    Abstract Background Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immuno...

  3. Detection of Rickettsia rickettsii antibodies in human sera by crossed immunoelectrophoresis.

    OpenAIRE

    Anacker, R. L.; Philip, R N; Wilfert, C M; Kleeman, K T; Turner, L; MacCormack, J. N.; Hechemy, K E

    1983-01-01

    To identify Rickettsia rickettsii antigens of immunological importance, we examined sera from patients with serologically confirmed cases of Rocky Mountain spotted fever by crossed immunoelectrophoresis for antibodies to antigens extracted from the R strain of R. rickettsii with the detergent Triton X-100. Sixteen antigens were identified in the detergent extract by crossed immunoelectrophoresis with a hyperimmune rabbit serum raised against whole rickettsiae. When the rabbit antiserum was pl...

  4. Detection of complement activation using monoclonal antibodies against C3d

    OpenAIRE

    Thurman, Joshua M.; Kulik, Liudmila; Orth, Heather; Wong, Maria; Renner, Brandon; Sargsyan, Siranush A.; Mitchell, Lynne M.; Hourcade, Dennis E.; Hannan, Jonathan P.; Kovacs, James M.; Coughlin, Beth; Woodell, Alex S.; Pickering, Matthew C.; Rohrer, Bärbel; Holers, V. Michael

    2013-01-01

    During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monocl...

  5. Detection of Mycoplasma pneumoniae in cerebrospinal fluid of children: Quantification of CSF-IgG antibody

    OpenAIRE

    Noorbakhsh S; Shekarabi M; Kalbasi Z; Tabatabaei A; Tonekaboni H; Afsharkhas L; Vafaei-shahi M

    2010-01-01

    "nBackground: M. pneumoniae infection in children is usual and diagnosis of its neurologic complications for rapid treatment is very important. To compare the CSF- M. pneumoniae antibody level between febrile children with acute neurologic signs (Menigoencephalitis, Guillan Barre Syndrome (GBS), Transverse myelitis, Ataxia and so on) with unaffected ones. "n"nMethods: A cross sectional/ case control study in pediatric wards of Rasoul-e-Akram & Mofid hospitals (2007-2009...

  6. An intracellular targeted antibody detects EGFR as an independent prognostic factor in ovarian carcinomas

    OpenAIRE

    Braicu Elena I; Sehouli Jalid; Buckendahl Ann-Christin; Darb-Esfahani Silvia; Weichert Wilko; Schwabe Michael; Noske Aurelia; Budczies Jan; Dietel Manfred; Denkert Carsten

    2011-01-01

    Abstract Background In ovarian cancer, the reported rate of EGFR expression varies between 4-70% depending on assessment method and data on patient outcome are conflicting. Methods: In this study we investigated EGFR expression and its prognostic value in a cohort of 121 invasive ovarian carcinomas, using a novel antibody against the intracellular domain of the receptor. We further evaluated an association between EGFR, the nuclear transporter CRM1 as well as COX-2. Furthermore, we evaluated ...

  7. Detection of antibodies to bacterial cell wall peptidoglycan in human sera. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Heymer, B.; Schleifer, K.H.; Read, S.; Zabriskie, J.B.; Krause, R.M.

    1976-07-01

    A radioimmunoassay has been developed for the measurement of antibodies to peptidoglycan in human sera including patients with rheumatic feaver and juvenile rheumatoid arthritis. The assay is based on the percentage of binding of the hapten /sup 125/I-L-Ala-..gamma..-D-Glu-L-Lys-D-Ala-D-Ala, the major peptide determinant of peptidoglycan. Because of differences in the avidity of the antibodies in different sera, the amount of antibody was expressed as pentapeptide hapten-binding capacity (pentapeptide-HBC in ng/ml of serum). Fourteen out of 105 normal blood donors had a pentapeptide-HBC value greater than or equal to 75 ng/ml serum. Values in healthy children 5 to 18 years of age were less than or equal to 50 ng/ml. Sixty-eight percent of the individuals with rheumatic fever had values greater than or equal to 75 ng/ml, an indication that streptococcal infections can stimulate an immune response to peptidoglycan. Thirty-five percent of the patients with juvenile rheumatoid arthritis had values greater than or equal to 75 ng/ml. Such a finding points to a possible association between bacterial infections and juvenile rheumatoid arthritis.

  8. Detection of antibodies to bacterial cell wall peptidoglycan in human sera

    International Nuclear Information System (INIS)

    A radioimmunoassay has been developed for the measurement of antibodies to peptidoglycan in human sera including patients with rheumatic feaver and juvenile rheumatoid arthritis. The assay is based on the percentage of binding of the hapten 125I-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala, the major peptide determinant of peptidoglycan. Because of differences in the avidity of the antibodies in different sera, the amount of antibody was expressed as pentapeptide hapten-binding capacity (pentapeptide-HBC in ng/ml of serum). Fourteen out of 105 normal blood donors had a pentapeptide-HBC value greater than or equal to 75 ng/ml serum. Values in healthy children 5 to 18 years of age were less than or equal to 50 ng/ml. Sixty-eight percent of the individuals with rheumatic fever had values greater than or equal to 75 ng/ml, an indication that streptococcal infections can stimulate an immune response to peptidoglycan. Thirty-five percent of the patients with juvenile rheumatoid arthritis had values greater than or equal to 75 ng/ml. Such a finding points to a possible association between bacterial infections and juvenile rheumatoid arthritis

  9. Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits.

    Science.gov (United States)

    Mattera, Lucia; Bhuckory, Shashi; Wegner, K David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J; Hildebrandt, Niko; Reiss, Peter

    2016-06-01

    A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter LOD) for PSA compared to commercially available hydrophilic QDs emitting at 605 and 705 nm, respectively. While the commercial QDs contain identical inorganic cores responsible for their fluorescence, they are coated with a comparably thick amphiphilic polymer layer leading to much larger hydrodynamic diameters (>26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL(-1) obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL(-1)) and demonstrate their direct applicability in clinical diagnostics. PMID:27188210

  10. Development of a monoclonal antibody detection assay for species-specific identification of abalone.

    Science.gov (United States)

    Lopata, Andreas L; Luijx, Thomas; Fenemore, Bartha; Sweijd, Neville A; Cook, Peter A

    2002-10-01

    Species identification based on biochemical and molecular techniques has a broad range of applications. These include compliance enforcement, the management and conservation of marine organisms, and commercial quality control. Abalone poaching worldwide and illegal trade in abalone products have increased mainly because of the attractive prices obtained and caused a sharp decline in stocks. Alleged poachers have been acquitted because of lack of evidence to correctly identify species. Therefore, a robust method is required that would identify tissue of abalone origin to species level. The aim of this study was to develop immunologic techniques, using monoclonal and polyclonal antibodies, to identify 10 different abalone species and subspecies from South Africa, the United States, Australia, and Japan. The combination of 3 developed monoclonal antibodies to South African abalone (Haliotis midae) enabled differentiation between most of the 10 species including the subspecies H. diversicolor supertexta and H. diversicolor diversicolor. In a novel approach, using antibodies of patients with allergy to abalone, the differentiation of additional subspecies, H. discus discus and H. discus hannai, was possible. A field-based immunoassay was developed to identify confiscated tissue of abalone origin. PMID:14961238

  11. Detection of anti-Neospora caninum antibodies in Iranian native cattle

    Directory of Open Access Journals (Sweden)

    Jamal Gharekhani

    2014-12-01

    Full Text Available Neospora caninum is an Apicomplexan parasite which may cause abortion in cattle. This study investigated occurrences of antibodies against N. caninum in Iranian native cattle. From September 2010 to September 2011, blood samples (n=768 of native cows were collected randomly from different rural regions of Hamedan (n=400 and Kurdistan provinces (n=368 located to the western part of Iran. All the samples were evaluated for IgG antibodies against N. caninum using Enzyme Linked Immunosorbent Assay. The IgG antibodies to N. caninum were found in 14.2% (n=109/768 of serum samples (95% CI: 11.74 - 16.66. There was a significant difference between seropositivity and abortion history (p<0.0001, OR=2.9, unlike to age groups (p=0.105. This is the first report of N. caninum infection in Iranian native cattle. In conclusion, N. caninum is an important factor in abortion in Iranian native cattle. Further comprehensive studies and designing control strategies for improving management in cattle farms are highly recommended.

  12. Failure of a novel, rapid antigen and antibody combination test to detect antigen-positive HIV infection in African adults with early HIV infection.

    Directory of Open Access Journals (Sweden)

    William Kilembe

    Full Text Available BACKGROUND: Acute HIV infection (prior to antibody seroconversion represents a high-risk window for HIV transmission. Development of a test to detect acute infection at the point-of-care is urgent. METHODS: Volunteers enrolled in a prospective study of HIV incidence in four African cities, Kigali in Rwanda and Ndola, Kitwe and Lusaka in Zambia, were tested regularly for HIV by rapid antibody test and p24 antigen ELISA. Five subgroups of samples were also tested by the Determine Ag/Ab Combo test 1 Antigen positive, antibody negative (acute infection; 2 Antigen positive, antibody positive; 3 Antigen negative, antibody positive; 4 Antigen negative, antibody negative; and 5 Antigen false positive, antibody negative (HIV uninfected. A sixth group included serial dilutions from a p24 antigen-positive control sample. Combo test results were reported as antigen positive, antibody positive, or both. RESULTS: Of 34 group 1 samples with VL between 5x105 and >1.5x107 copies/mL (median 3.5x106, 1 (2.9% was detected by the Combo antigen component, 7 (20.6% others were positive by the Combo antibody component. No group 2 samples were antigen positive by the Combo test (0/18. Sensitivity of the Combo antigen test was therefore 1.9% (1/52, 95% CI 0.0, 9.9. One false positive Combo antibody result (1/30, 3.3% was observed in group 4. No false-positive Combo antigen results were observed. The Combo antigen test was positive in group 6 at concentrations of 80 pg/mL, faintly positive at 40 and 20 pg/mL, and negative thereafter. The p24 ELISA antigen test remained positive at 5 pg/mL. CONCLUSIONS: Although the antibody component of the Combo test detected antibodies to HIV earlier than the comparison antibody tests used, less than 2% of the cases of antigen-positive HIV infection were detected by the Combo antigen component. The development of a rapid point-of-care test to diagnose acute HIV infection remains an urgent goal.

  13. Development and evaluation of two simple, rapid immunochromatographic tests for the detection of Yersinia pestis antibodies in humans and reservoirs.

    Directory of Open Access Journals (Sweden)

    Minoarisoa Rajerison

    Full Text Available BACKGROUND: Tools for plague diagnosis and surveillance are not always available and affordable in most of the countries affected by the disease. Yersinia pestis isolation for confirmation is time-consuming and difficult to perform under field conditions. Serologic tests like ELISA require specific equipments not always available in developing countries. In addition to the existing rapid test for antigen detection, a rapid serodiagnostic assay may be useful for plague control. METHODS/PRINCIPAL FINDINGS: We developed two rapid immunochromatography-based tests for the detection of antibodies directed against F1 antigen of Y. pestis. The first test, SIgT, which detects total Ig (IgT anti-F1 in several species (S (human and reservoirs, was developed in order to have for the field use an alternative method to ELISA. The performance of the SIgT test was evaluated with samples from humans and animals for which ELISA was used to determine the presumptive diagnosis of plague. SIgT test detected anti-F1 Ig antibodies in humans with a sensitivity of 84.6% (95% CI: 0.76-0.94 and a specificity of 98% (95% CI: 0.96-1. In evaluation of samples from rodents and other small mammals, the SlgT test had a sensitivity of 87.8% (95% CI: 0.80-0.94 and a specificity of 90.3% (95% CI: 0.86-0.93. Improved performance was obtained with samples from dogs, a sentinel animal, with a sensitivity of 93% (95% CI: 0.82-1 and a specificity of 98% (95% CI: 0.95-1.01. The second test, HIgM, which detects human (H IgM anti-F1, was developed in order to have another method for plague diagnosis. Its sensitivity was 83% (95% CI: 0.75-0.90 and its specificity about 100%. CONCLUSION/SIGNIFICANCE: The SIgT test is of importance for surveillance because it can detect Ig antibodies in a range of reservoir species. The HIgM test could facilitate the diagnosis of plague during outbreaks, particularly when only a single serum sample is available.

  14. Diagnosis of Helicobacter pylori Infection Using ELISA for Detection of Serum IgM, IgG and IgA Antibodies

    Directory of Open Access Journals (Sweden)

    MH K. Ansari

    2007-06-01

    Full Text Available Background: Helicobacter pylori is gram negative bacteria and is the etiologic agent of some gastrointestinal disease are such as chronic gastritis, gastric ulcers, lymphoma and aden carcinoma. Multiple invasive and noninvasive methods for detection of this infection are available, and among non invasive methods diagnostic value of specific IgM, IgG and IgA by ELISAis one of the best methods. The aim of this study to detect H. pylori antibodies for IgM, IgG and IgA by ELISA method. Methods: Blood samples From 131 referred patients (78 female and 53 male for detection of H.pylori was taken and serum separated for detection of IgM, IgG and IgA antibodies. IgG assessed by EIU units (42 was Positive .Also for IgM and IgA antibodies reported by U/ml unit (12 was Positive. Results: 131 Samples were tested for H. pylori IgM, IgG and IgA antibodies and showed 27.47%, 42% and 25.19% positive for IgM, IgG and IgA antibodies respectively. Age group 20-40 years old for IgM, IgG and IgA antibodies 23.3% and 28.3% and 16.7% positive respectively and for 41-80 years old IgM, IgG and IgA antibodies were 31%, 53.5% and 32%, respectively. The above data showing infection is higher at 41- 80 years old patients. Conclusion: The simple method for detection of H. pylori antibodies are available commercially and have sensitivity and specificity for H. pylori diseases. IgG, IgM and IgA have diagnostic importance, therefore regarding acceptable sensitivity and specificity, ease of work with ELISA, being economical and non invasive, it can be employed in diagnosis of H. pylori infections.

  15. Application of commercial enzyme linked immunosorbent assays (ELISA for the detection of antibodies for foot-and-mouth disease virus in wild boar and red deer

    Directory of Open Access Journals (Sweden)

    Terzić Svjetlana

    2012-01-01

    Full Text Available For detecting antibodies towards foot and mouth (FMD virus in sera collected from red deer hinds (Cervus elaphus and wild boars (Sus scrofa, three commercially available enzyme-linked immunosorbent assays (ELISA were used. Two ELISA kits (PrioCHECK FMDV NS and CHEKIT FMD-3ABC were used for the detection of antibodies towards non-structural proteins of FMD virus and one assay was based on the detection of antibodies for serotype O (PrioCHECK FMDV type O. All of the sera tested in our study were negative for antibodies against FMD virus. The aim of this study was to investigate the usefulness of commercially available ELISA kits given for marketing authorization in Croatia in testing the prevalence of FMD antibodies in wild boar and red deer populations. Since the producers of ELISA kits used in our study did not declare wild animals as a target species, we hypothesised that the same kits could be used for serological diagnosis of FMD in red deer and wild boars. Our study confirmed that the kits used are acceptable for detecting antibodies in both species tested, however, the investigation highlighted the problem of validating the kits due to the absence of available positive sera originating from red deer, as well as other susceptible species, especially artiodactyls.

  16. Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology

    Science.gov (United States)

    Kingston, Hugh W. F.; Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Laongnualpanich, Achara; Basnyat, Buddha; Day, Nicholas P. J.

    2015-01-01

    This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ≥1:1,600 versus 92% and 95% at ≥1:6,400, respectively. PMID:26291089

  17. Serodiagnosis of tuberculosis: specific detection of free and complex-dissociated antibodies anti-mycobacterium tuberculosis recombinant antigens

    Directory of Open Access Journals (Sweden)

    María Susana Imaz

    2008-06-01

    Full Text Available The diagnostic test characteristics of detecting free and complex-dissociated IgG to three recombinant antigens of Mycobacterium tuberculosis (38-kDa, Ag16 and Ag85B, singly and in combination, were evaluated in sera from 161 tuberculous patients [smear-positive pulmonary TB (50, smear-negative pulmonary TB (pTBsm- (60 and extrapulmonary TB (51 and 214 control patients (mycobacteriosis (14, mycoses(14, leprosy(4, other underlying diseases (82 and healthy people (100]. The individual antigens ranged from 25% to 42% in sensitivity and from 93% to 96% in specificity, while considering free IgG response. Addition of complex-dissociated antibodies against each individual antigen improved the sensitivity up to 55%. The number and levels of specific antibodies varied greatly from individual to individual. Combination of individual results for free and complex-dissociated IgG to 38-kDa, Ag16 and Ag85B offered 76% sensitivity and 83% specificity. When the three antigens were placed in the same well, the sensitivity was lower than that expected on the basis of single antigen (63% but with a good specificity (95%, even in the group of mycobacteriosis or mycoses. The highest contribution of complex-dissociated IgG results to free IgG results was seen for the diagnosis of pTBsm- patients. In conclusion, although neither single recombinant antigen was reactive with most sera from TB patients even after the measurement of both free and complex-dissociated antibodies, the use of multi-antigen cocktails improved the diagnostic utility of the ELISA assay, allowing the identification of almost 70% of pTBsm-, with a high level of specificity; the use of additional, well selected antigens should lead to the detection of almost all patients with TB.

  18. Comparison of dot-ELISA and standard ELISA for detection of Neisseria meningitidis outer membrane complex-specific antibodies

    Directory of Open Access Journals (Sweden)

    Elza FT Belo

    2010-02-01

    Full Text Available Dot-ELISA using the outer membrane complex antigens of Neisseria meningitidis as a target was standardized for rapid detection of meningococcal-specific antibodies in human serum. We investigated the level of meningococcal-specific IgG, IgA, and IgM in serum using dot-ELISA with outer membrane antigens prepared from Neisseria meningitidis serotype B:4.19:P1.15,3,7,9 (a strain isolated from a Brazilian epidemic. The dot-ELISA is based on the same principles as the standard ELISA and is useful for detection of anti-N. meningitidis B antibodies in serum of patients with meningococcal infections. For the assay, outer membrane complexes (OMCs were absorbed by nitrocellulose membrane and blocked with a 5% skim milk solution. Serum samples were drawn upon hospital admission and during convalescence from patients with meningococcal septicemia, and single samples were drawn from uninfected controls. We retrospectively examined a total of 57 serum samples: 35 from patients infected with N. meningitidis B, 12 from patients infected with Haemophilus influenzae b, and 10 from health individuals. When performed at room temperature, dot-ELISA took approximately four hours to perform, and the optimum antigen concentration was 0.42 µg per dot. The specificity of IgG, IgM, and IgA demonstrates that dot-ELISA using OMCs from N. meningitidis B as a target is suitable for serologic verification of clinically suspected meningococcal disease in patients and for titer determination of antibodies produced during different phases of natural infection. Furthermore, the sensitivity of dot-ELISA was comparable to that of standard ELISA. Overall, dot-ELISA is simple to perform, rapid, and low cost. Further validation of the test as a screening tool is required.

  19. Development of a immunochromatographic test with avidin-biotin for the detection of antibodies against antigen e of hepatitis B in human plasma

    International Nuclear Information System (INIS)

    The disappearance of antigen e of hepatitis B in the presence of the plasmatic antibodies against antigen e may indicate a satisfactory therapeutic response in patients with chronic hepatitis B. The immuno-chromatographic test carried out in the diagnosis of diseases use different antibody combinations and may employ the avidin or streptavidin-biotin technology to develop a rapid immuno-chromatographic test for the detection of antibodies anti-antigen e in the plasma. They were detected in the laboratory by means of two fast immuno-chromatographic tests when using in one of them the avidin-biotin technology. These tests are carried out with a one-step competitive inhibition format and amplified or not with avidin-biotin. Monoclonal antibodies against antigen e obtained by cellular hybridization were used. Forty-six plasmatic samples classified as positive and negative to the anti-antigen antibodies were evaluated with a reference immunochromatographic test Advanced QualityTM. The possible expiry time of the biological reagents forming part of these tests were studied with accelerated thermal-stability experiments. The possible interference in the plasma of some of the biochemical compounds used in these trials was analyzed. Four murine monoclonal antibodies anti-antigen e were obtained and only one of them was used in these immunochromatographic tests with an anti-antigen polyclonal antibody conjugated with gold. Both tests and their stable biological reagents discriminated the positive and negative samples to the antibodies anti-antigen e, as well as the commercial test. There was no interference in the biochemical compounds studied in these tests. Both immuno-chromatographic tests made in the laboratory are useful to detect antibodies anti-antigen e in the plasma. The avidin-biotin increased the analytical sensitivity of this type of fast immuno-chromatographic test without altering its performance features. (Author)

  20. Detection of plasma α-granule membrane protein (GMP-140) using 125I labeled monoclonal antibodies and its clinic applications

    International Nuclear Information System (INIS)

    Radioimmunoassay with two monoclonal antibodies to different epitopes of granule membrane protein (GMP-140) was used to measure the plasma GMP-140 level in patients suffering from acute myocardial infarction (AMI) or cerebral thrombosis and in patients during cardiopulmonary bypass (CPB). Monoclonal antibody (McAb) SZ-51 was used as a solid phase and 125I-labeled McAb S12 as a fluid phase. The assay was so sensitive that it could detect as little as 1 μg/L of purified GMP-140. The intra- and inter-assay coefficients of variation were 4.2% and 7.1% respectively. The concentration of plasma GMP-140 was found to be 10 +- 4.5 μg/L in normal subjects (n = 20). Patients undergoing CPB (n = 10) and patients with AMI (n = 16) or with acute cerebral thrombosis (n = 22) were also studied. The results suggest that plasma GMP-140 can be reliable detected by radioimmunoassay with two McAbs and plasma GMP-140 may provide a useful marker for thrombotic diseases

  1. Antibody-Free Colorimetric Detection of Total Aflatoxins in Rice Based on a Simple Two-Step Chromogenic Reaction.

    Science.gov (United States)

    Du, Bibai; Su, Xiaoou; Yang, Kunhao; Pan, Long; Liu, Qingju; Gong, Lingling; Wang, Peilong; Yang, Jingkui; He, Yujian

    2016-04-01

    The prevalently used immunoassays for fast screening of aftatoxins (AFs) usually cannot meet the requirement for simultaneous determination of total AFs (aflatoxin B1 + aflatoxin B2 + aflatoxin G1 + aflatoxin G2) due to the deficiency of highly group-specific antibodies. This paper describes a two-step chromogenic reaction based method to quantitatively detect total AFs in rice using colorimetric measurement without antibody. In the method, colorless AFs transform into green-colored indophenol products through the reaction with sodium hydroxide and 2,6-dibromoquinone-4-chloroimide (DBQC) successively, allowing selectively determining total AFs up to 3.9 μg/kg over other competitive mycotoxins under optimal conditions by a UV-vis spectrophotometer. In addition, the colorimetric measurement results of the rice samples agree well with that of a standard HPLC method, demonstrating the good reliability and applicability of the method. Uniquely, the method has potential for on-site detection of total AFs in rice when using a nylon membrane-based device. PMID:26938207

  2. Use of a monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens.

    Science.gov (United States)

    Ungar, B L; Yolken, R H; Quinn, T C

    1985-05-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Entamoeba histolytica in human feces, using both a monoclonal antibody and rabbit antisera. It detected from less than 1 to 57 trophozoites of 6 E. histolytica strains. Stool specimens were positive by ELISA in 18 of 22 (82%) patients with E. histolytica and in 3 of 186 (2%) of patients without demonstrable E. histolytica in their stools. The latter included one from a child living near an asymptomatic cyst carrier and another from a traveler with giardiasis who had recently taken antibiotics. One hundred eight of 183 microscopy-and ELISA-negative specimens contained other parasites including Giardia (49 specimens), Endolimax nana (24), Entamoeba coli (21), Iodamoeba butschlii (2), and Entamoeba hartmanni (1). This ELISA for E. histolytica is a simple, sensitive and specific diagnostic tool. PMID:2860814

  3. Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

    Directory of Open Access Journals (Sweden)

    Gentil Arthur Bentes

    2015-06-01

    Full Text Available An increasing amount of research has been conducted on immunoglobulin Y (IgY because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY reactive to hepatitis A virus (HAV antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

  4. Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens

    Directory of Open Access Journals (Sweden)

    Adele Caterino-de-Araujo

    1992-06-01

    Full Text Available The present report describes an alternative method for in vitro detection of HIV-1 -specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analized. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.

  5. Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment

    International Nuclear Information System (INIS)

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99mTc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. (orig.). With 3 figs., 1 tab

  6. Human monoclonal antibody {sup 99m}Tc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment

    Energy Technology Data Exchange (ETDEWEB)

    Krause, B.J. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Baum, R.P. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Staib-Sebler, E. [Department of General and Abdominal Surgery, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Lorenz, M. [Department of General and Abdominal Surgery, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Niesen, A. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany); Hoer, G. [Department of Nuclear Medicine, Johann Wolfgang Goethe University Medical Center, Frankfurt/Main (Germany)

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of {sup 99m}Tc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. (orig.). With 3 figs., 1 tab.

  7. Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment.

    Science.gov (United States)

    Krause, B J; Baum, R P; Staib-Sebler, E; Lorenz, M; Niesen, A; Hör, G

    1997-01-01

    This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99mTc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%. PMID:9044881

  8. Development of QCM Biosensor with Specific Cow Milk Protein Antibody for Candidate Milk Adulteration Detection

    OpenAIRE

    Sakti, Setyawan P.; Nur Chabibah; Ayu, Senja P.; Masdiana C. Padaga; Aulanni’am Aulanni’am

    2016-01-01

    Adulteration of goat milk is usually done using cow’s milk product. Cow milk is used as it is widely available and its price is cheaper compared to goat milk. This paper shows a development of candidate tools for milk adulteration using cow milk. A quartz crystal microbalance immunosensor was developed using commercial crystal resonator and polyclonal antibody specific to cow milk protein. A specific protein at 208 KDa is found only in cow milk and does not exist in goat milk. The existence o...

  9. Detection of anti-spermatozoal antibodies by a 125I-protein-A radioimmunological assay

    International Nuclear Information System (INIS)

    A newly developed solid-phase radioimmunobinding assay (RIBA) using detergent-solubilized sperm antigen has been used to evaluate anti-sperm antibodies. Results showed that none of the fertile females and males were positive in the RIBA, whereas approximately 6% of the females and 3% of the males with unexplained infertility were positive. These results are similar to those obtained using a chemically synthesized spermatozoal decapeptide antigen. The RIBA is simple to perform, requires no vital or intact spermatozoa, and large numbers of sera (up to 400) can be processed in one day with a total incubation time of 90 min. (Auth.)

  10. Early detection of Trypanosoma evansi infection and monitoring of antibody levels by ELISA following treatment

    OpenAIRE

    Yadav, S. C.; Kumar, Rajender; Manuja, Anju; Goyal, Liza; Gupta, A. K.

    2012-01-01

    In present communication, we report an outbreak of Trypanosoma evansi in equine herd n = 30 (horse and mules) which, were reared in fly proof stables as well as in open paddock maintained under semi-intensive system of management, and its effective control using trypanocidal drug. The infection was monitored by antibody ELISA up to 180 days post-treatment (PT). A total of 8 out of 14 equines (57.14 %) which were maintained only in open paddocks were found positive with T. evansi infection par...

  11. A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.

    Science.gov (United States)

    Birdsall, Robert E; McCarthy, Sean M; Janin-Bussat, Marie Claire; Perez, Michel; Haeuw, Jean-François; Chen, Weibin; Beck, Alain

    2016-01-01

    Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows. PMID:26651262

  12. Construction of a biotinylated cameloid-like antibody for lable-free detection of apolipoprotein B-100.

    Science.gov (United States)

    Li, Henan; Yan, Junrong; Ou, Weijun; Liu, Hong; Liu, Songqin; Wan, Yakun

    2015-02-15

    Nanobodies (Nbs), also known as the variable domain of the heavy-chain-only antibody (VHH), are single-domain antigen-binding fragments derived from heavy-chain antibodies that occur naturally in sera of camelids. Due to their unique properties of small size (15 kD), intrinsic stability, high affinity and specificity, Nbs are suitable for detecting clinical relevant antigens. Apolipoprotein B-100 (ApoB-100) is a highly predictive marker for coronary artery disease (CAD), which is frequently detected in clinical diagnosis. Herein, we successfully obtained anti-ApoB-100 Nbs for the first time and further fabricated a label-free and sensitive immunosensor for ApoB-100 based on isolated anti-ApoB-100 nanobody (Nb) using the electrochemical impedance spectroscopy (EIS) technique. We have generated an immunized phage display library against ApoB-100 and isolated four anti-ApoB-100 Nbs with high affinity and stability. The Nb with the highest affinity was biotinylated based on in vivo BirA system. Further, we developed a label-free electrochemical impedance immunosensor for ApoB-100 using this anti-ApoB-100 Nb. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5 ng mL(-1) with a detection limit of 0.03 ng mL(-1). This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis. PMID:25203942

  13. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

    OpenAIRE

    Broze, G J; S. Hickman; Miletich, J P

    1985-01-01

    Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

  14. The detection of bovine respiratory syncytial virus in formalin fixed bovine lung with commercially available monoclonal antibodies and avidin biotin complex immunohistochemistry.

    OpenAIRE

    Haines, D M; Clark, E.G.; Chelack, B J

    1989-01-01

    Eight commercially available monoclonal antibodies directed against respiratory syncytial virus antigens were tested for ability to detect bovine respiratory syncytial virus (BRSV) antigen in formalin fixed, paraffin embedded bovine lung using avidin-biotin complex immunohistochemical staining. Monoclonal antibodies from clone 18B2 purchased from Biosoft, Paris, France and those from clone 8G12 purchased from the Department of Veterinary Sciences, University of Nebraska, Lincoln, Nebraska sta...

  15. A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis

    OpenAIRE

    Ceribelli, A; Satoh, M; E.K.L. Chan

    2012-01-01

    Introduction Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies. Methods Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from...

  16. Sensitive Detection of Capsaicinoids Using a Surface Plasmon Resonance Sensor with Anti-Homovanillic Acid Polyclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Kiyoshi Toko

    2013-11-01

    Full Text Available Recently, highly functional biosensors have been developed in preparation for possible large-scale terrorist attacks using chemical warfare agents. Practically applicable sensors are required to have various abilities, such as high portability and operability, the capability of performing rapid and continuous measurement, as well as high sensitivity and selectivity. We developed the detection method of capsaicinoids, the main component of some lachrymators, using a surface plasmon resonance (SPR immunosensor as an on-site detection sensor. Homovanillic acid, which has a vanillyl group similar to capsaicinoids such as capsaicin and dihydrocapsaicin, was bound to Concholepas concholepas hemocyanin (CCH for use as an immunogen to generate polyclonal antibodies. An indirect competitive assay was carried out to detect capsaicinoids using SPR sensor chips on which different capsaicin analogues were immobilized. For the sensor chip on which 4-hydroxy-3-methoxybenzylamine hydrochloride was immobilized, a detection limit of 150 ppb was achieved. We found that the incubation time was not required and the detection can be completed in five minutes.

  17. Sensitive detection of capsaicinoids using a surface plasmon resonance sensor with anti-homovanillic Acid polyclonal antibodies.

    Science.gov (United States)

    Nakamura, Shingo; Yatabe, Rui; Onodera, Takeshi; Toko, Kiyoshi

    2013-01-01

    Recently, highly functional biosensors have been developed in preparation for possible large-scale terrorist attacks using chemical warfare agents. Practically applicable sensors are required to have various abilities, such as high portability and operability, the capability of performing rapid and continuous measurement, as well as high sensitivity and selectivity. We developed the detection method of capsaicinoids, the main component of some lachrymators, using a surface plasmon resonance (SPR) immunosensor as an on-site detection sensor. Homovanillic acid, which has a vanillyl group similar to capsaicinoids such as capsaicin and dihydrocapsaicin, was bound to Concholepas concholepas hemocyanin (CCH) for use as an immunogen to generate polyclonal antibodies. An indirect competitive assay was carried out to detect capsaicinoids using SPR sensor chips on which different capsaicin analogues were immobilized. For the sensor chip on which 4-hydroxy-3-methoxybenzylamine hydrochloride was immobilized, a detection limit of 150 ppb was achieved. We found that the incubation time was not required and the detection can be completed in five minutes. PMID:25586413

  18. Standardization of natural mycolic acid antigen composition and production for use in biomarker antibody detection to diagnose active tuberculosis.

    Science.gov (United States)

    Ndlandla, F L; Ejoh, V; Stoltz, A C; Naicker, B; Cromarty, A D; van Wyngaardt, S; Khati, M; Rotherham, L S; Lemmer, Y; Niebuhr, J; Baumeister, C R; Al Dulayymi, J R; Swai, H; Baird, M S; Verschoor, J A

    2016-08-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is characterized by the abundance of species specific, antigenic cell wall lipids called mycolic acids. These wax-like molecules all share an identical, amphiphilic mycolic motif, but have different functional groups in a long hydrophobic hydrocarbon mero-chain that divide them into three main classes: alpha-, keto- and methoxy-mycolic acids. Whereas alpha-mycolic acids constitutively maintain an abundance of around 50%, the ratio of methoxy- to keto-mycolic acid types may vary depending on, among other things, the growth stage of M. tuberculosis. In human patients, antibodies to mycolic acids have shown potential as diagnostic serum biomarkers for active TB. Variations in mycolic acid composition affect the antigenic properties and can potentially compromise the precision of detection of anti-mycolic acids antibodies in patient sera to natural mixtures. We demonstrate this here with combinations of synthetic mycolic acid antigens, tested against TB patient and control sera. Combinations of methoxy- and α-mycolic acids are more antigenic than combinations of keto- and α-mycolic acids, showing the former to give a more sensitive test for TB biomarker antibodies. Natural mixtures of mycolic acids isolated from mature cultures of M. tuberculosis H37Rv give the same sensitivity as that with synthetic methoxy- and α-mycolic acids in combination, in a surface plasmon resonance inhibition biosensor test. To ensure that the antigenic activity of isolates of natural mycolic acids is reproducible, we cultured M. tuberculosis H37Rv on Middlebrook 7H10 solid agar plates to stationary growth phase in a standardized, optimal way. The proportions of mycolic acid classes in various batches of the isolates prepared from these cultures were compared to a commercially available natural mycolic acid isolate. LC-MS/MS and NMR data for quantitation of mycolic acids class compositions show that the variation in batches

  19. Using of competitive ELISA in detection of brucella antibodies in cattle sera

    Directory of Open Access Journals (Sweden)

    M. I. Al-Farwachi

    2009-01-01

    Full Text Available Competitive ELISA test was used in this study to identify brucella antibodies in cattle sera in Mosul, and assessing the results that are given by Rose-Bengal test. Results of testing 126 cattle sera revealed that the seroprevalence was 16.7% using C-ELISA and 18.3% using RBT. There were differences in seroprevalence according to sex of animals, females showed higher seroprevalence than males, it was 19.1% and 20.2% in females and 9.4% and 12.5% in males by C-ELISA and RBT respectively. Also there were differences in seroprevalence according to age of animals and pregnancy status of females. Kappa value revealed that there was little agreement between C-ELISA and RBT, (Kappa value was 0.229. RBT was not accurate in giving real seroprevalence, since there were 13 false-negative samples from 21 samples positive with C-ELISA, and there were 15 false-positive samples from 105 samples negative with C-ELISA. Results of tube agglutination test and 2-Mercaptoethanol test indicated a decline of titers from those of previous studies, lower titer of TAT was 1/10 (19.05% and most of titers were lower than 1/40 (76.2% in 2-Me test. This study concluded that the brucella antibodies in cattle sera in Mosul was increased from previous years and most of cases were acute.

  20. Anti-NMDA-receptor antibody detected in encephalitis, schizophrenia, and narcolepsy with psychotic features

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    Tsutsui Ko

    2012-05-01

    Full Text Available Abstract Background Causative role of encephalitis in major psychotic features, dyskinesias (particularly orofacial, seizures, and autonomic and respiratory changes has been recently emphasized. These symptoms often occur in young females with ovarian teratomas and are frequently associated with serum and CSF autoantibodies to the NMDA receptor (NMDAR. Methods The study included a total of 61 patients from age 15 to 61 and was carried out between January 1, 2005, and Dec 31, 2010. The patients were divided into the following three clinical groups for comparison. Group A; Patients with typical clinical characteristics of anti-NMDAR encephalitis. Group B; Patients with narcolepsy with severe psychosis. Group C; Patients with schizophrenia or schizo-affective disorders. Results Ten out of 61 cases were anti-NMDAR antibody positive in typical encephalitis cases (group A: 3 of 5 cases and cases in a broader range of psychiatric disorders including narcolepsy (group B: 3 of 5 cases and schizophrenia (group C: 4 of 51 cases. Conclusion In addition to 3 typical cases, we found 7 cases with anti-NMDAR antibody associated with various psychotic and sleep symptoms, which lack any noticeable clinical signs of encephalitis (seizures and autonomic symptoms throughout the course of the disease episodes; this result suggest that further discussion on the nosology and pathophysiology of autoimmune-mediated atypical psychosis and sleep disorders is required.

  1. DETECTABLE ANTI-DENGUE VIRUS IGM ANTIBODIES AMONG HEALTHY INDIVIDUALS IN OGBOMOSO, OYO STATE, NIGERIA

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    E. K. Oladipo

    2014-01-01

    Full Text Available Dengue fever is a zoonosis maintained in nature by mosquitoes transmitting virus between non-human primate species, most of which develop clinically in apparent infection. It has been found to be endemic in Africa and beyond. A survey for Dengue virus IgM antibody was carried out in Ogbomoso (urban and rural areas using Enzyme Linked Immunosorbent Assay (WKEA Med Supplies Corp, Dengue fever virus ELISA Kit (China, to determine the seroprevalence and true incidence of dengue virus in Ogbomoso. A total of 186 apparently healthy individuals were recruited into the study. The sera of 93 subjects who consented to participate were collected. The mean age of the subjects tested was 37.6±1.67. Anti-Dengue virus IgM antibodies were found in 16/93, (17.2%. The highest prevalence of anti-Dengue 28.6% was found in persons whose ages were between 0-15 years, males (18.9%, civil servants (26.3% and urban dwellers (21.3%. The findings from this study show that there is primary infection of this virus in Ogbomoso and suggest the need for preventive and control measures against dengue fever virus.

  2. Detection of human leukocyte antigen compatibility and antibodies in liver transplantation in China

    Institute of Scientific and Technical Information of China (English)

    Xue-Qin Meng; Xuan Zhang; Jun Fan; Lin Zhou; Bing Hao; Xiao-Ming Chen; Wei-Hang Ma; Shu-Sen Zheng

    2009-01-01

    BACKGROUND: The exact roles of human leukocyte antigen (HLA) compatibility, HLA antibodies and underlying diseases in acute rejection of liver transplants are not clear. Moreover, cytomegalovirus (CMV) infection, one of the most common infections after transplantation, is related to HLA genotype and the incidence of acute rejection. METHODS: Since there are controversial reports, we analyzed the impact of HLA matching, HLA antibodies and underlying diseases in 38 liver transplant recipients in China, and assessed the association of CMV infection and HLA compatibility. RESULTS: The frequency of no HLA compatibility was high in patients without antigenemia (P=0.019). All 17 patients with HLA-A matching developed antigenemia (P0.05). In patients with acute rejection, no differences were found in the incidence of acute rejection in transplants for hepatitis B, tumors, or combined hepatitis B and tumors (P>0.05).CONCLUSIONS: There are fewer acute rejections in transplants with more HLA compatibilities. Speciifc investigations of underlying diseases and HLA typing may be necessary in liver transplantation. The mechanisms of CMV infection and HLA matching should be further studied. HLA before transplantation should be examined for the prevention of acute rejection and CMV infection.

  3. Detection of humoral antibodies to Renibacterium salmoninarum in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar challenged by immersion and in naturally infected populations.

    Science.gov (United States)

    Jansson, E; Ljungberg, O

    1998-06-19

    Humoral antibodies to heat-stable antigens of Renibacterium salmoninarum (Rs) were detected by enzyme-linked immunosorbent assay (ELISA) in rainbow trout Oncorhynchus mykiss and in Atlantic salmon Salmo salar challenged by immersion. A slow antibody response was found: 3% (1/30) was positive 4 wk after immersion and 72% (26/36) was positive after 8 wk. All 30 fish sampled after 4 wk were found to be infected, as determined by bacterial culture and/or the presence of soluble antigens in the kidney. At 6, 8 and 12 wk after immersion the proportion of positives indicated by ELISA was 58%. The Rs infection was detected by cultivation in 36% of sampled fish collected on the same occasion. Elevated antibody titres to Rs were detected in samples from both Atlantic salmon (59% in 1 farm) and from rainbow trout (20% in 1 of 5 sampled farms) in naturally exposed populations all of which classified positive for bacterial kidney disease (BKD). Elevated antibody titres were detected among sampled fish from populations of rainbow trout and salmon with clinical BKD. Samples collected from farm populations of rainbow trout, salmon and brown trout Salmo trutta, exposed to Rs but without clinical BKD, were negative in the ELISA, although Rs bacteria or soluble antigens were detected at the same sampling. The antibody ELISA method cannot be recommended for general fish health monitoring purposes, but may be a valuable tool for monitoring the disease progression during controlled experiments. PMID:9684315

  4. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    International Nuclear Information System (INIS)

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to

  5. A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Zu-Quan [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Li, He-Ping [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zhang, Jing-Bo [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Huang, Tao [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Liu, Jin-Long; Xue, Sheng [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); Wu, Ai-Bo [Institute for Agri-food Standards and Testing Technology, Laboratory of Quality and Safety Risk Assessment for Agro-products, Ministry of Agriculture, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai 201403 (China); Liao, Yu-Cai, E-mail: ycliao06@yahoo.com.cn [Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); National Center of Plant Gene Research, Wuhan 430070 (China)

    2013-02-18

    Graphical abstract: A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. Highlights: ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. -- Abstract: Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding

  6. Array-in-well platform-based multiplex assay for the simultaneous detection of anti-HIV- and treponemal-antibodies, and Hepatitis B surface antigen.

    Science.gov (United States)

    Talha, Sheikh M; Saviranta, Petri; Hattara, Liisa; Vuorinen, Tytti; Hytönen, Jukka; Khanna, Navin; Pettersson, Kim

    2016-02-01

    Multiplex assays detecting sets of related clinical analytes simultaneously can save considerable amount of time and resources. Array-in-well (AIW) is a powerful platform for the multiplex detection of different analytes where microarrays can be printed at the bottom of microtiter wells, thus combining the potential of microarrays with the ease of handling microtiter wells. We have developed a single-step AIW assay for the simultaneous screening of HIV, Treponema pallidum subspecies pallidum (causing syphilis) and Hepatitis B virus infections targeting the specific detection of anti-HIV- and treponemal-antibodies and Hepatitis B surface antigen (HBsAg), respectively, using two different fluorescent label technologies i.e. DyLight 633 and europium nanoparticle. Double-antigen assay formats were used for anti-HIV- and treponemal-antibody detection that can simultaneously detect both IgG and IgM, and thus reduce the window period of detection. AIW assay was evaluated with well characterized serum/plasma samples (n=111), and the qualitative results were in near complete agreement with those of the reference assays. The AIW assay exhibited 100% sensitivities for all three analytes, and 100% specificities for anti-HIV antibodies and HBsAg, and 98.6% specificity for treponemal antibodies. The limit of detection of HBsAg in AIW assay was 0.18 ng/ml. This high performing AIW assay has the potential to be used as a multiplex screening test for these three infections. PMID:26711310

  7. Simultaneous detection of IgG antibodies associated with viral hemorrhagic fever by a multiplexed Luminex-based immunoassay.

    Science.gov (United States)

    Wu, Wei; Zhang, Shuo; Qu, Jing; Zhang, Quanfu; Li, Chuan; Li, Jiandong; Jin, Cong; Liang, Mifang; Li, Dexin

    2014-07-17

    Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs including Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Andes virus (ANDV), Sin Nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and dengue virus (DENV) were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed Luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs. PMID:24631566

  8. Detection of experimental thrombi in rabbits with an 131I-labelled fibrin-specific monoclonal antibody

    International Nuclear Information System (INIS)

    The detection of thrombi in rabbits has been investigated with 131I-labelled DD-3B6/22, a monoclonal antibody (Mab) reactive at high affinity (Kd=2.68x10-10 M) with human D Dimer (DD). DD-3B6/22 bound well to both 'fresh' and 'aged' human clots in an in vitro assay but showed poor binding to rabbit clots. However, reactivity was restored to rabbit blood if it was seeded, before clotting, with human DD covalently coupled to Sepharose beads. Thus, a rabbit model was developed in which blood was allowed to clot around DD-Sepharose beads introduced into the jugular vein. Gamma camera imaging showed that intact 131I-labelled DD-3B6/22 localised to these clots within 24 h. Uptake at this time was 0.202%±0.012% injected dose per gram (%ID/g) compared with 0.086±0.018%ID/g after injection of control antibody. 131I-labelled F(ab')2 fragments of DD-3B6/22 allowed earlier scintigraphic detection of the clot which was evident 4 h after injection. Uptake in the clot at 24 h was 0.154±0.038% ID/g compared with 0.109±0.027% ID/g for a control F(ab')2. As antigen levels in the clot are estimated to be less than 300 μg DD, thus representing a very small human clot, the DD-3B6/22 Mab would appear to have a good potential for the sensitive detection of thrombi in a clinical setting. (orig.)

  9. A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera.

    Science.gov (United States)

    Osawa, T; Wastling, J; Maley, S; Buxton, D; Innes, E A

    1998-09-01

    Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle. The epidemiology of neosporosis is poorly understood, partly because accurate diagnosis of infection is difficult. In this paper we describe the development of a multiple antigen-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies to N. caninum in sera from cattle, sheep and goats as well as from bovine foetal fluids. A water-soluble fraction (wsf) of sonicated NC-1 strain tachyzoites was used as the antigen in the ELISA. Minimum optical density (OD) values that were considered to be Neospora antibody-positive, that is, the cut-off OD values were determined separately for bovine maternal sera, bovine foetal fluids, ovine sera and caprine sera; they were 0.40, 0.17, 0.23 and 0.41 OD, respectively. The ELISA gave a high signal/noise ratio, giving good sensitivity and specificity, correlating well with the indirect fluorescent antibody test (IFAT) currently used to diagnose Neospora infection in cattle, sheep and goats. In both the ELISA and immunoblot analysis using the same antigen, there was no significant cross-reactivity with sera from cattle, sheep or goats that had been infected with Toxoplasma gondii. The ELISA also showed no cross-reactivity in sera from cattle infected with Sarcocystis cruzi, Babesia divergens, B. bovis and B. bigemina. The wsf fraction of sonicated N. caninum tachyzoites used in this ELISA can be easily prepared and may be more sensitive than a single antigen ELISA, whilst still retaining good specificity. PMID:9777723

  10. Clinical Evaluation of BioPlex 2200 HIV Ag-Ab, an Automated Screening Method Providing Discrete Detection of HIV-1 p24 Antigen, HIV-1 Antibody, and HIV-2 Antibody

    OpenAIRE

    Salmona, Maud; Delarue, Severine; Delaugerre, Constance; Simon, François; Maylin, Sarah

    2014-01-01

    Early and accurate diagnosis is essential for optimal therapeutic outcomes in patients infected with HIV. Currently, none of the commercially available fourth-generation assays differentiate HIV-1 and HIV-2 antibodies (Ab) or the HIV-1 p24 antigen (Ag). The aim of this study was to evaluate the performance of a novel assay, the BioPlex 2200 HIV Ag-Ab. This assay uses a multiplex flow immunoassay design allowing the simultaneous detection and identification of antibodies to HIV-1 (groups M and...

  11. Whole-Blood Counting Immunoassay as a Short-Turnaround Test for Detection of Hepatitis B Surface Antigen, Anti-Hepatitis C Virus Antibodies, and Anti-Treponema pallidum Antibodies

    OpenAIRE

    Kudo, Toyoichiro; Kido, Aiko; Nishiyama, Yukiko; Koganeya, Hiroshi; Okuda, Takako; Nabeshima, Motoshige; Iinuma, Yoshitsugu; Ichiyama, Satoshi

    2004-01-01

    Whole-blood samples were used for a counting immunoassay (CIA) with the aim of developing a short- turnaround test. After optimization of the CIA, hepatitis B surface antigen (HBsAg), anti-hepatitis C virus antibodies (anti-HCV), and anti-Treponema pallidum antibodies (anti-TP) were detected as efficiently as by an enzyme immunoassay (EIA) with serum samples. The correlations between whole-blood CIA and serum EIA were 99.8, 97.1, and 99.4% for HBsAg, anti-HCV, and anti-TP, respectively. Whole...

  12. Utility of Direct Fluorescent Antibody Test for detection of Chlamydia trachomatis and its detection in male patients with non gonococcal urethritis in New Delhi

    Directory of Open Access Journals (Sweden)

    Agrawal S

    2003-03-01

    Full Text Available The purpose of this study was assessment of prevalence of Chlamydia trachomatis and utility of Direct Fluorescent Antibody (DFA test for its detection in male patients with non gonococcal urethritis in New Delhi , India Thirty male patients with symptoms of dysuria showing polymorphs in their gram stained urethral smears with no evidence of Neisseria gonorrhoeae, and negative for Trichomonas vaginalis and Candida albicans by wet mount were subjected to DFA test for detection of C. trachomatis in urethral samples. Microscopic examination of gram stained urethral smears revealed 5-7 polymorphs / HPF in 90% of the patients. Evidence of C. trachomatis with DFA (MicroTrak was detected in 11 cases (36.67% when a cut off of 10 elementary bodies was considered essential. It is concluded that C. trachomatis is an important cause of non gonococcal urethritis in male patients in New Delhi and DFA test is a useful diagnostic tool in its detection. Where facilities are not available for its detection antichlamydial therapy should be recommended emperically.

  13. Direct solid-phase radioimmunoassay for the detection of Aujeszky's disease antibodies

    International Nuclear Information System (INIS)

    A direct solid-phase radioimmunoassy (dRIA) was developed in order to demonstrate antibodies against Aujeszky's disease virus (ADV) in sera obtained from pigs and rabbits. In the presence of guinea-pig complement the above test is 160-fold to 1500-fold more sensitive than the neutralization test (NT) and 320-fold to 150 000-fold more sensitive than sera obtained from an ADV-infected farm, which were found to be negative in the complement assisted NT. It is possible to test a single dilution of unknown serum by dRIA by comparing same with a standard curve and to make a statement regarding its ADV-specific binding capacity to 125I-labelled ADV antigen. The advantages of dRIA in comparison to the indirect RIA and the advantages and disadvantages with regard to ELISA were discussed. (orig.)

  14. Developing a Noninvasive Procedure Using Labeled Monoclonal Antibody Anti-VEGF (Bevacizumab for Detection of Endometriosis

    Directory of Open Access Journals (Sweden)

    Daniel Escorsim Machado

    2015-01-01

    Full Text Available The off-label use of bevacizumab labeled with 99mTc as a new radiopharmaceutical for imaging of endometriosis is a promising noninvasive, new clinical procedure. The bevacizumab in monoclonal antibodies targeted at vascular endothelial growth factor (VEGF is superexpressed in cases of endometriosis. In this study we evaluate the imaging of endometriosis lesion in rats (induced to endometriosis using bevacizumab-99mTc. The results showed that bevacizumab-99mTc imaged the lesion and support his use for Nuclear Medicine applied to gynecology. Also the results appointed that this radiopharmaceutical has a hepatobiliary excretion. It is important to notice that the dose used was almost 0,01% of the usual dose for the bevacizumab.

  15. A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans.

    Science.gov (United States)

    Baumann, D; Gottstein, B

    1987-06-01

    A double-antibody sandwich ELISA was developed to detect detergent-solubilized antigens of Entamoeba histolytica in stool samples of humans. The test system was evaluated for its methodical and diagnostic sensitivity and specificity. In recovery experiments the lower limit of detection was 400 ng E. histolytica (HK9) protein/ml stool, corresponding to approximately 2000 amoebic trophozoites/ml stool. Samples of 97 patients with suspected intestinal amoebiasis were examined. Specific antigens were detected by ELISA (= positive reaction) in 14 (93%) out of 15 stool samples containing trophozoites of E. histolytica. In contrast, 68 (93%) of 73 samples with other protozoa, including Entamoeba coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii and Giardia lamblia, did not react in the test system (= negative reaction). The test was shown to detect only trophozoites of E. histolytica and not the cyst stage. This fact could facilitate the differentiation between cyst carriers and persons excreting trophozoites. The results of this preliminary study justify a further large scale evaluation of the test system. PMID:2888183

  16. Comparative Study of ELISA and IIFT in Detecting Chlamydia Pneumoniae Specific IgG Antibodies in Patients of Acute Coronary Syndrome (ACS

    Directory of Open Access Journals (Sweden)

    Bhuva S P, Bhuva P J, Javdekar T B, Jain M R, Mulla S A

    2012-01-01

    Full Text Available Background: Chlamydia pnaumoniae is reported to be associated with coronary heart disease (CHD. Efficacy of available serologic tests for detecting C.pneumoniae antibodies has been debated. The aim of present study was to compare Enzyme Linked Immunosorbant Assay (ELISA and Indirect Immunofluorescent Test (IIFT for detecting specific antichlamydial antibodies in patients of Acute Coronary Syndrome (ACS. Materials and Methods: Serum samples from 100 patients of ACS and 90 healthy controls were tested for the presence of Chlamydial IgG antibodies using ELISA and IIFT. To assess agreement between ELISA and IIFT, we used ‘nominal scale variables”. Agreement analysis was done using sensitivity, specificity, positive and negative predictive values (PPV and NPV and diagnostic accuracy of the test. Results: The ELISA and IIFT detected C.pneumoniae IgG antibodies in 66% and 48% respectively in patients of ACS, and 29% and 20% respectively of healthy controls. In patients of ACS, sensitivity and specificity of ELISA as compared to IIFT were 70.8% and 38.4% respectively. The PPV of ELISA for C.pneumoniae was 51.5% and NPV was 58.8%. The diagnostic accuracy of ELISA for C.pneumoniae was 54.6%. The two tests correlated in 54% of samples with a moderate agreement of =0.51. Conclusions: The results of present study indicate that ELISA test was inferior to IIFT in detecting C.pneumoniae antibodies in patients of ACS.

  17. An assay for the detection of grapevine leafroll-associated virus 3 using a single-chain fragment variable antibody.

    Science.gov (United States)

    Cogotzi, Laura; Giampetruzzi, Annalisa; Nölke, Greta; Orecchia, Martin; Elicio, Vito; Castellano, Maria Antonietta; Martelli, Giovanni P; Fischer, Rainer; Schillberg, Stefan; Saldarelli, Pasquale

    2009-01-01

    Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent C(L)-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The C(L)-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection. PMID:19082687

  18. Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase immunoprecipitation system.

    Science.gov (United States)

    Berguido, Francisco J; Bodjo, Sanne Charles; Loitsch, Angelika; Diallo, Adama

    2016-01-01

    Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR. PMID:26506137

  19. Detection of L1, infectious virions and anti-L1 antibody in domestic rabbits infected with cottontail rabbit papillomavirus.

    Science.gov (United States)

    Hu, Jiafen; Budgeon, Lynn R; Cladel, Nancy M; Culp, Timothy D; Balogh, Karla K; Christensen, Neil D

    2007-12-01

    Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections. PMID:18024897

  20. Diagnostic sensitivity of two radio receptor assays (TRAK Assay and TRAK Dyno human) for the detection of TSH receptor antibodies

    International Nuclear Information System (INIS)

    Radio receptor assays for the detection of TSH receptor antibodies in serum are typically based on binding the competition of TSH-R antibodies and 125I-labelled-TSH for membrane preparation of thyrocytes (TBII tests). The sensitivity of the available tests utilizing porcine cell membranes was found to be around 80%. A new test (TRAK Dyno human, BRAHMS) utilizes human recombinant TSH receptor and human standard material that is supposed to improve the performance of the test. We have compared the results of these two assays. The sensitivity of the TRAK Assay tested in 356 patients with untreated Grave's disease was found to be 85%, and 97.5% for TRAK Dyno human in 111 newly diagnosed patients. Both tests were performed from the same serum specimen for 60 of the investigated patients. The TRAK Assay was positive in 50 patients (83.2%) and TRAK Dyno human in 59 patients (98.3%). The specificity of the new radio receptor assay was also improved. (author)