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Sample records for blocking enzyme-linked immunosorbent

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Microwave-mediated enzyme-linked immunosorbent assay procedure.

    Science.gov (United States)

    Nahar, Pradip; Bora, Utpal; Sharma, Gainda L; Kannoujia, Dileep Kumar

    2012-02-15

    Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), human interleuken 1β (IL-1β), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diagnosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters. PMID:22033289

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

    OpenAIRE

    Corena de Beer; Monika Esser; Wolfgang Preiser

    2011-01-01

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

  4. Indirect enzyme-linked immunosorbent assay for the mycotoxin zearalenone.

    OpenAIRE

    Liu, M T; Ram, B P; Hart, L P; Pestka, J J

    1985-01-01

    A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone, an estrogenic mycotoxin. Zearalenone was converted to zearalenone-6'-carboxymethyloxime and conjugated to bovine serum albumin and poly-L-lysine for use as immunogen and solid-phase marker, respectively. Immunization of rabbits with the bovine serum albumin conjugate resulted in zearalenone antibody titers of 20,480 in 11 weeks. A competitive indirect ELISA was conducted by simulta...

  5. Enzyme-linked immunosorbent assay for shigella toxin.

    OpenAIRE

    Donohue-Rolfe, A; Kelley, M A; Bennish, M; Keusch, G T

    1986-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of shigella toxin. For the assay, a mouse monoclonal antibody against the B subunit of the toxin and a rabbit polyclonal antibody against the holotoxin were employed. The monoclonal antibody was used to coat wells of a microtiter plate, and the polyclonal antibody preparation was used as the detecting antibody. The amount of bound polyclonal antibody was determined by using a goat anti-rabbit immunoglobulin G-alkalin...

  6. Enzyme-linked immunosorbent assay for group B streptococcal antibodies.

    OpenAIRE

    Rote, N S; N. L. TAYLOR; Shigeoka, A O; Scott, J R; Hill, H R

    1980-01-01

    We report here on the development of enzyme-linked immunosorbent assays (ELISAs) for antibodies to types II and III group B streptococci. Streptococcal antigens were prepared by trichloroacetic acid extraction and fractional alcohol precipitation. Microtiter wells were coated with antigen in 0.1 M carbonate buffer at pH 9.6. Lyophilization was found to be an essential step for efficient binding of the streptococcal antigens. After incubation with antibody-containing rabbit serum, bound antibo...

  7. Enzyme-linked immunosorbent assays for Snow Mountain and Norwalk agents of viral gastroenteritis.

    OpenAIRE

    Madore, H P; Treanor, J J; Pray, K A; Dolin, R

    1986-01-01

    Enzyme-linked immunosorbent assays (ELISAs) for antigen detection and blocking ELISAs for serum antibody rises were developed for the Snow Mountain and Norwalk agents of viral gastroenteritis. The ELISAs were as sensitive as the existing radioimmunoassays and were specific for the Snow Mountain or Norwalk agent. The blocking ELISAs detected the same number of significant rises in antibodies to these agents as did the existing blocking radioimmunoassays.

  8. Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays.

    OpenAIRE

    Bryant, R. E.; Chamovitz, B N; Morse, S A; Apicella, M A; Morthland, V H

    1983-01-01

    The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat ant...

  9. Enzyme-linked immunosorbent assay for triclocarban in aquatic environments.

    Science.gov (United States)

    Zeng, Kun; Zou, Yanmin; Liu, Jianxia; Wei, Wei; Zhang, Meng; Zhou, Jun; Zhang, Zhen; Gai, Zikuan

    2015-01-01

    A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of triclocarban (TCC) in waters and sediments. Haptens were synthesized by derivatizing the paraposition of a phenyl moiety of TCC. The synthesized hapten was then coupled to bovine thyroglobulin to be used as an immunogen, based on which, a high affinity monoclonal antibody 4D5 was produced with the hybridoma technique. Under the optimized conditions, using the monoclonal antibody, excellent performances of the assay were obtained: satisfactory sensitivity (IC50 (50% inhibition concentration) value, 0.43 ng/mL; limit of detection, 0.05 ng/mL); good linear range (0.05-10 ng/mL); and satisfactory accuracy (recoveries 70.7-107% in waters; 74.8-98.3% in sediments). Furthermore, TCC was found with the concentration ranging from not detected to 422.12 ng/L in waters and from 6.68 ng/g to 78.67 ng/g in sediments in Yunliang River, Ancient Canal and Hongqiao Port in Zhenjiang City. In conclusion ELISA could be applied for monitoring TCC in aquatic environments. PMID:26540528

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

    Directory of Open Access Journals (Sweden)

    Corena de Beer

    2011-12-01

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

  11. Detection of plant viruses by enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Investigations were carried out to check the sensitivity, specificity and efficiency of the enzyme-linked immunosorbent assay (ELISA) test for the detection of potato leafroll virus, potato virus Y, potato virus S, potato virus M and beet necrotic yellow vein virus (BNYVV). For the identification of potato viruses, a comparative test of the conventional eye-shoot and ELISA techniques was performed on leaf and tuber material. A good correlation (88-100%) was achieved between the results obtained with these methods and the large differences between the extinction values of infected and healthy plant material (positive results were 13-49 times higher than negative ones). For detection of BNYVV in sugar-beets, the ELISA method and immune electron microscopy (the Derrick method) were compared. Correlation between the results obtained with the two assays was 78 and 87%, respectively. Owing to the low titre of immunized gammaglobulin and the long reaction time, after pipetting, of the p-nitrophenylphosphate, overlapping extinction values of the positive and negative samples occurred in classes 0.100-0.200. Investigations of the host range of BNYVV were carried out by ELISA and inoculation of test plants. Chenopodium quinoa was used as the indicator plant. Six days after mechanical inoculation, plants showed yellow-coloured local lesions with necrotic centres. If the concentration of BNYVV was low in the original plant material, inoculated indicator plants showed no symptoms of virus infection. After reinoculation of a second generation, specific lesions occurred. If high-quality test kits are available, ELISA is a very sensitive and rapid method for the detection of plant viruses. The ELISA technique is as reliable, or even more reliable, than the best conventional methods. (author)

  12. Enzyme-Linked Immunosorbent Assay for Immunoglobulin G Antibody to Encephalomyocarditis Virus

    OpenAIRE

    Shanley, John D.

    1980-01-01

    An enzyme-linked immunosorbent assay for immunoglobulin G antibody to encephalomyocarditis virus was developed. This assay was comparable to antibody assay by neutralization. Its adaptability should be useful for laboratory and epidemiological studies of infections due to encephalomyocarditis virus.

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

  14. Enzyme-linked immunosorbent assay for Salmonella typhimurium in food: feasibility of 1-day Salmonella detection.

    OpenAIRE

    Lee, H. A.; Wyatt, G M; Bramham, S; Morgan, M.R.

    1990-01-01

    A microtitration plate, antibody-capture, enzyme-linked immunosorbent assay was developed for detection of Salmonella typhimurium. The assay utilizes a monoclonal detector antibody which shows no cross-reactions with non-Salmonella species and only a slight cross-reaction with one other Salmonella serotype. By using only one cultural stage (in a nonselective, chemically defined medium) prior to the enzyme-linked immunosorbent assay, low numbers of cells in food (10 cells 25 g-1) were detected...

  15. A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

    Science.gov (United States)

    Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

    2012-01-01

    ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  17. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Science.gov (United States)

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

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

    DEFF Research Database (Denmark)

    Petersen, H. H.; Nielsen, J. P.; Jensen, A.L.; Heegaard, Peter M. H.

    2001-01-01

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

  19. USE OF ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA) FOR THE DETERMINATION OF TRITON X NONIONIC DETERGENTS

    Science.gov (United States)

    An enzyme-linked immunosorbent assay (ELISA) for 4-t-octylphenyl ethoxylates such as Triton X-100 was developed. Both the 4-t-octylphenyl and the ethoxylate moiety were required for antibody recognition since members of the Triton N series showed low cross-reactivity, and polyeth...

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

    DEFF Research Database (Denmark)

    Dziegiel, M; Borre, Mette; Jepsen, S;

    1991-01-01

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

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

    OpenAIRE

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

    1995-01-01

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

  2. 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...

  3. Development of an Enzyme-Linked Immunosorbent Assay for Immunoglobulin M Antibodies against Measles Virus

    OpenAIRE

    Roodbari, F.; Roustai, M. H.; A Mostafaie; Soleimanjdahi, H.; Foroshani, R. Sarrami; F Sabahi

    2003-01-01

    Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and th...

  4. 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...

  5. Conglutinating complex fixation test (CCFR) as a method alternate to enzyme linked immunosorbent assay (ELISA)

    International Nuclear Information System (INIS)

    An enzyme linked immunosorbent assay (ELISA) has proved to be the most versatile and sensitive method for the serological diagnosis of infectious animal diseases. However, other assays offer similar advantages. This has been shown by our research through the development of a conglutinating complex fixation test (CCFT). This is analogous to the complement fixation test (CFT) and is based on the phenomenon of conglutination. The limiting factor in this test is the source in cattle blood

  6. Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Dengue Capsid

    OpenAIRE

    Selvarajah, Suganya; Chatterji, Udayan; Kuhn, Richard; Kinney, Richard; Vasudevan, Subhash G.; Gallay, Philippe

    2012-01-01

    The astonishing speed with which Dengue has spread across the world and the severity of its infection make Dengue a prime threat to human life worldwide. Unfortunately, to date there are no effective vaccines or treatments against Dengue. Since only a few assays permit rapid and sensitive detection of Dengue, we developed a specific antigen capture enzyme-linked immunosorbent assay (ELISA) for the abundant structural Dengue-2 capsid protein. We showed that the ELISA allows rapid and sensitive...

  7. 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...

  8. Usefulness of an enzyme-linked immunosorbent assay for detection of Giardia antigen in feces.

    OpenAIRE

    Nash, T E; Herrington, D A; Levine, M M

    1987-01-01

    The usefulness of a recently developed enzyme-linked immunosorbent assay which detects Giardia lamblia antigen in feces was determined in experimentally infected humans. Giardia antigen was determined in serially collected fecal specimens from humans inoculated with two Giardia isolates, GS/M and Isr. A total of 277 stools from 18 volunteers were tested, 74 from Isr-inoculated volunteers and 203 from GS/M-inoculated volunteers. None of the five Isr-inoculated volunteers became infected, and n...

  9. Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

    OpenAIRE

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K.

    2015-01-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as t...

  10. Enzyme-linked immunosorbent assay for quantitation of attachment and ingestion stages of bacterial phagocytosis.

    OpenAIRE

    Athamna, A; Ofek, I

    1988-01-01

    Research on phagocytosis of bacteria is often hampered by the inability to distinguish quantitatively between bacteria that have been ingested by phagocytic cells and those which are attached to the surface of the cells. A method using the enzyme-linked immunosorbent assay technique to simply and accurately measure the rate of bacterial ingestion by phagocytic cells is described. The method is based on the ability of antibacterial antibodies to bind to bacteria attached to but not internalize...

  11. Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens.

    OpenAIRE

    Rigby, C E

    1984-01-01

    An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Tre...

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

    NARCIS (Netherlands)

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

    1991-01-01

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

  13. Use of an enzyme-linked immunosorbent assay (Elisa) for the determination of HSP 70

    International Nuclear Information System (INIS)

    It has been suggested that HSP 70 is a good indicator to quantify thermotolerance. The authors developed an enzyme-linked immunosorbent assay (Elisa) for measuring the total amount of HSP 70 in mammalian cells. Microtitration plates were incubated with various concentrations of cell lysate in PBS. Milk proteins were used to block non-specific protein binding sites in the wells. Monoclonal antibody against purified HSP 70 raised in a mouse was added, followed by peroxidase conjugated goat-anti-mouse antibody. After washing, the bound peroxidase activity was measured colorimetrically to calculate the original amount of HSP 70 antigen. Kinetic studies indicate that the linear range of the assay corresponds to the amount of HSP 70 from 103-104 HA-1 cells (0.1-2.0 μg cytosolic proteins in each well). Elisa is a highly sensitive method for quantifying HSP 70 and can be easily used to measure the total amount of HSP 70 instead of the synthesis rate of HSP 70 by pulse radiolabeling. The authors are currently developing the sensitive Elisa system for measurement of thermotolerance in human tumors. A comparison of measurement of HSP 70 by pulse labeling the cells with 35S-methionine and by Elisa has been done in HA-1 cells. HA-1 cells were exposed to 450C, 10 min, then incubated at 370C for 0-24 h. These results show that the rate of HSP synthesis increased several fold, although the total amount of HSP 70 is increased less than 2 fold during thermotolerance induction. These results are consistent with data in the literature obtained by other techniques

  14. 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...

  15. Enzyme-linked immunosorbent assay for measurement of antibody to type III group B streptococci.

    OpenAIRE

    Polin, R.A.; Douglas, S D; Kasper, D L; Baker, C J

    1982-01-01

    Neonates at risk for fulminant type III, group B streptococcal (III GBS) infection are those who lack antibody to the capsular polysaccharide. A newly developed enzyme-linked immunosorbent assay (ELISA) was compared with a standard radioactive antigen-binding assay (RABA) for quantitation of III GBS antibody in human sera. Although there was a significant correlation between the ELISA and RABA (r = 0.81; P less than 0.001) in general, the ELISA detected antibody both to core and native antige...

  16. 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...

  17. 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...

  18. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

    OpenAIRE

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae; Kang, Hwan-Goo

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate t...

  19. Use of Enzyme Linked Immunosorbent Assay for detection of aflatoxin M1 in milk powder

    OpenAIRE

    S. D. Al-Sawaf; O. A. Abdullah; O. H. Sheet

    2012-01-01

    Thirty five samples were collected from seven types of milk powder in Mosul city markets, and surveyed for the presence of aflatoxin M1 (AFM1), by using Enzyme Linked Immunosorbent Assay (ELISA) technique. Analytical results showed that 82.8% of the samples were contaminated with AFM1. The incidence of AFM1 in Al-mudhish, Angolac, Dielac, Lona, Nido, Melgro and Multi samples were 40, 50, 80, 100, 100, 100 and 100%, respectively. The occurrence of AFM1 in milk powder, 79.3% (23 out of 29) of p...

  20. Glycoprotein-based enzyme-linked immunosorbent assays for serodiagnosis of infectious laryngotracheitis.

    Science.gov (United States)

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K

    2015-05-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

  1. Enumeration of Treponema pallidum Cells Cultivated In Vitro by an Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Cox, D. L.; Moeckli, R A; Keaney, K M

    1984-01-01

    An enzyme-linked immunosorbent assay was developed to enumerate Treponema pallidum cells. The assay could detect from 2 X 10(7) to 4 X 10(8) treponemes per ml. Reactive rabbit serum and goat anti-rabbit immunoglobulin G (peroxidase conjugate) were used in the assay. Optimum results were obtained when 2,2'-azino-di(ethylbenzthiazolinesulfonic acid) was used as the dye for the enzyme reaction and the reactions were allowed to run for 45 min. Interestingly, assays in which in vivo-cultivated T. ...

  2. Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Machado Eleuza Rodrigues

    2003-01-01

    Full Text Available The present study was conducted to detected IgG antibodies using Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by the enzyme-linked immunosorbent assay (ELISA. Sera from 90 subjects were analyzed (30 with strongyloidiasis, 30 with other parasites and 30 healthy individuals. Results were expressed in antibody titers, which were considered as positive when titer was > 80. Sensibility and specificity of the assay were 100% and 96.7%, respectively. It can be concluded that the heterologous alkaline extract could be employed in ELISA as a diagnostic aid in human strongyloidiasis, due to its advantages as easiness of obtaining, practicability in preparing, and high indexes of sensitivity and specificity.

  3. Preparation of TSH kit by an enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    The method is established by the two-step with enzyme-linked immunosorbent assay (ELISA) using polyclonal antibody for coating, one monoclonal for labelling with horseradish peroxidase. The sensitivity is 0.06 mIU/L and analytical recovery of TSH is (98.4-101.4)%. The inter- and intra-assay CV are lower than 9.1% and 5.9% respectively. The average concentration of TSH in sera from health human (77) is 1.96 mIU/L. The correlation coefficient of results with TSH IRMA is 0.992. The assay is rapid, sensitive, simple and precise. It is suitable for clinical and research application

  4. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    Energy Technology Data Exchange (ETDEWEB)

    Muir, D.; Varon, S.; Manthorpe, M. (Univ. of California, San Diego, La Jolla (USA))

    1990-03-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

  5. Magnetic enzyme-linked immunosorbent assay (MELISA) for determination of specific IgG in paracoccidioidomycosis.

    Science.gov (United States)

    de Camargo, Z P; Guesdon, J L; Drouhet, E; Improvisi, L

    1984-01-01

    A magnetic solid phase enzyme-linked immunosorbent assay (MELISA) for quantification of IgG antibodies to somatic and metabolic antigens of Paracoccidioides brasiliensis was developed. Activation of magnetic polyacrylamide agarose beads with concanavalin A was superior to glutaraldehyde activation, and test sensitivity was higher for somatic than for metabolic antigens. Comparative MELISA, counterimmunoelectrophoresis and erythroimmunoassay tests with sera from 33 proven cases of paracoccidioidomycosis, 14 cases of histoplasmosis and 20 normal human sera showed the MELISA could distinguish antibody levels in paracoccidioidomycosis from those in normal sera; however two sera from histoplasmosis cases cross-reacted in the MELISA. MELISA is a rapid test (5-6 h) and the results suggest it has considerable potential value for assay of anti-P. brasiliensis antibodies. PMID:6438813

  6. Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

    Science.gov (United States)

    el Idrissi, A H; Ward, G E

    1992-06-15

    Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias. PMID:1496812

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

    DEFF Research Database (Denmark)

    Bak, H.; Sørensen, Vibeke

    2006-01-01

    tests. This QC panel was included as an internal QC to be tested every month. Besides the QC panel, 2 panels containing 100 samples of meat juice with OD above the positive cut-off value and 100 samples with OD below that value were prepared for quarterly control of the diagnostic sensitivity (DSe) and......In the Danish pig production system, an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies in meat juice is used for Salmonella surveillance. Quality control (QC) of this ELISA was previously based on repeated testing of control serum samples. The purpose of the study...... reported here was to collect, characterize, and implement a panel of meat juice pools for supplemental internal QC. Muscle samples for extraction of meat juice were collected from slaughter pigs of 5 herds infected with Salmonella spp. and from 4 herds without Salmonella infection. A QC panel with 39 pools...

  8. 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.

  9. Measurement of antibody titer to fowl pox virus by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Iritani, Y; Sawaguchi, K

    1994-12-01

    The usefulness of the measurement of antibody titer to fowl pox virus (FPV) by enzyme-linked immunosorbent assay (ELISA) was evaluated in SPF chickens with or without inoculation with FPV. The optimum concentration of purified antigen was 10 micrograms/ml of protein. The absorbance at 492 nm was less than 0.10 in the chickens negative to FPV from 1 to 63 days old. By contrast, a higher titer was detected in SPF chickens with various FPVs inoculated into the wing web than in non-inoculated chickens. Moreover, there was no cross response to chicken sera immunized with Haemophilus paragallinarum, Marek's disease virus, Newcastle disease virus or infectious bronchitis virus. The titers increased after vaccination were not increased after subsequent challenge with virulent FPV. These findings suggested the usefulness of the measurement of the antibody response to FPV vaccine by ELISA. PMID:7696418

  10. Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Battelli, M G; Abbondanza, A; Musiani, S; Buonamici, L; Strocchi, P; Tazzari, P L; Gramantieri, L; Stirpe, F

    1999-03-01

    Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range. PMID:10217635

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

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

    DEFF Research Database (Denmark)

    Bak, H.; Sørensen, Vibeke

    2006-01-01

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

  13. Determination of biotin concentration by a competitive enzyme-linked immunosorbent assay (ELISA) method.

    Science.gov (United States)

    Chang, Y S; Wu, C H; Chang, R J; Shiuan, D

    1994-12-01

    A method based on competitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of biotin concentrations which takes advantage of the extraordinarily high affinity between biotin and avidin. The biotin assay consisted of two steps, (i) a competition reaction between excess streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and solutions of known biotin concentrations or sample solutions and (ii) the measurement of the residual activities of the free form streptavidin-HRP which were correlated with the initial biotin concentrations. The procedure was modified by including an extra step of antibody-antigen interaction to assay biotin concentration unambiguously in more complex media. The entire assay was completed within 6 with sensitivities of approximately 1 pg/ml for biotin in a simple aqueous medium and 5 pg/ml in complex media. The method offers significant advantages in time, sensitivity and simplicity for determinations of biotin concentrations in various solutions. PMID:7699208

  14. A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for chlorpyrifos residue determination in Chinese agricultural smaples

    Science.gov (United States)

    A monoclonal antibody-based competitive antibody-coated enzyme-linked immunosorbent assay (ELISA) was developed and optimized for determining chlorpyrifos residue in agricultural products. The IC50 and IC10 of this ELISA were 3.3 ng/mL and 0.1 ng/mL respectively. The average recoveries recovery rate...

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  16. Enzyme-linked immunosorbent assay for the detection of bovine rennet whey powder in milk powder and buttermilk powder

    NARCIS (Netherlands)

    Bremer, M.G.E.G.; Kemmers-Voncken, A.; Boers, E.A.M.; Frankhuizen, R.; Haasnoot, W.

    2008-01-01

    An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of bovine rennet whey (BRW) solids in skim milk powders (SMP) and buttermilk powders is presented. The BRW content was determined in a neutralised trichloroacetic acid sample extract by binding of the dissolved caseinomacropep

  17. Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

    Science.gov (United States)

    Ott, Laura E.; Carson, Susan

    2014-01-01

    Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from...

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  20. Examination of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

    International Nuclear Information System (INIS)

    Fluorogenic and chromogenic substrates were used in direct and trapping enzyme linked immunosorbent assays for the detection of mouse IgG and foot and mouth disease virus (FMDV). The detection limits for both antigens were compared, using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results. (author). 3 refs, 6 figs, 5 tabs

  1. Mixed enzyme-linked immunosorbent assay (MELISA) for HLA class I antigen: a plasma membrane marker.

    Science.gov (United States)

    Bjerrum, O W; Borregaard, N

    1990-03-01

    This study introduces a simple, reproducible assay for HLA class I antigen using antibodies against beta 2-microglobulin and the heavy chain on HLA. The sandwich technique was named mixed enzyme-linked immunosorbent assay (MELISA), and was designed for identification of plasma membranes in neutrophil subcellular fractions. The subcellular localization of HLA was identical to that of other plasma membrane markers, [3H]concanavalin A and detergent-independent alkaline phosphatase, and was unchanged by stimulation of cells by weak and strong secretagogues. In addition to the presence as part of the HLA complex in the plasma membrane uncomplexed beta 2-microglobulin is present in the specific granules of neutrophils. However, the release of beta 2-microglobulin from intact neutrophils stimulated with formyl-methionylleucylphenylalanine was much higher than could be explained by exocytosis of specific granules. Subcellular fractionation studies demonstrated that beta 2-microglobulin is localized in fractions characterized by latent alkaline phosphatase and released from this novel secretory compartment in response to stimulation with formyl-methionylleucylphenylalanine. PMID:2181625

  2. Development of an enzyme-linked immunosorbent assay for bisphenol a using chicken immunoglobulins.

    Science.gov (United States)

    De Meulenaer, Bruno; Baert, Katleen; Lanckriet, Heikki; Van Hoed, Vera; Huyghebaert, Andre

    2002-09-11

    Bisphenol A was coupled, after derivatization into a suitable hapten, to bovine serum albumin and ovalbumin in order to produce immunizing and coating antigens. The immunizing antigens were injected into chickens, which allowed the isolation of specific bisphenol A immunoglobulins from the egg yolk. These antibodies were used in an indirect competitive enzyme-linked immunosorbent assay for the determination of bisphenol A in aqueous solutions. Various parameters, influencing the assay sensitivity, were evaluated. The applicability of the assay for the determination of bisphenol A in milk was also studied. The assay was not as sensitive as other analytical techniques used in bisphenol A analysis, since typical I(50) levels of 2.5 microM were reached in aqueous solutions. This study nevertheless illustrates the usefulness and the potency of chicken antibodies in the analysis of migration residues from packaging materials using immunochemical techniques. In addition, the assay showed to be quite specific for bisphenol A as well. Only for bisphenol A analogues, cross reactivities of about 40% were reached, enabling the use of the antibodies for the screening of bisphenol A and alike compounds. PMID:12207461

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

    Science.gov (United States)

    Akama, Kenji; Shirai, Kentaro; Suzuki, Seigo

    2016-07-19

    Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers. PMID:27322525

  4. Reliability of soluble IL-2 receptor measurements obtained with enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Using an enzyme-linked immunosorbent assay (ELISA), human soluble interleukin-2 receptors (IL-2R) were measured in the serum of patients with various autoimmune system diseases. To study the sensitivity and specificity of the assay, soluble IL-2Rs were measured in the culture supernatants and in the cell extracts of peripheral blood mononuclear cells activated with phytohemagglutinin (PHA), purified protein derivative of tuberculin, and allogeneic lymphocytes, as well as in the serum of patients with various collagen diseases. The results correlated well with reports from other laboratories. For example, when stimulated by PHA, the greatest amount of soluble IL-2Rs was produced at the fastest rate. In addition, soluble IL-2R levels in the serum of collagen disease patients were significantly higher than those in healthy persons, who themselves exhibited low levels of detectable soluble IL-2Rs. It is hoped that reliable ELISA measurements of soluble IL-2Rs in the serum of atomic bomb survivors will assist in the interpretation of data collected during the work described in RP 2-87, a study of autoimmunity and autoimmune diseases in the Adult Health Study. (author)

  5. Standardisation of an indirect enzyme-linked immunosorbent assay for the detection

    Directory of Open Access Journals (Sweden)

    Manuela Tittarelli

    2011-01-01

    Full Text Available An indirect enzyme-linked immunosorbent assay (ELISA was evaluated for the detection of Brucella antibodies in milk from water buffalo (Bubalus bubalis Linnaeus, 1758. The test accuracy was evaluated on milk samples from the Campania Region in Italy. A total of 100 negative samples were collected from 10 officially brucellosis-free herds in Salerno Province, while 30 positive samples were collected from 3 herds in Caserta Province, where animals gave positive results to the official tests and it was here that Brucella abortus biovar 1 had been isolated. Test sensitivity was 100%, with a confidence interval (CI of 90.8%-100%, while specificity was 98% (CI 93%-99.4% on individual milk samples. To simulate bulk milk samples from herds infected at various levels of infection, dilutions from 1:10 to 1:100 of positive milk samples in negative milk were also used. The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:50 in negative milk. Considering the average national water buffalo herd size, the probability of identifying infection in a water buffalo herd by bulk milk testing is 50% (CI 33.1%-66.9% in the worst case scenario of a single infected animal contributing to the bulk milk.

  6. A Review of Cry Protein Detection with Enzyme-Linked Immunosorbent Assays.

    Science.gov (United States)

    Albright, Vurtice C; Hellmich, Richard L; Coats, Joel R

    2016-03-23

    The widespread use of Cry proteins in insecticide formulations and transgenic crops for insect control has led to an increased interest in the environmental fate of these proteins. Although several detection methods are available to monitor the fate of Cry proteins in the environment, enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method, due to their cost-effectiveness, ease of use, and rapid results. Validation of ELISAs is necessary to ensure accurate measurements of Cry protein concentrations in the environment. Validation methodology has been extensively researched and published for the areas of sensitivity, specificity, accuracy, and precision; however, cross validation of ELISA results has been studied to a lesser extent. This review discusses the use of ELISAs for detection of Cry proteins in environmental samples and validation of ELISAs and introduces cross validation. The state of Cry protein environmental fate research is considered through a critical review of published literature to identify areas where the use of validation protocols can be improved. PMID:26949828

  7. Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin.

    Science.gov (United States)

    Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae; Kang, Hwan-Goo

    2015-01-01

    Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

  8. Fowl cholera immunity in broiler breeder chickens determined by the enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Dick, J W; Johnson, J W

    1985-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in broiler breeders vaccinated (wing web) with the CU fowl cholera vaccine. Birds were bled weekly from 9 to 26 weeks, every other week from 26 to 40 weeks, and every 4 weeks from 40 to 56 weeks of age. Overall mean ELISA antibody titers (9 to 56 weeks) reported as log10 values and survivability of the vaccinates after virulent challenge were as follows: unvaccinated--5.75, 48%; birds vaccinated once at 8 weeks--5.91, 78%; birds vaccinated twice at 8 and 14 weeks--6.11, 100%; birds vaccinated thrice at 8, 14, and 20 weeks--6.23, 100%; birds vaccinated twice at 8 and 20 weeks--6.12, 100%; and birds vaccinated twice at 8 and 20 weeks (plus fowl pox at 8 weeks)--6.08, 95%. Survivability in the vaccinates after virulent challenge with strain X-73 Pasteurella multocida was 100% in birds with ELISA antibody titers (log10) greater than 6.02. Under the conditions of this experiment, birds vaccinated two or three times between 8 and 20 weeks proved to be sufficiently immune at 56 weeks of age to withstand a virulent fowl cholera challenge. Birds not vaccinated or vaccinated only once at 8 weeks were not sufficiently immunized to withstand virulent challenge. PMID:4074239

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

    Directory of Open Access Journals (Sweden)

    Adin Priadi

    2006-10-01

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

  10. Use of Enzyme Linked Immunosorbent Assay for detection of aflatoxin M1 in milk powder

    Directory of Open Access Journals (Sweden)

    S. D. Al-Sawaf

    2012-01-01

    Full Text Available Thirty five samples were collected from seven types of milk powder in Mosul city markets, and surveyed for the presence of aflatoxin M1 (AFM1, by using Enzyme Linked Immunosorbent Assay (ELISA technique. Analytical results showed that 82.8% of the samples were contaminated with AFM1. The incidence of AFM1 in Al-mudhish, Angolac, Dielac, Lona, Nido, Melgro and Multi samples were 40, 50, 80, 100, 100, 100 and 100%, respectively. The occurrence of AFM1 in milk powder, 79.3% (23 out of 29 of positive samples were higher than the permissible limits, according to the European Commission (50 ng/kg, whereas 6.8% (2 out of 29 of positive samples were above the prescribed limit of US regulation (500 ng/kg. The level of AFM1 concentration in Melgro and Multi types was higher than Al-mudhish type and it had a low level of contamination, compared with other types of milk powder.

  11. The use of an enzyme-linked immunosorbent assay for rinderpest sero-monitoring in Kenya

    International Nuclear Information System (INIS)

    A recently introduced indirect enzyme-linked immunosorbent assay (ELISA) kit to detect antibodies to rinderpest virus in cattle sera was validated for use in Kenya. A negative test optical density (OD) value of 0.12 for Kenyan sera was established using 176 sera previously found to be free of neutralizing antibodies to rinderpest virus. The ELISA kit was subsequently used to assess the prevalence of antibodies in cattle populations before and after vaccination campaigns. Initially, only border districts were monitored but following outbreaks of rinderpest in three central districts more serum samples were collected in the affected areas. A total of 2,573 sera was tested between October 1987 and September 1989. Pre-vaccination sera obtained from the border districts of Busia, Bungoma and Marsabit in October and November 1987, had antibody prevalences of 41.9% 42.9% and 35.1% respectively. Post vaccination sera obtained four months later showed an increase in antibody prevalences in Busia (52.6%) and Bungoma (79.3%) districts while those for Marsabit (32.7%) district remained low. In central Kenya, sera were collected in Kiambu and Kajiado districts to assess the efficiency of emergency vaccinations following outbreaks of rinderpest in cattle. Similar levels of antibody prevalences were recorded in the two districts following the vaccination programme. However, clinical disease was controlled within one month in Kiambu while it persisted for 9 months in Kajiado. (author). 6 refs, 1 fig., 5 tabs

  12. Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody.

    Science.gov (United States)

    Alcorn, S W; Pascho, R J

    2000-05-01

    An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g. PMID:10826838

  13. Gluten determination by gliadin enzyme-linked immunosorbent assay kit: interlaboratory study.

    Science.gov (United States)

    Gabrovská, Dana; Rysová, Jana; Filová, Vanda; Plicka, Jan; Cuhra, Petr; Kubík, Martin; Barsová, Sona

    2006-01-01

    An interlaboratory study with 10 participants was performed to obtain validation and performance data for an enzyme-linked immunosorbent assay (ELISA) kit developed for quantitative gluten determination in foods. The ELISA kit used for this study is based on 2 monoclonal and 1 polyclonal antibody developed by Immunotech, a Beckman Coulter Co. This kit did not show any false positive results or cross-reactivity with oat, rice, maize, and buckwheat. The gliadin standard from the Working Group on Prolamin Analysis and Toxicity was included in the kit as reference material for calibration. All participants obtained a gliadin ELISA kit with Standard Operational Procedure and a form for recording test results. The study included 13 samples labeled as "gluten-free" and 2 samples spiked by wheat flour. Seven samples had gliadin content below the limit of quantitation (LOQ) of the method, and 1 sample exceeded the highest calibration level. Gliadin content in the range from 10 to 157 mg/kg (1st day) and from 11 to 183 mg/kg (2nd day) was found in 7 samples (including 2 spiked samples). Results of these samples were used for further statistical analysis and evaluation. The Cochran, Dixon, and Mandel statistical tests were applied for detection of outliers. The LOQ of the kit was estimated. PMID:16512241

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

    Directory of Open Access Journals (Sweden)

    Stef J. Koppelman

    2015-01-01

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

  15. Application of enzyme linked immunosorbent assays to the diagnosis of the pathogenic animal trypanosomoses

    International Nuclear Information System (INIS)

    The pathogenic animal trypanosomoses comprise a number of diseases caused by different species of vector borne trypanosomes. In Africa, trypanosomes transmitted by tsetse flies (Glossina spp.) constitute a major constraint on socioeconomic development; over ten million km2 of sub-Saharan Africa are infested by the fly and 400 million livestock are at risk from infection with Trypanosoma congolense, T. vivax and T. brucei. Diseases caused by trypanosomes also occur outside the tsetse belt. Two trypanosome species are transmitted mechanically by haematophagous biting flies, mainly tabanids, and thereby have achieved a wider geographical distribution; T. evansi occurs in North Africa, Asia and South America, while a non-tsetse-transmitted form of T. vivax is found in South America. Animal trypanosomoses are difficult to diagnose: there are no pathognomonic signs, and detection of parasites is problematic. Therefore, considerable effort has been expended to develop serological assays with improved diagnostic sensitivity for use in obtaining information on the incidence and prevalence of infection and in the management of disease in the individual animal. Many different tests have been used over the past 70 years, but it is only recently that attention has been paid to test standardization, principally because of the introduction of enzyme linked immunosorbent assays (ELISAs) for the detection of trypanosomal antibodies and antigens. Although a variety of serological diagnostic techniques have been available for many years, there is still a lack of reliable data on trypanosomosis prevalence that makes economic justification and planning of control difficult to support. Clearly, enzyme immunoassays could provide this basic information. Additionally, as the emphasis moves from eradication to control of the tsetse transmitted trypanosomoses, there will be a greater need for surveillance and diagnosis of infection in individual animals. Both antigen and antibody assays can

  16. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei

    Science.gov (United States)

    Suttisunhakul, Vichaya; Wuthiekanun, Vanaporn; Brett, Paul J.; Khusmith, Srisin; Day, Nicholas P. J.; Burtnick, Mary N.; Limmathurotsakul, Direk

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei. The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic. PMID:26912754

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

    Science.gov (United States)

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

    2002-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Priya K

    2002-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Sudarisman

    2006-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Michael J. Bangs

    2012-09-01

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

  1. Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii).

    Science.gov (United States)

    Selcer, Kyle W; Nespoli, Lisa M; Rainwater, Thomas R; Finger, Adam G; Ray, David A; Platt, Steven G; Smith, Philip N; Densmore, Llewellyn D; McMurry, Scott T

    2006-05-01

    The purpose of this study was to develop an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used for vitellogenin purification and samples from adult males were used for comparison. No differences were detected between males and females for plasma total protein concentration, as measured by Coomassie assay. However, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that female plasma contained a 210-kDa protein, presumably the vitellogenin monomer, that was absent in adult male plasma. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with a vitellogenin antiserum that was generated against a conserved vitellogenin peptide sequence. Crocodile vitellogenin was purified by two successive rounds of DEAE chromatography. The purified protein had an apparent molecular mass of 450 kDa, as determined by gel filtration chromatography, and 210 kDa on SDS-PAGE. An indirect enzyme-linked immunosorbent assay (ELISA) was then developed for C. moreletii vitellogenin. The detection limit of the assay was 20.0 ng/mL. The intra- and inter-assay coefficients of variation were 5.3% and 9.8%, respectively. The recovery of vitellogenin diluted into male plasma was 94.7%. The ELISA assay revealed that vitellogenin levels of adult female plasma during the breeding season ranged from 1.8 to 3.1 mg/mL with a mean of 2.5+/-0.25 mg/mL. No vitellogenin was detected in adult male plasma. Induction of vitellogenin in Morelet's crocodile may be a useful model system for field studies of crocodile reproduction and for investigations of endocrine disruption in this species. PMID:16448857

  2. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei.

    Science.gov (United States)

    Suttisunhakul, Vichaya; Wuthiekanun, Vanaporn; Brett, Paul J; Khusmith, Srisin; Day, Nicholas P J; Burtnick, Mary N; Limmathurotsakul, Direk; Chantratita, Narisara

    2016-05-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic. PMID:26912754

  3. The detection of canine autoantibodies to thyroid antigens by enzyme-linked immunosorbent assay, hemagglutination and indirect immunofluorescence.

    OpenAIRE

    Haines, D M; Lording, P M; Penhale, W. J.

    1984-01-01

    Autoantibodies to thyroid antigens in serums from 34 clinically hypothyroid dogs were detected by various methods. Antibodies to thyroglobulin were detectable by enzyme-linked immunosorbent assay (ELISA) in 59%, by chromic chloride hemagglutination in 53% and by indirect immunofluorescence on formalin-fixed, paraffin-embedded, trypsin-digested thyroid tissue in 73% of samples. Antibody to thyroid microsomal antigen was detectable by ELISA in 29% of serums. Indirect immunofluorescence showed c...

  4. 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...

  5. ENZYME-LINKED IMMUNOSORBENT ASSAY FOR SCREENING DIOXIN SOIL CONTAMINATION BY UNCONTROLLED COMBUSTION DURING INFORMAL RECYCLING IN SLUMS

    OpenAIRE

    Trindade, Mirta; Nording, Malin; Nichkova, Mikaela; Spinnel, Erik; Haglund, Peter; Last, Michael S.; Gee, Shirley; Hammock, Bruce; Last, Jerold A.; González-Sapienza, Gualberto; Brena, Beatriz M.

    2008-01-01

    Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELIS...

  6. 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...

  7. Electrochemical Enzyme-Linked Immunosorbent Assay (ELISA) for α-Fetoprotein Based on Glucose Detection with Multienzyme-Nanoparticle Amplification

    OpenAIRE

    Ning Gan; Bo Li; Li Lin; Bo Situ; Han-Kun Zhou; Xiao-Mao Yin; Xiao-Hui Yan; Qin-Lan Liu; Lei Zheng

    2013-01-01

    Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer....

  8. Evaluation of a Commercial Enzyme Linked Immunosorbent Assay (ELISA) for the Determination of the Neurotoxin BMAA in Surface Waters

    OpenAIRE

    Faassen, E.J.; Beekman-Lukassen, W.D.; Lurling, M.

    2013-01-01

    The neurotoxin β-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap s...

  9. Latex agglutination and enzyme-linked immunosorbent assays for cytomegalovirus serologic screening of transplant donors and recipients.

    OpenAIRE

    Chou, S W; Scott, K M

    1988-01-01

    The effectiveness of three serologic assays (two enzyme-linked immunosorbent assays [ELISAs] and latex agglutination) for cytomegalovirus (CMV) serologic matching of donors and recipients was assessed over a 2-year period in a major organ transplant program. Sera with equivocal test results were investigated by repeat testing of serum samples and additional specimens from the individuals involved and monitoring of CMV infection in recipients. An in-house ELISA identified all CMV-infective don...

  10. 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...

  11. Comparison of radioimmunoassay and enzyme-linked immunosorbent assay in measurement of antibodies to Neisseria meningitidis group A capsular polysaccharide.

    OpenAIRE

    Beuvery, E C; Kayhty, M H; Leussink, A B; Kanhai, V

    1984-01-01

    Antibodies to meningococcal group A polysaccharide were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) in serum samples from 16 adults vaccinated with bivalent meningococcal group A and C polysaccharide vaccine. The specific antibody levels in the serum samples were expressed as micrograms of antibody protein per milliliter of serum. For RIA the polysaccharide was radiolabeled extrinsically with 125I. Both native polysaccharide and polysaccharide labeled wi...

  12. 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...

  13. 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 ...

  14. Seroprevalence of human immunodeficiency virus in north Indian blood donors using third and fourth generation Enzyme linked immunosorbent assay

    OpenAIRE

    Sheetal Malhotra; Neelam Marwaha; Karan Saluja

    2013-01-01

    Background : The percentage of HIV cases attributable to blood transfusion has decreased significantly in the last decade. The newer 4 th generation Enzyme linked immunosorbent assay (ELISA) has been shown to have increased sensitivity compared to 3 rd generation ELISA. Objectives : To estimate the seroprevalence of HIV among blood donors using 4 th generation ELISA assay and to compare it with the 3 rd generation ELISA. Materials and Methods : This prospective study involved 10,200 blood don...

  15. Serological diagnosis of bovine, caprine, and ovine mastitis caused by Listeria monocytogenes by using an enzyme-linked immunosorbent assay.

    OpenAIRE

    Bourry, A; Cochard, T; POUTREL, B.

    1997-01-01

    An enzyme-linked immunosorbent assay for detecting Listeria monocytogenes antibodies in bovine (n = 35), caprine (n = 27), and ovine (n = 30) milk samples was evaluated by comparison with bacteriological examination. Microtiter plates were coated with proteins obtained from culture supernatant, and antibodies were revealed with a monoclonal antibody able to react with the immunoglobulins belonging to the three animal species. The arithmetic mean optical density (OD) of milk samples infected w...

  16. 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...

  17. Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots †

    OpenAIRE

    Levanony, Hanna; Bashan, Yoav; Kahana, Zvi E.

    1987-01-01

    The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 105 CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 108 CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows ...

  18. Enzyme-linked immunosorbent assay that uses labeled antigen for detection of immunoglobulin M and A antibodies in toxoplasmosis: comparison with indirect immunofluorescence and double-sandwich enzyme-linked immunosorbent assay.

    OpenAIRE

    van Loon, A M; Van der Logt, J. T.; Heessen, F W; van der Veen, J.

    1983-01-01

    A direct enzyme-linked immunosorbent assay (ELISA) is described that uses horseradish peroxidase-labeled antigen for detection of immunoglobulin M (IgM) and IgA antibodies to toxoplasma. In this assay, polystyrene microtiter plates were sensitized with anti-human IgM or IgA antibody to separate IgM or IgA from other classes of antibody. The presence of IgM or IgA antibodies to toxoplasma (Tox-IgM, Tox-IgA) was then detected by sequential addition of soluble horseradish peroxidase-labeled toxo...

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

    Science.gov (United States)

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

  20. Multicenter comparison of levels of antibody to the Neisseria meningitidis group A capsular polysaccharide measured by using an enzyme-linked immunosorbent assay.

    OpenAIRE

    Carlone, G M; Frasch, C E; Siber, G R; Quataert, S; Gheesling, L L; Turner, S H; Plikaytis, B D; Helsel, L O; Dewitt, W. E.; Bibb, W F

    1992-01-01

    There is no standard immunoassay for evaluating immune responses to meningococcal vaccines. We developed an enzyme-linked immunosorbent assay to measure total levels of antibody to Neisseria meningitidis group A capsular polysaccharide. Five laboratories measured the antibody levels in six paired pre- and postvaccination serum samples by using the enzyme-linked immunosorbent assay. Methylated human serum albumin was used to bind native group A polysaccharide to microtiter plate surfaces. The ...

  1. Application of an enzyme-linked immunosorbent assay (ELISA) to determine paraquat residues in milk, beef, and potatoes

    International Nuclear Information System (INIS)

    The USDA Food Safety and Inspection Service (FSIS) has included paraquat on its list of compounds to be considered for monitoring in foods. However, present methods do not easily accommodate the processing of large numbers of samples, thus limiting routine monitoring of the compound. The conventional method, based on spectrophotometry of reduced paraquat solutions, requires time-consuming sample preparation. Although the advantages of immunoassays for pesticide residue analysis have been pointed out, the reported immunoassays for paraquat have only been applied to cases of clinical poisoning or human exposure assessment. In this study, spiked milk, potato, and beef were analyzed directly, without prior cleanup, by an enzyme-linked immunosorbent assay (ELISA)

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

    OpenAIRE

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

    2005-01-01

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

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

    OpenAIRE

    Cho, Ki-Hyun; Kim, Jeongmi; Yoo, Hyun-ah; Kim, Dae-Hee; Park, Seung-Yong; Song, Chang-Seon; Choi, In-Soo; Lee, Joong-Bok

    2014-01-01

    Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conforma...

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

    OpenAIRE

    Pal, Subhamoy; Dauner, Allison L.; Valks, Andrea; Forshey, Brett M.; Long, Kanya C.; Thaisomboonsuk, Butsaya; Sierra, Gloria; Picos, Victor; Talmage, Sara; Morrison, Amy C.; Halsey, Eric S; Comach, Guillermo; Yasuda, Chadwick; Loeffelholz, Michael; Jarman, Richard G.

    2015-01-01

    We evaluated four dengue diagnostic devices from Alere, including the SD Bioline Dengue Duo (nonstructural [NS] 1 Ag and IgG/IgM), the Panbio Dengue Duo Cassette (IgM/IgG) rapid diagnostic tests (RDTs), and the Panbio dengue IgM and IgG capture enzyme-linked immunosorbent assays (ELISAs) in a prospective, controlled, multicenter study in Peru, Venezuela, Cambodia, and the United States, using samples from 1,021 febrile individuals. Archived, well-characterized samples from an additional 135 f...

  5. Comparison of Performance of Serum and Plasma in Panbio Dengue and Japanese Encephalitis Virus Enzyme-Linked Immunosorbent Assays

    OpenAIRE

    Blacksell, Stuart D; Lee, Sue J.; Chanthongthip, Anisone; Taojaikong, Thaksinaporn; Thongpaseuth, Soulignasack; Hübscher, Tanja; Newton, Paul N.

    2012-01-01

    We examined the comparative performance of serum and plasma (in dipotassium EDTA) in Panbio Dengue enzyme-linked immunosorbent assays (ELISAs) for detection of non-structural protein 1 (NS1), IgM, and IgG, and a dengue/Japanese encephalitis virus (JEV) combination IgM ELISA in a prospective series of 201 patients with suspected dengue in Laos. Paired comparisons of medians from serum and plasma samples were not significantly different for Dengue IgM, and NS1 which had the highest number of di...

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

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens...... in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test, The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high...

  7. 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...

  8. 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...

  9. A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

    International Nuclear Information System (INIS)

    Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10-3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

  10. Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 Capsid protein

    OpenAIRE

    Wu, Dai-Tze; Aiyer, Sriram; Villanueva, Rodrigo A.; Roth, Monica J

    2013-01-01

    Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was...

  11. The enzyme-linked immunosorbent assay for rapid screening and early detection of acute leptospirosis

    International Nuclear Information System (INIS)

    Full text: The enzyme-linked immunosorbent assay (ELISA) was compared to the microscopic agglutination test (MAT) in a serological survey for leptospiral infection in dogs. Results from two studies in the survey showed that the ELISA was more sensitive than the MAT: Study 1 had 81% (133/165) prevalence as detected by the ELISA whilst the MAT detected 33% (54/165) of the dogs had evidence of leptospiral infection. In another study, the ELISA detected 59% (17/29) whilst the MAT recognized only 10% (3/29). The ELISA using our own boiled antigen was found to be a useful screening technique. The antigen was easy to prepare and can be made available to any laboratory that has leptospiral cultures. The ELISA has the advantage of being a simple, rapid, sensitive and less hazardous assay compared to the established MAT. It will be a useful technique for screening serum samples for leptospiral infection. The serum samples were from stray dogs kept in a non-governmental organization. Stray dogs have been suspected of spreading leptospiral infection to other animals and humans. In certain countries livestock have been infected with closely related leptospiral serovars and both the ELISA and MAT have difficulties to identify the causal leptospiral serovar. In New Zealand, serovars hardjo and balcanica are closely related and have been placed in the same serogroup. Both serovars infect cattle in New Zealand and it is critical to identify the true causal serovar as their maintenance host and epidemiology are entirely different. In our study, using the type-specific main (TM) antigen, the ELISA was made to be more specific and was able to differentiate closely related leptospiral serovars such as between serovars hardjo and hebdomadis. The TM antigen which is the Fraction V protein was extracted from a serovar hardjo culture. The TM-ELISA was applied to a small number of serum samples obtained from local cattle and it was seen that the ELISA was more sensitive than the MAT. TM

  12. Diagnosis of selected tropical diseases (Shistosomiasis and Malaria) using the enzyme-linked immunosorbent assays

    International Nuclear Information System (INIS)

    Immunological reactions are commonly used in diagnostic procedures on the basis of their high levels of specificity and sensitivity. Antibodies or antigens labelled with various markers have been found to be particularly useful for assays of logical substances. The applications of Enzyme-Linked Immunoabsorbent Assays (ELISA) to research on various tropical and non-tropical diseases is now well established. The procedure depends on the labelling of one of the reactants with enzymes which can be detected accurately by an appropriate substrate. The detection mechanism depends on the labelling of one of the reactants in such a way that their their reactivity is not impaired or affected. In the present study, ELISA was applied to sera from kampumbu area of Isoka district in the Northern province of Zambia. The objective of this presentation is to show the relative positivity rate for antigen and antibody and the endemicity of schistosomiasis and malaria as assessed by classical parasitological procedures. (author)

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Luis Izquierdo

    2013-11-01

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

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

    DEFF Research Database (Denmark)

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

    1989-01-01

    A total of 228 stored serum samples from 140 high risk individuals was examined for serological markers of human immunodeficiency virus (HIV) infection by second generation enzyme-linked immunosorbent assay, immunoblot, and HIV antigen assay. All the samples were negative in first generation enzyme...... obtained. They were all negative by the tests employed. Further, 37 serum samples from 20 seroconverters were studied. Four patients had antigenaemia 6-12 months before seroconversion was detected by first generation ELISA. Our data do not support the notion that serological signs of HIV infection are...... common in high risk individuals seronegative by first generation ELISA. However, HIV infection do occur in subjects negative by first generation ELISA, which emphasises the need for more sensitive screening assays and/or the use of antigen detection as part of screening in high risk individuals. The...

  16. Evaluation of a covalent mix-enzyme linked immunosorbent assay for screening of Salmonella antibodies in pig serum

    DEFF Research Database (Denmark)

    Chow, E.Y.W.; Wu, J.T.Y.; Jauho, E.S.;

    2004-01-01

    .9% tested negative. The interlaboratory comparison study found a kappa value of 0.9 between our laboratory (using an automated system) and the manufacturer laboratory (using the manual method). Comparison of ELISA results from all 5 participating laboratories showed very good to excellent agreement, between......In this study, a commercial Salmonella covalent mix-enzyme linked immunosorbent assay (ELISA) for serological detection of Salmonella infection in swine was evaluated by comparing it with the conventional fecal culture method and inter-laboratory proficiency testing, using a panel of sera tested in...... 5 laboratories from Europe and North America. Comparison with culture results showed that 88.5% of 26 culture-positive animals were ELISA positive, as were 55% of 60 animals from 2 culture-positive pig herds. Of 90 animals from 2 high health farms with no clinical symptoms of salmonellosis, 98...

  17. Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins

    International Nuclear Information System (INIS)

    The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins. Furthermore, combining the production technique of the six-histidine tagged fusion protein with the competitive sandwich ELISA mode, using an enzyme labeled anti-6H antibody as a tracer, can be a universal immunochemical method to quantitate other polypeptides or proteins

  18. Serological survey for Lyme disease in sika deer (Cervus nippon yesoensis) by enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Isogai, E; Isogai, H; Masuzawa, T; Yanagihara, Y; Sato, N; Hayashi, S; Maki, T; Mori, M

    1991-01-01

    Seventy-six wild sika deer (Cervus nippon yesoensis) from areas endemic for Borrelia burgdorferi during 1988 to 1989 had the IgG antibody to the bacteria in enzyme-linked immunosorbent assay. The percentage of seropositive deer was 25.0% to strain HO14 and 22.4% to strain HP3, respectively. Specific IgG antibody titers were high in summer but low in winter. In summer, seropositive deer were 75.0%. Similar results were obtained in serum specimens obtained monthly from 4 farmed deer. It was suggested that the sika deer could be one of the wild reservoirs for B. burgdorferi in Hokkaido, Japan. The transmission risk of B. burgdorferi is considered to be greatest during late spring to early summer. PMID:1808467

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

    Science.gov (United States)

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

    2003-01-01

    Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis, a fatal human disease of the central nervous system often contracted after swimming in fresh water. Identifying sites contaminated by N. fowleri is important in order to prevent the disease. An Enzyme-Linked ImmunoSorbent Assay (ELISA) has been developed for the specific identification of N. fawleri in primary cultures of environmental water samples. Of 939 samples isolated from artificially heated river water and screened by ELISA, 283 were positive. These results were subsequently confirmed by isoelectric focusing, the established reference method. A sensitivity of 97.4% and a specificity of 97% were obtained. These results indicate that this ELISA method is reliable and can be considered as a powerful tool for the detection of N. fowleri in environmental water samples. PMID:12744523

  20. Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D.

    Science.gov (United States)

    Radolf, J D; Lernhardt, E B; Fehniger, T E; Lovett, M A

    1986-06-01

    An enzyme-linked immunosorbent assay (ELISA) for syphilis has been developed that detects IgG antibody to purified recombinant Treponema pallidum surface antigen 4D. The 4D ELISA was capable of detecting 25 ng of 4D antigen-specific antibody. Neither 172 nonsyphilitic sera nor 20 false-positive sera in the Venereal Disease Research Laboratory test reacted in the 4D ELISA. The sensitivity of the 4D ELISA was comparable to that of the adsorbed fluorescent treponemal antibody test in primary, secondary, and latent disease. Most sera from patients with yaws or pinta were also reactive, a result indicating that a 4D antigen-like molecule also exists in the closely related pathogenic treponemes Treponema pertenue and Treponema carateum. PMID:3517186

  1. Micro-light-pipe array with an excitation attenuation filter for lensless digital enzyme-linked immunosorbent assay

    Science.gov (United States)

    Takehara, Hironari; Nagasaki, Mizuki; Sasagawa, Kiyotaka; Takehara, Hiroaki; Noda, Toshihiko; Tokuda, Takashi; Ohta, Jun

    2016-03-01

    Digital enzyme-linked immunosorbent assay (ELISA) is used for detecting various biomarkers with hypersensitivity. We have been developing compact systems by replacing the fluorescence microscope with a CMOS image sensor. Here, we propose a micro-light-pipe array structure made of metal filled with dye-doped resin, which can be used as a fabrication substrate of the micro-reaction-chamber array of digital ELISA. The possibility that this structure enhances the coupling efficiency for fluorescence was simulated using a simple model. To realize the structure, we fabricated a 30-µm-thick micropipe array by copper electroplating around a thick photoresist pattern. The typical diameter of each fabricated micropipe was 10 µm. The pipes were filled with yellow-dye-doped epoxy resin. The transmittance ratio of fluorescence and excitation light could be controlled by adjusting the doping concentration. We confirmed that an angled excitation light incidence suppressed the leakage of excitation light.

  2. 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.

  3. 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.

  4. Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange.

    Science.gov (United States)

    Curtis, C F

    2001-02-24

    This study was designed to assess the accuracy of a commercial enzyme-linked immunosorbent assay for the diagnosis of canine scabies. Serum samples from 37 dogs were examined blind; 12 had sarcoptic mange confirmed by the identification of mites in skin scrapings, 12 were atopic (with positive intradermal reactions to one or more aeroallergens, including Dermatophagoides farinae), and 13 were healthy dogs with no history of skin disease. Optical density values of more than 0.16 were considered positive, 0.145 to 0.16 were considered questionable and less than 0.145 were considered negative. Ten of the 12 dogs with scabies were positive, all 12 atopic dogs were negative, and 11 of the 13 healthy dogs were negative and two were questionable. PMID:11289551

  5. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in dogs.

    Science.gov (United States)

    Lower, K S; Medleau, L M; Hnilica, K; Bigler, B

    2001-12-01

    Canine scabies is a challenging disease to diagnose because sarcoptic mites are hard to find on skin scrapings. The purpose of this study was to evaluate a serologic enzyme-linked immunosorbent assay (ELISA) as an aid in the diagnosis of canine scabies. In addition, serum samples were obtained post treatment to determine the duration and persistence of circulating scabies antibodies after resolution of natural infection. Nineteen dogs diagnosed with sarcoptic mange and 38 control dogs were tested. Sixteen scabies-infested dogs showed positive pretreatment ELISA results (84.2% sensitivity). Thirty-four control dogs showed negative ELISA results (89.5% specificity). In the 11 scabies dogs from which multiple post treatment serum samples were obtained, detectable antibodies were not present 1 month after treatment in four cases, but were present for 1-4.5 months post treatment in seven dogs. Our results suggest that this scabies ELISA test is useful in the diagnosis of canine scabies. PMID:11844220

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

    Science.gov (United States)

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

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

  7. Evaluation of the PANBIO Brucella Immunoglobulin G (IgG) and IgM Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Brucellosis

    OpenAIRE

    Araj, George F.; Kattar, Mireille M.; Fattouh, Layla G.; Bajakian, Kayane O.; Kobeissi, Sara A.

    2005-01-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis.

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

    DEFF Research Database (Denmark)

    Oviedo, J M; Valiño, F; Plasencia, I; Serrano, A G; Casals, C; Pérez-Gil, J

    2001-01-01

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

  9. Enzyme-linked immunosorbent assay detection of trichothecenes produced by the Bioherbicide Myrothecium verrucaria in cell cultures, extracts, and plant tissues

    Science.gov (United States)

    Commercially available enzyme linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross-reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L-2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of...

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

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    Levels of antibodies to the O antigens (0:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum...

  12. Comparison of enzyme-linked immunosorbent assay, surface plasmon resonance and biolayer interferometry for screening of deoxynivalenol in wheat and wheat dust

    Science.gov (United States)

    A sample preparation method was developed for the screening of deoxynivalenol (DON) in wheat and wheat dust. Extraction was carried out with water and was successful due to the polar character of DON. For detection, an enzyme-linked immunosorbent assay (ELISA) was compared to the sensor-based techni...

  13. EVALUATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY WITH RECOMBINANT RHOPTRY-ASSOCIATED PROTEIN-1 ANTIGEN AGAINST BABESIA BOVIS FOR THE DETECTION OF SPECIFIC ANTIBODIES IN CATTLE

    Science.gov (United States)

    The gene encoding Babesia bovis rhoptry-associated protein-1 (RAP-1) was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies against B. bovis. The B. bovis RAP-1 gene was subcloned into a baculovirus transfer vector and the RAP-1 protein was expressed in insec...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  15. Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Moller, T;

    1999-01-01

    The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L...

  16. Sensitivity of sandwich enzyme-linked immunosorbent assay for Cryptococcus neoformans polysaccharide antigen is dependent on the isotypes of the capture and detection antibodies.

    OpenAIRE

    S. Mukherjee; Casadevall, A.

    1995-01-01

    Immunoglobulin M (IgM) and IgG1 monoclonal antibody isotype switch variants to Cryptococcus neoformans capsular polysaccharide were used to study the sensitivity of a double sandwich enzyme-linked immunosorbent assay (ELISA). The most sensitive ELISA configurations used IgG1 monoclonal antibody absorbed on polystyrene plates or IgM immobilized with goat antisera for antigen capture.

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

    OpenAIRE

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

    2003-01-01

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

  18. Field assessment of the enzyme linked immunosorbent assay for foot-and-mouth disease virus diagnosis and typing

    International Nuclear Information System (INIS)

    The objective of the present study was to evaluate the enzyme linked immunosorbent assay (ELISA) in comparison with the complement fixation test (CFT) for the diagnosis and typing of foot-and-mouth disease (FMD) virus (FMDV). Diagnostic material was epithelium from either suspected cases of FMD or from animals experimentally infected with FMDV. Epithelial suspensions and supernatant fluids from cell culture passage were assayed by CFT and ELISA. The superiority of the ELISA over the CFT was demonstrated: 1) the detection rate was 23% higher than that of CFT on original (epithelial) suspensions (OS) submissions of all sample (positive and negative) and 30% higher on supernatant fluids from cell culture passage, 2) the detection rate of ELISA on OS of confirmed positive samples was 28% higher than that of CFT, 3) no significant differences were observed in the detection and typing rates between the PANAFTOSA and FAO/IAEA ELISA kits (P<0.05) and 4) the sensitivity of the ELISA was 16 to 85 times higher than that of CFT when serial dilutions of sample homogenates were examined. (author)

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

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    Yuanming Sun

    2011-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Gortázar Christian

    2008-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Shipra Singhal

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xian Zhang

    2015-10-01

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

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

    Science.gov (United States)

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

    2015-01-01

    A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R2 = 0.9904). The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R2 = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories. PMID:26492271

  4. Validation of an improved enzyme-linked immunosorbent assay for the diagnosis of trypanosomal antibodies in Ghanaian cattle

    International Nuclear Information System (INIS)

    The validation of an enzyme-linked immunosorbent assay (Ab-ELISA) for the detection of antibodies to pathogenic trypanosomes in cattle is described. Two hundred known negative sera obtained from the tsetse-free zone of Dori (Burkina Faso) were analyzed using microtitre plates pre-coated with crude antigen lysates of Trypanosoma congolense and T. vivax. A pre-test optimization was carried out and a percent positivity (PP) of 50% was chosen (specificity: >82%) for assaying field sera. A total of 440 serum samples collected from cattle in areas of known and unknown disease prevalence were assayed. For all animals the packed red cell volume (PCV) was determined and the buffy coat technique (BCT) and blood smears were examined to detect trypanosomes at the species level. A comparison of the BCT and Ab-ELISA results showed there was a much higher prevalence of antibodies to both species than the parasite prevalence as shown by the BCT (10 fold). The rate of agreement between BCT-positive and Ab-ELISA-positive samples for both species was low (<10%). No conclusion could be drawn from this finding because of the low number of known BCT positive cases that were identified. There was a better, albeit highly variable, agreement between BCT-negative and Ab-ELISA-negative samples (30-70%). Proposals for further improvement of the Ab-ELISA and prospects for the use of the assay in the monitoring of trypanosomosis control in Ghana are discussed. (author)

  5. 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)

  6. Presumptive diagnosis of subclinical infections utilizing computer-assisted analysis of sequential enzyme-linked immunosorbent assays against multiple antigens.

    Science.gov (United States)

    Mallinson, E T; Snyder, D B; Marquardt, W W; Russek-Cohen, E; Savage, P K; Allen, D C; Yancey, F S

    1985-09-01

    One-hundred-seventy-two serum samples, collected sequentially from four flocks of egg- and meat-type chickens, were evaluated for antibodies to multiple infectious agents by enzyme-linked immunosorbent assay (MELISA). The MELISA system used provided simultaneous measurement of antibody titers against avian infectious bronchitis (IB), infectious bursal disease (BD), Newcastle disease, avian encephalomyelitis and reovirus infections, and Mycoplasma gallisepticum. The use of computer-generated graphic print outs of relative MELISA titers provided immediate visulization of over 740 data points and convenient detection of any temporal changes in median titer class (MTC). The temporally changing MTC, or flock profiles obtained, indicated that negligible or waning IB immunity may be a common occurrence in previously vaccinated commercial chickens. These profiles further suggested that, despite no IB revaccination, these same flocks experienced episodes of reexposure to IB which otherwise may have been difficult to detect by conventional clinical or diagnostic laboratory protocols. MELISA profiles and sequential histologic examinations of bursas of Fabricius also provided evidence of a possible BD vaccination problem in young chickens that also experienced excessive losses from coccidiosis, ulcerative enteritis, and Marek's disease. Short sampling intervals were found to foster the detection and definition of fluctuations in MTC which otherwise may have been missed. PMID:4048057

  7. Analyses of mammalian sera in enzyme-linked immunosorbent assays with different strains of Borrelia burgdorferi sensu lato.

    Science.gov (United States)

    Magnarelli, L A; Anderson, J F; Johnson, R C

    1995-04-01

    Blood samples were collected from cottontail rabbits (Sylvilagus floridanus), raccoons (Procyon lotor), white-footed mice (Peromyscus leucopus), and white-tailed deer (Odocoileus virginianus) between 1977 and 1991 in southern Connecticut and New York State (USA) and were tested for antibodies against eight strains of Borrelia burgdorferi sensu lato in enzyme-linked immunosorbent assays. Among these spirochetes were six strains of B. burgdorferi sensu stricto, one strain of B. garinii (=IP90) and a strain (IPF) in group VS461. Sera from each study group reacted positively to all strains having origins in North America and Eurasia. Assay sensitivities normally ranged between 85% and 100% for all study groups. The lowest sensitivity (66%) was noted when mouse sera were tested with B. garinii, an isolate from Ixodes persulcatus in the former Soviet Union. Differences in serum reactivity to various strains were noted for all study groups, but because of multiple shared antigens among the closely related spirochetes tested, the selection of a particular North American strain of B. burgdorferi sensu stricto did not appear to be a critical factor for optimal assay performance. Locally obtained strains of this bacterium are preferred as coating antigens for serologic testing because of their availability. PMID:8583632

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

    Directory of Open Access Journals (Sweden)

    Andrea Cristine Koishi

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

  9. 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

  10. The role of iron in neurodegeneration - Mössbauer spectroscopy, electron microscopy, enzyme-linked immunosorbent assay and neuroimaging studies

    International Nuclear Information System (INIS)

    The possible role of iron in neurodegeneration was studied by various techniques: electron microscopy, enzyme-linked immunosorbent assay, Mössbauer spectroscopy, atomic absorption, ultrasonography and magnetic resonance imaging. The measurements were made on human tissues extracted from liver and from brain structures involved in diseases of the human brain: substantia nigra (Parkinson’s, PD), hippocampal cortex (Alzheimer’s, AD) and globus pallidus (progressive supranuclear palsy, PSP). The sizes of the iron cores of ferritin, the main iron storage compound in tissues, were found to be smaller in brain than in liver. Brain ferritin has a higher proportion of H to L chains compared to liver. A significant decrease of the concentration of L chains in PD compared to control was found. No increase in the concentration of iron in PD versus control was detected; however, there was an increase of labile iron, which constitutes only 2‰ of brain iron. In AD an increase in the concentration of ferritin was noticed, without a significant increase in iron concentration. In PSP an increase of total iron was observed. Our findings suggest that the mechanisms leading to the death of nerve cells in these three diseases may be different, although all may be related to iron mediated oxidative stress. (paper)

  11. Rapid detection of Nocardia amarae in the activated sludge process using enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Iwahori, K; Miyata, N; Morisada, S; Suzuki, N

    2000-01-01

    Nocardia amarae, a mycolic acid-containing bacterium, has often been reported to cause foaming of activated sludge in wastewater treatment plants. In this study, the number of N. amarae cells in the activated sludge process was estimated by enzyme-linked immunosorbent assay (ELISA) with anti-N. amarae polyclonal antibody. Use of the antibody enabled N. amarae to be detected at levels of 10(4) to 10(7) colony forming units. On the other hand, the antibody reacted with only a small portion of activated sludge, in which no N. amarae cells were detected by the plate count method. Competitive ELISA was employed to estimate the N. amarae cells in samples taken from a municipal wastewater treatment plant, including raw wastewater and activated sludge foam. The cell numbers estimated by competitive ELISA corresponded well with those obtained by plate counts. Hence, the antibody produced in this study was shown to be effective for the rapid monitoring of N. amarae in the activated sludge process. PMID:16232779

  12. ENZYME-LINKED IMMUNOSORBENT ASSAY FOR SCREENING DIOXIN SOIL CONTAMINATION BY UNCONTROLLED COMBUSTION DURING INFORMAL RECYCLING IN SLUMS

    Science.gov (United States)

    Trindade, Mirta; Nording, Malin; Nichkova, Mikaela; Spinnel, Erik; Haglund, Peter; Last, Michael S.; Gee, Shirley; Hammock, Bruce; Last, Jerold A.; González-Sapienza, Gualberto; Brena, Beatriz M.

    2010-01-01

    Uncontrolled combustion due to garbage recycling is a widespread activity among slum dwellers in distressed economy countries and has been indicated as a major source of dioxin contamination. However, because of the high cost and complexity of gas chromatography/high-resolution mass spectrometry (GC-HRMS) analysis, the magnitude of the problem remains largely unknown. The present study describes a first approach toward the use of a dioxin antibody-based enzyme-linked immunosorbent assay (ELISA) as the basis for a sustainable, simple, and low-cost monitoring program to assess the toxicological impact of uncontrolled combustion in slums. A panel of 16 samples was analyzed by GC-HRMS and ELISA on split extracts. Close to 20% of the analyzed samples showed dioxin concentrations up to almost twice the guidance level for residential soil in several countries, pointing out the need for performing a large-scale monitoring program. Despite the potential for variations in dioxin congener distribution due to the mixed nature of the incinerated material, there was a good correlation between the toxic equivalents as determined by GC-HRMS and ELISA. Furthermore, an interlaboratory ELISA validation showed that the capacity to perform the dioxin ELISA was successfully transferred between laboratories. It was concluded that the ELISA method performed very well as a screening tool to prioritize samples for instrumental analysis, which allows cutting down costs significantly. PMID:18522475

  13. Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.

    Science.gov (United States)

    Liu, Qin-Lan; Yan, Xiao-Hui; Yin, Xiao-Mao; Situ, Bo; Zhou, Han-Kun; Lin, Li; Li, Bo; Gan, Ning; Zheng, Lei

    2013-01-01

    Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies. PMID:24129276

  14. Comparison of Two Commercial Tick-Borne Encephalitis Virus IgG Enzyme-Linked Immunosorbent Assays.

    Science.gov (United States)

    Weissbach, Fabian H; Hirsch, Hans H

    2015-07-01

    Despite the availability of protective vaccines, tick-borne encephalitis virus (TBEV) infections have been increasingly reported to the European Centre for Disease Prevention and Control in the past 2 decades. Since the diagnosis of TBEV exposure relies on serological testing, we compared two commercial enzyme-linked immunosorbent assays (ELISAs), i.e., Immunozym FSME IgG assay (ELISA-1) and Euroimmun FSME Vienna IgG assay (ELISA-2). Both assays use whole TBEV antigens, but they differ in viral strains (Neudoerfl for ELISA-1 and K23 for ELISA-2) and cutoff values. In testing of samples from 398 healthy blood donors, ELISA-1 showed higher reactivity levels than ELISA-2 (P dengue virus in one case, suggesting that cross-reactivity contributed to the ELISA-1 results. We conclude that (i) cross-reactivity among flaviviruses remains a limitation of TBEV serological testing, (ii) ELISA-2 revealed reasonable sensitivity and specificity for anti-TBEV IgG population screening of human sera, and (iii) neutralization testing is most specific and should be reserved for selective questions. PMID:25924768

  15. Evaluation of a commercial enzyme linked immunosorbent assay (ELISA) for the determination of the neurotoxin BMAA in surface waters.

    Science.gov (United States)

    Faassen, Elisabeth J; Beekman, Wendy; Lürling, Miquel

    2013-01-01

    The neurotoxin β-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA. PMID:23762331

  16. Detection of thyroid stimulating hormone receptor antibodies (TRAb) by radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA)

    International Nuclear Information System (INIS)

    Thyroid stimulating hormone receptor antibodies (TRAb) were determined in 100 patients using radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of RRA and ELISA were found to be 70.6% and 88.2% respectively (n=51). The specificity of both assays were 100% (n=16). With RRA as the standard test the sensitivity and specificity of ELISA were 75.8% and 86.8%. In the untreated hyperthyroid the RRA result which expressed as % specific 125I-TSH inhibition was 33.6% (n=51), decline to 26.9% in the treated hyperthyroid (n=33) and 14.1% in the euthyroid (n=16). The mean 0.D492nm of TRAb-ELISA were 0.861 in untreated hyperthyroid, 0.437 in treated hyperthyroid and 0.135 in euthyroid Phi coefficient analysis show that the RRA was 60.4% correlated to hyperthyroidism where as TRAb-ELISA was 80.1%

  17. Restricted access supramolecular solvents for sample treatment in enzyme-linked immuno-sorbent assay of mycotoxins in food.

    Science.gov (United States)

    García-Fonseca, Sergio; Ballesteros-Gómez, Ana; Rubio, Soledad

    2016-09-01

    A restricted access supramolecular solvent (SUPRAS-RAM) made up of tetradecanoic acid reverse micelles is proposed as a wide-scope and low-cost strategy for the treatment of agrifood samples prior to enzyme-linked immunosorbent assays (ELISA). The approach was assessed for the determination of ochratoxin A (OTA) in wines and spices and aflatoxin B1 (AFB1) in cereals, two ubiquitous mycotoxins that were selected as representative contaminants for this study. The samples were selected to cover a variety of matrices in terms diverse composition and high complexity. Macromolecules such as proteins and carbohydrates were not-co-extracted due to the restricted access properties of the SUPRAS that are provided by chemical and physical mechanisms. In this sense, analyte extraction and clean-up were carried out in a single step. Parameters determining the extraction efficiency were studied and optimized. Certified reference materials were used for method validation. Recoveries of OTA ranged between 83% and 96% in wines (with relative standard deviation, RSD, of about 10%) and between 81% and 93% in spices (RSD 7%). Recoveries for AFB1 in wheat ranged from 75% to 85% (RSD 8%). The detection limits were all below the maximum levels established for OTA and for AFB1 by EU directives. This method offers a green and low-cost alternative to the organic solvent-based extraction and/or immunoaffinity columns-based cleanup of complex samples prior to ELISA. PMID:27543022

  18. Novel indirect enzyme-linked immunosorbent assay (ELISA) method to detect Total E. coli in water environment

    International Nuclear Information System (INIS)

    In order to establish ELISA (enzyme-linked immunosorbent assay) method to detect Total E. coli in water environment, E. coli multi-characters antigens in water environment were prepared according to the characters of kinds of E. coli serotypes, including antigen of whole cell, antigen of disrupted whole cell, somatic antigen, flagellar antigen and fimbrial antigen. Total E. coli polyclonal antibodies were obtained from the New Zealand rabbits immunized with these five antigens, respectively. Antibodies generated in this research are with high titers and good purity, can conjugate with antigens, specifically, stably and strongly. Indirect ELISA shows the titers of antibody of whole cell and antibody of disrupted whole cell are both over 1 x 105. The cross-reactivity of the antibody is from 12 to 30% which indicate the specificity of the antibody against Total E. coli. Based on these antibodies, we established indirect ELISA method to detect Total E. coli in water environment. The matrix effects were studied and the results show that there is no significant influence by all the factors. The ELISA result shows that the detection limitation could be 104 CFU (colony forming units) L-1. The indirect ELISA method developed in this study is well suited for Total E. coli analysis in real water samples as a rapid screen method

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

    Directory of Open Access Journals (Sweden)

    Malovrh Tadej

    2005-01-01

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

  20. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

    Science.gov (United States)

    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

    2016-05-01

    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-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China. PMID:26668175

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative...... splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects....... We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide as a...

  2. Rapid competitive enzyme-linked immunosorbent assay for detection of antibodies to peste des petits ruminants virus.

    Science.gov (United States)

    Choi, Kang-Seuk; Nah, Jin-Ju; Ko, Young-Joon; Kang, Shien-Young; Jo, Nam-In

    2005-04-01

    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 recombinant nucleocapsid protein (rPPRV-N). We tested 249 PPRV-positive serum samples and 733 PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be 512), even at dilutions > or = 512 in normal goat serum, and as early as 6 to 13 days postinfection from 12 goats, each of which was infected with one of the four PPRV lineages. Hyperimmune sera from animals experimentally vaccinated with rinderpest virus gave positive results by the rapid c-ELISA when the rinderpest virus VNT titers were >512, although the rapid c-ELISA titers were very low (2 to 16). However, the rapid c-ELISA was negative when the rinderpest virus VNT titer was < or = 128. The rapid c-ELISA developed in the present work provides a short turnaround time and could be a useful tool for the diagnosis of PPR and screening for PPRV in the field. PMID:15817764

  3. Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent for salmonid bacterial kidney disease

    Science.gov (United States)

    Pascho, R.J.; Mulcahy, D.

    1987-01-01

    A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of a soluble fraction of Renibacterium salmoninarum was developed from components extracted from the supernatant of an R. salmoninarum broth culture. The Costar® Serocluster™ EIA microplate gave the highest absorbance and signal-to-noise ratios among seven types tested. Including Tween 80 in the wash buffer resulted in higher absorbances than Tween 20 when antigen was present. Background absorbance did not increase when Tween 80 was added to the wash buffer, but did when Tween 80 replaced Tween 20 in antigen and conjugate diluents. Adsorption of coating antibody peaked within 4 h at 37 °C and 16 h at 4 °C. Antigen attachment to antibody-coated microplate wells depended more on incubation temperature than duration; we adopted a 3-h incubation at 25 °C. Conjugate incubation for longer than 1 h at 37 °C or 3 h at 25 °C resulted in unacceptable background levels. No cross-reactions resulted from heat-extracted antigens of 10 other species of bacteria. The optimized ELISA is a 6-h test that enables detection of levels of soluble antigen as low as 2–20 ng.

  4. Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 capsid protein.

    Science.gov (United States)

    Wu, Dai-Tze; Aiyer, Sriram; Villanueva, Rodrigo A; Roth, Monica J

    2013-11-01

    Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was developed using several goat-polyclonal serum against MuLV p30 generated by the NCI as primary antibody and a rat-monoclonal antibody to CA available from ATCC. The MuLV p30 CA antigen was standardized against recombinant MuLV p30 CA expressed from bacteria. The assay is sensitive, accurate and linear within a defined concentration range of CA. Comparison with different MuLV quantitative methods including reporter gene transfer, reverse transcriptase activity assay, and viral RNA quantitative PCR, showed this ELISA protocol to be highly quantifiable within defined ranges, which can be correlated with infectious viral titer. PMID:23810854

  5. Electrochemical Enzyme-Linked Immunosorbent Assay (ELISA for α-Fetoprotein Based on Glucose Detection with Multienzyme-Nanoparticle Amplification

    Directory of Open Access Journals (Sweden)

    Ning Gan

    2013-10-01

    Full Text Available Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1 and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2 to gold nanoparticles (AuNPs. After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA = 13.62033 − 2.86252 logCAFP (ng/mL, r = 0.99886 with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%–104%. This new method could be applied for detecting any protein biomarker with the corresponding antibodies.

  6. Assessment of two enzyme-linked immunosorbent assay tests marketed for detection of ruminant proteins in finished feed.

    Science.gov (United States)

    Myers, Michael J; Yancy, Haile F; Farrell, Dorothy E; Washington, Jewell D; Deaver, Christine M; Frobish, Russell A

    2007-03-01

    The performance characteristics of two enzyme-linked immunosorbent assay (ELISA) test kits, ELISA Technologies' MELISA-Tek test and Tepnel BioSystems' BioKit for (Cooked) Species Identification test, designed to detect ruminant proteins in animal feed, were evaluated. The test kits were evaluated by using acceptance criteria developed by the U.S. Food and Drug Administration's Center for Veterinary Medicine Office of Research for evaluating selectivity, sensitivity, ruggedness, and specificity. The acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response within a 95% confidence interval. In practice, this measure requires the test to achieve the correct response 58 times for every 60 samples evaluated, or a 96.7% accuracy rate. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods presently used by the U.S. Food and Drug Administration. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of this same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% BMBM. The MELISA-Tek test passed the acceptance set-point criteria for selectivity assessment but failed the sensitivity assessment at all levels except at the 2% level. The MELISA-Tek test came close to passing at the 1% level, detecting true-positive findings at a rate of 93%, but failed at lower levels, in spite of the label claim of 0.5% sensitivity. The BioKit for (Cooked) Species Identification test detected only 2 of 17 samples fortified at the 2% BMBM level and failed to detect any other BMBM-fortified samples. The results of this evaluation indicate that neither test is adequate for regulatory use. PMID:17388061

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

    Directory of Open Access Journals (Sweden)

    Bibiana Gonzales Santana

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

  8. Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita, Osamu; Mizoguchi, Toshio; Shindo, Junji; Park, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; Oyamada, Toshifumi; Yamada, Akio; Morikawa, Shigeru; Tanabayashi, Kiyoshi

    2014-04-01

    Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild. PMID:24689989

  9. Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis.

    Science.gov (United States)

    Flannery, B; Costa, D; Carvalho, F P; Guerreiro, H; Matsunaga, J; Da Silva, E D; Ferreira, A G; Riley, L W; Reis, M G; Haake, D A; Ko, A I

    2001-09-01

    There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (P > 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis. PMID:11526167

  10. An enzyme-linked immunosorbent assay for lipovitellin quantification in copepods: a screening tool for endocrine toxicity.

    Science.gov (United States)

    Volz, David C; Chandler, G Thomas

    2004-02-01

    Vitellogenin (VTG) has been widely used as a biomarker of estrogenic exposure in fish, leading to the development of standardized assays for VTG quantification. However, standardized quantitative assays for invertebrate, particularly crustacean, lipovitellin (also known as vitellin [VTN]) are lacking. In this study, a fluorescence-based VTN enzyme-linked immunosorbent assay (ELISA) was developed to quantify microquantities of VTN in the estuarine, sediment-dwelling copepod Amphiascus tenuiremis. This ELISA utilizes a VTN-specific polyclonal antibody developed against amphipod (Leptocheirus plumulosus) embryo VTN and exhibits specificity toward female copepod proteins. In routine assays, the working range of the ELISA was 31.25 to 1,000 ng/ml (75-25% specific binding/maximum antibody binding [B/B0]) with a 50% B/B0 intra- and interassay variation of 3.9% (n = 9) and 12.5% (n = 26), respectively. This ELISA is capable of detecting VTN as low as 2 ng/ml, and can accurately detect VTN in as few as four copepods. The ELISA significantly discriminated positive (gravid female) and negative (male) samples, and was suitable for screening endocrine toxicity in copepods. Stage-I juvenile copepods were individually reared to adults in aqueous microvolumes of the phenylpyrazole insecticide, fipronil, and whole-body homogenate extracts were assayed for VTN levels. Fipronil-exposed virgin adult females, but not males, exhibited significantly higher levels of VTN relative to control males and females. This crustacean VTN ELISA is likely useful for evaluating endocrine activity of environmental toxicants in copepods and other crustacean species. PMID:14982375

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

    International Nuclear Information System (INIS)

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

  12. Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Baszler, T V; Knowles, D P; Dubey, J P; Gay, J M; Mathison, B A; McElwain, T F

    1996-06-01

    Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle. PMID:8735092

  13. Evaluation by enzyme-linked immunosorbent assay (ELISA) of Renibacterium salmoninarum bacterins affected by persistence of bacterial antigens

    Science.gov (United States)

    Pascho, R.J.; Goodrich, T.D.; McKibben, C.L.

    1997-01-01

    Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with a bacterin containing killed Renibacterium salmoninarum cells delivered alone or in an oil-based adjuvant. We evaluated the relative abilities of the batterins to prevent the initiation or progression of infection in fish challenged by waterborne exposure to R. salmoninarum. Sixty-one days after vaccination, fish were held for 24 h in water containing either no bacteria or approximately 1.7 x 103, 1.7 x 105, or 5.3 x 106 live R. salmoninarum cells/mL. An enzyme-linked immunosorbent assay (ELISA) was used to monitor changes in the levels of R. salmoninarum antigen in live fish before and after the immersion challenges. High levels of R. salmoninarum antigens were detected by ELISA in kidney-spleen tissue homogenates from vaccinated fish immediately before the challenges. Levels of those antigens remained high in the tissues of unchallenged fish throughout the study. We found that the ELISA used in this study may be unsuitable for evaluating the efficacy of batterins because it did not distinguish antigens produced by the challenge bacteria during an infection from those of the bacterins. Groups of control and vaccinated fish also were injected with either 1.7 x 104 or 1.7 x 106 R. salmoninarum cells and served as R. salmoninarum virulence controls. Relative survival among the various subgroups in the injection challenge suggests that adverse effects might have been associated with the adjuvant used in this study. The lowest survival at both injection challenge levels was among fish vaccinated with bacteria in adjuvant.

  14. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the herbicide chlorimuron-ethyl.

    Science.gov (United States)

    Zhao, Jing; Yi, Guo-Xiang; He, Su-Ping; Wang, Bao-Min; Yu, Cai-Xia; Li, Gang; Zhai, Zhi-Xi; Li, Zhao-Hu; Li, Qing X

    2006-07-12

    Hybridomas secreting a monoclonal antibody (mAb) against the herbicide chlorimuron-ethyl (CE) were produced by fusing the mouse myeloma cell line (SP2/0) with splenocytes from a mouse immunized against the conjugate of the sulfonamide moiety of CE and bovine serum albumin (BSA). The mAb, designated 1F5C5A10, had very weak affinity with metsulfuron, ethametsulfuron, pyrazosulfuron, bensulfuron, and chlorsulfuron. Two mAb-based indirect competitive enzyme-linked immunosorbent assays (icELISA) were developed. A conventional icELISA (icELISA-I) showed a concentration of half-maximum inhibition (IC(50)) of 11.6 ng/mL with a dynamic range of 1.6-84 ng/mL. A simplified icELISA (icELISA-II) had an IC(50) of 28.7 ng/mL and a dynamic range of 2.2-372 ng/mL. The two assays were tested on spiked water and soil samples. CE (1-500 ng/mL) fortified in water samples could be analyzed directly without any sample preparation by both immunoassays with an average recovery between 74 and 114%. icELISA-II, but not icELISA-I, was able to accurately analyze the herbicide residues in the crude soil extracts with recoveries between 99 and 129% without obvious matrix effects due to its lesser amount of sample used. In contrast to icELISA-I, icELISA-II is more convenient, whereas it consumes more reagents of coating antigen and goat anti-mouse IgG-peroxidase. PMID:16819901

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-02-06

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

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

    Directory of Open Access Journals (Sweden)

    Marco Tamba

    2010-01-01

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

  17. Evaluation of the PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays for diagnosis of human brucellosis.

    Science.gov (United States)

    Araj, George F; Kattar, Mireille M; Fattouh, Layla G; Bajakian, Kayane O; Kobeissi, Sara A

    2005-11-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. PMID:16275951

  18. Measurement of immunoglobulin M, immunoglobulin G, and immunoglobulin A antibodies against Yersinia enterocolitica by enzyme-linked immunosorbent assay: comparison of lipopolysaccharide and whole bacterium as antigen.

    OpenAIRE

    Granfors, K; Viljanen, M K; Toivanen, A

    1981-01-01

    An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of human immunoglobulin M (IgM), IgG, and IgA antibodies against Yersinia enterocolitica by using lipopolysaccharides as antigens is described. The results obtained with the lipopolysaccharide ELISA were compared with the results of the whole bacterium ELISA. The correlations observed were good for each immunoglobulin class. Cross-reactions between Y. enterocolitica serotypes O:3 and O:9, Yersinia pseudotuberculos...

  19. Enzyme-Linked Immunosorbent Assay for Detecting Herpesvirus Exposure in Mediterranean Tortoises (Spur-Thighed Tortoise [Testudo graeca] and Hermann's Tortoise [Testudo hermanni])†

    OpenAIRE

    Origgi, F. C.; Klein, P A; Mathes, K.; Blahak, S; Marschang, R. E.; Tucker, S J; Jacobson, E R

    2001-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to a herpesvirus associated with an upper respiratory tract disease in Mediterranean tortoises [spur-thighed tortoise (Testudo graeca) and Hermann's tortoise (Testudo hermanni)]. This serodiagnostic test was validated through a hyperimmunization study. The mean of the A405 readings of the plasma samples collected at time zero of the hyperimmunization study plus three times the standard deviation was use...

  20. Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria.

    OpenAIRE

    Glatman-Freedman, A; Martin, J.M.; Riska, P F; Bloom, B R; Casadevall, A

    1996-01-01

    Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to ...

  1. Comparison of Two Commercial Enzyme-Linked Immunosorbent Assays with an Immunofluorescence Assay for Detection of Legionella pneumophila Types 1 to 6

    OpenAIRE

    Malan, Annette K.; Martins, Thomas B; Jaskowski, Troy D; Hill, Harry R.; Litwin, Christine M.

    2003-01-01

    Members of the genus Legionella are characterized as gram-negative, motile, freshwater-dwelling bacteria that were responsible for a pneumonia outbreak among American Legion members in 1976. Because clinicians routinely order serologic testing for Legionella pneumophila serogroups 1 to 6 as a screen for possible L. pneumophila infections, we evaluated the Wampole Laboratories L. pneumophila type 1 to 6 immunoglobulin G (IgG) and IgM combined enzyme-linked immunosorbent assay (ELISA) and the Z...

  2. Modified Indirect Porcine Circovirus (PCV) Type 2-Based and Recombinant Capsid Protein (ORF2)-Based Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to PCV

    OpenAIRE

    Nawagitgul, Porntippa; Harms, Perry A.; Morozov, Igor; Thacker, Brad J.; Sorden, Steven D.; Lekcharoensuk, Chalermpol; Paul, Prem S.

    2002-01-01

    Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PC...

  3. Field evaluation of an immunoglobulin G anti-F1 enzyme-linked immunosorbent assay for serodiagnosis of human plague in Madagascar.

    OpenAIRE

    Rasoamanana, B; Leroy, F.; Boisier, P.; Rasolomaharo, M; Buchy, P.; Carniel, E; Chanteau, S.

    1997-01-01

    Bacteriological isolation of Yersinia pestis is the reference test for confirming plague infection, but recovery of the pathogen from human samples is usually very poor. When the etiology of the disease cannot be bacteriologically confirmed, it may be useful to possess alternative tests such as detection of specific circulating antibodies to help guide the diagnosis. In the present study, the immunoglobulin G (IgG) anti-F1 enzyme-linked immunosorbent assay (ELISA) has been applied to various ...

  4. Optimasi Deteksi Dini Potato Virus Y (PVY) Pada Kentang Solanum tuberosum L. varietas Kalosi Dengan Teknik ELISA (Enzyme Linked Immunosorbent Assay)

    OpenAIRE

    Zainuddin, Masniawati; Kuswinanti, Tutik; Gobel, Risco B.; Toyyibah, Indah

    2011-01-01

    A research about Optimization of Early Detection of Potato Virus Y (PVY) in Potato Solanum tuberosum L. varieties Kalosi by ELISA Technique (Enzyme linked immunosorbent assay) was done. The purpose of this research was to determine the level of sensitivity of the combination of antibody and antigen dilutions with ELISA technique in detecting the virus PVY in potato plants. The research was carried out in the Laboratory of Agricultural Biotechnology, Research Center of Hasanuddin University, M...

  5. Evaluation of the specificity of three enzyme-linked immunosorbent assays for detection of antibodies against Salmonella in bovine bulk milk

    OpenAIRE

    Nyman, Ann-Kristin J; Ågren, Estelle CC; Bergström, Karin; Wahlström, Helene

    2013-01-01

    Background The Swedish Salmonella control program has been running for decades and has resulted in a low prevalence of Salmonella in Swedish food producing animals. Routine bacteriology is used to detect Salmonella, however, bacteriology is time consuming, costly and has a low sensitivity. Different enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of antibodies against Salmonella Dublin and S. Typhimurium in bovine bulk milk, individual milk samples as well as in ...

  6. Diagnosis and Clinical Virology of Lassa Fever as Evaluated by Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent-Antibody Test, and Virus Isolation

    OpenAIRE

    Bausch, D. G.; Rollin, P E; Demby, A H; Coulibaly, M.; Kanu, J.; Conteh, A. S.; Wagoner, K. D.; McMullan, L. K.; Bowen, M. D.; Peters, C. J.; Ksiazek, T. G.

    2000-01-01

    The Lassa virus (an arenavirus) is found in West Africa, where it sometimes causes a severe hemorrhagic illness called Lassa fever. Laboratory diagnosis has traditionally been by the indirect fluorescent-antibody (IFA) test. However, enzyme-linked immunosorbent assays (ELISAs) for Lassa virus antigen and immunoglobulin M (IgM) and G (IgG) antibodies have been developed that are thought to be more sensitive and specific. We compared ELISA and IFA testing on sera from 305 suspected cases of Las...

  7. Detection of rubella-specific immunoglobulin G: comparison of the enzyme-linked immunosorbent assay and an automated microparticle enzyme immunoassay (IMx).

    OpenAIRE

    Skurrie, I J; Head, J L; Garland, S M

    1991-01-01

    An automated microparticle enzyme immunoassay (IMx Rubella IgG Antibody Assay; Abbott Laboratories, North Chicago, Ill.) was compared with a conventional enzyme-linked immunosorbent assay (ELISA) for detection of rubella-specific immunoglobulin G (IgG) in 400 consecutive antenatal patients. There was complete agreement between the two tests in this population, which had a positivity rate of 99% for rubella-specific IgG antibodies. The performance of the IMx was also evaluated at the cutoff zo...

  8. Analysis of Benzo[a]pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISPE) Coupled with Enzyme-Linked Immunosorbent Assay (ELISA)

    OpenAIRE

    Michael Pschenitza; Rudolf Hackenberg; Reinhard Niessner; Dietmar Knopp

    2014-01-01

    This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE) method coupled with enzyme-linked immunosorbent assay (ELISA) for determination of the PAH benzo[a]pyrene (B[a]P) in vegetable oils. Different molecularly imprinted polymers (MIPs) were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene ...

  9. Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of bovine viral diarrhea virus antibody in cattle.

    OpenAIRE

    Cho, H J; Masri, S A; Deregt, D; Yeo, S G; Thomas, E J

    1991-01-01

    A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum a...

  10. Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

    OpenAIRE

    Alonso-Urmeneta, B; Marin, C.M.; Aragon, V. (V.); Blasco, J.M. (José); Diaz, R.; Moriyon, I. (Ignacio)

    1998-01-01

    Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, an...

  11. Novel hapten synthesis for antibody production and development of an enzyme-linked immunosorbent assay for determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

    OpenAIRE

    Xu, Z.-L.; Shen, Yu-Dong; Sun, Yuan-Ming; Campbell, Katrina; Tian, Yaun-Xin; Zhang, Shi-Wei; Lei, Hong-Tao; Jiang, Yue-ming

    2013-01-01

    A heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) was developed. AMOZ was derivatised with 2-(4-formylphenoxy) acetic acid or 2-(3-formylphenoxy) acetic acid to obtain two novel immunizing haptens. The ability of these haptens in producing specific polyclonal antibodies against the nitrophenyl derivative of AMOZ (NPAMOZ) was compared with that of traditional immu...

  12. Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

    OpenAIRE

    Ge, Meng; Luo, Wei; Jiang, Daliang; Li, Runcheng; Zhao, Wenwei; Chen, Guoliang; Yang, Xingdong; Yu, Xinglong

    2012-01-01

    A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 9...

  13. Serodiagnostic comparison of enzyme-linked immunosorbent assay and surface plasmon resonance for the detection of antibody to Porcine circovirus type 2

    OpenAIRE

    Cho, Ho-Seong; Kim, Tae-Jung; Lee, Jae-Il; Park, Nam-Yong

    2006-01-01

    This paper describes the cloning and expression of the capsid protein of Porcine circovirus type 2 (PCV2) in an Escherichia coli expression system that was used to produce a fusion protein for subsequent immunologic studies: enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Polymerase chain reaction was used to amplify the gene encoding the capsid protein from the DNA of PCV2. The protein was then cloned into a pRSET prokaryotic expression vector. Western blot ana...

  14. An investigation of enzootic bovine leucosis (EBL) infection by agar gel immunodiffusion (AGID), enzyme linked immunosorbent assay (ELISA) tests and hematological applications on the dairy cows in the

    OpenAIRE

    Kale M.; Öztürk F.

    2004-01-01

    Haematological tests (alfa nafthyl acetate esterase ANAE activity, May Grünwald Giemsa staining and total leucocyte counts) were applied to 469 dairy cows, where the enzootic form of bovine leucosis was investigated. In the same 469 dairy cows, a search for antibodies directed against bovine leucosis virus (BLV) was carried out using agar gel immunodiffusion (AGID) in blood samples and enzyme linked immunosorbent assay (ELISA) in milk samples. Among the 469 animals screened, 90 were positive ...

  15. Comparison of indirect immunofluorescent-antibody assay, enzyme-linked immunosorbent assay, and Western immunoblot for the diagnosis of Lyme disease in dogs.

    OpenAIRE

    Lindenmayer, J; M. Weber; Bryant, J; Marquez, E; Onderdonk, A.

    1990-01-01

    Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent-antibody assay (IFA), and Western immunoblot were used to test serum samples from 128 dogs for the presence of antibody to Borrelia burgdorferi. Sera included 72 samples from dogs suspected of having Lyme disease, 32 samples from dogs residing in areas in which Lyme disease was not considered endemic, and 24 samples from dogs with clinical and serologic evidence of immune-mediated disease (n = 10), Rocky Mountain spotted fe...

  16. Comparison of monoclonal antibody-based sandwich enzyme-linked immunosorbent assay and virus isolation for detection of peste des petits ruminants virus in goat tissues and secretions.

    OpenAIRE

    Saliki, J T; House, J A; MEBUS, C.A.; Dubovi, E. J.

    1994-01-01

    A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed for specific detection of peste des petits ruminants virus. Compared with virus isolation in Vero cell cultures using 89 paired tissue and secretion samples from six experimentally infected goats, S-ELISA was significantly more sensitive (71.9% versus 65.2%; P < 0.05). The S-ELISA is a suitable alternative to virus isolation.

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

  18. Multicountry prospective clinical evaluation of two enzyme-linked immunosorbent assays and two rapid diagnostic tests for diagnosing dengue fever.

    Science.gov (United States)

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

    2015-04-01

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

  19. Development of an enzyme-linked immunosorbent assay to detect chicken serum antibody to glycoprotein G of infectious laryngotracheitis virus.

    Science.gov (United States)

    Shil, Niraj K; Markham, Philip F; Noormohammadi, Amir H; O'Rourke, Denise; Devlin, Joanne M

    2012-09-01

    Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction

  20. The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

    OpenAIRE

    Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F. W.

    1987-01-01

    The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("ex...

  1. Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces

    International Nuclear Information System (INIS)

    Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy. (author)

  2. Entwicklung und Validierung eines Enzyme-linked Immunosorbent Assays (ELISA) für die Quantifizierung von Carbamazepin in Abwasser, Oberflächenwasser und Trinkwasser

    OpenAIRE

    Bahlmann, Arnold

    2013-01-01

    Ein kompetitiver ELISA (Enzyme-linked Immunosorbent Assay) für den Nachweis von Carbamazepin (CBZ) mit einer Bestimmungsgrenze von ca. 30 ng/L wurde entwickelt und validiert. Dieser in Gewässern häufig auftretende anthropogene Marker wurde anschließend in einer Vielzahl an Proben aus Abwässern, Oberflächengewässern und Trinkwässern nachgewiesen. Der ELISA zeigte eine exzellente Präzision und erbrachte in allen Matrizes geringfügig höhere Analysenergebnisse als die Referenzmethode HPLC-MS/MS. ...

  3. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    OpenAIRE

    Yan Zhang; FengXia Wang; Li Fang; Shuo Wang; GuoZhen Fang

    2009-01-01

    To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extract...

  4. Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

    International Nuclear Information System (INIS)

    The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems. (author)

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

    Directory of Open Access Journals (Sweden)

    Joyce Matie Kinoshita da Silva

    2014-06-01

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

  6. Indirect enzyme-linked immunosorbent assay for measurement of human immunoglobulins E and G to purified cow's milk proteins: application in diagnosis of cow's milk allergy.

    OpenAIRE

    Campbell, D E; Ngamphaiboon, J; Clark, M M; Harris, M C; Kolski, G B; Douglas, S D

    1987-01-01

    An indirect double-antibody enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human immunoglobulin E (IgE) and IgG to the cow's milk proteins (CMP) alpha-casein, alpha-lactalbumin, and beta-lactoglobulin. Human serum albumin was used as the negative-antigen control. Rabbit anti-human IgE or IgG served as the primary antibody, and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin served as the secondary antibody. Positive control sera were obtained...

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

    OpenAIRE

    Hoorfar, J; Lind, P.; Bitsch, V

    1995-01-01

    Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from a...

  8. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

    OpenAIRE

    Walsh, Robert B.; Kelton, David F.; Hietala, Sharon K.; Duffield, Todd F.

    2013-01-01

    Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identifi...

  9. Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to Caprine Arthritis-Encephalitis Virus: Diagnostic Tool for Successful Eradication

    OpenAIRE

    Herrmann, Lynn M.; Cheevers, William P.; McGuire, Travis C.; Adams, D. Scott; Hutton, Melinda M.; Gavin, William G.; Knowles, Donald P

    2003-01-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Özyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGu...

  10. Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Herrmann, Lynn M.; Cheevers, William P.; Marshall, Katherine L.; McGuire, Travis C.; Hutton, Melinda M.; Lewis, Gregory S.; Knowles, Donald P

    2003-01-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of bi...

  11. Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus

    OpenAIRE

    Saijo, Masayuki; Qing, Tang; Niikura, Masahiro; Maeda, Akihiko; Ikegami, Tetsuro; Prehaud, Christophe; Kurane, Ichiro; Morikawa, Shigeru

    2002-01-01

    The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni2+ column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previous...

  12. Kinetics of specific immunoglobulins M, E, A, and G in congenital, primary, and secondary cytomegalovirus infection studied by antibody-capture enzyme-linked immunosorbent assay.

    OpenAIRE

    Nielsen, S L; Sørensen, I; Andersen, H K

    1988-01-01

    Antibody-capture enzyme-linked immunosorbent assay (ELISA) using enzyme-labeled cytomegalovirus (CMV) nuclear antigen is a reliable and easily performed test suitable for routine use. As the serologic response to CMV infection may, however, vary considerably among patients, we have studied the kinetics of CMV-specific immunoglobulin M (IgM), IgE, IgA, and IgG antibodies in 352 sera from 61 patients by using antibody-capture ELISA and complement fixation (CF) tests. In a CMV mononucleosis grou...

  13. Detection of cytomegalovirus in urine samples by an enzyme-linked immunosorbent assay using a monoclonal antibody against the viral 150-kilodalton protein.

    OpenAIRE

    Yamanaka, T.(International Center for Elementary Particle Physics, Department of Physics, The University of Tokyo, Tokyo, Japan); Kiyotani, K; Sakaguchi, T.; Y. Fukuda(Miyagi University of Education); Dohi, K.; Yamada, M.; Yoshida, M; Nii, S.; Yoshida, T.

    1992-01-01

    McKeating et al. (J.A. McKeating, P.D. Griffiths, and J.E. Grundy, J. Gen. Virol. 68:785-792, 1987; J. A. McKeating, J. E. Grundy, Z. Varghese, and P. D. Griffiths, J. Med. Virol. 18:341-348, 1986; J. A. McKeating, S. Stagno, P. R. Stirk, and P. D. Griffiths, J. Med. Virol. 16:367-373, 1985) reported previously that beta 2 microglobulin inhibits the detection of human cytomegalovirus (CMV) in urine specimens by an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody against th...

  14. Modification of a direct enzyme-linked immunosorbent assay for the detection of immunoglobulin G and M antibodies to pneumococcal capsular polysaccharide.

    OpenAIRE

    Messina, J P; Hickox, P G; Lepow, M. L.; Pollara, B; Venezia, R A

    1985-01-01

    In contrast to the usual indirect enzyme-linked immunosorbent assay (ELISA) method for detection of antibody responses, a modified direct ELISA technique was used to measure immunoglobulin G (IgG) and IgM responses to pneumococcal capsular types 1, 3, 9N, and 23F in humans. Individual capsular polysaccharides were covalently bound to poly-L-lysine before adsorption to the solid phase. The coupling reaction was enhanced by maintenance of a constant pH of 8.2 after the addition of all reactants...

  15. 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...

  16. Evaluation of enzyme-linked immunosorbent assays and a haemagglutination inhibition tests for the detection of antibodies to Newcastle disease virus in village chickens using a Bayesian approach.

    Science.gov (United States)

    Chaka, H; Thompson, P N; Goutard, F; Grosbois, V

    2015-04-01

    Newcastle disease (ND) is an endemic disease in village chickens in Ethiopia with substantial economic importance. The sensitivity (Se) and specificity (Sp) of a blocking enzyme-linked immunosorbent assay (bELISA, Svanova Biotech), indirect ELISA (iELISA, Laboratoire Service International) and haemagglutination inhibition (HI) test for ND virus (NDV) antibody detection were evaluated in a Bayesian framework in the absence of a gold standard test, on sera collected from unvaccinated chickens kept under the village production system in household flocks and at markets in two woredas (i.e. districts) of the Eastern Shewa zone, Ethiopia. The outcomes of the iELISA test differed dramatically from those of the two other tests with 92% of the samples testing positive as compared with less than 15% for bELISA and HI. iELISA results were also inconsistent with previous estimations of Newcastle serological prevalence. The information provided by the iELISA test was thus considered as highly unreliable, probably due to an extremely low specificity, and thus not considered in the Bayesian models aiming at estimating serological prevalence and test performance parameters. Bayesian modelling of HI and bELISA test results suggested that bELISA had both the highest Se (86.6%; 95% posterior credible interval (PCI): 61.8%; 98.5%), and the highest Sp (98.3%; 95% PCI: 97.2%; 99.5%), while HI had a Se of 80.2% (95% PCI: 59.1%; 94.3%), and a Sp of 96.1% (95% PCI: 95.1%; 97.4%). Model selection and the range of the posterior distribution of the correlation between bELISA and HI test outcomes for truly seropositive animals (median at 0.461; PCI: -0.055; 0.894) suggested a tendency for bELISA and HI to detect the same truly positive animals and to fail to detect the same truly positive animals. The use of bELISA in screening and surveillance for NDV antibodies is indicated given its high Se and Sp, in addition to its ease of automation to handle large numbers of samples compared to HI. The

  17. 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...

  18. 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).

  19. Use of circumsporozoite protein enzyme-linked immunosorbent assay compared with microscopic examination of salivary glands for calculation of malaria infectivity rates in mosquitoes (Diptera: Culicidae) from Cameroon.

    Science.gov (United States)

    Fontenille, D; Meunier, J Y; Nkondjio, C A; Tchuinkam, T

    2001-05-01

    A survey in Cameroon compared the usefulness of the circumsporozoite protein enzyme-linked immunosorbent assay (CSP ELISA) to dissection and microscopic examination of anopheline salivary glands for measuring infectivity rates in anopheline mosquitoes. The salivary glands of 375 females, belonging to four species were examined for sporozoites. After microscopic examination, the glands as well as all the remaining heads and thoraces were tested by ELISA. The sensitivity of ELISA was 100% (18/18), confidence interval (CI) (78.1-100) and the specificity was 99.7% (357/358), CI (98.2 100). The Kappa value, agreement between examination of the glands and salivary gland ELISA, was 0.97. The head thorax CSP ELISA overestimated the true salivary gland infection rate by 12.0%. The results obtained in Central Africa in a village with perennial transmission highly justified the use of the ELISA for measuring the entomological inoculation rate. PMID:11372973

  20. An investigation of enzootic bovine leucosis (EBL infection by agar gel immunodiffusion (AGID, enzyme linked immunosorbent assay (ELISA tests and hematological applications on the dairy cows in the

    Directory of Open Access Journals (Sweden)

    Kale M.

    2004-01-01

    Full Text Available Haematological tests (alfa nafthyl acetate esterase ANAE activity, May Grünwald Giemsa staining and total leucocyte counts were applied to 469 dairy cows, where the enzootic form of bovine leucosis was investigated. In the same 469 dairy cows, a search for antibodies directed against bovine leucosis virus (BLV was carried out using agar gel immunodiffusion (AGID in blood samples and enzyme linked immunosorbent assay (ELISA in milk samples. Among the 469 animals screened, 90 were positive by ELISA and the haematological tests, while 23 were positive by the AGID test. From these results we would advise heavily infected farms to be eliminated. ELISA and haematological methods, in which ANAE activity, May-Grünwald Giemsa staining and total leucocyte counts were used together, were more reliable and sensitive than the AGID test for detecting EBL.

  1. Use of radio-immuno-assay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques to diagnose some tick-borne hemo-parasitic diseases

    International Nuclear Information System (INIS)

    Improved serology is essential for the diagnosis of tick-borne diseases in epidemiological studies. Sero-diagnosis of hemo-protozoan diseases is an important adjust to the use of vaccines for the control of these infections. These tests must meet a number of criteria, including sensitivity, specificity and the ability to detect long term infections. Enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) techniques were developed for the diagnosis of both Babesia bovis and B.bigemina antibodies. Whilst the RIA technique is more sensitive than the ELISA, the latter has the advantage of stability and relative safety of the detection system. Long term single infections can be diagnosed with these systems and they are ideal for detecting low levels of antibody. Further development of the B.bigemina ELISA is necessary, especially as the antigen is quite labile and must be freshly prepared. This is the one disadvantage in an otherwise highly sensitive and specific test system

  2. 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.

  3. Standardization of the indirect enzyme-linked immunosorbent assay for detection of antibodies against Newcastle disease virus in chickens

    International Nuclear Information System (INIS)

    Newcastle disease is the major viral disease of poultry causing significant economic losses in most countries except Australia and New Zealand. Serological monitoring of poultry has traditionally been carried out using the haemagglutinin-inhibition (HI) test. More recently, ELISA has been used for the same purpose. This paper described the use of an indirect ELISA for assay of antibodies in chickens against Newcastle disease viruses and compares some of the parameters for this test. The sucrose density gradient purified, inactivated, antigen enabled performance of the test without the addition of any blocking agents other than the usual Tween 20. A range of plates was compared and the most suitable plate was found to be a polystyrene haemagglutination plate giving an excellent positive to negative ratio of 33.2, compared with some expensive ELISA plates which gave very low +ve/-ve ratios. Various incubation conditions for the steps in the ELISA were compared and incubation with shaking at room temperature (24 to 28 deg. C) gave adequate reactivity whilst simplifying incubation conditions and speeding up the test. The negative cut-off value was determined by testing 1632 HI negative specific pathogen free sera from birds of a wide age range. The reactivity of sera in the ELISA was standardized using a standard curve on every plate and converting the readings to ELISA units (EUs) in the range of 16 to 512 EUs. The EU values of these sera were not normally distributed and so the 95% cut-off was determined by ranking the values in descending order and retaining only the top 5% of the values as false positives. This resulted in a cut-off value of 33.6 EUs, with few of HI positive sera having values lower than this cut-off. The use of a standard curve on each plate is recommended in order to standardize the assay and to determine the ELISA units for the test sera. (author). 14 refs, 2 figs, 1 tab

  4. Distribution of Murine Tissue Specific γ-Glutamyltransferase: Comparison of six monoclonal antibody applications in Enzyme linked immunosorbent assay, radioimmunoassay, immunohistochemistry, and autoradiography

    International Nuclear Information System (INIS)

    γ-Glutamyltransferase (GGT: E.C. 2.3.2.2.) is a glycoprotein enzyme which is involved in glutathione metabolism and amino acid transport through the plasma membrane. It is distributed widely in several organs including liver and kidney. Several isozymes of GGT have been reported and some of the isozymes may be associated with hepatocarcinogenesis. We have produced six monoclnal antibodies (mAbs) against GGT purified from the liver of 2-acetamidofluorene (AAF) treated rats. All of the six mAbs were obtained by immunizing mice with liver GGT. Six hybridomas which produced anti-GGT Abs were extensively subcloned and injected into the peritoneal cavity of BALB/c mice to obtain large quantities of Abs. These mAbs were purified from ascites by ammonium sulfate precipitation and protein A sepharose CL-4B column chromatography. Using these mAbs we performed enzyme-linked immunosorbent assay (ELISA), radioimmvnoassay (RIA), immunohistochemistry (IHC), and autoradiography (ARG) to study the distribution of GGT isozyme in tissue. The results indicate that GGT-mAb 1 is specific for the AAF treated liver GGT, GGT-mAb 5 for the normal liver GGT, and GGT-mAb 6 for the normal kidney GGT. These mAbs may be used to evaluate the distribution of GGT isozymes in different tissues.

  5. Use of chemiluminescence for the serological diagnosis of bovine and ovine brucellosis with indirect and competitive enzyme-linked immunosorbent assays

    Directory of Open Access Journals (Sweden)

    Manuela Tittarelli

    2008-06-01

    Full Text Available The official methods specified in the national bovine and ovine/caprine brucellosis eradication plan are the Rose Bengal and complement fixation tests. In the current phase of the eradication plan, it is often difficult to interpret the results obtained with the official tests. Consequently, additional tests that offer greater sensitivity and specificity are thus required. For this reason, two methods, the indirect chemiluminescence enzyme-linked immunosorbent assay (i-ELISA CL and the competitive chemiluminescence ELISA (c-ELISA CL that use a chemiluminescent substrate to determine anti-Brucella antibodies in bovine and ovine serum were validated. The methods are based on the detection of anti-Brucella antibodies in serum by catalysis of a chemiluminescent enzyme substrate (luminol/ H2O2/enhancer system by peroxidase conjugated to secondary anti IgG antibodies in i-ELISA CL and to monoclonal anti-lipopolysaccharide (LPS antibodies in c-ELISA CL. From the results obtained, a cut-off of 60% for bovine serum and 37.5% for ovine serum, expressed as positivity rate (PR, were established Using these cut-off values, for the i-ELISA CL, 100% sensitivity and specificity was obtained for bovine serum and 100% sensitivity and 99.8% specificity for ovine serum. Cut-off values of 30% for bovine serum and 40% for ovine serum, expressed as inhibition rate, were selected for c-ELISA CL, which ensured 100% sensitivity and specificity in both cases.

  6. Parvovirus B19 infections in state of Rio de Janeiro, Brasil: 526 sera analyzed by IgM-enzyme-linked immunosorbent assay and polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    MCL Mendonça

    2005-12-01

    Full Text Available In this study were analyzed 526 sera; the patients aged from two days to 65 years old presenting exanthema, which was the most frequent symptom observed, besides fever, adenomegaly, and arthralgia. These sera were negative by enzyme-linked immunosorbent assay (IgM-ELISA for either rubella (495, toxoplasma (41, cytomegalovirus (12, measles (40, dengue (56, and they were submitted to nested polymerase chain reaction (PCR for B19 DNA and commercial IgM-ELISA for B19. In 39 abortion cases, IgM or DNA were not detected, therefore they were not took into account for analysis. Specific DNA and IgM were detected respectively in 71 (14.5% and IgM in 62 (12.7% sera from 487 sera analyzed. IgM and DNA were simultaneously detected in 43 (8.8%, while agreement among the results by PCR and IgM-ELISA was observed in 440 (90.4%. The sera were collected from January 1999 to December 2000, most of them in 1999 (325, during winter and spring. The major number of clinical cases was observed in the age group from one to ten years old. IgM or DNA were detected in 23 from 51 municipal districts of the state of Rio de Janeiro, where the samples were collected.

  7. Indirect enzyme-antibody sandwich enzyme-linked immunosorbent assay for quantification of TAXI and XIP type xylanase inhibitors in wheat and other cereals.

    Science.gov (United States)

    Beaugrand, Johnny; Gebruers, Kurt; Ververken, Cedric; Fierens, Ellen; Dornez, Emmie; Goddeeris, Bruno M; Delcour, Jan A; Courtin, Christophe M

    2007-09-19

    To quantify Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP) type proteins in cereals in general and wheat ( T. aestivum) in particular, a robust enzyme-linked immunosorbent assay (ELISA) using an uncommon enzyme-antibody sandwich format was developed. Bacillus subtilis glycoside hydrolase family (GH) 11 and Aspergillus oryzae GH 10 xylanases were selected for coating ELISA plate wells to capture TAXI and XIP, respectively, prior to probing with antibodies. The detection threshold of the developed ELISA was much lower than that of the currently used xylanase inhibitor assay and the recently described Western blot approach. Because of its broad dynamic range (TAXI, 30-600 ng/mL, and XIP, 3-60 ng/mL), one proper standard extract dilution can be used for analyzing different wheat varieties, whereas for the currently used colorimetric assay, often different dilutions need to be analyzed. The TAXI ELISA for wheat was successfully adapted for barley ( Hordeum vulgare) and could also be used for other cereals. PMID:17715986

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

    Science.gov (United States)

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

    2015-10-21

    A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples. PMID:26416205

  9. Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial enzyme-linked immunosorbent assay kits.

    Science.gov (United States)

    Lee, Hyun Jung; Meldrum, Alexander D; Rivera, Nicholas; Ryu, Dojin

    2014-10-01

    Ochratoxin A (OTA), a nephrotoxic mycotoxin, naturally occurs in wide range of agricultural commodities. Typical screening of OTA involves various enzyme-linked immunosorbent assay (ELISA) methods. Pistachio (Pistacia vera L.) is a rich source of phenolic compounds that may result in a false positive due to structural similarities to OTA. The present study investigated the cross-reactivity profiles of phenolic compounds using two commercial ELISA test kits. High-performance liquid chromatography was used to confirm the concentration of OTA in the pistachio samples and compared with the results obtained from ELISA. When the degree of interaction and 50 % inhibitory concentration of phenolic compounds were determined, the cross-reactivity showed a pattern similar to that observed with the commercial ELSIA kits, although quantitatively different. In addition, the degree of interaction increased with the increasing concentration of phenolic compounds. The ELISA value had stronger correlations with the content of total phenolic compound, gallic acid, and catechin (R(2) = 0.757, 0.732, and 0.729, respectively) compared with epicatechin (R(2) = 0.590). These results suggest that phenolic compounds in pistachio skins may cross-react with the OTA antibody and lead to a false positive or to an overestimation of OTA concentration in ELISA-based tests. PMID:25285493

  10. Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red

    International Nuclear Information System (INIS)

    To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01 ng mL-1 in phosphate-buffered saline (PBS) buffer and 0.5 ng g-1 in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products

  11. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

    Science.gov (United States)

    Walsh, Robert B.; Kelton, David F.; Hietala, Sharon K.; Duffield, Todd F.

    2013-01-01

    Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection. PMID:24082160

  12. Use of a commercial enzyme-linked immunosorbent assay for rapid detection of Giardia duodenalis in dog stools in the environment: a Bayesian evaluation.

    Science.gov (United States)

    Papini, Roberto; Carreras, Giulia; Marangi, Marianna; Mancianti, Francesca; Giangaspero, Annunziata

    2013-05-01

    Giardia duodenalis is considered a potentially zoonotic protozoan. Some animal species, including infected dogs, may play an important role in the spread of Giardia cysts through environmental contamination with their feces. In the present study, a commercial enzyme-linked immunosorbent assay (ELISA) was used to examine 143 samples of dog feces collected in urban areas as an indicator of the risk of field contamination. Using a Bayesian statistical approach, the ELISA showed a sensitivity of 88.9% and a specificity of 95.8% with positive and negative predictive values of 89.6% and 95.4%, respectively. The test affords the advantage of rapid processing of fecal samples without a complex technical structure and extensive costly labor. Moreover, the present results show that the assay provides public health veterinarians with a practical tool that can be used in screening programs, as a valid alternative or as an adjunct to other tests, in order to assess the biological risk of exposure to G. duodenalis cysts from dogs in human settlements. However, the test may not be completely accurate for human health risk evaluation, as it does not discriminate between zoonotic and non-zoonotic isolates. PMID:23604261

  13. Immunodiagnosis of bovine trypanosomiasis in Anambra and Imo states, Nigeria, using enzyme-linked immunosorbent assay: zoonotic implications to human health

    Directory of Open Access Journals (Sweden)

    M.C. Ezeani

    2008-11-01

    Full Text Available Background & objectives: The prevalence of trypanosomiasis was studied in cattle, being a major source of animal protein in Nigeria, thus, a very likely means of spread of Human African Trypano-somosis (HAT. Methods: Enzyme-linked immunosorbent assay (ELISA was used to diagnose bovine trypanosomiasis in 264 samples collected from adult cattle of mixed breeds, age and sex, in Anambra and Imo states, Nigeria. Results: Out of 264 samples analysed, 21 (7.96% were seropositive for Trypanosoma congolense while 20 (7.58% were seropositive for T. vivax and 8 (3.03% were seropositive for T. brucei infections in both the states. Interpretation & conclusion: The predominant species was found to be T. congolense. Mixed infection of three species, T. vivax, T. congolense and T. brucei was found to dominate other mixed infections in both the states. ELISA detected the infection of the three species of trypanosomes in the same group of animals. The usefulness of antigen capture ELISA in the diagnosis of human or animal trypanosomiasis was established, and the possibility of the spread of HAT caused by T. brucei gambiense and T.b. rhodesiense through cattle was expressed.

  14. Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

    Science.gov (United States)

    Heller, Martin; Gicheru, Nimmo; Tjipura-Zaire, Georgina; Muriuki, Cecilia; Yu, Mingyan; Botelho, Ana; Naessens, Jan; Jores, Joerg; Liljander, Anne

    2016-06-01

    Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP. PMID:27053669

  15. Mutations in the rpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Garcia, Lucia; Alonso-Sanz, Mercedes; Rebollo, Maria J.; Tercero, Juan C.; Chaves, Fernando

    2001-01-01

    Genetic alterations in the rpoB gene were characterized in 50 rifampin-resistant (Rifr) clinical isolates of Mycobacterium tuberculosis complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of rpoB mutations was evaluated with isolates of the M. tuberculosis complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different rpoB mutations among the Rifr isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rifr allele within each strain, 10 of the 11 Rifr genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of M. tuberculosis complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rifr organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods. PMID:11325996

  16. Enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody as a new tool to detect Sudan dyes and Para red

    Energy Technology Data Exchange (ETDEWEB)

    Ju Chunmei [College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642 (China); Tang Yong [Center of Antibody Engineering, Department of Bioengineering, Jinan University, Guangzhou 510632 (China); Fan Huiying [College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642 (China); Chen Jinding [College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642 (China)], E-mail: jdchen@scau.edu.cn

    2008-07-28

    To set up an immunoassay-based method to detect Sudan dyes and Para red, we generated a monoclonal antibody (Mab) using a specially designed carboxyl derivative of Sudan I (CSD I) as the immunogen. CSD I was synthesized by azocoupling reaction using 2-naphthol and diazotised 4-aminobenzoic acid. The antibody was obtained from a hybridoma, which was derived from the fusion of the mouse myeloma SP2/0 cells and the splenocytes from the mice immunized with the CSD I-bovine serum albumin (BSA) conjugate. In addition, we showed that the Mab was highly specific for Sudan I, III and Para red. The limit of detection was approximately 0.01 ng mL{sup -1} in phosphate-buffered saline (PBS) buffer and 0.5 ng g{sup -1} in chilli tomato sauce. The recoveries of Sudan I, III and Para red for the chilli tomato sauce were from 84% to 99% and coefficients of variation were from 14.9% to 33.3%. Thus, the enzyme-linked immunosorbent assay (ELISA) method is a rapid and high throughput screening tool to detect Sudan dyes and Para red in food products.

  17. Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

    Science.gov (United States)

    Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

    2015-06-01

    Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac. PMID:25928837

  18. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    2009-01-01

    Full Text Available To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver, we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2>0.95, which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods.

  19. Rapid Determination of Ractopamine Residues in Edible Animal Products by Enzyme-Linked Immunosorbent Assay: Development and Investigation of Matrix Effects

    Science.gov (United States)

    Zhang, Yan; Wang, FengXia; Fang, Li; Wang, Shuo; Fang, GuoZhen

    2009-01-01

    To determine ractopamine residues in animal food products (chicken muscle, pettitoes, pig muscle, and pig liver), we established a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody generated from ractopamine-linker-BSA. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 0.6 ng/mL, and the limit of detection was 0.04 ng/mL. The matrix effect of the samples was easily eliminated by one-step extraction with PBS, without any organic solution or clean-up procedure such as SPE or liquid-liquid extraction, making it a much more simple and rapid method than previously reported ones. The detection limit in blank samples was 0.2 μg/kg. To validate this new RAC (ractopamine hydrochloride) ELISA, a RAC-free pig liver sample spiked at three different concentrations was prepared and analyzed by HPLC and ELISA. The results showed a good correlation between the data of ELISA and HPLC (R2 > 0.95), which proves that the established ELISA is accurate enough to quantify the residue of RAC in the animal derived foods. PMID:19826637

  20. 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.

  1. Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus.

    Science.gov (United States)

    de Rezende, N A; Dias, M B; Campolina, D; Chavéz-Olortegui, C; Amaral, C F

    1995-01-01

    The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD) corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients. PMID:7569644

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

    Science.gov (United States)

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

    2016-06-01

    For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

  3. Enzyme-linked immunosorbent assay with worm vomit and cercarial secretions of Schistosoma mansoni to detect infections in an endemic focus of Burkina Faso.

    Science.gov (United States)

    Bahgat, M; Sorgho, H; Ouédraogo, J B; Poda, J N; Sawadogo, L; Ruppel, A

    2006-03-01

    Cercariae and adult Schistosoma mansoni were used to prepare, respectively, cercarial secretions (CS) and worm vomit (WoV). These were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to test the IgG-reactivity of sera obtained in an S. mansoni-endemic area of Burkina Faso. Among the egg-excreting individuals (n = 240), 94.6% reacted positively with WoV, but only 62.9% with CS, thus suggesting a high diagnostic sensitivity of WoV, but not of CS. Among those individuals without detectable eggs in two Kato-Katz thick smears from different stool specimens (n = 215), the respective percentages of positive IgG reactivity were 78.1% and 63.3%. These positive reactions in the absence of detectable eggs are interpreted in terms of limited sensitivity of parasitological stool examinations. Optical density values in ELISA with CS, but not with WoV, correlated negatively with age, which may reflect decreasing exposure to cercariae in older individuals. PMID:16469168

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

    Science.gov (United States)

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

    2010-05-26

    Prickly pear cactus has been an important food source in Mexico since ancient times due to its economical and ecological benefits and potential nutraceutical value. Nevertheless, studies on the nutritional aspects and health benefits have been scarce. The purpose of this study was to assess, apparently for the first time, the folate contents of cladodes of nopal by a microbiological assay, using Lactobacillus casei (ATCC 7469) in extracts that were enzymatically treated to release the bound vitamin, employing single, dual, and trienzymatic procedures, and using the enzyme-linked immunosorbent assay (ELISA). We used Opuntia cladodes of different length sizes. The microbiological assay showed some differences among enzyme treatments and sizes of nopal; the trienzyme treatment (alpha-amylase-protease-conjugase) was more efficient in determining the folate content in nopal, giving 5.0 ng/g in the small size cladodes at 54 h of testing time, while ELISA showed no significant differences in the folate content among sizes of cladodes (5.5-5.62 ng/g at 0 min testing time). Both techniques may be used for the assessment of folate content in cladodes, but ELISA is more rapid and reliable. PMID:20441169

  5. Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

    Institute of Scientific and Technical Information of China (English)

    Yi; Huang; Youjie; Zhu; Mengshi; Yang; Zhenqing; Zhang; Donglin; Song; Zhiming; Yuan

    2014-01-01

    Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

  6. Application of 3D Printing Technology in Increasing the Diagnostic Performance of Enzyme-Linked Immunosorbent Assay (ELISA for Infectious Diseases

    Directory of Open Access Journals (Sweden)

    Harpal Singh

    2015-07-01

    Full Text Available Enzyme-linked Immunosorbent Assay (ELISA-based diagnosis is the mainstay for measuring antibody response in infectious diseases and to support pathogen identification of potential use in infectious disease outbreaks and clinical care of individual patients. The development of laboratory diagnostics using readily available 3D printing technologies provides a timely opportunity for further expansion of this technology into immunodetection systems. Utilizing available 3D printing platforms, a ‘3D well’ was designed and developed to have an increased surface area compared to those of 96-well plates. The ease and rapidity of the development of the 3D well prototype provided an opportunity for its rapid validation through the diagnostic performance of ELISA in infectious disease without modifying current laboratory practices for ELISA. The improved sensitivity of the 3D well of up to 2.25-fold higher compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization and Lab-On-a-Chip platforms to reduce time, volume of reagents and samples needed for such assays in the laboratory diagnosis of infectious and other diseases including applications in other disciplines.

  7. Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis.

    Science.gov (United States)

    Vanasco, N B; Jacob, P; Landolt, N; Chiani, Y; Schmeling, M F; Cudos, C; Tarabla, H; Lottersberger, J

    2016-04-01

    Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations. PMID:26867967

  8. Comparative Evaluation of Fast Enzyme Linked Immunosorbent Assay (Fast-ELISA and Standard-ELISA For The Diagnosis Of Human Hydatidosis

    Directory of Open Access Journals (Sweden)

    MB Rokni

    2006-06-01

    Full Text Available Fast enzyme linked immunosorbent assay (Fast-ELISA was compared with the standard ELISA for the diagnosis of human hydatidosis. Seventy serum samples including 30 from hydatidosis patients (surgically confirmed, healthy control individuals not infected with any parasitic diseases (n=/20 and from others with different parasitic infections including, toxocariosis (n=5, fasciolosis (n=5, trichostrongylosis (n=5, and strongyloidosis (n=5 were analysed for anti-hydatid IgG antibodies using sheep hydatid cyst fluid antigen. The sensitivity, specificity, positive and negative predictive values, as well as validity of the test were found as 96.7%, 95.2%, 93.7%, 97.5% and 96% for conventional ELISA, while these paramters for fast-ELISA were respectively as follows: 100%, 97.5%, 96.7%, 100% and 98.8%. Regarding standard-ELISA 3µg/ml of antigen, serum dilution of 1:500, conjugate dilution of 1:3000 and 30 min incubation were found optimal, while for fast-ELISA 3µg/ml of antigen, serum dilution of 1:125, conjugate dilution of 1:1000 and 5 min incubation were utilized. The present study indicates that fast ELISA can easily be performed in place of the standard ELISA for the serodiagnosis of human hydatidosis with the advantage of minimising consumed time and manpower hours. Moreover, this test can be utilized in screening tests to diagnos human hydatidosis.

  9. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of 6-benzylaminopurine and its ribose adduct in bean sprouts.

    Science.gov (United States)

    Zhang, Wei; He, Lishan; Zhang, Rui; Guo, Suqin; Yue, Huanfang; Ning, Xiangxue; Tan, Guiyu; Li, Qing X; Wang, Baomin

    2016-09-15

    6-Benzylaminopurine (6-BA), a cytokinin plant growth regulator, has been banned for use in bean sprout production in China. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with a specific monoclonal antibody (mAb 3E5). The assay showed a half-maximum inhibition concentration (IC50) and detection range of 18.9ng/mL and 3.6-106ng/mL, respectively. Recoveries of 6-BA spiked in home cultured bean sprout samples averaged from 75% to 89% with a correlation coefficient (R(2)) of 0.998 between the results determined by icELISA and those by liquid chromatography-electrospray ionization quadrupole Orbitrap mass spectrometry (LC-ESI-MS). LC-ESI-MS showed that 6-BA had been partially metabolized to 6-benzylaminopurine riboside (6-BAR) in the positive samples. The content of 6-BA determined by icELISA was about 5-70 times higher than that of LC-ESI-MS because mAb 3E5 had 315% cross-reactivity with 6-BAR. Such icELISA being ultra-sensitive to 6-BAR would allow quick monitoring of 6-BA by detecting 6-BAR as a potential biomarker. PMID:27080901

  10. Analysis of benzo[a]pyrene in vegetable oils using molecularly imprinted solid phase extraction (MISPE) coupled with enzyme-linked immunosorbent assay (ELISA).

    Science.gov (United States)

    Pschenitza, Michael; Hackenberg, Rudolf; Niessner, Reinhard; Knopp, Dietmar

    2014-01-01

    This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE) method coupled with enzyme-linked immunosorbent assay (ELISA) for determination of the PAH benzo[a]pyrene (B[a]P) in vegetable oils. Different molecularly imprinted polymers (MIPs) were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q) of ~32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v)) was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v)) for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values. PMID:24887045

  11. Analysis of Benzo[a]pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISPE Coupled with Enzyme-Linked Immunosorbent Assay (ELISA

    Directory of Open Access Journals (Sweden)

    Michael Pschenitza

    2014-05-01

    Full Text Available This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE method coupled with enzyme-linked immunosorbent assay (ELISA for determination of the PAH benzo[a]pyrene (B[a]P in vegetable oils. Different molecularly imprinted polymers (MIPs were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q of ~32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values.

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

    Science.gov (United States)

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

    2016-06-01

    Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies. PMID:27053675

  13. Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

    Science.gov (United States)

    Lally, N C; Jenkins, M C; Dubey, J P

    1996-05-01

    Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta. PMID:8705668

  14. Monoclonal antibody production and indirect competitive enzyme-linked immunosorbent assay development of 3-methyl-quinoxaline-2-carboxylic acid based on novel haptens.

    Science.gov (United States)

    Li, Guopeng; Zhao, Liang; Zhou, Feng; Li, Jiaying; Xing, Yuan; Wang, Tiangang; Zhou, Xilong; Ji, Baoping; Ren, Wanpeng

    2016-10-15

    Two novel immunizing haptens of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) were synthesized and conjugated with cationized bovine serum albumin. Female BALB/c mice were immunized with above conjugates, splenocytes were fused with Sp2/0 cells to produce monoclonal antibody. Compared with previous studies, antibodies raised in this work showed higher sensitivity. Meantime, a novel heterologous coating hapten was also prepared. The indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the optimum condition showed an IC50 of 3.1μg/kg (ppb), and the linear range of 0.46-10.5ppb for MQCA. The limit of detect (LOD) of MQCA in swine muscle, swine liver and chicken was 0.32, 0.54, and 0.28ppb, respectively. The LOD of this assay can satisfy the minimum required performance levels (4ppb) for MQCA. These results indicated that the proposed ELISA, with high sensitivity and specificity, as well as good reproducibility and accuracy, is suitable for determination of MQCA residues in food samples. PMID:27173564

  15. Quantitative Analysis of Free 15-F2t-Isoprostane from Plasma of Obstructive Sleep Apnea Patients Using Enzyme Linked Immunosorbe

    Directory of Open Access Journals (Sweden)

    Bertha Rusdi

    2012-03-01

    Full Text Available 15-F2t-isoprostane is a biomarker in assessment of oxidative stress status that due to its relatively low concentration in biological fluid and also has many isomers, the 15-F2t-isoprostane sample need to be extracted prior to the quantifying processes. Extraction techniques commonly used to extract 15-F2t-isoprostane are solid phase extraction (SPE and immunoaffinity extraction. Improvements to the SPE and immunoaffinity extraction techniques had been conducted, and the recovery results was then compared. The quantification of 15-F2t-isoprostane then was conducted using Enzyme Linked Immunosorbent Assay (ELISA method. Then followed by the examination of the plasma recovery results. Extraction technique which had the highest recovery then was used to quantify 15-F2t-isoprostane from plasma of Obstructive Sleep Apnea (OSA patients. Immunoaffinity extraction technique has a good recovery result. OSA patients have the tendency to have high 15-F2t-isoprostane concentrations in the plasma, therefore have a potential risk to get diseases related to the biological activities of 15-F2t-isoprostane, such as arteriosclerosis.

  16. A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA Detection Platform for a Point-of-Care Dengue Detection System on a Lab-on-Compact-Disc

    Directory of Open Access Journals (Sweden)

    Aung Thiha

    2015-05-01

    Full Text Available The enzyme-linked Immunosorbent Assay (ELISA is the gold standard clinical diagnostic tool for the detection and quantification of protein biomarkers. However, conventional ELISA tests have drawbacks in their requirement of time, expensive equipment and expertise for operation. Hence, for the purpose of rapid, high throughput screening and point-of-care diagnosis, researchers are miniaturizing sandwich ELISA procedures on Lab-on-a-Chip and Lab-on-Compact Disc (LOCD platforms. This paper presents a novel integrated device to detect and interpret the ELISA test results on a LOCD platform. The system applies absorption spectrophotometry to measure the absorbance (optical density of the sample using a monochromatic light source and optical sensor. The device performs automated analysis of the results and presents absorbance values and diagnostic test results via a graphical display or via Bluetooth to a smartphone platform which also acts as controller of the device. The efficacy of the device was evaluated by performing dengue antibody IgG ELISA on 64 hospitalized patients suspected of dengue. The results demonstrate high accuracy of the device, with 95% sensitivity and 100% specificity in detection when compared with gold standard commercial ELISA microplate readers. This sensor platform represents a significant step towards establishing ELISA as a rapid, inexpensive and automatic testing method for the purpose of point-of-care-testing (POCT in resource-limited settings.

  17. Serological analysis by enzyme-linked immunosorbent assay using recombinant antigen LipL32 for the diagnosis of swine leptospirosis.

    Science.gov (United States)

    Hartleben, Cláudia P; Leal, Fernanda M A; Monte, Leonardo G; Hartwig, Daiane D; Seixas, Fabiana K; Vasconcellos, Sílvio A; Brihuega, Bibiana; Dellagostin, Odir A

    2013-02-01

    Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14-60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys. PMID:23064970

  18. An electric detection of immunoglobulin G in the enzyme-linked immunosorbent assay using an indium oxide nanoparticle ion-sensitive field-effect transistor

    International Nuclear Information System (INIS)

    Semiconducting nanoparticle ion-sensitive field-effect transistors (ISFETs) are used to detect immunoglobulin G (IgG) in the conventional enzyme-linked immunosorbent assay (ELISA). Indium oxide and silica nanoparticles were layer-by-layer self-assembled with the oppositely charged polyelectrolyte as the electrochemical transducer and antibody immobilization site, respectively. The assay was conducted on a novel platform of indium oxide nanoparticle ISFETs, where the electric signals are generated in response to the concentration of target IgG using the labeled detecting antibody. The sandwiched ELISA structure catalyzed the conversion of the acidic substrate into neutral substance with the aid of horseradish peroxidase. The pH change in the substrate solution was detected by nanoparticle ISFETs. Normal rabbit IgG was used as a model antigen whose detection limit of 0.04 ng ml−1 was found. The facile electric detection in the conventional assay through the semiconducting nanoparticle ISFET has potential applications as a point-of-care detection or a sensing element in a lab-on-a-chip system

  19. Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows.

    Science.gov (United States)

    Walsh, Robert B; Kelton, David F; Hietala, Sharon K; Duffield, Todd F

    2013-04-01

    Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection. PMID:24082160

  20. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication.

    Science.gov (United States)

    Herrmann, Lynn M; Cheevers, William P; McGuire, Travis C; Adams, D Scott; Hutton, Melinda M; Gavin, William G; Knowles, Donald P

    2003-03-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Ozyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGuire, M. Hutton, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 8:44-51, 2001). Two hundred serum samples from goats in the United States were used to determine the sensitivity and specificity of cELISA based on the immunoprecipitation (IP) of [(35)S]methionine-labeled viral antigens as a standard of comparison. A positive cELISA was defined as >33.2% inhibition of MAb 74A binding based on 2 standard deviations above the mean percent inhibition of 140 IP-negative serum samples. At this cutoff value, there were 0 of 60 false-negative sera (100% sensitivity) and 5 of 140 false-positive sera (96.4% specificity). Additional studies utilized IP-monitored cELISA to establish a CAEV-free herd of 1,640 dairy goats. PMID:12626453

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    Aim:  The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100...... were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P < 0.001) and correlated to the amount of hepatic type IV collagen...... expression in BDL rats (r = 0.49, P < 0.05) in contrast to sham (r = -0.12). P4NP 7S also correlated to total collagen in CCL4 treated livers (P < 0.001, r = 0.67), however, not in controls (r = 0.04). Conclusions:  This newly developed serum assay specific for P4NP 7S was highly related to liver fibrosis...

  2. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

    Science.gov (United States)

    Pascho, R.J.; Chase, D.; McKibben, C.L.

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 3 109 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 3 104 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

  3. Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmonid ovarian fluid.

    Science.gov (United States)

    Pascho, R J; Chase, D; McKibben, C L

    1998-01-01

    Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 x 10(9) cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 x 10(4) cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods. PMID:9526862

  4. Relative efficiency of polymerase chain reaction and enzyme-linked immunosorbant assay in determination of viral etiology in congenital cataract in infants

    Directory of Open Access Journals (Sweden)

    Shyamala G

    2008-01-01

    Full Text Available Background: Perinatal viral infections of fetus are among the leading causes of congenital cataract and identifying the viral etiology is important. Objectives: To detect the presence of Rubella virus (RV, herpes simplex virus (HSV and cytomegalovirus (CMV in lens aspirate specimens obtained from patients with congenital cataract and relate the results with serology. Setting and Design: Prospective study carried out in tertiary care hospital. Materials and Methods: Fifty lens aspirates from 50 infants with congenital cataract were subjected to HSV, RV isolation and polymerase chain reaction (PCR for detection of HSV and CMV. Reverse transcription polymerase chain reaction (RT-PCR was applied for RV detection. Peripheral blood specimens were screened for anti-HSV, RV and CMV antibodies by enzyme-linked immunosorbant assay (ELISA. Results: Rubella virus was detected in nine (18% lens aspirates, by nRT-PCR which includes six positive by culture. HSV-2 DNA was detected in nine other lens aspirates, while CMV was not detected by PCR. Serological results did not correlate with the presence of viruses in the lens aspirates. This is the first report of detection of HSV-2 DNA in cases of congenital cataract. Conclusions: Cytomegalovirus may not be playing a significant role in causation of congenital cataract. The role of serology in identifying causative viral infection for congenital cataract needs to be re-evaluated.

  5. Milk progesterone enzyme-linked immunosorbent assay as a tool to investigate ovarian cyclicity of water buffaloes in relation to body condition score and milk production

    Directory of Open Access Journals (Sweden)

    Banu Turgish A

    2012-05-01

    Full Text Available Abstract Background Application of assisted reproductive technologies in buffaloes is limited to some extent by farmers’ inability to detect oestrus because of its poor expression. The present study aimed at investigating reliability of a milk progesterone enzyme-linked immunosorbent assay (ELISA to assess the ovarian cyclicity during post partum, oestrus and post-breeding periods in water buffaloes. Methods Progesterone concentrations were measured by an ELISA in milk of 23 postpartum buffaloes in relation to oestrus, pregnancy, body condition score (BCS and milk production. Two milk samples were taken at 10 days intervals, every month starting from day 30 and continued to day 150 post partum. BCS and milk production were recorded during sample collection. Milk samples from bred buffaloes were collected at Day 0 (day of breeding, Days 10–12 and Days 22–24. Defatted milk was preserved at −80°C until analysis. Pregnancy was confirmed by palpation per rectum on Days 70–90. Results Seventeen buffaloes had 47 ovulatory cycles, one to four in each, 13 were detected in oestrus once (28 % oestrus detection rate. Progesterone concentration ≥1 ng/ml in one of the two 10-day-interval milk samples reflected ovulation and corpus luteum formation. The intervals between calving to first luteal activity and to first detected oestrus varied from 41 to 123 days (n = 17 and 83 to 135 (n = 13 days, respectively. Eight buffaloes were bred in the course of the study and seven were found pregnant. These buffaloes had a progesterone profile of low (P P  Conclusions Milk progesterone ELISA is a reliable tool for monitoring ovarian cyclicity and good BCS may be an indicator of resuming cyclicity in water buffalo.

  6. Evaluation of an IgG Enzyme-Linked Immunosorbent Assay as a Serological Assay for Detection of Mycoplasma bovis Infection in Feedlot Cattle.

    Science.gov (United States)

    Wawegama, Nadeeka K; Markham, Philip F; Kanci, Anna; Schibrowski, Meghan; Oswin, Sally; Barnes, Tamsin S; Firestone, Simon M; Mahony, Timothy J; Browning, Glenn F

    2016-05-01

    Mycoplasma bovis is a pathogen of emerging significance in cattle throughout the world that is causing a range of diseases, including mastitis, arthritis, and pneumonia. The limited availability and efficacy of current diagnostic and prophylactic tools for its control and its increasing antimicrobial resistance are contributing to its increasing importance in beef and dairy cattle. We have developed an indirect IgG enzyme-linked immunosorbent assay (ELISA) based on a recombinant fragment of the MilA protein and have shown its potential as an effective diagnostic tool. To more comprehensively estimate the diagnostic sensitivity and specificity of this IgG ELISA for detection of infection with M. bovis in cattle and to define a suitable cutoff for use in the field, we further assessed its performance in experimentally infected calves in a closed beef herd and by applying Bayesian latent class modeling to laboratory testing results from 7,448 cattle entering Australian feedlots. The most effective cutoff points were estimated to be 68.6 antibody units (AU) for experimentally infected calves and to be 58.7 AU for a closed adult herd. Under field conditions, in feedlot cattle the globally optimal cutoff was estimated to be 105 AU. At this cutoff, the diagnostic sensitivity was 94.3% (95% probability interval [PI], 89.9% to 99.6%) with a diagnostic specificity of 94.4% (95% PI, 90.3% to 99.6%). Applying this 105 AU cutoff, 13.1% of cattle were seropositive for infection with M. bovis on entry into feedlots, and 73.5% were seropositive when followed up approximately 6 weeks later suggesting a high risk of infection shortly after entry into feedlots. PMID:26912757

  7. Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

  8. Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

    Institute of Scientific and Technical Information of China (English)

    WU Gang; CHEN LiHua; ZHONG Shan; LI Qi; SONG ChaoJun; JIN BoQuan; ZHU ZuoYan

    2008-01-01

    The aim of the present study was to purify the common native carp growth hormon.e (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries.Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1 -7, 9 and 10) were used in fluorescent staining and immunohistochemIstry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9- and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

  9. A protein A/G indirect enzyme-linked immunosorbent assay for the detection of anti-Brucella antibodies in Arctic wildlife.

    Science.gov (United States)

    Nymo, Ingebjørg H; Godfroid, Jacques; Åsbakk, Kjetil; Larsen, Anett K; das Neves, Carlos G; Rødven, Rolf; Tryland, Morten

    2013-05-01

    A species-independent indirect enzyme-linked immunosorbent assay (iELISA) based on chimeric protein A/G was established for the detection of anti-Brucella antibodies in Arctic wildlife species and compared to previously established brucellosis serological tests for hooded seals (Cystophora cristata), minke whales (Balaenoptera acutorostrata), sei whales (Balaenoptera borealis), fin whales (Balaenoptera physalus), and polar bears (Ursus maritimus), as well as bacteriology results for reindeer and caribou (Rangifer tarandus sp.). The protein A/G iELISA results were consistent with the other serological tests with Cohen kappa values between 0.47 and 0.92, and the protein A/G iELISA can thus offer a technically simple method for these species yielding results consistent with established brucellosis serological tests. Receiver operator characteristics analysis proved that the reindeer and caribou protein A/G iELISA results were consistent with the bacteriological gold standard with an area under the curve of 0.99, and the protein A/G iELISA was thus validated as a sensitive and specific serological method for the detection of anti-Brucella antibodies in reindeer and caribou. The binding of the antibodies from the respective species to protein A and G were also evaluated in the iELISA. The antibodies from hooded seals and polar bears reacted stronger to protein A than to G. The sei whale, fin whale, reindeer, and caribou antibodies reacted stronger to protein G than to A. The minke whale antibodies reacted to both protein A and G. There was a strong correlation (r s = 0.88-0.98) between the optical density results obtained with the iELISA with protein A/G and protein A or G, showing that protein A/G is as well suited as protein A or G for the detection of anti-Brucella antibodies in these species with the iELISA. PMID:23572454

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

    Science.gov (United States)

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

    2010-11-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A₄₀₅ values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations. PMID:20810678

  11. Improved Enzyme-Linked Immunosorbent Assay To Reveal Mycoplasma agassizii Exposure: a Valuable Tool in the Management of Environmentally Sensitive Tortoise Populations▿

    Science.gov (United States)

    Wendland, Lori D.; Zacher, Laurie A.; Klein, Paul A.; Brown, Daniel R.; Demcovitz, Dina; Littell, Ramon; Brown, Mary B.

    2007-01-01

    The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory tract disease, has relied on the detection of specific M. agassizii antibody by enzyme-linked immunosorbent assay (ELISA). We report here substantive refinements in the diagnostic assay and discuss the implications of its use in wildlife conservation and management. The ELISA has been refined to include more stringent quality control measures and has been converted to a clinically more meaningful titer reporting system, consistent with other diagnostic serologic tests. The ELISA results for 5,954 desert and gopher tortoises were plotted, and a subset of these serum samples (n = 90) was used to determine end-point titers, to establish an optimum serum dilution for analyzing samples, and to construct a standard curve. The relationship between titer and A405 was validated using 77 serum samples from known positive (n = 48) and negative (n = 29) control tortoises from prior transmission studies. The Youden index, J, and the optimal cut point, c, were estimated using ELISA results from the 77 control sera. Based on this evaluation, the refinement has substantially improved the performance of the assay (sensitivity of 0.98, specificity of 0.99, and J of 0.98), thus providing a clinically more reliable diagnostic test for this important infection of tortoises. PMID:17626160

  12. Mycoplasma agassizii Strain Variation and Distinct Host Antibody Responses Explain Differences between Enzyme-Linked Immunosorbent Assays and Western Blot Assays ▿

    Science.gov (United States)

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

    2010-01-01

    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A405 values were significantly correlated (r2 goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations. PMID:20810678

  13. Improved enzyme-linked immunosorbent assay to reveal Mycoplasma agassizii exposure: a valuable tool in the management of environmentally sensitive tortoise populations.

    Science.gov (United States)

    Wendland, Lori D; Zacher, Laurie A; Klein, Paul A; Brown, Daniel R; Demcovitz, Dina; Littell, Ramon; Brown, Mary B

    2007-09-01

    The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory tract disease, has relied on the detection of specific M. agassizii antibody by enzyme-linked immunosorbent assay (ELISA). We report here substantive refinements in the diagnostic assay and discuss the implications of its use in wildlife conservation and management. The ELISA has been refined to include more stringent quality control measures and has been converted to a clinically more meaningful titer reporting system, consistent with other diagnostic serologic tests. The ELISA results for 5,954 desert and gopher tortoises were plotted, and a subset of these serum samples (n = 90) was used to determine end-point titers, to establish an optimum serum dilution for analyzing samples, and to construct a standard curve. The relationship between titer and A405 was validated using 77 serum samples from known positive (n = 48) and negative (n = 29) control tortoises from prior transmission studies. The Youden index, J, and the optimal cut point, c, were estimated using ELISA results from the 77 control sera. Based on this evaluation, the refinement has substantially improved the performance of the assay (sensitivity of 0.98, specificity of 0.99, and J of 0.98), thus providing a clinically more reliable diagnostic test for this important infection of tortoises. PMID:17626160

  14. Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool.

    Science.gov (United States)

    Song, Linan; Zhao, Mingwei; Duffy, David C; Hansen, Joshua; Shields, Kelsey; Wungjiranirun, Manida; Chen, Xinhua; Xu, Hua; Leffler, Daniel A; Sambol, Susan P; Gerding, Dale N; Kelly, Ciarán P; Pollock, Nira R

    2015-10-01

    The currently available diagnostics for Clostridium difficile infection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenic C. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinical C. difficile isolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity. PMID:26202120

  15. Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

    Directory of Open Access Journals (Sweden)

    Zoheiry Mona K

    2011-09-01

    Full Text Available Abstract Background This research was carried out to develop a reliable monoclonal antibody (MoAb-based sandwich enzyme linked immunosorbent assay (ELISA for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes. Methods From a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags, a pair (12B/11D/3F and 10A/9D/10G was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA. Results The two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p Conclusions These data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

  16. Field evaluation of an immunoglobulin G anti-F1 enzyme-linked immunosorbent assay for serodiagnosis of human plague in Madagascar.

    Science.gov (United States)

    Rasoamanana, B; Leroy, F; Boisier, P; Rasolomaharo, M; Buchy, P; Carniel, E; Chanteau, S

    1997-09-01

    Bacteriological isolation of Yersinia pestis is the reference test for confirming plague infection, but recovery of the pathogen from human samples is usually very poor. When the etiology of the disease cannot be bacteriologically confirmed, it may be useful to possess alternative tests such as detection of specific circulating antibodies to help guide the diagnosis. In the present study, the immunoglobulin G (IgG) anti-F1 enzyme-linked immunosorbent assay (ELISA) has been applied to various human sera to evaluate its large-scale applicability in the high-endemicity plague foci of Madagascar. The sensitivity of the test was found to be 91.4%, and its specificity was 98.5%. The positive and negative predictive values were 96 and 96.6%, respectively. Seroconversion was observed on day 7 after onset of the disease. Patients with a positive ELISA result could be separated into high (82%) and low (18%) IgG anti-F1 responders. Cross-reactions with eight other infectious diseases prevalent in Madagascar were scarce and were found in 1 of 27 Mycobacterium tuberculosis-, 3 of 34 Schistosoma haematobium-, and 1 of 41 Salmonella-infected patients. Finally, the efficiency of the IgG anti-F1 ELISA was evaluated during the Mahajanga, Madagascar, plague outbreak of 1995. When the number of ELISA-positive patients was added to the number of bacteriologically confirmed and probable cases, the number of positive patients was increased by 35%. In conclusion, although it does not replace bacteriology, IgG anti-F1 ELISA is a useful and powerful tool for retrospective diagnosis and epidemiological surveillance of plague outbreaks. PMID:9302210

  17. Diagnosis and Clinical Virology of Lassa Fever as Evaluated by Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent-Antibody Test, and Virus Isolation

    Science.gov (United States)

    Bausch, D. G.; Rollin, P. E.; Demby, A. H.; Coulibaly, M.; Kanu, J.; Conteh, A. S.; Wagoner, K. D.; McMullan, L. K.; Bowen, M. D.; Peters, C. J.; Ksiazek, T. G.

    2000-01-01

    The Lassa virus (an arenavirus) is found in West Africa, where it sometimes causes a severe hemorrhagic illness called Lassa fever. Laboratory diagnosis has traditionally been by the indirect fluorescent-antibody (IFA) test. However, enzyme-linked immunosorbent assays (ELISAs) for Lassa virus antigen and immunoglobulin M (IgM) and G (IgG) antibodies have been developed that are thought to be more sensitive and specific. We compared ELISA and IFA testing on sera from 305 suspected cases of Lassa fever by using virus isolation with a positive reverse transcription-PCR (RT-PCR) test as the “gold standard.” Virus isolation and RT-PCR were positive on 50 (16%) of the 305 suspected cases. Taken together, Lassa virus antigen and IgM ELISAs were 88% (95% confidence interval [CI], 77 to 95%) sensitive and 90% (95% CI, 88 to 91%) specific for acute infection. Due to the stringent gold standard used, these likely represent underestimates. Diagnosis could often be made on a single serum specimen. Antigen detection was particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. Detection by ELISA of IgG antibody early in the course of illness helped rule out acute Lassa virus infection. The presence of IFA during both acute and convalescent stages of infection, as well as significant interobserver variation in reading the slides, made interpretation difficult. However, the assay provided useful prognostic information, the presence of IFA early in the course of illness correlating with death. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever. PMID:10878062

  18. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Adungo, Ferdinard; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu

    2016-01-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  19. Identification and expression of Babesia ovis secreted antigen 1 and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Sevinc, Ferda; Cao, Shinuo; Xuan, Xuenan; Sevinc, Mutlu; Ceylan, Onur

    2015-05-01

    In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis. PMID:25694531

  20. Development and use of an indirect enzyme-linked immunosorbent assay for detection of iridovirus exposure in gopher tortoises (Gopherus polyphemus) and eastern box turtles (Terrapene carolina carolina).

    Science.gov (United States)

    Johnson, April J; Wendland, Lori; Norton, Terry M; Belzer, Bill; Jacobson, Elliott R

    2010-05-19

    Iridoviruses, pathogens typically associated with fish and amphibians, have recently been shown to cause acute respiratory disease in chelonians including box turtles, red-eared sliders, gopher tortoises, and Burmese star tortoises. Case reports of natural infections in several chelonian species in the United States have been reported, however the prevalence remains unknown in susceptible populations of free-ranging chelonians. To determine the prevalence of iridovirus exposure in free-ranging gopher tortoises (Gopherus polyphemus) in the southeast United States, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate plasma samples from wild gopher tortoises (G. polyphemus) from: Alabama (n=9); Florida (n=658); Georgia (n=225); Louisiana (n=12); Mississippi (n=28); and unknown locations (68) collected between 2001 and 2006. Eight (1.2%) seropositive tortoises were identified from Florida and seven (3.1%) from Georgia for an overall prevalence of 1.5%. Additionally, a population of eastern box turtles was sampled from a private nature sanctuary in Pennsylvania that experienced an outbreak of iridovirus the previous year, which killed 16 turtles. Only 1 turtle out of 55 survivors tested positive (1.8%). Results suggest a low exposure rate in chelonians to this pathogen; however, it is suspected that this is an underestimate of the true prevalence. Since experimental transmission studies and past outbreaks have shown a high rate of mortality in infected turtles, turtles may die before they develop an antibody response. Further, the duration of the antibody response is unknown and may also cause an underestimate of the true prevalence. PMID:19931321

  1. Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits

    Directory of Open Access Journals (Sweden)

    Raba Julio

    2011-10-01

    Full Text Available Abstract Background Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA for quantification of B. cinerea in apple (Red Delicious, table grape (pink Moscatel, and pear (William's tissues. Results The method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 μg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation. Conclusions The developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide.

  2. Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples.

    Science.gov (United States)

    Brooks, B W; Devenish, J; Lutze-Wallace, C L; Milnes, D; Robertson, R H; Berlie-Surujballi, G

    2004-10-01

    A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples. PMID:15381269

  3. Seroprevalence studies of bovine brucellosis using indirect enzyme-linked immunosorbent assay (i-ELISA at organized and unorganized farms in three different states of India

    Directory of Open Access Journals (Sweden)

    Ramesh Somavanshi

    2013-06-01

    Full Text Available Aim: The present study was undertaken to know the seroprevalence of brucellosis in cattle and buffaloes. The work was carried out during the period 2011 through 2013 at Centre for Animal Diseases, Research and Diagnostics, IVRI, Izatnagar, Uttar Pradesh, India. Materials and Methods: A total of 1005 sera samples were collected and tested for bovine brucellosis using ELISA Kit; IDEXX, CHEKIT, Brucellose serum, Brucella abortus Antibody Test Kit. Results: A total of 1005 serum samples were collected from Karnataka, Uttar Pradesh and Uttarakhand during the period of 2011 to 2013 and were screened for bovine brucellosis using i-ELISA (indirect enzyme linked immunosorbent assay. The prevalence of bovine brucellosis was compared between organized and unorganized farms in order to find the epidemiology of the disease among the animal population. Sera from 5 organized farms in Karnataka were collected for seroprevalence studies. Out of 417 animals, 191 (45.80% animals were found positive by i-ELISA. A total of 361 serum samples were collected from 5 unorganized farms or villages, of which 82 (22.71% were positive. From Uttar Pradesh, bovine serum samples were collected from 3 organized farms. Out of 192 animals, 43 (22.39% animals were found positive for brucellosis. Similarly, sera collected from a single organized farm from Uttarakhand, showed 3 (8.57% positivity among 35 animals. On the whole, 319 (31.74% animals were found positive for brucellosis among the 3 states taken for study, which includes 138 (27.21% cattle and 181 (36.34% buffaloes. Conclusion: It has been found that Brucella infections are widely prevalent in organized and unorganized dairy farms in investigated states of India. [Vet World 2013; 6(8.000: 550-553

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  5. Impact of grey zone sample testing by enzyme-linked immunosorbent assay in enhancing blood safety: Experience at a tertiary care hospital in North India

    Directory of Open Access Journals (Sweden)

    Archana Solanki

    2016-01-01

    Full Text Available Background: Enzyme-linked immunosorbent assay (ELISA used for screening blood donors for transfusion transmitted infections (TTIs can sometimes fail to detect blood donors who are recently infected or possessing the low strength of pathogen. Estimation of a grey zone in ELISA testing and repeat testing of grey zone samples can further help in reducing the risks of TTI in countries where nucleic acid amplification testing for TTIs is not feasible. Materials and Methods: Grey zone samples with optical density (OD lying between cut-off OD and 10% below the cut-off OD (cut-off OD × 0.9 were identified during routine ELISA testing. On performing repeat ELISA testing on grey zone samples in duplicate, the samples showing both OD value below grey zone were marked nonreactive, and samples showing one or both OD value in the grey zone were marked indeterminate. The samples on repeat testing showing one or both OD above cut-off value were marked positive. Results: About 119 samples (77 for hepatitis B virus [HBV], 23 for human immunodeficiency virus [HIV], and 19 for hepatitis C virus [HCV] were found to be in grey zone. On repeat testing of these samples in duplicate, 70 (58.8% samples (45 for HBV, 12 for HIV, and 13 for HCV were found to be reactive. Six (5% samples (four for HBV, one for HIV, and one for HCV were found to be indeterminate. Conclusion: Seventy donors initially screened negative, were found out to be potentially infectious on repeat grey zone testing. Thus, estimation of grey zone samples with repeat testing can further enhance the safety of blood transfusion.

  6. A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients

    OpenAIRE

    Kragstrup, Tue W; Vorup-Jensen, Thomas; Deleuran, Bent; Hvid, Malene

    2013-01-01

    Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are chronic diseases characterized by activation of the immune system and production of antibodies. Thus, rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies in plasma samples from arthritis patients can interfere with immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) systems often used in arthritis research. However, standard methodologies on how to test for false results caused by these antibod...

  7. Differentiation of foot-and-mouth disease virus-infected from vaccinated pigs by enzyme-linked immunosorbent assay using nonstructural protein 3AB as the antigen and application to an eradication program

    DEFF Research Database (Denmark)

    Chung, Wen Bin; Sørensen, Karl Johan; Liao, Pei Chih;

    2002-01-01

    Baculovirus-expressed foot-and-mouth disease virus (FMDV) nonstructural protein 3AB was used as the antigen in an enzyme-linked immunosorbent assay. This assay allowed the differentiation of vaccinated from infected pigs. Serial studies were performed using sera collected from pigs in the field...... in Taiwan showed that the positive reactors steadily decreased over time in both finishers and sows, indicating that the pig population risk of infection by FMDV has decreased....

  8. Application of the Ceditest FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against Foot-and-mouth disease virus in selected livestock and wildlife species in Uganda

    OpenAIRE

    Ayebazibwe, Chrisostom; Mwiine, Frank Norbert; Balinda, Sheila Nina; Tjørnehøj, Kirsten; Alexandersen, Søren

    2012-01-01

    Diagnosis and control of Foot-and-mouth disease virus (FMDV) requires rapid and sensitive diagnostic tests. Two antibody enzyme-linked immunosorbent assay (ELISA) kits, Ceditest FMDV-NS for the detection of antibodies against the nonstructural proteins of all FMDV serotypes and Ceditest FMDV type O for the detection of antibodies against serotype O, were evaluated under African endemic conditions where the presence of multiple serotypes and the use of nonpurified vaccines complicate serologic...

  9. Development and Characterization of a Stable Vero Cell Line Constitutively Expressing Peste des Petits Ruminants Virus (PPRV) Hemagglutinin Protein and Its Potential Use as Antigen in Enzyme-Linked Immunosorbent Assay for Serosurveillance of PPRV▿

    OpenAIRE

    V. Balamurugan; Sen, A; Saravanan, P; Rasool, T. J.; Yadav, M. P.; Bandyopadhyay, S. K.; Singh, R. K.

    2006-01-01

    We developed and characterized a stable Vero cell line constitutively expressing Peste des petits ruminants virus (PPRV) hemagglutinin (H) protein and assessed its potential use as diagnostic antigen in enzyme-linked immunosorbent assay (ELISA). PPRV H gene of the vaccine strain (Sungri-96) was amplified by reverse transcription (RT)-PCR, cloned into a eukaryotic expression vector (pTarget), and subsequently transfected and expressed in Vero cells. A stable Vero cell line was developed after ...

  10. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar.

    OpenAIRE

    Mirelman, D; Nuchamowitz, Y; Stolarsky, T

    1997-01-01

    A comparison of the use of three commercially available enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes to detect and differentiate Entamoeba histolytica from E. dispar was carried out. Only the Techlab kit did not cross-react with E. dispar antigens, but it was 100 times less sensitive than PCR in detection of and differentiation between the two types of Entamoeba.

  11. Comparison of Galactomannan Detection, PCR-Enzyme-Linked Immunosorbent Assay, and Real-Time PCR for Diagnosis of Invasive Aspergillosis in a Neutropenic Rat Model and Effect of Caspofungin Acetate

    OpenAIRE

    Scotter, Jennifer M.; Chambers, Stephen T

    2005-01-01

    The performance of different in vitro diagnostic tests for the diagnosis of invasive aspergillosis (IA) was investigated in a transiently neutropenic rat model. Rats were immunosuppressed with cyclophosphamide and then inoculated intravenously with 1.5 × 104 CFU Aspergillus fumigatus spores. Animals were then either treated with caspofungin acetate, 1 mg/kg/day for 7 days, or not treated. PCR-enzyme-linked immunosorbent assay (ELISA), real-time PCR, and galactomannan (GM) detection were perfo...

  12. Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows ▿

    OpenAIRE

    Pajuaba, Ana C. A. M.; Deise A O Silva; Mineo, José R.

    2010-01-01

    This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegati...

  13. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    OpenAIRE

    Anju Mohan; Hari Mohan Saxena; Puneet Malhotra

    2016-01-01

    Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT), microtiter plate agglutination test (MAT), indirect hemagglutination assay (IHA), and indirect enzyme-linked immunosorbent assay (iELISA) as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cat...

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

    OpenAIRE

    J.J.N. Ngeranwa; S.P. Shompole; E.H. Venter; Wambugu, A.; J.E. Crafford; Penzhorn, B.L.

    2008-01-01

    The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife / livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA), using a recombinant antigen (MSP-5) from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was ...

  15. Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

    Institute of Scientific and Technical Information of China (English)

    XIN Jiu-qing; GAO Yun-long; LI Yuan; WANG Yan-fan; QIAN Ai-dong

    2007-01-01

    Mycoplasma mycoides subsp mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA (Trp) codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI Ⅹ strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL-1, and coated to microtiter enzyme-linked immunosorbent assay (ELISA) plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 (0.934/0.193), the sensitivity to be 95.8% (46/48), and the specificity to be 98.9% (161/163). 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

  16. Detection of serum antibodies to ovine progressive pneumonia virus in sheep by using a caprine arthritis-encephalitis virus competitive-inhibition enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Herrmann, Lynn M; Cheevers, William P; Marshall, Katherine L; McGuire, Travis C; Hutton, Melinda M; Lewis, Gregory S; Knowles, Donald P

    2003-09-01

    A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity. PMID:12965917

  17. 酶联免疫法检测水中的伏草隆%Determination of Fluometuron in Water by Enzyme-Linked Immunosorbent Assay

    Institute of Scientific and Technical Information of China (English)

    张琳; 生威; 李伶伶; 那宇; 王硕

    2013-01-01

      Fluometuron is one kind of benzene urea herbicides ,it can effectively control weeds in cotton fields. Because of surface runoff, these pesticides in irrigation water may constitute a significant source of contamination of water and soil, ELISA to detect fluometuron did not report before. In this work, an enzyme-linked immunosorbent assay was established for detecting fluometuron in water , the assay sensitivity was 22.1 μg/L and the limit of detection was 0.9 μg/L, liner range is between 0.06 μg/L and 81 μg/L. The recoveries range from 76.95%to 117.27%and the coefficients of variation range from 8.95%to 17.90%in tap water samples. The assay could be used to detect fluometuron in tap water quickly and accurately.%  伏草隆是一种苯脲类除草剂,可有效防除棉田中的杂草,这类农药可由地表径流,进入地下水和土壤,容易造成地下水污染。目前国内外鲜有报道检测伏草隆的酶联免疫方法(ELISA)。本研究建立的伏草隆间接竞争ELISA方法,方法的灵敏度(IC50)为22.1μg/L,检测限(IC15)为0.9μg/L,检测范围在0.06μg/L~81μg/L之间。本方法用来检测自来水中的伏草隆,加标回收率在76.95%到117.27%之间,变异系数在8.95%到17.90%之间。本文所建立的方法可以用于快速、准确的检测自来水体中伏草隆的残留量。

  18. A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Aboge, Gabriel Oluga; Jia, Honglin; Terkawi, Mohamad Alaa; Goo, Younkyoung; Kuriki, Ken; Nishikawa, Yoshifumi; Igarashi, Ikuo; Suzuki, Hiroshi; Xuan, Xuenan

    2007-10-21

    We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection. PMID:17706873

  19. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Science.gov (United States)

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  20. Evaluation of an enzyme-linked immunosorbent assay for serological surveillance of infection with Actinobacillus pleuropneumoniae serotype 5 in pig herds

    DEFF Research Database (Denmark)

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

    2002-01-01

    An indirect enzyme-linked immunoassay for serological surveillance of infection of pigs with Actinobacillus pleuropneumoniae (Ap) serotype 5 was developed. The antigen used was prepared from Ap serotype 5b strain L20. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis...

  1. Monoclonal Antibody-Based Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Sensitivity of the Nucleocapsid Protein in Acute-Phase Sera of Severe Acute Respiratory Syndrome Patients

    OpenAIRE

    Di, Biao; Hao, Wei; Gao, Yang; Wang, Ming; Wang, Ya-di; QIU, LI-WEN; Wen, Kun; Zhou, Duan-Hua; Wu, Xin-wei; Lu, En-Jie; Liao, Zhi-yong; Mei, Ya-bo; Zheng, Bo-Jian; Che, Xiao-Yan

    2005-01-01

    Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum sa...

  2. Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera.

    Science.gov (United States)

    Ozyörük, F; Cheevers, W P; Hullinger, G A; McGuire, T C; Hutton, M; Knowles, D P

    2001-01-01

    Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection. PMID:11139194

  3. Application of the Ceditest FMDV type O and FMDV-NS enzyme-linked immunosorbent assays for detection of antibodies against Foot-and-mouth disease virus in selected livestock and wildlife species in Uganda

    DEFF Research Database (Denmark)

    Ayebazibwe, Chrisostom; Mwiine, Frank Norbert; Balinda, Sheila Nina;

    2012-01-01

    Diagnosis and control of Foot-and-mouth disease virus (FMDV) requires rapid and sensitive diagnostic tests. Two antibody enzyme-linked immunosorbent assay (ELISA) kits, Ceditest FMDV-NS for the detection of antibodies against the nonstructural proteins of all FMDV serotypes and Ceditest FMDV type O......, and selected samples were tested not only in serotype-specific ELISAs for antibodies against primarily FMDV serotype O, but also against other serotypes. The FMDV-NS assay detected far more positive samples (93%) than the FMDV type O assay (30%) in buffalo (P ... the South African Territories (SAT) serotypes, while the seroprevalence was generally comparable in cattle with antibodies against serotype O elicited by infection and/or vaccination. However, some districts had higher seroprevalence using the FMDV type O assay indicating vaccination without infection...

  4. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    Science.gov (United States)

    Mohan, Anju; Saxena, Hari Mohan; Malhotra, Puneet

    2016-01-01

    Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT), microtiter plate agglutination test (MAT), indirect hemagglutination assay (IHA), and indirect enzyme-linked immunosorbent assay (iELISA) as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (pBrucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. PMID:27536032

  5. Quality assurance/quality control of foot and mouth disease solid phase competition enzyme-linked immunosorbent assay--Part II. Quality control: comparison of two charting methods to monitor assay performance.

    Science.gov (United States)

    Goris, N; De Clercq, K

    2005-12-01

    Diagnostic laboratories are increasingly required to meet stringent quality standards, and validated assays are needed to achieve formal accreditation. Validation of test methods is often considered to be finalised when the assay parameters and characteristics have been established. However, like any process, diagnostic assays are subject to random variation resulting in shifts in the mean test values. Continuous monitoring of assays using control charts will alert the interpreter of changes in performance. For this purpose, several charting methods have been developed and implemented. This paper compares the Shewhart and the exponentially weighted moving average (EWMA) control charts with respect to the day to day monitoring of internal quality control samples for the foot and mouth disease solid phase competition enzyme-linked immunosorbent assay. Both chart types are equally sensitive to shifts, but the EWMA method seems to provide the best balance between false rejection and false acceptance. PMID:16642771

  6. Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection: correlation of results with those of the plaque reduction neutralization test.

    Science.gov (United States)

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Yueh, Yi-Yun; Chow, Ling; Chien, Li-Jung; Chin, Chuan; Yang, Hui-Hua; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2002-05-01

    An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping. PMID:11980973

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

    Directory of Open Access Journals (Sweden)

    J.J.N. Ngeranwa

    2008-09-01

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

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

    Science.gov (United States)

    Ngeranwa, J J N; Shompole, S P; Venter, E H; Wambugu, A; Crafford, J E; Penzhorn, B L

    2008-09-01

    The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife/livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA), using a recombinant antigen (MSP-5) from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA), as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers. PMID:19040134

  9. Synthesis of an oxytetracyline-tolidin-BSA immunogen and antibodies production of anti-oxytetracyline developed for oxytetracyline residue detection with enzyme-linked immunosorbent assays technique

    Directory of Open Access Journals (Sweden)

    Widiastuti R

    2013-06-01

    Full Text Available An oxytetracycline-tolidin-bovine serum albumin (OTC-tolidin-BSA-conjugate was synthezed as immunogen for producing specific antibodies in immunized rabbits that would be used as reagent for development of OTC residue detection with enzym-linked immunoassays technique. The immunogen was prepared through diazotization tolidin and subsequently reacted with OTC. The red purple immunogen of OTC-tolidin-BSA absorbed at wave lengths of 277 nm and 488 nm under UV screening absorbances and confirmation with the high performance liquid chromatography (HPLC showed the absence of peak at retention time of 3.46 minutes. Characaterized result with SDS-PAGE showed the molecular weight of the OTC-tolidin-BSA at 69.79 kDA. Subsequently, the immunogen was immunized into New Zealand rabbits in order to produce the polyclonal antibodies. The antibodies were purified using a protein A sepharose column. The OD optimum responses of 0.92 to 1.20 were obtained from the second fractionation at dilution of 1/1000 by titrating the antibodies and OTC-tolidin-BSA coating antigen at concentration of 10 µg/mL on several bleeding times.

  10. [Diagnosis of cerebral amyloid angiopathy with cerebral hemorrhage by enzyme linked immunosorbent assay (ELISA) of cystatin C in the cerebrospinal fluid].

    Science.gov (United States)

    Shimode, K; Fujihara, S; Nakamura, M; Kobayashi, S; Tsunematsu, T

    1990-03-01

    The lower level of cystatin C in cerebrospinal fluid (CSF) is one of the useful diagnostic markers of hereditary cerebral hemorrhage with amyloidosis in Iceland. We attempted to establish an assay to determine the level of cystatin C in CSF for diagnosis of CAA due to the deposition of cystatin C in CSF for diagnosis of CAA due to the deposition of cystatin C. We carried out the sandwich enzyme immunosorbent assay with the use of monoclonal mouse anti-cystatin C and polyclonal rabbit anti-cystatin C antibodies. CSF from nine cases of cerebral hemorrhage and fifty reference cases with other neurological diseases were examined. Four patients with cerebral hemorrhage showed a low level of cystatin C and clinical manifestations suggestive of CAA. Our study showed the feasibility of using ELISA for the diagnosis of cerebral amyloid angiopathy that causes cerebral hemorrhage with the deposition of cystatin C. PMID:2364630

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

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Enøe, Claes; Stockmarr, Anders;

    2015-01-01

    eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR® BVDV-Ab indirect ELISA. The latter has...... successfully been used to eradicate BVD in Sweden. Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples...... BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level...

  12. 酶联免疫吸附技术在食品检测分析中的研究进展%Advances in food determination and analysis of enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    石超; 吕长鑫; 冯叙桥; 李萌萌; 刘苏苏

    2014-01-01

    Enzyme-linked immunosorbent assay (ELISA) is the most widely used technique in the enzyme immunoassay techniques. The antigen or antibody is adsorbed on the surface of the solid phase carrier, anti-gen-antibody reaction on the surface of the carrier, produce a color reaction by the enzyme and substrate, then quantitative determination after washed the free ingredients of the liquid phase. The ELISA technique is mainly divided into sandwich method, the indirect method and competition law. This paper describes the common me-thod and basic mechanism of action of the enzyme-linked immunosorbent assay, and details their specific ap-plication and development in detection of pesticide residues, pathogenic microorganisms, biotoxins, genetically modified food, heavy metal residues, allergic residues and illegal additives, and finally summarizes and ana-lyzes its prospects.%酶联免疫吸附技术(ELISA)是酶免疫测定技术中应用最广泛的技术,是将已知的抗原或抗体吸附在固相载体表面,使酶标记的抗原抗体反应在固相表面进行,通过酶与底物产生颜色反应,用洗涤法将液相中的游离成分除去后再进行定量测定。主要分为夹心法、间接法和竞争法。本文简要介绍了几种常用方法及其基本作用机制,详述了ELISA检测技术在食品检测中具体应用及发展历史与现状,对其在食品中农药残留、病原微生物、生物毒素、转基因食品、重金属残留、过敏性残留物及违规添加成分检测等方面的应用情况进行系统总结,并对其发展前景进行了分析。

  13. 动物性食品中氨苯砜dcELISA的检测%Development of a direct competitive enzyme-linked immunosorbent assay for detection of dapsone in animal foods

    Institute of Scientific and Technical Information of China (English)

    郑小娇; 张晶; 张亚卓; 史晨杉; 王向红

    2015-01-01

    A direct competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect Dapsone in animal foods.Hapten dapsone was conjugated with Bovine Sera Albumin (BSA) by diazotization to produce immunogen BSA-DDS used for immunizing rabbit.The rabbit antiserum was purified with ProteinA-Sephros 4B to prepare polyclonal antibody against dapsone.A direct enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantitate dapsone.The bewt synthesis of BSA-DDS and the conjugated-ratio was 1 ∶ 10.The IC50 was 4.78 ng/mL,visual detection limit was 0.03 ng/mL,and recoveries of dapsone in spiked samples ranged from 75.45% ~ 91.75%.The artificial antigens can be used for preparing specificity anti-dapsone antibody.The method is sensitive and the procedure of sample pretreatment is simple and quick.It is suitable to use in the detection of DDS residues in animal foods on site.%建立了动物性食品中氨苯砜残留快速检测技术.将氨苯砜重氮化后连接到牛血清蛋白(BSA)上制得免疫原BSA-DDS,免疫新西兰大耳白兔获得多克隆抗体,经ProteinA-Sephros 4B对抗体进行纯化,在此基础上建立直接竞争ELISA方法.结果显示:该方法可制备目标抗原BSA-DDS和抗体,氨苯砜与载体蛋白偶联比可达到1:10;建立的直接竞争ELISA方法的IC50为4.78 ng/mL,最低检测限达0.03 ng/mL,加标回收率为75.45% ~91.75%.

  14. A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Enosse, Sonia; Pearce, Richard;

    2005-01-01

    . However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA...

  15. The use of enzyme-linked immunosorbent assay (ELISA) for the diagnosis and monitoring of foot-and-mouth disease in the Philippines

    International Nuclear Information System (INIS)

    The establishment and use of the indirect sandwich ELISA for the detection of foot-and-mouth disease (FMD) virus antigen sero-types O, A and C and the liquid phase blocking ELISA (LPB-ELISA) for antibody levels against similar FMD sero-types has been adopted for routine diagnosis at the FMD diagnostic laboratory, PAHC. A total of 552 epithelial samples and 4401 serum samples were tested starting 1995 to 1998. Out of 552, 84 (17.9%) were found negative and 468 (84.78%) diagnosed as positive for sero-types O and C (42% of the total positives). Within 4 years, 62 representative samples were sent to WRL for FMD for confirmation diagnosis. From 62 samples sent 54 (87%) were diagnosed as positive and 8 (12.9%) were negative. Serum samples received were either for diagnosis (71 samples), surveillance (3002 serum), post vaccination titre (1303 serum) and for the FAO/IAEA external quality assurance programme (25 samples) by the FAO/IAEA Co-ordinated Research Project on FMD. The assay has been a useful tool in the fast diagnosis and confirmation of FMD suspect cases and in the measurement of antibodies against FMDV in serum samples from all animals either vaccinated or infected. In the future, the assay will be use for potency testing of imported vaccines and for monitoring and surveillance purposes to show freedom from disease for the support documentation from OIE. (author)

  16. Glycidyl methacrylate-co-N-vinyl-2-pyrrolidone coated polypropylene strips: Synthesis, characterization and standardization for dot-enzyme linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi, Charu; Tomar, Lomas [Centre for Biomedical Engineering, Indian Institute of Technology, Delhi 110016 (India); Singh, Harpal [Centre for Biomedical Engineering, Indian Institute of Technology, Delhi 110016 (India)], E-mail: tyagicharu11@rediffmail.com

    2009-01-26

    Glycidyl methacrylate and N-vinyl-2-pyrrolidone (GMA-co-NVP) copolymers with various GMA:NVP ratios were synthesized by solution polymerization technique in toluene using 2,2'-azobisisobutyronitrile (AIBN) as free radical initiator and dip coated onto polypropylene strips. The copolymer composition in polymeric coatings was confirmed by proton NMR spectroscopy. Various techniques like FTIR, SEM and contact angle were used for surface characterization of the polymer coatings. These polymer coated strips were evaluated and standardized for their application in dot-ELISA in two steps. In first step, specificity, sensitivity and reproducibility of the assay on developed polymer coated strips was evaluated through a model system using rabbit anti-goat IgG, goat anti-rabbit IgG and goat anti-rabbit IgG HRP (horseradish peroxidase)-conjugate. Polymer coating with GMA-NVP mol% ratio of 78:22 was able to detect rabbit anti-goat IgG antibody at a concentration as low as 2 ng mL{sup -1} with 1% BSA as blocking agent using antispecies IgG peroxidase conjugate diluted 1500 times. In the second step, the sensitivity and specificity of the developed system was established with human blood and finally used to identify the source of mosquito blood meal which is an important parameter in epidemiological studies, particularly in determining the role of mosquito in malaria transmission. The time duration of standardized assay with developed polymer coated strips was cut down to one hour compared to the 3-4 h required in usual dot-ELISA.

  17. A study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test.

    Science.gov (United States)

    Oliveira, Trícia Maria F de Sousa; Furuta, Patrícia I; de Carvalho, Débora; Machado, Rosangela Z

    2008-01-01

    To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT) and Enzyme-linked immunosorbent assay (ELISA). All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis by IFAT. None of the serum samples from non-endemic areas were positive for Leishmania sp., by IFAT, but 67% were positive for B. canis and 78% for E. canis using the same test. When tested by ELISA for Leishmania sp., four samples from non-endemic area were positive. These dogs were then located and no clinical signs, parasites or antibody was detected in new tests for a six month period. Only one of these 4 samples was positive for B. canis by IFAT and ELISA and three for E. canis by IFAT. The results of the work suggest a co-infection in the endemic area and no serological cross-reaction among these parasites by IFAT and ELISA. PMID:18554433

  18. Enzyme linked immunosorbent assay (ELISA for the detection of IgG, IgM, IgE and IgA against Cysticercus cellulosae in cerebrospinal fluid of patients with neurocysticercosis

    Directory of Open Access Journals (Sweden)

    Odashima Newton Satoru

    2002-01-01

    Full Text Available The objective of this study was to analyze different immunoglobulins classes (IgG, IgM, IgE and IgA against Cysticercus cellulosae in the cerebrospinal fluid (CSF, through enzyme linked immunosorbent assay (ELISA, correlating them to clinical and tomographic profiles in patients with neurocysticercosis (NCC. Eighty-five specimens of CSF were obtained from 43 cases with NCC (26 with the active form and 17 with the inactive form and from 42 patients with other neurological diseases. The inactive form of NCC presented a profile in CSF similar to the group without NCC. The active form of NCC presented elevation of specific immunoglobulins (IgG, IgM, IgE, and IgA in decreasing order, with the highest values being detected among the cases with intraventricular cysts, or with inflammation signs in CSF or in those with multiple clinical manifestations. The highest sensitivity and specificity were obtained with ELISA-IgG (88.5% and 93.2%, respectively. This study confirmed the importance of ELISA in the immunologic diagnosis of NCC.

  19. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran

    Directory of Open Access Journals (Sweden)

    Mehrangiz Rajaii

    2015-01-01

    Full Text Available Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%, 102 (26%, 74 (18.92% in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF, enzyme-linked immunosorbent assay (ELISA and IgG Avidity techniques. Results: We have found 111 (28.38% seronegative and 280 (71.61% seropositive cases by IIF and 124 (31.70% seronegative, 267 (68.28% seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23% and 7 (10.76% doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.

  20. Evaluation of two Taenia solium cysticercal antigenic preparations (vesicular fluid and a glycoprotein fraction with affinity for lentil lectin for the immunodiagnosis of neurocysticercosis by enzyme-linked immunosorbent assay (ELISA

    Directory of Open Access Journals (Sweden)

    Lisandra Akemi Suzuki

    2011-06-01

    Full Text Available OBJECTIVE: To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. METHOD: Fifty-six cerebrospinal fluid (CSF samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA. RESULTS: The VF ELISA showed 100% sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9% and 97.1%, with the CSF samples and 95.5% and 100% with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. CONCLUSION: Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.

  1. Sensitivity and specificity enhanced enzyme-linked immunosorbent assay by rational hapten modification and heterogeneous antibody/coating antigen combinations for the detection of melamine in milk, milk powder and feed samples.

    Science.gov (United States)

    Cao, Biyun; Yang, Hong; Song, Juan; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-11-15

    The adulteration of food products with melamine has led to an urgent requirement for sensitive, specific, rapid and reliable quantitative/screening methods. To enhance the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the detection of melamine in milk, milk powder and feed samples, rational hapten modification and heterogeneous antibody/coating antigen combinations were adopted. Three melamine derivatives with different length of carboxylic spacer at the end were synthesized and linked to carrier proteins for the production of immunogens and coating antigens. Monoclonal antibody against melamine was produced by hybridoma technology. Under optimal experimental conditions, the standard curves of the ELISAs for melamine were constructed in range of 0.1-100 ng mL(-1). The sensitivity was 10-300 times enhanced compared to those in the published literatures. The cross-reactivity values of the ELISAs also demonstrated the assays exhibited high specificity. Five samples were spiked with melamine at different concentrations and detected by the ELISA. The recovery rates of 72.8-123.0% and intra-assay coefficients of variation of 0.8-18.9% (n=3) were obtained. The ELISA for milk sample was confirmed by high-performance liquid chromatography with a high correlation coefficient of 0.9902 (n=6). The proposed ELISA was proven to be a feasible quantitative/screening method for melamine analysis. PMID:24148389

  2. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

    Science.gov (United States)

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-09-01

    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. PMID:20592135

  3. Evaluation of Chicken IgY Generated Against Canine Parvovirus Viral-Like Particles and Development of Enzyme-Linked Immunosorbent Assay and Immunochromatographic Assay for Canine Parvovirus Detection.

    Science.gov (United States)

    He, Jinxin; Wang, Yuan; Sun, Shiqi; Zhang, Xiaoying

    2015-11-01

    Immunoglobulin Y (IgY) antibodies were generated against canine parvovirus virus-like particles (CPV-VLPs) antigen using chickens. Anti-CPV-VLPs-IgY was extracted from hen egg yolk and used for developing enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay (ICA) for the detection of CPV in dog feces. The cutoff negative values for anti-CPV-VLPs-IgY were determined using negative fecal samples (already confirmed by polymerase chain reaction [PCR]). In both ELISA and ICA, there was no cross-reaction with other diarrheal pathogens. Thirty-four fecal samples were collected from dogs with diarrhea, of which 26.47% were confirmed as CPV-positive samples by PCR, while 29.41% and 32.35% of the samples were found to be positive by ELISA and ICA, respectively. The developed ELISA and ICA exhibited 97.06% and 94.12% conformity with PCR. Higher sensitivity and specificity were observed for IgY-based ELISA and ICA. Thus, they could be suitable for routine use in the diagnosis of CPV in dogs. PMID:26469376

  4. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL−1 with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO2 particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors

  5. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay.

    Science.gov (United States)

    Blacksell, Stuart D; Lim, Cherry; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L; Paris, Daniel H; Limmathurotsakul, Direk; Day, Nicholas P J

    2016-06-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus. PMID:27008880

  6. Improved methods for urinary atrazine mercapturate analysis-Assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

    Energy Technology Data Exchange (ETDEWEB)

    Koivunen, Marja E. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Dettmer, Katja [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Vermeulen, Roel [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); Bakke, Berit [Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD (United States); National Institute of Occupational Health, Oslo (Norway); Gee, Shirley J. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States); Hammock, Bruce D. [Department of Entomology and the UC Davis Cancer Center, University of California, Davis (United States)]. E-mail: bdhammock@ucdavis.edu

    2006-07-21

    Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis[reg] HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL{sup -1}), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis[reg] HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL{sup -1}) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R {sup 2} values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R {sup 2} = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine.

  7. Improved methods for urinary atrazine mercapturate analysis-Assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography-mass spectrometry (LC-MS) method utilizing online solid phase extraction (SPE)

    International Nuclear Information System (INIS)

    Elimination of interfering substances in urine by solid phase extraction (SPE) prior to analysis resulted in 10-fold improvement in the sensitivity of atrazine mercapturate (AM) enzyme-linked immunosorbent assay (ELISA) compared to previous reports. Of the two tested SPE systems, Oasis[reg] HLB and MCX, the mixed-mode MCX gave good recoveries (82%) of AM in spiked samples measured by ELISA, whereas the reverse-phase HLB phase was not compatible with the immunochemical method. At relatively high concentrations of urinary AM (>20 ng mL-1), sample dilution was effective enough for the elimination of interfering substances. The new liquid chromatography-mass spectrometry (LC-MS) method developed for AM utilizes online-SPE with Oasis[reg] HLB, column switching and a stable-isotope internal standard. The limit of quantification (0.05 ng mL-1) indicates improved sensitivity compared with most previously published LC-MS methods for AM. Validation of all three methods, LC-MS, ELISA + SPE and ELISA + dilution with spiked urine samples showed good correlation between the known and measured concentrations with R 2 values of 0.996, 0.957 and 0.961, respectively. When a set (n = 70 plus 12 blind duplicates) of urine samples from farmers exposed to atrazine was analyzed, there was a good agreement (R 2 = 0.917) between the log normalized data obtained by ELISA + SPE and LC-MS. High correlation among the data obtained by the two tested methods and the LC-MS method by the Center of Disease Control and Prevention (CDC), together with low variability among the blind duplicates, suggests that both methods reported here would be suitable for the analysis of urinary AM as a biomarker for human exposure of atrazine

  8. Faunal distribution of fleas and their blood-feeding preferences using enzyme-linked immunosorbent assays from farm animals and human shelters in a new rural region of southern Iran.

    Science.gov (United States)

    Moemenbellah-Fard, Mohammad Djaefar; Shahriari, Bahador; Azizi, Kourosh; Fakoorziba, Mohammad Reza; Mohammadi, Jalal; Amin, Masoume

    2016-03-01

    Blood sucking insects, such as fleas, are responsible for the transmission of many infectious disease-causing agents which impose an intolerable burden on the health of people living particularly in endemic parts of the world. Fleas (Insecta: Siphonaptera) are found in many parts of the world including Iran. Both adult male and female fleas are obligatory ectoparasites. They are one of the main public health concerns as a result of their nuisance or the potential to act as vectors of a number of medically-important pathogens. The current study was conducted to examine the geographical distribution and fauna of fleas and their anthropophagic index in part of Fars province, southern Iran. This study was the first to be done in Iran. A total of 20 villages were randomly selected. From October 2011 to May 2012, adult fleas were collected by direct hand catch from human to animal shelters. Overall 848 fleas, most of which were blood-fed, were captured from the floor or the body of farm animal hosts (cattle, sheep, goat and hens). Only two different genera of fleas were identified, the main species (99.76 %) was human flea, Pulex irritans. The village of Shamsabad was the most heavily infested area. P. irritans had an anthropophagic index of 15 % using indirect enzyme-linked immunosorbent assays (ELISA). It could be concluded that P. irritans is widely distributed in this area. Based on their blood feeding activity, fleas thus posed a serious health threat to residents and their economically important livestock in this part of Iran. PMID:27065620

  9. Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.

    Science.gov (United States)

    Otsuyama, Ken-Ichiro; Tsuneoka, Hidehiro; Kondou, Kaori; Yanagihara, Masashi; Tokuda, Nobuko; Shirasawa, Bungo; Ichihara, Kiyoshi

    2016-04-01

    The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD. PMID:26865692

  10. Enhanced expression of the Erns protein of classical swine fever virus in yeast and its application in an indirect enzyme-linked immunosorbent assay for antibody differentiation of infected from vaccinated animals.

    Science.gov (United States)

    Luo, Yuzi; Li, Lin; Austermann-Busch, Sophia; Dong, Mei; Xu, Jingjing; Shao, Lina; Lei, Jianlin; Li, Na; He, Wen-Rui; Zhao, Bibo; Li, Su; Li, Yongfeng; Liu, Lihong; Becher, Paul; Sun, Yuan; Qiu, Hua-Ji

    2015-09-15

    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating disease of swine worldwide. Although a mandatory vaccination with the modified live vaccine C-strain has been implemented in China for decades, CSF remains a serious threat to the swine industry. To facilitate the control and eradication of CSF in China, the E2-based marker vaccine rAdV-SFV-E2, an adenovirus-delivered, alphavirus replicon-vectored vaccine, has been developed. Accordingly, an accompanying discriminatory test that allows differentiating infected from vaccinated animals (DIVA) is required. Here, the enhanced expression of E(rns) protein of CSFV was achieved in the methyltropic yeast Pichia pastoris by codon-optimization of the E(rns) gene, and an indirect enzyme-linked immunosorbent assay (iELISA) based on the yeast-expressed E(rns) (yE(rns)) was developed and evaluated. The optimized iELISA was able to detect CSFV-specific antibodies in the serum samples from the CSFV-infected pigs as early as 6 days post-infection, and discriminate the CSFV-infected pigs from those vaccinated with rAdV-SFV-E2. The iELISA was evaluated using a panel of swine sera, and showed comparable sensitivity (94.6%) and specificity (97.1%), and the consistence rates with the virus neutralization test were 96.8% for CSFV-infected swine sera, 83.3% for C-strain-vaccinated swine sera, and 95.0% for field swine sera. In addition, the iELISA showed higher sensitivity (90.4%) compared with PrioCHECK CSFV E(rns) (59.6%). Taken together, the yE(rns)-based iELISA is specific and sensitive, representing a promising DIVA test for E2-based marker vaccines against CSF. PMID:26005003

  11. Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus-infected pigs from pigs vaccinated with CP7_E2alf.

    Science.gov (United States)

    Pannhorst, Katrin; Fröhlich, Andreas; Staubach, Christoph; Meyer, Denise; Blome, Sandra; Becher, Paul

    2015-07-01

    Infections with Classical swine fever virus (CSFV) are a major economic threat to pig production. To combat CSF outbreaks and to maintain trade, new marker vaccines were developed that allow differentiation of infected from vaccinated animals (DIVA principle). The chimeric pestivirus CP7_E2alf was shown to be safe and efficacious. Its DIVA strategy is based on the detection of CSFV E(rns)-specific antibodies that are only developed on infection. However, for the new marker vaccine to be considered a valuable control tool, a validated discriminatory assay is needed. One promising candidate is the already commercially available enzyme-linked immunosorbent assay, PrioCHECK CSFV E(rns) ELISA (Prionics BV, Lelystad, The Netherlands). Four laboratories of different European Union member states tested 530 serum samples and country-specific field sera from domestic pigs and wild boar. The ELISA displayed a good robustness. However, based on its reproducibility and repeatability, ranges rather than single values for diagnostic sensitivity and specificity were defined. The ELISA displayed a sensitivity of 90-98% with sera from CSFV-infected domestic pigs. A specificity of 89-96% was calculated with sera from domestic pigs vaccinated once with CP7_E2alf. The ELISA detected CSFV infections in vaccinated domestic pigs with a sensitivity of 82-94%. The sensitivity was lower with sera taken ≤21 days post-challenge indicating that the stage of CSFV infection had a considerable influence on testing. Taken together, the PrioCHECK CSFV E(rns) ELISA can be used for detection of CSFV infections in CP7_E2alf-vaccinated and nonvaccinated domestic pig populations, but should only be applied on a herd basis by testing a defined number of animals. PMID:26179095

  12. Erysipelothrix rhusiopathiae and Mycoplasma hyopneumoniae: the sensitivities of enzyme-linked immunosorbent assays for detecting vaccinated sows of unknown disease status using serum and colostrum, and the correlation of the results for sow serum, colostrum, and piglet serum.

    Science.gov (United States)

    Jenvey, Caitlin J; Reichel, Michael P; Cockcroft, Peter D

    2015-03-01

    Due to relatively high concentrations of immunoglobulins, colostrum has the potential to improve the sensitivity of diagnostic tests for diseases in pigs when compared with serum. It is possible that colostrum could improve the sensitivity of the antibody enzyme-linked immunosorbent assay (ELISA) compared with serum. Colostrum is also essential for piglets, providing protection against infections in the first few weeks and months of life. The sensitivity of 2 commercially available ELISAs, one for the detection of Erysipelothrix rhusiopathiae and the second for Mycoplasma hyopneumoniae antibodies, when used with sow colostrum in comparison with serum was investigated. The correlation of maternal E. rhusiopathiae- and M. hyopneumoniae-specific antibody levels with specific-antibody serum levels in the piglet was also determined. The sensitivity was defined as the proportion of vaccinated sows that were correctly identified as vaccinated at a given cutoff point. The true disease status of the sows with regard to the 2 infections was unknown. Blood and colostrum samples were collected from 20 sows, 10 primiparous and 10 multiparous, and blood samples were also collected from the piglets of each sow, 48-72 hr post-farrowing. The sensitivities of both ELISAs were significantly improved when using colostrum compared with serum. Sow serum and colostrum optical density (OD) values were significantly correlated. The mean sow OD values for serum for E. rhusiopathiae and M. hyopneumoniae and colostrum for E. rhusiopathiae were significantly correlated with piglet serum OD levels. If the improved sensitivity of colostrum can be demonstrated in infected animals, this will increase the ability of the test to identify infected animals using both individual and pooled colostrum. Testing serum and/or colostrum using ELISA can be useful predictors of piglet disease-specific OD values. PMID:25613041

  13. Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein.

    Science.gov (United States)

    Khamehchian, S; Madani, R; Rasaee, M J; Golchinfar, F; Kargar, R

    2007-06-01

    A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance. PMID:17668032

  14. Identification of high-affinity anti-IL-1 α autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1 α

    International Nuclear Information System (INIS)

    A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 α (rhIL-1 alpha) has been developed. Results from this ELISA have demonstrated that the concentration of rhIL-1 α added to normal human serum (NHS) decreased by 16.3% after 3 h and 24.9% after 6 h at room temperature. Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 α added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation. The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 α in NHS. Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 α binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 α-specific IgG. The binding of 125I-labeled IL-1 α to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 7.4 x 10(-11) M) but not by unlabeled IL-1 β. Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 α-specific IgGs was 4.7 x 10(10) M-1. These results suggest that these autoantibodies may interfere with the detection of IL-1 α in human serum by various assay systems and also could be a regulator of circulating IL-1 α

  15. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Anju Mohan

    2016-07-01

    Full Text Available Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT, microtiter plate agglutination test (MAT, indirect hemagglutination assay (IHA, and indirect enzyme-linked immunosorbent assay (iELISA as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001. The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005. The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002. However, the difference in mean iELISA titers of infected cattle (1.3678±0.014 and healthy vaccinated cattle (1.367±0.014 was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.

  16. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail: hyhan@mail.hzau.edu.cn

    2015-08-05

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL{sup −1} with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

  17. Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum.

    Science.gov (United States)

    Muleme, Michael; Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M; Nguyen, Chelsea; Stevenson, Mark A; Wilks, Colin R; Firestone, Simon M

    2016-06-01

    Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. PMID:27122484

  18. Generation of E. coli-derived virus-like particles of porcine circovirus type 2 and their use in an indirect IgG enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Zhang, Yan; Wang, Zhanfeng; Zhan, Yang; Gong, Qian; Yu, Wanting; Deng, Zhibang; Wang, Aibing; Yang, Yi; Wang, Naidong

    2016-06-01

    Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes. PMID:26973229

  19. Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus Padronização de um teste imunoenzimático (ELISA) para detectar antígenos tóxicos circulantes do veneno em pacientes picados pelo escorpião Tityus serrulatus

    OpenAIRE

    Nilton Alves de Rezende; Mariana Borges Dias; Délio Campolina; Carlos Chavéz-Olortegui; Carlos Faria Santos Amaral

    1995-01-01

    The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who...

  20. Evaluation of indirect enzyme-linked immunosorbent assays and IgG avidity assays using a protein A-peroxidase conjugate for serological distinction between Brucella abortus S19-vaccinated and -infected cows.

    Science.gov (United States)

    Pajuaba, Ana C A M; Silva, Deise A O; Mineo, José R

    2010-04-01

    This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle. PMID:20147498

  1. A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients.

    Science.gov (United States)

    Kragstrup, Tue W; Vorup-Jensen, Thomas; Deleuran, Bent; Hvid, Malene

    2013-12-01

    Rheumatoid arthritis (RA) and spondyloarthritis (SpA) are chronic diseases characterized by activation of the immune system and production of antibodies. Thus, rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies in plasma samples from arthritis patients can interfere with immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) systems often used in arthritis research. However, standard methodologies on how to test for false results caused by these antibodies are lacking. The objective of this study was to design a simple set of steps to validate a sandwich ELISA before using it for measuring analytes in plasma from arthritis patients. An interleukin-24 (IL-24) sandwich ELISA system was prepared with a monoclonal mouse capture antibody and a polyclonal goat detection antibody and tested for interference by rheumatoid factor, anti-animal IgG antibodies and heterophilic antibodies. Plasma samples from 23 patients with RA and SpA were used. No differences were found between plasma samples measured in wells coated with anti-IL-24 specific antibody and in wells coated with isotype control antibody (false positive results), and recombinant human IL-24 was not recovered in spiked samples (false negative results). This interference was removed after preincubating the plasma samples from patients with arthritis with goat or bovine IgG, suggesting that anti-animal IgG antibodies found in the plasma of the arthritis patients caused the false results. Additional testing showed that the signal-to-noise ratio could be increased by titration of the capture and detection antibodies and by using the ELAST amplification system. Finally, the calculated concentration of IL-24 was increased in ethylenediaminetetraacetic acid (EDTA) plasma compared to heparin plasma and serum and decreased with repetitive freeze/thaw cycles of the samples illustrating how sample handling could additionally contribute to the variations reported by different

  2. 草甘膦残留的酶联免疫分析方法的建立%Establishment of the Enzyme-linked Immunosorbent Assay for Glyphosate Residues

    Institute of Scientific and Technical Information of China (English)

    潘熙萍; 楼佳俊; 张高精; 张丽莎; 施曼玲

    2012-01-01

    通过碳化二亚胺法将草甘膦(Glyphosate,GLY)分别与卵清蛋白(OVA)和牛血清白蛋白(BSA)偶联制备免疫原和包被原.基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)检测表明,草甘膦与牛血清白蛋白和卵清蛋白的偶联比分别为11∶1和9∶1.合成的免疫原作用新西兰大白兔制备出草甘膦多克隆抗体,ELISA检测抗体溶液效价为1∶1×107,该抗体不与草甘膦的结构类似物增甘膦和双甘膦起交叉反应.以此抗体建立了草甘膦间接竞争ELISA检测方法(ciELISA),其线性范围为0.78~100 μg/mL,线性回归方程为y=15.851 0 lnx+7.780 5,R2=0.993 8.草甘膦的检测限为IC10=1.15μg/mL,IC50=14.35 μg/mL.在添加回收试验中,草甘膦添加值5和10 μg/g时,玉米粉中的草甘膦回收率为104.12%和81.37%,小麦粉中的回收率则分别为118.94%和为109.39%.结果表明,采用所制备的多克隆抗体而建立的草甘膦ciELISA方法可有效地应用于玉米粉和小麦粉中草甘膦的残留检测.%The diimine carbonization methods were used to synthesize immunogen and coating antigen by glyphosate being conjugated to ovalbumin (OVA) and bovine serum albumin (BSA), respectively. The results showed the ratios of hapten to BSA and OVA were 11:1 and 9:1 by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The polyclonal antibodies against glyphosate were produced through immunizing New Zealand white rabbits with the immunogen. The titer of antibody against glyphosate was 1:1×107 by the test of the enzyme-linked immunosorbent assay (ELISA). The glyphosate polyclonal antibodies did not cross-react with glyphosine and N-(Phosphonomethyl) iminodiacetic. An indirect competitive ELISA (ciELISA) was developed for glyphosate detection, which has a detection limit (IC10) of 1.15 μg/mL and a linear working range of 0.78-100 μg /mL with an IC50 value of 14.35 μg/mL. The regression equation was y= 15.8510 ln

  3. 抗恩诺沙星抗血清的制备及其ELISA检测%Preparation of enrofloxacin antiserum and its determination by enzyme - linked immunosorbent assay (ELISA)

    Institute of Scientific and Technical Information of China (English)

    刘兵; 曾华金; 杨冉; 雷利芳; 屈凌波

    2011-01-01

    Objective;To develop a rapid and sensitive enzyme - linked immunosorbent assay(ELISA) for determination of enrofloxacin( ENR). Method; Immunogen enrofloxacin - bovine serum albumin( Enrofloxacin - BSA) and coating antigen enrofloxcin -ovum albumin (Enrofloxacin - OVA) were prepared by a succinic anhydride method. By subcutaneous injection, the polyclonal antiserum was produced from big ear rabbits immunized with conjugates ENR -BSA. Result; After the assay procedure was optimized,the standard curve of ENR was established and the practical measuring range of the ELISA extended from 5 ng ? mL-1 to 2. 5 |xg ? mL '' ( R2 =0. 9935 ) . In a comparison of the assay results obtained by ELISA and HPLC, there is a good correlation between these two methods (R2 = 0. 9892, n = 9 ) . The proposed method has been satisfactorily applied for the determination of ENR in rat plasma and Pharmaceuticals with recoveries in the range of 98. 4% to 105. 8% for milk sample,91. 7% to 101. 2% for rat plasma and 97.0% to 110.3% for rat urine. Conclusion; The experimental data indicated that in some extent the ELISA method was more suitable for high throughput and real - time ENR analysis in biological samples and animal food with lower detection limit,low background and no requirement of sample pre -treatment.%目的:建立快速、灵敏的恩诺沙星残留酶联免疫检测方法(ELISA).方法:采用混合酸酐法,将恩诺沙星与牛血清白蛋白和卵清蛋白分别合成免疫抗原(Enrofloxacin-BSA)和包被抗原(Enrofloxacin-OVA).通过背部多点免疫法免疫日本大白耳兔,制备抗恩诺沙星的特异性抗血清.结果:利用所获得的特异性抗血清,建立的恩诺沙星ELISA检测方法,其线性范围为5 ng·mL.~2.5μg·mL(R=0.9935),且与HPLC方法具有良好的相关性(R=0.9892,n=9).牛奶、大鼠血浆和尿液中的加样回收率分别为98.4%~105.8%,91.7%~101.2%,97.0%~110.3%.运用所建立的方法,对大鼠体内血浆及药品

  4. Comparison of Three Enzyme-Linked Immunosorbent Assay Methods for Quantitative Determination of Ricin%3种酶联免疫分析法在蓖麻毒素定量测定中的比较

    Institute of Scientific and Technical Information of China (English)

    马小溪; 刘合珠; 唐吉军; 郭磊; 谢剑炜

    2011-01-01

    Three determination methods in enzyme-linked immunosorbent assay, namely optical absorption, fluorescent and chemiluminescent immunoassay, were established for quantitation of biotoxins protein ricin in various matrices. The detection conditions were systematically optimized.The results showed the best signal-to-noise (SNR) could be obtained for CLIA when dilution factor was 1:8000; and the chemiluminescence signal was stable at around 3 minutes after horseradish peroxidase reacted with its substrate. Whereafter, the three methods were compared for determination of ricin under individually optimized conditions. The comparative results indicated that chemiluminescent immunoassay could provide wider linear range (from 0.02 μg/L to 5.5 μg/L with correlation coefficient of 0.999), higher sensitivity (limit of detection was 0.005 μg/L), and could be adopted as a simple, rapid and robust approach. The CLIA method was applied to measure the spiked ricin in water, carbonated beverage, milk powder, coffee and human serum samples. The limits of detection and the recoveries were from 0.005 to 0.08 μg/L and from 89.6% to 108.8%, respectively.The method was quite suitable for quantitative determination of trace amounts of ricin in contaminated or poisoned samples.%建立了蓖麻毒素的3种酶联免疫定量分析法,即光吸收、荧光和化学发光免疫分析法,并系统优化了各项实验条件.结果显示:对于化学发光检测法,当酶标抗体HRP-4C13稀释倍数为1:8000时可获得最佳的信噪比,且酶与底物反应3 min后信号趋于稳定.随后在各自优化的条件下将3种方法用于毒素的检测.比较结果表明:化学发光酶联免疫分析法除具有线性范围宽(0.02~5.5 μg/L,r(2)=0.999)、灵敏度高(检出限为5 ng/L)的特点外,还具有简单、快速、体系稳定性好等优点.将本方法用于不同实际样品基质,如饮用水、碳酸饮料、奶粉、咖啡和血清中添加的蓖麻毒

  5. A novel enzyme-linked immunosorbent assay for quantitative detection of anti-thyroid peroxidase antibodies in serum%血清抗甲状腺过氧化物酶抗体ELISA定量方法的建立

    Institute of Scientific and Technical Information of China (English)

    孙颖; 李会强; 陈寅; 仁杰; 李婵

    2011-01-01

    Objective To establish a novel enzyme-linked immunosorbent assay (ELISA) for quantitative detection of the concentra tion of anti-thyroid peroxidase antibody (TPOAb) in serum. Methods The microtiter plate was coated with biotinylated bovine serum albumin (BSA) and streptavidin. The biotinylated TPO antigen and standardized anti-TPOAb or test sera were successively added into the wells of plate. The HRP-anti-IgG was then added into the plate for colorization. The optimal concentrations of biotinylated TPO an tigen and HRP-anti-IgG were screened by chessboard titration, and the reaction conditions were optimized for the method evaluation. Results In the indirect-coating mode, the amount of coated antigen was 0. 083 μg/mL. The sensitivity of the assay was 0. 165 IU/mL. The coefficient of variations (CV) of inter-assay in high and low concentration of serum mixture were 9.2% and 9.0% , and the CV of intra-assay were 4.6% and 5.6% respectively. The recovery rate was between 96% and 104%. The coated ELISA plate re mained stable for 5 days at 37 ℃. The rate of cross-reaction with anti-thyroid globulin (TGAb) was 0. 22%. The reference range in serum was less than 65. 7 IU/mL. The correlation coefficient of the experimental results with those of Abbott kit was 0. 985 (P < 0.01). Conclusion In the developed ELISA of indirect-coated mode, the amount of needed purified antigen significantly reduced. The sensitivity and specificity of the assay were satisfied. The method is simple and cost-saving, so it should be very suitable for anti TPOAb detection in primary hospitals.%目的 建立一种ELISA方法用于定量分析血清抗甲状腺过氧化物酶(TPO)抗体(TPOAb).方法 用生物素化牛血清清蛋白(BSA)和链霉亲合素包被微孔板,同时加入生物素化TPO抗原和待检血清,再加入酶标记抗人IgG,建立间接包被模式酶联免疫法测定抗TPO抗体.经方阵滴定确定生物素化抗原和酶标抗体的最适浓度,优化反应条件,

  6. Establishment of ComplementFixation-Enzyme Linked Immunosorbent Assay(CF-ELISA) for the Detection of Brucellosis%补体结合酶联免疫吸附试验方法的建立

    Institute of Scientific and Technical Information of China (English)

    毛开荣; 王楠; 李文平; 蒋颖; 程君生; 刘轶秋; 蒋玉文; 丁家波

    2011-01-01

    A new complement fixation-enzyme linked immunosorbent assay(CF-ELISA)method was established to improve accuracy rate of immunological test.CF-ELISA was test used ELISA mothed,that antibody conjugated Horse Radish Peroxidase(HRP) is anti-complement C3 extracted and purified with inulin from the serum of guinea pigsand the enzyme-substrate system be used detect complement fixation with antibody-antigen complex.The CF-ELISA method could detect 0.01 IU antibody,which had the same sensitivity with iELISA,and about 5 000 times higher than that of RBPT、SAT for brucellosis.The positive rate of 349 serum samples from Brucella-infected flocks of goats/sheep and bovines with CF-ELISA,iELISA,CFT,SAT and RBPT method was 35.82%,36.39%,31.81%,30.09% and 36.1%,respectively.The positive coincidence rate of CF-ELISA to iELISA,CFT,SAT,RBPT was 98.4%,88.8%,80.0% and 90.6%.The negative rate of 490 samples collected from Brucella-free flocks with CF-ELISA,iELISA,CFT,SAT and RBPT was 100%,99.6%,100%,99.4% and 99.8%,respectively.The negative coincidence rate of CF-ELISA to iELISA,CFT,SAT,RBPT was 99.6%,100%,99.4% and 99.8%.The results showed that CF-ELISA was a promising candidate method for the detection of brucellosis with high specificity and sensitivity.%为改进免疫学诊断技术的准确性,研究了一种基于补体结合的免疫学检测新技术———补体结合酶联免疫吸附试验(CF-ELISA)。CF-ELISA技术采用酶标记抗菊糖纯化豚鼠补体C3抗体及其酶显色系统作为补体参与反应的指示系统,用ELISA方法进行补体结合试验。经对布氏菌病抗体检测的初步试验结果显示,CF-ELISA技术可检测到0.01 IU的布氏菌病抗体,灵敏度与间接酶联免疫吸附试验(iELISA)相当,是虎红平板凝集试验(RBPT)试管凝集试验(SAT)的5 000倍、补体结合试验(CFT)的10 000倍。对349份确诊布氏菌病感染群牛、羊血清的检测结果显示,CF-ELISAi、ELISA、CFT

  7. Evaluation of a Caprine Arthritis-Encephalitis Virus/Maedi-Visna Virus Indirect Enzyme-Linked Immunosorbent Assay in the Serological Diagnosis of Ovine Progressive Pneumonia Virus in U.S. Sheep

    Science.gov (United States)

    Serological diagnostic testing of sheep and goats using enzyme immunosorbent assays (ELISAs) is the most common method of determining small ruminant lentivirus (SRLV) infection. A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect (i) ELISA, which utilizes MVV EV1 capsid a...

  8. Study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test Estudo da reatividade cruzada em amostras de soro de cães positivos para Leishmania sp., Babesia canis e Ehrlichia canis, pelo ensaio imunoenzimático indireto e pela reação de imunofluorescência indireta

    OpenAIRE

    Trícia Maria F. de Sousa Oliveira; Patrícia I. Furuta; Débora Carvalho; Rosangela Z. Machado

    2008-01-01

    To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT) and Enzyme-linked immunosorbent assay (ELISA). All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis ...

  9. Evaluation of a commercial blocking enzyme-linked immunosorbent assay to detect avian influenza virus antibodies in multiple experimentally infected avian species

    Science.gov (United States)

    Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoir for avian influenza (AI) viruses. Traditionally, AI surveillance in wild birds has relied on virus detection strategies including virus isolation and polymerase chain reaction. To evaluate the efficacy of a commerc...

  10. Detection of Anti-Influenza A Nucleoprotein Antibodies in Pigs Using a Commercial Influenza Epitope-Blocking Enzyme-Linked Immunosorbent Assay Developed for Avian Species

    Science.gov (United States)

    Influenza virus causes acute respiratory disease in pigs and is of concern for its potential public health significance. Many subtypes of influenza virus have been isolated from pigs and the virus continues to evolve in swine populations. Current antibody assays have limited antigenic recognition ...

  11. Field trial of brucellosis competitive enzyme linked immunosorbent assay (ELISA)

    International Nuclear Information System (INIS)

    The purpose of this study was to compare the diagnostic performance characteristics of the competitive (C) ELISA for the diagnosis of bovine brucellosis and the differentiation of Brucella infected from Brucella vaccinated cattle to the indirect (I) ELISA and the conventional serological techniques used in Argentina. The diagnostic specificity and relative sensibility of ELISA was comparable to CFT and 2ME, which are the official complementary tests in Argentina. Besides, C-ELISA showed the best performancein analysing vaccinated animals. The application of the ELISA test is largely feasible because of its reproducibility and easy standardization. In addition, although CFT has a great performance, its application is cumbersom; the 2ME test lasted for 48 h and is toxic. Both the C-ELISA, using MAb M84, and the I-ELISA, using MAb M23, have been demonstrated to have the best performance. Owing to our field conditions (brucellosis prevalence and mandatory vaccination of female calves) the competitive ELISA should be applied as a complement of the official screening tests such as BP. These results suggest that the ELISA test would be very useful in contributing to the control and eradication programme of bovine brucellosis in Argentina

  12. Sandwich enzyme-linked immunosorbent assay for naringin.

    Science.gov (United States)

    Qu, Huihua; Wang, Xueqian; Qu, Baoping; Kong, Hui; Zhang, Yue; Shan, Wenchao; Cheng, Jinjun; Wang, Qingguo; Zhao, Yan

    2016-01-15

    Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA. PMID:26709308

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

  14. and Indigenous Indirect Enzyme-Linked Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    Annapurna S. Agasthya

    2012-01-01

    Full Text Available Brucellosis is one of the most important reemerging zoonoses in many countries. Brucellosis is caused by Gram-negative coccobacillus belonging to genus Brucella. Human brucellosis often makes the diagnosis difficult. The symptoms and clinical signs most commonly reported are fever, fatigue, malaise, chills, sweats headaches, myalgia, arthralgia, and weight loss. Some cases have been presented with only joint pain, lower backache, and involuntary limb movement, burning feet, or ischemic heart attacks. The focus of this work was to develop a highly sensitive and specific indirect ELISA by using smooth lipopolysaccharide antigen of Brucella abortus 99 to detect anti-Brucella antibodies at Project Directorate on Animal Disease Monitoring and Surveillance. Serum samples collected from 652 individuals in whom fever was not the major symptom but the complaint was of joint pain, headache, lower backache, and so forth, were screened by Rose Bengal plate agglutination test (RBPT and standard tube agglutination test (STAT. Subsequent testing of sera by indigenous indirect ELISA detected 20 samples positive (3.6% seroprevalence, and indirect ELISA was found to be more sensitive than RBPT and STAT. The seroprevalence in South Karnataka was 2.14%, and in North Karnataka it was 0.92%.

  15. Establishment of an Enzyme Linked Immunosorbent Assay for Albumin

    International Nuclear Information System (INIS)

    A sensitive and specific ELISA for Albumin was established by using Albumin antibody coated on the microtiter plate, Albumin labeled horseradish peroxidase(HRP). The sensitivity of the assay was 0.28 mg/L. The intra-and inter-assay CVs were 2.63%-5.28% and 2.40%-4.26% respectively. The analytical recovery was 95.0%-106.4%, the coefficient correlation was 0.9974. This method for measuring Alb was rapid, sensitive and convenient. It is suitable for clinical detection and research application. (authors)

  16. Quantitative analysis of cholinesterase from Daphnia magna by indirect and non-competitive enzyme-linked immunosorbent assay%大型溞胆碱酯酶间接非竞争酶联免疫吸附定量分析法的建立

    Institute of Scientific and Technical Information of China (English)

    刘洪翠; 杨艳霞; 李少南

    2012-01-01

    用达到电泳纯的大型溞(Daphnia magna)胆碱酯酶(cholinesterase,ChE)免疫BALB/c小鼠,得到效价达1∶8000的鼠抗大型溞ChE多克隆抗血清;通过优化抗原抗体反应条件,建立定量分析大型溞ChE含量的间接非竞争酶联免疫吸附法(indirect and non-competitive enzyme-linked immunosorbent assay).结果表明:此方法检测灵敏度为24 ng·mL-1;经免疫交叉反应鉴定,所获抗血清与5、10 μg·mL-1标准蛋白质和其他生物如花翅摇蚊、尖额溞、斑马鱼(脑、脑除外的整体)、家蚕、意大利蜜蜂、赤子爱胜蚯蚓、非洲爪蟾(蝌蚪)等来源的ChE交叉反应率分别为:<0.25%、<0.25%、2.13%、10.19%、3.88%、3.56%、2.81%、3.93%、5.00%和2.75%,具较高的特异性.说明用本试验得到的抗血清建立的间接非竞争酶联免疫吸附法可用于大型溞体内ChE含量的测定,以利于在农药暴露环境下大型溞体内ChE活性的精确检测.%Polyclonal anti-serum of cholinesteiase with dilution ratio of 1 :8 000 was obtained by immunizing BALB/c mice with purified cholinesterase (ChE) from Daphnia magna. An indirect and non-competitive enzyme-linked immunosorbent assay (ELISA), which used mice anti-Daphnia ChE as antibodies to react with the Daphnia ChE, was established to quantify immunoreactive content of ChE in D. magna. The minimal detection limit of the method was estimated to be 24 ng-mL"'. Specificity of anti-serum was high, with cross reaction of <0. 25%, <0. 25%, 2.13%, 10.19%, 3.88%, 3.56%, 2.81%, 3.93%, 5.00% and 2.75% towards non-ChE marker (5. 10 μg·mL-1), Chironomus kiinensis, Alona sp. , Brachydanio rerio (the brain, the whole body without brain), Bombyx mori, Apis mellifera L., Eisenia foetida , and tadpoles of Xenopus laevis, respectively. It is indicated that the established method can be used to quantify immunoreactive content of ChE in D. magna so as to measureme accurately in vivo activity of the ChE in pesticide

  17. 结核分枝杆菌RD1区编码蛋白ELISPOT辅助诊断活动性结核病的应用价值%The Diagnostic Value of Enzyme-linked immunosorbent assay by RD1 of Mycobacterium tuberculosis encoded protein in Detection of Active Tuberculosis

    Institute of Scientific and Technical Information of China (English)

    杜凤娇; 傅瑜; 吴雪琼; 李亮; 高静韬; 张宗德

    2012-01-01

    Objective To compare T-SPOT. TB and Enzyme-linked immunosorbent assay(ELISPOT) using ESAT-6/CFP-10 fusion proteinas( E/C) and Rv3879c as antigens,and explore their roles in the auxiliary diagnosis of active tuberculosis. Methods Totally 106 patients with initial pulmonary tuberculosis(tuberculosis group) and 48 healthy person(healthy control group) and 43 patients with non-tuberculosis pulmonary disease(disease control group) were enrolled. The peripheral blood mononuclear cells of all participants were detected by ELISPOT and T-SPOT. TB, Then enumerated the spot forming cells(SFCs). All participants were also performed TST simultaneously. Results The sensitivity of ELISPOT and T-SPOT. TB were 90.6% and 93.4% in tuberculosis group and specificity were 91.5% in TST negative control group. The positive rate were 18. 8% and 16.7% in healthy person by these two methods, There wre not statistically significance difference between two methods(P >0.05). Conclusions Enzyme-linked immunosorbent assay( ELISPOT) using ESAT-6/CFP-10 fusion proteinas( E/C) and Rv3879c as antigens can be a assistant to diagnosis active tuberculosis. It offers certain diagnostic evidences for latent tuberculosis infection.%目的 比较E/C联合Rv3879c蛋白作为抗原的ELISPOT和T-SPOT.TB试剂盒,探讨其在活动性结核病诊断中的应用价值.方法 选取初治结核病患者106例作为结核组,43例肺部其它疾病患者做为疾病对照组和48例健康者做为健康对照组,应用T-SPOT.TB和ELISPOT检测受试者外周血单个核细胞的斑点形成细胞数量,同时对受试者行T研.结果 T-SPOT.TB和ELISPOT在结核病组中的敏感度分别为92.5%和93.4%,TST阴性对照组中的特异度均为91.5%,在健康者中的阳性率分别为18.8%和16.7%,且两种方法差异无统计学意义(均P>0.05).结论 E/C联合Rv3879c蛋白作为抗原的ELISPOT能够诊断活动性结核病,并为结核感染诊断提供一定的依据.

  18. Padronização da técnica imunoenzimática do ELISA de captura, no sistema avidina-biotina para a identificação de sangue ingerido por Lutzomyia (Lutzomyia longipalpis (Lutz & Neiva, 1912 Enzyme-linked Immunosorbent Assay biotin/avidin method standardization, for identification of Lutzomyia (Lutzomyia longipalpis bloodmeals (Lutz & Neiva, 1912

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    Ana Maria Marassá

    2004-12-01

    Full Text Available A identificação de sangue ingerido pelos insetos é um importante parâmetro para elucidar aspectos ligados à transmissão de zoonoses, dentre elas, as leishmanioses. Dos métodos empregados para esclarecer a atração de vetores por animais que possam atuar como reservatórios dessas parasitoses, destacam-se os imunológicos. O estudo teve como objetivo, padronizar a técnica imunoenzimática de captura e titular amostras de sangue ingerido em fêmeas de flebotomíneos ingurgitadas de Lutzomyia longipalpis criadas em laboratório e alimentadas experimentalmente em rato. Em vista da alta sensibilidade, favorecida pelo sistema avidina-biotina, foi possível a realização de pelo menos noventa testes, de cada uma das amostras em duplicata, e constatar a presença de sangue para todas as amostras com períodos de 12 e 24 horas pós-ingestão, observando-se diferença significativa entre os respectivos títulos.Bloodmeals taken by insects constitute an important parameter for clarifying aspects of the transmission of zoonoses, including leishmaniases. Immunological assays can be used to investigate the attraction of vectors to animals, which may be hosts of these parasitoses. The objective of this study was to standardize a sandwich enzyme-linked immunosorbent assay and titer samples with different time periods of digestion, in laboratory-bred Lutzomyia longipalpis fed on rats. In the light of the high sensitivity that the biotin-avidin method permits, the technique provided at least ninety repeat tests for each sample and identified recent bloodmeals taken by these insects. Bloodmeals were detectable up to 12 and 24h after blood ingestion, and a significant difference between these titers was observed.

  19. Fluorescence polarization assay, competitive enzyme-linked immunosorbent assay (ELISA-C and indirect ELISA for the diagnosis of brucellosis in buffaloes (Bubalus bubalis Teste de polarização fluorescente, teste imunoenzimático competitivo (ELISA-C e ELISA indireto para o sorodiagnóstico da brucelose em búfalos (Bubalus bubalis

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    Lília Márcia Silva Paulin

    2012-09-01

    Full Text Available The objective of the present study was to compare the performance of three serological tests for diagnosis of Brucella abortus infections in buffaloes (Bubalus bubalis. Serum samples collected from 696 adult females were submitted to the competitive enzyme-linked immunosorbent assay (ELISA-C, (I-ELISA, fluorescence polarization test (FPA, 2-mercaptoethanol test (2-ME and complement fixation test (CFT. The gold standard was the combination of CFT and 2-ME, considering as positive the reactors in both CFT and 2-ME, and as negative those non-reactors. ROC analyses were done for C-ELISA, I-ELISA and FPA and the Kappa agreement index were also calculated. The best combinations of relative sensitivity (SEr and relative specificity (SPr and Kappa were given by C-ELISA (96.9%, 99.1%, and 0.932, respectively and FPA (92.2%, 97.6 and 0.836, respectively. The C-ELISA and FPA were the most promising confirmatory tests for the serological diagnosis of brucellosis in buffaloes, and for these tests, cut-off values for buffaloes may be the same as those used for bovines.O presente estudo objetivou comparar o desempenho de três testes para o sorodiagnóstico da Brucella abortus em búfalos (Bubalus bubalis. Soros de 696 fêmeas bubalinas adultas foram submetidos aos testes: teste imunoenzimático indireto (ELISA-I, teste imunoenzimático competitivo (ELISA-C, teste de polarização fluorescente (TPF, 2-mercaptoetanol (2-ME e teste de fixação do complemento (FC. Foi empregada, como gold standard, a combinação de dois testes, FC e 2-ME. A curva ROC foi construída para os três testes: ELISA-I, ELISA-C e PF e, com base nos resultados desta análise, foi calculado o índice de concordância Kappa para cada teste. As melhores combinações de sensibilidade (Sr e especificidade (Er e os melhores resultados de Kappa foram alcançados pelo ELISA-C (96,9%, 99,1% e 0,932, respectivamente, seguido pelo PF (92,2%, 97,6%, e 0,836, respectivamente. Concluiu-se que os

  20. 动物组织中呋喃妥因代谢物残留酶联免疫试剂盒的研制%Development of an enzyme linked immunosorbent assay kit for furantoin metabolite residues determination in animal tissue

    Institute of Scientific and Technical Information of China (English)

    罗晓琴; 方华林; 张德红; 孙震; 赵正苗; 崔廷婷

    2013-01-01

    通过对呋喃妥因代谢物分子结构进行改造,制备了半抗原及人工抗原,免疫动物,制备特异性强的单克隆抗体在筛选呋喃妥因代谢物单克隆抗体的基础上,建立酶联免疫检测方法,并研制出检测动物组织中呋喃妥因代谢物残留的试剂盒该试剂盒的IC50值为0.094μg/L,最低检测限为0.1μg/kg,样本添加回收率为78.2%~ 102.7%,变异系数为5.1%~11.4%;与呋喃妥因的交叉反应率为13%,与其他药物的交叉反应率均小于1%;对实际样本的检测结果与液相色谱-串联质谱法基本一致 该方法快速、灵敏、可靠,为动物组织中呋喃妥因代谢物的残留检测提供了便捷、准确的分析检测手段.%Artificial hapten and immunogen were synthesized,after which monoclonal antibodies against furantoin metabolite was raised. On the basis of furantoin metabolite monoclonal antibody,rapid test enzyme linked immunosorbent assay(ELISA) kit for furantoin metabolite residues in animal tissue was developed. The sensitivity and detection limit of ELISA kit were 0.05μg/L and 0.1μg/kg, respectively. Sample recoveries were 78.2%~102.7%,and the coefficients of variation were 5.1%~11.4%. The cross-reactivity towards furantoin was 13%. As for other drugs,the cross -reactivity was below 1%. The results of ELISA kit and liquid chromatographic tandem mass spectrometry(LC-MS/MS) were in accordance. This method was rapid,sensitive and reliable, which provided a convenient and accurate way for detection of furantoin metabolite residues.

  1. Avaliação do desempenho do método do ensaio por enzimas imuno-adsorvidas (ELISA em leite em pó reconstituído contaminado experimentalmente com aflatoxina M1 Evaluation of competitive enzyme-linked immunosorbent assay (ELISA in milk powder contaminated with known concentrations of aflatoxin M1

    Directory of Open Access Journals (Sweden)

    Carlos Augusto Fernandes de Oliveira

    1996-12-01

    Full Text Available O desempenho do ensaio por enzimas imuno-adsorvidas (ELISA, mediante o emprego de conjuntos de reativos produzidos em escala comercial, para determinação de aflatoxina, foi avaliado em condições experimentais, através de análises repetidas, em amostras de leite em pó reconstituído contaminadas com concentrações conhecidas da fração M1 da toxina. Para os níveis de 0,10; 0,20; 0,50; e, 1,00 ng/ml, os percentuais de recuperação foram: 83,0%; 87,5%; 103,0%; e, 111,8%, respectivamente. O desvio-padrão relativo, para as referidas concentrações, foi, respectivamente, 65,5%; 31,8%; 10,9% e 13,6% (n=10, para cada nível de contaminação. Os resultados obtidos demonstram que o método é apropriado para pesquisas e levantamentos sobre a ocorrência de aflatoxina M1 em leite, sobretudo nas faixas de concentração entre 0,20 - 1,00 ng/ml.The evaluation of commercially available test systems of competitive enzyme-linked immunosorbent assay (ELISA, for Aflatoxin M1 (AFM1 was performed in experimental conditions, through repeated analysis, in samples of milk powder contamined with known concentrations of the toxin. Recoveries of AFM1 added to milk at levels of 0.10, 0.20, 0.50 and 1.00 ng/ml were 83.0%, 87.5%, 103.0% and 111.8% respectively. Relative standard deviations for the above mentioned concentrations were 65.5%, 31.8%, 10.9% and 13.6%, respectively (n =10, per spiking level. According to these results the ELISA is appropriate for AFM1 research and/or surveying, mainly for concentrations between 0.20 - 1.00 ng/ml.

  2. QuEChERS-酶联免疫快速检测法测定茶叶中黄曲霉毒素B1%Simultaneous determination for aflatoxin B1 in tea leaf by QuEChERS -enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    刘辉; 张燕

    2015-01-01

    目的:建立QuEChERS-酶联免疫快速检测茶叶中黄曲霉毒素B1的方法,并对样品前处理条件进行优化。方法茶叶样品用70%乙腈水提取溶液进行提取目标物,离心后取上清液并用PSA+MgSO4进行净化处理后,采用酶联免疫快速检测方法进行分析测定。结果本方法的检出限为0.078µg/kg,线性范围为0.125~0.854µg/kg, IC50值为0.327 ng/mL。在三个不同添加水平下,样品的平均回收率为87.66%~97.17%,相对标准偏差为4.89%~7.16%。检测结果与高效液相色谱法(high performance liquid chromatography, HPLC)方法的相关系r2=0.9854,线性相关性良好。结论该方法更加简便、快速、高效,能够用于茶叶中黄曲霉毒素B1的检测。%Objective To establish a QuEChERS-enzyme-linked immunosorbent assay (ELISA) method for determining the aflatoxin B1 (AFB1) in tea leaf rapidly, and optimizing the sample pre-treatment conditions. Methods The samples were extracted with 70% acetonitrile in water, and the supernatant liquid was directly purified with PSA+MgSO4 for detection of ELISA.Results The limit of detection (LOD) of method was 0.078 µg/kg, with linear range between 0.125 and 0.854 µg/kg, and IC50 value was 0.327 ng/mL. Average recovery rate of samples was between 87.66% and 97.17% at 3 adding levels, and the relative standard deviation (RSD) was 4.89%~7.16%. The results showed a good coefficient with high performance liquid chromatography (HPLC) (r2=0.9854).Conclusion This method proved to be suitable for the screening of tea leaf samples for the presence of AFB1, which was easier, faster and more efficient.

  3. Evaluation of the Sensibility and Specificity of the Complement Fixation Enzyme Linked Immunosorbent Assay Kit for Detecting Brucellosis Antibodies%布鲁氏菌病补体结合酶联免疫吸附试验抗体检测试剂盒敏感性和特异性评价

    Institute of Scientific and Technical Information of China (English)

    王楠; 姚学军; 马立峰; 王秀丽; 程君生; 蒋玉文; 赵心力; 毛开荣

    2014-01-01

    To analysis the sensibility, specificity and coincidence rate of the complement fixation enzyme linked immunosorbent assay kit ( CF-ELISA kit ) for detecting brucellosis antibodies, 400 bovine and sheep serum specimens were collected from brucellosis-affected areas selecting CF-ELISA kit to detect brucellosis antibodies, where cases had already been identified. Another total of 200 bovine and sheep serum specimens for detecting antibodies were collected from no brucellosis-affected areas, where no cases had been identified. Results showed that there was no significant difference (P>0.05) among the CF-ELISA kit, iELISA kit and CGS for detecting antibodies of brucellosis-affected areas serum. Kappa statistics result showed that the method of CF-ELISA and iELISA had highly coincidence rate. The specificity and coincidence rate were 100% between the CF-ELISA kit and iELISA kit for detecting antibodies of no brucellosis-affected areas serum.%为评价布鲁氏菌病补体结合酶联免疫吸附试验抗体检测试剂盒( CF-ELISA试剂盒)的敏感性、特异性及与其他试剂盒的符合率,用CF-ELISA试剂盒对布病感染地区牛、羊血清各200份,布病净化地区牛、羊群血清各100份进行抗体检测,同时与其他商品化检测试剂进行了比较。结果表明,感染地区牛、羊血清抗体的McNemar检验结果表明CF-ELISA试剂盒与间接酶联免疫吸附试验( iELISA)试剂盒和CGS的敏感性和特异性差异均不显著( P>0.05),Kappa一致性检验分析结果表明,CF-ELISA试剂盒与iELISA试剂盒检测结果有高度的符合性。检测净化地区牛、羊群血清抗体产品间的特异性和符合率均为100%。

  4. Standardization of an enzyme linked immunosorbent assay (ELISA for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus Padronização de um teste imunoenzimático (ELISA para detectar antígenos tóxicos circulantes do veneno em pacientes picados pelo escorpião Tityus serrulatus

    Directory of Open Access Journals (Sweden)

    Nilton Alves de Rezende

    1995-02-01

    Full Text Available The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients.Neste trabalho foram determinadas a sensibilidade e a especificidade da técnica imunoenzimática (ELISA desenvolvida por CHAVÉZ-OLORTEGUI et al. para detectar antígenos circulantes de veneno em pacientes picados po Tityus serrulatus. A média mais dois desvios padrão da observância do soro de 100 pacientes controles foi utilizada como limite entre teste positivo e teste negativo ("cutoff". A especificidade do ELISA foi igual a 97,0%. A sensibilidade do método, quando incluidos pacientes classificados como casos leves, moderados e graves de escorpionismo, foi de 39,3% e aumentou para 94,7% quando considerados apenas os casos moderados e graves. Estes resultados mostram que o ELISA pode ser utilizado para detecção de antígenos tóxicos circulantes em pacientes

  5. Enzyme-linked immunosorbent assay (ELISA for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests Reação imunoenzimática (ELISA para detecção de anticorpos para o vírus do sarampo: comparação com reações de inibição da hema-glutinação, imunofluorescência indireta e neutralização de placas

    Directory of Open Access Journals (Sweden)

    Vanda Akico Ueda Fick de Souza

    1991-02-01

    Full Text Available An enzyme-linked immunosorbent assay (ELISA for measles antibodies was compared with Plaque Neutralization (PRN, Haemagglutination inhibition (HI and Fluorescent antibody (IFA tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%. IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.A reação imunoenzimática (ELISA para determinação de anticorpos para o vírus do sarampo foi comparada com a reação de neutralização de placas (RNP, inibição da hemaglutinação (RIH e imunofluorescência indireta (RIF. Das 179 amostras positivas pela RNP, somente 2, com títulos iguais a 8, se apresentaram negativas por ELISA (copositividade de 98.9%. A RIF e RIH apresentaram, respectivamente, copositividade de 93.3 e 82.7%. ELISA apresentou sensibilidade equivalente à complexa RNP, boa reprodutibilidade e representa uma alternativa para a detecção de baixos títulos de anticorpos contra o sarampo.

  6. Avaliação do método imunoenzimático (ELISA para diagnóstico da infecção por Helicobacter pylori em crianças e adolescentes Evaluation of enzyme-linked immunosorbent assay for the diagnosis of Helicobacter pylori infection in children and adolescents

    Directory of Open Access Journals (Sweden)

    Aurea Portorreal

    2002-07-01

    Full Text Available RACIONAL: A infecção por Helicobacter pylori é reconhecida como a causa mais freqüente de gastrite crônica em adultos e crianças. Seu diagnóstico é realizado com métodos invasivos em fragmentos de mucosa gástrica obtidos com pinça endoscópica e os não-invasivos. O método imunoenzimático constitui exame simples, rápido e de baixo custo, apresentando alta sensibilidade em pacientes adultos. OBJETIVO: Avaliou-se o método ELISA prospectivamente em 111 crianças e adolescentes. MATERIAL E MÉTODOS: Utilizou-se o kit "Cobas Core II" (Roche. Considerou-se Helicobacter pylori positivo quando o teste rápido da urease e a histologia resultaram ambos positivos ou quando a cultura foi positiva, e Helicobacter pylori negativo quando todos os testes foram negativos. RESULTADOS: A idade dos 111 pacientes variou de 3 meses a 16 anos, (mediana = 9a 5m; média = 8a 7m ± 4.0. Infecção por Helicobacter pylori foi diagnosticada em 47,7% (53/111. A sensibilidade da sorologia foi de 83,0% e 86,0% e a especificidade foi de 70,6% e 71,0%, utilizando o ponto de corte de 7 U/mL e 5 U/mL, respectivamente. Em pacientes maiores de 10 anos de idade, a sensibilidade foi de 90,6% e 96,8% e a especificidade 71,0% e 61,9%, com ponto de corte de 7 U/mL e 5 U/mL, respectivamente. Quando foi utilizada somente a cultura positiva como padrão ouro e ponto de corte em 5 U/mL, a sensibilidade foi de 93,3%. CONCLUSÃO: O método ELISA apresentou boa sensibilidade em crianças maiores de 10 anos, utilizando o ponto de corte de 5 U/mL, porém a especificidade foi menor.BACKGROUND: Helicobacter pylori infection is recognized as the most frequent cause of chronic gastritis in adults and children. The diagnosis is accomplished with invasive methods in fragments of endoscopic gastric biopsies and non-invasive methods. The enzyme-linked immunosorbent assay constitutes a simple, fast exam and of low cost with high sensibility in adult patients. AIM: The purpose of this study

  7. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens.

    OpenAIRE

    Ungar, B L

    1990-01-01

    Cryptosporidium sp. is a ubiquitous 4- to 6-micron protozoan parasite infecting the intestinal tract of humans. It causes mild to fulminant diarrhea in patients, especially immunocompromised persons, and it may be hard to detect by microscopic fecal examination. An indirect, double-antibody enzyme-linked immunosorbent assay (ELISA) was developed using specifically produced goat and rabbit antisera to detect Cryptosporidium antigens in human feces. Of 62 frozen stools from patients with crypto...

  8. Determinação da infecção por Entamoeba histolytica em residentes da área metropolitana de Belém, Pará, Brasil, utilizando ensaio imunoenzimático (ELISA para detecção de antígenos Determination of Entamoeba histolytica infection in patients from Greater Metropolitan Belém, Pará, Brazil, by enzyme-linked immunosorbent assay (ELISA for antigen detection

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    Mônica Cristina de Moraes Silva

    2005-06-01

    Full Text Available O status epidemiológico da amebíase está sendo reavaliado desde que a Entamoeba histolytica (patogênica foi considerada espécie distinta de Entamoeba dispar (não patogênica. Em nosso estudo, realizamos pesquisa de antígenos de E. histolytica em amostras fecais de pacientes residentes na cidade de Belém, Pará, Brasil, utilizando ensaio imunoenzimático (E. histolytica Test, TechLab Inc., Blacksburg, Estados Unidos disponível comercialmente. Foram analisadas 845 amostras, com positividade em 248 (29,35%. A infecção por E. histolytica foi maior no grupo etário acima de 14 anos (30,36% que no grupo de 0-14 anos (28,28%, porém sem significância estatística (p The epidemiological status of amebiasis has been reevaluated since Entamoeba histolytica (pathogenic was considered a distinct species from Entamoeba dispar (non-pathogenic. We investigated E. histolytica antigens in stool samples from residents of Belém, Pará State, Brazil, with commercially available enzyme-linked immunosorbent assay (E. histolytica Test, TechLab Inc., Blacksburg, USA. A total of 845 samples were analyzed, of which 248 were positive (29.35%. E. histolytica infection was more frequent in the over-14-year age group (30.36% than in the 0-14-year group (28.28%, but the difference was not statistically significant (p < 0.05. Of all the samples, 334 were also submitted to parasitological methods (direct, Hoffman, and Faust et al.. There were discordant results between ELISA and parasitological methods in 83 samples (24.85%, with more positive results using ELISA. Our results thus suggest that intestinal amebiasis is an important public health problem in Greater Metropolitan Belém.

  9. Detection of Aspergillus galactomannan: comparison of an enzyme-linked immunoassay and a europium-linked time-resolved fluoroimmunoassay.

    Science.gov (United States)

    Paugam, A; Sarfati, J; Romieu, R; Viguier, M; Dupouy-Camet, J; Latgé, J P

    1998-10-01

    With a view to improving the sensitivity of serological detection of Aspergillus galactomannan (GM), a europium-linked time-resolved fluoroimmunoassay was developed. This method was compared to an enzyme-linked immunosorbent assay using a peroxidase-conjugated detector antibody. No increase in the sensitivity of the detection of GM standards was seen with the europium-based fluoroimmunoassay. PMID:9738075

  10. Detection of Aspergillus Galactomannan: Comparison of an Enzyme-Linked Immunoassay and a Europium-Linked Time-Resolved Fluoroimmunoassay

    OpenAIRE

    Paugam, A; Sarfati, J; Romieu, R.; Viguier, M; Dupouy-Camet, J; Latgé, J P

    1998-01-01

    With a view to improving the sensitivity of serological detection of Aspergillus galactomannan (GM), a europium-linked time-resolved fluoroimmunoassay was developed. This method was compared to an enzyme-linked immunosorbent assay using a peroxidase-conjugated detector antibody. No increase in the sensitivity of the detection of GM standards was seen with the europium-based fluoroimmunoassay.

  11. Study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test Estudo da reatividade cruzada em amostras de soro de cães positivos para Leishmania sp., Babesia canis e Ehrlichia canis, pelo ensaio imunoenzimático indireto e pela reação de imunofluorescência indireta

    Directory of Open Access Journals (Sweden)

    Trícia Maria F. de Sousa Oliveira

    2008-03-01

    Full Text Available To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT and Enzyme-linked immunosorbent assay (ELISA. All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis by IFAT. None of the serum samples from non-endemic areas were positive for Leishmania sp., by IFAT, but 67% were positive for B. canis and 78% for E. canis using the same test. When tested by ELISA for Leishmania sp., four samples from non-endemic area were positive. These dogs were then located and no clinical signs, parasites or antibody was detected in new tests for a six month period. Only one of these 4 samples was positive for B. canis by IFAT and ELISA and three for E. canis by IFAT. The results of the work suggest a co-infection in the endemic area and no serological cross-reaction among these parasites by IFAT and ELISA.Para verificar a existência de reação cruzada entre leishmaniose visceral, erliquiose e babesiose, nos testes sorológicos utilizados em programas de controle da leishmaniose visceral humana, amostras de soro canino provenientes de áreas endêmicas e não endêmicas para essa enfermidade, foram testadas pela Reação de Imunofluorescência (RIFI e Ensaio imunoenzimático (ELISA. Todos os soros provenientes de área endêmica foram positivos para Leishmania sp pelo ELISA e RIFI, 51% para Babesia canis e 43% para Ehrlichia canis pela RIFI. Pela RIFI, nenhum dos soros provenientes de área não endêmica foi positivo para Leishmania sp, sendo 67% positivos para B. canis e 78% para E. canis pelo mesmo teste. Quando testados pelo ELISA para Leishmania sp., quatro soros da área não endêmica foram positivos. Os cães foram localizados

  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. Padronização de ensaio imunoenzimático para pesquisa de anticorpos das classes IgM e IgG anti-Toxoplasma gondii e comparação com a técnica de imunofluorescência indireta Standardization of enzyme-linked immunosorbent assay ELISA to detect anti-Toxoplasma gondii IgM and IgG antibodies, and comparison with the indirect immunofluorescence technique

    Directory of Open Access Journals (Sweden)

    Cláudia Maria Antunes Uchôa

    1999-12-01

    Full Text Available A sorologia tem sido o método de escolha para o diagnóstico da toxoplasmose. Devido a isto, padronizamos um ensaio imunoenzimático (ELISA e comparamos seus resultados com a técnica de imunofluorescência indireta (IFI. A técnica padronizada apresentou na pesquisa de IgG sensibilidade (S de 96,7% e especificidade (E de 75%, com valor de predição de positividade (VPP de 83,3% e de negatividade (VPN de 94,7%, com uma concordância ajustada (K de 73,5%. A IFI apresentou S de 83,8%, E de 79,1% com VPP de 83,8 % e VPN de 79,1% com K de 63%. A concordância bruta entre os dois testes (ELISA/IFI foi de 88,3% para pesquisa de IgG e de 81,5% para pesquisa de IgM, sendo o K de 70,8% para IgG e de 1,3% para IgM, sendo o índice de correlação (r de 0,556 para IgG e de -0,023 para IgM. Podemos concluir que a ELISA-IgG padronizada é indicada nos processos de triagem sorológica, sendo a ELISA-IgM desaconselhada uma vez que apresentou baixos índices de concordância ajustada com a técnica de referência, sugerindo pouca confiabilidade dos resultados.Serology has been the most popular method to diagnose toxoplasmosis. Accordingly, this study standardizes an enzyme-linked immunosorbent assay (ELISA and compares its results with the IFI technique. In the IgG detection test, the standardized technique presented a sensibility (S of 96.77%, a specificity (SP of 75%, with a positive predictive value (PPV of 83.33%, a negative predictive value (NPV of 94.74%, and an adjusted concordance (K of 73.50%. The IFI exhibited 83.87% for S, 79.16% for SP, 83.81% for PPV, 79.16% for NPV, and 63% for K. The rough concordance between these two tests (ELISA/IFI was 88.35% for the IgG detection test and 81.55% for the IgM detection test. K was 70.82% and 1.31% for IgG and IgM, respectively, the correlation index (r being 0.556 for IgG and -0.023 for IgM. We can conclude that standardized ELISA-IgG is indicated in serologic selection processes, whereas the ELISA-IgM is

  14. Enzyme linked immunosorbent assay (ELISA) used for the detection of Cysticercus bovis injection in cattle

    International Nuclear Information System (INIS)

    Sensitivity and specificity of the ELISA for the diagnosis of T. saginata cysticercosis were studied. The specificity of the ELISA, by using ThFAS-L antigen, was found to be quite high when the hyperimmune serum samples of animals contained relatively high numbers of metacestodes. There was a direct relationship between the intensity of infection and the ELISA values of hyperimmune serum samples. Although ThFAS-L did not cross-react with F. hepatica and E. granulosus and gave no false positive reactions, it is not possible to regard this test as sensitive enough to detect naturally and weakly infected animals. Sensitivity and specificity of the ELISA may be improved by elution of the protein of MW 21800, which showed a diagnostic significance during immunoblot analysis. At present, the ELISA using ThFAS-L antigen has limited use in the diagnosis of cysticercosis infections in cattle

  15. Development of an Enzyme-Linked Immunosorbent Assay for Dibutyl Phthalate in Liquor

    Directory of Open Access Journals (Sweden)

    Libing Wang

    2013-06-01

    Full Text Available A monoclonal antibody specifically recognizing dibutyl phthalate (DBP was prepared based on a hapten (di-n-butyl-4-aminophthalate. After optimizing various parameters such as concentrations of antibody, coating antigen and composition of the assay buffer, an inhibition curve was plotted with the 50% inhibition concentration value (IC50 33.6 ± 2.5 ng/mL. A low level of cross-reactivity (<5% was found for other phthalate esters. Recovery tests were conducted using liquor simulant (a mixture of water and ethanol at two fortification levels (100 ng/mL and 300 ng/mL. The recovery rates ranged from 84.7% to 94.5% with a coefficient of variation between 7.1% and 12.8%. Nine liquor samples of different alcoholic strengths were detected using the proposed measure and confirmatory analysis was performed using liquid chromatography-mass spectroscopy (LC-MS. The detection results showed good consistency between the two measures and all the data above indicated that the proposed ELISA could be applied in DBP screening.

  16. Nonradioactive PCR-enzyme-linked immunosorbent assay method for detection of human cytomegalovirus DNA.

    OpenAIRE

    Allen, R. D.; Pellett, P E; Stewart, J A; Koopmans, M

    1995-01-01

    We developed a rapid, sensitive, and specific PCR-based assay for human cytomegalovirus (HCMV). The assay includes primer and probe sequences derived from conserved HCMV nucleotide sequences and nonradioactive hybridization-confirmation. The assay detected between 10 and 100 viral genomes. All HCMV clinical isolates tested (39 of 39) gave positive reactions.

  17. An enzyme-linked immunosorbent assay for the detection of Entamoeba histolytica antigens in faecal material.

    Science.gov (United States)

    Grundy, M S; Voller, A; Warhurst, D

    1987-01-01

    This paper describes a method for the detection of Entamoeba histolytica antigens in stool samples using a multi-layer ELISA. The method is sensitive and specific, showing no interference with other intestinal parasites, e.g. E. coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii, Hymenolepis nana, Giardia lamblia, Trichomonas and Ascaris. The method provides a rapid and simple screening assay for E. histolytica infections and should assist in diagnosis and epidemiological studies of the disease. PMID:2895514

  18. Enzyme-linked immunosorbant assays for glucosidases involved in processing of N-linked glycoproteins

    International Nuclear Information System (INIS)

    The conventional biological assays for glucosidase I and II are to determine the release of 14C-glucose from radiolabeled Glc3Man9(GlcNAc)2 and Glc/sub 1-2/Man9(GlcNAc)2, respectively by the actions of glucosidases. They have developed ultrasensitive ELISA's for these two glycosidases by using highly specific polyclonal antibodies prepared against purified glucosidase I and II from the bovine mammary tissue. Anti-glucosidase I and anti-glucosidase II antibodies are shown to be monospecific and are cross-reactive towards glucosidase I and II from liver, mammary gland, kidney and heart tissues of cow, guinea pig, mouse and rat, respectively. Western blot analysis revealed that anti-glucosidase I and anti-glucosidase II are very specific and are not cross-reactive towards any other protein present in solubilized microsomal extracts. Using these antibodies, conditions for ELISA's for glucosidase I and II were standardized. Dose response curves for purified enzymes and crude extracts were developed. A correlation between biological activities of these enzymes and immunoreactive glucosidase I and II, present in the solubilized microsomal preparations of liver, mammary gland, kidney and heart tissues from cow, guinea pig, mouse and rat, is made. The sensitivity of ELISA's for glucosidase I and II are in the range of 10 - 1000 ng of immunoreactive species

  19. Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Welner, Simon; Trier, Nicole Hartwig; Morten Frisch, Morten;

    2013-01-01

    Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation...

  20. Detection of ultraviolet-induced DNA damages by ELISA (Enzyme-linked immunosorbent assay)

    International Nuclear Information System (INIS)

    Authors described the procedure for detection by ELISA of cyclobutane-type dimers and (6-4)-type dimers in DNA of cultured human cells irradiated by ultraviolet light (UV) using specific monoclonal antibody TDM-2 and 64M-2, respectively. The procedure included; 1. coating of micro-titre plates with protamine sulfate, 2. cell culture and UV irradiation (WI-38 VA13 cells were irradiated by UV lamp mainly consisting of 254 nm at 0.4 J/m2/s which was measured by UV-dosimeter UVR-1), 3. extraction of DNA from the irradiated cells (DNA was extracted just after or at various times of continued culture after irradiation to see the degree of repair), 4. adsorption of the UV-irradiated DNA onto the protamine sulfate-coated micro-titre plates (DNA was made single-strand by heat before adsorption). 5. measurement of UV-induced DNA damage by ELISA using the specific monoclonal antibodies mentioned and peroxidase-streptavidin, and 6. an example of given results with brief discussion on the speculation of those and on the repair. (H.O.)

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

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Borre, M B; Jepsen, S; Hogh, B; Petersen, E; Vuust, J

    1991-01-01

    . falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant...... New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control of...

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

    Directory of Open Access Journals (Sweden)

    Zhenlin Xu

    2008-04-01

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

  3. Seroprevalence Study of Human Brucellosis by Conventional Tests and Indigenous Indirect Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Agasthya, Annapurna S.; Srikrishna Isloor; Prabhudas Krishnamsetty

    2012-01-01

    Brucellosis is one of the most important reemerging zoonoses in many countries. Brucellosis is caused by Gram-negative coccobacillus belonging to genus Brucella. Human brucellosis often makes the diagnosis difficult. The symptoms and clinical signs most commonly reported are fever, fatigue, malaise, chills, sweats headaches, myalgia, arthralgia, and weight loss. Some cases have been presented with only joint pain, lower backache, and involuntary limb movement, burning feet, or ischemic heart ...

  4. Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Reimert, C M; Venge, P; Kharazmi, A;

    1991-01-01

    Eosinophil cationic protein (ECP) is a highly basic and potent cytotoxic single-chain zinc-containing protein present in the granules of the eosinophilic granulocytes. ECP appears to be involved in defence against parasites and in the tissue damage seen in subjects with allergic and inflammatory...

  5. Cross-reactivity of designer drugs, including cathinone derivatives, in commercial enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Swortwood, Madeleine J; Hearn, W Lee; DeCaprio, Anthony P

    2014-01-01

    Since the introduction of synthetic heroin, designer drugs have been increasing in prevalence in the United States drug market over the past few decades. Recently, 'legal highs' sold as 'bath salts' have become a household term for one such class of designer drugs. While a number of federal and state bans have been enacted, the abuse of these designer drugs still continues. Few assays have been developed for the comprehensive detection of such compounds, so it is important to investigate how they may or may not react in presumptive screens, i.e. pre-existing commercial immunoassays. In this experiment, 16 different ELISA reagents were evaluated to determine the cross-reactivity of 30 designer drugs, including 24 phenylethylamines (including 8 cathinone derivatives), 3 piperazines, and 3 tryptamines. Cross-reactivity towards most drugs was <4% in assays targeting amphetamine or methamphetamine. Compounds such as MDA, MDMA, ethylamphetamine, and α-methyltryptamine demonstrated cross-reactivities in the range of 30-250%, but data were consistent with both manufacturer's inserts and published literature. When tested against the Randox Mephedrone/Methcathinone kit, cathinone derivatives demonstrated cross-reactivity at concentrations as low as 150 ng/ml. Since this same reagent did not cross-react with other amphetamine-like compounds, it opens the possibility to screen post-mortem specimens without the interference of putrefactive amines. All other assays demonstrated essentially no cross-reactivity towards any of the analytes evaluated. Given these results, a great need exists for more broad-range screening techniques to be applied when analyzing biological specimens by immunoassays for drugs of abuse, specifically the more recent designer drugs. PMID:23677923

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

    Indian Academy of Sciences (India)

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

    2003-02-01

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

  7. Enzyme-linked immunosorbent assay for coproantigen detection of Trichuris vulpis in dogs.

    Science.gov (United States)

    Elsemore, David A; Geng, Jinming; Flynn, Laurie; Cruthers, Larry; Lucio-Forster, Araceli; Bowman, Dwight D

    2014-03-26

    Infections with Trichuris vulpis, the canine whipworm, may be challenging to diagnose even though characteristic bipolar eggs are shed by mature worms and may be recovered from feces. Decreased detection sensitivities because of using flotation solutions with specific gravities vulpis infections is reported herein. Whipworm-specific coproantigens were evidenced in feces from experimentally infected dogs using the newly developed ELISA starting as early as day 23 postinfection, while eggs were not detected in feces until day 69. In addition, 1,156 field fecal samples were tested using fecal flotation methods and the newly developed whipworm ELISA. Of these, 27 samples were found by flotation to be whipworm egg positive, while 35 had detectable antigen on the ELISA. Discrepant results were obtained in 12 samples; 2 egg-positive samples tested ELISA negative, and 10 ELISA-positive samples did not contain detectable egg levels. Using the fecal ELISA for the detection of whipworms in dogs should allow for earlier detection of infection, aid the identification of cases in the face of low egg shedding, and increase detection sensitivity as most commercial laboratories are using flotation solutions not optimal for T. vulpis egg detection. PMID:24670954

  8. Consequences of Interference of Milk with Chemoattractants for Enzyme-Linked Immunosorbent Assay Quantifications▿

    OpenAIRE

    RAINARD, P

    2010-01-01

    Concentrations of the chemoattractants CXCL1, CXCL2, CXCL3, CXCL8, and C5a in milk were reduced by the preparation of milk whey by high-speed centrifugation or with rennet. About half of the chemoattractants (35 to 65%) were associated with the casein micelle sediment, except when whey was prepared by acidification. Consequently, quantification of chemoattractants should be carried out preferentially with skimmed milk samples or, whenever whey is needed, with acidic whey samples. The interfer...

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

  11. Evaluation of an enzyme linked immunosorbent assay (ELISA) in the serodiagnosis of swine trypanosomosis

    International Nuclear Information System (INIS)

    In this study, the T. evansi antigen used in the ELISA was specific and sensitive for the detection of antibodies against T. evansi in pigs, the antibody being detected in an experimentally infected pig on day 17 post-infection and until the end of the study. In pigs naturally infected with T. evansi, the assay yielded also positive results with high OD values. In uninfected pigs from countries free from trypanosomosis, it only gave a low post positive value. There were no cross-reactions in pigs infected with Toxoplasma and Eperythrozoon. Even though peroxidase recombinant protein G conjugate, instead of specific anti-IgC swine conjugate, was used, it was still effective in ELISA. The antigen was characterized by western blot, and it was found that the predominant polypeptide band was a 67 kD protein. In Thailand, trypanosomosis is found not only in pigs, but also in cattle, buffalo, horses and dogs. Trypanosomosis is widespread in Thailand and difficult to eliminate, so that Ab-ELISA will be an important tool for the diagnosis and epidemiological study of the disease in different animal species, including pigs

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    procedure was assessed previously. The recovery and linearity percentages, the standard curve and parallelism were determined. Results. The standard curve showed a high correlation coefficient (r2 = 0.998). The cortisol concentration of two samples fluctuated between 64 and 72 ng.ml-1. Only the 200 ng.ml-1...

  13. Seroepidemiology of rinderpest in bovines in Sri Lanka using the enzyme linked immunosorbent assay (ELISA) technique

    International Nuclear Information System (INIS)

    This is the first inland wide study on seroprevalence of rinderpest in Sri Lanka. This study shows the advantages of using a serologically sensitive test, such as ELISA, in studying the seroepidemiology of a disease with low prevalence. The prevalence was highest in the northern, eastern and north-central provinces. The spread of rinderpest from the first location of the outbreak, which was in the eastern province in 1987, is attributed to the movement of bovines for slaughtering purposes. It appears that the spread of rinderpest could be reduced by controlling animal movement. Apparently, rinderpest had shifted from an epidemic form in the 1987-1989 period to an epidemic form from 1990 onwards, towards areas with high bovine density (>0.3 bovines/hectare). Furthermore, the extensive management system mostly practiced in the DL regions, in which animal-to-animal contact is more frequent, had contributed to the spread of rinderpest. The prevalence was higher in older bovines, probably because of exposure to natural infection during the last epidemic

  14. A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals

    OpenAIRE

    Ulrich, Sophia Arlena; Lehnert, Kristina; Siebert, Ursula; Strube, Christina

    2015-01-01

    Background Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is...

  15. The Role of Maternal Thyroid Stimulating Hormone Receptor Blocking Antibodies in the Etiology of Congenital Hypothyroidism in Isfahan, Iran

    OpenAIRE

    Mahin Hashemipour; Shima Salehi Abari; Neda Mostofizadeh; Shaghayegh Haghjooy-Javanmard; Nafiseh Esmail; Silva Hovsepian; Amini Masoud; Roya Kelishadi; Akbar Hasanzadeh; Mehrdad Mirouliaei

    2012-01-01

    Background: Considering the role of maternal thyroid stimulating hormone (TSH) receptor blocking antibody (TRAb) in the etiology of congenital hypothyroidism (CH), this study aimed to determine TRAb among patients with CH in Isfahan, Iran. Methods: In this case-control study, patients with CH and their mothers were compared with a group of healthy neonates and their mothers. Venous blood samples were obtained for measurement of TRAb using enzyme-linked immunosorbent assay (ELISA) method a...

  16. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    Science.gov (United States)

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  17. Application Of Laser Fluorimetry To Enzyme-Linked Immunoassay

    Science.gov (United States)

    Hinsberg, William D.; Milby, Kristin H.; Lidofsky, Steven D.; Zare, Richard N.

    1981-09-01

    An enzyme-linked sandwich immunoassay for insulin is described. Horseradish peroxidase is employed as an enzyme label for antibody, and enzyme activity is measured via the fluorogenic substrate, p-hydroxyphenylacetic acid. The product is detected by excitation of fluorescence with the 325 nm line of a cw helium-cadmium ion laser on-line with reverse phase high performance liquid chromatography. The method requires a total incubation time of 45 minutes, and the limit of insulin detection is 1.1 μU/ml (6.6 pM). This assay is applicable to the analysis of human serum samples.

  18. Enzyme-linked small-molecule detection using split aptamer ligation.

    Science.gov (United States)

    Sharma, Ashwani K; Kent, Alexandra D; Heemstra, Jennifer M

    2012-07-17

    Here we report an aptamer-based analogue of the widely used sandwich enzyme-linked immunosorbent assay (ELISA). This assay utilizes the cocaine split aptamer, which is comprised of two DNA strands that only assemble in the presence of the target small molecule. One split aptamer fragment is immobilized on a microplate, then a test sample is added containing the second split aptamer fragment. If cocaine is present in the test sample, it directs assembly of the split aptamer and promotes a chemical ligation between azide and cyclooctyne functional groups appended to the termini of the split aptamer fragments. Ligation results in covalent attachment of biotin to the microplate and provides a colorimetric output upon conjugation to streptavidin-horseradish peroxidase. Using this assay, we demonstrate detection of cocaine at concentrations of 100 nM-100 μM in buffer and 1-100 μM human blood serum. The detection limit of 1 μM in serum represents an improvement of two orders of magnitude over previously reported split aptamer-based sensors and highlights the utility of covalently trapping split aptamer assembly events. PMID:22715870

  19. Immunosorbent analysis of ricin contamination in milk using colorimetric, chemiluminescence, and electrochemiluminescence detection

    Science.gov (United States)

    Analytical methodology to detect ricin in food matrices is important because of the potential use of foodborne ricin as a terrorist weapon. Monoclonal antibodies (mAbs) that bind ricin were used for both capture and detection in sandwich enzyme-linked immunosorbent assay (ELISA) and electrochemilumi...

  20. 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.

  1. An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

    DEFF Research Database (Denmark)

    Hansen, Soren; Schmidt, Vivi; Steffensen, Maria Abildgaard;

    2008-01-01

    Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of othe...

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

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Ajjouri, Gitte; Handberg, Kurt;

    2013-01-01

    BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of...

  3. An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

    DEFF Research Database (Denmark)

    Selman, L; Henriksen, M L; Brandt, J;

    2012-01-01

    be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269-299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated...... freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes....

  4. Enzyme linked immunosorbent assay for the diagnosis of cerebral cysticercosis in Reunion island: comparison with computerized tomography scan

    International Nuclear Information System (INIS)

    An immunoenzymologic (Elisa) serodiagnosis of cysticercosis is evaluated in 75 encephalic cysticercotic patients whose diagnosis of the disease and its progression is assessed by tomodensitometry. A Taenia solium antigen is used. Only Ig G are investigated. The sensibility of serodiagnosis is 85 % and specificity 87 % when there is a progression of the disease; no difference is noticed in the patients without any progression of the disease and in control normal subjects. This serodiagnosis of cysticercosis appears of value for the evaluation of the activity of the disease

  5. Application of enzyme linked immunosorbent assays for the diagnosis of bovine brucellosis in Rio Grande do Sul, Brazil

    International Nuclear Information System (INIS)

    The results of an indirect ELISA (I-ELISA) and a competitive ELISA (C-ELISA) for detection of antibody to Brucella abortus in cattle were compared with those of conventional serological tests. The sensitivity of I-Elisa using 230 sera from infected animals was 98.6% and the specificity in 720 sera from brucellosis-free animals was 98.2%. The C-ELISA when tested in 94 positive sera and 91 negative sera was 100% sensitive and specific. The relative sensitivity and specificity of I-ELISA compared to C-ELISA in 582 sera from groups of animals vaccinated with S19 by different routes and doses was respectively 64% and 100%. When comparing five serological tests on sera from vaccinated animals, the specificity was 94% for C-ELISA, 93% for 2-mercaptoethanol (2ME), 88% for Rose Bengal Plate test (RBPT), 84% for Slow Tube Agglutination test (SAT) and 34% for I-ELISA. It can be concluded from this study that the RBPT gave less false positive results than the I-ELISA in vaccinated animals and the C-ELISA is a good confirmatory test with the advantage of distinguishing the antibody response due to vaccination from that resulting from infection with Brucella abortus. (author)

  6. False-Positive Aspergillus Galactomannan Enzyme-Linked Immunosorbent Assay Results In Vivo during Amoxicillin-Clavulanic Acid Treatment

    OpenAIRE

    Mattei, Daniele; Rapezzi, Davide; Mordini, Nicola; Cuda, Federica; Lo Nigro, Cristiana; Musso, Maura; Arnelli, Aldo; Cagnassi, Sebastiano; Gallamini, Andrea

    2004-01-01

    Positive Platelia Aspergillus test results were observed in consecutive serum samples from an immunocompromised host during amoxicillin-clavulanic acid treatment, and a correlation between plasmatic amoxicillin concentration and galactomannan optical density index was observed. Amoxicillin-clavulanic acid vials tested positive for galactomannan but were negative for Aspergillus DNA.

  7. Comparison between magnetic enzyme-linked immunosorbent assay (MELISA) and complement fixation test (CF) in the diagnosis of paracoccidioidomycosis.

    Science.gov (United States)

    Pires de Camargo, Z; Unterkircher, C; Drouhet, E

    1986-02-01

    MELISA and CF were compared using sera from paracoccidioidomycosis patients before treatment and patients undergoing antimycotic treatment. With MELISA it was possible to distinguish different antibody levels in both groups of patients whereas such distinction was not observed by using CF tests. MELISA is thus an advantageous alternative to CF in the diagnosis of paracoccidioidomycosis, including the possibility of testing sera with anticomplementary activity. PMID:3701544

  8. IgM capture enzyme linked immunosorbent assay (ELISA) used as diagnostic tool for Rift Valley fever in Sudan

    International Nuclear Information System (INIS)

    Rift Valley fever (RVF) is a mosquito-borne viral disease. It causes abortion in sheep, goats and cattle, and deaths of young animals. Humans can acquire infection from contact with infected tissues of livestock, or less frequently from mosquito bite. This study is aimed to use RVFV IgM ELISA as rapid serological techniques to detect IgM immunoglobulin in 323 blood samples collected from different species caprine, ovine and bovine from different region Gizera, White Nile, Sinnar and Blue Nile States where the environment and climate suited occurrence of the disease. The study was conducted in White Nile, Gizera, Blue Nile and Sinnar states. These States are agricultural areas where we found frequent rainfall, water reserves and dams. We took animal presence in the area as essential element in disease happening. Blood sample were collected from different animals caprine, ovine, and bovine and different ages so as to study the development of disease taking into account the affect of intrinsic factors species, breed, sex and age. Serum samples were prepared, preserved in cooled containers at 4 deg C and transported to laboratory. The ELISA kits were purchased from the special pathogen unit, National institute of Virology, Johannesburg, South Africa. The kit contains the following: Rabbit anti-sheep IgM (capture antibody), freeze-dried 2 x 250μl; RVFV antigen (RVF Ag), freeze-dried, 2 x 300μl; Control antigen (control Ag), freeze-dried, 2 x 300μl; anti-RVFV serum (detection antibody), freeze-dried 2 x 100μl; Goat anti-mouse IgG horseradish peroxidase (HPRO) conjugate, 1 x 100μl; Control sera: high positive (C++) and negative control (C-), freeze-dried 1 x 200V each; low positive (C+) control serum, freeze-dried 2 x 100μl; Phosphate-buffered saline (PBS) powder, 20 x sachets; Skim milk powder, 2 x 50g; Tween 20, 1 x100ml; Immunoplates, 25 x; ABTS-substrate, 3 x 100ml; 10% SDS stop solution ('Electran') 1 x 100ml. The ELISA is based on a capture format in which the plates were coated with rabbit anti-sheep IgM capture antibody and then reacted with test sera. Anti sheep capture antibody can be used for detection of IgM in sheep, goats and cattle. The captured IgM antibody was reacted with RVF antigen, and the bounded antigen was then detected with mouse anti-RVF antibody anti-mouse horseradish peroxidase (HRPO) conjugate plus ABTS substrate. Total of 323 serum samples from different animals caprine, ovine and bovine in ALgazira, White Nile, Sinnar Blue Nile states were examined for rift valley fever virus IgM antibodies. 63(61%)caprine, 38(51%)ovine and 10 (23%) bovine were positive in Algazira state. 4(18%) caprine , 6(50%) ovine and 5(17%) bovine were positive in White Nile state .In Sinnar and Blue Nile state no caprines sample were tested , but 1(14%) ovine, 7(22%)bovine were positive. RVF was previously reported in Sudan based on serological survey that indicated presence of antibodies to RVF virus in animals and human sera, it seems that RVF was prevalent in subclinical form, but the lack or poor of reporting system of the clinical cases did not reflect the real situation, because indigenous animals may not show clinical signs due to innate resistance.the virus activity may be revealed by random isolation from mosquitoes or by occasional human disease. in study area. Gizera state showed highest morbidity rate in comparison to White Nile, Sinnar and Blue Nile states .presence of IgM antibodies is indicative of active and circulating virus in study area , but no virus isolation is done

  9. Serosurveillance for Francisella tularensis Among Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

    OpenAIRE

    Sharma, Neekun; Hotta, Akitoyo; Yamamoto, Yoshie; Uda, Akihiko; Fujita,Osamu; MIZOGUCHI, Toshio; Shindo, Junji; PARK, Chun-Ho; Kudo, Noboru; Hatai, Hitoshi; OYAMADA, Toshifumi; Yamada, Akio; Morikawa, Shigeru; Tanabayashi, Kiyoshi

    2014-01-01

    Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularens...

  10. Use of enzyme linked immunosorbent assay (ELISA) for the diagnosis of brucellosis in cattle in Yucatan, Mexico

    International Nuclear Information System (INIS)

    Sera (247) from non-vaccinated brucellosis negative herds, 328 negative sera from Brucella abortus strain 19 vaccinated herds (brucellosis free), and 95 sera positive to the Rose Bengal test (RBT) and Complement Fixation test (CFT) from Brucella abortus-infected herds, were used to determine the relative sensitivity and specificity of a FAO/IAEA I-ELISA kit and the Rivanol Agglutination Test (RAT), using the CFT as a 'gold standard'. A threshold value for the I-ELISA was determined to be 37 PP using the mean plus 3 standard deviations of the negative sera from vaccinated animals. The I-ELISA showed a high relative sensitivity (100%) and a good relative specificity (92.5%), using the threshold determined for local conditions. The RAT gave a lower sensitivity value than the CFT (97.8%) and good specificity (99.3%). The I-ELISA could be used as a screening test under Yucatan conditions or as a confirmatory test in places where vaccination is not carried out. The RAT lacks sensitivity and is therefore not recommended for use in final stages of eradication programs but could be used in control programmes or early stages of eradication campaigns as a confirmatory test. (author)

  11. Enzyme linked immunosorbent assay for the diagnosis of cerebral cysticercosis in Reunion island: comparison with computerized tomography scan

    Energy Technology Data Exchange (ETDEWEB)

    Michault, A.; Coubes, P.; Laporte, J.P.; Bouillan-Linet, E.; Leroy, D.

    1988-03-01

    An immunoenzymologic (Elisa) serodiagnosis of cysticercosis is evaluated in 75 encephalic cysticercotic patients whose diagnosis of the disease and its progression is assessed by tomodensitometry. A Taenia solium antigen is used. Only Ig G are investigated. The sensibility of serodiagnosis is 85 % and specificity 87 % when there is a progression of the disease; no difference is noticed in the patients without any progression of the disease and in control normal subjects. This serodiagnosis of cysticercosis appears of value for the evaluation of the activity of the disease.

  12. Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Geiger Stefan Michael

    2001-01-01

    Full Text Available Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2% and 91.1%, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis.

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

    Science.gov (United States)

    Bovine viral diarrhea virus is a costly disease of cattle that can be controlled by vaccination, biosecurity, and removal of persistently infected cattle. Development and proficiency testing of assays to identify persistently infected cattle substantial quantities of known positive and negative samp...

  14. 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 ...

  15. Use of an indirect enzyme linked immunosorbent assay for the diagnosis and epidemiological studies of Brucella abortus in Chile

    International Nuclear Information System (INIS)

    It is well known that traditional brucellosis diagnostic tests have a lack of sensitivity and specificity depending on the area or country where they have been used. Seeking for better alternatives, we undertook an evaluation of an ELISA test for bovine brucellosis diagnosis, for its possible use in Chile. We collected 2 groups of sera from negative animals: one from an area free of S19 vaccination (n=491) and a second from vaccinated herds (n=349). These groups were used for determination of cut-offs and diagnostic specificity calculations. A positive group of sera was collected from animals (n=186) from infected herds which were all reactors in the Rose Bengal (RB) and Rivanol (RIV) tests; some were also bacteriological culture positive and positive to an immunodiffusion test. This group was used for sensitivity calculation. A final test group of 385 sera from herds with brucellosis, either negative or positive in the RB or RIV tests, were used for relative sensitivity and specificity calculations. The calculated cut-off values were 26% and 61% of Percentage Positivity (PP) for the free area and vaccinated negative groups, respectively. Real sensitivity was 100% and real specificity was 98.6% when a 26% cut-off was used. Using the 61% cut-off value, the relative sensitivity respect of the RB and RIV test were 95.7% and 100%, respectively. In addition, relative specificity with respect to the RB and RIV tests were 82.4% and 81.8%, respectively. (author)

  16. Technical note: Development of an enzyme-linked immunosorbent assay for the determination of florfenicol and thiamphenicol in swine feed.

    Science.gov (United States)

    Luo, P J; Jiang, W X; Chen, X; Shen, J Z; Wu, Y N

    2011-11-01

    One polyclonal antibody against florfenicol and thiamphenicol was produced and a competitive ELISA was developed for the detection of florfenicol and thiamphenicol in swine feed. The ELISA gave a 50% inhibiting concentration of 1.02 ng/mL for florfenicol. For swine feed fortified with 0.05 to 3.0 mg/kg, the interassay recoveries of florfenicol and thiamphenicol ranged from 86.4 to 118.6%, whereas intraassay recoveries of both drug ranged from 90.1 to 126.5% with less than 15% CV. Results obtained from HPLC-tandem mass spectrometry indicated this ELISA procedure could be used as a convenient method for rapid screening of florfenicol and thiamphenicol in swine feed. PMID:21724944

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

    DEFF Research Database (Denmark)

    Sækmose, Susanne Gjørup; Schlosser, Anders; Holst, René; Johansson, Sofie Lock; Wulf-Johansson, Helle; Tornøe, Ida; Vestbo, Jørgen; Kyvik, Kirsten Ohm; Barington, Torben; Holmskov, Uffe; Sørensen, Grith Lykke

    2013-01-01

    Microfibrillar-associated protein 4 (MFAP4) is a systemic biomarker that is significantly elevated in samples from patients suffering from hepatic cirrhosis. The protein is generally localized to elastic fibers and other connective tissue fibers in the extracellular matrix (ECM), and variation in...

  18. Comparison of Metallothionein Detection by Using Brdicka Reaction and Enzyme-Linked Immunosorbent Assay Employing Chicken Yolk Antibodies

    Czech Academy of Sciences Publication Activity Database

    Křížková, S.; Bláhová, P.; Nakielna, J.; Fabrik, I.; Adam, V.; Eckschlager, T.; Beklová, M.; Svobodová, Z.; Horák, Vratislav; Krížek, R.

    2009-01-01

    Roč. 21, č. 23 (2009), s. 2575-2583. ISSN 1040-0397 Institutional research plan: CEZ:AV0Z50450515 Keywords : Metallothionein * Differential pulse voltammetry * ELISA Subject RIV: CG - Electrochemistry Impact factor: 2.630, year: 2009

  19. Apparent seroprevalence of Salmonella spp. in harp seals in the Greenland Sea as determined by enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Aschfalk, A; Folkow, L; Rud, H; Denzin, N

    2002-10-01

    An indirect ELISA was developed as a possible tool for surveillance of the seroprevalence of Salmonella spp. in harp seals. This species is hunted for human consumption and thus transmission of disease to humans cannot be excluded. To cover a broad spectrum of serogroups, a mixture of the lipopolysaccharides (LPS) of S. typhimurium and S. choleraesuis was used as the antigen in this pilot study. Chicken anti-harp-seal immunoglobulin horseradish peroxidase conjugate served as the immunoconjugate. Sera from four captive harp seals, which were Salmonella culture-negative and had no clinical or historical evidence of salmonellosis, were used as negative controls. After immunization with an inactivated S. typhimurium vaccine, further sera from these seals were used as positive controls, as no serum from naturally infected animals was available. Serum samples from 93 harp seals caught in the Greenland sea in 1999 were examined, and anti-Salmonella antibodies were found in the samples from two individuals (seroprevalence 2.2%). Although Salmonella has been isolated from other pinniped species, this is the first documentation of Salmonella-seropositive harp seals. This study contributes to the evaluation of the importance of salmonellosis in arctic marine mammals and thus to the prevention of potential outbreaks of this important zoonosis. PMID:12416866

  20. ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR SCREENING OF MILK SAMPLES FOR SALMONELLA-TYPHIMURIUM IN DAIRY HERDS

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Wedderkopp, A.

    1995-01-01

    We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples. Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S typhimurium epsiodes within the previous 7...... positive (herd specificity, 0.9 and herd sensitivity, 1.0). A sig nificant correlation (P <0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis. It was concluded that ELISA testing of individual milk sam ples can be used for surveillance of...... months, and 220 herds of unknown disease status, were tested. A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3. Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA...

  1. Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

    Science.gov (United States)

    A competitive ELISA (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C-terminus of Babesia bigemina rhoptry-associated protein-1a was validated for international use. Receiver Operating Characteristic (ROC) analysis revealed 16% inhibition as the threshold for a neg...

  2. 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.

  3. Development of an enzyme-linked immunosorbent assay for equine neutrophil elastase measurement in blood: preliminary application to colic cases.

    Science.gov (United States)

    de la Rebière de Pouyade, Geoffroy; Franck, Thierry; Salciccia, Alexandra; Deby-Dupont, Ginette; Grulke, Sigrid; Heyden, Laurent Vander; Sandersen, Charlotte; Serteyn, Didier

    2010-06-15

    Equine neutrophil elastase (NE) is a protease released in inflammatory diseases and participating in tissue destruction. To measure NE in horse plasma to assess its role in pathological conditions, we purified elastase from equine neutrophils by a double step chromatography and obtained a pure protein of 27 kDa, 4 kDa smaller than the NE 2A previously purified (Scudamore et al., 1993; Dagleish et al., 1999), which was likely to be NE 2B. We developed an ELISA by using two specific polyclonal antibodies obtained from rabbit and guinea pig. The sandwich complex was detected using a secondary antibody conjugated to alkaline phosphatase. The ELISA showed good precision and accuracy, with intra- and inter-assay coefficients of variation below 10% for equine NE concentrations ranging from 1.875 to 60 ng/ml. A stable plasma NE value, unaffected by the delay of centrifugation (over 4h), was obtained with plasma from EDTA anticoagulated blood. The mean value (+/-SEM) measured in 37 healthy horses was 32.53+/-4.6 ng/ml. NE level in plasma of horses with colic at the time of admission was significantly higher than in healthy horses. Our results indicate that the ELISA technique we developed to measure plasmatic NE is a powerful tool for studying the role of elastase in equine inflammatory disease. In future, the application will be extended to other equine biological fluids. PMID:19932512

  4. Enzyme-linked immunosorbent assays for doping control of 5alpha-reductase inhibitors finasteride and dutasteride.

    Science.gov (United States)

    Brun, Eva M; Torres, Ana; Ventura, Rosa; Puchades, Rosa; Maquieira, Angel

    2010-06-25

    Finasteride and dutasteride are 5alpha-reductase inhibitors included in the World Anti-Doping Agency's list of banned substances. Two highly sensitive and selective ELISA assays were developed for these compounds. Polyclonal rabbit antibodies were raised using synthesized haptens and other commercial products. The best immunoassay obtained, based on an antibody-coated format, showed a limit of detection of 0.01 microg L(-1) and an IC(50) of 0.75 microg L(-1) for finasteride (cross-reactivity with dutasteridefinasteride and dutasteride determination, with limits of detection of 0.013 and 0.021 microg L(-1), and IC(50) values 0.18 and 1.18 microg L(-1), respectively. Both assays were highly selective to a set of anabolic steroids, but they showed 37% and 30% cross-reactivity with the major urinary metabolite of finasteride, allowing its determination. The developed ELISA had better sensitivity than HPLC/MS/MS method and was applied as a screening technique to quantify dutasteride, finasteride, and its main metabolite in human urine without sample pre-treatment. Moreover, the analysis of dutasteride's excretion urines by ELISA was used to obtain its human excretion rate, essential to improve the analytical strategies about this type of drugs (permitted as medicines and prohibited in sport) and to establish an effective anti-doping policy. PMID:20541645

  5. Generation of antibody and development of an enzyme-linked immunosorbant assay for the feed additive roxarsone

    Science.gov (United States)

    Roxarsone (3-nitro-4-hydroxy-phenyl arsonic acid) has been used in the poultry and swine industries as a feed additive to treat coccidiosis and other intestinal disorders as well as to improve feed efficiencies and weight gain. In animals, roxarsone is eliminated mostly as parent compound which may ...

  6. The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

    OpenAIRE

    Bibiana Gonzales Santana; Dalton, John P.; Fabio Vasquez Camargo; Michael Parkinson; Momar Ndao

    2013-01-01

    BACKGROUND: Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, the...

  7. Direct Agglutination Test and Enzyme Linked Immunosorbent Assay with Urine Samples for the Diagnosis of Visceral Leishma-niasis

    Directory of Open Access Journals (Sweden)

    Sarkari B

    2007-07-01

    Full Text Available Background: Visceral leishmaniasis (VL or Kala azar is an infectious disease caused by various species of Leishmania parasites. The aim of this study was to detect and compare the presence of anti-Leishmania antibodies in the urine of vis-ceral leishmaniasis patients using ELISA and DAT methods."nMethods: A total of 30 urine samples were collected from VL patients referred to Shiraz (southeast of Iran hospitals. Moreover 31 urine samples were collected from healthy individuals and patients with other diseases such as malaria, brucellosis, hydatidosis and cutaneous leishmaniasis. Collected samples were examined to detect anti-Leishmania antibod-ies in urine, using ELISA and DAT."nResults: Anti-Leishmania antibody was detected in urine of 18 out of 30 (60% VL patients by DAT while ELISA detected anti-Leishmania antibodies in urine of 28 out of 30 (93.3% of VL cases. Sensitivity and specificity of urine-based DAT was 60% and 83.9%, respectively while sensitivity and specificity of urine-based ELISA were 93.3% and 93.5%, corre-spondingly. "nConclusion: Urine-based DAT and ELISA have a reasonable specificity and sensitivity in diagnosis of VL. Accordingly, urine-based ELISA might be a suitable alternative for serum based assays for diagnosis of VL.

  8. Immunocapture Enzyme-Linked Immunosorbent Assay for Assessment of In Vitro Potency of Recombinant Hepatitis B Vaccines▿

    OpenAIRE

    Shanmugham, Rajalakshmi; Thirumeni, Nagarajan; Rao, Varaprasada Sankarashetty; Pitta, Vidyasagar; Kasthuri, Saranyarevathy; Singanallur, Nagendrakumar Balasubramanian; Lingala, Rajendra; Mangamoori, Lakshmi Narsu; Villuppanoor, Srinivasan Alwar

    2010-01-01

    Quantification of hepatitis B surface antigen (HBsAg) or relative in vitro potency in the final vaccines is a prerequisite for hepatitis B vaccine batch release. The commercial kit for automated analysis (AxSYM) is expensive, and an alternative is required for the estimation of HBsAg in hepatitis B vaccines. Mouse monoclonal antibodies (MAbs) specific for HBsAg were developed and characterized. One of the monoclonal antibodies (HBs06) was used in development of an immunocapture ELISA (IC-ELIS...

  9. Trypanosomosis surveillance on Zanzibar island, using the trypanosomal antigen detection ELISA (enzyme-linked immunosorbent assay) technique

    International Nuclear Information System (INIS)

    The effectiveness of trypanosomosis control programs depends greatly on prior knowledge of basic data of the epidemiological situation of the disease, which in turns depends, among others, on the use of techniques that give a fairly quick and accurate diagnosis. An antigen-detection (Ag) ELISA was first introduced into Tanzania and validated at the Animal Disease Research Institute (ADRI) through the FAO/IAEA Research Contract (RC) No. 5030/NL. Incorporation of the Ag-ELISA technique into a FAO animal disease control project (1986-1993) on Unguja island, in 1992, revealed useful information of high trypanosomosis prevalence in an area previously declared free of the disease using just stained blood smears and buffy coat examinations. This triggered further efforts into more intensive surveys of the tsetse and trypanosomosis situation on Unguja island. The present study is a continuation of previous work in an effort to confirm the practical application of Ag-ELISA in trypanosomosis control operations. Results obtained from a known tsetse and trypanosomosis-free area, on Pemba island, showed a high specificity of the test for Trypanosoma congolense, T. vivax and T. brucei. A preliminary cut-off value of 10% (Percent Positivity = PP) was used. When the PP of 10 was used on sera of trypanosomosis-endemic areas (Mangapwani, Ndijani, Dunga and Kikungwi) on Unguja island, the results reflected the 'real' trypanosomis situation in the affected area. This was most strongly felt in the Mangapwani area, where tsetse and trypanosomosis were considered under control by 1994 (no tsetse flies were caught and no samples were encountered positive by the buffy coat technique). However, it should be stressed that the buffy coat technique and the Ag-ELISA complement each other and should be used in conjunction. (author). 8 refs, 1 fig., 5 tabs

  10. Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

    OpenAIRE

    Kim, Sung-Hee; Cha, Sang-Ho; Karyn, Bischoff; Park, Sung-Won; Son, Seong-Wan; KANG, HWAN-GOO

    2011-01-01

    Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody de...

  11. Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay.

    Science.gov (United States)

    Kim, Sung-Hee; Cha, Sang-Ho; Karyn, Bischoff; Park, Sung-Won; Son, Seong-Wan; Kang, Hwan-Goo

    2011-06-01

    Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R(2) > 0.99) and from 1 to 100 ng/ml (R(2) > 0.99), respectively. The intra- and inter-assay precision CVs were extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed. PMID:24278561

  12. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Bremer, Hanna D; Lattwein, Erik; Renneker, Stefanie; Lilliehöök, Inger; Rönnelid, Johan; Hansson-Hamlin, Helene

    2015-12-15

    Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lübeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT). The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and/or anti-Smith (Sm) antibodies. Reactivity against Sjögren's syndrome related antigens (SS)-A (including the Ro-60 and Ro-52 subcomponents), SS-B, histidyl tRNA synthetase (Jo-1), topoisomerase I antigen (Scl-70), polymyositis-scleroderma antigen (PM-Scl) and proliferating cell nuclear antigen (PCNA) was also noted in individual dogs. In conclusion, by using a commercial LIA and different ELISAs originally developed for detection of human ANA, we identified several specific ANA in serum samples from dogs sampled for IIF-ANA testing. Further, we found that the types of IIF-ANA pattern were associated with reactivity against some particular nuclear antigens. PMID:26547884

  13. ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR SCREENING OF MILK SAMPLES FOR SALMONELLA-TYPHIMURIUM IN DAIRY HERDS

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Wedderkopp, A.

    1995-01-01

    positive (herd specificity, 0.9 and herd sensitivity, 1.0). A sig nificant correlation (P <0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis. It was concluded that ELISA testing of individual milk sam ples can be used for surveillance of...

  14. Use of Polymerase Chain Reaction Enzyme Linked Oligonucleotide Sorbent Assay (Pcr-Elosa for Detection of Disease Agents

    Directory of Open Access Journals (Sweden)

    Simson Tarigan

    2016-03-01

    Full Text Available Diagnostic tool comprises one of the vital components in the control of infectious diseases. One of the most common techniques in the diagnosis of infectious disease currently available is the polymerase chain reaction (PCR because this technique is very sensitive, specific, and rapid. This technique requires an adjunct technique to indicate the formation of the right reaction product. Agarose gel electrophoresis has been the most common technique to visualise the PCR product or amplicon. Enzyme linked oligonucleotide sorbent assay (ELOSA is an alternative technique which is more sensitive and gives more important identity of the amplicon. This technique can be more than 100 times as sensitive as a gel agarose electrophoresis, and very specific since confirmation of the amplicon is carried out by DNA hibridisation. The capacity of the ELOSA can also be extended to the detection of disease-causal agent at subtype level, or detection of mutation at particular location in a gene. Since the equipment used for ELOSA is similar to that for ELISA (enzyme linked immunosorbent assay, a large number of samples can be accomplished rapidly. As in ELISA, a number of variation can be made in ELOSA depend on the requirement. Nucleotide can be immobilised on the microwell plate either by passive adsorbtion, by first conjugation of nucleotide with biotin then immobilisation on streptavidin-coated microwell plate, or immobilisaion by covalent bonding. The PCR and ELOSA can be performed at separate or in a single tube by first immobilising the PCR primers on the surface of microwell plates.

  15. 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.

  16. High-sensitivity electrochemical enzyme-linked assay on a microfluidic interdigitated microelectrode

    OpenAIRE

    Chen, I-Jane; White, Ian M.

    2011-01-01

    A novel enzyme-linked DNA hybridization assay on an interdigitated array (IDA) microelectrode integrated into a microfluidic channel is demonstrated with sub-nM detection limit. To improve the detection limit as compared to conventional electrochemical biosensors, a recyclable redox product, 4-aminophenol (PAP) is used with an IDA microelectrode. The IDA has a modest and easily fabricated inter-digit spacing of 10 μm, yet we were able to demonstrate 97% recycling efficiency of PAP due to the ...

  17. Detection of Pneumocystis carinii in respiratory specimens by PCR-solution hybridization enzyme-linked immunoassay.

    OpenAIRE

    Ortona, E; Margutti, P; Tamburrini, E; Mencarini, P; Visconti, E; Zolfo, M; Siracusano, A.

    1997-01-01

    By using a recently developed PCR-solution hybridization enzyme-linked assay (PCR-SHELA), we investigated Pneumocystis carinii in bronchoalveolar lavage fluid samples and induced sputa of patients with pneumocystosis. In detecting P. carinii, PCR-SHELA proved more sensitive than immunofluorescence staining or a single PCR and significantly more diagnostically specific than a nested PCR. Our data suggest that PCR-SHELA could be used to detect P. carinii organisms in respiratory samples, partic...

  18. In-house validation and quality control of commercial enzyme-linked immunosorbnet assays for screening of nitrofuran metabolites in food of animal origin

    Directory of Open Access Journals (Sweden)

    Dimitrieska-Stojkovic Elizabeta

    2012-01-01

    Full Text Available Application of nitrofuran antimicrobials at food production animals was prohibited by Commission Regulation 2003/181/EC because of their potential carcinogenic and mutagenic effects on humans. Main protein-bound metabolites of nitofurans are 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ, 1-aminohydantoin (AHD, semicarbazide (SEM and 3-amino-2-oxazolidinone (AOZ. Since then numerous costly liquid chromatography with tandem mass spectrometry (LC/MS/MS methods have been developed for screening and confirmation of nitrofuran metabolites in line with the EU requirements for performing official controls. As an inexpensive and less time consuming alternative, enzyme-immunoassay methods were developed for screening of the respective compounds. In this study validation and evaluation of four commercial enzyme-linked immunosorbent assay (ELISA has been performed. According to the requirements of Commission Decision 2002/657/EC, different performance characteristics (specificity, detection capability, precision for various matrices (liver, eggs, honey have been determined for each kit. The validation study has confirmed that the methods studied possess suitable characteristics: detectionlimits between 0.126 and 0.240 μg/kg, detection capabilities ≤1.0 μg/kg and the inter-day precision in the range from 16.20% to 22.11 %. The validation study was finalized by participation in FAPAS Proficiency testing scheme in 2011, and the obtained results have confirmed the capability of applied methods for unambiguous discrimination between negative and positive sample.

  19. An in-house enzyme-linked immunoabsorbant assay for human growth hormone.

    Science.gov (United States)

    Nazaimoon, W; Ng, M L; Bak, K

    1993-06-01

    A simple, non-isotopic in-house enzyme-linked immunoabsorbant assay (ELISA) for human growth hormone (GH) was developed. The assay involved using in-house polyclonal anti-GH adsorbed onto 96-well microtitre plates, commercially prepared mouse monoclonal anti-GH, and goat anti-mouse IgG horseradish peroxidase detection system. Results of recovery and parallelism studies ranged from 95%-106% and 98%-101% respectively, of the expected values. The detection limit of the assay was 0.008 mIU/well or the equivalent to 0.4 mIU/L of undiluted serum. Intra- and interassay coefficients of variations were 4.8%-7.9% and 6.5%-8.7% respectively. Serum GH levels measured in this assay correlated well with those measured in established in-house radioimmunoassays (r = 0.985, p NETRIA (r = 0.984, p < 0.001). PMID:8277795

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

    DEFF Research Database (Denmark)

    Damgaard, Dres; Palarasah, Yaseelan; Skjødt, Karsten; Catrina, Anca I; Hensen, Sanne M M; Pruijn, Ger J M; Nielsen, Claus H

    2014-01-01

    The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (m...... antibodies, mAbs DN2 and DN6. The assay had a lower detection limit of 200pg/mL in serum and plasma samples, and showed dilution linearity and recovery ranging from 95 to 106%. The mAbs and the ELISA showed isotype specificity for PAD2. Circulating PAD2 was found in 8/28 (29%) serum samples from healthy...

  1. Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies.

    Science.gov (United States)

    Koestel, Carole; Simonin, Céline; Belcher, Sandrine; Rösti, Johannes

    2016-08-01

    Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 μg/L and a LOQ of 56.4 μg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices. PMID:27356183

  2. Development of a two-site enzyme-linked immunosorbent assay for alpha-amylase from Aspergillus oryzae based on monoclonal antibodies.

    Science.gov (United States)

    Sander, I; Neuhaus-Schröder, C; Borowitzki, G; Baur, X; Raulf-Heimsoth, M

    1997-12-15

    A two-site monoclonal antibody ELISA was developed to quantify the allergen Asp o 2 (alpha-amylase from Aspergillus oryzae). Two mAbs recognizing distinct epitopes were selected, enriched by in vitro production in a modular minifermenter and affinity-purified. The first antibody was bound to microtiter plates which were then incubated with samples containing the allergen. Bound allergen was detected using a biotinylated second antibody and peroxidase-polymer-labelled streptavidin. The assay had a sensitivity of 0.6 ng/ml and did not react to high concentrations of wheat and rye flour or yeast proteins. The mAb ELISA will be useful in individual or epidemiological studies of baker's asthma to assess workplace allergen concentrations and the efficacy of allergen exposure prevention. It can be used as a standard assay for the quantification of alpha-amylase and the establishment and control of threshold limits in European bakeries. PMID:9502588

  3. Improved Enzyme-Linked Immunosorbent Assay To Reveal Mycoplasma agassizii Exposure: a Valuable Tool in the Management of Environmentally Sensitive Tortoise Populations▿

    OpenAIRE

    Wendland, Lori D.; Zacher, Laurie A.; Klein, Paul A.; Brown, Daniel R.; Demcovitz, Dina; Littell, Ramon; Brown, Mary B.

    2007-01-01

    The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory trac...

  4. Childhood Epidermolysis Bullosa Acquisita: Confirmation of Diagnosis by Skin Deficient in Type VII Collagen, Enzyme-linked Immunosorbent Assay, and Immunoblotting.

    Science.gov (United States)

    Goyal, Nupur; Rao, Raghavendra; Balachandran, C; Pai, Sathish; Bhogal, Balbir S; Schmidt, Enno; Zillikens, Detlef

    2016-01-01

    Epidermolysis bullosa acquisita (EBA) is an acquired subepidermal bullous disorder characterized by autoantibodies against Type VII collagen. It usually affects adults; childhood EBA is rare. We describe a 10-year-old girl presenting with recurrent tense blisters predominantly on legs, dorsa of hands and feet accompanied by oral erosions since the age of 5 years. Direct immunofluorescence (IF) microscopy showed linear deposition of IgG and C3 along the basement membrane zone (BMZ); indirect IF microscopy on salt-split skin revealed staining of IgG to the dermal side of the split. The patient's serum did not show BMZ staining in recessive dystrophic epidermolysis bullosa skin deficient for Type VII collagen, thus confirming autoantibody reactivity against Type VII collagen. Circulating antibodies against the immunodominant noncollagenous 1 domain of Type VII collagen were detected by ELISA and immunoblotting studies. The patient was treated with oral corticosteroids and dapsone with good improvement. PMID:27293257

  5. Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

    Energy Technology Data Exchange (ETDEWEB)

    Boucas, Rodrigo Ippolito [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Trindade, Edvaldo S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Departamento de Biologia Celular, Universidade Federal do Parana, Curitiba, Parana (Brazil); Tersariol, Ivarne L.S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP (Brazil); Dietrich, Carl P. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Nader, Helena B. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil)], E-mail: hbnader.bioq@epm.br

    2008-06-23

    Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

  6. A supersensitive in-house enzyme-linked immunosorbent assay (ELISA) for measurement of thyroid-stimulating hormone (TSH) and its clinical applications.

    Science.gov (United States)

    Goh, K H; Ng, M L; Thean, E T; Khalid, B A; Goh, M L

    1992-12-01

    A supersensitive ELISA was developed for measurement of thyroid-stimulating hormone (TSH) concentrations in serum using in-house rabbit polyclonal antisera and a commercial monoclonal antibody. The assay was optimised and validated by recovery, linearity and cross-reactivity experiments and further compared to other available assays and EQAS samples. Good precision was obtained with a working assay range of 0.2 to 100 mIU/L with NETRIA IRMA (r + 0.995; p < 0.001; n = 76). The normal reference range established was 0.4 to 4.0 mIU/L (n = 76). TSH levels in serum of thyrotoxic patients (n = 83) were significantly lower (0.07 to 0.20 mIU/L, p < 0.0001) and completely distinct from normal values thereby obviating the requirement of a TRH-stimulation test. Stability studies showed that coated wells can be stored at 4 degrees C for at least 2 months. This highly sensitive in-house hTSH ELISA which is cheap, stable and readily available is useful for diagnosis and management of patients with various thyroid disorders. PMID:1303476

  7. Comparison of an enzyme-linked immunosorbent assay (ELISA) to gas chromatography (GC) - measurement of polychlorinated biphenyls (PCBs) in selected US fish extracts

    Science.gov (United States)

    Zajicek, J.L.; Tillitt, D.E.; Schwartz, T.R.; Schmitt, C.J.; Harrison, R.O.

    2000-01-01

    The analysis of PCBs in fish tissues by immunoassay methods was evaluated using fish collected from a US monitoring program, the National Contaminant Biomonitoring Program of the US Department of Interior, Fish and Wildlife Service. Selected composite whole fish samples, which represented widely varying concentrations and sources of PCBs, were extracted and subjected to congener PCB analysis by gas chromatography (GC) and total PCB analysis using an ELISA (ePCBs) calibrated against technical Aroclor 1248. PCB congener patterns in these fishes were different from the patterns found in commercial Aroclors or their combinations as demonstrated by principal component analysis of normalized GC congener data. The sum of the PCB congeners measured by GC (total-PCBs) ranged from 37 to 4600 ng/g (wet weight). Concentrations of PCBs as determined by the ELISA method were positively correlated with total-PCBs and the ePCBs/total-PCBs ratios for individual samples ranged from 1 to 6. Ratios of ePCBs/total-PCBs for dilutions of Aroclors 1242, 1254, and 1260 and for matrix spikes range from 0.6 for 1242 to 2.5 for 1254 and 1260. These results suggest that higher chlorinated PCB congeners have higher affinity for the anti-PCB antibodies. Partial least squares with latent variable analysis of GC and ELISA data of selected Aroclors and fish samples also support the conclusion that ELISA derived PCB concentrations are dependent on the degree on chlorination.

  8. Quantifying 8-hydroxy-2′-deoxyguanosine with enzyme-linked immunosorbent assay (ELISA) as a measure of DNA damage in the eutardigrade Richtersius coronifer

    OpenAIRE

    Mortensen, Louise Hindborg; Nilsson, Line Lynge; Nyby, Camilla; Vilstrup, Annika

    2010-01-01

    Under desiccated conditions tardigrades can enter a state known as anhydrobiosis, which enables them to survive without metabolism until favourable conditions resume. Studies have suggested that desiccation can cause accumulation of DNA damage induced by reactive oxygen species (ROS). This is due to the metabolic arrest and hereby the cessation of DNA repair systems during anhydrobiosis. In order to quantify this accumulation of DNA damage, previous studies have used comet assay. This method ...

  9. [Enzyme-linked immunosorbent assay for detection of antibodies to porcine reproductive and respiratory syndrome virus, by using recombinant nucleocapsid protein N].

    Science.gov (United States)

    Bogdanova, V S; Tsibezov, V V; Grabovetskiĭ, V V; Eliseeva, O V; Grebennikova, T V; Verkhovskiĭ, O A; Zabereshchnyĭ, A D; Aliper, T I

    2007-01-01

    Recombinant nucleocapsid (rN) protein N of porcine reproductive and respiratory syndrome virus (PRRSV) was prepared, by using the E. coli expressiom system. Insertion of a polyhistidine marker into the structure of the protein allowed the latter to be purified by metal-chelate affinity chromatography. The purity of protein was confirmed by PAAG electrophoresis and its immunospecificity was verified by immunoblotting using rN-specific monoclonal antibodies. The protein was used as an antigen to develop indirect ELISA of PRRSV antibodies. ELISA was shown to be highly sensitive and specific. PMID:17500240

  10. Comparison of IgM Capture Enzyme- Linked Immunosorbent Assay (ELISA) using Inhouse method and commercially available MRL kit for serological confirmation of dengue infection

    International Nuclear Information System (INIS)

    Laboratory diagnosis of dengue infection is important for the management of the patients. In this study igM capture ELISA using an inhouse method and commercially available kit (MRL diagnostics,USA) was compared to detect diagnostic capability of Inhouse IgM ELISA for provision of diagnostic facilities to the public at an affordable cost. Eighty acute and convalescent serum samples were collected from serologically confirmed dengue patients. Serological confirmation of patients were performed by Haemagglutination Inhibition (HI) assay, gold standard assay for dengue on paired serum samples. All collected acute and convalescent sera were tested by IgM ELISA using the inhouse method and MRL kit. Antigen and conjugate for the inhouse IgM method were prepared in the laboratory. A cocktail of four dengue antigens containing 25 Antigen ELISA units of each type was prepared and used as the assay antigen. Conjugate was prepared using a serum sample with high dengue Anti flavi IgG antibody titre conjugated with Horseradish peroxidase. A prospective study of both IgM ELISA assays were performed using 113 acute sera collected from dengue suspected cases. Overall results showed that 46% and 52% acute sera collected from dengue confirmed patients were positive by inhouse ELISA assay and MRL kits respectively. In the prospective study done using acute sera collected from dengue suspected patients showed that 44% and 52% were positive by inhouse ELISA assay and MRL kits. There was no significant difference in positivity between these two assays. (P=0.18). Inhouse IgM ELISA can be used for provision of laboratory diagnosis of dengue virus infection more than 5 days. The assay is 10 times less costly than using MRL kits as assay antigen and conjugate can be prepared easily in the laboratory

  11. Childhood epidermolysis bullosa acquisita: Confirmation of diagnosis by skin deficient in Type VII Collagen, enzyme-linked immunosorbent assay, and immunoblotting

    Directory of Open Access Journals (Sweden)

    Nupur Goyal

    2016-01-01

    Full Text Available Epidermolysis bullosa acquisita (EBA is an acquired subepidermal bullous disorder characterized by autoantibodies against Type VII collagen. It usually affects adults; childhood EBA is rare. We describe a 10-year-old girl presenting with recurrent tense blisters predominantly on legs, dorsa of hands and feet accompanied by oral erosions since the age of 5 years. Direct immunofluorescence (IF microscopy showed linear deposition of IgG and C3 along the basement membrane zone (BMZ; indirect IF microscopy on salt-split skin revealed staining of IgG to the dermal side of the split. The patient's serum did not show BMZ staining in recessive dystrophic epidermolysis bullosa skin deficient for Type VII collagen, thus confirming autoantibody reactivity against Type VII collagen. Circulating antibodies against the immunodominant noncollagenous 1 domain of Type VII collagen were detected by ELISA and immunoblotting studies. The patient was treated with oral corticosteroids and dapsone with good improvement.

  12. Childhood Epidermolysis Bullosa Acquisita: Confirmation of Diagnosis by Skin Deficient in Type VII Collagen, Enzyme-linked Immunosorbent Assay, and Immunoblotting

    Science.gov (United States)

    Goyal, Nupur; Rao, Raghavendra; Balachandran, C; Pai, Sathish; Bhogal, Balbir S; Schmidt, Enno; Zillikens, Detlef

    2016-01-01

    Epidermolysis bullosa acquisita (EBA) is an acquired subepidermal bullous disorder characterized by autoantibodies against Type VII collagen. It usually affects adults; childhood EBA is rare. We describe a 10-year-old girl presenting with recurrent tense blisters predominantly on legs, dorsa of hands and feet accompanied by oral erosions since the age of 5 years. Direct immunofluorescence (IF) microscopy showed linear deposition of IgG and C3 along the basement membrane zone (BMZ); indirect IF microscopy on salt-split skin revealed staining of IgG to the dermal side of the split. The patient's serum did not show BMZ staining in recessive dystrophic epidermolysis bullosa skin deficient for Type VII collagen, thus confirming autoantibody reactivity against Type VII collagen. Circulating antibodies against the immunodominant noncollagenous 1 domain of Type VII collagen were detected by ELISA and immunoblotting studies. The patient was treated with oral corticosteroids and dapsone with good improvement. PMID:27293257

  13. Childhood Epidermolysis Bullosa Acquisita: Confirmation of Diagnosis by Skin Deficient in Type VII Collagen, Enzyme-linked Immunosorbent Assay, and Immunoblotting

    OpenAIRE

    Nupur Goyal; Raghavendra Rao; C. Balachandran; Sathish Pai; Bhogal, Balbir S.; Enno Schmidt; Detlef Zillikens

    2016-01-01

    Epidermolysis bullosa acquisita (EBA) is an acquired subepidermal bullous disorder characterized by autoantibodies against Type VII collagen. It usually affects adults; childhood EBA is rare. We describe a 10-year-old girl presenting with recurrent tense blisters predominantly on legs, dorsa of hands and feet accompanied by oral erosions since the age of 5 years. Direct immunofluorescence (IF) microscopy showed linear deposition of IgG and C3 along the basement membrane zone (BMZ); indirect I...

  14. Estimation of immune complexes by a microplate-adapted C1q-Protein A enzyme-linked-immunosorbent-assay (C1q-PA-ELISA)

    DEFF Research Database (Denmark)

    Bjerrum, L; Glikmann, G; Jensenius, J C;

    1983-01-01

    . Bound IC was measured by use of alkaline phosphatase-labelled Protein A followed by the substrate para-nitro-phenyl-phosphate. A dose response was found for both delta IgG and BSA anti-BSA complexes, while variations in the concentration of monomer IgG did not affect the optical density. Elevated levels...

  15. Added value of use of a purified protein derivative-based enzyme-linked immunosorbent spot assay for patients with Mycobacterium bovis BCG infection after intravesical BCG instillations.

    Science.gov (United States)

    Heemstra, Karen A; Bossink, Ailko W J; Spermon, Roan; Bouwman, John J M; van der Kieft, Robert; Thijsen, Steven F T

    2012-06-01

    In this case series, we describe four cases in which the use of gamma interferon release assays with purified protein derivative (PPD) as a stimulating antigen was able to demonstrate PPD-specific immune activation. This may help to improve the adequate diagnosis of (systemic) Mycobacterium bovis BCG infections after intravesical BCG instillations for bladder carcinoma. PMID:22461529

  16. Added Value of Use of a Purified Protein Derivative-Based Enzyme-Linked Immunosorbent Spot Assay for Patients with Mycobacterium bovis BCG Infection after Intravesical BCG Instillations

    OpenAIRE

    Heemstra, Karen A.; Bossink, Ailko W. J.; Spermon, Roan; Bouwman, John J. M.; van der Kieft, Robert; Thijsen, Steven F. T.

    2012-01-01

    In this case series, we describe four cases in which the use of gamma interferon release assays with purified protein derivative (PPD) as a stimulating antigen was able to demonstrate PPD-specific immune activation. This may help to improve the adequate diagnosis of (systemic) Mycobacterium bovis BCG infections after intravesical BCG instillations for bladder carcinoma.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    -verified Legionnaires' disease and sera from 12 patients with pneumonia and a diagnostic rise in titre by a microagglutination test (MA) was studied. Our results indicated that the SON IgA assay was the most sensitive test in both groups of patients. The LPS IgG and IgM assays, however, were the most specific tests......Currently recommended methods in Legionnaires' disease serology are based upon crude whole-cell antigenic preparations. To investigate whether purified antigens would perform better in a given diagnostic test for antibodies against Legionella pneumophila, we compared the performance of three......, closely followed by the corresponding SON tests. By combining two individual assays, a maximum nosographic sensitivity of 85% could be obtained. Whereas no benefit of using purified outer membrane protein or flagella instead of a sonic extract in the indirect ELISAs was found, the LPS antigen provided...

  18. Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

    Directory of Open Access Journals (Sweden)

    Eliza Yoshie do Rosário

    2005-04-01

    Full Text Available The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2% were positive in parasitological exams (skin and/or spleen. These animals were compared to healthy animals (n = 25, negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%. Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%. Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00. High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97. Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

  19. Polyvinylpolypyrrolidone reduces cross-reactions between antibodies and phenolic compounds in an enzyme-linked immunosorbent assay for the detection of ochratoxin A.

    Science.gov (United States)

    Robinson, Andrew L; Lee, Hyun Jung; Ryu, Dojin

    2017-01-01

    Ochratoxin A (OTA) is a fungal metabolite and putative carcinogen which can contaminate a variety of foods such as cereals, wine, and nuts. Commercial ELISA kits are known to give false-positive results for OTA concentrations when phenolic compounds are present. Pistachios represent a food matrix rich in phenolic compounds potentially contaminated with OTA, and were used to model OTA cross-reactivity. Polyvinylpolypyrrolidone (PVPP) was incorporated during extraction of OTA using a commercial ELISA protocol. HPLC methods were used to confirm that PVPP does not interact with OTA and levels of gallic acid and catechin remaining in pistachio extracts decreased with increasing PVPP application. Cross-reactivity of extracts also decreased with increasing PVPP application, and color loss was used as an indicator of anthocyanin removal. Incorporating PVPP into ELISA protocols allows for the continued use of rapid immunological methods in food matrices containing phenolic compounds. PMID:27507446

  20. Evaluation of the INOVA Diagnostics Enzyme-Linked Immunosorbent Assay Kits for Measuring Serum Immunoglobulin G (IgG) and IgA to Deamidated Gliadin Peptides

    OpenAIRE

    Prince, Harry E.

    2006-01-01

    New assays for antibodies to deamidated gliadin peptides (DGP) expressing celiac disease-specific epitopes were evaluated using 154 sera previously tested for endomysial immunoglobulin A (IgA) (EMA), transglutaminase IgA (TGA), and conventional gliadin antibodies. DGP antibody results showed 97% concordance with EMA and TGA results. Of 56 sera negative for EMA and TGA but positive for conventional gliadin antibodies, 54 (96%) were negative for DGP antibodies.

  1. Heterophile Antibodies to Bovine and Caprine Proteins Causing False-Positive Human Immunodeficiency Virus Type 1 and Other Enzyme-Linked Immunosorbent Assay Results

    OpenAIRE

    Willman, Joseph H.; Martins, Thomas B; Jaskowski, Troy D; Hill, Harry R.; Litwin, Christine M.

    1999-01-01

    Heterophile antibodies are a well-recognized cause of erroneous results in immunoassays. We describe here a 22-month-old child with heterophile antibodies reactive with bovine serum albumin and caprine proteins causing false-positive results to human immunodeficiency virus type 1 and other infectious serology testing.

  2. Avian influenza in wild waterfowl and shorebirds in the Donana National Park: Serological survey using the enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Astorga, R J; Leon, L; Cubero, M J; Arenas, A; Maldonado, A; Tarradas, M C; Perea, A

    1994-06-01

    The indirect ELISA was used to detect antibodies to influenzavirus A in the sera of wildfowl from the Donana National Park. Of the 712 birds examined, 44 (6.2%) were seropositive. Positive birds belonged to 10 of the 13 species studied. Infection rates varied widely: spoonbill (Platalea leucorodia, 32.2%), mallard (Anas platyrhynchos, 9.9%), gadwall (Anas strepera, 8.6%), red-crested pochard (Netta rufina, 8.1%), pochard (Aythya ferina, 6.4%), shoveler (Anas clypeata, 5%), great crested grebe (Podiceps cristatus, 4.3%), avocet (Recurvirostra avosetta, 3.1%), grey heron (Ardea cinerea, 3.1%) and coot (Fulica atra, 0.8%). Although infection rates were not high, the wide range of avian species susceptible to influenzavirus A suggests circulation of the virus amongst wildfowl at Donana. PMID:18671098

  3. Analysis of pirlimycin residues in beef muscle, milk and honey by a biotin-streptavidin-amplified enzyme-linked immunosorbent assay

    Science.gov (United States)

    Food contamination caused by veterinary drug residues is a world-wide public health concern and requires continuous monitoring. In this paper, we describe a biotin–streptavidin-amplified ELISA (BA-ELISA) for detecting pirlimycin residues in beef, milk, and honey. The IC50 value of the BA-ELISA was...

  4. Efficacy of an enzyme-linked immunosorbent assay as a diagnostic tool for schistosomiasis mansoni in individuals with low worm burden

    Directory of Open Access Journals (Sweden)

    Edward José de Oliveira

    2005-07-01

    Full Text Available IgM-ELISA is an immunoenzymatic method useful for detection of IgM antibodies against a fraction of Schistosoma mansoni adult worm antigen (AWA that is soluble in trichloroacetic acid (AWA-TCA. This method was applied to three groups of individuals with different clinical and epidemiological characteristics, and the results compared with those obtained by other diagnostic methods: immunofluorescence test for detection of IgM antibodies (IgM-IFT or IgG antibodies (IgG-IFT, ELISA for detection of IgG antibodies (IgG-ELISA, and two parasitological methods, Kato-Katz and miracidium hatching. The IgM-ELISA presented a sensitivity of 98%, when the parasitologic fecal examination was defined as reference diagnostic method, and a specificity of 98 and 97.3%, respectively for the group of clinically healthy individuals and other helminth carriers. A comparative analysis between the results of IgM-ELISA and those obtained by other serologic tests showed a good degree of agreement, with Kappa indices ranging from 0.95 to 0.98. The diagnostic efficacy of 97.8%, as determined with schistosomiasis patients with low parasitic burden, suggests the excellent performance of the IgM-ELISA and its usefulness for the diagnosis of schistosomiasis when applied in low endemic areas.

  5. Investigation of the induction of oxidative DNA damage by means of an enzyme-linked immunosorbent assay (ELISA) for thymine glycol containing DNA

    International Nuclear Information System (INIS)

    The report explains an ELISA test system for the detection and quantification of toxic effects on genes, induced by mutagenic or carcinogenic chemicals introduced by way of reactive oxygen species. Sensitivity and reproducibility are defined, and the system's applicability to the detection of oxidative DNA damage as a result of the metabolism of chemicals in cellular systems is discussed. (TRV)

  6. Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

    OpenAIRE

    Cha, Sang-Ho; Kim, Sung-Hee; Bischoff, Karyn; Kim, Hyun-Jeong; Son, Seong-Wan; Kang, Hwan-Goo

    2012-01-01

    A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were fr...

  7. Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax

    Directory of Open Access Journals (Sweden)

    N Ghosh

    2015-01-01

    Full Text Available Background & objectives: Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA in human cutaneous anthrax cases. Methods: Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. Results: The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] =0.9982; slope=0.9186; intercept = 0.1108. Interpretation & conclusions: The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

  8. Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN). Detection by enzyme-linked immunosorbent assay and purification from normal human urine

    DEFF Research Database (Denmark)

    Reimert, C M; Minuva, U; Kharazmi, A; Bendtzen, K

    1991-01-01

    Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) is one of the cationic proteins found in the granules of the human eosinophilic granulocytes. EPX was purified from extracts of granules isolated from blood buffy coat cells of healthy donors. Polyclonal anti-EPX antibodies were subsequ......, but they differed slightly on SDS-PAGE probably due to differences in glycosylation. Our results support the findings that EPX/EDN is identical to a nonsecretory ribonuclease isolated from urine....... were 6.5% and 8.2%, respectively, and the mean recoveries of 25 and 50 pg of purified EPX added to diluted serum samples were 106 +/- 16% (mean +/- SD; n = 12) and 112 +/- 14%, respectively. Using this assay we found high amounts of EPX in normal human urine (U-EPX). U-EPX was purified by a two step...

  9. Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

    DEFF Research Database (Denmark)

    Nielsen, Ragnhild; van den Bosch, Johannes F.; Plambeck, Tamara; Sørensen, Vibeke; Nielsen, Jens Peter

    The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apr I, Apr II and Apr III. The toxins are important virulence factors. In the present study, ELISAs with purified Apr I, Apr II and Apr III, respectively, as...

  10. Diagnosis of Lymphoid Leukosis (LL) by ELISA (Enzyme-Linked Immunosorbent Assay) and Marek's Disease (MD) by IF (Immunofluorescence) Test in Chickens

    OpenAIRE

    ÖZBİLGİN, Selda; ŞEN, Ayşin; ÜLGEN, Mihriban; ÇARLI, K. Tayfun

    2001-01-01

    In this study, our aim was to diagnose lymphoid leukosis and Marek's disease, which are neoplastic diseases of chickens, by ELISA and Indirect Immunofluorescence Test (IIF), respectively. Samples (eggs and tissues) were selectively taken from the flocks suspected of having lymphoid leukosis and Marek's disease. The tissues were macroscopically and microscopically examined. Tissues belonging to some flocks showed typical lesions for lymphoid leukosis and Marek's disease. Seve...

  11. Competitive inhibition enzyme-linked immunosorbent assay for antibody in sheep and other ruminants to a conserved epitope of malignant catarrhal fever virus.

    OpenAIRE

    Li, H.; Shen, D T; Knowles, D P; Gorham, J. R.; Crawford, T B

    1994-01-01

    Malignant catarrhal fever (MCF) is a severe, usually fatal, acute systemic disease syndrome of certain domestic and wild ruminants caused by members of the family Gammaherpesvirinae. Two distinct but closely related viruses cause clinically indistinguishable syndromes: one that is indigenous to the widebeest and the other that apparently is indigenous to domestic sheep. Neither the pathogenesis nor the epidemiology of sheep-associated MCF (SA-MCF) is understood, primarily because of a lack of...

  12. Comparison of Surface Plasmon Resonance, Resonant Waveguide Grating Biosensing and Enzyme Linked Immunosorbent Assay (ELISA in the Evaluation of a Dengue Virus Immunoassay

    Directory of Open Access Journals (Sweden)

    Joe Buechler

    2013-07-01

    Full Text Available Two label-free biosensor platforms, Resonance Waveguide Grating (RWG and Surface Plasmon Resonance (SPR, were used to rank a large panel of anti-dengue virus NS1 antibodies. Dengue non-structural 1 (NS1 protein is an established serological marker for the early detection of dengue infection. A variety of commercial dengue NS1 antigen capture immunoassays are available in both ELISA and lateral flow format. However, there is a significant scope to improve both the sensitivity and the specificity of those tests. The interactions of antibody (Ab-antigen (Ag were profiled, with weak interactions (KD = 1–0.1 μM able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR. There were significant differences in the absolute affinities determined by the two technologies, and there was a poor correlation between antibodies best ranked by RWG and the lower limit of detection (LLOD found by ELISA. Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats.

  13. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.; Marks, James D.; Varnum, Susan M.

    2012-11-15

    Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.

  14. "Comparison of Adult Somatic and Cysteine Proteinas Antigens of Fasciola gigantica in Enzyme Linked Immunosorbent Assay for Serodiagnosis of Human Fasciolosis"

    Directory of Open Access Journals (Sweden)

    MB Rokni

    2002-08-01

    Full Text Available Fasciolosis caused by Fasciola hepatica and F.gigantica is one of the major public health problems in the world and in Iran. Considering that stool examination for Fasciola eggs is not a sensitive method and only 25% of infected patients pass the eggs in the faeces , and immunodiagnosis methods are more applicable for this purpose, the present study was conducted to compare the somatic (S and cysteine proteinase (CP antigens of F.gigantica in IgG-ELISA to diagnose human fasciolosis. This has been the first report on this case so far in Iran. Serum samples obtained from 178 individuals collected during the fasciolosis outbreak in 1999 in the Gilan province, northern Iran, that were coprologically positive for fasciolosis, were analyzed by IgG-ELISA for total antibody responses against (S and CP antigens from Fasciola gigantica. The cut-off points for (S and CP were 0.38 and 0.33, respectively. All cases that showed clinical manifestations of fasciolosis, were also seropositive using both (S and CP antigens whereas all 25 non-infected controls were seronegative. Therefore, the sensitivity of the test was 100% for both antigens. On the other hand the specificity of (S and CP antigens were calculated as 96.4% and 98.1%, respectively. The positive and negative predictive values of the test regarding (S antigen were 97.8% and 100%, whereas these values as for CP antigen were 98.9% and 100% correspondingly. Two individuals with hydatidosis and two with toxocariasis had antibodies against (S antigen whereas concerning CP antigen, one individual with hydatidosis and another with toxocariasis showed cross reactivity against it. We have demonstrated that altogether CP antigen provide a more conclusive diagnosis as possessing lower cut-off and enabling better to discriminate between seronegative and seropositive subpopulations.This study may be useful to implement a reliable test to diagnose human fasciolosis and for seroepidmiological objectives.

  15. Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

    International Nuclear Information System (INIS)

    Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture

  16. Performance and diagnostic usefulness of commercially available enzyme linked immunosorbent assay and rapid kits for detection of HIV, HBV and HCV in India

    Directory of Open Access Journals (Sweden)

    Maity Susmita

    2012-11-01

    Full Text Available Abstract Background HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. Results A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.; ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd. gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd., Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd. did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100% except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.. The kit efficiency didn’t vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p  Conclusions ELISA is a good screening assay for markers of HIV, HBV and HCV infections. Rapid tests are useful for further detection of false positive samples. ELISA seems the appropriate assay in blood bank. For availability of quality commercial diagnostic assays, evaluation of kit may be helpful.

  17. Performance of a Redesigned HIV Selectest Enzyme-Linked Immunosorbent Assay Optimized To Minimize Vaccine-Induced Seropositivity in HIV Vaccine Trial Participants

    OpenAIRE

    Penezina, Oksana; Krueger, Neil X; Rodriguez-Chavez, Isaac R.; Busch, Michael P; Hural, John; Kim, Jerome H.; O'Connell, Robert J.; Hunter, Eric; Aboud, Said; Higgins, Keith; Kovalenko, Victor; Clapham, David; Crane, David; Levin, Andrew E

    2014-01-01

    Vaccine-induced seropositivity (VISP) or seroreactivity (VISR), defined as the reaction of antibodies elicited by HIV vaccines with antigens used in HIV diagnostic immunoassays, can result in reactive assay results for vaccinated but uninfected individuals, with subsequent misclassification of their infection status. The eventual licensure of a vaccine will magnify this issue and calls for the development of mitigating solutions in advance. An immunoassay that discriminates between antibodies...

  18. Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

    Science.gov (United States)

    Guan, Weihua; Chen, Liben; Rane, Tushar D.; Wang, Tza-Huei

    2015-09-01

    We present a continuous-flow droplet-based digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for sensitive detection and absolute quantification of RNA molecules. Droplet digital ELOHA incorporates direct hybridization and single enzyme reaction via the formation of single probe-RNA-probe (enzyme) complex on magnetic beads. It enables RNA detection without reverse transcription and PCR amplification processes. The magnetic beads are subsequently encapsulated into a large number of picoliter-sized droplets with enzyme substrates in a continuous-flow device. This device is capable of generating droplets at high-throughput. It also integrates in-line enzymatic incubation and detection of fluorescent products. Our droplet digital ELOHA is able to accurately quantify (differentiate 40% difference) as few as ~600 RNA molecules in a 1 mL sample (equivalent to 1 aM or lower) without molecular replication. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria gonorrhoeae 16S rRNA to show its potential value in real complex samples.

  19. Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

    Directory of Open Access Journals (Sweden)

    William H Roldán

    2009-05-01

    Full Text Available To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis. Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%, whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

  20. Enzyme-catalyzed reaction of voltammetric enzyme-linked immunoassay system based on OAP as substrate

    Institute of Scientific and Technical Information of China (English)

    张书圣; 陈洪渊; 焦奎

    1999-01-01

    The o-aminophenol (OAP)-H2O2-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay new system has extremely high sensitivity. HRP can be measured with a detection limit of 6.0×10-(10) g/L and a linear range of 1.0×10-9—4.0×10-6 g/L. The pure product of H2O2 oxidizing OAP catalyzed by HRP was prepared with chemical method. The enzyme-catalyzed reaction has been investigated with electroanalytical chemistry, UV/Vis spectrum, IR spectrum, 13C NMR, 1H NMR, mass spectrum, elemental analysis, etc. Under the selected enzyme-catalyzed reaction conditions, the oxidation product of OAP with H2O2 catalyzed by HRP is 2-aminophe-noxazine-3-one. The processes of the enzyme-catalyzed reaction and the electroreduction of the product of the enzymecatalyzed reaction have been described.

  1. Real-time monitoring of extracellular adenosine using enzyme-linked microelectrode arrays.

    Science.gov (United States)

    Hinzman, Jason M; Gibson, Justin L; Tackla, Ryan D; Costello, Mark S; Burmeister, Jason J; Quintero, Jorge E; Gerhardt, Greg A; Hartings, Jed A

    2015-12-15

    Throughout the central nervous system extracellular adenosine serves important neuroprotective and neuromodulatory functions. However, current understanding of the in vivo regulation and effects of adenosine is limited by the spatial and temporal resolution of available measurement techniques. Here, we describe an enzyme-linked microelectrode array (MEA) with high spatial (7500 µm(2)) and temporal (4 Hz) resolution that can selectively measure extracellular adenosine through the use of self-referenced coating scheme that accounts for interfering substances and the enzymatic breakdown products of adenosine. In vitro, the MEAs selectively measured adenosine in a linear fashion (r(2)=0.98±0.01, concentration range=0-15 µM, limit of detection =0.96±0.5 µM). In vivo the limit of detection was 0.04±0.02 µM, which permitted real-time monitoring of the basal extracellular concentration in rat cerebral cortex (4.3±1.5 µM). Local cortical injection of adenosine through a micropipette produced dose-dependent transient increases in the measured extracellular concentration (200 nL: 6.8±1.8 µM; 400 nL: 19.4±5.3 µM) [P<0.001]. Lastly, local injection of dipyridamole, which inhibits transport of adenosine through equilibrative nucleoside transporter, raised the measured extracellular concentration of adenosine by 120% (5.6→12.3 µM) [P<0.001]. These studies demonstrate that MEAs can selectively measure adenosine on temporal and spatial scales relevant to adenosine signaling and regulation in normal and pathologic states. PMID:26183072

  2. A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide

    DEFF Research Database (Denmark)

    Barington, T; Sparholt, S; Juul, L;

    1992-01-01

    A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary...

  3. Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

    Directory of Open Access Journals (Sweden)

    Jian Wang

    Full Text Available A novel enzyme-linked receptor assay (ELRA based on β2-adrenergic receptor (β2-AR has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists in urine. Human embryonic kidney cells (HEK293 were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.

  4. Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

    Science.gov (United States)

    Wang, Jian; She, Yongxin; Wang, Miao; Jin, Maojun; Li, Yongfei; Wang, Jing; Liu, Yuan

    2015-01-01

    A novel enzyme-linked receptor assay (ELRA) based on β2-adrenergic receptor (β2-AR) has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists) in urine. Human embryonic kidney cells (HEK293) were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine. PMID:26422475

  5. Accuracy of bronchoalveolar lavage enzyme-linked immunospot assay to diagnose smear-negative tuberculosis: a meta-analysis

    OpenAIRE

    Li, Zhenzhen; Qin, Wenzhe; Li, Lei; WU, QIN; Chen, Xuerong

    2015-01-01

    Purpose: While the bronchoalveolar lavage enzyme-linked immunospot assay (BAL-ELISPOT) shows promise for diagnosing smear-negative tuberculosis, its accuracy remains controversial. We meta-analyzed the available evidence to obtain a clearer understanding of the diagnostic accuracy. Methods: Studies of the diagnostic performance of ELI-SPOT on smear-negative tuberculosis were identified through systematic searches of the PubMed and EMBASE databases. Pooled data on sensitivity, specificity and ...

  6. Fluobodies against Bioactive Natural Products and their Application in Fluorescence-Linked Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    Seiichi Sakamoto

    2012-09-01

    Full Text Available An enzyme-linked immunosorbent assay (ELISA using monoclonal antibody (MAb, Fab antibody, and single-chain variable fragment (scFv antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA. In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article.

  7. Naked-eye detection as a universal approach to lower the limit of detection of enzyme-linked immunoassays.

    Science.gov (United States)

    O'Connor, Erin F; Paterson, Sureyya; de la Rica, Roberto

    2016-05-01

    Colorimetric biosensors for the detection of analytes with the naked eye are required in environmental monitoring, point-of-care diagnostics, and analyses in resources constrained settings, where detection instruments may not be available. However, instrument-based detection methods are usually more adequate for detecting small variations in the signal compared to naked-eye detection schemes, and consequently the limit of detection of the latter is usually higher than the former. Here, we demonstrate that the limit of detection of colorimetric enzyme-linked immunoassays can be decreased several orders of magnitude when using naked-eye detection instead of a spectrophotometer for detecting the signal. The key step to lower the limit of detection is adding a small volume of chromogenic substrate during the signal generation step. This generates highly colored solutions that can be easily visualized with the naked eye and recorded with the camera of a mobile phone. The proposed method does not require expensive equipment or complex protocols to enhance the signal, and therefore it is a universal approach to lower the limit of detection of colorimetric enzyme-linked immunoassays. PMID:26970749

  8. Mitigation of microtiter plate positioning effects using a block randomization scheme.

    Science.gov (United States)

    Roselle, Christopher; Verch, Thorsten; Shank-Retzlaff, Mary

    2016-06-01

    Microtiter plate-based assays are a common tool in biochemical and analytical labs. Despite widespread use, results generated in microtiter plate-based assays are often impacted by positional bias, in which variability in raw signal measurements are not uniform in all regions of the plate. Since small positional effects can disproportionately affect assay results and the reliability of the data, an effective mitigation strategy is critical. Commonly used mitigation strategies include avoiding the use of outer regions of the plate, replicating treatments within and between plates, and randomizing placement of treatments within and between plates. These strategies often introduce complexity while only partially mitigating positional effects and significantly reducing assay throughput. To reduce positional bias more effectively, we developed a novel block-randomized plate layout. Unlike a completely randomized layout, the block randomization scheme coordinates placement of specific curve regions into pre-defined blocks on the plate based on key experimental findings and assumptions about the distribution of assay bias and variability. Using the block-randomized plate layout, we demonstrated a mean bias reduction of relative potency estimates from 6.3 to 1.1 % in a sandwich enzyme-linked immunosorbent assay (ELISA) used for vaccine release. In addition, imprecision in relative potency estimates decreased from 10.2 to 4.5 % CV. Using simulations, we also demonstrated the impact of assay bias on measurement confidence and its relation to replication strategies. We outlined the underlying concepts of the block randomization scheme to potentially apply to other microtiter-based assays. PMID:27116421

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

    Directory of Open Access Journals (Sweden)

    Haifa Al-Dubai

    2010-10-01

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

  10. Accuracy of the Bronchoalveolar Lavage Enzyme-Linked Immunospot Assay for the Diagnosis of Pulmonary Tuberculosis: A Meta-analysis.

    Science.gov (United States)

    Pang, Caishuang; Wu, Yanqiu; Wan, Chun; Shen, Konglong; Hu, Yuzhu; Yang, Ting; Shen, Yongchun; Wen, Fuqiang

    2016-03-01

    Assessing of local immune response may improve the accuracy of pulmonary tuberculosis (PTB) diagnosis. Many studies have investigated diagnosing PTB based on enzyme-linked immunospot (ELISPOT) assay of bronchoalveolar lavage (BAL) fluid, but the results have been inconclusive. We meta-analyzed the available evidences on overall diagnostic performance of ELISPOT assay of BAL fluid for diagnosing PTB.A systematic literature search was performed using PubMed, Embase, Wangfang, Weipu, and CNKI. Data were pooled on sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). Overall test performance was summarized using summary receiver operating characteristic curves and the area under the curve (AUC). Deeks test was used to test for potential publication bias.Seven publications with 814 subjects met our inclusion criteria and were included in this meta-analysis. The following pooled estimates for diagnostic parameters were obtained: sensitivity, 0.90 (95% CI: 0.85-0.94); specificity, 0.80 (95% CI: 0.77-0.84); PLR, 5.08 (95% CI: 2.70-9.57); NLR, 0.13 (95% CI: 0.06-0.28); DOR, 49.12 (95% CI: 12.97-186.00); and AUC, 0.96. No publication bias was identified.The available evidence suggests that ELISPOT assay of BAL fluid is a useful rapid diagnostic test for PTB. The results of this assay should be interpreted in parallel with clinical findings and the results of conventional tests. PMID:27015211

  11. The E. coli immunosorbent as used in serodiagnosis of Legionella infections studied by crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Friis-Møller, A; Rechnitzer, C;

    1988-01-01

    In this study we investigated an immunosorbent, E. coli blocking fluid (BF), proposed for use in the Legionella Indirect Fluorescent Antibody Test (IFA). With crossed immunoelectrophoresis (CIE) of clinically relevant Legionella species, only one heat-stable antigen (no. 1) cross-reacted with the...

  12. Linear quantification of a streptavidin-alkaline phosphatase probe for enzyme-linked immuno mass spectrometric assay.

    Science.gov (United States)

    Florentinus-Mefailoski, Angelique; Marshall, John G

    2016-06-15

    The alkaline phosphatase-streptavidin (AP-SA) probe released adenosine (∼267.2 Da) from the substrate adenosine monophosphate (AMP), where a signal may be detected from as little as 0.5 μl of a 0.1-pg/ml dilution of the probe (2.6 × 10(-22) mol). The signal from the AP-SA probe was linear from 1 to 50 pg/ml by monitoring adenosine release at 268 m/z (M + H) with liquid chromatography, electrospray ionization, and quadrupole mass spectrometry (LC-ESI-MS). The safe limit of detection and quantification of the AP-SA probe was approximately 0.5 pg/well or 5 pg/ml. Enzyme-linked immuno mass spectrometric assay (ELIMSA) using the AP-SA probe provided a linear signal response for prostate-specific antigen (PSA) against external standards from 1 to 500 pg/ml. The ELIMSA showed a safe limit of detection and quantification at 5 pg PSA/well or 50 pg/ml (false positive detection rate P ≤ 0.01). Female samples of 100 μl plasma/well were read against standards and blanks made in normal female plasma, and the lowest sample quantified was approximately 9.8 pg/well or 98 pg/ml. Here ELIMSA was applied to measure PSA in plasma from female, normal male, prostatectomy patient, and cancer patient samples that showed significant differences by analysis of variance (ANOVA). PMID:26944413

  13. Enzyme-Linked Electrochemical Detection of PCR-Amplified Nucleotide Sequences Using Disposable Screen-Printed Sensors. Applications in Gene Expression Monitoring

    Directory of Open Access Journals (Sweden)

    Miroslav Fojta

    2008-01-01

    Full Text Available Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE, followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.

  14. Filter paper blood spot enzyme linked immunoassay for insulin and application in the evaluation of determinants of child insulin resistance.

    Directory of Open Access Journals (Sweden)

    Richard M Martin

    Full Text Available BACKGROUND: In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA for quantifying insulin from dried blood spots and demonstrate its application in a large trial. METHODS: We adapted an existing commercial kit (Mercodia Human Insulin ELISA, 10-1113-01 to quantify insulin from two 3-mm diameter discs (≈6 µL of blood punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3-11.8 years from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry. RESULTS: Mean intra-assay (n = 157 coefficients of variation were 15% and 6% for 'low' (6.7 mU/L and 'high' (23.1 mU/L values, respectively; the respective inter-assay values (n = 33 were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95. Bloodspot insulin was stable for at least 31 months at -80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97% children. The geometric mean insulin (log standard deviation concentrations amongst 12,812 children were 3.0 mU/L (1.1 in boys and 4.0 mU/L (1.0 in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose. CONCLUSIONS: Our simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin.

  15. Inhibitory effect of some tropical green leafy vegetables on key enzymes linked to Alzheimer’s disease and some pro-oxidant induced lipid peroxidation in rats’ brain

    OpenAIRE

    Oboh, Ganiyu; Akinyemi, Ayodele Jacobson; Ademiluyi, Adedayo Oluwaseun; Bello, Fatai Olumide

    2011-01-01

    This study sought to investigate the inhibitory effect of some commonly consumed Nigerian green leafy vegetables (raw and blanched) on acetylcholinesterase and butyrylcholinesterase (key enzyme linked to Alzheimer’s disease) activities and some pro-oxidants (FeSO4, Sodium nitroprusside and Quinolinic acid) induced lipid peroxidation in rat brain in vitro. Three commonly consumed green leafy vegetables in Nigeria [Amarantus cruentus (Arowojeja), Struchium sparganophora (Ewuro-odo) and Telfairi...

  16. Inhibition of Key Enzymes Linked to Type 2 Diabetes and Sodium Nitroprusside Induced Lipid Peroxidation in Rats’ Pancreas by Phenolic Extracts of Avocado Pear Leaves and Fruit

    OpenAIRE

    Oboh, Ganiyu; Isaac, Adelusi Temitope; Akinyemi, Ayodele Jacobson; Ajani, Richard Akinlolu

    2014-01-01

    Persea americana fruit and leaves had been known in folk medicine for their anti-diabetic prowess. Therefore, this study sought to investigate the inhibitory effect of phenolic extract from avocado pear (Persea americana) leaves and fruits on some key enzymes linked to type 2 diabetes (α-amylase and α-glucosidase); and sodium nitroprusside (SNP) induced lipid peroxidation in rats’ pancreas in vitro. The phenolic extracts of Persea americana fruit and leaves were extracted using methanol and 1...

  17. IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

    Directory of Open Access Journals (Sweden)

    Tuanne Rotti ABRANTES

    2016-01-01

    Full Text Available After the report of a second case of canine visceral leishmaniasis (CVL in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA, enzyme-linked immunosorbent assay (ELISA, and rapid chromatographic immunoassay based on dual-path platform (DPP(r were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r, with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

  18. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg-1 almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg-1. We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg-1 almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg-1. Further, between 100 and 100,000 mg kg-1 spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals.

  19. Comparison of enzyme linked immunosorbent assay (ELISA), indirect haemagglutination test (IHA) and slide agglutination test (SAT) for screening of Pasteurella multocida associated with haemorrhagic septicaemia in cattle and buffalo of Pakistan

    International Nuclear Information System (INIS)

    Haemorrhagic septicaemia (HS) is an acute infectious epidemic disease of cattle and buffalo caused by Pasteurella multocida. The disease is prevalent sporadically throughout the year but may occur in epizootic form during the rainy season. The organism associated with this disease is heterogeneous in its characteristics. Many attempts have been made to subdivide these organisms into types which bear some relation to host species. Some isolates obtained from the clinical cases have been identified as Carter's type B. However, no systematic studies of the organism as it occurred during the subsequent years has been made hitherto. The knowledge of occurrence of various serotypes of P. multocida is important for the determination of reservoirs of infection and the geographical spread of this organism. This is also important for the production of autogenous vaccine and the recognition of the prevalence of new serotypes. Keeping in view the above considerations, the present work was planned to categorize the strains of P. multocida isolated from cattle and buffalo in order to learn about the prevalence of serotypes. In this investigation, an attempt was also made to compare the status of efficiency and sensitivity of SAT, IHA and ELISA

  20. Establishment of Detection Method for the Residues of Nitrofurazone Metabolite by Enzyme-Linked Immunosorbent Assay%呋喃西林代谢物酶联免疫检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    吴鹏; 万宇平; 陶光灿; 冯才茂; 赵正苗; 刘琳; 励建荣

    2013-01-01

    [目的]建立动物性食品中呋喃西林代谢物ELISA检测方法.[方法]通过制备特异性强的单克隆抗体,采用间接竞争ELISA法建立动物性食品中呋喃西林代谢物ELISA检测方法,并研制出对动物性食品中呋喃西林代谢物残留检测的试剂盒.[结果]该试剂盒标准曲线的IC50值为0.267 7 μg/L,最低检测限为0.1μg/kg,样本加标回收率范围为79.5%~95.6%,变异系数为7.6% ~9.7%.在交叉反应试验中,对呋喃西林的交叉反应率为25%,与其他药物的交叉反应率均小于1%,表明该方法具有很强的特异性.[结论]该方法灵敏度、准确度、精密度均较高,能满足兽药残留的检测要求,且检测时间短(45 min),样本前处理简单、检测成本低,适合于大量样本中呋喃西林代谢物残留检测的快速筛选.%[ Objective ] The research aimed to establish an ELJSA method for detecting nitrofurazone metabolites in animal food. [ Method ] Monoclonal antibodies with high specificity were prepared. And an ELISA method for detecting nitrofurazone metabolites in animal food was established by indirect competitive ELJSA. The kit for detecting the residues of nitrofurazone metabolites in animal food was developed. [ Result] IC50 of the standard curve for the kit was 0.267 7 μg/L,the lowest detection limit was 0.1 μg/kg,the addition recovery was 79.5% -95.6% and the coefficients of variation ranged from 7.6% to 9.7%. In the cross reactivity test,the cross reactivity rate of nitrofurazone was 25% and its cross reactivity rate with other drugs was less than 1 % , which indicated that this method had higher specificity. [ Conclusion ] This method had higher sensitivity .accuracy and precision and could meet the detection demands of residues in veterinary medicine. This method had the characteristics of short detection time(45 min) .simple pre-treatment.low cost and it was suitable for rapid screening for detecting the residues of nitrofurazone metabolites in a large amount of samples.