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Sample records for detectables por elisa

  1. A rapid method to improve protein detection by indirect ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measureable substrate. Numerous incarnations of th...

  2. Plasmonic ELISA for the ultrasensitive detection of Treponema pallidum.

    PubMed

    Nie, Xin-Min; Huang, Rong; Dong, Cai-Xia; Tang, Li-Juan; Gui, Rong; Jiang, Jian-Hui

    2014-08-15

    In this report, we have developed a plasmonic ELISA strategy for the detection of syphilis. Plasmonic ELISA is an enzyme-linked immunoassay combined with enzyme-mediated surface plasmon resonance (SPR) of gold nanoparticles (AuNPs). Immune response of the Treponema pallidum (T. pallidum) antibodies triggers the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine to produce abundant thiocholine. The positive charged thiol, in turn, alters the surface charge distribution the AuNPs and leads to the agglomeration of the AuNPs. The induced strong localized SPR effect of the agglomerate AuNPs can, thus, allow the quantitative assay of T. pallidum antibodies due to the remarkable color and absorption spectral response changes of the reaction system. The plasmonic ELISA exhibited a quasilinear response to the logarithmic T. pallidum antibody concentrations in the range of 1pg/mL-10ng/mL with a detection limit of 0.98pg/mL. Such a low detection limit was 1000-fold improvements in sensitivity over a conventional ELISA. The results of plasmonic ELISA in syphilis assays of serum specimens from 60 patients agreed with those obtained using a conventional ELISA method. The plasmonic ELISA has characteristics (analyte specific, cost-effective, ease of automatic, low limit of detection) that provide potential for diagnosis and therapeutic monitoring of syphilis. PMID:24662060

  3. An ELISA detecting antibody to conserved pestivirus epitopes.

    PubMed

    Paton, D J; Ibata, G; Edwards, S; Wensvoort, G

    1991-01-01

    A monoclonal antibody based competition-ELISA is described for the detection of pestivirus antibodies directed against conserved epitopes on the p80 viral protein. The ELISA detected increases in serum antibody following experimentally induced infections of pigs, cattle and sheep with a wide range of pestiviruses, although the sensitivity of the test was not uniform for the different viruses studied. The ELISA was compared with virus neutralization tests for the assessment of porcine, bovine and ovine field sera. At a cut-off value of 50% inhibition, the ELISA showed a high specificity relative to virus neutralization tests, but appeared less sensitive for the detection of some weakly positive samples from pigs. Sera from both ruminants and pigs could be assessed without any modification of the test. PMID:1713919

  4. ELISA for brucellosis detection based on three Brucella recombinant proteins.

    PubMed

    Thepsuriyanont, Pikun; Intarapuk, Apiradee; Chanket, Panita; Tunyong, Wittawat; Kalambaheti, Thareerat

    2014-01-01

    Control of brucellosis among farm animals, wildlife and humans require reliable diagnosis. Rose Bengal serological test (RBT) is based on lipopolysaccharide antigen of Brucella, which may cross react with other gram-negative bacteria and produce false positive result. Immunoreactive proteins, such as outer-membrane protein BP26, ribosome recycling factor protein CP24 and Brucella lumazine synthase (BLS), previously reported to be recognized by infected sheep sera, were selected for production of recombinant proteins for use in an ELISA in order to investigate immune response among goats and cows, in comparison with commercial RBT. Cut-off value for ELISA was based on the immune response of in vitro fertilized goats and cows. Goats positive for Brucella culture or by RBT were ELISA positive for either IgG or IgM against at least one recombinant protein. For animals with negative RBT, animals with positive ELISA could be detected, and 61.6% possessed ELISA values as high as in infected animals. Thus, this ELISA procedure is proposed as an alternative to RBT for screening of brucellosis in farm animals. PMID:24964662

  5. Detection of Clostridium perfringens epsilon toxin by ELISA.

    PubMed

    Naylor, R D; Martin, P K; Sharpe, R T

    1987-03-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed as an alternative to neutralisation tests in mice to detect Clostridium perfringens type D epsilon toxin in the intestinal contents of animals which have died from suspected enterotoxaemia. The test was sensitive and quantitative and gave excellent agreement with the mouse protection test. PMID:2884704

  6. Development of an ELISA for the Detection of Azaspiracids.

    PubMed

    Samdal, Ingunn A; Løvberg, Kjersti E; Briggs, Lyn R; Kilcoyne, Jane; Xu, Jianyan; Forsyth, Craig J; Miles, Christopher O

    2015-09-01

    Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium spinosum and accumulate in shellfish. Ovine polyclonal antibodies were produced and used to develop an ELISA for quantitating AZAs in shellfish, algal cells, and culture supernatants. Immunizing antigens were prepared from synthetic fragments of the constant region of AZAs, while plate coating antigen was prepared from AZA-1. The ELISA provides a sensitive and rapid analytical method for screening large numbers of samples. It has a working range of 0.45-8.6 ng/mL and a limit of quantitation for total AZAs in whole shellfish at 57 μg/kg, well below the maximum permitted level set by the European Commission. The ELISA has good cross-reactivity to AZA-1-10, -33, and -34 and 37-epi-AZA-1. Naturally contaminated Irish mussels gave similar results whether they were cooked or uncooked, indicating that the ELISA also detects 22-carboxy-AZA metabolites (e.g., AZA-17 and AZA-19). ELISA results showed excellent correlation with LC-MS/MS analysis, both for mussel extract spiked with AZA-1 and for naturally contaminated Irish mussels. The assay is therefore well suited to screening for AZAs in shellfish samples intended for human consumption, as well as for studies on AZA metabolism. PMID:26245830

  7. Fate of Estrogens in Soils and Detection by ELISA

    NASA Astrophysics Data System (ADS)

    Caron, Emmanuelle; Sheedy, Claudia; Farenhorst, Annemieke; Zvomuya, Francis; Gaultier, Jeanette; Goddard, Tom

    2010-05-01

    Land application of manure can contribute to the release of estrogenic compounds in the environment. Estrogens may move from soils to water by processes such as runoff and leaching. The objectives of the present study were to determine the fate of estrogens in soils and to develop a detection method for these compounds. The sorption (soil sorption coefficient (Kd) and sorption coefficient per unit organic carbon (Koc)) of 17β-estradiol, estrone, estriol and equol were studied, using batch equilibrium experiments, in 121 surface soils from Alberta, Canada. The mineralization of [4-14C] 17β-estradiol was determined in soil microcosms in a subset of 36 samples. Quantitative relationships at the regional level were explored using partial least squares regression (PLS) (between Kd or Koc values and soil properties) and by ordinary least squares regression (between Kd or Koc values of different estrogens). Soil properties (r2 0.51-0.87 for Kd and 0.32-0.44 for Koc) provided better prediction models than using the data of different estrogens (r2 0.38-0.71 for Kd and 0.18-0.40 for Koc). PLS regression models for mineralization parameters of 17ß-estradiol had lower predictive power (lower r2)than models developed for sorption parameters. In addition, it has become of primary importance to develop sensitive detection methods that are able to detect low estrogen concentrations (ng L-1) in a wide variety of environmental matrices in order to validate the prediction of their fate and to study their presence in affected ecosystems. Conjugates were synthesized using a mixed anhydride reaction and two Enzyme-Linked Immunosorbent Assays (ELISAs) were developed using polyclonal antibodies. One ELISA was highly specific for 17β-estradiol (with an IC50 of 243 ng mL-1) and the second allowed for the broader detection of 17β-estradiol, estrone and estriol (with an IC50 of 18 ng mL-1 for 17β-estradiol). The cross-reactivity of both ELISAs was studied against 13 compounds (natural

  8. Impact of thermal processing on ELISA detection of peanut allergens.

    PubMed

    Fu, Tong-Jen; Maks, Nicole

    2013-06-19

    This study examined the effect of heat treatment on the solubility of peanut proteins and compared the performances of two commercial ELISA kits (Veratox Quantitative Peanut Allergen Test and BioKits Peanut Assay Kit) for quantitation of peanut residues as affected by different heat treatments (moist and dry heat) and detection targets (mixture of proteins vs specific protein). Both laboratory-prepared and commercial peanut flour preparations were used for the evaluation. The two ELISA kits tended to underestimate the levels of protein in samples that were subjected to elevated heat, respectively, by more than 60- or 400-fold lower for the autoclaved samples and by as much as 70- or 2000-fold lower for the dark-roast commercial flour samples. The BioKits test, which employs antibodies specific to a heat labile protein (Ara h 1), in general exhibited a greater degree of underestimation. These results suggest that commercial ELISA kits may not be able to accurately determine the amount of proteins present in thermally processed foods due to changes in the solubility and immunoreactivity of the target proteins. Users need to be aware of such limitations before applying ELISA kits for evaluation of food allergen control programs. PMID:23473340

  9. A micro-capture ELISA for detecting Mycoplasma pneumoniae IgM: comparison with indirect immunofluorescence and indirect ELISA.

    PubMed Central

    Wreghitt, T. G.; Sillis, M.

    1985-01-01

    A mu-capture ELISA was developed for detecting Mycoplasma pneumoniae-specific IgM, and compared with an indirect immunofluorescent antibody (IFA) technique and an indirect ELISA. mu-capture ELISA and IFA compared well and were found to be the most sensitive assays. The IFA test can be completed in 2 h whilst the results of the mu-capture ELISA can be available in 24 h. Both tests are amenable to routine diagnostic use and have similar sensitivity. Indirect ELISA was found to be less sensitive and less specific, giving high assay values with several sera having undetectable M. pneumoniae CF antibody or CF antibody in low titre. Serum samples obtained from 11 patients at various times after M. pneumoniae infection showed maximum antibody levels within the first month by all assays, with a gradual fall in amount of IgM with time when assayed by mu-capture ELISA, a more gradual decline by IFA and hardly any decline with indirect ELISA. It was concluded that the indirect ELISA is unsuitable for the investigation of possible M. pneumoniae infection because the sustained high assay values with serum samples taken many months after infection, make interpretation of the test results very difficult. PMID:3921607

  10. Detection of cow milk adulteration in yak milk by ELISA.

    PubMed

    Ren, Q R; Zhang, H; Guo, H Y; Jiang, L; Tian, M; Ren, F Z

    2014-10-01

    In the current study, a simple, sensitive, and specific ELISA assay using a high-affinity anti-bovine β-casein monoclonal antibody was developed for the rapid detection of cow milk in adulterated yak milk. The developed ELISA was highly specific and could be applied to detect bovine β-casein (10-8,000 μg/mL) and cow milk (1:1,300 to 1:2 dilution) in yak milk. Cross-reactivity was <1% when tested against yak milk. The linear range of adulterant concentration was 1 to 80% (vol/vol) and the minimum detection limit was 1% (vol/vol) cow milk in yak milk. Different treatments, including heating, acidification, and rennet addition, did not interfere with the assay. Moreover, the results were highly reproducible (coefficient of variation <10%) and we detected no significant differences between known and estimated values. Therefore, this assay is appropriate for the routine analysis of yak milk adulterated with cow milk. PMID:25151876

  11. Duration of Loxosceles reclusa Venom Detection by ELISA from Swabs

    PubMed Central

    McGLASSON, DAVID L; GREEN, JONATHON A; STOECKER, WILLIAM V; BABCOCK, JAMES L; CALCARA, DAVID A

    2011-01-01

    BACKGROUND Diagnosis of Loxosceles reclusa envenomations is currently based upon clinical presentation. An enzyme-linked immunosorbent assay (ELISA) can detect surface Loxosceles venom at the envenomation site, allowing diagnostic confirmation. The length of time that venom on the skin is recoverable non-invasively is unknown. MATERIALS AND METHODS To investigate duration of recoverable venom antigen, whole venom and fractionated sphingomyelinase D venom aliquots were injected subcutaneously in New Zealand White rabbits. Cotton and Dacron swabs were compared for venom recovery over a 21-day period using a surface swab technique. RESULTS Significant amounts of Loxosceles reclusa antigen were found on the surface of rabbit skin after experimental injection of whole venom and sphingomyelinase D. The duration of recoverable antigen using this experimental model appears to be at least two weeks and as long as 21 days in some cases. CONCLUSIONS Because the duration of the recoverable antigen is seen to be at least two weeks, the ELISA venom test appears capable of detecting venom on most patients presenting with Loxosceles envenomations. This detection system will allow the physician more accurate determination of whether the lesion is from a brown recluse spider or some other agent that can cause this type of necrotic ulcer. PMID:19967916

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

    PubMed

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

    2011-01-01

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

  13. Sensitive detection of Escherichia coli O157:H7 based on cascade signal amplification in ELISA.

    PubMed

    Shan, Shan; Liu, Daofeng; Guo, Qi; Wu, Songsong; Chen, Rui; Luo, Kai; Hu, Liming; Xiong, Yonghua; Lai, Weihua

    2016-09-01

    In this study, cascade signal amplification in ELISA involving double-antibody sandwich ELISA and indirectly competitive ELISA was established to sensitively detect Escherichia coli O157:H7. In the double-antibody sandwich ELISA, a complex was formed comprising anti-E. coli O157:H7 polyclonal antibody, E. coli O157:H7, biotinylated anti-E. coli O157:H7 monoclonal antibody, streptavidin, and biotinylated β-lactamase. Penicillin solution was then added into the ELISA well and hydrolyzed by β-lactamase. Afterward, the penicillin solution was transferred to indirectly competitive ELISA. The concentration of penicillin can be sensitively detected in indirectly competitive ELISA. In the cascade signal amplification system, increasing the amount of added E. coli O157:H7 resulted in more β-lactamase and less penicillin. The detection sensitivity of E. coli O157:H7, which was 20cfu/mL with the cascade signal amplification in ELISA, was 1,000-fold higher than that of traditional ELISA. Furthermore, the novel method can be used to detect E. coli O157:H7 in milk (2cfu/g). Therefore, this new signaling strategy will facilitate analyses of highly sensitive foodborne pathogens. PMID:27394946

  14. Immunofluorescence versus ELISA for the detection of antinuclear antigens.

    PubMed

    Rondeel, Jan M M

    2002-05-01

    Determining the presence and specificity of antinuclear antigens (ANA) is a challenge to a laboratory involved in the diagnosis of connective tissue disease (CTD). The immunofluorescent technique (IF), once considered the gold standard, is more and more displaced by ELISA. ELISA can be fully automated and the interpretation does not require the extensive experience needed in IF. However, literature in which both techniques are compared does not give unequivocal conclusions that ELISA indeed performs better. The clue as to which technique is best in the cascade testing of ANA, is given by its clinical value, not only by its technical and logistic performance. Selective test ordering is strongly recommended to increase the predictive value of these tests. The pros and cons of both techniques are discussed. PMID:12050861

  15. Evaluation of a competitive ELISA for antibody detection against avian influenza virus.

    PubMed

    Song, Dae Sub; Lee, Youn Jeong; Jeong, Ok Mi; Kim, Yong Joo; Park, Chan Hee; Yoo, Jung Eun; Jeon, Woo Jin; Kwon, Jun Hun; Ha, Gun Woo; Kang, Bo Kyu; Lee, Chul Seung; Kim, Hye Kwon; Jung, Byeong Yeal; Kim, Jae Hong; Oh, Jin Sik

    2009-12-01

    Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity. PMID:19934598

  16. Comparison of DOT-ELISA and Standard-ELISA for Detection of the Vibrio cholerae Toxin in Culture Supernatants of Bacteria Isolated from Human and Environmental Samples.

    PubMed

    Meza-Lucas, Antonio; Pérez-Villagómez, María-Fernanda; Martínez-López, José-Patricio; García-Rodea, Ricardo; Martínez-Castelán, María-Guadalupe; Escobar-Gutiérrez, Alejandro; de-la-Rosa-Arana, Jorge-Luis; Villanueva-Zamudio, Altagracia

    2016-09-01

    A comparison of DOT-ELISA and Standard-ELISA was made for detection of Vibrio cholerae toxin in culture supernatants of bacteria isolated from human and environmental samples. A total of 293 supernatants were tested in a double blind assay. A correlation of 100 % was obtained between both techniques. The cholera toxin was found in 20 Inaba and 3 Ogawa strains. Positive samples were from seafood (17 samples), potable water (1 sample) and sewage (5 samples). The DOT-ELISA was useful as the standard-ELISA to confirm the presence of cholera toxin in the environmental samples. PMID:27407304

  17. ELISA assays for the detection of Bothrops lanceolatus venom in envenomed patient plasmas.

    PubMed

    Rodriguez-Acosta, A; Uzcategui, W; Azuaje, R; Giron, M E; Aguilar, I

    1998-01-01

    A double antibody sandwich enzyme linked immunosorbant assay (ELISA) was carried out to detect Bothrops Ianceolatus venom in plasma from envenomed patients at various time intervals (0, 6, 12, 18 and 24 hrs). The test could detect Bothrops lanceolatus levels up to 12 ng/mL of envenomed patient plasmas. Elaboration of an easy, fast and species-diagnostic based on this ELISA technique useful to physicians is discussed. PMID:11845439

  18. A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs

    PubMed Central

    Liu, Baoyuan; Wang, Lizhen; Sun, Yani; Li, Huixia; Wang, Xinjie; Syed, Shahid Faraz; Zhang, Gaiping; Zhou, En-Min

    2016-01-01

    Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no “gold standard” assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs. PMID:27023902

  19. A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs.

    PubMed

    Chen, Yiyang; Zhao, Qin; Liu, Baoyuan; Wang, Lizhen; Sun, Yani; Li, Huixia; Wang, Xinjie; Syed, Shahid Faraz; Zhang, Gaiping; Zhou, En-Min

    2016-01-01

    Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no "gold standard" assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 μg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs. PMID:27023902

  20. Further evaluation and validation of a commercially available competitive ELISA (cELISA) for the detection of antibodies specific to equine arteritis virus (EAV).

    PubMed

    Pfahl, K; Chung, C; Singleton, M D; Shuck, K M; Go, Y Y; Zhang, J; Campos, J; Adams, E; Adams, D S; Timoney, P J; Balasuriya, U B R

    2016-01-23

    The purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (EAV) competitive ELISAs (original and enhanced cELISAs) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. First, the original and subsequently enhanced cELISAs were compared with the virus neutralisation test (VNT) using a panel of archived serum samples from experimentally inoculated animals. Then, the enhanced cELISA was compared with the VNT using a large panel of archived serum samples. The total number of equine sera tested was 3255, which included sera against 25 different EAV strains. The study confirmed that the enhanced cELISA was more sensitive than the original cELISA. Based on testing sera from experimentally inoculated animals and field sera, the enhanced cELISA had an estimated sensitivity (98.9 percent and 99.6 percent, respectively) and specificity (98.3 percent and 98.7 percent, respectively). The currently marketed enhanced VMRD EAV antibody cELISA test kit (VMRD Inc., Pullman, Washington, USA) has high sensitivity and specificity relative to the VNT. Based on the findings of this study, the authors would propose that the enhanced cELISA should be considered as an alternative approved method to the VNT for the detection of antibodies to EAV. PMID:26733051

  1. A PCR-ELISA for detecting Shiga toxin-producing Escherichia coli.

    PubMed

    Ge, Beilei; Zhao, Shaohua; Hall, Robert; Meng, Jianghong

    2002-03-01

    A sensitive and specific PCR-ELISA was developed to detect Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primer specific for Shiga toxin genes during PCR amplification. The labeled PCR products were bound to streptavidin-coated wells of a microtiter plate and detected by an ELISA. The specificity of the PCR was determined using 39 bacterial strains, including STEC, enteropathogenic E. coli, E. coli K12, and Salmonella. All of the STEC strains were positive, and non-STEC organisms were negative. The ELISA detecting system was able to increase the sensitivity of the PCR assay by up to 100-fold, compared with a conventional gel electrophoresis. The detection limit of the PCR-ELISA was 0.1-10 CFU dependent upon STEC serotypes, and genotypes of Shiga toxins. With the aid of a simple DNA extraction system, PrepMan, the PCR-ELISA was able to detect ca. 10(5) CFU of STEC per gram of ground beef without any culture enrichment. The entire procedure took about 6 h. Because of its microtiter plate format, PCR-ELISA is particularly suitable for large-scale screening and compatible with future automation. PMID:11909738

  2. Prospects of eLISA for detecting Galactic binary black holes similar to GW150914

    NASA Astrophysics Data System (ADS)

    Seto, Naoki

    2016-07-01

    We discuss the prospects of eLISA for detecting gravitational waves (GWs) from Galactic binary black holes (BBHs) similar to GW150914. For a comoving merger rate that is consistent with current observation, eLISA is likely to identify at least one BBH with a sufficient signal-to-noise ratio. In addition, eLISA has a potential to measure the eccentricity of the BBH as small as e ˜ 0.02, corresponding to the residual value e ˜ 10-6 at 10 Hz. Therefore, eLISA could provide us with a crucial information to understand the formation processes of relatively massive BBHs like GW150914. We also derive a simple scaling relation for the expected number of detectable Galactic BBHs.

  3. Parts Per Trillion Detection of 7-Aminonitrazepam by Nano-Enhanced ELISA

    PubMed Central

    Peng, Chifang; Duan, Xiaohui; Song, Shanshan; Xue, Feng

    2013-01-01

    It is challenging to detect 7-aminonitrazepam (7-ANZP) residue in animal tissues simply and sensitively by the enzyme-linked sorbent immunoassay (ELISA) method. This paper demonstrates that utilizing a bioconjugate of gold nanoparticles and enzyme-labeled antibody as a signal probe increases the sensitivity of a traditional ELISA for 7-ANZP by nearly 20 times. The sensitivity of this ELISA for 7-ANZP was 5.6 pg/mL in buffer, and the limit of detection (LOD) of 0.18 μg/kg for 7-ANZP in urine could be achieved after the urine samples were simply hydrolyzed and diluted by buffer. This simple and sensitive method has potential application for improving the sensitivity of ELISA methods against various small molecules. PMID:24071944

  4. Evaluation of commercial ELISA kits for the detection of antibodies against bluetongue virus.

    PubMed

    Niedbalski, W

    2011-01-01

    The aim of this study was to estimate the diagnostic value of different commercially available ELISA kits for the detection of bluetongue virus (BTV) antibodies in infected and vaccinated animals. The relative specificity of ELISA kits was evaluated using a panel of sera originating from healthy cattle, never vaccinated nor exposed to BTV. All ELISA kits applied had a high relative specificity (99.3 - 100%). The relative sensitivity of ELISA kits assessed using a panel of sera collected from BTV infected cattle was also high and similar for all the kits (97.3 - 100%). However, the relative sensitivity evaluated on the basis of testing vaccinated animals was different: the highest sensitivity was found for Ingenasa, PrioCHECK and ID VET ELISAs (96.5 - 98.3%). Slightly lower sensitivity was calculated for Pourquier and LSI kits (82.8% and 85.4%, respectively) and much lower sensitivity was found for VMRD ELISA kit (69.5%). The repeatability of BTV ELISA kits was expressed as a coefficient of variation (CV) of results of sera tested 5 times in the same day and in different days by the period of 2 months, by the same person, in the same conditions, and by using the same equipment. The CVs of sera tested in all ELISA kits ranged from 6.1 to 9.8% and were below 10% threshold adopted as a maximum for the acceptable repeatability of the method. In conclusion, it can be stated that the applied ELISA kits can be a valuable diagnostic tool for the serological monitoring studies in the BTV contaminated premises. All the methods are very specific and sensitive when testing BTV infected animals. Nevertheless, the Ingenasa and PrioCHECK can be the most useful in sero-surveillance of livestock following vaccination. PMID:22439333

  5. Evaluation of Fas2-ELISA for the serological detection of Fasciola hepatica infection in humans.

    PubMed

    Espinoza, Jose R; Maco, Vicente; Marcos, Luis; Saez, Sandra; Neyra, Victor; Terashima, Angelica; Samalvides, Frine; Gotuzzo, Eduardo; Chavarry, Elizabeth; Huaman, Maria Cecilia; Bargues, M Dolores; Valero, M Adela; Mas-Coma, Santiago

    2007-05-01

    The performance of Fas2-ELISA for the diagnosis of Fasciola hepatica infection in children living in areas of high endemicity for fascioliasis in the Peruvian Andes is analyzed. Fas2-ELISA is based on the detection of circulating IgG antibodies elicited in infected individuals against a F. hepatica antigen termed Fas2. The study was conducted in three Andean localities, Huertas-Julcan in Junin, Asillo in Puno, and Cajamarca, with a total population of 634 children in an age range 1 to 16 years old. Child fascioliasis prevalence was 21.1% in Huertas-Julcan, 25.4% in Asillo, and 24% in Cajamarca, estimated by coprological inspection. The seroprevalence of F. hepatica infection, determined by Fas2-ELISA, was 27.8% in Huertas-Julcan, 44.6% in Asillo, and 29.1% in Cajamarca. The overall sensitivity of Fas2-ELISA was 92.4%, the specificity 83.6%, and the negative predictive value 97.2%. No association between OD(450) Fas2-ELISA and infection intensity measured by egg counting was observed. Results show that Fas2-ELISA is a highly sensitive immunodiagnostic test for the detection of F. hepatica infection in children living in human fascioliasis endemic areas. PMID:17488926

  6. Nanospherical Brush as Catalase Container for Enhancing the Detection Sensitivity of Competitive Plasmonic ELISA.

    PubMed

    Huang, Xiaolin; Chen, Rui; Xu, Hengyi; Lai, Weihua; Xiong, Yonghua

    2016-02-01

    Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth shows great potential for the determination of disease-related biomarkers at ultralow concentrations by using sandwich formats. However, the relatively low sensitivity of this strategy using competitive formats limits its adoption for hapten detection. Herein, we present an improved competitive pELISA for ultrasensitive detection of ochratoxin A (OTA), where silica nanoparticles carrying poly(acrylic acid) brushes (SiO2@PAA) were used to decrease the affinity of competing antigens to anti-OTA monoclonal antibodies and amplify the signal as a "CAT container" (SiO2@PAA@CAT). The developed competitive pELISA exhibits extremely high sensitivity for OTA with detection limits of 10(-18) and 5 × 10(-20) g/mL by the naked eye and microplate reader, respectively. These values are at least 7 orders of magnitude lower than that of competitive CAT-based pELISA (10(-11) g/mL by the naked eye) and 8 orders of magnitude lower than that of horseradish peroxidase-based conventional ELISA (10(-11) g/mL by the microplate reader), respectively. Reliability and robustness of the proposed method were evaluated using actual agricultural products and human serum samples. This study demonstrated the potential of this modified method in practical applications involving the ultrasensitive detection of mycotoxins or other haptens. PMID:26719076

  7. Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts.

    PubMed

    Lemons, Angela R; Bledsoe, Toni A; Siegel, Paul D; Beezhold, Donald H; Green, Brett J

    2013-11-29

    Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals commonly used in the manufacturing industry. Occupational exposures to dNCOs have been shown to elicit allergic sensitization and occupational asthma. Among the most commonly used dNCOs in industry are the aromatic dNCOs, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). This study aimed to develop enzyme linked immunosorbent assays (ELISA) utilizing aromatic dNCO-specific monoclonal antibodies (mAbs) for the detection of aromatic dNCO adducts. Two sandwich ELISAs were developed. The first sandwich ELISA utilized mAb 60G2 along with an anti-human serum albumin (HSA) polyclonal antibody. This assay detected MDI-, 2,4- and 2,6-TDI-HSA adducts with limits of detection (LOD) of 2.67, <0.10, and 1.70 ng/mL, respectively. When spiked into human serum, the LOD of this ELISA increased to 34.37, 7.64 and 24.06 ng/mL, respectively. The second ELISA utilized mAbs 62G5 and 60G2 for capture and detection. This assay was capable of detecting 2,4- and 2,6-TDI-HSA adducts with LODs of <4.90 and 26.92 ng/mL, respectively, and when spiked in human serum, <4.90 and 95.93 ng/mL, respectively. This 62G5-60G2 sandwich assay was also able to detect dNCO adducted transferrin, hemoglobin, keratin and actin, but with less sensitivity than dNCO-HSA. The results of this study demonstrate potential application of these ELISAs in the identification and characterization of aromatic dNCO adducts as well as in biomonitoring occupational and environmental dNCO exposures. PMID:24012971

  8. Detection of hazelnut in foods using ELISA: challenges related to the detectability in processed foodstuffs.

    PubMed

    Cucu, Tatiana; Devreese, Bart; Trashin, Stanislav; Kerkaert, Barbara; Rogge, Maarten; De Meulenaer, Bruno

    2012-01-01

    Hazelnuts are widely used nowadays, and can pose a serious threat to allergic consumers due to cross-contamination that may occur during processing. This might lead to the presence of hidden hazelnut in foods. Therefore, reliable tests are needed to detect hazelnut, especially in processed foods. A hazelnut-specific indirect competitive ELISA based on polyclonal chicken antibodies was developed. The polyclonal antibodies were raised against modified hazelnut proteins in order to improve the detectability of hazelnut proteins in processed foods. The assay showed a detection limit of 1.36 microg hazelnut protein/mL of 5 mM urea in phosphate-buffered saline buffer (pH 7.4). Limited cross-reactivity with walnut and pecan nut was observed; no cross-reactivity was observed with other food ingredients. Blank cookies spiked before analysis showed recoveries of 73-107%. However, cookies spiked before baking showed that the detectability was severely decreased. Addition of lactose to the cookies, which led to more severe modification through the Maillard reaction, led to an increase in the detectability. These results indicate that using antibodies developed toward allergens modified through food processing-simulating reactions is a better approach for detection. PMID:22468353

  9. [Comparative study of indirect immunofluorescence (IIF) and ELISA techniques in the detection of parvovirus B19].

    PubMed

    González, M; Hassanhi, M; Rivera, S; Bracho, M P

    2000-03-01

    To determine the seroprevalence of IgG and IgM antibodies against Parvovirus B19 (P. B19), we studied the sera of 53 patients with different hematologic disorders and the sera of 15 controls using indirect immunofluorescence (IFI) and the ELISA method. The prevalence of IgG in the control group was 46.6%, in patients with aplastic crisis was 83.3% (IFI) and 66.7% (ELISA) and, in patients without crisis was 68.9% (IFI) and 72.4% (ELISA). IgM was negative except for patients with crisis: 8.3% (IFI) and 29.1% (ELISA). The higher seroprevalence (IgG) found in patients in comparison with controls might be due to a greater exposure of of patients to the virus. The agreement for both techniques was 81%(IgG) and 93% (IgM) however ELISA technique was more sensitive for detecting IgM of P. B19. In spite of serologic evidence and evaluating a simple serum sample per patient, we could establish an association between aplastic crisis and viral infection for IgM ELISA but not for IgG between hematologic disorders and infection for the P. B19. PMID:10758696

  10. Sandwich-ELISA detection of venom antigens in envenoming by Phoneutria nigriventer spider.

    PubMed

    Chávez-Olórtegui, C; Bohórquez, K; Alvarenga, L M; Kalapothakis, E; Campolina, D; Maria, W S; Diniz, C R

    2001-06-01

    Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Phoneutria nigriventer spider venom. Horse anti-P. nigriventer immunoglobulins were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with P. nigriventer venom from those in mice inoculated with Lycosa sp. and Loxosceles intermedia spider venoms, Tityus serrulatus scorpion venom and Apis mellifera bee venom. Measurable absorbance signals were obtained with 0.8ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice and to detect antigens in the sera of patients envenomed by P. nigriventer. PMID:11137553

  11. Comparative Evaluation of RUT, PCR and ELISA Tests for Detection of Infection with Cytotoxigenic H. pylori

    PubMed Central

    Jalalypour, Farzaneh; Farajnia, Safar; Somi, Mohammad Hossein; Hojabri, Zoya; Yousefzadeh, Rana; Saeedi, Nazli

    2016-01-01

    Purpose: Helicobacter pylori is one of the most prevalent infectious agents in the world which causes a variety of gastrointestinal diseases including gastritis, peptic ulcer and gastric carcinoma. The objective of this study was to comparatively evaluate invasive (rapid urease test and polymerase chain reaction) and non-invasive (enzyme-linked immunosorbent assay) tests in diagnosis of infection with cytotoxigenic H. pylori. Methods: Biopsy specimens and sera were collected from 105 patients with gastric disorders. The presence of H. pylori infection in gastric biopsies was evaluated by RUT and PCR methods using chemotaxis signal transduction protein gene (CSTP), Urea C and HP-16srRNA primers. Serum samples were used for the ELISA test. Detection of infection with cag A-positive strains was performed by PCR and cag A-IgG ELISA kit. Results: Patients with at least two out of three positive results were regarded as infected. The sensitivity, specificity, predictive value and accuracy of the three different methods were evaluated. Of the 105 gastric biopsies, H. pylori were positive in 51 patients (48.57%). The best sensitivity (92.16%) belonged to RUT. The sensitivities of other tests including PCR and ELISA test were 88.24% and 90.20%, respectively. PCR showed the best specificity (94.44%), and the specificities of the other tests including RUT and ELISA test, were 90.74 % and 61.11%, respectively. Furthermore, results of PCR and cag A-IgG ELISA showed high prevalence of cag A-positive strain in the study population. Conclusion: Based on our findings, serum ELISA is a rapid noninvasive test for screening of H. pylori infection in the absence of endoscopy indication. In addition, considering the high prevalence of cytotoxigenic H. pylori strains, cag A is suggested as a promising target for PCR and non- invasive ELISA tests for detection of infection with toxigenic strains. PMID:27478790

  12. Amplified ELISA to detect autoantibodies to N-glycolyl-GM3 ganglioside.

    PubMed

    Iznaga, N; Carr, A; Fernández, L E; Solozabal, J; Núñez, G; Perdomo, Y; Morales, A

    1996-01-01

    Numerous immunochemical methods are now available for the detection of antibodies to gangliosides. An amplified ELISA method for detection of autoantibodies to NGcGM3 ganglioside in the sera of patients with various type of renal diseases was developed. IgM antibodies were found in 39 out of 53 sera of patients using 30 normal healthy blood donor as a negative control. For human IgG conjugate no reactivity to NGcGM3 was seen in the sera. Positive ELISA results were confirmed by TLC-immunostaining using GM3, NGcGM3, NGcGM2 and Standard bovine gangliosides (GM1, GD1a, GD1b and GT1b). All sera were also assayed for reactivity with GM3 in ELISA to determine the line specificity of these antibodies. Based on these results, a protocol for a sensitive and reproducible amplification ELISA system for serum anti-NGcGM3 antibodies in patients with renal or other diseases is presented. The ELISA method described here in appear to be useful adjunt to measure antiNGcGM3 antibodies in sera of patients with various type of renal or other diseases. PMID:16296265

  13. False positive detection of peanut residue in liquid caramel coloring using commercial ELISA kits.

    PubMed

    Stelk, T; Niemann, L; Lambrecht, D M; Baumert, J L; Taylor, S L

    2013-07-01

    Initial food industry testing in our laboratory using enzyme-linked immunosorbent assay (ELISA) methods indicated that the darkest caramel color (class IV) unexpectedly contained traces of peanut protein, a potential undeclared allergen issue. Caramel production centers on the heating of sugars, often glucose, under controlled heat and chemical processing conditions with other ingredients including ammonia, sulfite, and/or alkali salts. These ingredients should not contain any traces of peanut residue. We sought to determine the reliability of commercially available peanut allergen ELISA methods for detection of apparent peanut residue in caramel coloring. Caramel color samples of classes I, II, III, and IV were obtained from 2 commercial suppliers and tested using 6 commercially available quantitative and qualitative peanut ELISA kits. Five lots of class IV caramel color were spiked with a known concentration of peanut protein from light roasted peanut flour to assess recovery of peanut residue using a spike and recovery protocol with either 15 ppm or 100 ppm peanut protein on a kit-specific basis. A false positive detection of peanut protein was found in class IV caramel colors with a range of 1.2 to 17.6 parts per million recovered in both spiked and unspiked liquid caramel color samples. ELISA kit spike/recovery results indicate that false negative results might also be obtained if peanut contamination were ever to actually exist in class IV caramel color. Manufacturers of peanut-free products often test all ingredients for peanut allergen residues using commercial ELISA kits. ELISA methods are not reliable for the detection of peanut in class IV caramel ingredients and their use is not recommended with this matrix. PMID:23647653

  14. Transmission of a Viral Disease (AIDS) Detected by a Modified ELISA Reaction: A Laboratory Simulation.

    ERIC Educational Resources Information Center

    Grimes, William J.; Chambers, Linda; Kubo, Kenneth M.; Narro, Martha L.

    1998-01-01

    Describes a laboratory exercise that simulates the spread of an infectious agent among students in a classroom. Uses a modified Enzyme Linked ImmunoSorbent Assay (ELISA) to provide students with experience using an authentic diagnostic tool for detecting human infections. (DDR)

  15. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    EPA Science Inventory

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

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

    PubMed

    Lee, P-W; Hefle, S L; Taylor, S L

    2008-05-01

    Undeclared mustard residues in food products could trigger allergic reactions in mustard-allergic consumers. Our objective was to develop and validate a sandwich-type ELISA for the detection of mustard residues in foods. A mixture of yellow, brown, and oriental mustard seeds was used to immunize 3 rabbits and 1 sheep. Two mustard ELISAs were developed by utilizing the reciprocal combination of rabbit and sheep polyclonal antimustard sera as the capture and detector reagents. Binding was visualized by addition of rabbit antisheep or goat antirabbit IgG antibody labeled with alkaline phosphatase and subsequent addition of substrate. The optimized ELISAs have limits of quantification (LOQ) of 1 and 3 ppm (mug of ground, whole mustard seeds/mL) for the sheep capture and rabbit capture formats, respectively. Only rapeseed cross-reacted in the rabbit and sheep capture mustard ELISAs at a level equivalent to 12300 and 16900 ppm of mustard. The mean percent recovery for cooked frankfurters spiked with 0 to 1000 ppm mustard flour was 95.3%+/- 10.7%. A limited retail survey of 29 foods revealed that, of 15 samples having mustard declared on the ingredient list, 2 baked bean products contained no detectable mustard, possibly owing to a decrease in extractability and detectability of mustard proteins after subjecting to thermal processing. For the remaining 14 samples without mustard declared on the label, 3 samples contained detectable mustard, presumably due to the labeling of mustard as "spice" or inadvertent cross-contamination. This sandwich-type ELISA can serve as a powerful tool for food manufacturers and regulatory agencies to detect and quantify mustard residues in processed foods. PMID:18460147

  17. Apparent ELISA detection times for albuterol after administration with the torpex equine inhaler device.

    PubMed

    Dirikolu, Levent; Mollett, Brad A; Troppmann, Amy; Woods, William E; Bratton, Calvert; Cashman, Conor P; Schroedter, Dwight; Mayer, Brent; Lehner, Andreas F; Karpiesiuk, Wojciech; Hughes, Charlie; Boyles, Jeff; Harkins, John D; Tobin, Thomas

    2002-01-01

    Single doses of one, three, and six actuations (120 micro g albuterol/actuation) and multiple daily doses (six actuations per dose four times daily) for 5 days of aerosol albuterol sulfate were sequentially administered to each of six horses using an equine inhaler device (Torpex, 3M Animal Care Products, St. Paul, MN [corrected] and Boehringer Ingleheim Vetmedica, Inc., St. Joseph, MO [corrected]). A 2-week washout period was allowed between each dose. ELISA testing revealed no evidence of albuterol in urine at 24 hours after any single-dose administration. Results indicated that 48 hours or longer should be allowed for albuterol to be cleared from urine after single doses. When given at the maximum recommended rate of six actuations per dose four times a day for 5 days, urine samples tested by ELISA showed no evidence of albuterol at 48 hours after the final dose. Testing of nasal swabs by ELISA demonstrated the presence of albuterol for 8 hours after each single dose, and some horses might have detectable levels of albuterol in nasal swabs for several days following administration of multiple doses. As a guideline for withdrawal time, 72 hours or longer should be allowed after administration of aerosol albuterol sulfate to horses before participation in equestrian competitions that are regulated for detection of certain performance-enhancing substances. However, these recommendations were based on a small sample of horses and the specific ELISA test used and interpreted as described. Factors specific to individual horses may influence these detection times. PMID:12447838

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

    PubMed

    Gaskin, Ferdelie E; Taylor, Steve L

    2011-01-01

    The presence of undeclared cashew can pose a health risk to cashew-allergic consumers. The food industry has the responsibility to declare the presence of cashews on packaged foods even when trace residues are or might be present. The objective of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) for the detection of cashew residues. Raw and roasted cashews were defatted and used separately to immunize sheep, goats, and rabbits. The cashew ELISA was developed using sheep and rabbit polyclonal anti-roasted cashew sera as capture and detector reagents, respectively, with visualization through an alkaline phosphatase-mediated substrate reaction. The cashew ELISA was shown to have a limit of quantification of 1 ppm (1 μg cashew/g). The ELISA was highly specific except that substantial cross-reactivity was noted with pistachio and a lesser degree of cross-reactivity was noted with hazelnut. The performance of the ELISA was assessed by manufacturing cookies, ice cream, and milk chocolate with added known amounts (0 to 1000 ppm) of cashew. The mean percent recoveries for ice cream, cookies, and milk chocolate were 118%± 2.9%, 84.3%± 4.0%, and 104%± 3.0%, respectively. In a limited retail survey, 4/5 retail samples with cashew declared on ingredient labels tested positive for cashew compared to 5/36 samples of foods with precautionary labels indicating the possible presence of one or more tree nuts and 0/18 samples without cashew declared on the label in any manner. The cashew ELISA can be used to detect undeclared cashew residue in foods and as a potential tool for the food industry to assess the effectiveness of allergen control strategies and to guarantee compliance with food labeling regulatory requirements. PMID:22416731

  19. Plasmonic ELISA for the detection of gp120 at ultralow concentrations with the naked eye

    NASA Astrophysics Data System (ADS)

    Cecchin, D.; de La Rica, R.; Bain, R. E. S.; Finnis, M. W.; Stevens, M. M.; Battaglia, G.

    2014-07-01

    The technique of plasmonic ELISA is utilised here to detect the HIV-1 protein gp120 with the ultralow limit of detection of 8 × 10-20 M (10-17 g mL-1) in an independent laboratory. It was corroborated that changes in the concentration of hydrogen peroxide as small as 0.05 μM could lead to nanoparticle solutions of completely different tonality.The technique of plasmonic ELISA is utilised here to detect the HIV-1 protein gp120 with the ultralow limit of detection of 8 × 10-20 M (10-17 g mL-1) in an independent laboratory. It was corroborated that changes in the concentration of hydrogen peroxide as small as 0.05 μM could lead to nanoparticle solutions of completely different tonality. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr06167a

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

    PubMed

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

    2013-06-01

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

  1. Immune response against Treponema spp. and ELISA detection of digital dermatitis.

    PubMed

    Gomez, A; Anklam, K S; Cook, N B; Rieman, J; Dunbar, K A; Cooley, K E; Socha, M T; Döpfer, D

    2014-01-01

    The objective of this longitudinal study was to evaluate the immune response against Treponema spp. infection in dairy heifers affected with digital dermatitis (DD). In addition, the accuracy of an indirect ELISA detecting anti-Treponema IgG antibodies in identifying clinical DD status has been assessed. A cohort of 688 pregnant Holstein heifers was evaluated at least 3 times before calving during a period of 6 mo. Complete clinical assessment of DD presence on the back feet of each heifer and blood extraction were performed in a stand-up chute. Digital dermatitis cases were characterized by the M-stage classification system and size and level of skin proliferation. An ELISA was performed on blood serum samples obtained from a subcohort of 130 heifers. For description purposes, the animals were classified by the number of clinical cases experienced during the study period as type I (no clinical cases were observed), type II (only 1 acute clinical case diagnosed), and type III (at least 2 acute clinical cases diagnosed). Multivariable repeated-measures models were used to evaluate the immune response against Treponema spp. infection. A binormal Bayesian model for the ELISA data without cut-point values was used to assess the accuracy of the ELISA as a diagnostic tool. Animals that never experienced a DD event throughout the study kept a constant low level of antibody titer. A 56% increase in mean ELISA titer was observed in heifers upon a first clinical DD case diagnosis. After topical treatment of an acute DD case with oxytetracycline, the antibody titer decreased progressively in type II heifers, achieving mean levels of those observed in healthy cows after a mean of 223 d. Surprisingly, antibody titer was not increased in the presence of M1 (DD lesion <20mm in diameter surrounded by healthy skin) and M4.1 (DD lesion <20mm in diameter embedded in a circumscribed dyskeratotic or proliferative skin alteration) DD stages. Type III cows showed a slight increase in

  2. Detection of Q Fever Specific Antibodies Using Recombinant Antigen in ELISA with Peroxidase Based Signal Amplification

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Glennon, Erin; Ching, Wei-Mei

    2014-01-01

    Currently, the accepted method for Q fever serodiagnosis is indirect immunofluorescent antibody assay (IFA) using the whole cell antigen. In this study, we prepared the recombinant antigen of the 27-kDa outer membrane protein (Com1) which has been shown to be recognized by Q fever patient sera. The performance of recombinant Com1 was evaluated in ELISA by IFA confirmed serum samples. Due to the low titers of IgG and IgM in Q fever patients, the standard ELISA signals were further amplified by using biotinylated anti-human IgG or IgM plus streptavidin-HRP polymer. The modified ELISA can detect 88% (29 out of 33) of Q fever patient sera collected from Marines deployed to Iraq. Less than 5% (5 out of 156) of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies. PMID:26904739

  3. Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor

    PubMed Central

    Weng, Xuan; Gaur, Gautam; Neethirajan, Suresh

    2016-01-01

    The risks associated with the presence of hidden allergens in food have increased the need for rapid, sensitive, and reliable methods for tracing food allergens in commodities. Conventional enzyme immunosorbent assay (ELISA) has usually been performed in a centralized lab, requiring considerable time and sample/reagent consumption and expensive detection instruments. In this study, a microfluidic ELISA platform combined with a custom-designed optical sensor was developed for the quantitative analysis of the proteins wheat gluten and Ara h 1. The developed microfluidic ELISA biosensor reduced the total assay time from hours (up to 3.5 h) to 15–20 min and decreased sample/reagent consumption to 5–10 μL, compared to a few hundred microliters in commercial ELISA kits, with superior sensitivity. The quantitative capability of the presented biosensor is a distinctive advantage over the commercially available rapid methods such as lateral flow devices (LFD) and dipstick tests. The developed microfluidic biosensor demonstrates the potential for sensitive and less-expensive on-site determination for rapidly detecting food allergens in a complex sample system. PMID:27338488

  4. Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor.

    PubMed

    Weng, Xuan; Gaur, Gautam; Neethirajan, Suresh

    2016-01-01

    The risks associated with the presence of hidden allergens in food have increased the need for rapid, sensitive, and reliable methods for tracing food allergens in commodities. Conventional enzyme immunosorbent assay (ELISA) has usually been performed in a centralized lab, requiring considerable time and sample/reagent consumption and expensive detection instruments. In this study, a microfluidic ELISA platform combined with a custom-designed optical sensor was developed for the quantitative analysis of the proteins wheat gluten and Ara h 1. The developed microfluidic ELISA biosensor reduced the total assay time from hours (up to 3.5 h) to 15-20 min and decreased sample/reagent consumption to 5-10 μL, compared to a few hundred microliters in commercial ELISA kits, with superior sensitivity. The quantitative capability of the presented biosensor is a distinctive advantage over the commercially available rapid methods such as lateral flow devices (LFD) and dipstick tests. The developed microfluidic biosensor demonstrates the potential for sensitive and less-expensive on-site determination for rapidly detecting food allergens in a complex sample system. PMID:27338488

  5. Interlaboratory Study of ELISA Kits for the Detection of Egg and Milk Protein in Processed Foods.

    PubMed

    Kato, Shigeki; Yagi, Takahiro; Kato, Ayako; Yamamoto, Shunsuke; Akimoto, Masanobu; Arihara, Keizo

    2015-01-01

    The labeling of seven specific allergenic ingredients (egg, milk, wheat, buckwheat, peanut, shrimp, and crab) is mandatory in Japan. To ensure proper labeling, two kinds of ELISA kits using polyclonal antibodies have been developed. However, we developed two novel ELISA kits using monoclonal antibodies with improved specificity, the Allergeneye ELISA Egg (AE-Egg) and Allergeneye ELISA Milk (AE-Milk) Kits, to detect egg and milk proteins in processed foods, respectively. Five types of processed food containing 10 mg/kg of egg or milk soluble protein were prepared for an interlaboratory study of the performance of these kits. The kits showed a relatively high reproducibility level of interlaboratory precision (AE-Egg RSDR, 3.7-5.7%; AE-Milk RSDR, 6.8-10.5%) and satisfied the recovery rate stipulated by Japanese guidelines (AE-Egg, 61.6-89.3%; AE-Milk, 52.1-67%) for all processed foods. Our results suggest that the AE-Egg and AE-Milk Kits are precise and reliable tools for detecting egg or milk proteins in processed foods. PMID:26086260

  6. Isoelectric focusing and ELISA for detecting adulteration of donkey milk with cow milk.

    PubMed

    Pizzano, Rosa; Salimei, Elisabetta

    2014-06-25

    Donkey milk has been recently revalued intensely due to its nutritional properties. Moreover, donkey milk has been proposed as an effective alternative food for some infants with cow milk allergy. Two fast analytical methods were proposed to detect the fraudulent practice of blending cow milk to donkey milk. Detection of cow αs1-casein bands along the profiles of experimental donkey-cow milk mixtures analyzed by isoelectric focusing was adequate to estimate cow milk used as adulterant of donkey milk starting from 5% (v/v). An ELISA-based method using the antipeptide antibodies raised against the 1-28 sequence stretch of cow β-casein was also developed for an accurate definition of composition of donkey-cow milk mixtures. The presence of cow milk at levels as low as 0.5% (v/v) was detected in donkey-cow milk mixtures prepared at laboratory scale and assayed by ELISA. PMID:24892189

  7. Development and Optimization of a Homemade ELISA Kit for Detection of Antibodies Against Haemophilus influenzae Type b

    PubMed Central

    Mousavi, Seyed Fazlolah; Fatemi, Sara; Siadat, Seyed Davar; Zahraei, Seyed Mohsen; Nikanpour, Elnaz; Malekan, Mohamad Ali; Khabiri, Ali Reza; Janani, Ali Reza

    2016-01-01

    Background: Haemophilus influenzae type b (Hib) infection has high morbidity and mortality rate, especially in children under 5 years of age. Enzyme-linked immunosorbent assay (ELISA) technique is the most used method to detect antibodies against H. influenzae. Available commercial ELISA kits are expensive and not always readily available, particularly for epidemiological studies. Objectives: This study was performed to develop and optimize a homemade ELISA kit for the detection of Hib anti-polyribosylribitol phosphate (PRP) antibodies in children. Materials and Methods: To develop and optimize an indirect ELISA method, pure PRP was prepared. The PRP was coupled to bovine serum albumin, using sodium periodate. Then optimal conditions for ELISA, including coating antigen concentration and peroxidase labeled conjugate concentrations, incubation temperature and incubation time, were determined. To confirm the efficacy of optimized kit, 83 serum samples from non-vaccinated children, aged less than 6 years were collected and analyzed, using homemade and commercial ELISA. Results: The optimal conditions were considered to perform ELISA. Comparison between results obtained from optimized ELISA kit and commercial ELISA kit showed a good agreement. Conclusions: Taking into account these data, we elaborated a homemade ELISA kit that is an efficacious and cost-effective substitute for commercial kit, in disease control and diagnosis. PMID:27540453

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

    PubMed

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

    2016-01-01

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

  9. Development of a lipovitellin-based goldfish (Carassius auratus) vitellogenin ELISA for detection of environmental estrogens.

    PubMed

    Wang, Jun; Wang, Wei; Zhang, Xiaona; Tian, Hua; Ru, Shaoguo

    2015-08-01

    The susceptibility of vitellogenin (Vtg) to degradation is a major problem affecting the robustness of enzyme-linked immunosorbent assay (ELISA) for goldfish (Carassius auratus) Vtg. In this study, a phospholipoglycoprotein with molecular mass of ∼420kDa was purified from goldfish egg extracts and it produced a single band corresponding to ∼112kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the amino acid composition of the purified protein was comparable to that of lipovitellin (Lv) from other fish species. Thus, the purified protein was identified as goldfish Lv. Purified Lv and anti-Lv polyclonal antiserum were used to develop an ELISA with a detection range between 31.25 and 1000ngmL(-)(1). The intra- and inter-assay coefficients of variation were 6.45% and 7.08%, respectively. The immunological similarity between goldfish Vtg and Lv was confirmed by immunoelectrophoresis and Western blot. Goldfish Lv showed higher stability than Vtg after -80°C storage, multiple freeze/thaw cycles, and heat treatment. Moreover, the use of treated Lv in the ELISA did not change the slopes of standard curves. Parallelism between the Lv standard curve and plasma dilution curves of vitellogenic females confirmed the validity of the assay for quantifying plasma Vtg. The Lv-based Vtg ELISA was further applied to evaluate the estrogenic activity of monocrotophos pesticide. PMID:25855009

  10. Evaluation of an inhouse rapid ELISA test for detection of giardia in domestic sheep (Ovis aries).

    PubMed

    Wilson, Jolaine M; Hankenson, F Claire

    2010-11-01

    Sheep (Ovis aries) are increasingly used at our institution as models of human disease. Within the research environment, routine husbandry and handling of sheep has potential for transmission of zoonotic agents, including Giardia. The prevalence of Giardia in sheep may approach 68%. Classic diagnostic testing involves microscopic examination for fecal cysts or trophozoites; however, limitations of microscopy include time, labor, and potential false-negative results due to intermittent shedding. We wished to determine whether a commercial rapid ELISA used for Giardia detection in dogs and cats could be used in sheep. Fecal samples collected from sheep (n = 93) were tested with a combination of 6 methods: reference laboratory fecal flotation, reference laboratory ELISA, inhouse fecal flotation, and commercially available tests (enzyme immunoassay, direct fluorescence antibody assay, and rapid ELISA). Prevalence of Giardia infection in facility sheep was 11.8% (11 of 93 animals). Of the 11 samples considered positive, 3 were confirmed by multiple testing methods, and 5 were positive by microscopy alone. Inhouse fecal flotation for 8 samples was positive on only 1 of 2 consecutive testing days. The rapid ELISA test exhibited 0% sensitivity for sheep giardiasis. Overall, the examined methods had low sensitivities and low positive predictive values. Despite limitations, microscopic analysis of repeat fecal samples remained the most accurate diagnostic method for ovine giardiasis among the methods tested. PMID:21205445

  11. Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens

    PubMed Central

    Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A.; González-Warleta, Marta; Mezo, Mercedes; Ubeira, Florencio M.

    2016-01-01

    ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1–10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis. PMID:27438470

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

    PubMed Central

    2010-01-01

    Background Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection. Results In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples. Conclusions Considering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry. PMID:21192824

  13. Detection of expressed IL-32 in human stomach cancer using ELISA and immunostaining.

    PubMed

    Seo, Eun-Hee; Kang, Jeongwoo; Kim, Ki-Hong; Cho, Min-Chul; Lee, Sojung; Kim, Hee-Jong; Kim, Jung-Hee; Kim, Eun-Jin; Park, Dong-Ki; Kim, Soo-Hyun; Choi, Yang Kyu; Kim, Jin Man; Hong, Jin Tae; Yoon, Do-Young

    2008-09-01

    Interleukin (IL)-32 is a recently identified proinflammatory cytokine that is one of the IL-18 inducible genes, and plays an important role in autoimmune and inflammatory diseases. We produced antibodies against IL-32 and studied the expression of IL-32 in human stomach cancer. We detected IL-32 secreted from K-562 cells that werw stably transfected with IL-32 and in the sera of stomach cancer patients, by a sandwich ELISA using a monoclonal antibody KU32-52 and a polyclonal antibody. In order to optimize a sandwich immunoassay, recombinant IL-32alpha was added, followed by the addition of a biotinylated KU32-52 into microtiter plate wells precoated with a goat anti-IL-32 antibody. The bound biotinylated KU32-52 was probed with a streptavidin conjugated to HRP. This sandwich ELISA was highly specific and had a minimal detection limit of 80 pg/ml (mean+/-SD of zero calibrator) and measuring up to 3,000 pg/ml. This ELISA showed no cross-reaction with other cytokines such as hIL-1alpha, hIL-1beta, hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. Intra-assay coefficients of variation were 18.5% to 4.6% (n=10), and inter-assay coefficients were 23% to 9% (n=10). The average IL-32 level in the sera of 16 stomach cancer patients (189 pg/ml) was higher than that of 12 healthy control men (109 pg/ml). Our results indicate that serum IL-32 level can be detected by using an established ELISA, and that this immunoassay and mAb KU32-09 specific for immunohistochemistry can be used in the detection of expressed and secreted IL- 32 in stomach cancer patients. PMID:18852519

  14. Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens.

    PubMed

    Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A; González-Warleta, Marta; Mezo, Mercedes; Ubeira, Florencio M

    2016-07-01

    ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1-10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis. PMID:27438470

  15. Detection of tetracosactide in plasma by enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Martin, Laurent; Chaabo, Ayman; Lasne, Françoise

    2015-06-01

    As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme-linked immunosorbent assay (ELISA) method is proposed for detection of this drug in plasma. Since its structure corresponds to the 24 N-terminal of the 39 amino acids of the natural endogenous peptide ACTH, tetracosactide can be detected with a commercial ELISA kit for ACTH that uses antibodies, the epitopes of which are located in the 1-24 part of ACTH. However, an essential condition for detection specificity is the preliminary total clearance of endogenous ACTH in the plasma samples. This is achieved by a preparative step based on cation-exchange chromatography before ELISA. The method is specific and sensitive (LOD: 30 pg/mL) and may be used as a screening analysis in anti-doping control. The pre-analytical conditions are shown to be of the upmost importance and recommendations for blood collection (EDTA tubes), sample transport (4 °C) and plasma sample storage (-20 °C) are presented. PMID:25219545

  16. Development of an ELISA and Immunochromatographic Strip for Highly Sensitive Detection of Microcystin-LR

    PubMed Central

    Liu, Liqiang; Xing, Changrui; Yan, Huijuan; Kuang, Hua; Xu, Chuanlai

    2014-01-01

    A monoclonal antibody for microcystin–leucine–arginine (MC-LR) was produced by cell fusion. The immunogen was synthesized in two steps. First, ovalbumin/ bovine serum albumin was conjugated with 6-acetylthiohexanoic acid using a carbodiimide EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)/ NHS (N-hydroxysulfosuccinimide) reaction. After dialysis, the protein was reacted with MC-LR based on a free radical reaction under basic solution conditions. The protein conjugate was used for immunization based on low volume. The antibodies were identified by indirect competitive (ic)ELISA and were subjected to tap water and lake water analysis. The concentration causing 50% inhibition of binding of MC-LR (IC50) by the competitive indirect ELISA was 0.27 ng/mL. Cross-reactivity to the MC-RR, MC-YR and MC-WR was good. The tap water and lake water matrices had no effect on the detection limit. The analytical recovery of MC-LR in the water samples in the icELISA was 94%–110%. Based on this antibody, an immunochromatographic biosensor was developed with a cut-off value of 1 ng/mL, which could satisfy the requirement of the World Health Organization for MC-LR detection in drinking water. This biosensor could be therefore be used as a fast screening tool in the field detection of MC-LR. PMID:25120158

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

    PubMed

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

    2014-09-01

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

  18. Validation and comparison of a sandwich ELISA, two competitive ELISAs and a real-time PCR method for the detection of lupine in food.

    PubMed

    Ecker, Christina; Ertl, Anna; Pulverer, Walter; Nemes, Albert; Szekely, Pal; Petrasch, Angelika; Linsberger-Martin, Gertrud; Cichna-Markl, Margit

    2013-11-01

    Methods applied in food allergen analysis should be specific, sensitive and applicable to both raw and highly processed foods. The performance of the most commonly used methods, ELISA and real-time PCR, may, however, be influenced by food processing steps, e.g., heat treatment. The present study compares the applicability of four in-house developed methods, one sandwich ELISA, two competitive ELISAs and a real-time PCR method, for the detection of lupine in four different food matrices, comprising bread, biscuits, rice patties and noodles. In order to investigate the influence of food processing on the detectability, not only the heat treated model foods but also the corresponding doughs were analysed. The sandwich ELISA proved to be the most sensitive method. The LOD was found to be 10 ppm lupine, independent from the food matrix and independent if the dough or the heat treated food was analysed. In addition, the methods were applied to the analysis of commercial foodstuffs differing in their labelling. PMID:23768374

  19. Detecting polysaccharide antigen of Neisseria meningitidis group C in cerebrospinal fluid by dot-ELISA assay.

    PubMed

    Correia Barbosa, S F; Alkmin, M G; Landgraf, I M

    2000-06-01

    Cerebrospinal fluid (CSF) samples from 210 patients (200 with clinical evidence of bacterial meningitis, 10 with other clinical neurologic disease) were tested by a Dot-ELISA assay for detection of polysaccharide antigen of N. meningitidis group C. CSF samples were treated with EDTA 0.1 M, at pH 7.5 and heated to 90>C for 10 min. Polyclonal antiserum was purified by use of ethanol fractionation. The results were compared to those using bacterial culture (BC), latex agglutination (LA), counterimmunoelectrophoresis (CIE), and direct microscopy (DM) methods. Test results showed a correlation of 93.3%, 94.3%, 91.0% and 69.5% respectively, and sensitivity of 0.947 and specificity of 0.930. This study suggests that the dot-ELISA assay of CSF is a useful alternative technique for the diagnosis of group C meningitis. PMID:10934498

  20. Sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of lupine residues in foods.

    PubMed

    Kaw, C H; Hefle, S L; Taylor, S L

    2008-10-01

    Lupine has been increasingly used in food applications due to its high nutritional value and excellent functional properties. However, lupine provokes allergic reactions in susceptible individuals. The presence of undeclared lupine residues in foods can pose a serious health risk to lupine-allergic individuals. Therefore, the objective of this research was to develop a sandwich-type ELISA for the detection of lupine residues in foods. Lupine flour derived from Lupinus albus was used to immunize 3 rabbits and a sheep. Pooled lupine-specific antibodies were partially purified from the sera by ammonium sulfate precipitation. A sandwich lupine ELISA with a limit of quantification (LOQ) of 1 ppm was developed by utilizing the rabbit antisera as the capture reagent and the sheep antiserum as the detector reagent. The binding of the antigen-antibody complex was visualized by the addition of commercial rabbit antisheep IgG antibody labeled with alkaline phosphatase with subsequent addition of p-nitrophenyl phosphate substrate to produce a colored product for quantification. Minor cross-reactivity was observed with soy (Glycine max) and black bean (Castanospermum australe). The performance of the lupine ELISA was evaluated in reference food standards (beef frankfurter and apple cinnamon muffin) and laboratory-prepared cooked frankfurters and corn muffins. The mean percent recovery for lupine spiked-frankfurters and corn muffins were 108.4%+/- 8.8% and 103.1%+/- 11.5%, respectively. The sandwich-type lupine ELISA developed in this study provides food manufacturers and regulatory agencies with an effective analytical tool to detect and quantify lupine residues in processed foods. PMID:19019135

  1. High Sensitive Detection of Carbohydrate Binding Proteins in an ELISA-Solid Phase Assay Based on Multivalent Glyconanoparticles

    PubMed Central

    Chiodo, Fabrizio; Marradi, Marco; Tefsen, Boris; Snippe, Harm; van Die, Irma; Penadés, Soledad

    2013-01-01

    Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers. PMID:24014084

  2. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye

    NASA Astrophysics Data System (ADS)

    de La Rica, Roberto; Stevens, Molly M.

    2012-12-01

    In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye. The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates coloured solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10-18 g ml-1. p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid-based test.

  3. An ELISA kit with two detection modes for the diagnosis of lymphatic filariasis.

    PubMed

    Wongkamchai, S; Satimai, W; Loymek, S; Nochot, H; Boitano, J J

    2015-09-01

    The aim of this study was to develop a low-cost antifilarial immunoglobulin (Ig) G4 detection kit for the diagnosis of lymphatic filariasis. The kit was designed to be used by minimally trained personnel without the constraints of expensive laboratory equipment. We provide a description of the development and validation of a single-serum-dilution based enzyme-linked immunosorbent assay (ELISA) kit with ready-to-use reagents for measuring antifilarial IgG4 antibodies. The kit was tested on residents in Brugia malayi-endemic areas in southern Thailand. Detection was performed by naked-eye observation of the resultant colour of the immunological reactivity. The coefficient of variation (CV) was used to assess the reproducibility of the results. Long-term stability was measured over a 6-month period. Sensitivity of the test kit was 97% when compared with microfilariae detection in thick blood smears. Specificity was 98.7% based on the sera of 57 patients living outside the endemic areas who were infected with other parasites and 100 parasite-free subjects. All positive CVs were < 10%. The test kit was remarkably stable over 6 months. Field validation was performed by the detection of antifilarial IgG4 in 4365 serum samples collected from residents of brugian filariasis-endemic areas and compared with outcome colours of the test samples by the naked eye. Subsequent ELISA evaluation of these results using an ELISA reader indicated high agreement by the kappa statistic. These results demonstrate that the test kit is efficient and useful for public health laboratories as an alternative tool for the diagnosis of lymphatic filarial infection. PMID:24916386

  4. A blocking ELISA for the detection of antibodies to psittacine beak and feather disease virus (BFDV).

    PubMed

    Shearer, Patrick L; Sharp, Margaret; Bonne, Nicolai; Clark, Phillip; Raidal, Shane R

    2009-06-01

    Currently, the only diagnostic test available routinely for the sero-diagnosis of BFDV is the haemagglutination-inhibition (HI) assay. This test, whilst useful and applicable to samples from a wide range of psittacine birds, is not an ideal assay; it requires erythrocytes from live animals, virus purified from the feathers of infected birds and polyclonal antibody preparations in order to perform the assay. Variations in these reagents make consistency between tests difficult to achieve, underscoring the need for a new test with standardised reagents for the sero-diagnosis of BFDV infection which has led to the development of an antibody response. The methods used to develop a novel "blocking" (or "competitive") ELISA (bELISA) for the detection of anti-BFDV antibodies in psittacine sera are presented in this paper. The assay was developed using a baculovirus-expressed recombinant BFDV capsid protein and a newly developed monoclonal antibody raised against this protein. The assay was then validated with 160 samples from eastern long-billed corellas (Cacatua tenuiostris) vaccinated with the recombinant capsid protein and challenged with live virus and samples from 82 cockatiels known to be HI negative. The bELISA described in this study is a sensitive and specific diagnostic test and should have wide application for the sero-diagnosis of BFDV. PMID:19428582

  5. Transferability of antibody pairs from ELISA to fiber optic surface plasmon resonance for infliximab detection

    NASA Astrophysics Data System (ADS)

    Van Stappen, Thomas; Lu, Jiadi; Bloemen, Maarten; Geukens, Nick; Spasic, Dragana; Delport, Filip; Verbiest, Thierry; Lammertyn, Jeroen; Gils, Ann

    2015-03-01

    Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine up-regulated in inflammatory bowel disease, rheumatoid arthritis and psoriasis. The introduction of anti-TNF drugs such as infliximab has revolutionized the treatment of these diseases. Recently, therapeutic drug monitoring (TDM) of infliximab has been introduced in clinical decision making to increase cost-efficiency. Nowadays, TDM is performed using radio-immunoassays, homogeneous mobility shift assays or ELISA. Unfortunately, these assays do not allow for in situ treatment optimization, because of the required sample transportation to centralized laboratories and the subsequent assay execution time. In this perspective, we evaluated the potential of fiber optic-surface plasmon resonance (FO-SPR). To achieve this goal, a panel of 55 monoclonal anti-infliximab antibodies (MA-IFX) was developed and characterized in-house, leading to the identification of nine different clusters. Based on this high diversity, 22 antibody pairs were selected and tested for their reactivity towards IFX, using one MA-IFX as capture and one MA-IFX for detection, in a sandwich type ELISA and FO-SPR. This study showed that the reactivity towards IFX of each antibody pair in ELISA is highly similar to its reactivity on FO-SPR, indicating that antibody pairs are easily transferable between both platforms. Given the fact that FO-SPR shows the potential for miniaturization and fast assay time, it can be considered a highly promising platform for on-site infliximab monitoring.

  6. Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

    PubMed

    Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

    2008-06-22

    Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats. PMID:18182256

  7. Direct ELISA.

    PubMed

    Lin, Alice V

    2015-01-01

    First described by Engvall and Perlmann, the enzyme-linked immunosorbent assay (ELISA) is a rapid and sensitive method for detection and quantitation of an antigen using an enzyme-labeled antibody. Besides routine laboratory usage, ELISA has been utilized in medical field and food industry as diagnostic and quality control tools. Traditionally performed in 96-well or 384-well polystyrene plates, the technology has expanded to other platforms with increase in automation. Depending on the antigen epitope and availability of specific antibody, there are variations in ELISA setup. The four basic formats are direct, indirect, sandwich, and competitive ELISAs. Direct ELISA is the simplest format requiring an antigen and an enzyme-conjugated antibody specific to the antigen. This chapter describes the individual steps for detection of a plate-bound antigen using a horseradish peroxidase (HRP)-conjugated antibody and luminol-based enhanced chemiluminescence (ECL) substrate. The methodological approach to optimize the assay by chessboard titration is also provided. PMID:26160564

  8. Preparation and identification of monoclonal antibody against Citreoviridin and development of detection by Ic-ELISA.

    PubMed

    Jin, Ni; Ling, Sumei; Yang, Chi; Wang, Shihua

    2014-11-01

    Citreoviridin (CIT), a neurotoxic mycotoxin produced by Penicillium citreonigrum is generally detected in cereal grains and agricultural products worldwide, and has numerous toxicological effects on human and animal health. Therefore, it is necessary to develop a rapid, sensitive, and reliable immunoassay method for CIT. In this study, artificial antigen CIT-KLH and CIT-BSA was successfully prepared via succinic anhydride and carbodiimide two-step method. CIT-KLH conjugates were injected into Balb/c mice, and titer of the antiserum against CIT was determined using CIT-BSA as coating antigen by ELISA method. A hybridoma cell line 8D8 stably secreting monoclonal antibody against CIT was generated by fusing SP2/0 myeloma cells with the splenocytes from the immunized mice. The titer of 8D8 mAb reached 1: 1.28 × 10(5) after purified by caprylic/ammonium sulfate precipitation (CA-AS) method. The 8D8 mAb was identified as IgG1 subtype. The cross-reactivity results indicated that anti-CIT mAb was highly specific to Citreoviridin, and the average affinity 4.57 × 10(8) L/mol. A sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for CIT was established. Under optimal condition, the linear range to detect CIT was 11.02-2370.48 ng/mL with IC50 of 161.66 ng/mL and the limit of detection of the ic-ELISA was 11.86 ng/mL. With the mean coefficient of variation lowing 5%, the mean recovery in intra-assay and inter-assay were (90.06 ± 1.60)% and (89.65 ± 1.69)%, respectively. Therefore, the anti-CIT mAb secreted by 8D8 hybridoma cell line is useful for analysis of food contaminated with CIT. PMID:25157801

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

    PubMed

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

    2013-02-01

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

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

    PubMed

    Chen, Hao-tai; Peng, Yun-hua; Zhang, Yong-guang; Liu, Xiang-tao

    2012-12-01

    An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Further, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suitable for diagnosis of FMDV serotype O infection in field stations. PMID:23244327

  11. Development of an ELISA to detect clenbuterol in swine products using a new approach for hapten design.

    PubMed

    Bui, Quoc Anh; Vu, Thi Huynh Han; Ngo, Vo Ke Thanh; Kennedy, Ivan R; Lee, N Alice; Allan, Robin

    2016-09-01

    This research outlines the application of an enzyme-linked immunosorbent assay (ELISA) for the analysis of clenbuterol in animal products. Our assay showed good sensitivity for clenbuterol (0.4 ng/g or 0.4 ppb) and low detection limit (0.09 ng/g or 0.09 ppb). A low cross-reactivity for other β2-agonist drugs such as salbutamol, terbutaline, and epinephrine led to formatting an ELISA kit considered to have a high specificity for clenbuterol. A survey of Ho Chi Minh City pork market was conducted as part of the validation of our ELISA. ELISA results showed a surprisingly high value of contamination. However, it will be necessary to conduct a more statistically valid replicated survey with evaluation by other instrumental methods to obtain a definite conclusion. This ELISA kit will be used to monitor growth promoter residues in Vietnam's animal products. PMID:27481170

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

    PubMed Central

    Stanker, Larry H.; Scotcher, Miles C.; Cheng, Luisa; Ching, Kathryn; McGarvey, Jeffery; Hodge, David; Hnasko, Robert

    2013-01-01

    Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A – H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10−11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested. PMID:24253240

  13. Development of a fast ELISA for the specific detection of both leucomalachite green and malachite green

    NASA Astrophysics Data System (ADS)

    Jiang, Yousheng; Chen, Li; Hu, Kun; Yu, Wenjuan; Yang, Xianle; Lu, Liqun

    2015-04-01

    Malachite green (MG), a dye, is an antifungal agent that has been used to treat and prevent fish diseases. It is metabolized into reduced leucomalachite green forms (LMG) that may reside in fish muscles for a long period, thus being harmful to human health. The aim of this study was to develop a competitive and direct enzyme-linked immunosorbent assay (ELISA) to detect MG and LMG specifically. The monoclonal antibody (mAb) to LMG was generated using a hybridoma technique. The obtained mAb showed good cross-reactivity (CR) to malachite green (MG), but not to crystal violet (CV) and Brilliant Green (BG). The mAb was used to develop a fast detecting ELISA of MG and LMG in fish. By introducing the conjugation LMG-HRP, the detection capability was 0.37 ng mL-1 for MG and LMG. The mean recovery from spiked grass carp tissues ranged from 76.2% to 82.9% and the coefficients of variation varied between 1.8% and 7.5%. The stable and efficient monoclonal cell line obtained is a sustainable source of sensitive and specific antibody to MG and LMG.

  14. Detection of Legionella pneumophila by PCR-ELISA method in industrial cooling tower water.

    PubMed

    Soheili, Majid; Nejadmoghaddam, Mohammad Reza; Babashamsi, Mohammad; Ghasemi, Jamileh; Jeddi Tehrani, Mahmood

    2007-11-15

    Water supply and Cooling Tower Water (CTW) are among the most common sources of Legionella pneumophila (LP) contamination. A nonradio active method is described to detect LP in industrial CTW samples. DNA was purified and amplified by nested -PCR with amplimers specific for the 16s rRNA gene of LP. The 5' end biotinylated oligomer probe was immobilized on sterptavidin B coated microtiter plates. The nested-PCR product was labeled with digoxigenin and then hybridized with 5'-biotinylated probes. The amplification products were detected by using proxidase-labled anti dioxygenin antibody in a colorimetric reaction. The assay detected LP present in 1 L of 5 CTW samples examined. All of the samples were Legionella positive in both culture and PCR-ELISA methods. The PCR-ELISA assay appears to exhibit high specificity and is a more rapid technique in comparison with bacterial culture method. Thus could prove suitable for use in the routine examination of industrial CTW contamination. PMID:19090273

  15. Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer

    PubMed Central

    2014-01-01

    Background The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA [EVELISA]), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer. Methods By using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 [uninfected, n = 40; naturally infected, n = 22]) and white-tailed deer (n = 41 [uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9]). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting. Results Among the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively. Conclusion The results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species. PMID:24992970

  16. [Definitive ability of Stamp-staining, antigen-ELISA, PCR and cell culture for the detection of Coxiella burnetii].

    PubMed

    Henning, Klaus; Sting, Reinhard

    2002-01-01

    Many assays are used for the detection of the aetiological agent of Q fever, Coxiella burnetii, i.e. staining according to the method of Stamp, capture ELISA, PCR or isolation by cell culture. In this study the results of these four assays are compared for their sensitivity and specificity. Staining smears according to the method of Stamp gave many false positive or false negative results. The capture ELISA seems to be a very sensitive assay for the detection of Coxiella burnetii but it has a lack in specificity. It is a useful test system for the screening of large scales of samples. Positive ELISA results should be confirmed by PCR, a very sensitive and specific method for the detection of Coxiella burnetii but more time consumptive than the ELISA. Isolation of the agent using cell cultures was not completely satisfactory because of its lack in sensitivity. Therefore it should only be used in special cases. PMID:12357676

  17. Microchip-based enzyme-linked immunosorbent assay (microELISA) system with thermal lens detection.

    PubMed

    Sato, Kiichi; Yamanaka, Maho; Hagino, Tomokazu; Tokeshi, Manabu; Kimura, Hiroko; Kitamori, Takehiko

    2004-12-01

    A microchip-based enzyme-linked immunosorbent assay (microELISA) system was developed and interferon-gamma was successfully determined. The system was composed of a microchip with a Y-shaped microchannel and a dam structure, polystyrene microbeads, and a thermal lens microscope (TLM). All reactions required for the immunoassay were done in the microchannel by successive introduction of a sample and regents. The enzyme reaction product, in a liquid phase, was detected downstream in the channel using the TLM as substrate solution was injected. The antigen-antibody reaction time was shortened by the microchip integration. The limit of the determination was improved by adopting the enzyme label. Moreover, detection procedures were greatly simplified and required time for the detection was significantly cut. The system has good potential to be developed as a small and automated high throughput analyzer. PMID:15570367

  18. A novel inhibition ELISA for the detection and monitoring of Penicillium marneffei antigen in human serum.

    PubMed

    Prakit, K; Nosanchuk, J D; Pruksaphon, K; Vanittanakom, N; Youngchim, S

    2016-04-01

    The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment. PMID:26838686

  19. Development and Application of an ELISA for the Detection of Porcine Deltacoronavirus IgG Antibodies

    PubMed Central

    Thachil, Anil; Gerber, Priscilla F.; Xiao, Chao-Ting; Huang, Yao-Wei; Opriessnig, Tanja

    2015-01-01

    Porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was first detected in North America in early 2014 and associated with enteric disease in pigs, resulting in an urgent need to further investigate the ecology of this virus. While assays detecting nucleic acids were implemented quickly, assays to detect anti-PDCoV antibodies have not been available. In this study, an indirect anti-PDCoV IgG enzyme-linked immunosorbent assay (ELISA) based on the putative S1 portion of the spike protein was developed and utilized to determine the prevalence of anti-PDCoV IgG in U.S. pigs. The diagnostic sensitivity of the PDCoV ELISA was 91% with a diagnostic specificity of 95%. A total of 968 serum samples were tested including samples with confirmed infection with PDCoV, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus or porcine respiratory coronavirus. There was no cross-reactivity with any of the other coronaviruses. Among 355 arbitrarily selected serum samples collected in 2014 and originating from 51 farms across 18 U.S. states, anti-PDCoV IgG antibodies were detected in 8.7% of the samples and in 25.5% of the farms whereas anti-PEDV IgG was detected in 22.8% of the samples and in 54.9% of the farms. In addition, anti-PDCoV IgG antibodies were detected in archived samples collected in 2010, perhaps indicating an earlier undetected introduction into the U.S. pig population. Overall, the obtained data suggest that PDCoV seroprevalence in U.S. pigs is lower compared to PEDV and PDCoV may have been introduced to the U.S. prior to PEDV. PMID:25881086

  20. An ELISA method detecting the active form of suPAR.

    PubMed

    Zhou, Xiaolei; Xu, Mingming; Huang, Hailong; Mazar, Andrew; Iqbal, Zafar; Yuan, Cai; Huang, Mingdong

    2016-11-01

    Urokinase plasminogen activator receptor (uPAR) exists in a number of formats in human plasma, including soluble uPAR (suPAR) and uPAR fragments. We developed an ELISA method to detect specifically the active form suPAR, which binds to its natural ligand uPA. The intra CV and inter CV of this ELISA assay is 8.5% and 9.6% respectively, and the assay can recover 99.74% of added recombinant suPAR from 10% plasma. This assay is quite sensitive, capable of detecting down to 15pg/ml of suPAR, and can measure suPAR concentrations in the range of 0.031-8ng/ml with high linear relationship. Plasma samples from pregnant women were also measured for the active form of suPAR with this assay, giving an averaged level of 1.39ng/ml, slightly higher than the level of pooled plasma from healthy donors (0.96ng/ml). This study demonstrates the feasibility to measure the active form of suPAR, which will likely have value in clinical applications. PMID:27591605

  1. Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin*

    PubMed Central

    Zhang, Hai-tang; Jiang, Jin-qing; Wang, Zi-liang; Chang, Xin-yao; Liu, Xing-you; Wang, San-hu; Zhao, Kun; Chen, Jin-shan

    2011-01-01

    Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method was employed to synthesize the artificial antigen of enrofloxacin (ENR), and New Zealand rabbits were used to produce anti-ENR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (ELISA) standard curve was established. This assay was sensitive and had a linear range from 0.6 to 148.0 μg/kg (R 2=0.9567), with the half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 9.4 μg/kg and 0.2 μg/kg, respectively. Of all the competitive analogues, the produced pAb exhibited a high cross-reactivity to ciprofloxacin (CIP) (87%), the main metabolite of ENR in tissues. After optimization, the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork. The overall recoveries and coefficients of variation (CVs) were in the ranges of 86%–109% and 6.8%–13.1%, respectively. It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues. PMID:22042652

  2. Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA.

    PubMed

    Wilson, T; Carson, J; Bowman, J

    2002-10-01

    A simple, high-throughput, low-cost polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) protocol that detects the presence of 4 fg of DNA from four bacterial fish pathogens Yersinia ruckeri, Tenacibaculum maritimum (formerly Flexibacter maritimus), Lactococcus garvieae and Aeromonas salmonicida was developed. DNA amplification was undertaken in a biphasic system with free and bound PCR that are achieved in the one NucleoLink tube. Solid-phase amplicons were detected using biotin labelled hybridization probes and visualised colourimetrically with streptavidin-alkaline phosphatase and p-nitrophenylphosphate as substrate. PCR and hybridization took less than 8 h to perform with maximum signal output for femtogram amounts of template DNA achieved within 24 h. Implementation and optimization of the protocol is discussed. PMID:12133608

  3. Development of dual-function ELISA for effective antigen and antibody detection against H7 avian influenza virus

    PubMed Central

    2013-01-01

    Background Outbreaks in poultry involving influenza virus from H7 subtype have resulted in human infections, thus causing a major concern for public health, as well as for the poultry industry. Currently, no efficient rapid test is available for large-scale detection of either antigen or antibody of H7 avian influenza viruses. Results In the present study, a dual function ELISA was developed for the effective detection of antigen and antibody against H7 AIVs. The test was established based on antigen-capture-ELISA and epitope blocking ELISA. The two Mabs 62 and 98 which were exploited in the assay were identified to recognize two conformational neutralizing epitopes on H7 HA1. Both of the epitopes exist in all of the human H7 strains, including the recent H7N9 strain from China and > 96.6% of avian H7 strains. The dual ELISA was able to detect all of the five H7 antigens tested without any cross reaction to other influenza subtypes. The antigen detection limit was less than 1 HA unit of H7. For antibody detection, the sensitivity and specificity of the dual ELISA was evaluated and compared to HI and microneutralization using immunized animal sera to different H7 strains and different subtypes of AIVs. Results indicated that antibodies to H7 were readily detected in immunized animal sera by the dual ELISA whereas specimens with antibodies to other AIVs yielded negative results. Conclusions This is the first dual-function ELISA reported for either antigen or antibody detection against H7 AIVs. The assay was highly sensitive and 100% specific in both functions rendering it effective for H7 diagnosis. PMID:24083616

  4. Development of an antigen-capture ELISA for the detection of avian leukosis virus p27 antigen.

    PubMed

    Yun, Bingling; Li, Delong; Zhu, Haibo; Liu, Wen; Qin, Liting; Liu, Zaisi; Wu, Guan; Wang, Yongqiang; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei; Gao, Yulong

    2013-02-01

    An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs. PMID:23201286

  5. Subattomole detection of adiponectin in urine by ultrasensitive ELISA coupled with thio-NAD cycling

    PubMed Central

    Morikawa, Mika; Naito, Rina; Mita, Koichi; Watabe, Satoshi; Nakaishi, Kazunari; Yoshimura, Teruki; Miura, Toshiaki; Hashida, Seiichi; Ito, Etsuro

    2015-01-01

    Adiponectin is a hormone secreted from adipocytes, and it demonstrates antidiabetic, anti-atherosclerotic, antiobesity and anti-inflammatory effects. However, the patterns of change in urinary adiponectin levels in various diseases remain unknown, because only trace amounts of the hormone are present in urine. In the present study, we applied an ultrasensitive ELISA coupled with thio-NAD cycling to measure urinary adiponectin levels. Spikeand-recovery tests using urine confirmed the reliability of our ultrasensitive ELISA. The limit of detection for adiponectin in urine was 2.3×10−19 moles/assay (1.4 pg/mL). The urinary adiponectin concentration ranged between 0.04 and 5.82 ng/mL in healthy subjects. The pilot study showed that the urinary adiponectin levels, which were corrected by the creatinine concentration, were 0.73±0.50 (ng/mg creatinine, N=6) for healthy subjects, versus 12.02±3.85 (ng/mg creatinine, N=3) for patients with diabetes mellitus (DM). That is, the urinary adiponectin levels were higher (P<0.05) in DM patients than in healthy subjects. Further, these urinary adiponectin levels tended to increase with the progression of DM accompanied with nephropathy. Our method is thus expected to provide a simple, rapid and reasonably priced test for noninvasive monitoring of the progression of DM without the requirement of special tools.

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

    PubMed

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

    2016-01-01

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

  7. Compact USB-powered mobile ELISA-based pathogen detection: design and implementation challenges

    NASA Astrophysics Data System (ADS)

    Starodubov, Dmitry; Asanbaeva, Anya; Berezhnyy, Ihor; Chao, Chung-Yen; Koziol, Richard; Miller, David; Patton, Edward; Trehan, Sushma; Ulmer, Chris

    2011-05-01

    Physical Optics Corporation (POC) presents a novel Mobile ELISA-based Pathogen Detection system that is based on a disposable microfluidic chip for multiple-threat detection and a highly sensitive portable microfluidic fluorescence measurement unit that also controls the flow of samples and reagents through the microfluidic channels of the chip. The fluorescence detection subsystem is composed of a commercial 635-nm diode laser, an avalanche photodiode (APD) that measures fluorescence, and three filtering mirrors that provide more than 100 dB of excitation line suppression in the signal detection channel. Special techniques to suppress the fluorescence and scattering background allow optimizing the dynamic range for a compact package. Concentrations below 100 ng/mL can be reliably identified. The entire instrument is powered using a USB port of a notebook PC and operates as a plug-and-play human-interface device, resulting in a truly peripheral biosensor. The operation of the system is fully automated, with minimal user intervention through the detection process. The resolved challenges of the design and implementation are presented in detail in this publication.

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

    PubMed Central

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

    2016-01-01

    Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 104–2.8 × 106 cells/mL with a detection limit (LOD) of 0.9 × 103 cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens. PMID:26976753

  9. Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-γ detection ex vivo.

    PubMed

    Dai, Hua; Xu, Zheng-Zhong; Wang, Meiling; Chen, Jun-Hua; Chen, Xiang; Pan, Zhi-Ming; Jiao, Xin-An

    2016-04-01

    The aim of the present work was to develop reagents to set up a chicken interferon-γ (ChIFN-γ) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-γ were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-γ, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-γ, and also has an excellent capacity for detecting native ChIFN-γ. This ELISA was then used to detect ChIFN-γ level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-γ. PMID:27127340

  10. Design and synthesis of immunoconjugates and development of an indirect ELISA for rapid detection of 3, 5-dinitrosalicyclic Acid hydrazide.

    PubMed

    Shen, Yu-Dong; Zhang, Shi-Wei; Lei, Hong-Tao; Wang, Hong; Xiao, Zhi-Li; Jiang, Yue-Ming; Sun, Yuan-Ming

    2008-01-01

    In this study novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive indirect ELISA method to directly detect residues of 3,5-dinitrosalicyclic acid hydrazide (DNSH), a toxic metabolite of nifursol present in chicken tissues. The hapten DNSHA was first designed and used to covalently couple to BSA to form an immunogen which was immunized to rabbits to produce a polyclonal antibody against DNSH. Furthermore, a novel 3,5-dinitrosalicylic acidovalbumin (DNSA-OVA) immunoconjugate structurally different from DNSHA-OVA was designed and used as a "substructural coating antigen" to improve the sensitivity of an indirect ELISA analysis for a direct DNSH detection. Based on the "substructural coating antigen" concept, an optimized indirect ELISA method was established that exhibited good specificity and high sensitivity for detecting DNSH, with a cross-reactivity of less than 0.1% (excluding the parent compound nifursol), IC(50) of 0.217 nmol/mL and detection limit of 0.018 nmol/mL. Finally, a simple and efficient analysis of DNSH samples in chicken tissues showed that the average recovery rate of the indirect ELISA analysis was 82.3%, with the average coefficient of variation 15.9%. Thus, the developed indirect ELISA method exhibited the potential for a rapid detection of DNSH residues in tissue. PMID:18830153

  11. Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA.

    PubMed

    Amani, Jafar; Ahmadpour, Askary; Imani Fooladi, Abbas Ali; Nazarian, Shahram

    2015-01-01

    Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins. PMID:25911087

  12. ELISA for the detection of toxic antigens in experimental and clinical envenoming by Tityus serrulatus scorpion venom.

    PubMed

    Chávez-Olórtegui, C; Fonseca, S C; Campolina, D; Amaral, C F; Diniz, C R

    1994-12-01

    An ELISA was developed for identification of circulating toxic antigens from Tityus serrulatus scorpion venom. The toxic fraction from the scorpion venom was purified by Sephadex G-50 chromatography and immunoaffinity techniques were used for identifying antibodies that reacted with this fraction. These antibodies were used to develop a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity for identifying mice that were experimentally inoculated with T. serrulatus venom from those inoculated with Phoneutria nigriventer spider venom, Apis mellifera bee venom and Bothrops atrox, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.1 ng of venom per assay. The ELISA also detected antigens in the sera of patients systemically envenomed by T. serrulatus. Therefore, this ELISA could be a valuable tool for clinicians and epidemiologists, owing to its sensitivity and specificity. PMID:7725332

  13. An indirect ELISA for detection of Theileria spp. antibodies using a recombinant protein (rTlSP) from Theileria luwenshuni.

    PubMed

    He, Haining; Li, Youquan; Liu, Junlong; Liu, Zhijie; Yang, Jifei; Liu, Aihong; Chen, Ze; Ren, Qiaoyun; Guan, Guiquan; Liu, Guangyuan; Luo, Jianxun; Yin, Hong

    2016-07-01

    Theileria is a tick-borne, intracellular protozoan parasite of worldwide economic and veterinary importance in small ruminants. Here, an enzyme-linked immunosorbent assay (ELISA) was developed based on Theileria luwenshuni recombinant surface protein (rTlSP) and was used in the standardization and validation of an ELISA for the detection of circulating antibodies against ovine and caprine theileriosis. A total of 233 sera samples were used for the calculation of the cut-off value which served as a threshold between the positive and the negative sera. When the positive threshold was chosen as 19% of the specific mean antibody rate, the specificity was 97.9%, and the sensitivity was 97.1%. There was a cross-reaction with sera against Theileria uilenbergi and Theileria ovis, and no cross-reaction with sera against Babesia spp. in the ELISA and Western blotting. Two hundred forty samples collected from sheep in Gansu province were detected with blood smears and ELISA, respectively. The results showed that the positive rate of Theileria infection in Gansu province were 63.75% with rTlSP-ELISA, and 46.67% with blood smears, respectively. Our test proved that the rTlSP ELISA is suitable to diagnose Theileria infection and could be used in serological surveys to map out the prevalence of ovine and caprine theileriosis. PMID:27048941

  14. [Comparative studies on detection of Chlamydophila psittaci and Chlamydophila abortus in meat turkey flocks using cell culture, ELISA, and PCR].

    PubMed

    Sting, R; Lerke, E; Hotzel, H; Jodas, S; Popp, C; Hafez, H M

    2006-02-01

    The prevalence of chlamydia in 10 meat turkey flocks was investigated. As samples served of each moment of collection and sex of the animals 10 cloacal swabs which were taken at the age of 1, 4, 8 and 12 (females) or 16 weeks (males) and at the time of slaughter at the age of 16 or 20 weeks. Spleen samples were taken at the time of slaughter, additionally. These were pooled making 1 pool out of 5 individual samples. The cloacal and spleen pools were examined by nested PCR (nPCR), Capture-ELISA and Capture Blocking-ELISA directly as well as after isolation attempts in cell cultures. The most sensitive method to detect chlamydia, with 6 isolates proved to be the isolation by cell culture followed by detection using nPCR. Not corresponding to the results of the nPCR were 4 positive reactions found by the Capture-ELISA which could in no case be affirmed by Capture-Blocking-ELISA. The direct examination of cloacal swab pools by nPCR proved positive in only 2 cases. In contrast to this the examination of these samples by Capture-ELISA showed a high percentage of 71.9% positive results, of which only 2 cases were confirmed by nPCR and none by Capture-Blocking-ELISA. Of the 8 Chlamydia positive results in the nPCR 7 could be classified by DNA sequencing to Cp. abortus and only one to Cp. psittaci. PMID:16555483

  15. A sensitive enhanced chemiluminescent-ELISA for the detection of Plasmodium falciparum circumsporozoite antigen in midguts of Anopheles stephensi mosquitoes.

    PubMed

    Grabias, Bryan; Zheng, Hong; Mlambo, Godfree; Tripathi, Abhai K; Kumar, Sanjai

    2015-01-01

    Efforts to develop a successful malaria vaccine are hampered due to lack of assays that are predictive of protective immunity without conducting large clinical studies. The effect of experimental vaccines and drugs on malaria transmission is yet more difficult to measure. Knowledge on the Plasmodium infection rate in mosquito populations will aid the measurement of effects from intervention measures for malaria control. Here, we report the development of a chemiluminescent sandwich ELISA (ECL-ELISA) that can detect Plasmodium falciparum circumsporozoite protein (Pf CSP) produced in recombinant form at concentrations of 4.4pg and in P. falciparum sporozoites (Pf SPZ) derived from mosquito salivary glands at levels corresponding to 5 Pf SPZ. Most importantly, we demonstrate reliable Pf CSP-based detection of 0.056day 8 P. falciparum oocysts developing inside mosquito midguts in whole mosquito lysates. Cumulatively, the ECL-ELISA is 47× more sensitive for the detection of Pf CSP than a colorimetric ELISA while greatly simplifying sample preparation, obviating the need for cumbersome midgut dissections and allowing high throughput screening of Plasmodium infection in mosquito populations. The ECL-ELISA may also have broader application in diagnosis of infectious diseases and the prognostic value in cancer and other diseases such as auto-immunity and genetic disorders based on antigen detection, or quality validation of biological vaccine components. PMID:25455023

  16. Development of enzyme linked immunosorbent assay (ELISA) for the detection of root-knot nematode Meloidogyne incognita.

    PubMed

    Kapur-Ghai, J; Kaur, M; Goel, P

    2014-09-01

    Root-knot nematodes (Meloidogyne incognita) are obligate, sedentary plant endoparasites that are extremely polyphagous in nature and cause severe economic losses in agriculture. Hence, it is essential to control the parasite at an early stage. For any control strategy to be effective, an early and accurate diagnosis is of paramount importance. Immunoassays have the inherent advantages of sensitivity and specificity; have the potential to identify and quantify these plant-parasitic nematodes. Hence, in the present studies, enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of M.incognita antigens. First an indirect ELISA was developed for detection and titration of anti-M.incognita antibodies. Results indicated as high as 320 K titre of the antisera. Finally competitive inhibition ELISA was developed employing these anti-M.incognita antibodies for detection of M.incognita antigens. Sensitivity of ELISA was 10 fg. Competitive inhibition ELISA developed in the present studies has the potential of being used as an easy, rapid, specific and sensitive diagnostic tool for the detection of M.incognita infection. PMID:25035590

  17. Development of A Sensitive and Specific Epitope-Blocking ELISA for Universal Detection of Antibodies to Human Enterovirus 71 Strains

    PubMed Central

    He, Fang; Kiener, Tanja K.; Lim, Xiao Fang; Tan, Yunrui; Raj, Kattur Venkatachalam Ashok; Tang, Manli; Chow, Vincent T. K.; Chen, Qingfeng; Kwang, Jimmy

    2013-01-01

    Background Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. Methodology/Principal Finding In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6) that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. Conclusion The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera. PMID:23383215

  18. Dose-dependent inhibition of BrdU detection in the cell proliferation ELISA by culture medium proteins.

    PubMed

    Padet, Lauriane; St-Amour, Isabelle; Aubin, Eric; Proulx, Dominic Paquin; Bazin, Renée; Lemieux, Réal

    2009-01-01

    Determination of the proliferation rate of cultured mammalian cells is widely done using incorporation of 5-bromo-2-deoxyuridine into replicating DNA followed by quantitative detection in ELISA using a specific monoclonal antibody. However, we noted that the BrdU ELISA results did not correlate with viable cell counts when increasing concentrations of proteins were added to test their effects on proliferating cells. This observation suggested that proteins could interfere with BrdU incorporation or detection in the commercial BrdU ELISA used. We show here that the presence of exogenous proteins during cell fixation and DNA denaturation significantly inhibited BrdU detection presumably by coating the extracted DNA by a concentration-dependent protein film. A simple modification to the manufacturer's protocol (cell washing) permitted to avoid this interference and resulted in a significant increase of the assay sensitivity. PMID:19591047

  19. Development of a sandwich ELISA-type system for the detection and quantification of hazelnut in model chocolates.

    PubMed

    Costa, Joana; Ansari, Parisa; Mafra, Isabel; Oliveira, M Beatriz P P; Baumgartner, Sabine

    2015-04-15

    Hazelnut is one of the most appreciated nuts being virtually found in a wide range of processed foods. The simple presence of trace amounts of hazelnut in foods can represent a potential risk for eliciting allergic reactions in sensitised individuals. The correct labelling of processed foods is mandatory to avoid adverse reactions. Therefore, adequate methodology evaluating the presence of offending foods is of great importance. Thus, the aim of this study was to develop a highly specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of hazelnut in complex food matrices. Using in-house produced antibodies, an ELISA system was developed capable to detect hazelnut down to 1 mg kg(-1) and quantify this nut down to 50 mg kg(-1) in chocolates spiked with known amounts of hazelnut. These results highlight and reinforce the value of ELISA as rapid and reliable tool for the detection of allergens in foods. PMID:25466021

  20. Development of a sandwich enzyme-linked immunosorbent assay (ELISA) for detection of buckwheat residues in food.

    PubMed

    Panda, Rakhi; Taylor, Steve L; Goodman, Richard E

    2010-08-01

    Buckwheat is a pseudocereal (an eudicot with seed qualities and uses similar to those of monocot cereals, family Poaceae) that is consumed in some Asian countries as a staple, and in some western countries as a health food. Allergic reactions to buckwheat are common in some countries. The objective was to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to detect traces of buckwheat that might inadvertently contaminate other foods in order to assure accurate labeling and consumer protection. Buckwheat-specific antibodies produced in 3 species of animals were tested for specificity and titer by direct ELISA and immunoblot. A sandwich ELISA was developed utilizing pooled rabbit antibuckwheat sera to capture buckwheat proteins and pooled goat antibuckwheat sera, followed by enzyme-labeled rabbit antigoat immunoglobulin G (IgG), to detect bound buckwheat proteins. The lower limit of quantification (LOQ) of the sandwich ELISA was 2 parts per million (ppm) of buckwheat in the presence of complex food matrices. The ELISA is highly specific with no cross-reactivity to any of 80 food ingredients and matrices tested. Validation studies conducted with buckwheat processed into noodles and muffins showed greater than 90% and 60% recovery, respectively. The percent recovery of buckwheat from noodles was similar to that achieved with a commercial buckwheat ELISA kit (ELISA Systems Pty. Ltd., Windsor, Queensland, Australia) at high buckwheat concentrations. However, the sensitivity of this ELISA was greater than the commercial ELISA. This newly developed ELISA is sufficiently specific and sensitive to detect buckwheat residues in processed foods to protect buckwheat-allergic subjects from potential harm. Practical Application: Buckwheat is becoming a common food ingredient in a number of processed foods due to potentially beneficial nutritional properties, without the celiac disease inducing glutenin proteins of wheat and related cereals. However

  1. Ultra-sensitive detection of biomarker using localized surface plasmon resonance (LSPR) enhanced by ELISA

    NASA Astrophysics Data System (ADS)

    Shin, Yong-Beom; Jo, Na rae; Lee, Ki joong

    2015-07-01

    We demonstrate a highly sensitive detection of AFP (α-fetoprotein) protein (liver cancer marker) in human serum using the LSPR biosensor. Gold metal nanodot array (MNA) on a glass wafer were fabricated by UV nanoimprint lithography (NIL). After the NIL process using a film stamp and the removal of residual layer via oxygen plasma etching, metal films were deposited using an electron-beam evaporator, followed by the lift-off step. Consequently, the gold MNA was realized on 5-inch glass wafer and the pitch, diameter and height of MNA were 300nm, 150 nm and 20 nm, respectively. We employed observation of LSPR spectra via back-reflection, which provides a stable measurement of LSPR because a probe light does not pass a bio-sample. In addition, one channel among two flow channels was used a control channel, the MNA surface in which was modified with bovine serum albumin, not antibody. After antigen-antibody reaction, the enzyme/precipitation was employed on the MNA (Nano-ELISA). As a result, we could detect AFP in 50 L human serum with limit of detection (LOD) of 0.7 zeptomole (10-21 mole).

  2. Diagnosis of typhoid fever by the detection of anti-LPS & anti-flagellin antibodies by ELISA.

    PubMed

    Jesudason, M V; Sridharan, G; Arulselvan, R; Babu, P G; John, T J

    1998-05-01

    In a developing country like ours where typhoid fever is endemic and there are very few microbiology laboratories to provide diagnosis by culture, a search for non culture techniques for rapid and reliable diagnosis continues. The Widal test has a low sensitivity. We have attempted to adapt the well established enzyme linked immunosorbent assay (ELISA) technique to provide a serological test of better sensitivity and specificity. The ELISA described here to detect anti-LPS antibodies was found to have a sensitivity of 89.4 per cent and a specificity of 94.9 per cent. The sensitivity for the antiflagellin ELISA was 68.1 per cent and specificity was 97.4 per cent. The likelihood ratio was 17.5 for anti-LPS ELISA and 26.2 for the anti-flagellin ELISA. Considering the positivity of either one ELISA as diagnostic, the sensitivity was 93.6 per cent and specificity was 94.9 per cent. PMID:9670617

  3. Comparison between a soluble antigen-based ELISA and IFAT in detecting antibodies against Babesia canis in dogs.

    PubMed

    Furuta, Patrícia Iriê; Oliveira, Tricia Maria Ferreira de Sousa; Theixeira, Márcia Cristina Alves; Rocha, Artur Gouveia; Machado, Rosangela Zacarias; Tinucci-Costa, Mirela Gouveia

    2009-01-01

    An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-B. canis antibodies in the sera of dogs using, indirect fluorescent antibody test (IFAT) as a reference test. ELISA uses a soluble antigenic preparation of B. canis and the optimal dilutions of the antigen, serum and conjugate were determined by check board titration, using positive and negative reference serum. The soluble antigen preparation of B. canis merozoites was 10 microg x mL(-1), with reference sera from positive and negative in a single dilution of 1:100, and conjugated to 1:4.000. A total of 246 serum samples were collected from dogs during the rabies vaccination campaign in Jaboticabal, São Paulo, Brazil and examined for the presence of antibodies against B. canis by ELISA and IFAT. Under these conditions, the average absorbance of negative serum was 0.129 + or - 0.025, resulting in a cut-of value of 0.323 (ELISA level 3) and the average absorbance of positive reference serum was 2.156 + or - 1.187. The serological positive samples tested for B. canis by ELISA and IFAT were 67.89% (n = 167) and 59.35% (n = 146), respectively. These results suggest that ELISA described may prove to be an effective serological test to diagnose canine babesiosis. PMID:19772774

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

    PubMed Central

    Thiha, Aung; Ibrahim, Fatimah

    2015-01-01

    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. PMID:25993517

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

    PubMed

    Thiha, Aung; Ibrahim, Fatimah

    2015-01-01

    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. PMID:25993517

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

    PubMed

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

    2012-01-01

    This investigation was aimed at using superparamagnetic particles to enzyme-linked immunosorbent assay (SPIO-ELISA) of human chorionic gonadotropin (hCG) to enhance detection sensitivity of hCG. We found that N-(3-dimethyl aminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was the best cross-linking reagent to link anti hCG α antibody to superparamagnetic particle (SPIO-anti hCG α antibody immunomagnetic particle). To improve the specificity of the assay, a horse radish peroxidase (HRP)-labeled anti-hCG beta monoclonal antibody was used to detect captured hCG using double antibody sandwich ELISA assay. SPIO-ELISA application to determine hCG increased the sensitivity to 1 mIU/mL, which is a level of sensitivity enabling the diagnosis of pregnancy during the early gestational period. PMID:22963487

  7. Comparing Rapid and Specific Detection of Brucella in Clinical Samples by PCR-ELISA and Multiplex-PCR Method

    PubMed Central

    Mohammad Hasani, Sharareh; Mirnejad, Reza; Amani, Jafar; Vafadar, Mohamad javad

    2016-01-01

    Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value <0.05. Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis. PMID:27499776

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

    PubMed

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2014-01-01

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

  9. Application of Microwave Irradiation and Heat to Improve Gliadin Detection and Ricin ELISA Throughput with Food Samples

    PubMed Central

    Garber, Eric A. E.; Thole, Joseph

    2015-01-01

    The utility of microwave irradiation to accelerate the onset of equilibrium and improve ELISA performance was examined using ELISAs for the detection of the plant toxin ricin and gliadin. The ricin ELISA normally requires several one hour incubations at 37 °C, a total assay time of approximately five hours, and employs a complex buffer containing PBS, Tween-20®, and non-fat milk. Different energy levels and pulse designs were compared to the use of abbreviated incubation times at 37 °C for the detection of ricin in food. The use of microwave irradiation had no significant advantage over the application of heat using an oven incubator and performed worse with some foods. In contrast, a gliadin ELISA that relied on 30 min incubation steps at room temperature and a salt-based buffer performed better upon irradiation but also displayed improvement upon incubating the microtiter plate at 37 °C. Whether microwave irradiation was advantageous compared to incubation in an oven was inconclusive. However, by abbreviating the incubation time of the ricin ELISA, it was possible to cut the assay time to less than 2 hours and still display LOD values < 10 ppb and recoveries of 78%–98%. PMID:26110503

  10. Application of Microwave Irradiation and Heat to Improve Gliadin Detection and Ricin ELISA Throughput with Food Samples.

    PubMed

    Garber, Eric A E; Thole, Joseph

    2015-06-01

    The utility of microwave irradiation to accelerate the onset of equilibrium and improve ELISA performance was examined using ELISAs for the detection of the plant toxin ricin and gliadin. The ricin ELISA normally requires several one hour incubations at 37 °C, a total assay time of approximately five hours, and employs a complex buffer containing PBS, Tween-20®, and non-fat milk. Different energy levels and pulse designs were compared to the use of abbreviated incubation times at 37 °C for the detection of ricin in food. The use of microwave irradiation had no significant advantage over the application of heat using an oven incubator and performed worse with some foods. In contrast, a gliadin ELISA that relied on 30 min incubation steps at room temperature and a salt-based buffer performed better upon irradiation but also displayed improvement upon incubating the microtiter plate at 37 °C. Whether microwave irradiation was advantageous compared to incubation in an oven was inconclusive. However, by abbreviating the incubation time of the ricin ELISA, it was possible to cut the assay time to less than 2 hours and still display LOD values < 10 ppb and recoveries of 78%-98%. PMID:26110503

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

    PubMed

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

    2014-04-01

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

  12. Comparison of FT-NIR Spectroscopy and ELISA for Detection of Adulteration of Goat Cheeses with Cow's Milk.

    PubMed

    Dvorak, Lukas; Mlcek, Jiri; Sustova, Kvetoslava

    2016-01-01

    This study investigated the ability of two methods to detect adulteration of goat cheeses via the addition of cow's milk, with a negligible effect on the raw materials. Cheeses were produced from a mixture of goat's and cow's milk and were then analyzed by Fourier transform near-IR (FT-NIR) spectroscopy and competitive ELISA. The cheese spectra were scanned in the spectroscope in reflectance mode on an integrating sphere at 80 scans and a resolution of 4 cm(-1). The spectra were evaluated via discriminant analysis, and a calibration was created via a partial least-squares algorithm to quantify the cow's milk admixture. A correlation coefficient of R = 0.999 was reached with a standard error of calibration of 0.0407. The results were statistically processed to a median value via a t-test. Adulteration detection by the ELISA method was performed using a commercial Milk Fraud/Bovine ELISA kit. It was found that the FT-NIR spectroscopy method is capable of detecting an admixture of cow's milk in goat cheese as small as 1%. The ELISA method did not return satisfactory results for the detection of adulteration with cow's milk. PMID:26822518

  13. Elevated Background in DAS-I ELISA for the Detection of Citrus Tristeza Virus in Mandarin Varieties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Healthy tissue extracts from mandarin orange varieties occasionally resulted in elevated absorbance which can exceed twice that of healthy sweet orange tissue extracts in DAS-I-ELISA detection with an expressed coat protein polyclonal antiserum of Citrus tristeza virus (CTV). This problem occurs se...

  14. A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera.

    PubMed

    Osawa, T; Wastling, J; Maley, S; Buxton, D; Innes, E A

    1998-09-01

    Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle. The epidemiology of neosporosis is poorly understood, partly because accurate diagnosis of infection is difficult. In this paper we describe the development of a multiple antigen-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies to N. caninum in sera from cattle, sheep and goats as well as from bovine foetal fluids. A water-soluble fraction (wsf) of sonicated NC-1 strain tachyzoites was used as the antigen in the ELISA. Minimum optical density (OD) values that were considered to be Neospora antibody-positive, that is, the cut-off OD values were determined separately for bovine maternal sera, bovine foetal fluids, ovine sera and caprine sera; they were 0.40, 0.17, 0.23 and 0.41 OD, respectively. The ELISA gave a high signal/noise ratio, giving good sensitivity and specificity, correlating well with the indirect fluorescent antibody test (IFAT) currently used to diagnose Neospora infection in cattle, sheep and goats. In both the ELISA and immunoblot analysis using the same antigen, there was no significant cross-reactivity with sera from cattle, sheep or goats that had been infected with Toxoplasma gondii. The ELISA also showed no cross-reactivity in sera from cattle infected with Sarcocystis cruzi, Babesia divergens, B. bovis and B. bigemina. The wsf fraction of sonicated N. caninum tachyzoites used in this ELISA can be easily prepared and may be more sensitive than a single antigen ELISA, whilst still retaining good specificity. PMID:9777723

  15. Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA.

    PubMed

    Aguiar, Daniel M; Zhang, Xiaofeng; Braga, Isis A; Taques, Isis I G G; McBride, Jere W

    2016-02-01

    We recently characterized a novel genotype of Ehrlichia canis based on the tandem repeat (TR) sequence of the TRP36 gene in Brazil. The TR amino acid sequence of the Brazilian (Br) genotype (ASVVPEAE) was divergent from the previously described US genotype (TEDSVSAPA) of E. canis. In this study, we developed an ELISA based on TRP36 TR synthetic peptides from both Br and US E. canis TRP36 genotypes to serologically detect and distinguish infections caused by these genotypes. Sera from 30 Brazilian dogs naturally infected with E. canis, sera from dogs experimentally infected E. canis (Jake and Cuiabá #1 strains) and E. chaffeensis (Arkansas strain) and 12 seronegative E. canis dogs were evaluated. Fifteen naturally infected Brazilian dogs had antibodies that reacted with the US TRP36 (n=9) or Br TRP36 (n=6) only, and 13 dogs had antibodies that reacted with both TPR36 peptides suggesting that these dogs were exposed to both genotypes. Most dogs (n=28) had antibodies that reacted with the highly conserved E. canis TRP19 peptide; however, two dogs had antibodies to E. canis TRP19, but did not have TRP36 antibodies, raising the possibility that another novel TRP36 genotype is circulating in Brazil. Our results demonstrate that synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil. PMID:26482949

  16. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    SciTech Connect

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  17. Allergens of Arachis hypogaea and the effect of processing on their detection by ELISA.

    PubMed

    Iqbal, Amjad; Shah, Farooq; Hamayun, Muhammad; Ahmad, Ayaz; Hussain, Anwar; Waqas, Muhammad; Kang, Sang-Mo; Lee, In-Jung

    2016-01-01

    Food allergies are an emerging public health problem in industrialized areas of the world. They represent a considerable health problem in these areas because of the relatively high number of reported cases. Usually, food allergens are proteins or glycoproteins with a molecular mass ranging from 10 to 70 kDa. Among the food allergies, peanut is accounted to be responsible for more than 50% of the food allergy fatalities. Threshold doses for peanut allergenic reactions have been found to range from as low as 100 µg to 1 g of peanut protein, which equal to 400 µg to 4 g peanut meal. Allergens from peanut are mainly seed storage proteins that are composed of conglutin, vicilin, and glycinin families. Several peanut proteins have been identified to induce allergic reactions, particularly Ara h 1-11. This review is mainly focused on different classes of peanut allergens, the effect of thermal and chemical treatment of peanut allergens on the IgY binding and detectability of these allergens by enzyme linked immunosorbent assay (ELISA) to provide knowledge for food industry. PMID:26931300

  18. Allergens of Arachis hypogaea and the effect of processing on their detection by ELISA

    PubMed Central

    Iqbal, Amjad; Shah, Farooq; Hamayun, Muhammad; Ahmad, Ayaz; Hussain, Anwar; Waqas, Muhammad; Kang, Sang-Mo; Lee, In-Jung

    2016-01-01

    Food allergies are an emerging public health problem in industrialized areas of the world. They represent a considerable health problem in these areas because of the relatively high number of reported cases. Usually, food allergens are proteins or glycoproteins with a molecular mass ranging from 10 to 70 kDa. Among the food allergies, peanut is accounted to be responsible for more than 50% of the food allergy fatalities. Threshold doses for peanut allergenic reactions have been found to range from as low as 100 µg to 1 g of peanut protein, which equal to 400 µg to 4 g peanut meal. Allergens from peanut are mainly seed storage proteins that are composed of conglutin, vicilin, and glycinin families. Several peanut proteins have been identified to induce allergic reactions, particularly Ara h 1–11. This review is mainly focused on different classes of peanut allergens, the effect of thermal and chemical treatment of peanut allergens on the IgY binding and detectability of these allergens by enzyme linked immunosorbent assay (ELISA) to provide knowledge for food industry. PMID:26931300

  19. Usefulness of serological ELISA assay for Taenia saginata to detect naturally infected bovines.

    PubMed

    Paulan, Silvana de Cássia; Gonzáles, Rutilia Marisela Hernándes; Peralta, Laura Adalid; Vicentini-Oliveira, Josy Campanhã; Biondi, Germano Francisco; Conde, Edda Sciuto; Parkhouse, Robert Michael Evans; Nunes, Cáris Maroni

    2013-01-01

    Bovine cysticercosis, a cosmopolitan disease caused by Taenia saginata, leads to economic losses due to carcass devaluation at slaughter. Sanitary inspection at slaughterhouses, the routine diagnostic method in Brazil, lacks the necessary sensitivity to detect the mildly infected cattle that are typically encoutered in Brazil. In this study we have tested cattle sera from animals diagnosed as positive and negative by veterianry inspection for (1) anti-parasite antibodies using metacestodes antigens (T. solium vesicular fluid and T. saginata secretions) and (2) the HP10 secreted antigen of viable metacestodes. The cut-off values were calculated by ROC curve for intense and mild infections conditions, and by the classical method ( for negative samples). The sensitivity and specificity of these diagnostic tests were different depending on the assumed cut-off value and, importantly, whether the infection was mild or intense. In spite of these observations, however, such ELISA assays for serum antibodies and parasite antigens constitute an important tool for epidemiological porposes, and in establishing priorities for the control of bovine cysticercosis. PMID:23802239

  20. Importance of competitive binding in the detection of antigen specific bovine isotypes and subisotypes by the micro ELISA.

    PubMed

    Townsend, J; Duffus, W P; Lammas, D A

    1982-11-01

    An enzyme-linked immunosorbent assay was developed to detect and quantify the specific bovine immunoglobulin class response to Trypanosoma theileri, Dictyocaulus viviparus and infectious bovine rhinotracheitis virus. Comparative measurement of the specific immunoglobulin classes in whole serum was achieved using monospecific rabbit antibovine IgG1, IgG2 and IgM, followed by a goat anti-rabbit Ig-enzyme conjugate (antiglobulin ELISA). The results obtained in the antiglobulin ELISA compared favourably with the standard (or indirect) ELISA using the purified immunoglobulins. Competitive inhibition between specific immunoglobulins of different isotypes and subisotypes was the major disadvantage of the antiglobulin ELISA. This latter assay failed to detect specific IgG2 against T theileri antigen in both calf and adult whole serum. The inability to detect the specific IgG2 was a result of competitive inhibition by specific IgG1. However, competitive inhibition between specific immunoglobulins was not observed in either of the other test systems using D viviparus and infectious bovine rhinotracheitis virus antigens. PMID:6296953

  1. A Simple and Rapid ELISA for Detecting Aflatoxin Contamination in Corn

    ERIC Educational Resources Information Center

    Weck, Robert; Van Putte, Robb

    2006-01-01

    Learn how to use biotechnology to investigate a serious agricultural problem. The exercise presented here provides an inexpensive way to introduce students to ELISA techniques in an economically and agriculturally important context.

  2. Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.

    PubMed

    Thaitrong, Numrin; Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Karoonuthaisiri, Nitsara

    2013-01-01

    Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation. PMID:24376668

  3. Synthesis and processing of ELISA polymer substitute: The influence of surface chemistry and morphology on detection sensitivity

    NASA Astrophysics Data System (ADS)

    Hosseini, Samira; Ibrahim, Fatimah; Djordjevic, Ivan; Rothan, Hussin A.; Yusof, Rohana; van der Marel, Cees; Koole, Leo H.

    2014-10-01

    Despite the known drawbacks of enzyme-linked immunosorbent assay (ELISA), one of the deficiencies that have relatively been ignored is the performance of ELISA substrate itself. Polystyrene (PS), as the cost effective material of choice for mass production of ELISA well-plates, has shown obvious lacks of suitable physical and chemical properties for protein attachment. The general concept of this work was to develop a potential substrate that can be suggested as a material of choice for production of a new generation of ELISA analytical kits. Spin-coated thin films of polymethyl methacrylate-co-methacrylic acid (PMMA-co-MAA) on silicon surfaces were designed and processed for detection of dengue virus. Coated surfaces of different molar ratios have been investigated as carboxyl-functionalized layers for obtaining platform for biomolecule immobilization with high level of protein activity. To improve the sensitivity of detection, we have used amine functional "spacers", hexamethylenediamine (HMDA) and polyethyleneimine (PEI), which were covalently bonded to the surfaces of PMMA-co-MAA coatings. Results demonstrate that the variation of surface concentration of carboxyl groups of PMMA-co-MAA can be used to control the amine surface concentration after carbodiimide coupling with HMDA and PEI spacers. The presence of amine spacers increases hydrophilicity of the coatings and significantly impacts the polymer surface morphology. In particular, protein immobilization via amine-bearing spacers has been achieved in two effective steps: (1) carbodiimide bonding between amine spacer molecules and PMMA-co-MAA polymer coatings; and (2) covalent immobilization of antibody via glutaraldehyde reaction with amine groups from amine-treated surfaces. The application of PEI spacer in comparison to HMDA has shown much higher intensity of detection signal in ELISA experiment, indicating better immobilization efficiency and preservation of antibody activity upon attachment to the

  4. Comparsion of an immunochromatographic strip with ELISA for simultaneous detection of thiamphenicol, florfenicol and chloramphenicol in food samples.

    PubMed

    Guo, Lingling; Song, Shanshan; Liu, Liqiang; Peng, Juan; Kuang, Hua; Xu, Chuanlai

    2015-09-01

    Rapid and sensitive indirect competitive enzyme-linked immunosorbent assays (ic-ELISA) and gold nanoparticle immunochromatographic strip tests were developed to detect thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in milk and honey samples. The generic monoclonal antibody for TAP, FF and CAP was prepared based on a hapten [D-threo-1-(4-aminophenyl)-2- dichloroacetylamino-1,3-propanediol], and the haptenwas linked to a carrier protein using the diazotization method. After the optimization of several parameters (coating, pH, sodium chloride content and methanol content), the ic-ELISA was established. The quantitative working range for TAP was 0.11-1.36 ng/mL, with an IC50 of 0.39 ng/mL. The optimized ELISA showed cross-reactivity to CAP (300%) and FF (15.6%), with IC50 values of 0.13 and 2.5 ng/mL, respectively. The analytical recovery of TAP, FF and CAP in milk and honey samples in the ic-ELISA ranged from 81.2 to 112.9%. Based on this monoclonal antibody, a rapid and sensitive immunochromatographic test strip was also developed. This strip had a detection limit of 1 ng/mL for TAP, FF and CAP in milk and honey samples. Moreover, the test was completed within 10 min. Our results showed that the proposed ic-ELISA and immunochromatographic test strip method are highly useful screening tools for TAP, FF and CAP detection in milk and honey samples. PMID:25675893

  5. Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces

    PubMed Central

    Gomes, Luciana Inácia; dos Santos Marques, Letícia Helena; Enk, Martin Johannes; de Oliveira, Maria Cláudia; Coelho, Paulo Marcos Zech; Rabello, Ana

    2010-01-01

    Background A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed to overcome the need for sensitive techniques for the efficient diagnosis of Schistosoma infection in endemic settings with low parasitic burden. Methodology/Principal Findings This system amplifies a 121-base pair tandem repeat DNA sequence, immobilizes the resultant 5′ biotinylated product on streptavidin-coated strip-well microplates and uses anti-fluorescein antibodies conjugated to horseradish peroxidase to detect the hybridized fluorescein-labeled oligonucleotide probe. The detection limit of the Schistosoma PCR-ELISA system was determined to be 1.3 fg of S. mansoni genomic DNA (less than the amount found in a single cell) and estimated to be 0.15 S. mansoni eggs per gram of feces (fractions of an egg). The system showed good precision and genus specificity since the DNA target was found in seven Schistosoma DNA samples: S. mansoni, S. haematobium, S. bovis, S. intercalatum, S. japonicum, S. magrebowiei and S. rhodaini. By evaluating 206 patients living in an endemic area in Brazil, the prevalence of S. mansoni infection was determined to be 18% by examining 12 Kato-Katz slides (41.7 mg/smear, 500 mg total) of a single fecal sample from each person, while the Schistosoma PCR-ELISA identified a 30% rate of infection using 500-mg of the same fecal sample. When considering the Kato-Katz method as the reference test, artificial sensitivity and specificity rates of the PCR-ELISA system were 97.4% and 85.1%, respectively. The potential for estimating parasitic load by DNA detection in feces was assessed by comparing absorbance values and eggs per gram of feces, with a Spearman correlation coefficient of 0.700 (P<0.0001). Conclusions/Significance This study reports the development and field evaluation of a sensitive Schistosoma PCR-ELISA, a system that may serve as an alternative for diagnosing Schistosoma infection. PMID:20421918

  6. Activity-dependent release of endogenous brain-derived neurotrophic factor from primary sensory neurons detected by ELISA in situ.

    PubMed

    Balkowiec, A; Katz, D M

    2000-10-01

    To define activity-dependent release of endogenous brain-derived neurotrophic factor (BDNF), we developed an in vitro model using primary sensory neurons and a modified ELISA, termed ELISA in situ. Dissociate cultures of nodose-petrosal ganglion cells from newborn rats were grown in wells precoated with anti-BDNF antibody to capture released BDNF, which was subsequently detected using conventional ELISA. Conventional ELISA alone was unable to detect any increase in BDNF concentration above control values following chronic depolarization with 40 mM KCl for 72 hr. However, ELISA in situ demonstrated a highly significant increase in BDNF release, from 65 pg/ml in control to 228 pg/ml in KCl-treated cultures. The efficacy of the in situ assay appears to be related primarily to rapid capture of released BDNF that prevents BDNF binding to the cultured cells. We therefore used this approach to compare BDNF release from cultures exposed for 30 min to either continuous depolarization with elevated KCl or patterned electrical field stimulation (50 biphasic rectangular pulses of 25 msec, at 20 Hz, every 5 sec). Short-term KCl depolarization was completely ineffective at evoking any detectable release of BDNF, whereas patterned electrical stimulation increased extracellular BDNF levels by 20-fold. In addition, the magnitude of BDNF release was dependent on stimulus pattern, with high-frequency bursts being most effective. These data indicate that the optimal stimulus profile for BDNF release resembles that of other neuroactive peptides. Moreover, our findings demonstrate that BDNF release can encode temporal features of presynaptic neuronal activity. PMID:11007900

  7. Comparison of two commercial kits and an in-house ELISA for the detection of equine rotavirus in foal feces.

    PubMed

    Miño, S; Kern, A; Barrandeguy, M; Parreño, V

    2015-09-15

    Group A rotaviruses (RVA) are important infectious agents associated with diarrhea in the young of several animal species including foals. Currently, a variety of diagnosis methods are commercially available, like ELISA, latex agglutination and immunochromatographic assays. These commercial tests are mainly designed for the detection of human RVA; its applicability in veterinary diagnosis has been poorly studied. The aim of this study was to compare the sensitivity and specificity of two commercial diagnostic kits, Pathfinder™ Rotavirus and FASTest Rota® strip, with an in-house KERI ELISA, for the detection of equine RVA. A total of 172 stool samples from Thoroughbred foals with diarrhea were analyzed. The presence of equine RVA in samples in which only one of the three methods showed positive results was confirmed by RT-PCR. A sample was considered "true positive" when RVA was detected by at least two of the methods, and "true negative" when it tested negative by the three assays. Following these criteria, 50 samples were found positive and 122 were found negative, and were handled as reference population for the assay validation. Pathfinder™ Rotavirus assay showed 32% sensitivity and 97% specificity, FASTest Rota® strip, 92% sensitivity and 97% specificity, and KERI ELISA, 76% sensitivity and 93% specificity. Pathfinder™ Rotavirus showed 77%, FASTest Rota® strip 95%, and KERI ELISA 88% accuracy to correctly classify the samples as equine RVA positive or negative. Pathfinder failed specifically to detect equine RVA G3P12I6 genotype; such performance might be related to the specificity of the monoclonal antibody included in this kit. According to our results, differences among VP6 genotypes could influence the sensitivity to detect equine RVA in foal feces, and thus assay validation of diagnostic kits for each species is necessary. In conclusion, FASTest Rota® strip is more suitable than ELISA Pathfinder™ Rotavirus for the screening of rotavirus

  8. Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin.

    PubMed

    Arora, S; Agarwal, R K; Bist, B

    2006-02-01

    The present study was conducted to assess the best method of the most commonly used methods for detection of aeromonads in foods of animal origin. With this objective an OMP based indirect plate ELISA and a duplex-PCR using primers targeting aerolysin gene and 16S rRNA gene and yielding amplicons of 252 bp and 599 bp, respectively, were standardized. The standardized protocols and the conventional cultural method were then compared for their respective sensitivities and specificities for detecting aeromonads from chicken and milk samples. Both the standardized assays were found to be highly specific for Aeromonas. The efficiency of the standardized indirect-ELISA and duplex-PCR protocols was assessed by artificial inoculation studies with varying concentrations of Aeromonas cells inoculated in chicken and milk samples followed by enrichment in Alkaline Peptone Water supplemented with 10 mg/ml cephalothin (APW-C) for 12 h. The results revealed that indirect-ELISA was able to detect a minimum of 10(3) cells/ml or g of Aeromonas cells in spiked milk and chicken samples, respectively. Whereas, duplex-PCR and cultural method were able to detect as low as 1 cell/ml or g of Aeromonas cells in spiked milk and chicken samples. The developed assays were also tested for their efficiency to detect Aeromonas spp. in naturally contaminated milk and chicken samples. Out of a total 50 milk samples screened for presence of Aeromonas by the three methods viz., indirect-ELISA, duplex-PCR and cultural method only 1 (2%) turned out to be positive showing positive results by all three methods. Similarly, 50 samples of chicken were tested by all three methods. Three samples (6%) turned out to be positive and here again by all the three methods. PMID:16216375

  9. Improved performance and quantitative detection of copro-antigens by a monoclonal antibody based ELISA to diagnose human opisthorchiasis.

    PubMed

    Watwiengkam, Nattaya; Sithithaworn, Jiraporn; Duenngai, Kunyarat; Sripa, Banchob; Laha, Thewarach; Johansen, Maria Vang; Sithithaworn, Paiboon

    2013-12-01

    Copro-antigen detection has been advocated as a promising method for diagnosis of opisthorchiasis, particularly in people that harbored light infection or have had recent drug treatment. This study aimed to improve performance of a monoclonal antibody-based enzyme-linked immunosorbent assay (Mab-ELISA) for detection of Opisthorchis viverrini copro-antigen and assess the correlation between copro-antigen and intensity of infection. Four different treatment methods of 71 samples from the Lawa endemic area, Khon Kaen province were assessed for copro-antigen detection, namely (1) phosphate buffer saline (PBS), (2) heating (70°C 30min), (3) alkaline (pH 9.6 in carbonate buffer), and (4) trichloroacetic acid (TCA) treatment. Comparison of these protocols showed that the TCA method gave the best performance in discriminating O. viverrini positive and negative samples with high sensitivity (97.9%) and moderate specificity (54.2%) compared with other methods. Application of TCA-based Mab-ELISA method for antigen detection in fecal samples obtained from an endemic area of opisthorchiasis revealed that 86 of 141 samples (61.0%) were positive compared with 68 (48.2%) by PBS-based protocol, while the formalin ethyl-acetate concentration technique (FECT) yielded a positive proportion of 71.6%. Among 40 egg-negative samples confirmed by a gold standard parasitological method (FECT) from the same endemic area, 19 (47.5%) were positive by the TCA-based while only 6 (15%) were positive by PBS-based Mab-ELISA protocol. In addition, levels of antigen detection significantly correlated with intensity of infection (egg per gram feces). The results show that the improved Mab-ELISA method has high sensitivity and also quantifiable diagnosis of opisthorchiasis. PMID:24055716

  10. Development of a Cell Marker ELISA for the Detection of Goose T Cell Surface CD8α Molecules.

    PubMed

    Cheng, Beibei; Zhang, Wei; Chen, Shun; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue; Cheng, Anchun

    2016-06-01

    CD8 molecule is a key marker on T cell surface and is connected with the antigen recognition and activation of T lymphocytes. In order to provide a detection method for quantifying goose CD8α expression, this study raised the protein and antibody for goose CD8α and developed a feasible cell marker enzyme-linked immunoabsorbent assay (ELISA) method. Recombinant protein of the extracellular region gene of goCD8α was expressed in prokaryotic expression system, and specific polyclonal antibodies for goCD8α were raised and purified, which was further confirmed by Western-blot, immunofluorescence assay (IFA), and immunohistochemistry (IHC). A cell marker ELISA was established and optimized to detect the change of goCD8α expression between goose parvovirus (GPV)-infected and mock-infected goose peripheral blood mononuclear cells (PBMCs), which is consistent with our previously results of real-time quantitative PCR (qPCR). Cell marker ELISA can provide a new method to detect goCD8α in protein level and in a sensitive, specific, and simple way. This may provide a convenient and novel method for the detection of goCD8α expression. PMID:26879976

  11. Immunodiagnostic monoclonal antibody-based sandwich ELISA of fasciolosis by detection of Fasciola gigantica circulating fatty acid binding protein.

    PubMed

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-09-01

    Up to now, parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Hence, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In the present study, a monoclonal antibody (MoAb) against recombinant Fasciola gigantica fatty acid binding protein (rFgFABP) has been produced. As well, a reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) has been developed for the detection of circulating FABP in the sera of mice experimentally and cattle naturally infected with F. gigantica. MoAb 3A3 and biotinylated rabbit anti-recombinant FABP antibody were selected due to their high reactivities and specificities. The lower detection limit of sandwich ELISA was 5 pg mL-1, and no cross-reaction with other parasite antigens was observed. This assay could detect F. gigantica infection from day 1 post infection. In experimental mice, the sensitivity, specificity and accuracy of this assay were 93·3, 100 and 98·2%, while in natural cattle they were 96·7, 100 and 99·1%. Hence, this sandwich ELISA method showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:27312522

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

    PubMed

    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

  13. An ELISA to Detect Serum Antibodies to the Salivary Gland Toxin of Ixodes holocyclus Neumann in Dogs and Rodents

    PubMed Central

    Hall-Mendelin, S.; O'Donoghue, P.; Atwell, R. B.; Lee, R.; Hall, R. A.

    2011-01-01

    The Ixodes holocyclus tick causes paralysis in up to 10,000 companion and domestic animals each year in Australia. Treatment requires the removal of the parasite and the administration of a commercial tick antiserum that is prepared from hyperimmune dogs. Each batch of this serum is initially tested for toxin-neutralising potency in a mouse bioassay that is expensive, time consuming, and subjective. With the aim of developing a rapid in vitro assay to replace the bioassay, we used a partially purified antigen prepared from I. holocyclus salivary glands to develop an ELISA to detect toxin-reactive antibodies in hyperimmune dog sera. The optimised ELISA reliably detected antibodies reactive to I. holocyclus salivary gland antigens. Parallel testing of sera with a negative control antigen prepared from the salivary glands of the nontoxic tick Rhipicephalus (Boophilus) microplus provided further evidence that we were detecting toxin-specific antibodies in the assay. Using the ELISA, we could also detect antibodies induced in rats after experimental infestation with I. holocyclus. This assay shows promise as an alternative means of assessing the potency of batches of hyperimmune dog serum and to screen for toxin-reactive monoclonal antibodies produced from immunised rodents. PMID:21687655

  14. International ring trial to detect anti-Trichinella IgG by ELISA on pig sera.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the present study, the sensitivity and specificity of the ELISA assay determined by the CRLP validation was 100% and 98.29%, respectively. The assay was reproducible, moreover, based on the receiver-operator characteristic (ROC) curve, the sensitivity and specificity of the assay reached 97.5% an...

  15. Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background The use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed...

  16. Development and validation of an ELISA kit for the detection of ricin toxins from biological specimens and environmental samples.

    PubMed

    Chen, Hsiao Ying; Tran, Hung; Foo, Ling Yann; Sew, Tracey Wenhui; Loke, Weng Keong

    2014-08-01

    Ricin is a toxin that can be easily extracted from seeds of Ricinus communis plants. Ricin is considered to be a major bio-threat as it can be freely and easily acquired in large quantities. A deliberate release of such toxin in civilian populations would very likely overwhelm existing public health systems, resulting in public fear and social unrest. There is currently no commercially available or FDA-approved prophylaxis such as vaccines, or therapeutic antitoxins or antidotes, available for ricin intoxication. Patient treatment is typically supportive care based on symptoms, often designed to reinforce the body's natural response. This paper describes the development and validation of a robust ELISA test kit, which can be used to screen for ricin in biological specimens such as whole blood and faeces. Faecal specimens are shown in this study to have better diagnostic sensitivity and a wider diagnostic window compared to whole blood. From these results, it is concluded that faeces is the most suitable clinical specimen for diagnosis of ricin poisoning via the oral route. The ELISA test kit can also detect ricin in environmental samples. An advantage of this ELISA kit over other commercial off-the-shelf (COTS) detection kits currently on the market that are developed to screen environmental samples only is its ability to diagnose ricin poisoning from clinical specimens as well as detect ricin from environmental samples. PMID:24928113

  17. Detection of Clonorchis sinensis Circulating Antigen in Sera from Chinese Patients by Immunomagnetic Bead ELISA Based on IgY

    PubMed Central

    Nie, Ge; Wang, Ting; Lu, Shengjun; Liu, Wenqi; Li, Yonglong; Lei, Jiahui

    2014-01-01

    Background Clonorchiasis, caused by Clonorchis sinensis, is widely distributed in Southeast Asia including China. Clonorchiasis is included in control programs of neglected tropical diseases by World Health Organization (WHO) because it is one of the major health problems in most endemic areas. Diagnosis of clonorchiasis plays a key role in the control programs. However, so far, there is no satisfactory method for clonorchiasis because of low sensitivity, poor practicality and high false positivity of available diagnostic tools. Methodology/Principal Findings We developed an immunomagnetic bead enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) against cysteine proteinase of C. sinensis for detection of circulating antigen in serum samples of patients infected with C. sinensis. The polyclonal IgY, coated with magnetic beads, was used as a capture antibody and a monoclonal IgG labeled with horseradish peroxidase as a detection antibody in the IgY-based immunomagnetic bead ELISA system (IgY-IMB-ELISA). The results showed that the sensitivity of IgY-IMB-ELISA was 93.3% (14 of 15) in cases of heavy infection (5000 to 9999 eggs per gram feces, i.e, EPG 5000–9999), 86.7% (13 of 15) in cases of moderate infection (EPG 1000–4999) and 75.0% (9 of 12) in cases of light infection (EPG <1000) of clonorchiasis. Together 36 of total 42 (85.7%) serum samples of human clonorchiasis gave a positive reaction. There was a significant correlation between ELISA optical density and egg counts (EPG) with a correlation coefficient of 0.83 in total 42 patients. There were no positive results in patients with trichinosis (n = 10) or cysticercosis (n = 10). Cross-reactivity was 6.7% (2 of 30) with schistosomiasis japonica and 10.0% (3 of 30) with paragonimiasis, respectively. No positive reaction was found in 20 healthy persons. Conclusions Our findings suggest that IgY-IMB-ELISA appears to be a sensitive and specific assay for detection of

  18. Development of an Aquaporin-4 Orthogonal Array of Particle-Based ELISA for Neuromyelitis Optica Autoantibodies Detection

    PubMed Central

    Pisani, Francesco; Settanni, Paolo; Rosito, Stefania; Mola, Maria Grazia; Iorio, Raffaele; Tortorella, Carla; Ruggieri, Maddalena; Trojano, Maria; Svelto, Maria; Frigeri, Antonio; Nicchia, Grazia Paola

    2015-01-01

    Serological markers of Nuromyelitis Optica (NMO), an autoimmune disorder of the central nervous system, are autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4). We have previously demonstrated that the main epitopes for these autoantibodies (AQP4-IgG) are generated by the supramolecular arrangement of AQP4 tetramers into an Orthogonal Array of Particles (OAPs). Many tests have been developed to detect AQP4-IgG in patient sera but several procedural issues affect OAP assembly and consequently test sensitivity. To date, the protein based ELISA test shows the lowest sensitivity while representing a valid alternative to the more sensitive cell based assay (CBA), which, however, shows economic, technical and interpretation problems. Here we have developed a high perfomance ELISA in which native OAPs are used as the molecular target. To this aim a native size exclusion chromatography method has been developed to isolate integral, highly pure and AQP4-IgG-recognized OAPs from rat brain. These OAPs were immobilized and oriented on a plastic plate by a sandwich approach and 139 human sera were tested, including 67 sera from NMO patients. The OAP-ELISA showed a 99% specificity and a higher sensitivity (91%) compared to the CBA test. A comparative analysis revealed an end-point titer three orders of magnitude higher than the commercial ELISA and six times higher than our in-house CBA test. We show that CNS-extracted OAPs are crucial elements in order to perform an efficient AQP4-IgG test and the OAP-ELISA developed represents a valid alternative to the CBA currently used. PMID:26599905

  19. Development of an Aquaporin-4 Orthogonal Array of Particle-Based ELISA for Neuromyelitis Optica Autoantibodies Detection.

    PubMed

    Pisani, Francesco; Settanni, Paolo; Rosito, Stefania; Mola, Maria Grazia; Iorio, Raffaele; Tortorella, Carla; Ruggieri, Maddalena; Trojano, Maria; Svelto, Maria; Frigeri, Antonio; Nicchia, Grazia Paola

    2015-01-01

    Serological markers of Nuromyelitis Optica (NMO), an autoimmune disorder of the central nervous system, are autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4). We have previously demonstrated that the main epitopes for these autoantibodies (AQP4-IgG) are generated by the supramolecular arrangement of AQP4 tetramers into an Orthogonal Array of Particles (OAPs). Many tests have been developed to detect AQP4-IgG in patient sera but several procedural issues affect OAP assembly and consequently test sensitivity. To date, the protein based ELISA test shows the lowest sensitivity while representing a valid alternative to the more sensitive cell based assay (CBA), which, however, shows economic, technical and interpretation problems. Here we have developed a high perfomance ELISA in which native OAPs are used as the molecular target. To this aim a native size exclusion chromatography method has been developed to isolate integral, highly pure and AQP4-IgG-recognized OAPs from rat brain. These OAPs were immobilized and oriented on a plastic plate by a sandwich approach and 139 human sera were tested, including 67 sera from NMO patients. The OAP-ELISA showed a 99% specificity and a higher sensitivity (91%) compared to the CBA test. A comparative analysis revealed an end-point titer three orders of magnitude higher than the commercial ELISA and six times higher than our in-house CBA test. We show that CNS-extracted OAPs are crucial elements in order to perform an efficient AQP4-IgG test and the OAP-ELISA developed represents a valid alternative to the CBA currently used. PMID:26599905

  20. Evaluation of a Western Blot and ELISA for the detection of anti-Trichinella-IgG in pig sera.

    PubMed

    Nöckler, K; Reckinger, S; Broglia, A; Mayer-Scholl, A; Bahn, P

    2009-08-26

    Human trichinellosis is a foodborne disease caused by ingestion of infective Trichinella muscle larvae via pork or meat of other food animals which are susceptible to this zoonotic parasite. There are new approaches for a risk-oriented meat inspection for Trichinella in pigs which are accompanied by monitoring programmes on herd level to control freedom from this parasite. For this purpose, testing schemes utilizing serological tests with a high sensitivity and specificity are required. This study aimed at the evaluation of an ELISA and a Western Blot (WB) for the detection of anti-Trichinella-IgG in terms of sensitivity and specificity taking results of artificial digestion as gold standard. For this purpose, 144 field sera from pigs confirmed as Trichinella-free as well as 159 sera from pigs experimentally infected with T. spiralis (123), T. britovi (19) or T. pseudospiralis (17) were examined by ELISA (excretory-secretory antigen) and WB (crude worm extract). Sera from pigs experimentally infected with four other nematode species were included to investigate the cross-reactivity of the antigen used in the WB. For all Trichinella-positive pig sera, band pattern profiles were identified in the WB and results were analysed in relation to ELISA OD% values. Testing of pig sera revealed a sensitivity of 96.8% for the ELISA and 98.1% for the WB whereas the methods showed a specificity of 97.9 and 100%, respectively. WB analysis of Trichinella-positive pig sera revealed five specific band patterns of 43, 47, 61, 66, and 102 kDa of which the 43 kDa protein was identified as the predominant antigen. The frequency of the band pattern profile was irrespective of the dose and the period of infection as well as the Trichinella species investigated. In conclusion, monitoring in swine farms for Trichinella antibodies should be based on screening pig sera by means of ELISA followed by confirmatory testing through WB analysis. PMID:19473770

  1. Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti.

    PubMed

    Schares, G; Basso, W; Majzoub, M; Rostaher, A; Scharr, J C; Langenmayer, M C; Selmair, J; Dubey, J P; Cortes, H C; Conraths, F J; Haupt, T; Pürro, M; Raeber, A; Buholzer, P; Gollnick, N S

    2011-01-10

    Bovine besnoitiosis is an economically important disease in cattle caused by the protozoan parasite Besnoitia besnoiti, which occurs endemically in many countries of Africa and Asia and is spreading in Europe. Serological identification of subclinically infected cattle is important to avoid the introduction of infected animals into naive herds. Here we determine the sensitivity and specificity of the PrioCHECK(®) Besnoitia Ab, a serological test recently introduced into the European market. Analytical specificity was examined using sera from animals experimentally infected with parasites related to B. besnoiti (n=27). Three animals experimentally infected with Neospora caninum or Toxoplasma gondii showed inconclusive reactions in the ELISA (percent positivity relative to the positive control [PP] 10% ≤ 20%) while all other sera reacted negative (PP<10%). An estimate of the diagnostic specificity was obtained by analysing field sera from bovine herds without besnoitiosis but with abortion problems associated to N. caninum (n=403). The analysis revealed a specificity of 94.3% or 96.8% depending on the applied cut-off (PP 10% or 20%, respectively). Sensitivity was assessed with sera from 110 animals of a herd in Germany where clinical bovine besnoitiosis was first diagnosed in September 2008. A positive serological reference standard was defined regarding sera from animals as reference positive, if these animals had tested positive in at least two of a panel of three other serological tests (two different B. besnoiti immunoblots and one immunofluorescence antibody test) on both of two sampling dates, November 2008 and April 2009. A diagnostic sensitivity of 91.8% or 75.5% was determined for sera collected in November 2008 and a sensitivity of 82.7% or 50% for sera collected in April 2009 (cut-off PP 10% or PP 20%, respectively). The marked drop in sensitivity from November 2008 to April 2009 was predominantly observed in reference-positive cattle without clinical

  2. Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using Competitive-Inhibition ELISA and PCR.

    PubMed

    Rosales, Romel; Rangel-Rivas, Ariadna; Escalona, América; Jordan, Luis Segundo; Gonzatti, Mary Isabel; Aso, Pedro Maria; Perrone, Trina; Silva-Iturriza, Adriana; Mijares, Alfredo

    2013-09-01

    The focus of this study was the detection of equine piroplasmosis in Distrito Capital, Miranda, Aragua, Guárico and Apure States from Venezuela, using two methods: Competitive-Inhibition ELISA and multiplex PCR and the analysis of the possible differences in occurrence in relation to the primary purpose of the horses, which is related to varied degrees of exposure to tick. Antibody levels to Babesia caballi and Theileria equi were assessed in 694 equine serum samples using Competitive-Inhibition ELISA, while PCR assays were performed in 136 horses, using two sets of oligonucleotides to establish the presence of T. equi, B. caballi or both. The overall seroprevalence of equine piroplasmosis was 50.2%, antibodies to B. caballi were found in 161 horses (23.2%), whereas 97 (14.0%) were seropositive to T. equi and 90 (13.0%) were positives to both parasites (mixed infections). PCR determinations (n=136) showed a prevalence of 66.2%, distributed in 84 (61.8% positives) for T. equi and, 6 (4.4%) were positive to both parasites. The cELISA showed higher levels of prevalence of B. caballi and mixed infections, as compared to the PCR method. This discrepancy can be explained by the different parameters that are evaluated by each technique, PCR detect the parasite itself, while cELISA detects antibodies to the parasite. By PCR, the highest prevalence was found in Apure state, where 92.3% of the samples were positive to T. equi infections. In this locality, free grazing animals are used for livestock management. This high prevalence may be linked to the tick species present in that area. More epidemiological studies will be necessary to assess the epidemiological status of equine piroplasmosis in Venezuela. PMID:23582233

  3. Silver nanoprism etching-based plasmonic ELISA for the high sensitive detection of prostate-specific antigen.

    PubMed

    Liang, Jiajie; Yao, Cuize; Li, Xiuqing; Wu, Ze; Huang, Caihong; Fu, Qiangqiang; Lan, Caifeng; Cao, Donglin; Tang, Yong

    2015-07-15

    Ultrasensitive and quantitative detection using simple and low-cost assays is critical in clinical diagnostics. In this report, we developed a triangular silver nanoprism (AgNPRs) etching-based plasmonic biosensor for the detection of cancer biomarkers. The triangular AgNPRs-based plasmonic biosensor is an enzyme-linked immunosorbent assay combined with the enzyme-mediated surface plasmon resonance (SPR) of triangular AgNPRs. Triangular AgNPRs uses the immune response of prostate-specific antigen (PSA) to trigger the glucose oxidase (GOx)-catalysed oxidation of glucose (Glu), producing hydrogen peroxide. Hydrogen peroxide acts as an oxidant to etch the triangular AgNPRs into smaller spherical silver nanoparticles, which is accompanied by a substantial blueshift of the SPR peak and a colourimetric blue-to-purple change that can be observed by the naked eye. The SPR peak shift enables the quantitative assessment of PSA due to the remarkable colour change. The triangular AgNPRs-based plasmonic ELISA approach exhibited a quasilinear response to logarithmic PSA concentrations in the range of 10fg/mL to 100pg/mL with a limit of detection (LOD) of 4.1fg/mL. In addition, the LOD of PSA in this approach exceeds that of the conventional HRP-based ELISA (1.25ng/mL) approach by more than 5 orders of magnitude. Patient serum samples from 16 donors were assayed with triangular AgNPRs-based plasmonic ELISA. The results from the triangular AgNPRs-based immunoassay and the time-resolved fluorescence immunoassay showed excellent correlation, and there were no significant differences in the quantified amounts of PSA. The triangular AgNPRs-based plasmonic ELISA approach has advantages (ultrasensitive, cost-effective, ease of operation) that are expected to be of great interest in diagnostics and to be suitable for a point-of-care test. PMID:25721976

  4. ELISA: Methods and Protocols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antibody is central to the performance of an ELISA providing the basis of analyte selection and detection. It is the interaction of antibody with analyte under defined conditions that dictates the outcome of the ELISA and deviations in those conditions will impact assay performance. The aim of...

  5. Superiority of radiobinding assay over ELISA for detection of IAAs in newly diagnosed type I diabetic children

    SciTech Connect

    Levy-Marchal, C.; Bridel, M.P.; Sodoyez-Goffaux, F.; Koch, M.; Tichet, J.; Czernichow, P.; Sodoyez, J.C. )

    1991-01-01

    Liquid- or solid-phase assays have been used for insulin autoantibody (IAA) determination, and the method of IAA measurement has not been standardized. IAAs were determined by radiobinding assay (RBA) and enzyme-linked immunosorbent assay (ELISA) in two large age-matched groups of nondiabetic and newly diagnosed insulin-dependent (type I) diabetic children. Positivity for IAA by RBA (greater than or equal to nondiabetic mean + 3SD) was 2 of 178 (1.1%) and 55 of 173 (32%) in nondiabetic and diabetic children, respectively. Prevalence of IAA by RBA was significantly higher in the youngest age-group (63% between 0-4 yr). Positivity for IAA by ELISA was 1 of 178 (0.6%) and 8 of 169 (4.7%) in nondiabetic and diabetic children, respectively. Concordance rates between both assays were 0 of 3 (0%) in control subjects and 5 of 58 (8.6%) in diabetic children. We conclude that RBA is more appropriate than ELISA for IAA detection at the onset of the disease. In addition, because available data suggest that IAAs detected by RBA only are high-affinity antibodies, it is tempting to speculate that IAAs reflect a mature immune reaction against endogenous insulin.

  6. A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish, and mammalian body fluid.

    PubMed Central

    Naar, Jerome; Bourdelais, Andrea; Tomas, Carmelo; Kubanek, Julia; Whitney, Philip L; Flewelling, Leanne; Steidinger, Karen; Lancaster, Johnny; Baden, Daniel G

    2002-01-01

    We developed a competitive enzyme-linked immunosorbent assay (ELISA) to analyze brevetoxins, using goat anti-brevetoxin antibodies obtained after immunization with keyhole limpet hemocyanin-brevetoxin conjugates, in combination with a three-step signal amplification process. The procedure, which used secondary biotinylated antibodies, streptavidine-horseradish peroxidase conjugate, and chromogenic enzyme substrate, was useful in reducing nonspecific background signals commonly observed with complex matrices. This competitive ELISA detected brevetoxins in seawater, shellfish extract and homogenate, and mammalian body fluid such as urine and serum without pretreatment, dilution, or purification. We investigated the application of this technique for shellfish monitoring by spiking shellfish meat with brevetoxins and by analyzing oysters from two commercial shellfish beds in Florida that were exposed to a bloom of Karenia brevis (formerly Gymnodinium breve). We performed brevetoxin analysis of shellfish extracts and homogenates by ELISA and compared it with the mouse bioassay and receptor binding assay. The detection limit for brevetoxins in spiked oysters was 2.5 microg/100 g shellfish meat. This assay appears to be a useful tool for neurotoxic shellfish poisoning monitoring in shellfish and seawater, and for mammalian exposure diagnostics, and significantly reduces the time required for analyses. PMID:11836147

  7. Metal-linked Immunosorbent Assay (MeLISA): the Enzyme-Free Alternative to ELISA for Biomarker Detection in Serum

    PubMed Central

    Yu, Ru-Jia; Ma, Wei; Liu, Xiao-Yuan; Jin, Hong-Ying; Han, Huan-Xing; Wang, Hong-Yang; Tian, He; Long, Yi-Tao

    2016-01-01

    Determination of disease biomarkers in clinical samples is of crucial significance for disease monitoring and public health. The dominating format is enzyme-linked immunosorbent assay (ELISA), which subtly exploits both the antigen-antibody reaction and biocatalytic property of enzymes. Although enzymes play an important role in this platform, they generally suffer from inferior stability and less tolerant of temperature, pH condition compared with general chemical product. Here, we demonstrate a metal-linked immunosorbent assay (MeLISA) based on a robust signal amplification mechanism that faithfully replaces the essential element of the enzyme. As an enzyme-free alternative to ELISA, this methodology works by the detection of α-fetoprotein (AFP), prostatic specific antigen (PSA) and C-reactive protein (CRP) at concentrations of 0.1 ng mL-1, 0.1 ng mL-1 and 1 ng mL-1 respectively. It exhibits approximately two magnitudes higher sensitivity and is 4 times faster for chromogenic reaction than ELISA. The detection of AFP and PSA was further confirmed by over a hundred serum samples from hepatocellular carcinoma (HCC) and prostate cancer patients respectively. PMID:27446504

  8. Metal-linked Immunosorbent Assay (MeLISA): the Enzyme-Free Alternative to ELISA for Biomarker Detection in Serum.

    PubMed

    Yu, Ru-Jia; Ma, Wei; Liu, Xiao-Yuan; Jin, Hong-Ying; Han, Huan-Xing; Wang, Hong-Yang; Tian, He; Long, Yi-Tao

    2016-01-01

    Determination of disease biomarkers in clinical samples is of crucial significance for disease monitoring and public health. The dominating format is enzyme-linked immunosorbent assay (ELISA), which subtly exploits both the antigen-antibody reaction and biocatalytic property of enzymes. Although enzymes play an important role in this platform, they generally suffer from inferior stability and less tolerant of temperature, pH condition compared with general chemical product. Here, we demonstrate a metal-linked immunosorbent assay (MeLISA) based on a robust signal amplification mechanism that faithfully replaces the essential element of the enzyme. As an enzyme-free alternative to ELISA, this methodology works by the detection of α-fetoprotein (AFP), prostatic specific antigen (PSA) and C-reactive protein (CRP) at concentrations of 0.1 ng mL(-1), 0.1 ng mL(-1) and 1 ng mL(-1) respectively. It exhibits approximately two magnitudes higher sensitivity and is 4 times faster for chromogenic reaction than ELISA. The detection of AFP and PSA was further confirmed by over a hundred serum samples from hepatocellular carcinoma (HCC) and prostate cancer patients respectively. PMID:27446504

  9. Evaluation of a Commercial ELISA for the Detection of Antibodies to Sarcoptes scabiei in Wild Boar (Sus scrofa).

    PubMed

    Haas, Chloé; Rossi, Sophie; Meier, Roman; Ryser-Degiorgis, Marie-Pierre

    2015-07-01

    Sarcoptic mange occurs in free-ranging wild boar (Sus scrofa) but has been poorly described in this species. We evaluated the performance of a commercial indirect enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of sarcoptic mange in domestic swine when applied to wild boar sera. We tested 96 sera from wild boar in populations without mange history ("truly noninfected") collected in Switzerland between December 2012 and February 2014, and 141 sera from free-ranging wild boar presenting mange-like lesions, including 50 live animals captured and sampled multiple times in France between May and August 2006 and three cases submitted to necropsy in Switzerland between April 2010 and February 2014. Mite infestation was confirmed by skin scraping in 20 of them ("truly infected"). We defined sensitivity of the test as the proportion of truly infected that were found ELISA-positive, and specificity as the proportion of truly noninfected that were found negative. Sensitivity and specificity were 75% and 80%, respectively. Success of antibody detection increased with the chronicity of lesions, and seroconversion was documented in 19 of 27 wild boar sampled multiple times that were initially negative or doubtful. In conclusion, the evaluated ELISA has been successfully applied to wild boar sera. It appears to be unreliable for early detection in individual animals but may represent a useful tool for population surveys. PMID:26161723

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

    PubMed

    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

  11. Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of Gentamicin residues.

    PubMed

    He, Jinxin; Hu, Jianjun; Thirumalai, Diraviyam; Schade, Ruediger; Du, Enqi; Zhang, Xiaoying

    2016-01-01

    Gentamicin (Gent) is an aminoglycoside antibiotic being used in livestock sector. Gent residues could cause some genetic disorders by nonsense mutations. This study aimed to develop IgY-based ELISA for the detection of Gent in animal products. Gent was conjugated with Bovine serum albumin (BSA) by carbodiimide method for further immunization in the laying chickens. PEG-6000 extraction method was employed to extract IgY from the egg yolk. The titer of anti-Gent-IgY attained the peak of 1:256,000 after the 5(th) booster immunization. Checkerboard titration confirmed that, anti-Gent IgY in 1:2,000 dilution could give an Optical Density (OD) 1.0 at 2 µg mL(-1) of Gent-OVA coating concentration. IgY-based indirect competitive ELISA (Ic-ELISA) showed that, the IC50 value of anti-Gent IgY was 2.69 ng mL(-1) and regression curve equation was y = -16.27x + 56.97 (R(2) = 0.95, n = 3), confirming that, the detection limit (LOD, IC10 value) was 0.01 ng mL(-1). Recoveries from fresh milk, pork and chicken samples were ranged from 69.82% to 94.32%, with relative standard deviation lower than 10.88%. Our results suggested that generated anti-Gent IgY antibodies can be used in routine screening analysis of Gent residues in food samples. PMID:26513166

  12. Detection of subtle differences in analogous viral capsid proteins by allowing unrestricted specific interaction in solution competition ELISA.

    PubMed

    Cao, Lu; Wang, Xin; Fang, Mujin; Xia, Ningshao; Zhao, Qinjian

    2016-10-01

    Assay artifacts were reported in plate-based immuoassays during the assessment of specific molecular interactions owing to the surface induced aggregation/conformational changes. To circumvent surface adsorption and associated artifacts, we used a solution competition ELISA by allowing unrestricted interaction between binding partners to occur in solution for better discrimination between epitopes with subtle differences. A difference of two orders of magnitude in binding to neutralizing antibodies for two truncated versions of the hepatitis E virus capsid protein was observed, while other assays showed comparable antigenicity with the same monoclonal antibodies. Discrimination of epitopes with high degree resemblance in analogous viral capsid proteins was demonstrated quantitatively based on their specific interactions. Therefore, the solution competition ELISA is a method of choice when the detection of subtle differences of two highly analogous proteins is desired. PMID:27321427

  13. Clinical utility of a competitive ELISA to detect antibodies against Treponema pallidum.

    PubMed

    Gutiérrez, J; Vergara, M J; Soto, M J; Piédrola, G; Maroto, M d

    2000-01-01

    Screening for Treponema pallidum infection is carried out on a large human population. To reduce costs, fewer tests which still offer adequate sensitivity and specificity could be performed. We studied the reliability of a novel indirect ELISA method to test for this infection. Several panels of sera were used that corresponded to 40 primary infections (group 1), 13 recurrences (group 2), 348 latent infections (group 3), 5 samples with anticardiolipin antibodies (group 4), 15 samples from patients with Lyme borreliosis (group 5), and 400 samples from blood donors and healthy pregnant women (group 6). The ELISA showed a global sensitivity and specificity of 100 and 99.5%, respectively. Our evaluation shows that Enzygnost Syphilis is a sensitive, specific, and simple test to screen for this infection. PMID:10683619

  14. Development of recombinant antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of skatole.

    PubMed

    Leivo, Janne; Mäkelä, Joonas; Rosenberg, Jaana; Lamminmäki, Urpo

    2016-01-01

    The occurrence of boar taint and the European Commission recommendation to discontinue the surgical castration of pigs by the year 2018 creates an urgent need for new analytical methods that are simple, affordable, and suitable for field testing. We describe the generation and engineering of a skatole-specific antibody derived from a synthetic antibody library and the development of ELISA for its detection. The immunoassay is capable of detecting skatole with IC50 of 222 μg L(-1), which is within the analytical threshold level suggested for skatole, and with low cross-reactivity interference from other indolic compounds. PMID:26410338

  15. Development of an Indirect ELISA Using Different Fragments of Recombinant Ncgra7 for Detection of Neospora caninum Infection in Cattle and Water Buffalo

    PubMed Central

    HAMIDINEJAT, Hossein; SEIFI ABAD SHAPOURI, Massoud Reza; NAMAVARI, Mohammad Mehdi; SHAYAN, Parviz; KEFAYAT, Marzieh

    2015-01-01

    Background: Dense granules are immunodominant proteins for the standardization of immunodiagnostic procedures to detect neosporosis. In the presented study different fragment of a dense-granule protein was evaluated for serodiagnosis of Neospora caninum in cattle and water buffalo. Methods: NcGRA7, from N. caninum tachyzoites was amplified. PCR product and pMAL-c2X plasmid were digested with EcoR1 restriction enzyme and expressed in Escherichia coli to evaluate its competence for detection of anti- N. caninum antibodies with ELISA in comparison with commercial IDEXX ELISA. Furthermore, 230 sera of presumably healthy cattle and water buffaloes (108 cattle and 122 water buffaloes) were analyzed by both tests to determine the agreement of these two procedures. Results: Sensitivities and specificities of NcGRA7-based ELISA were 94.64% and 90.38% respectively using sera of cattle, but were 98.57% and 86.54% in the case of buffaloes respectively. A good correlation between the results of IDEXX ELISA and ELISA based on recombinant NcGRA7 for detecting N. caninum antibodies was appeared. Analyzing by Mc Nemar′s showed that NcGRA7-based ELISA has acceptable capability to differentiate the positive results in comparison with IDEXX ELISA. Conclusion: NcGRA7-based ELISA considering utilized new fragment of genomic DNA is a good tool for serodiagnosis of anti- N. caninum antibodies for screening and epidemiological purposes on cattle herd and water buffaloes as well. PMID:25904948

  16. Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein

    PubMed Central

    Yuan, Han; Tank, Mihir; Alsofyani, Abeer; Shah, Naman; Talati, Nishant; LoBello, Jaclyn C; Kim, Jin Ryoun; Oonuki, Yoji; de la Motte, Carol A; Cowman, Mary K

    2013-01-01

    Hyaluronan (HA) is widely detected in biological samples and its concentration is most commonly determined by the use of a labeled specific HA binding protein (aggrecan G1-IGD-G2, HABP), employing membrane blotting and sandwich enzyme-linked immunosorbent assay (ELISA)-like methods. However, the detected signal intensity or the quantified value obtained by using these surface-based methods is related to the molecular mass (M) of HA, especially for HA in the low M range below ∼150 kDa. At the same mass or mass concentration, higher M HA gives a higher signal than lower M HA. We have experimentally determined the quantitative relationship between the M of HA (in the range 20–150 kDa) and the relative signal intensity in comparison with a standard HA, in a sandwich ELISA-like assay. An M-dependent signal correction factor (SCF) was calculated and used to correct the signal intensity, so that the corrected concentration value would more accurately reflect the true HA concentration in solution. The SCF for polydisperse low M HA was also calculated and compared with experimental results. When the molecular mass distribution of an HA sample is determined by a method such as gel electrophoresis, then its appropriately averaged SCF can be calculated and used to correct the signal in sandwich ELISA to obtain a more accurate concentration estimation. The correction method works for HA with M between ∼150 and 20 kDa, but lower M HA is too poorly detected for useful analysis. The physical basis of the M-dependent detection is proposed to be the increase in detector-accessible fraction of each surface-bound molecule as M increases. PMID:23964097

  17. The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA.

    PubMed

    Stearns, Nancy A; Zhou, Shuxia; Petri, Michelle; Binder, Steven R; Pisetsky, David S

    2016-01-01

    Antibodies to nuclear antigens (antinuclear antibodies or ANAs) are the serological hallmark of systemic lupus erythematosus (SLE). These antibodies bind diverse nuclear antigens that include DNA, histones and non-histone proteins as well as complexes of proteins with DNA and RNA. Because of the frequency of ANA expression in SLE, testing is an important component of clinical evaluation as well as determination of eligibility for clinical trials or utilization of certain therapies. Immunofluorescence assays have been commonly used for this purpose although this approach can be limited by issues of throughput, variability and difficulty in determining positivity. ELISA and multiplex assays are also useful approaches although these assays may give an incomplete picture of antibodies present. To develop a sensitive and quantitative ANA assay, we have explored an ELISA platform in which plates are pre-coated with a positively charged nucleic acid binding polymer (NABP) to increase adherence of antigens containing DNA or RNA. As a source of antigens, we have used supernatants of Jurkat cells undergoing apoptosis in vitro. As results presented show, a poly-L-lysine (PLL) pre-coat significantly enhances detection of antibodies to DNA as well as antigens such as histones, SSA, SSB and RNP. Comparison of the ELISA assay with the PLL pre-coat with a multiplex assay using the BioPlex® 2200 system indicated good agreement in results for a panel of lupus sera. Together, these studies indicate that a pre-coat with a positively charged polymer can increase the sensitivity of an ANA ELISA using as antigens molecules released from dead and dying cells. This assay platform may facilitate ANA testing by providing an ensemble of antigens more similar in composition and structure with antigens present in vivo, with a NABP promoting adherence via charge-charge interactions. PMID:27611194

  18. Development and validation of a blocking ELISA test for the detection of avian influenza antibodies in poultry species.

    PubMed

    Henriques, Ana Margarida; Fagulha, Teresa; Barros, Sílvia Carla; Ramos, Fernanda; Duarte, Margarida; Luís, Tiago; Fevereiro, Miguel

    2016-10-01

    Avian influenza (AI) is an infectious viral disease usually asymptomatic in wild birds that can spread to domestic poultry and cause large-scale outbreaks. Some of the AI viruses (AIV) can cross the species barrier and induce fatal disease to humans, a matter of great concern worldwide. Early detection of AIV is of major importance for disease control, since prompt implementation of adequate measures can prevent spread of the virus and therefore further outbreaks. This paper reports the development and validation of a blocking ELISA using a monoclonal antibody against a conserved structural protein for the serologic diagnosis of all AI virus subtypes from domestic bird species, allowing the quick, easily automated and low-cost screening of a high number of farms. The test will be of great value not only for surveillance, but also for monitoring the efficiency of vaccination programs. Cut-off values were established in 20% of inhibition for turkey sera and in 50% of inhibition for chicken and duck sera. Estimations of 100% specificity and 100% sensitivity were obtained based on the results of known AI positive (n=130) and negative (n=208) sera, including serum samples from birds infected with other common avian viral pathogens (n=7). ROC analysis showed an area under curve (AUC) of 1.0 for chicken, duck and turkey sera, indicating that the blocking ELISA was able to perfectly discriminate between negative and positive samples of any of the poultry species tested. Inter- and intra-assay coefficients of variation were above the acceptance threshold. Furthermore, the ELISA titers were similar to the known hemagglutination inhibition titers of three positive reference sera indicating sensitivity comparable with the golden standard HI method. The method here described is an economically attractive alternative to the commercial ELISAs currently available. PMID:27421625

  19. A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans.

    PubMed

    Baumann, D; Gottstein, B

    1987-06-01

    A double-antibody sandwich ELISA was developed to detect detergent-solubilized antigens of Entamoeba histolytica in stool samples of humans. The test system was evaluated for its methodical and diagnostic sensitivity and specificity. In recovery experiments the lower limit of detection was 400 ng E. histolytica (HK9) protein/ml stool, corresponding to approximately 2000 amoebic trophozoites/ml stool. Samples of 97 patients with suspected intestinal amoebiasis were examined. Specific antigens were detected by ELISA (= positive reaction) in 14 (93%) out of 15 stool samples containing trophozoites of E. histolytica. In contrast, 68 (93%) of 73 samples with other protozoa, including Entamoeba coli, E. hartmanni, Endolimax nana, Iodamoeba buetschlii and Giardia lamblia, did not react in the test system (= negative reaction). The test was shown to detect only trophozoites of E. histolytica and not the cyst stage. This fact could facilitate the differentiation between cyst carriers and persons excreting trophozoites. The results of this preliminary study justify a further large scale evaluation of the test system. PMID:2888183

  20. Development of a highly sensitive monoclonal antibody based ELISA for detection of benzo[a]pyrene in potable water.

    PubMed

    Matschulat, Diana; Deng, Anping; Niessner, Reinhard; Knopp, Dietmar

    2005-07-01

    In Europe, a limit value of 10 ng L(-1) was set by the European Commission for benzo[a]pyrene (B[a]P) in water intended for human consumption (Council Directive 98/83/EC) and, therefore, sensitive and reliable methods are needed to evaluate its presence. We report here on the development of a highly sensitive indirect competitive ELISA for the detection of B[a]P in potable water. Fourteen monoclonal antibodies were generated in mice using novel B[a]P derivatives. The immunoassay with the least interference and the best sensitivity was optimized and characterized. As co-solvent, ten percent methanol (v/v) was determined as the optimum concentration for B[a]P solubilization for use with the developed ELISA. With the purified antibody (clone 22F12) the average IC50 for B[a]P and corresponding detection limit at a signal:noise (S/N) ratio of 3 was 65 ng L(-1) and 24 ng L(-1), respectively. From the 16 EPA-designated PAHs, only chrysene, indeno[1,2,3-cd]pyrene, and benzo[b]fluoranthene showed a cross-reactivity (CR) higher than 20%. No CR was observed for two- and three-ringed aromatics as well as dibenz[ah]anthracene and benzo[ghi]perylene. The effect of pH value (range 6.5-9.5), ionic strength (specific electric conductivity 1 microS cm(-1)-2.5 mS cm(-1)), and inorganic ions (sodium, copper, iron, aluminium, manganese, chloride, sulfate, nitrate, and nitrite at maximum permissible levels according to the Council Directive) on both signal and sensitivity of the ELISA was studied. No significant influence of these parameters on the ELISA competition curve was found. We suggest that the optimized ELISA can be used to monitor potable water samples without previous extraction from the samples. The assay should facilitate the cleanup of B[a]P contaminated sites where B[a]P levels fall close to the limit value of the new drinking water directive. PMID:15965533

  1. A new multi-host species indirect ELISA using protein A/G conjugate for detection of anti-Toxoplasma gondii IgG antibodies with comparison to ELISA-IgG, agglutination assay and Western blot.

    PubMed

    Al-Adhami, Batol H; Gajadhar, Alvin A

    2014-02-24

    Toxoplasma gondii is a zoonotic protozoan parasite which can cause significant disease and losses in livestock and wild animals. It is increasingly recognized as an important foodborne pathogen in a broad range of food animals and products. Effective control strategies require rapid, reliable and cost-effective detection methods for large scale surveys and diagnostic applications in a broad range of warm-blooded animals. To overcome one or more of these shortcomings in the currently available detection methods for T. gondii infection a non-species-specific protein A/G conjugate was used in the development of an indirect ELISA (ELISA-A/G) for the detection of IgG antibodies in serum samples obtained from experimentally infected pigs. The performance of the assay was evaluated using serum samples from pigs, cats, mice and seals with known positive or negative status for T. gondii infection. Results of the ELISA-A/G obtained with pig serum samples were compared with those generated by traditional ELISA using host specific IgG conjugate (ELISA-IgG), modified agglutination test (MAT) and Western blot analysis (WB). Using protein A/G conjugate, comparative analysis of results from 77 samples obtained from T. gondii infected pigs showed excellent agreement between the ELISA-A/G and in-house ELISA-IgG (0.917 κ). Similar agreements were also observed when these samples were tested by a commercial ELISA kit (0.816 κ), MAT (0.816 κ) and WB (0.79 κ). A total of 86 serum samples obtained from cats, mice and seals experimentally infected with T. gondii and tested by the ELISA-A/G as well as MAT for the presence of anti-Toxoplasma IgG antibodies yielded Kappa value of 1.0 for cats and mice and 0.79 for seals. These results show that the ELISA-A/G is a suitable method for serological detection of T. gondii infection in multiple host species and has the potential for testing samples from a broad range of domestic, wild, and aquatic mammalian host species. Simultaneous testing

  2. Development of a Blocking ELISA for Detection of Serum Neutralizing Antibodies against Newly Emerged Duck Tembusu Virus

    PubMed Central

    Li, Xuesong; Li, Guoxin; Teng, Qiaoyang; Yu, Lei; Wu, Xiaogang; Li, Zejun

    2012-01-01

    Background Since April 2010, domesticated ducks in China have been suffering from an emerging infectious disease characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death. The causative agent was identified as a duck Tembusu virus (DTMUV), a member of the Ntaya virus (NTAV) group within the genus Flavivirus, family Flaviviridae. DTMUV is highly contagious and spreads rapidly in many species of ducks. More than 10 million shelducks have been infected and approximately 1 million died in 2010. The disease remains a constant threat to the duck industry; however, it is not known whether DTMUV can infect humans or other mammalians, despite the fact that the virus has spread widely in southeast China, one of the most densely populated areas in the world. The lack of reliable methods to detect the serum antibodies against DTMUV has limited our ability to conduct epidemiological investigations in various natural hosts and to evaluate the efficiency of vaccines to DTMUV. Methodology/Principal Findings A neutralizing monoclonal antibody (mAb) 1F5 binding specifically to the E protein was developed. Based on the mAb, a blocking enzyme-linked immunosorbent assay (ELISA) was developed for the detection of neutralizing antibodies against DTMUV. The average value of percent inhibition (PI) of 350 duck serum samples obtained from DTMUV-free farms was 1.0% ±5.8% (mean ± SD). The selected cut-off PI values for negative and positive sera were 12.6% (mean +2SD) and 18.4% (mean +3SD), respectively. When compared with a serum neutralizing antibody test (SNT) using chicken embryonated eggs, the rate of coincidence was 70.6% between the blocking ELISA and SNT, based on the titration of 20 duck DTMUV-positive serum samples. Conclusions/Significance The blocking ELISA based on a neutralizing mAb allowed rapid, sensitive, and specific detection of neutralization-related antibodies against DTMUV. PMID:23300851

  3. Amebiasis and comparison of microscopy to ELISA technique in detection of Entamoeba histolytica and Entamoeba dispar.

    PubMed Central

    Nesbitt, Rose A.; Mosha, Franklin W.; Katki, Hormuzd A.; Ashraf, Mohammad; Assenga, Charles; Lee, Clarence M.

    2004-01-01

    The analysis of records of amoebal infection in various hospitals in Kilimanjaro indicated frequent occurrence of amebiasis. The population over the age of five years had higher rate of amoebal infection compared to less than that of a five-year-old population; however, both age groups had similar patterns of amebiasis during January 1999 to June 2001. To investigate misdiagnosis of amebiasis, 226 patients (passive cases) in three hospitals and 616 individuals (active cases) from three different localities in Kilimanjaro were examined. In passive cases, the prevalences of Entamoeba histolytica and Entamoeba dispar were 1% and 7.3%, respectively. Among active cases, 1% were infected with E. histolytica, and 15% were infected with E. dispar. There were no significant differences in amoebal infection between the male and female populations. A pool of 842 stool samples was used for diagnosis of E. histolytica and E. dispar by microscopic examination or ELISA kits. The microscopic examination indicated 8.7% amoebal infection; however, using ELISA as the gold standard, the prevalence of histolytica/dispar was 0.8% and 7.4%, respectively. This study indicated that E. dispar infection was 14.5 times more prevalent than E. histolytica infection. PMID:15160983

  4. Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater.

    PubMed

    Meyers, T R; Thrower, F; Short, S; Lipson, K; Joyce, J; Farrington, C; Doherty, S

    1999-01-29

    Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater. The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA. Possible causes for this difference are discussed. Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs. PMID:10092972

  5. Development of an ELISA detecting Tumor Protein 53-Induced Nuclear Protein 1 in serum of prostate cancer patients.

    PubMed

    Saadi, Houda; Seillier, Marion; Sandi, Maria José; Peuget, Sylvain; Kellenberger, Christine; Gravis, Gwenaëlle; Dusetti, Nelson J; Iovanna, Juan L; Rocchi, Palma; Amri, Mohamed; Carrier, Alice

    2013-01-01

    Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) plays an important role during cell stress response in synergy with the potent "genome-keeper" p53. In human, the gene encoding TP53INP1 is expressed at very high level in some pathological situations, such as inflammation and prostate cancer (PC). TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse, making TP53INP1 a relevant specific target for molecular therapy of Castration Resistant (CR) PC. In that context, detection of TP53INP1 in patient biological fluids is a promising diagnostic avenue. We report here successful development of a new Enzyme-Linked Immunosorbent Assay (ELISA) detecting TP53INP1, taking advantage of molecular tools (monoclonal antibodies (mAbs) and recombinant proteins) generated in the laboratory during the course of basic functional investigations devoted to TP53INP1. The ELISA principle is based on a sandwich immunoenzymatic system, TP53INP1 protein being trapped by a first specific mAb coated on microplate then recognized by a second specific mAb. This new assay allows specific detection of TP53INP1 in serum of several PC patients. This breakthrough paves the way towards investigation of a large cohort of patients and assessment of clinical applications of TP53INP1 dosage. PMID:24600558

  6. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.

    PubMed

    Tang, Yu Qian; Han, Shuang Yan; Zheng, Hong; Wu, Lin; Ueda, Mitsuyoshi; Wang, Xiao Ning; Lin, Ying

    2008-07-01

    In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen. PMID:18542951

  7. Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

    NASA Astrophysics Data System (ADS)

    Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

    2014-05-01

    Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

  8. ELISA Detection of Phenazepam, Etizolam, Pyrazolam, Flubromazepam, Diclazepam and Delorazepam in Blood Using Immunalysis® Benzodiazepine Kit.

    PubMed

    O'Connor, Lauren C; Torrance, Hazel J; McKeown, Denise A

    2016-03-01

    Phenazepam and etizolam were the first uncontrolled benzodiazepines available for sale in the UK. Pyrazolam, flubromazepam and diclazepam are not used medicinally anywhere in the world; they are produced exclusively for the uncontrolled, recreational market. It is important to know whether potentially abused drugs like these can be detected in routine toxicological screening tests. The purpose of this study was to evaluate whether the Immunalysis® Benzodiazepines ELISA kit could detect phenazepam, etizolam, pyrazolam, flubromazepam, diclazepam and its metabolite delorazepam. Their cross-reactivity was assessed by comparing the absorbance of the drug with that of oxazepam, the reference standard. This study found that these uncontrolled benzodiazepines cross-react sufficiently to produce a positive result with the Immunalysis® Benzodiazepine ELISA kit. Cross-reactivity ranged from 79 to 107% for phenazepam, etizolam, pyrazolam, flubromazepam, diclazepam and delorazepam fortified into blood. The results show that it is possible to detect these newer benzodiazepines with traditional forensic toxicology laboratory tools and it is important to include these benzodiazepines in the confirmation tests. PMID:26518230

  9. Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA

    PubMed Central

    Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi

    2014-01-01

    Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings. PMID:24786974

  10. Validation and comparison of two commercial ELISA kits and three in-house developed real-time PCR assays for the detection of potentially allergenic mustard in food.

    PubMed

    Palle-Reisch, Monika; Hochegger, Rupert; Štumr, Stepan; Korycanova, Kveta; Cichna-Markl, Margit

    2015-05-01

    The study compares the applicability of two commercial mustard ELISA kits (Mustard ELISA Kit-specific and Mustard ELISA Kit-total) and three in-house developed real-time PCR assays (singleplex assay for white mustard, singleplex assay for black/brown mustard and duplex assay for the detection of white, black and brown mustard). Analyses of raw and brewed model sausages containing white and black/brown mustard in the range from 1 to 50 ppm indicate that both ELISAs and the three real-time PCR assays allow the detection of traces of mustard in raw and in brewed sausages. The ELISAs were found to be more sensitive than the real-time PCR assays. When the ELISAs and real-time PCR assays were applied to the analysis of 15 commercial foodstuffs differing in their labelling concerning mustard, in one sample mustard was detected with both ELISAs and the three real-time PCR assays although mustard was not indicated on the food ingredient list. PMID:25529654

  11. Detection of Tuberculosis in HIV Co-infected Individuals: Use of Multiple ELISA Responses to 38kDa, Lipoarabinomannan and ESAT– 6 of M. tuberculosis

    PubMed Central

    Gutlapalli, Ravi; Sykam, Aparna; Tenali, Sandeep P; Chandran, Priscilla; Suneetha, Sujai

    2016-01-01

    Introduction There is a constant search for more sensitive and specific laboratory markers for tuberculosis (TB) infection. The early detection of TB in HIV co infected individuals is a diagnostic challenge. This is further compounded in those harbouring extrapulmonary disease. Aim To evaluate the use of multiple Enzyme Linked Immunosorbent Assays (ELISA) quantifying antibody responses to 38kDa, LAM and ESAT-6 M.tb antigens in detection of TB in patients with TB and HIV-TB co-infection. Materials and Methods This is a cross-sectional study carried out in Hyderabad, India. Patient groups included 124 HIV-TB {62 with pulmonary TB (PTB) and 62 with extrapulmonary TB (ETB)}, 39 TB, 56 HIV and 57 healthy subjects (HS). A combination of anti 38kDa and LAM ELISAs measuring IgG, IgM and IgA levels and another ELISA measuring anti ESAT-6 combined antibody levels of IgG, IgM and IgA were evaluated. One-way ANOVA was performed to compare antibody responses among groups. To assess the efficacy of multiple ELISAs in detecting TB, concomitant seropositivity of an individual for all four ELISAs were evaluated for sensitivity and specificity. Results A single ELISA carried out to detect TB in HIV patients showed a sensitivity ranging from 39% to 72%. The sensitivities of concomitant evaluation of multiple ELISAs were 92% for any single, 72% for any two, 44% for any three and 14% for any four. Based on the specificities, a simple algorithm for TB detection can be deduced. When four ELISAs are positive (specificity 100%) in a patient-confirmed TB; when three ELISAs are positive (specificity 98%) – probably TB; when two ELISAs are positive (specificity 95%) – possibly TB; and when one ELISA is positive (specificity 70%) – suspicion of TB. Conclusion The present study establishes the value of combining two or more M.tb antigen based ELISAs to enhance the sensitivity and specificity of TB detection in patients with tuberculosis as well as in those co-infected with HIV. PMID

  12. Development of an ELISA for sensitive and specific detection of IgA autoantibodies against BP180 in pemphigoid diseases

    PubMed Central

    2011-01-01

    Background Pemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/BP180. An entity of the pemphigoid group is the lamina lucida-type of linear IgA disease (IgA pemphigoid) characterized by IgA autoantibodies against BP180. While for the detection of IgG and IgE autoantibodies specific to collagen XVII several ELISA systems have been established, no quantitative immunoassay has been yet developed for IgA autoantibodies. Therefore, the aim of the present study was to develop an ELISA to detect IgA autoantibodies against collagen XVII in the sera of patients with pemphigoids. Methods We expressed a soluble recombinant form of the collagen XVII ectodomain in mammalian cells. Reactivity of IgA autoantibodies from patients with IgA pemphigoid was assessed by immunofluorescence microscopy and immunoblot analysis. ELISA test conditions were determined by chessboard titration experiments. The sensitivity, specificity and the cut-off were determined by receiver-operating characteristics analysis. Results The optimized assay was carried out using sera from patients with IgA pemphigoid (n = 30) and healthy donors (n = 105). By receiver operating characteristics (ROC) analysis, an area under the curve of 0.993 was calculated, indicating an excellent discriminatory capacity. Thus, a sensitivity and specificity of 83.3% and 100%, respectively, was determined for a cut-off point of 0.48. As additional control groups, sera from patients with bullous pemphigoid (n = 31) and dermatitis herpetiformis (n = 50), a disease associated with IgA autoantibodies against epidermal transglutaminase, were tested. In 26% of bullous pemphigoid patients, IgA autoantibodies recognized the ectodomain of collagen XVII. One of 50 (2%) of dermatitis herpetiformis patients sera slightly topped the cut-off value. Conclusions We developed the first ELISA for the specific and sensitive detection of serum IgA autoantibodies specific to collagen XVII in patients

  13. Evaluation of ELISA screening test for detecting aflatoxin in biogenic dust samples

    SciTech Connect

    Durant, J.T.

    1996-05-01

    Aflatoxin is a carcinogenic chemical that is sometimes produced when agricultural commodities are infested by the fungi Aspergillus flavus and A. Parasiticus. Aflatoxin has been found to be present in air samples taken around persons handling materials likely to be contaminated. The purpose of this investigation was to demonstrate the feasibility of using an Enzyme Linked Immunosorbent Assay (ELISA) test kit that was developed to screen for aflatoxin in bulk agricultural commodities, to an air sample. Samples were taken from two environments likely to be contaminated with aflatoxin, a dairy farm feed mixing operation and a peanut bagging operation. The dust collected from these environments was considered to be biogenic, in that it originated primarily from biological materials.

  14. Modification of a bovine ELISA to detect camelid antibodies to Mycobacterium paratuberculosis.

    PubMed

    Kramsky, J A; Miller, D S; Hope, A; Collins, M T

    2000-12-20

    Mycobacterium avium subsp. paratuberculosis infection, or Johne's disease, reportedly has a low prevalence in South American camelid populations. Recently, however, single cases in the United States as well as an outbreak of the disease in Australian alpacas (Lama pacos) have been described. To provide a rapid and cost-effective method of diagnosing Johne's disease in this species, the bovine Parachek((R)) Johne's Absorbed EIA (CSL, Vic., Australia) was modified to create a camelid-specific serum antibody assay. An anti-llama IgG conjugated to horseradish peroxidase replaced the anti-bovine immunoglobulin. Checkerboard titration of principal reagents was performed using serum from nine tissue and/or fecal culture-positive camelids. Optimal dilutions of key components were determined in order to provide clear discrimination between positive and negative controls. Completion of a kinetic assay determined the optical density at which the enzyme-substrate reaction should be stopped. A herd of 100 camelids with no history of disease or exposure to M. a. paratuberculosis, a subset of which were tissue and/or fecal culture-negative, was tested to establish a cut-off value. Sample results were expressed as a percentage of the results for control sera by transforming optical density values to ELISA values (EV%). A preliminary EV% cut-off of 20 was established. Using this prototype assay, culture-positive animals showed significantly different antibody responses from culture-negative animals. These results indicate that this camelid-specific ELISA, once refined, may be a useful tool for screening camelid herds for M. a. paratuberculosis infection. PMID:11118718

  15. Assessment of diagnostic accuracy of a commercial ELISA for the detection of Toxoplasma gondii infection in pigs compared with IFAT, TgSAG1-ELISA and Western blot, using a Bayesian latent class approach.

    PubMed

    Basso, Walter; Hartnack, Sonja; Pardini, Lais; Maksimov, Pavlo; Koudela, Bretislav; Venturini, Maria C; Schares, Gereon; Sidler, Xaver; Lewis, Fraser I; Deplazes, Peter

    2013-06-01

    Serological methods are the most commonly used diagnostic tools to detect Toxoplasma gondii infections in pigs. In the absence of a readily available 'gold standard', an estimation of diagnostic accuracy is difficult to assess. A commercial ELISA (PrioCHECK® Toxoplasma Ab porcine ELISA, Prionics, Schlieren, Switzerland) for the diagnosis of T. gondii infection in pigs was evaluated in naturally infected animals from two distinct populations; indoor and outdoor living animals. An assessment of diagnostic accuracy, using a Bayesian latent class approach with adjustment for within indoor and outdoor farm clustering using random effects, was performed. Tests used for comparison were: IFAT; ELISA using native affinity-purified P30 (SAG1) T. gondii tachyzoite surface antigen (TgSAG1-ELISA); and Western blot with T. gondii tachyzoites lysate. The data set comprised 297 pig serum samples across outdoor (n=149) and indoor (n=148) farms in Argentina. The estimated sensitivity and specificity for the commercial ELISA were 98.9% (95% credible interval: 96.2; 100) and 92.7% (95% credible interval: 87.7; 96.6), respectively. The analysis of sera and plasma from pigs (n=6) experimentally inoculated with 5,000 T. gondii oocysts revealed a pronounced antibody response beginning 2 weeks p.i. until the end of the observation period (11 weeks p.i.) in all animals. Meat juice obtained from inoculated animals after euthanasia also tested positive. These results suggest that the PrioCHECK® Toxoplasma Ab porcine ELISA may be a useful tool to perform serological diagnosis of T. gondii infections in pigs to control Toxoplasma infection in pigs and humans. PMID:23538054

  16. Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5'-bromo-2'-deoxyuridine

    USGS Publications Warehouse

    Gauthier, David T.; Cartwright, Deborah D.; Densmore, Christine L.; Blazer, Vicki; Ottinger, Christopher A.

    2003-01-01

    In this study we present a method for the measurement of in vitro mitogenesis in fish leucocytes that is based on the incorporation of the thymidine analogue 5′-bromo-2′-deoxyuridine (BrdU) into the DNA of replicating cells, followed by ELISA-based detection. This technique, adapted from methods developed for mammalian cells, operates on a similar biological principle to 3H-thymidine incorporation, but circumvents the logistical and safety issues inherent with the radioactive label. Because it directly measures DNA proliferation, the assay has advantages over other colorimetric methods that may be strongly influenced by leucocyte metabolic status. Using BrdU incorporation followed by ELISA, we evaluate the responsiveness of rainbow trout (Oncorhynchus mykiss [Walbaum]) leucocytes to the mammalian T-cell mitogen Concanavalin A (Con A) as well as the differential response of white perch (Morone americana [Gmelin]) leucocytes to Con A and pokeweed mitogen. Specific considerations intrinsic to the assay system are discussed, including the implications of utilising enzyme-based detection.

  17. Development of an ELISA to detect the humoral immune response to Trichinella zimbabwensis in Nile crocodiles (Crocodylus niloticus).

    PubMed

    Ludovisi, Alessandra; La Grange, Louis Jacobus; Gómez Morales, Maria Angeles; Pozio, Edoardo

    2013-05-20

    Crocodiles are known reservoir hosts of Trichinella papuae and Trichinella zimbabwensis, two zoonotic parasites that also infect mammals. Since commercial crocodile farming represents a key source of income in several countries, it is important to monitor this nematode infection in both farmed crocodiles and in breeding stocks which are frequently introduced from the wild. For this purpose, an indirect ELISA was developed to detect the anti-Trichinella immune response in crocodile sera. New Zealand rabbits were immunized with pooled sera from non-infected farmed crocodiles in the presence of Freund's complete adjuvant. The anti-crocodile serum was then conjugated with horseradish peroxidase. Serum samples from four Nile crocodiles (Crocodylus niloticus) experimentally infected with T. zimbabwensis and from four uninfected crocodiles were used to set up the ELISA. The larval burden per gram of muscle tissue was determined by muscle biopsy. The test was performed on serum samples from an additional 15 experimentally infected crocodiles as well as eight wild Nile crocodiles. Among the 19 experimentally infected crocodiles, seroconversion was observed in 11 animals. The highest antibody response was observed six weeks post infection (p.i.), but in most of these animals, antibodies were not detectable after six weeks p.i. even though live larvae were present in the muscles up to six months p.i. PMID:23433644

  18. Evaluation of an enzyme-linked immunosorbent assay (ELISA) kit for the detection of botulinum neurotoxins A, B, E, and F in selected food matrices.

    PubMed

    Singh, Ajay; Datta, Shomik; Sachdeva, Amita; Maslanka, Susan; Dykes, Janet; Skinner, Guy; Burr, Donald; Whiting, Richard C; Sharma, Shashi K

    2015-01-01

    The mouse bioassay (MBA) is the only accepted standard method for detection of botulinum neurotoxins (BoNTs) in foods. The ELISA method has several advantages over the MBA and is therefore widely used for in vitro detection of BoNTs. The US Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) conducted a precollaborative study to evaluate the applicability of Botulinum Toxin ELISA kits for the detection of BoNT serotypes A, B, E, and F in a variety of food matrices. In this study, food samples (e.g., broccoli, salami, smoked salmon, green beans, orange juice, tomato juice, low-fat plain yogurt, whole milk, liquid infant formula milk, and liquid eggs) were spiked with high, medium, and low concentration BoNT serotypes A, B, E, and F. Samples (unspiked and spiked) were tested at both laboratories by the ELISA kits. All food samples were positive for BoNTs by ELISA in both laboratories at medium and high spiking levels; a positive ELISA result in low spiked samples was both serotype and laboratory dependent. Overall, the ELISA method appears to be an effective preliminary screening method for BoNT detection in food matrices. PMID:25812427

  19. Ovatoxin-a, A Palytoxin Analogue Isolated from Ostreopsis cf. ovata Fukuyo: Cytotoxic Activity and ELISA Detection.

    PubMed

    Pelin, Marco; Forino, Martino; Brovedani, Valentina; Tartaglione, Luciana; Dell'Aversano, Carmela; Pistocchi, Rossella; Poli, Mark; Sosa, Silvio; Florio, Chiara; Ciminiello, Patrizia; Tubaro, Aurelia

    2016-02-01

    This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC50 = 1.1 × 10(-9) M vs 1.8 × 10(-11) M, MTT test) in agreement with a reduced binding affinity (Kd = 1.2 × 10(-9) vs 2.7 × 10(-11) M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in O. cf. ovata extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program. PMID:26714047

  20. Application of total error approach to assess the performance of a biological method (ELISA) to detect nicarbazin residues in eggs.

    PubMed

    Gaudin, V; Laurentie, M

    2009-08-01

    Nicarbazin, a coccidiostat, is used as a feed additive in poultry but not in laying hens. Feed contamination may however occur resulting in residues being present in eggs. As a Maximum Residue Limit (MRL) does not exist for nicarbazin residues in eggs a "Differential Action Level" (DAL) of 100 microg/kg has been established by the Veterinary Medicines Directorate (VMD). We have studied a commercial ELISA kit validated to detect and quantify nicarbazin in eggs with a sensitivity of 3 microg/kg. We used the total error approach to assess the performance of and validate the kit at the DAL level. The accuracy profile has been successfully obtained for the ELISA kit. The method cannot however be validated as a semi-quantitative method and we have consequently determined a cut-off based on 5% false negative rate according to European Decision 2002/657 on blank and spiked samples (70 microg/kg). The cut-off value established was 20 microg/kg using the 95th percentile. PMID:19345618

  1. A field and laboratory evaluation of a commercial ELISA for the detection of Giardia coproantigens in humans and dogs.

    PubMed

    Hopkins, R M; Deplazes, P; Meloni, B P; Reynoldson, J A; Thompson, R C

    1993-01-01

    A capture enzyme linked immunosorbent assay (CELISA) was evaluated for its ability to detect Giardia coproantigens in the faeces of humans and dogs in the Perth metropolitan area and Aboriginal communities in Fitzroy Crossing, Western Australia. Using zinc sulphate flotation and light microscopy, Giardia cysts and/or trophozoites were observed in 8 of 57 (14%) human stool samples from Perth and 21 of 55 (38%) stool samples from Fitzroy Crossing, after 2 separate examinations. Analysis of diagnostic sensitivity using the ELISA revealed that coproantigens were detected in all 29 human samples (100%) in which Giardia cysts and/or trophozoites were also present. Coproantigens were detected in one further sample from Perth and in 3 samples from Fitzroy Crossing in which no Giardia cyst or trophozoite was observed. The specificity of the test, as defined using Fitzroy Crossing samples free from Giardia, was 91%. The assay did not cross-react with Giardia-free stool samples containing Hymenolepis nana, Entamoeba coli, E. hartmanni, Chilomastix mesnili or Ancylostoma duodenale. Giardia cysts and/or trophozoites were also observed in 11 of 32 dog faecal samples (34%) in Perth and 11 of 29 dog samples (38%) in Fitzroy Crossing, after one zinc sulphate examination. The sensitivity of the ELISA for dogs was 64% and 55% for Perth and Fitzroy Crossing specimens respectively. The specificity was 95% when Fitzroy Crossing samples were used. Other parasites observed in Giardia-free faecal samples from dogs which did not produce a positive reaction with the kit were Ancylostoma caninum, Sarcocystis sp. and Isospora sp.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8465392

  2. Comparative Diagnosis of Serum IgG1 and Coproantigen ELISA for Fasciolosis Detection of Goats in Mexico

    PubMed Central

    Molina-Mendoza, Pedro; Hernández-Guzmán, Karina; Olivares-Pérez, Jaime; Sarracent-Pérez, Jorge; Zumaquero-Ríos, José

    2016-01-01

    The objective of present study was to determine the prevalence of natural caprine fasciolosis in the Mixteca region of Mexico using coproantigen and serum IgG1 ELISA tests for comparative purposes. A total of 1070 serum and faecal samples were analyzed for IgG1 antibodies and coproantigens, using ELISA with E/S products as antigen and a monoclonal antibody-based sandwich ELISA. Prevalence of 73.46% was found using the serological ELISA and a percentage of 77.20 was found for coproantigen ELISA. The diagnostic sensitivity and specificity for serum ELISA were 86.7% and 96.4%, and for the coproantigen ELISA they were 93.1% and 97.8%, respectively. The seropositive samples were further categorized as low, medium, or high positivity. Results show a great proportion of low and medium positive goats when the serum ELISA test was used. Correlation coefficients between coproantigens and seropositivity were statistically significant (P < 0.01) for low seropositivity (r = 0.93) and medium seropositivity (r = 0.84). The accuracy of faecal antigen ELISA was higher compared to indirect ELISA serological test. Two ELISAs were shown to be useful for demonstrating the current status of F. hepatica infection in the endemic areas and can be employed in studies on epidemiology as well as anthelmintics treatment for preventing economic loss and the risk of transmission to humans. PMID:27563665

  3. Comparative Diagnosis of Serum IgG1 and Coproantigen ELISA for Fasciolosis Detection of Goats in Mexico.

    PubMed

    Villa-Mancera, Abel; Molina-Mendoza, Pedro; Hernández-Guzmán, Karina; Olivares-Pérez, Jaime; Sarracent-Pérez, Jorge; Zumaquero-Ríos, José

    2016-01-01

    The objective of present study was to determine the prevalence of natural caprine fasciolosis in the Mixteca region of Mexico using coproantigen and serum IgG1 ELISA tests for comparative purposes. A total of 1070 serum and faecal samples were analyzed for IgG1 antibodies and coproantigens, using ELISA with E/S products as antigen and a monoclonal antibody-based sandwich ELISA. Prevalence of 73.46% was found using the serological ELISA and a percentage of 77.20 was found for coproantigen ELISA. The diagnostic sensitivity and specificity for serum ELISA were 86.7% and 96.4%, and for the coproantigen ELISA they were 93.1% and 97.8%, respectively. The seropositive samples were further categorized as low, medium, or high positivity. Results show a great proportion of low and medium positive goats when the serum ELISA test was used. Correlation coefficients between coproantigens and seropositivity were statistically significant (P < 0.01) for low seropositivity (r = 0.93) and medium seropositivity (r = 0.84). The accuracy of faecal antigen ELISA was higher compared to indirect ELISA serological test. Two ELISAs were shown to be useful for demonstrating the current status of F. hepatica infection in the endemic areas and can be employed in studies on epidemiology as well as anthelmintics treatment for preventing economic loss and the risk of transmission to humans. PMID:27563665

  4. A highly sensitive sandwich ELISA for the determination of glycomacropeptide to detect liquid whey in raw milk.

    PubMed

    Chávez, Norma A; Jauregui, Juan; Palomares, Laura A; Macías, Karla E; Jiménez, Mariela; Salinas, Eva

    2012-03-01

    Milk processing industries and distributors have problems with adulteration of liquid milk by the addition of bovine cheese whey. Recently, the detection of fraudulent manipulation of milk with whey has focused on the identification of glycomacropeptide (GMP). Current non-immunological methods to detect GMP in dairy products are expensive and time-consuming or have low sensitivity. In this study, a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of whey in raw milk was developed, using a polyclonal rabbit anti-GMP antibody. Calibration curves were constructed by analyzing raw milk standards containing different known concentrations of liquid cheese whey (0.02-20%). The method had a detection limit of 0.047% (v/v) and a quantification limit of 0.14% (v/v). The antibody showed high specificity and no cross-reaction with milk components (other than κ-casein) and was successful in detecting GMP in dairy commercial products. The recovery ratio was between 95.62% and 113.88% for all matrices tested. The intra-assay and interassay coefficients of variation were <6% and <7%, respectively. Finally, it can be stored for 3 months in the form of a ready-to-use kit, while maintaining its accuracy and reproducibility. PMID:22662290

  5. Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care.

    PubMed

    Wang, Shuqi; Zhao, Xiaohu; Khimji, Imran; Akbas, Ragip; Qiu, Weiliang; Edwards, Dale; Cramer, Daniel W; Ye, Bin; Demirci, Utkan

    2011-10-21

    Ovarian cancer is asymptomatic in the early stages and most patients present with advanced levels of disease. The lack of cost-effective methods that can achieve frequent, simple and non-invasive testing hinders early detection and causes high mortality in ovarian cancer patients. Here, we report a simple and inexpensive microchip ELISA-based detection module that employs a portable detection system, i.e., a cell phone/charge-coupled device (CCD) to quantify an ovarian cancer biomarker, HE4, in urine. Integration of a mobile application with a cell phone enabled immediate processing of microchip ELISA results, which eliminated the need for a bulky, expensive spectrophotometer. The HE4 level detected by a cell phone or a lensless CCD system was significantly elevated in urine samples from cancer patients (n = 19) than healthy controls (n = 20) (p < 0.001). Receiver operating characteristic (ROC) analyses showed that the microchip ELISA coupled with a cell phone running an automated analysis mobile application had a sensitivity of 89.5% at a specificity of 90%. Under the same specificity, the microchip ELISA coupled with a CCD had a sensitivity of 84.2%. In conclusion, integration of microchip ELISA with cell phone/CCD-based colorimetric measurement technology can be used to detect HE4 biomarker at the point-of-care (POC), paving the way to create bedside technologies for diagnostics and treatment monitoring. PMID:21881677

  6. Development of monoclonal antibody-based galactomannoprotein antigen-capture ELISAs to detect Aspergillus fumigatus infection in the invasive aspergillosis rabbit models.

    PubMed

    Wang, Z-Y; Cai, J-P; Qiu, L-W; Hao, W; Pan, Y-X; Tung, E T K; Lau, C C Y; Woo, P C Y; Lau, S K P; Yuen, K-Y; Che, X-Y

    2012-11-01

    Aspergillus fumigatus is one of the most prominent opportunistic fungal pathogens in immunocompromised hosts. Early recognition of this infection along with prompt antifungal therapy may increase the survival rate. We expressed two potential bio-markers of A. fumigatus infection-galactomannoprotein Afmp1p and Afmp4p in Pichia pastoris. We generated 33 monoclonal antibodies (MAbs), 20 against recombinant Afmp1p (rAfmp1p) and the other 13 against recombinant Afmp4p (rAfmp4p). Subsequently, we developed two antigen-capture enzyme-linked immunosorbent assays (ELISAs) which employed MAbs as both the capture and the detection antibodies for rAfmp1p and rAfmp4p. The two antigen-capture ELISAs specifically detected Afmp1p/Afmp4p in cultures of A. fumigatus and had no cross-reaction with other tested pathogenic fungi, including Penicillium marneffei and other pathogenic Aspergillus species. The Afmp1p-captured ELISA would be positive even when the culture supernatant of A. fumigatus had been diluted to 128-fold of its original concentration. The two antigen ELISAs could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus-challenged animal models. We developed two antigen-capture ELISAs for the laboratory diagnosis of A. fumigatus infection. These two antigen-capture ELISAs may be useful in the clinical diagnosis of aspergillosis. PMID:22669560

  7. Detection of cyclic di-AMP using a competitive ELISA with a unique pneumococcal cyclic di-AMP binding protein

    PubMed Central

    Underwood, Adam J.; Zhang, Yang; Metzger, Dennis W.; Bai, Guangchun

    2014-01-01

    Cyclic di-AMP (c-di-AMP) is a signaling molecule that has been shown to play important roles in bacterial physiology and infections. Currently, c-di-AMP detection and quantification relies mostly on the use of high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed, which utilizes a novel pneumococcal c-di-AMP binding protein (CabP) and a newly commercialized c-di-AMP derivative. With this new method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. Furthermore, this assay is much more efficient than current methods as it requires less overall cost and training while processing many samples at once. Therefore, this assay can be extensively used in research into c-di-AMP signaling. PMID:25239824

  8. Antigen sandwich ELISA predicts RT-PCR detection of dengue virus genome in infected culture fluids of Aedes albopictus C6/36 cells.

    PubMed

    Buerano, Corazon C; Natividad, Filipinas F; Contreras, Rodolfo C; Ibrahim, Ima Nurisa; Mangada, Marlou Noel M; Hasebe, Futoshi; Inoue, Shingo; Matias, Ronald R; Igarashi, Akira

    2008-09-01

    Antigen detection by sandwich ELISA was evaluated to predict RT-PCR detection of dengue viral genome in infected culture fluid of Aedes albopictus clone C6/36 cells. Serum specimens collected from dengue patients within 5 days from onset of fever in 2 hospitals in Metro Manila, Philippines, were inoculated into C6/36 cells, and incubated at 28 degrees C. A total of 282 infected culture fluid specimens were harvested and examined by sandwich ELISA and RT-PCR to detect dengue viral antigen and genome, respectively. In the sandwich ELISA, the P/N ratio was calculated by dividing optical density (OD) of a given test specimen by the OD of the standard negative specimen. Samples with a P/N ratio > or = 4.001 were positive for viral genome detection by RT-PCR. The sensitivity and specificity of antigen sandwich ELISA with RT-PCR as the standard, were 90.4% and 100%, respectively. Although antigen sandwich ELISA is less sensitive than RT-PCR, its usefulness lies in its capability to screen a large number of samples at a minimum cost, especially during an outbreak. Samples that meet a set cutoff value can undergo confirmation by RT-PCR for further epidemiological studies. PMID:19058574

  9. Development of a direct competitive ELISA for the detection of Mycoplasma bovis infection based on a monoclonal antibody of P48 protein

    PubMed Central

    2014-01-01

    Background Mycoplasma bovis (M. bovis) is a major, but often overlooked, pathogen documented to cause respiratory disease, mastitis, and arthritis in cattle throughout China since 2008. Here, we report the development of a direct competitive enzyme-linked immunosorbent assay (Dc-ELISA) to detect M. bovis antibody. Results We used a recombinant P48 protein and monoclonal antibody (mAb) 10E. MAb 10E, prepared against the recombinant P48 protein of M. bovis, identified all M. bovis strains with no cross-reactivity with other related pathogens. Coating micro plates with P48 protein instead of whole M. bovis cells as well as the use of mAb 10E produced a specific and sensitive Dc-ELISA for M. bovis antibody detection with a cut-off percent inhibition (PI) value of 32%. Compared with two commercial indirect ELISA (i-ELISA) kits, our Dc-ELISA offered a higher positive detection rate in 165 clinical bovine serum samples. Conclusions A rapid, sensitive, and reliable serological diagnosis method was developed for M. bovis, which can facilitate M. bovis surveillance, assisting researchers in understanding the ecology and epidemiology of M. bovis. PMID:24533468

  10. Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

    PubMed

    Pochechueva, Tatiana; Jacob, Francis; Goldstein, Darlene R; Huflejt, Margaret E; Chinarev, Alexander; Caduff, Rosemarie; Fink, Daniel; Hacker, Neville; Bovin, Nicolai V; Heinzelmann-Schwarz, Viola

    2011-12-01

    Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection. PMID:21948103

  11. Detection of canine vector-borne diseases in eastern Poland by ELISA and PCR.

    PubMed

    Dzięgiel, Beata; Adaszek, Łukasz; Carbonero, Alfonso; Łyp, Paweł; Winiarczyk, Mateusz; Dębiak, Piotr; Winiarczyk, Stanisław

    2016-03-01

    The aim of the study was to establish the prevalence of Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi in dogs in eastern Poland and to determine the factors associated with exposure (seroposity) or infection (PCR). Anti-A. phagocytophilum, anti-B. burgdorferi and anti-E. canis antibodies were determined in 400 dogs, using the SNAP 4Dx ® test (IDEXX Laboratories). In addition, PCRs were performed for the detection of E. canis, A. phagocytophilum and B. burgdorferi DNA. In reference to the risk factor analysis, a regression logistic model was determined for each aetiological agent. The overall seroprevalence was highest for B. burgdorferi (11.0 %), followed by A. phagocytophilum (8.0 %) and E. canis (1.5 %). Eleven healthy dogs were found to be infected with A. phagocytophilum, as determined by PCR, while the remainder were seronegative. For B. burgdorferi, the DNA of the spirochetes was detected in the blood of 20 dogs, while the presence of anti-B. burgdorferi IgG was detected in the sera of ten of these. For E. canis, none of the dogs tested positive by PCR. Tick control was included as a protective factor for A. phagocytophilum and B. burgdorferi, while the origin (rural) was included as a risk factor for B. burgdorferi and A. phagocytophilum infection. In addition, breed (pure) was a risk factor for B. burgdorferi infection, and sex (female) was a risk factor for E. canis. PMID:26581374

  12. Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata

    PubMed Central

    Bakırcı, Serkan; Tait, Andrew; Kinnaird, Jane; Eren, Hasan

    2016-01-01

    Tropical or Mediterranean theileriosis, caused by the protozoan parasite Theileria annulata, remains an economically important bovine disease in North Africa, Southern Europe, India, the Middle East and Asia. The disease affects mainly exotic cattle and imposes serious constraints upon livestock production and breed improvement programmes. While microscopic and molecular methods exist which are capable of detecting T. annulata during acute infection, the identification of animals in the carrier state is more challenging. Serological tests, which detect antibodies that react against parasite-encoded antigens, should ideally have the potential to identify carrier animals with very high levels of sensitivity and specificity. However, assays developed to date have suffered from a lack of sensitivity and/or specificity and it is, therefore, necessary to identify novel parasite antigens, which can be developed for this purpose. In the present study, genes encoding predicted antigens were bioinformatically identified in the T. annulata genome. These proteins, together with a panel of previously described antigens, were assessed by western blot analysis for immunoreactivity, and this revealed that four novel candidates and five previously described antigens were recognised by immune bovine serum. Using a combination of immunoprecipitation and mass spectrophotometric analysis, an immunodominant protein (encoded by TA15705) was identified as Ta9, a previously defined T cell antigen. Western blotting revealed another of the five proteins in the Ta9 family, TA15710, also to be an immunodominant protein. However, validation by Enzyme-Linked Immunosorbent Assay indicated that due to either allelic polymorphism or differential immune responses of individual hosts, none of the novel candidates can be considered ideal for routine detection of T. annulata-infected/carrier animals. PMID:27270235

  13. Identification and Analysis of Immunodominant Antigens for ELISA-Based Detection of Theileria annulata.

    PubMed

    Bilgic, Huseyin Bilgin; Karagenc, Tulin; Bakırcı, Serkan; Shiels, Brian; Tait, Andrew; Kinnaird, Jane; Eren, Hasan; Weir, William

    2016-01-01

    Tropical or Mediterranean theileriosis, caused by the protozoan parasite Theileria annulata, remains an economically important bovine disease in North Africa, Southern Europe, India, the Middle East and Asia. The disease affects mainly exotic cattle and imposes serious constraints upon livestock production and breed improvement programmes. While microscopic and molecular methods exist which are capable of detecting T. annulata during acute infection, the identification of animals in the carrier state is more challenging. Serological tests, which detect antibodies that react against parasite-encoded antigens, should ideally have the potential to identify carrier animals with very high levels of sensitivity and specificity. However, assays developed to date have suffered from a lack of sensitivity and/or specificity and it is, therefore, necessary to identify novel parasite antigens, which can be developed for this purpose. In the present study, genes encoding predicted antigens were bioinformatically identified in the T. annulata genome. These proteins, together with a panel of previously described antigens, were assessed by western blot analysis for immunoreactivity, and this revealed that four novel candidates and five previously described antigens were recognised by immune bovine serum. Using a combination of immunoprecipitation and mass spectrophotometric analysis, an immunodominant protein (encoded by TA15705) was identified as Ta9, a previously defined T cell antigen. Western blotting revealed another of the five proteins in the Ta9 family, TA15710, also to be an immunodominant protein. However, validation by Enzyme-Linked Immunosorbent Assay indicated that due to either allelic polymorphism or differential immune responses of individual hosts, none of the novel candidates can be considered ideal for routine detection of T. annulata-infected/carrier animals. PMID:27270235

  14. Development and application of an indirect ELISA test for the detection of antibodies to Mycoplasma crocodyli infection in crocodiles (Crocodylus niloticus).

    PubMed

    Dawo, Fufa; Mohan, Krishna

    2007-01-31

    Non-availability of a standardized rapid serodiagnostic test for quick and accurate diagnosis of Mycoplasma crocodyli (M. crocodyli) infection in crocodiles was the underlining reason for conducting the present study. An indirect enzyme-linked immunosorbent assay (iELISA) for the detection of antibodies (Ab) to M. crocodyli infection in crocodile sera was developed using sonicated antigen (Ag) and anti-crocodile conjugate. The iELISA test was optimised with different reagents and at different steps. A cut-off value of percent positive greater than or equal to 53.47% resulted in an estimated sensitivity and specificity of 85.67 and 100%, respectively. The developed iELISA could be used for detection of Abs to M. crocodyli infection in crocodiles and may enable to understand the transmission of the disease. PMID:17014973

  15. Comparison of ELISA and capillary electrophoresis with laser-induced fluorescence detection in the analysis of Ochratoxin A in low volumes of human blood serum.

    PubMed

    Köller, Gábor; Wichmann, Gunnar; Rolle-Kampczyk, Ulrike; Popp, Peter; Herbarth, Olf

    2006-08-18

    In this paper the determination of Ochratoxin A (OTA) in low volumes of human blood serum by enzyme-linked immunosorbent assay (ELISA) is compared with an appropriate capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) method. In order to use ELISA for high-throughput analysis in epidemiological studies no sample cleanup was performed. Both methods showed a limit of detection (LOD) of 0.5 ng/mL. Comparing the precisions of both methods, the data show that the quantified concentrations in ELISA are higher than the corresponding concentrations in the CE-LIF method. Using a matrix calibration curve instead of a standard calibration curve the reproducibilities of both methods are comparable. No additional matrix effect could be observed by adding phenylalanine as probable matrix compound to the serum. PMID:16731055

  16. Development of sandwich dot-ELISA for specific detection of Ochratoxin A and its application on to contaminated cereal grains originating from India

    PubMed Central

    Venkataramana, M.; Rashmi, R.; Uppalapati, Siva R.; Chandranayaka, S.; Balakrishna, K.; Radhika, M.; Gupta, Vijai K.; Batra, H. V.

    2015-01-01

    In the present study, generation and characterization of a highly specific monoclonal antibody (mAb) against Ochratoxin A (OTA) was undertaken. The generated mAb was further used to develop a simple, fast, and sensitive sandwich dot-ELISA (s-dot ELISA) method for detection of OTA from contaminated food grain samples. The limit of detection (LOD) of the developed enzyme-linked immunosorbent assay (ELISA) method was determined as 5.0 ng/mL of OTA. Developed method was more specific toward OTA and no cross reactivity was observed with the other tested mycotoxins such as deoxynivalenol, fumonisin B1, or aflatoxin B1. To assess the utility and reliability of the developed method, several field samples of maize, wheat and rice (n = 195) collected from different geographical regions of southern Karnataka region of India were evaluated for the OTA occurrence. Seventy two out of 195 samples (19 maize, 38 wheat, and 15 rice) were found to be contaminated by OTA by s-dot ELISA. The assay results were further co-evaluated with conventional analytical high-performance liquid chromatography (HPLC) method. Results of the s-dot ELISA are in concordance with HPLC except for three samples that were negative for OTA presence by s-dot ELISA but found positive by HPLC. Although positive by HPLC, the amount of OTA in the three samples was found to be lesser than the accepted levels (>5 μg/kg) of OTA presence in cereals. Therefore, in conclusion, the developed s-dot ELISA is a better alternative for routine cereal based food and feed analysis in diagnostic labs to check the presence of OTA over existing conventional culture based, tedious analytical methods. PMID:26074899

  17. A novel enzyme-linked immunosorbent assay (ELISA) for the detection of beryllium antibodies.

    PubMed

    Clarke, S M

    1991-03-01

    A novel immunological method has been developed for detecting antibodies (IgG molecules) specific to beryllium, a light metal used in industry and capable of causing chronic beryllium disease. Beryllium metal was vacuum deposited onto commercially available immunological microsticks, which were then exposed to test plasma containing the putative antibodies. Antigen-antibody complexes were located using a biotin-avidin amplification method. One employee diagnosed with chronic beryllium disease and one diagnosed as "sensitized" (lymphocyte transformation positive) exhibited antibody titers graphically and statistically different and higher than a pooled baseline control population. Plasma from these two employees (former beryllium workers) was used in four different approaches to validate the presence of beryllium antibodies. The assay proved to be reproducible. PMID:2010619

  18. [Detection of genetic modification in maize and maize products by ELISA-test].

    PubMed

    Urbanek-Karłowska, Bogumiła; Sawilska-Rautenstrauch, Dorota; Jedra, Małgorzata; Badowski, Paweł

    2003-01-01

    Enzyme immunoassay methods--TRAIT Test--was applied for detection of genetic modification in maize seeds and foodstuffs, which have been produced from this crop. TRAIT Test is based on the identification GMO protein Cry 1Ab produced by a gene derived from Bacillus thuringiensis (Bt) incorporated into insect resistant corn grain. The experiment was carried out on maize standards and foodstuffs from Warsaw market. The positive result was obtained for one maize product, which was not labelled as GMO. The presence of GMO material was approximately equal to 1%. In conclusion, this test is proper for fast routine qualitative (yes/no) determination GMO material in maize seeds and unprocessed food products. PMID:15052732

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

    FUKAI, Katsuhiko; NISHI, Tatsuya; MORIOKA, Kazuki; YAMADA, Manabu; YOSHIDA, Kazuo; KITANO, Rie; YAMAZOE, Reiko; KANNO, Toru

    2015-01-01

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

  1. Development and evaluation of a DAS-ELISA for rapid detection of Tembusu virus using monoclonal antibodies against the envelope protein.

    PubMed

    Chen, Hao; Ou, Quanbin; Tang, Yi; Gao, Xuhui; Wu, Lili; Xue, Cong; Yu, Chunmei; Cui, Jingteng; Diao, Youxiang

    2014-01-01

    Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin. PMID:24797141

  2. Development and Evaluation of a DAS-ELISA for Rapid Detection of Tembusu Virus Using Monoclonal Antibodies against the Envelope Protein

    PubMed Central

    Chen, Hao; Ou, Quanbin; Tang, Yi; Gao, Xuhui; Wu, Lili; Xue, Cong; Yu, Chunmei; Cui, Jingteng; Diao, Youxiang

    2014-01-01

    Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin. PMID:24797141

  3. Novel fluorescent ELISA for the sensitive detection of zearalenone based on H2O2-sensitive quantum dots for signal transduction.

    PubMed

    Zhan, Shengnan; Huang, Xiaolin; Chen, Rui; Li, Juan; Xiong, Yonghua

    2016-09-01

    A direct competitive fluorescent enzyme-linked immunosorbent assay (ELISA) was developed for the detection of zearalenone (ZEN) using ZEN labeled catalase (CAT) as a competing antigen with H2O2-sensitive CdTe quantum dots (QDs) for signal transduction. The novel fluorescent ELISA showed very high sensitivity for ZEN detection because it combined the high catalytic activity of CAT to H2O2 and H2O2-sensitive property of QDs. Under optimal conditions, the developed method showed a good dynamic linear detection for ZEN in the range of 2.4pg/mL to 1.25ng/mL with a detection limit of 4.1pg/mL. The median inhibition concentration (IC50) of ZEN was 75pg/mL, which was approximately 17-fold lower than that of horseradish peroxidase-based conventional ELISA. Moreover, our developed method also showed a high reproducibility and an excellent selectivity. In brief, the novel fluorescent ELISA shows great potential for the sensitive and economic detection of mycotoxins and other analytes in food analysis, clinical diagnosis and environmental monitoring. PMID:27343577

  4. Development of an antigen-capture ELISA for the detection of the p27-CA protein of HERV-K(HML-2).

    PubMed

    Hohn, Oliver; Mostafa, Saeed; Norley, Stephen; Bannert, Norbert

    2016-08-01

    The detection or quantification of retroviruses is often achieved using an antigen-capture ELISA (AC-ELISA) that targets the Gag capsid (CA) protein. We report here the development of an AC-ELISA specific for the p27-CA protein of HERV-K(HML-2). A monoclonal p27-specific antibody is used for capture and a polyclonal anti-p27-CA immune serum generated in rabbits serves for detection. The assay was shown to be specific for HERV-K(HML-2), showing no evidence of cross reactivity with the human retroviruses HIV-1, HIV-2 and HTLV-1 or with XMRV (as a model non-human gammaretrovirus). Using purified recombinant antigen, the limit of detection was shown to be 130pg/ml. The AC-ELISA can be used to quantify HERV-K(HML-2) expression in teratocarcinoma cell lines and to normalize HERV particles generated by transfecting HEK 293T cells with full-length molecular clones. This novel AC-ELISA also proved useful in studies of virus regulation, for example in demonstrating that HERV-K(HML-2) expression is dramatically enhanced by overexpression of Staufen-1, a binding partner of the HERV-K(HML-2) Rec protein. This specific and sensitive HERV-K(HML-2) AC-ELISA will be a useful tool for investigating many aspects of endogenous retroviruses, from basic research to the role they may play in human diseases or as a surrogate marker for particular diseases. PMID:27142113

  5. Fluorescence ELISA for sensitive detection of ochratoxin A based on glucose oxidase-mediated fluorescence quenching of CdTe QDs.

    PubMed

    Liang, Yi; Huang, Xiaolin; Yu, Ruijin; Zhou, Yaofeng; Xiong, Yonghua

    2016-09-14

    The present study described a novel fluorescence enzyme-linked immunosorbent assay (ELISA) used to detect ochratoxin A (OTA) by using the glucose oxidase (GOx)-mediated fluorescence quenching of mercaptopropionic acid-capped CdTe quantum dots (MPA-QDs), in which GOx was used as an alternative to horseradish peroxidase (HRP) for the oxidization of glucose into hydrogen peroxide (H2O2) and gluconic acid. The MPA-QDs were used as a fluorescent signal output, whose fluorescence variation was extremely sensitive to the presence of H2O2 or hydrogen ions in the solution. Under the optimized conditions, the proposed fluorescence ELISA demonstrated a good linear detection of OTA in corn extract from 2.4 pg mL(-1) to 625 pg mL(-1) with a limit of detection of 2.2 pg mL(-1), which was approximately 15-fold lower than that of conventional HRP-based ELISA. Our developed fluorescence immunoassay was also similar to HRP-based ELISA in terms of selectivity, accuracy, and reproducibility. In summary, this study was the first to use the GOx-mediated fluorescence quenching of QDs in immunoassay to detect OTA, offering a new possibility for the analysis of other mycotoxins and biomolecules. PMID:27566355

  6. ABILITY OF AN ELISA-BASED SEED HEALTH TEST TO DETECT ERWINIA STEWARTII IN MAIZE SEED TREATED WITH FUNGICIDES AND INSECTICIDES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISA-based seed health test detected E. stewartii in infected maize seed treated with 11 combinations of captan, carboxin, clothianidin, imidacloprid, thiamethoxam, fludioxonil, mefenoxam, or thiram. Treated healthy seeds were blended with treated E. stewartii-infected seed of Jubilee or A632 t...

  7. ABILITY OF AN ELISA-BASED SEED HEALTH TEST TO DETECT ERWINIA STEWARTII IN MAIZE SEED TREATED WITH FUNGICIDES AND INSECTICIDES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISA-based seed health test detected E. stewartii in infected maize seed treated with 11 combinations of captan, carboxin, clothianidin, imidacloprid, thiamethoxam, fludioxonil, mefenoxam, or thiram. Treated, healthy seeds were blended with treated, E. stewartii-infected seeds of Jubilee or A632...

  8. Evaluation of a commercial ELISA kit for detection of antibodies against Toxoplasma gondii in serum, plasma and meat juice from experimentally and naturally infected sheep

    PubMed Central

    2013-01-01

    Background Toxoplasmosis is one of the most common food borne zoonoses worldwide, and can be a serious life-threatening disease in the congenitally infected fetus and in immunosupressed patients. Among food animals, sheep along with goats and pigs possess the highest incidence of T. gondii cysts in meat, and play a major role as a source of human infection. Methods In this study, a new commercial ELISA kit (PrioCHECK® Toxoplasma Ab SR, Prionics Schlieren-Zurich, Switzerland) for the detection of anti-T. gondii antibodies in serum, plasma and meat juice of sheep, was evaluated by comparing it with the indirect fluorescent antibody test (IFAT), indirect haemagglutination test (IHA) and real-time PCR, on samples from experimentally inoculated and naturally exposed sheep. Results The commercial ELISA detected the infection status in 50% and 100% of sheep orally inoculated with 10,000 T. gondii oocysts (n = 6), from two or three weeks post infection (wpi), respectively, both on serum and plasma samples. Meat juice from all experimentally inoculated sheep collected at slaughter (12 wpi) showed positive ELISA values. In naturally exposed sheep (n = 396), the ELISA showed a very good agreement with IFAT (kappa = 0.91-1.0) and IHA (kappa = 0.96-1.0) performed on serum; and a positive correlation was observed between ELISA values and IFAT titers. By a Receiver Operating Characteristics (ROC) curve analysis, the commercial ELISA had relative sensitivities between 93.33% and 100%, and relative specificities between 96.87% and 100% respect to IFAT and IHA, depending on the considered cut-off value and animal groups tested. Furthermore, the ELISA correctly recognized all animals reacting positive in real-time PCR. The ELISA results on meat juice agreed with those on serum samples in all experimentally inoculated animals, and in 94 out of 96 (97.9%) naturally exposed sheep, when meat juice was tested at a 1:10 dilution. Conclusion The commercial ELISA kit

  9. Determination of the IgG index for the detection of intrathecal immunoglobulin synthesis in dogs using an ELISA.

    PubMed

    Tipold, A; Pfister, H; Vandevelde, M

    1993-01-01

    The IgG index is a calculated quotient using IgG and albumin contents of cerebrospinal fluid (CSF) and serum to detect intrathecal IgG synthesis, which is important in the diagnosis of inflammatory infectious diseases of the central nervous system (CNS). An ELISA to measure IgG and albumin contents in blood and CSF was developed to determine the IgG index. Twenty-three normal dogs and 98 dogs with various neurological diseases were examined. In most dogs with infectious inflammatory diseases, with the exception of the acute form of nervous canine distemper, there was an elevation of the IgG index. Tumours of the CNS had an IgG index within the normal range. An elevation was found in lymphoid tumours or meningiomas with secondary cellular infiltration. Degenerative diseases, spinal cord compression and ischaemic myelopathy due to fibrocartilagenous embolism had normal or only slightly elevated IgG indices. In seven cases with inflammatory lesions there was no obvious CSF pleocytosis but the IgG index was elevated. In 17 cases in which infectious inflammatory disease was suspected, based on elevated CSF cell counts, inflammation could be excluded after determination of the IgG index. The reproducibility of the technique was found to be good and, considering the limited amount of time, expense and effort required, the determination of the IgG index in dogs is a useful extension of the conventional CSF examination. PMID:8434146

  10. Efficacy of Dot-ELISA using different antigens in detecting anti-schistosome antibodies among bovines in field conditions.

    PubMed

    Lakshmanan, Bindu; Devada, K; Joseph, Siju; Binu, M B; Kuttan, Karthik

    2016-03-01

    Schistosomosis has been recognised as one of the major parasitic diseases of livestock and human beings. Schistosoma spindale is the major cause of visceral schistosomosis among bovines of Kerala State. Besides pathology in animals, it has been long known that cercariae of S. spindale are a common cause of dermatitis in human beings in Asia. However, detection of this disease based on coprology has underestimated the prevalence of this economically important disease among cattle of the State. An efficient diagnostic tool providing unequivocal evidence of infection in living animals is perhaps, the key to formulate and deliver control measures to the target population. It is also crucial for an enhanced understanding of parasite epidemiology. The utility of excretory-secretory proteins as diagnostic and vaccine candidates for schistosomosis has been a focus of medical research since long. There exists a paucity of information with regard to analysis of ES proteins of S. spindale and their incorporation to develop sensitive and specific serodiagnostic tool. Hence a study was designed to evaluate the efficacy of Dot-ELISA incorporating different antigens of S. spindale and to validate the test under field conditions. PMID:27065623

  11. Improving the detection of IgM antibodies against glycolipids complexes of GM1 and Galactocerebroside in Multifocal Motor Neuropathy using glycoarray and ELISA assays.

    PubMed

    Delmont, Emilien; Halstead, Susan; Galban-Horcajo, Francesc; Yao, Denggao; Desnuelle, Claude; Willison, Hugh

    2015-01-15

    Antibodies against complexes of GM1:GalC are detected in multifocal motor neuropathy. Previous studies used different techniques, explaining disparities in the results. Antibodies against GM1 and GM1:GalC with different proportions of GalC were measured with both glycoarray and ELISA in 20 multifocal motor neuropathies, and 45 controls. The 1:5 ratio and the 1:1 ratio of GM1:GalC (weight ratio) were respectively the most effective for glycoarray and for ELISA. Testing for anti-GM1:GalC antibodies increased the sensitivity from 40% with anti-GM1 antibodies to 65% with array and 60% with ELISA without loss in specificity (above 91%). Anti-GM1:GalC antibodies are effective biological tools to diagnose multifocal motor neuropathy. PMID:25468269

  12. Development of a RT-PCR ELISA for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants and its evaluation on clinical samples.

    PubMed

    Dubey, Pooja; Mishra, N; Rajukumar, K; Behera, S P; Kalaiyarasu, S; Nema, R K; Prakash, A

    2015-03-01

    The aim of this study was to develop a reverse transcription polymerase chain reaction ELISA (RT-PCR ELISA) for detection of ruminant pestiviruses and to evaluate its diagnostic performance on clinical samples obtained from cattle, sheep and goats. Optimization was carried out by serial dilution of home-made digoxygenin-labelled RT-PCR product standards obtained from pestivirus isolates and pestivirus infected animals. The detection limit of the assay was 10TCID50/ml, similar to virus isolation and real-time RT-PCR but 10-fold higher than RT-PCR. The assay had high analytical specificity along with a good reproducibility. When the assay was evaluated on the samples obtained from animals infected experimentally with BVDV and from the field using virus isolation as standard, it showed a high diagnostic sensitivity (95.9%) and specificity (98.6%) and there was strong agreement (97.5% concordance) between the two tests. However, it displayed an increased diagnostic specificity and sensitivity over RT-PCR. Additionally, when a few samples (n=26) were tested by RT-PCR ELISA and real-time RT-PCR, 100% concordance was obtained between them. Our results showed that RT-PCR ELISA can be a rapid, cost effective and alternative molecular diagnostic test for simultaneous detection of BVDV-1, BVDV-2 and BDV in ruminants in ordinary laboratory settings. PMID:25486086

  13. Pretreatment of serum samples to reduce interference of colostrum-derived specific antibodies with detection of Bovine viral diarrhea virus antigen by ELISA in young calves.

    PubMed

    Lanyon, Sasha R; Reichel, Michael P

    2016-05-01

    Antigen enzyme-linked immunosorbent assay (ELISA) is used for the detection of Bovine viral diarrhea virus persistently infected (BVDV PI) cattle; however, colostrum-derived antibodies may interfere with antigen detection in serum from young PI calves. Our study aimed to assess serum pretreatment methods for reducing such interference. Dilution of PI serum with serum containing specific antibody showed that antibody levels equivalent to those observed in colostrum-fed calves were able to eliminate all antigen signals in a serum sample. Serum was treated with ethylenediamine tetra-acetic acid at pH 4.5, 5.5, 6.5, and 7.5, then boiled, centrifuged, and the supernatant-recovered. BVDV antibody was undetectable by ELISA in supernatants from treated samples, and the antigen ELISA signal was improved. Maximum antigen signal recovery of >90% was achieved at pH 5 ± 0.5. When this optimal treatment method was applied to field samples from 3 PI calves (which were negative in the antigen-capture ELISA without treatment), the antigen signal improved and gave a positive result in each case. Pretreatment may provide an improvement in the detection of young PI calves. PMID:27016723

  14. Detection of preformed type A botulinal toxin in hash brown potatoes by using the mouse bioasssay and a modified ELISA test.

    PubMed

    Ferreira, J L; Eliasberg, S J; Harrison, M A; Edmonds, P

    2001-01-01

    A foodborne illness caused by type A toxin-producing Clostridium botulinum was investigated by using the standard mouse bioassay and a rapid invitro test for toxin detection. The patient, who consumed improperly stored hash brown potatoes that contained the preformed toxin, was diagnosed with type A botulism. C. botulinum type A toxin was detected in the hash brown potatoes as well as in the tryptone-peptone-glucose-yeast extract (TPGY) medium subcultures of this food using the mouse bioassay and an amplified ELISA technique. The mouse bioassay revealed preformed toxin at 10,000 minimum lethal dose (MLD)/g uncooked product and the amplified ELISA an equivalent 50,000 MLD/g. The cultural toxin from the uncooked product killed mice at the 10(6) dilution and a modification of the ELISA procedure was positive at the 10(3) dilution. Cooked food obtained from the consumer's waste can contained 100 MLD/g and the ELISA was also positive at the same dilution of the product. The culture of the cooked product obtained from the waste can was lethal for mice at the 10(7) dilution and positive using the modified ELISA at the 10(4) dilution. The unmodified amplified ELISA method indicated a toxin level of approximately 1 ng/mL (equivalent to 5 x 10(5) MLD/mL) in diluted culture fluid from the uncooked food and the culture of cooked food obtained from the waste can. The hash brown potatoes were negative for types B, E, and F preformed and cultural botulinal toxins using both assays. PMID:11601465

  15. Enzyme-linked immunosorbent assay (ELISA) for brown trout vitellogenin for use in the detection of environmental estrogens

    SciTech Connect

    Gamble, A.; Solomon, K.; Sherry, J.; Fielden, M.; Hodson, P.

    1995-12-31

    Vitellogenin (Vg) is a phospholipoprotein egg yolk precursor found in females of most oviparous vertebrates. Vitellogenin induction in male teleosts is known to result from exposure to xenoestrogens. Using antisera for brown trout Vg, the authors adapted an (ELISA) for in vivo Vg in brown trout (Salmo trutta). The authors will use the Vg bioassay to detect the estrogenic effects of effluents, contaminated sediments and suspect chemicals. Vg production in male fish was induced by intraperitoneal injection of 11-B estradiol. Harvested and purified Vg was used in an assay based on competition between soluble Vg and Vg adsorbed in microtitre wells for binding sites on rabbit anti-Vg antibody. Adsorbed Vg-antibody complexes were subsequently revealed by an alkaline phosphatase technique. The reaction intensity was measured as absorbance and used to quantify the amount of antibody bound to adsorbed Vg. The amount of bound antibody was inversely proportional to the amount of vitellogenin in the fish plasma. Preliminary results show that the assay`s working range, corresponding to 80--20% binding, was 14--355 ng/ml, with 50% binding (IC{sub 50}) around 71 ng/mi. Calculated at 50% binding, the intra-assay variation was less than 8% (n = 9) and the inter-assay variation was also less than 8% (n =4). The assay`s sensitivity was 0.067 ng/mi and the limit of detection was 0.134 ng/ml. The authors will present the results of Vg bioassays for the presence of estrogen mimics in industrial effluent.

  16. Specific serum immunoglobulin D, detected by antibody capture enzyme-linked immunosorbent assay (ELISA), in cytomegalovirus infection.

    PubMed Central

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

    1989-01-01

    An antibody capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of immunoglobulin D (IgD) antibodies to cytomegalovirus (CMV) in sera from blood donors and various groups of patients infected with CMV. This method has previously been found especially valuable in detecting specific antibodies of the IgM, IgE, IgA and IgG class in patients with CMV infection. Specific CMV IgD antibodies were found in 37% of CMV seropositive blood donors and in 47 (88%) of the 53 patients investigated, including bone marrow transplant and renal allograft transplant patients, patients with CMV mononucleosis, neonates with CMV infection and AIDS patients with CMV infection. The highest IgD reactivity was found in patients having either a primary post-transplant CMV infection or CMV mononucleosis. The IgD reactivity in patients with AIDS and in neonates was low. It was also found that in the acute phase of CMV infection the development of CMV antibodies of the IgD class was similar to the development of antibodies of the other classes. The maintenance of IgD activity in some patients together with the presence of CMV IgD antibodies in a great proportion of the blood donors indicates that the development of CMV IgD antibodies resembles that of the IgG class. Determination of specific IgD antibodies offered no advantage over determination of specific antibodies of the IgM, IgE and IgA classes in the diagnosis of CMV infection. PMID:2539278

  17. Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains

    PubMed Central

    Fu, Xiaoxiang; Wang, Ali; Wang, Xiaohan; Lin, Fengke; He, Lishan; Lai, Daowan; Liu, Yang; Li, Qing X.; Zhou, Ligang; Wang, Baoming

    2015-01-01

    Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC50) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples. PMID:26343725

  18. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

    PubMed Central

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Background Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Methods In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. Results The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402

  19. Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs.

    PubMed

    Kulkarni, Diwakar D; Venkatesh, Govindarajalu; Tosh, Chakradhar; Patel, Priyanka; Mashoria, Anita; Gupta, Vandana; Gupta, Sourabh; D, Senthilkumar

    2016-01-01

    The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus. PMID:26327601

  20. Correlation between ELISA and pseudovirion-based neutralisation assay for detecting antibodies against human papillomavirus acquired by natural infection or by vaccination

    PubMed Central

    Zhao, Hui; Lin, Zhi-Jie; Huang, Shou-Jie; Li, Juan; Liu, Xiao-Hui; Guo, Meng; Zhang, Jun; Xia, Ning-Shao; Pan, Hui-Rong; Wu, Ting; Li, Chang-Gui

    2014-01-01

    A pseudovirion-based neutralisation assay (PBNA) has been considered the gold standard for measuring specific antibody responses against human papillomavirus (HPV). However, this assay is labor intensive and therefore very difficult to implement in large-scale studies. Previous studies have evaluated the agreement between virus-like particle (VLP)-based ELISA and PBNA for measuring HPV vaccine-induced antibodies. However, the concordance of these assays to detect antibodies induced by natural infection has not yet been fully elucidated. In this study, the results of an Escherichia coli (E. coli)-expressed VLP-based ELISA were found to be highly concordant with those of a baculovirus-expressed VLP-based ELISA (r = 0.96 and 0.97 for HPV-16 and HPV-18) when detecing HPV vaccine induced antibodies and the concordance was medium (r = 0.68 and 0.68 for HPV-16 and HPV-18) when assessing natural infection induced antibodies. The results of the E. coli expressed VLP-based ELISA correlated well with those of the PBNA when testing 1020 post-vaccination human sera collected at one month after vaccination with the E. coli expressed VLP-based bivalent HPV vaccine (r = 0.83 and 0.81 for HPV-16 and HPV-18). The agreement and correlation were moderate (kappa < 0.3 for both HPV types 16 and 18, r = 0.59 and 0.68 for HPV-16 and HPV-18, respectively) when assessing 1600 serum samples from unvaccinated women of age 18–25 years. In conclusion, the VLP-based ELISA is an acceptable surrogate for the neutralizing antibody assay in measuring vaccine responses. However, the use of the VLP-based ELISA in epidemiological studies should be carefully considered. PMID:24384608

  1. Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

    PubMed

    Reddy, Prakash; Ramlal, Shylaja; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

    2014-06-01

    In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate. PMID:24941881

  2. Evaluation of a PCR-ELISA to detect Wuchereria bancrofti in Culex pipiens from an Egyptian village with a low prevalence of filariasis.

    PubMed

    Kamal, I H; Fischer, P; Adly, M; El Sayed, A S; Morsy, Z S; Ramzy, R M

    2001-12-01

    The programmes for the elimination of bancroftian filariasis that have been implemented in the Nile delta of Egypt are expected to lead to substantial reductions in filarial loads in the treated populations. Better methods than those currently available are needed for monitoring the efficacy of these and similar efforts at intervention. A PCR-ELISA was therefore evaluated as an epidemiological tool for the detection of the Wuchereria-bancrofti-specific SspI repeat in pools of Culex pipiens collected in a village with a low prevalence of filarial infection in its human residents (2.1%). Indoor-resting mosquitoes were collected by aspiration from 114 randomly selected houses (during one to nine visits/house) and separated into 673 pools, each of which held the mosquitoes collected during one night from one house. Although 18 (2.7%) of the pools showed PCR inhibition and had to be excluded, filarial DNA was detected, using the PCR-ELISA, in 91 (13.9%) of the 655 remaining mosquito pools. The minimum prevalence of W. bancrofti infection in the mosquitoes caught (assuming one infected mosquito/positive pool) was 2.8%. The mean (S.D.) number of mosquitoes/pool did not vary significantly between positive [5.5 (3.4)] and negative [4.9 (3.5)] pools. The assay detected parasite DNA in mosquitoes from 19.3% of 114 houses when only the first visit was considered and from 73.9% of the 88 houses visited more than once. The PCR-ELISA yielded results comparable with those of the regular PCR-SspI assay. The latter assay is recommended for the routine examination, in laboratories in endemic areas, of mosquito pools from randomly selected houses, as the ELISA component of the PCR-ELISA is exceedingly time-consuming, expensive and requires special equipment. PMID:11784438

  3. PCR and ELISA vis-à-vis Microscopy for Detection of Bovine Anaplasmosis: A Study on Associated Risk of an Upcoming Problem in North India

    PubMed Central

    Singla, L. D.; Kaur, Paramjit; Bal, M. S.

    2015-01-01

    This investigation demonstrates the status of bovine anaplasmosis caused by A. marginale in bovines from Submountain and Undulating Zone of Punjab. Out of 184 suspected animals, 25 (19.51%), 47 (31.71%), and 78 (68.75%) were positive by microscopy, indirect ELISA, and PCR assay, respectively. The microscopy showed 29% sensitivity and 99% specificity, while ELISA showed 32% sensitivity and 79% specificity in concordance with PCR assay. Five false negative samples by msp1β PCR were reconfirmed for Anaplasma spp. targeting 16S rRNA gene. The sequence analysis showed the presence for A. marginale specific restriction site, indicating variation in the local strains of the organism resulting in no amplification with msp1β gene primers. Of 82 samples positive by PCR, 57 were negative by ELISA indicating lower efficacy of ELISA to detect early anaplasmosis. The assessment of risk factor with results of PCR technique indicated that cattle (Odds ratio = 2.884), particularly those of age > 1 years (Odds ratio = 2.204) of district Pathankot (Odds ratio = 3.182) of Submountain Zone (Odds ratio = 2.086), were at high risk of anaplasmosis. All three districts of Submountain Zone are at higher risk indicating the impact of biotic and abiotic factors on the incidence of disease. PMID:25811041

  4. Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs.

    PubMed

    van Wyngaardt, Wouter; Mashau, Cordelia; Wright, Isabel; Fehrsen, Jeanni

    2013-01-01

    There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping. PMID:23388433

  5. Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis.

    PubMed

    Barrs, V R; Ujvari, B; Dhand, N K; Peters, I R; Talbot, J; Johnson, L R; Billen, F; Martin, P; Beatty, J A; Belov, K

    2015-03-01

    Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1). Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA. PMID:25634077

  6. A peptide ELISA to detect antibodies against Pythium insidiosum based on predicted antigenic determinants of exo-1,3-beta-glucanase.

    PubMed

    Keeratijarut, Angsana; Lohnoo, Tassanee; Yingyong, Wanta; Sriwanichrak, Kanchana; Krajaejun, Theerapong

    2013-07-01

    Human pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Diagnosis of pythiosis relies on culture identification, serodiagnosis, and molecular-based assay. Preparation of a serodiagnostic test requires culture filtrate antigen (CFA) extracted from the live pathogen. A 74-kDa immunoreactive protein of P. insidiosum, is encoded by the exo-1,3-beta-glucanase gene (PinsEXO1). PinsEXO1 protein is recognized by sera from pythiosis patients but not by sera from uninfected patients; therefore, this protein could be used to detect anti-P. insidiosum antibodies. In this study we aimed to: identify, synthesize, and evaluate an antigenic determinant (epitope) of PinsEXO1 to be used to serodiagnose pythiosis based on peptide ELISA, and to compare the diagnostic performance of that test with the current CFA-based ELISA. Two antigenic determinants of PinsEXO1 (Peptide-A and -B) were predicted using the PREDITOP program. The sera from 34 pythiosis patients and 92 control subjects were evaluated. Peptide-A, Peptide-B, and CFA-based ELISAs all had a specificity of 100%. Peptide-B ELISA had a sensitivity of 91% and an accuracy of 98% and both Peptide-A and CFA-based ELISAs had a sensitivity of 100% and an accuracy of 100%. Peptide-A is a more efficient epitope than Peptide-B, and can be used as an alternative antigen to develop a serodiagnostic assay for pythiosis. PMID:24050102

  7. Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR.

    PubMed

    Arif, Muhammad; Ali, Murad; Rehman, Anayatur; Fahim, Muhammad

    2014-01-01

    The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in Nicotiana debneyi plants grown in putative viruliferious soils from 20/26 locations. The results were confirmed by back inoculation of sap from both roots and leaves of bait plant on indicator hosts (N. debneyi, Nicotiana benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26 locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566bp were amplified from coat protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait plant bioassay, infectivity assay, ELISA and RT-PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea and tubers of 10 potato cultivars commercially grown in the region. PMID:24161813

  8. Comparative study of three screening tests, two microbiological tube tests, and a multi-sulphonamide ELISA kit for the detection of antimicrobial and sulphonamide residues in eggs.

    PubMed

    Gaudin, V; Hedou, C; Rault, A; Sanders, P; Verdon, E

    2009-04-01

    The screening of antimicrobial residues in eggs is an especially important subject. Three different commercial kits for the screening of sulphonamides and other antimicrobials in eggs were validated in accordance with Decision 2002/657/EC: one enzyme-linked immunoabsorbant assay (ELISA) kit multi-sulphonamides (from RAISIO Diagnostics) and two microbiological tests (a Premi test from DSM and an Explorer kit from Zeu-Inmunotec). The false-positive rates were lower than 2% for all kits. The detection capabilities (CCbeta) have to be as low as possible for banned substances and lower than the maximum residue limit (MRL) when MRLs have been set. The sensitivity of the Premi test was better than that of the Explorer test, probably because of the dilution of the eggs before the Explorer test was used. The CCbeta values towards most of the tested sulphonamides were satisfactory with the Premi test (< or = 100 microg kg(-1)). Performance in a proficiency test for the detection of sulphonamides in eggs with the Premi test confirmed these results. The detection capabilities of tetracycline and doxycycline were at the level of the MRL or twice the MRL maximum. The detection capabilities for chlortetracycline and oxytetracycline were higher (four to six times the MRL). The detection capabilities for amoxicillin, neomycin, tylosin and erythromycin were lower than their respective MRLs. Detection capabilities for sulphonamides were much lower for the ELISA kit than for microbiological tests. The ELISA kit could be recommended for the targeted screening of sulphonamides in eggs. On the other hand, the Explorer and Premi tests could be used as wide screening tests allowing the detection of most of the antimicrobial families. PMID:19680917

  9. Improved detection of equine antibodies against Sarcocystis neurona using polyvalent ELISAs based on the parasite SnSAG surface antigens.

    PubMed

    Yeargan, Michelle R; Howe, Daniel K

    2011-02-28

    Equine protozoal myeloencephalitis (EPM) is a common neurologic disease of horses that is caused by the apicomplexan pathogen Sarcocystis neurona. To help improve serologic diagnosis of S. neurona infection, we have modified existing enzyme-linked immunosorbent assays (ELISAs) based on the immunogenic parasite surface antigens SnSAG2, SnSAG3, and SnSAG4 to make the assays polyvalent, thereby circumventing difficulties associated with parasite antigenic variants and diversity in equine immune responses. Two approaches were utilized to achieve polyvalence: (1) mixtures of the individual recombinant SnSAGs (rSnSAGs) were included in single ELISAs; (2) a collection of unique SnSAG chimeras that fused protein domains from different SnSAG surface antigens into a single recombinant protein were generated for use in the ELISAs. These new assays were assessed using a defined sample set of equine sera and cerebrospinal fluids (CSFs) that had been characterized by Western blot and/or were from confirmed EPM horses. While all of the polyvalent ELISAs performed relatively well, the highest sensitivity and specificity (100%/100%) were achieved with assays containing the rSnSAG4/2 chimera (Domain 1 of SnSAG4 fused to SnSAG2) or using a mixture of rSnSAG3 and rSnSAG4. The rSnSAG4 antigen alone and the rSnSAG4/3 chimera (Domain 1 of SnSAG4 fused to Domain 2 of SnSAG3) exhibited the next best accuracy at 95.2% sensitivity and 100% specificity. Binding ratios and percent positivity (PP) ratios, determined by comparing the mean values for positive versus negative samples, showed that the most advantageous signal to noise ratios were provided by rSnSAG4 and the rSnSAG4/3 chimera. Collectively, our results imply that a polyvalent ELISA based on SnSAG4 and SnSAG3, whether as a cocktail of two proteins or as a single chimeric protein, can give optimal results in serologic testing of serum or CSF for the presence of antibodies against S. neurona. The use of polyvalent SnSAG ELISAs will

  10. ELISA-based detection of C4d after liver transplantation--a helpful tool for differential diagnosis between acute rejection and HCV-recurrence?

    PubMed

    Schmeding, Maximilian; Kienlein, Stefan; Röcken, Christoph; Neuhaus, Ruth; Neuhaus, Peter; Heidenhain, Christoph; Neumann, Ulf P

    2010-08-01

    Hepatitis-C is the most common indication for liver transplantation. Recurrence of HCV is universal leading to graft failure in up to 40% of all patients. The differentiation between acute rejection and recurrent hepatitis-C is crucial as rejection treatments are likely to aggravate HCV-recurrence. Histological examination of liver biopsy remains the gold standard for diagnosis of acute rejection but has failed in the past to distinguish between acute rejection and recurrent hepatitis-C. In a retrospective study we have recently reported that C4d as a marker of the activated complement cascade is detectable in a hepatic specimen in acute rejection after liver transplantation and may serve as a valuable tool in differential diagnosis between ACR and HCV-recurrence. We performed a prospective analysis by ELISA measurement of C4d concentration in cryo-preserved liver biopsies of LTX patients who had either experienced acute rejection, hepatitis-C recurrence or displayed no pathological alterations (controls). Opposed to our immunohistologically based findings in paraffinized tissue we were unable to detect significant differences of C4d concentration in ELISA of cryo-preserved liver tissue. Consequently the role and potential value of C4d as a diagnostic marker may not be determined using ELISA-based tissue evaluation. PMID:20558292

  11. Evaluation of a commercial ELISA for H5 low pathogenic avian influenza virus antibody detection in duck sera using Bayesian methods.

    PubMed

    Schmitz, Audrey; Le Bras, Marie-Odile; Guillemoto, Carole; Pierre, Isabelle; Rose, Nicolas; Bougeard, Stéphanie; Jestin, Véronique

    2013-10-01

    Following the emergence of highly pathogenic avian influenza (AI), active surveillance of infections due to the H5 and H7 subtypes in poultry has increased and been made compulsory in Europe since 2002, by means of annual serological surveys using the haemagglutination inhibition (HI) test. Domestic anseriforms, particularly ducks and geese, are more frequently infected by H5 low pathogenic AI virus, often subclinically, and represent a threat for other terrestrial poultry. 1783 sera, mainly from ducks, have been used to evaluate and compare a commercial ELISA kit detecting H5 antibodies with the currently recommended HI test. Different approaches to calculating specificity and sensitivity have been used, including the original Bayesian method. Results were similar when data were analyzed at the individual and batch levels, and when using different methods of calculation. However, results showed that H5 ELISA had both a higher sensitivity and a lower specificity than the HI test. Given that sensitivity is the most important factor for a screening test, H5 ELISA could therefore be recommended for AI surveillance, followed in cases of positivity by molecular tests aimed at detecting the virus gene. PMID:23727545

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

    PubMed

    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

  13. Simultaneous detection of multi-allergens in an incurred food matrix using ELISA, multiplex flow cytometry and liquid chromatography mass spectrometry (LC-MS).

    PubMed

    Gomaa, Ahmed; Boye, Joyce

    2015-05-15

    Food allergy is a public health concern and an important food safety issue. Food allergies affect up to 6% of infants and children and 4% of adults. The objective of this work was to determine differences in the detection of single and multiple allergens (i.e., casein, soy protein, and gluten) in an incurred food matrix before and after baking. Cookies were used as a model food system. Three methods, namely, multiplex assay (a new optimized method based on flow cytometry for multiple allergen analysis), enzyme-linked immunosorbent assay (ELISA) using commercial kits and LC-MS were used to detect allergens in the samples before and after baking. The ELISA kits performed well in detecting allergens in the raw samples with recoveries of 91-108%, 88-127% and 85-108% for casein, soy protein and gluten, respectively. Recoveries were poor for the baked cookies (67-90%, 66-95% and 66-88% for casein, soy protein and gluten, respectively). The multiplex flow cytometry assay permitted multiple allergen detection in the raw samples, with the following recoveries based on soluble protein: casein, 95-107%; soy protein, 92-97%, and gluten, 96-99%. Data for the baked cookies were as follows: casein, 84-90%; soy protein, 80-88%, and gluten, 80-90%. The LC-MS technique detected marker peptides that could be used to identify allergens in the baked food samples up to concentrations of 10 ppm for casein and soy protein, and 100 ppm for gluten. To the best of our knowledge, the current study is the first to compare ELISA, LC-MS and multiplex flow cytometry methods for the detection of multiple allergens simultaneously incurred in a model food system. PMID:25577123

  14. Detection of cyclic di-AMP using a competitive ELISA with a unique pneumococcal cyclic di-AMP binding protein.

    PubMed

    Underwood, Adam J; Zhang, Yang; Metzger, Dennis W; Bai, Guangchun

    2014-12-01

    Cyclic di-AMP (c-di-AMP) is a recently recognized bacterial signaling molecule. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed using a novel pneumococcal c-di-AMP binding protein (CabP). With this method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. PMID:25239824

  15. Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection.

    PubMed

    Bai, Xiaofei; Shaozhou, Wulin; Zhang, Qingshan; Li, Chenxi; Qiu, Na; Meng, Runzhe; Liu, Ming; Zhang, Yun

    2015-03-01

    The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds. PMID:25588821

  16. Development of an ELISA using a recombinant 41 kDa partial protein (P45N') for the detection of Riemerella anatipestifer infections in ducks.

    PubMed

    Huang, Bin; Kwang, Jimmy; Loh, Hilda; Frey, Joachim; Tan, Hai-Meng; Chua, Kim-Lee

    2002-09-24

    Riemerella anatipestifer, a gram-negative bacillus, is the causative agent of duck septicemia, a disease which could incur much economic loss in the duck industry. An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to facilitate early detection of R. anatipestifer infection in ducks. The antigen used was a recombinant 41 kDa N-terminal fragment (rP45N') of a newly characterized R. anatipestifer potential surface protein, P45, which was expressed in Escherichia coli as an N-terminal GST fusion protein. The rP45N'-based ELISA successfully detected P45 antibodies in the sera of 20 ducks immunized with bacterin preparations of R. anatipestifer serotypes 1, 10 15, 19 and the ATCC11845 strain. Antibodies to P45 were also detected in the sera of 25% (75/296) of White Pekin ducks which were imported into Singapore from three different farms. Successful discrimination was obtained between sera from infected ducks and that of specific-pathogen free ducks (p<0.01). The rP45N'-GST antigen did not cross-react with antibodies in sera from guinea pigs which were infected with other gram-negative and gram-positive bacterial pathogens, including Aeromonas hydrophila, Citrobacter freundii, E. coli, Klebsiella pneumoniae, Pastuerella multocida, Proteus mirabilis, Salmonella spp., Serratia maccescens, Shigella sonnei and Yersinia enterocolitica. In addition, the DNA sequence encoding P45 was detected in R. anatipestifer serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 18, 19 and the ATCC11845 strain, suggesting that P45 is probably also universally expressed in these R. anatipestifer serotypes. Thus, the ELISA described is applicable to the detection of R. anatipestifer infection in ducks. PMID:12220809

  17. Evaluation of the IDS One-Step ELISA kits for the detection of illicit drugs in hair.

    PubMed

    Pujol, Marie-Laure; Cirimele, Vincent; Tritsch, Pierre Julien; Villain, Marion; Kintz, Pascal

    2007-08-01

    This work presents the validation of a new immunological assay, the One-Step enzyme-linked immunosorbent assay (ELISA) tests from International Diagnostic Systems Corp. for the screening of drugs of abuse (cannabis, amphetamines, opiates, and cocaine) in human hair, with subsequent GC-MS confirmation. After decontamination and segmentation into small pieces, 50 mg of hair sample were incubated in 1 ml of methanol during 16 h at 40 degrees C. A 100 microL aliquot was collected and evaporated to dryness in presence of 100 microL of methanol/hydrochloric acid (99:1, v/v) to avoid amphetamines loss. The dried extract was dissolved in 100 microL of the "sample and standard diluent" solution included in the kit. This solution was submitted to analysis according to the recommended instructions of the manufacturer. During the validation phase, GC-MS confirmations were conducted according to our fully validated and published methods for opiates, cocaine, cannabis, and amphetamines determinations in hair. In a last development step, these procedures were slightly modified to directly confirm ELISA results by GC-MS using the methanolic extract. Ninety-three specimens were simultaneously screened by the ELISA tests (103 for tetrahydrocannabinol (THC)) and confirmed by GC-MS. Twenty were found positive for cannabis (THC: 0.10-6.50 ng/mg), 21 for cocaine (0.50-55.20 ng/mg), 24 for opiates (6-acetylmorphine (6-AM): 0.20-11.60 ng/mg, MOR: 0.20-8.90 ng/mg, codeine (COD): 0.20-5.90 ng/mg), and 13 for amphetamines (AP: 0.20 and 0.27 ng/mg, methamphetamine (MAP): 0.30 and 1.10 ng/mg, methylenedioxymethamphetamine (MDMA): 0.22-17.80 ng/mg). No false negative results were observed according to the Society of Hair Testing's (SoHT) cutoffs (0.5 ng/mg for cocaine, 0.2 ng/mg for opiates and amphetamines, and 0.1 ng/mg for THC). The One-Step ELISA kits appear suitable due to their sensitivity and specificity for drug of abuse screening in hair. This technology should find interest in

  18. Significance of quantitative enzyme-linked immunosorbent assay (ELISA) results in evaluation of three ELISAs and Western blot tests for detection of antibodies to human immunodeficiency virus in a high-risk population.

    PubMed Central

    Nishanian, P; Taylor, J M; Korns, E; Detels, R; Saah, A; Fahey, J L

    1987-01-01

    The characteristics of primary (first) tests with three enzyme-linked immunosorbent assay (ELISA) kits for human immunodeficiency virus (HIV) antibody were determined. The three ELISAs were performed on 3,229, 3,130, and 685 specimens from high-risk individuals using the Litton (LT; Litton Bionetics Laboratory Products, Charleston, S.C.), Dupont (DP; E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.), and Genetic Systems (GS; Genetic Systems, Seattle, Wash.) kits, respectively. Evaluation was based on the distribution of quantitative test results (such as optical densities), a comparison with Western blot (WB) results, reproducibility of the tests, and identification of seroconverters. The performances of the GS and the DP kits were good by all four criteria and exceeded that of the LT kit. Primary ELISA-negative results were not always confirmed with repeat ELISA and by WB testing. The largest percentage of these unconfirmed negative test results came from samples with quantitative results in the fifth percentile nearest the cutoff. Thus, supplementary testing was indicated for samples with test results in this borderline negative range. Similarly, borderline positive primary ELISA results that were quantitatively nearest (fifth percentile) the cutoff value were more likely to be antibody negative on supplementary testing than samples with high antibody values. In this study, results of repeated tests by GS ELISA showed the least change from first test results. DP ELISA showed more unconfirmed primary positive test results, and LT ELISA showed more unconfirmed primary negative test results. Designation of a specimen with a single ELISA quantitative level near the cutoff value as positive or negative should be viewed with skepticism. A higher than normal proportion of specimens with high negative optical densities by GS ELISA (fifth percentile nearest the cutoff) and also negative by WB were found to be from individuals in the process of seroconversion. PMID

  19. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples.

    PubMed

    Wang, Yuzhen; Wei, Dapeng; Yang, Hong; Yang, Yuan; Xing, Weiwei; Li, Yuan; Deng, Anping

    2009-03-15

    The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ngmL(-1). The values of IC(50) for nine standard curves were in the range of 1.1-2.0 ngmL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N=3) was 0.07-0.14 ngmL(-1). The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC(50) value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n=9). The mAb-based ELISA proven to be a feasible quantitative

  20. Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA.

    PubMed

    Giménez-Lirola, Luis G; Mur, Lina; Rivera, Belen; Mogler, Mark; Sun, Yaxuan; Lizano, Sergio; Goodell, Christa; Harris, D L Hank; Rowland, Raymond R R; Gallardo, Carmina; Sánchez-Vizcaíno, José Manuel; Zimmerman, Jeff

    2016-01-01

    In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence. PMID:27611939

  1. A microELISA assay for detection of anti-HLA activity of mouse monoclonal antibodies using an Astroscan 2100 automated plate reader.

    PubMed

    Sadler, A M; Krausa, P; Marsh, S G; Heyes, J M; Bodmer, J G

    1992-04-27

    A microenzyme-linked immunosorbent assay (microELISA) method has been developed using an Astroscan 2100 system automated plate reader which was initially designed for tissue typing by a two colour fluorescent microcytotoxicity assay. A 96-well plate ELISA used for screening mouse monoclonal antibodies raised against surface HLA antigens has been modified for use with the Astroscan plate reader and 72-well typing trays. The existing substrate 4-methylumbelliferyl-beta-D-galactoside (4MUG) has been replaced with fluorescein-di-beta-D-galactopyranoside (FDG), to provide a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes have also been reduced tenfold for use with Terasaki microtest plates. The assay now has the major advantage of requiring only 5 microliters of test supernatant allowing hybridomas to be screened earlier during a fusion and on a wider cell panel. The use of the large panel which includes B lymphoblastoid cell lines (B-LCL) and mouse L cell transfectants expressing HLA genes, reduces the length of time the hybridomas need to be kept in tissue culture before selection. Other advantages include the reduction in the number of target cells required, smaller volumes of reagents throughout the assay and the ability to screen cytotoxic as well as non-cytotoxic monoclonal antibodies. The sensitivity of this microELISA proved to be comparable with the original assay and so provides an efficient screening method for monoclonal antibodies. PMID:1583310

  2. [Detection of anti-Brucella spp. antibodies in swine by agglutination techniques and indirect ELISA in the Buenos Aires and La Pampa provinces, Argentina].

    PubMed

    Castro, H A; González, S R; Prat, M I; Baldi, P C

    2006-01-01

    Porcine brucellosis is one of the most important zoonoses in this country. Currently, there is no control program for porcine brucellosis in Argentina and the epidemiological situation is still unknown. The purpose of our study was to detect anti-Brucella spp. antibodies in swine in the southwest of the Buenos Aires province and the east of the La Pampa province. Blood samples were obtained when animals were slaughtered. The presence of anti-brucella antibodies was studied by the buffered plate agglutination test (BPA), the tube agglutination test (SAT), the 2-mercaptoethanol (2-ME) agglutination test and indirect ELISA tests, using the cytosolic fraction from Brucella abortus S19 (CYT), and lipopolysaccharide (LPS)-free cytosolic proteins (CP). Out of a total of 325 samples analyzed, 17.8% reacted positively to BPA, 13.8% to SAT, 8.0% to 2-ME, 21.0% to ELISA-CYT and 10.0% to ELISA-CP. These results agree with the few data available in our country and suggest that brucellosis screening should be extended to other regions. PMID:17037254

  3. Magnetic particles functionalized with PAMAM-dendrimers and antibodies: a new system for an ELISA method able to detect Ara h3/4 peanut allergen in foods.

    PubMed

    Speroni, Francesca; Elviri, Lisa; Careri, Maria; Mangia, Alessandro

    2010-08-01

    An innovative enzyme-linked immunosorbent assay (ELISA) format based on antibody-coated magnetic micro-particles (MPs) for the sensitive detection of Ara h3/4 allergen in food is described. The immunosupport is suspended in the incubation solutions and the MPs with the captured allergen can be easily harvested on a magnet, separated from the solutions, and washed using an easy-to-use, fast and selective approach that allows its detection and quantification. Two differently coated MPs, ProteinA-Pn-b and MP-NH(2)-PAMAM G 1.5-Pn-b immunosupports, were tested. The functionalization of the MPs with PAMAM-sodium carboxylate dendrimers elicits a major stability on the immunoglobulin activity resulting in a threefold enhancement of the analytical sensitivity for the assay with respect to a ProteinA immobilization. Validation was carried out on two different matrices: corn flakes and biscuits. In the case of MP-NH(2)-PAMAM G 1.5 -Pn-b immunosupport, limit of detection was found to be 0.2 mg peanuts/kg matrix in both matrices; the linear response range was demonstrated from 2.5 to 15 mg peanuts/kg matrix by performing statistical tests (homoscedasticity and Mandel fitting tests). Good accuracy and recovery (>80 +/- 2%) were obtained. Different food samples were tested and the results were compared with those obtained with a commercially available ELISA kit. The results obtained in this work demonstrated the applicability of the immunomagnetic ELISA methods on real samples and the possibility to perform the assay with significantly reduced reagent and sample consumption. PMID:20607526

  4. Immunodiagnosis of Human Fascioliasis by an Enzyme-Linked Immunosorbent Assay (ELISA) and a Micro-ELISA

    PubMed Central

    Carnevale, Silvana; Rodríguez, Mónica I.; Santillán, Graciela; Labbé, Jorge H.; Cabrera, Marta G.; Bellegarde, Enrique J.; Velásquez, Jorge N.; Trgovcic, Jorge E.; Guarnera, Eduardo A.

    2001-01-01

    Enzyme-linked immunosorbent assay (ELISA) and micro-ELISA were evaluated for their ability to detect anti-Fasciola hepatica antibodies in humans by using excretory-secretory antigen. The sensitivity of each method was 100%, but the specificity was 100% for ELISA and 97% for micro-ELISA. The micro-ELISA could be used as a screening assay and ELISA could be used as a confirmatory method for the serodiagnosis of human fascioliasis. PMID:11139214

  5. An electrochemical ELISA-like immunosensor for miRNAs detection based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes.

    PubMed

    Tran, H V; Piro, B; Reisberg, S; Huy Nguyen, L; Dung Nguyen, T; Duc, H T; Pham, M C

    2014-12-15

    We design an electrochemical immunosensor for miRNA detection, based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes. An original immunological approach is followed, using antibodies directed to DNA.RNA hybrids. An electrochemical ELISA-like amplification strategy was set up using a secondary antibody conjugated to horseradish peroxidase (HRP). Hydroquinone is oxidized into benzoquinone by the HRP/H2O2 catalytic system. In turn, benzoquinone is electroreduced into hydroquinone at the electrode. The catalytic reduction current is related to HRP amount immobilized on the surface, which itself is related to miRNA.DNA surface density on the electrode. This architecture, compared to classical optical detection, lowers the detection limit down to 10 fM. Two miRNAs were studied: miR-141 (a prostate biomarker) and miR-29b-1 (a lung cancer biomarker). PMID:24973539

  6. Using mass spectrometry to detect hydrolysed gluten in beer that is responsible for false negatives by ELISA.

    PubMed

    Colgrave, Michelle L; Goswami, Hareshwar; Blundell, Malcolm; Howitt, Crispin A; Tanner, Gregory J

    2014-11-28

    Gluten is the collective name for a class of proteins found in wheat, rye, barley and oats. Eating gluten triggers an inappropriate auto-immune reaction in ∼70 million people globally affected by coeliac disease, where the gut reacts to gluten proteins and this triggers an immune response, resulting in intestinal inflammation and damage. Gluten-free foods are now commonplace, however, it is difficult to accurately determine the gluten content of products claiming to be gluten-free using current methodologies as the antibodies are non-specific, show cross-reactivity and have different affinities for the different classes of gluten. The measurement of gluten in processed products is further confounded by modifications to the proteins that occur during processing and in some case hydrolysis of the proteins. In this study, LC-MS/MS was used to profile whole beer, and two beer fractions representing hydrolysed hordeins (<30 kDa) and hordein peptide fragments (<10 kDa). Subsequently, multiple reaction monitoring (MRM) MS enabled the relative quantification of selected peptide fragments in beer and revealed that certain classes of hordein were prone to hydrolysis (B- and D-hordein). Furthermore, select beers contained very high levels of gluten-derived fragments. Strikingly, those beers that contained high levels of B-hordein fragments gave near zero values by ELISA. The hydrolysed fragments that persist in beer show a dose-dependent suppression of ELISA measurement of gluten despite using a hordein standard for calibration of the assay. The development of MS-based methodology for absolute quantification of gluten is required for the accurate assessment of gluten, including hydrolysed forms, in food and beverages to support the industry, legislation and to protect consumers suffering from CD. PMID:25454134

  7. Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA.

    PubMed

    Thornton, Christopher R; Slaughter, David C; Davis, R Michael

    2010-10-15

    Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes. PMID:20850192

  8. Serological detection of bovine ephemeral fever virus using an indirect ELISA based on antigenic site G1 expressed in Pichia pastoris.

    PubMed

    Zheng, Fu-Ying; Lin, Guo-Zhen; Qiu, Chang-Qing; Zhou, Ji-Zhang; Cao, Xiao-An; Gong, Xiao-Wei

    2010-08-01

    An indirect ELISA for the serological detection of bovine ephemeral fever virus (BEFV) infection in cattle is described in which a glycosylated protein of approximately 25 kDa (including the G1 antigenic site of the virus glycoprotein) expressed in Pichia pastoris GS115 was used as the coating antigen. The optimal concentration of coated antigen was 0.3 microg/well at a serum dilution of 1:40. The optimal positive threshold value of the assay was 1.88, as derived from receiver operating characteristic curve analysis. The test had 100% sensitivity and 96.7% specificity when compared with a micro-neutralisation test using 336 positive and 180 negative sera to BEFV, respectively. The inter-assay and intra-assay coefficients of variation for 15 sera were both <5.8% and there was no evidence of cross-reactivity between the tested coating antigen and antibodies to related rabies virus. The ELISA is an inexpensive and rapid serological detection method that would be suitable for screening for BEFV infection on a large scale. PMID:19586786

  9. Detection of Toxoplasma gondii in raw caprine, ovine, buffalo, bovine, and camel milk using cell cultivation, cat bioassay, capture ELISA, and PCR methods in Iran.

    PubMed

    Dehkordi, Farhad Safarpoor; Borujeni, Mohammad Reza Haghighi; Rahimi, Ebrahim; Abdizadeh, Rahman

    2013-02-01

    This study was conducted to determine the presence of Toxoplasma gondii in animal milk samples in Iran. From a total of 395 dairy herds in three provinces of Iran, 66 bovine, 58 ovine, 54 caprine, 33 buffalo, and 30 camel herds were studied, and from these parts of Iran, 200 bovine, 185 ovine, 180 caprine, 164 buffalo, and 160 camel milk samples were collected from various seasons. Samples were tested for Toxoplasma gondii by cell line culture, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) technique. Only the results of cell line cultivation were confirmed by bioassay in cat. Results indicated that all herds were infected with Toxoplasma gondii. The culture method showed that 51 out of 889 milk samples (5.73%) were positive for Toxoplasma gondii, and all 51 positive culture results were positive with bioassay in cat. The Fars province had the highest prevalence of Toxoplasma gondii (6.84%). The ELISA test showed that 41 milk samples (4.61%) were positive for the presence of Toxoplasma gondii, while the PCR showed that 46 milk samples were positive for Toxoplasma gondii. The results showed higher sensitivity of PCR and higher specificity of ELISA. Caprine had the highest (10%) and camel had the lowest (3.12%) prevalence rate of parasite. The summer season had the highest (76.47%) but winter (3.92) had the lowest incidence of Toxoplasma gondii. This study is the first prevalence report of direct detection of Toxoplasma gondii in animal milk samples in Iran. PMID:23441913

  10. Detection of rabies antibody by ELISA and RFFIT in unvaccinated dogs and in the endangered Simien jackal (Canis simensis) of Ethiopia.

    PubMed

    Mebatsion, T; Sillero-Zubiri, C; Gottelli, D; Cox, J H

    1992-05-01

    Varying levels of rabies antibody have been detected both by Enzyme Linked Immunosorbent Assay (ELISA) and Rapid Fluorescent Focus Inhibition Test (RFFIT) in the sera collected from wild and domestic canids in the Bale Mountains National Park (BMNP) of Southern Ethiopia. Rabies antibody was detected in 80% (8 out of 10) of domestic dog samples, 13.3% (2 out of 15) of Simien jackal samples and in one common jackal. Rabies virus was isolated from one dog in an area where contact with the Simien jackal could possibly occur. All samples examined from wild rodents as possible reservoir hosts for rabies were found negative. The presence of large proportion of susceptible Simien jackals in the population should be a cause of great concern in saving this endangered species from the ravages of rabies. PMID:1642078