Science.gov

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. [Detection of Toxoplasma antibodies using the c-ELISA method].

    PubMed

    Sýkora, J; Pokorný, J; Zástĕra, M; Frühbauer, Z; Svandová, E

    1989-05-01

    Sensitive and high capacity ELISA techniques introduced for the detection of toxoplasmatic antibodies so far do not correlate very well with standard serological examination systems used in common diagnosis of Toxoplasmosis. The most frequently used method in the CSSR - SFT using tween-ether soluble antigen extract, and displaying very satisfactory agreement with the Sabin-Feldman test, was compared with the system of direct ELISA, using the same antigen extract and the described c-ELISA method. The latter method uses for antibody detection whole non-disintegrated formol-treated trophozoites of Toxoplasma gondii and thus in the reaction in particular membrane antigens, incl. latent ones, are involved. Parallel examination of 160 sera of subjects with clinical suspicion of toxoplasma infection by the standard CFT, method and methods ELISA and c-ELISA revealed a better correlation between CFT and c-ELISA (r = 0.8031) than between CFT and ELISA with soluble antigen (r = 0.6016). Other positive specific features of the c-ELISA method are the use of commercially produced antigen for the immunofluorescence test, considerable elimination of selective adsorption of the polystyrene carrier and the perspective of the long-term preservation of plates with the fixed antigen.

  3. Detection of hazelnuts and almonds using commercial ELISA test kits.

    PubMed

    Garber, Eric A E; Perry, Jesse

    2010-03-01

    Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.

  4. 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

  5. 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.

  6. 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

  7. Paper-based ELISA to rapidly detect Escherichia coli.

    PubMed

    Shih, Cheng-Min; Chang, Chia-Ling; Hsu, Min-Yen; Lin, Jyun-Yu; Kuan, Chen-Meng; Wang, Hsi-Kai; Huang, Chun-Te; Chung, Mu-Chi; Huang, Kui-Chou; Hsu, Cheng-En; Wang, Chun-Yuan; Shen, Ying-Cheng; Cheng, Chao-Min

    2015-12-01

    Escherichia coli is a generic indicator of fecal contamination, and certain serotypes cause food- and water-borne illness such as O157:H7. In the clinic, detection of bacteriuria, which is often due to E. coli, is critical before certain surgical procedures or in cases of nosocomial infection to prevent further adverse events such as postoperative infection or sepsis. In low- and middle-income countries, where insufficient equipment and facilities preclude modern methods of detection, a simple, low-cost diagnostic device to detect E. coli in water and in the clinic will have significant impact. We have developed a simple paper-based colorimetric platform to detect E. coli contamination in 5h. On this platform, the mean color intensity for samples with 10(5)cells/mL is 0.118±0.002 (n=4), and 0.0145±0.003 (P<0.01⁎⁎) for uncontaminated samples. This technique is less time-consuming, easier to perform, and less expensive than conventional methods. Thus, paper-based ELISA is an innovative point-of-care diagnostic tool to rapidly detect E. coli, and possibly other pathogens when customized as appropriate, especially in areas that lack advanced clinical equipment.

  8. ELISA for the detection of antibodies to Ebola viruses.

    PubMed

    Ksiazek, T G; West, C P; Rollin, P E; Jahrling, P B; Peters, C J

    1999-02-01

    EIAs for IgG and IgM antibodies directed against Ebola (EBO) viral antigens have been developed and evaluated using sera of animals and humans surviving infection with EBO viruses. The IgM capture assay detected anti-EBO (subtype Reston) antibodies in the sera of 5 of 5 experimentally infected animals at the time they succumbed to lethal infections. IgM antibodies were also detected in the serum of a human who was infected with EBO (subtype Reston) during a postmortem examination of an infected monkey. The antibody was detectable as early as day 6 after infection in experimentally infected animals and persisted for <90 days. The IgG response was less rapid; however, it persisted for >400 days in 3 animals who survived infection, and it persisted for approximately 10 years after infection in the sera of 2 humans. Although these data are limited by the number of sera available for verification, the IgM assay seems to have great promise as a diagnostic tool. Furthermore the long-term persistence of the IgG antibodies measured by this test strongly suggests that the ELISA will be useful in field investigations of EBO virus. PMID:9988184

  9. Evaluation of Monoclonal Antibody-Based Sandwich Direct ELISA (MSD-ELISA) for Antigen Detection of Foot-and-Mouth Disease Virus Using Clinical Samples

    PubMed Central

    Morioka, Kazuki; Fukai, Katsuhiko; Sakamoto, Kenichi; Yoshida, Kazuo; Kanno, Toru

    2014-01-01

    A monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) method was previously developed for foot-and-mouth disease (FMD) viral antigen detection. Here we evaluated the sensitivity and specificity of two FMD viral antigen detection MSD-ELISAs and compared them with conventional indirect sandwich (IS)-ELISA. The MSD-ELISAs were able to detect the antigen in saliva samples of experimentally-infected pigs for a longer term compared to the IS-ELISA. We also used 178 RT-PCR-positive field samples from cattle and pigs affected by the 2010 type-O FMD outbreak in Japan, and we found that the sensitivities of both MSD-ELISAs were about 7 times higher than that of the IS-ELISA against each sample (P<0.01). In terms of the FMD-positive farm detection rate, the sensitivities of the MSD-ELISAs were about 6 times higher than that of the IS-ELISA against each farm (P<0.01). Although it is necessary to conduct further validation study using the other virus strains, MSD-ELISAs could be appropriate as a method to replace IS-ELISA for FMD antigen detection. PMID:24736560

  10. A comparison of two antigen-detection ELISA for detecting infection of Dirofilaria immitis in dogs.

    PubMed

    Euclid, J M; Copeman, D B

    1997-09-01

    A survey on 87 dogs necropsied in the Townsville region revealed 34 (39%) were infected with Dirofilaria immitis. Infected dogs had an average of 6.1 adult worms in the heart and associated blood vessels. The VetRED assay detected 23 of the 34 infected dogs (sensitivity 65%) and the Og4C3 ELISA detected 27 (sensitivity 80%). Sensitivity of the VetRED and Og4C3 ELISA increased to 88 and 94% respectively in dogs with three or more worms. Both tests detected correctly all uninfected dogs. Despite the higher accuracy of the Og4C3 ELISA, compared to the VetRED assay, it is unlikely to be used widely as a field test for heartworm unless it can be modified from its present plate ELISA format which takes 4 hours, into one which is more rapid and convenient. However, as a reference ELISA, it may well be worthwhile in situations which require considerable accuracy for detecting D. immitis infection.

  11. [Sensitivity and specificity of the ELISA Kit for the detection of antidobies to Junin virus].

    PubMed

    Pirozhkov, A P; Timofeev, M A; Borisevich, I V; Syromiatnikova, S I; Shatokhina, I V; Pantyukhov, V B; Kovalchuk, A V; Borisevich, S V

    2015-01-01

    The goal of this work was to describe methodological approaches to determination of sensitivity and specificity of the enzyme-linked immunosorbent assay kit (ELISA Kit) for detection of the specific anti-Junin virus (JV) antibody. Comparison of ELISA to plaque reduction neutralization test (PRNT) showed direct relationship between antibody titers in the samples of serum of immunized animals, determined by either PRNT or ELISA methods. The obtained results provided an opportunity to form the panels of positive and negative serum samples to determine the sensitivity and specificity of the ELISA Kit. Sensitivity of the ELISA Kit was at least 98% when studying the samples of serum of immunized guinea pigs and rabbits (determined as positive in PRNT). The sensitivity of the ELISA Kit was at least 68% when studying the samples determined by PNRT as uncertain positive. The specificity was 98%. The specificity of the ELISA Kit was 98%.

  12. 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.

  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. Comparing electron microscopy and a competitive blocking ELISA in the detection of rotaviruses in porcine faeces.

    PubMed

    Rodák, Ladislav; Smítalová, Radka; Šmíd, Bedřich; Nevoránková, Zora; Valíček, Lubomír; Pšikal, Ivan

    2011-02-01

    Analysis of 476 faecal samples from diarrhoeic piglets was performed using electron microscopy (EM) and a competitive blocking (CB)-ELISA based on monoclonal antibodies to the VP6 protein of group A rotavirus. Rotavirus was detected by EM and/or CB-ELISA in 111 (23.3%) samples. Of these, all groups of rotavirus were identified in 83 (74.8%) samples by EM (EM+), while group A rotavirus was identified in 90 (81.1%) samples by CB-ELISA (ELISA+). However, only 62 (55.9%) of samples were positive using both detection methods. The finding of 28 (25.2%) EM-/CB-ELISA+ samples illustrated the high sensitivity of the CB-ELISA method. On PCR analysis, groups B and C rotavirus was found in 3 and 16 of 19 EM+/CB-ELISA- samples, respectively. Although the study illustrates the high sensitivity of a CB-ELISA in rotavirus detection, the findings highlight the need to use a range of diagnostic methods in detecting these viruses in clinical samples.

  15. Applicability of ELISA detection of statherin for forensic identification of saliva.

    PubMed

    Akutsu, Tomoko; Watanabe, Ken; Fujinami, Yoshihito; Sakurada, Koichi

    2010-09-01

    Statherin is a low molecular-weight phosphoprotein secreted from the parotid gland. Statherin mRNA was previously reported to be a useful marker for mRNA-based saliva identification. In this study, applicability of ELISA detection of statherin for forensic identification of saliva was investigated. The specificity and sensitivity of ELISA for detection of statherin were compared with those of ELISA for α-amylase and the Phadebas® amylase test. Statherin was specifically detected in saliva but not in other body fluids. In addition, statherin was successfully detected in aged saliva stains, mixed body fluids-saliva stains, and simulated casework samples. On the other hand, although ELISA for α-amylase showed higher sensitivity than ELISA for statherin, it was not specific enough to identify saliva. The Phadebas® amylase test also showed positive results in other body fluids that are known to have α-amylase activity; however, it is easy to use for screening forensic casework samples. In conclusion, ELISA for detection of statherin developed in this study could be an effective tool for the forensic identification of saliva because of its specificity for saliva among other body fluids. Forensic casework samples should be tested by ELISA detection or mRNA-based analysis for statherin, depending on the condition of the sample, to supplement presumptive tests for α-amylase, such as the Phadebas® amylase test.

  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.

  17. 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

  18. 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

  19. A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

    SciTech Connect

    Varnum, Susan M.

    2007-06-01

    An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

  20. Microchip ELISA coupled with cell phone to detect ovarian cancer HE4 biomarker in urine.

    PubMed

    Wang, ShuQi; Akbas, Ragip; Demirci, Utkan

    2015-01-01

    Ovarian cancer is a leading cause of death from gynecologic cancers in the USA, and early diagnosis can potentially increase 5-year survival rate. Detection of biomarkers derived from hyperplasia of epithelial tissue by enzyme-linked immunosorbent assay (ELISA) proves to be a practical way of early diagnosis of ovarian cancer. However, ELISA is commonly performed in a laboratory setting, and it cannot be used in a clinical setting for on-site consultation. We have shown a microchip ELISA that detects HE4, an ovarian cancer biomarker, from urine using a cell phone integrated with a mobile application for imaging and data analysis. In microchip ELISA, HE4 from urine was first absorbed on the surface; the primary and secondary antibodies were subsequently anchored on the surface via immuno-reaction; and addition of substrate led to color development because of enzymatic labeling. The microchip after color development was imaged using a cell phone, and the color intensity was analyzed by an integrated mobile application. By comparing with an ELISA standard curve, the concentration of HE4 was reported on the cell phone screen. The presented microchip ELISA coupled with a cell phone is portable as opposed to traditional ELISA, and this method can facilitate the detection of ovarian cancer at the point-of-care (POC). PMID:25626535

  1. Microchip ELISA coupled with cell phone to detect ovarian cancer HE4 biomarker in urine.

    PubMed

    Wang, ShuQi; Akbas, Ragip; Demirci, Utkan

    2015-01-01

    Ovarian cancer is a leading cause of death from gynecologic cancers in the USA, and early diagnosis can potentially increase 5-year survival rate. Detection of biomarkers derived from hyperplasia of epithelial tissue by enzyme-linked immunosorbent assay (ELISA) proves to be a practical way of early diagnosis of ovarian cancer. However, ELISA is commonly performed in a laboratory setting, and it cannot be used in a clinical setting for on-site consultation. We have shown a microchip ELISA that detects HE4, an ovarian cancer biomarker, from urine using a cell phone integrated with a mobile application for imaging and data analysis. In microchip ELISA, HE4 from urine was first absorbed on the surface; the primary and secondary antibodies were subsequently anchored on the surface via immuno-reaction; and addition of substrate led to color development because of enzymatic labeling. The microchip after color development was imaged using a cell phone, and the color intensity was analyzed by an integrated mobile application. By comparing with an ELISA standard curve, the concentration of HE4 was reported on the cell phone screen. The presented microchip ELISA coupled with a cell phone is portable as opposed to traditional ELISA, and this method can facilitate the detection of ovarian cancer at the point-of-care (POC).

  2. 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.

  3. 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

  4. 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

  5. An antigen trapping ELISA for the detection of capripoxvirus in tissue culture supernatant and biopsy samples.

    PubMed

    Carn, V M

    1995-01-01

    A trapping ELISA for the detection of capripoxvirus antigen in tissue culture supernatant and biopsy material was developed, using a guinea-pig polyclonal detector antiserum raised against a recombinant capripoxvirus specific antigen, expressed in Escherichia coli using the plasmid vector pGEX-2T. The ELISA detected antigen in tissue culture samples that on virus titration contained equal to or in excess of 10(2.8) TCID50/ml. Virus isolation and ELISA were compared for the detection of capripoxvirus in skin biopsy samples from sheep, goats and cattle. The ELISA compared well with virus isolation, and has applications as a diagnostic test. This assay reduces the reliance of diagnostic laboratories on tissue culture facilities, and can be used to confirm the presence of capripoxvirus in tissue culture.

  6. 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.

  7. 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.

  8. Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods.

    PubMed

    Le Blay, Gwenaëlle; Fliss, Ismaïl; Lacroix, Christophe

    2004-11-01

    Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.

  9. An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces.

    PubMed

    Rodák, L; Valícek, L; Smíd, B; Nevoránková, Z

    2005-01-01

    Monoclonal antibodies to porcine epidemic diarrhoea virus (PEDV) membrane protein M were prepared and used for the comparative assessment of three blocking ELISA variants to detect PEDV. The competitive blocking ELISA (CB-ELISA) format showed the highest sensitivity, allowing detection of 10(2.5) plaque-forming units of PEDV/ml in culture medium. Its specificity was verified by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and rotavirus A in each analysis. Eighty porcine field samples of faeces obtained from 38 herds affected with diarrhoea were examined, and PEDV was found in 15 (19%) samples from 6 (16%) herds. The suitability of the CB-ELISA for the screening herds in epizootiologic situations is discussed.

  10. 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

  11. 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.

  12. Detection of antibodies to Newcastle disease virus in ostriches (Struthio camelus) by an indirect ELISA.

    PubMed

    Williams, R; Boshoff, C H; Verwoerd, D; Schoeman, M; van Wyk, A; Gerdes, T H; Roos, K

    1997-01-01

    A two-graph receiver operating characteristic analysis, performed on the hemagglutination-inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) test results of a Newcastle disease virus (NDV)-positive and NDV-negative control group of ostrich sera, proved that the ELISA was superior to the HI in both sensitivity and specificity. Comparison of results of the two assays performed on a panel of simulated positive sera ranging from very weak to very strong showed that the ELISA was at least 10 times more sensitive than the HI in detecting low levels of ostrich antibodies to NDV. The ELISA also has the advantage of using untreated serum in a single dilution as opposed to the HI test, which uses pretreated serum in titration.

  13. Qualitative and quantitative detection of T7 bacteriophages using paper based sandwich ELISA.

    PubMed

    Khan, Mohidus Samad; Pande, Tripti; van de Ven, Theo G M

    2015-08-01

    Viruses cause many infectious diseases and consequently epidemic health threats. Paper based diagnostics and filters can offer attractive options for detecting and deactivating pathogens. However, due to their infectious characteristics, virus detection using paper diagnostics is more challenging compared to the detection of bacteria, enzymes, DNA or antigens. The major objective of this study was to prepare reliable, degradable and low cost paper diagnostics to detect viruses, without using sophisticated optical or microfluidic analytical instruments. T7 bacteriophage was used as a model virus. A paper based sandwich ELISA technique was developed to detect and quantify the T7 phages in solution. The paper based sandwich ELISA detected T7 phage concentrations as low as 100 pfu/mL to as high as 10(9) pfu/mL. The compatibility of paper based sandwich ELISA with the conventional titre count was tested using T7 phage solutions of unknown concentrations. The paper based sandwich ELISA technique is faster and economical compared to the traditional detection techniques. Therefore, with proper calibration and right reagents, and by following the biosafety regulations, the paper based technique can be said to be compatible and economical to the sophisticated laboratory diagnostic techniques applied to detect pathogenic viruses and other microorganisms.

  14. 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.

  15. 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

  16. ELISA assays and alcohol: increasing carbon chain length can interfere with detection of cytokines.

    PubMed

    von Maltzan, Kristine; Pruett, Stephen B

    2011-02-01

    Enzyme-linked immunosorbent assays (ELISAs) are frequently used in studies on cytokine production in response to treatment of cell cultures or laboratory animals. When an ELISA assay is performed on cell culture supernatants, samples often contain the treatment agents. The purpose of the present study was to determine if some of the agents evaluated might inhibit cytokine detection by interfering with the ELISA, leaving the question of whether cytokine production was inhibited unanswered. Mouse and human cytokine ELISA kits from BD Biosciences were used according to the manufacturer's instructions. Cytokine proteins were subjected to one to five carbon alcohols at 86.8mM (methanol, ethanol, 1-propanol, 2-propanol, n-butanol, and n-pentanol). After treating cell cultures with alcohols of different carbon chain lengths, we found that some of the alcohols interfered with measurement of some cytokines by ELISA, thus making their effects on cytokine production by cells in culture unclear. Increasing carbon chain length of straight chain alcohols positively correlated with their ability to inhibit detection of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10), but not with the detection of interleukin 6 (IL-6), interleukin 8, (IL-8), and interleukin 12 (IL-12). To avoid misinterpretation of treatment effects, ELISA assays should be tested with the reference protein and the treatment agent first, before testing biological samples. These results along with other recent results we obtained using circular dichroism indicate that alcohols with two or more carbons can directly alter protein conformation enough to disrupt binding in an ELISA (shown in the present study) or to inhibit ligand-induced conformational changes (results not shown). Such direct effects have not been given enough consideration as a mechanism of ethanol action in the immune system.

  17. Monoclonal antibodies and an indirect ELISA for detection of psychrotrophic bacteria in refrigerated milk.

    PubMed

    Gutiérrez, R; González, I; García, T; Carrera, E; Sanz, B; Hernández, P E; Martín, R

    1997-01-01

    Monoclonal antibodies generated against live cells of Pseudomonas fluorescens have been used in an indirect ELISA format for the detection of Pseudomonas spp. and related psychrotrophic bacteria in refrigerated milk. The immunorecognition of monoclonal antibodies adsorbed to bacteria bound to the wells of a microtiter plate was performed with rabbit anti-mouse immunoglobulins conjugated to horseradish peroxidase. Subsequent enzymic conversion of the substrate resulted in distinct absorbance differences when assaying milk samples containing psychrotrophic bacteria in the range 10(5) to 10(9) CFU ml(-1) . The detection threshold for the ELISA assay developed in this work is 10(5) CFU ml(-1).

  18. Quantitative detection of hazelnut (Corylus avellana) in cookies: ELISA versus real-time PCR.

    PubMed

    Platteau, Céline; De Loose, Marc; De Meulenaer, Bruno; Taverniers, Isabel

    2011-11-01

    Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.

  19. Competitive PCR-ELISA protocols for the quantitative and the standardized detection of viral genomes.

    PubMed

    Musiani, Monica; Gallinella, Giorgio; Venturoli, Simona; Zerbini, Marialuisa

    2007-01-01

    Competitive PCR-ELISA combines competitive PCR with an ELISA to allow quantitative detection of PCR products. It is based on the inclusion of an internal standard competitor molecule that is designed to differ from the target by a short sequence of nucleotides. Once such a competitor molecule has been designed and constructed, target and competitor sequences are concurrently PCR-amplified, before hybridization to two different specific probes and determination of their respective OD values by ELISA. The target can be quantified in relation to a titration curve of different dilutions of the competitor. The competitor can alternatively be used at a unique optimal concentration to allow for standardized detection of the target sequence. PCR-ELISA can be performed in 1 d in laboratories without access to a real-time PCR thermocycler. This technique is applied in diagnostics to monitor the course of infections and drug efficacy. Competitive PCR-ELISA protocols for the quantitative and for the standardized detection of parvovirus B19 are detailed here as an example of the technique.

  20. Validation of ELISA for the detection of African horse sickness virus antigens and antibodies.

    PubMed

    Rubio, C; Cubillo, M A; Hooghuis, H; Sanchez-Vizcaino, J M; Diaz-Laviada, M; Plateau, E; Zientara, S; Crucière, C; Hamblin, C

    1998-01-01

    The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y Produccion Animal (LSPA) in Algete, Spain was charged with the responsibility of co-ordinating and supplying samples of viruses and antisera to the participating laboratories in Spain, France and the United Kingdom. The panels comprised 76 antigen samples for assay by indirect sandwich ELISAs and 53 serum samples for antibody detection by either indirect or competitive ELISAs. Results generated by ELISA for each laboratory were analysed in LSPA in terms of their relative sensitivities and specificities. There was a good agreement between the ELISAs used for either antigen or antibody detection. The participating groups agreed that any field sample giving a doubtful result would always be retested by ELISA and an alternative assay.

  1. A liquid-phase blocking ELISA for the detection of antibodies to rabies virus.

    PubMed

    Esterhuysen, J J; Prehaud, C; Thomson, G R

    1995-01-01

    A liquid-phase blocking ELISA was adapted to the detection and titration of antibodies to principally the nucleoprotein of rabies virus. Sera from animals that had either been vaccinated against rabies or inoculated with street rabies viruses, as well as sera from animals that had no recorded contact with rabies, were tested. These included sera from people, cattle, sheep, goats, dogs, laboratory mice, rabbits, yellow mongooses, wild dogs and lions. Where possible, the results were compared with those obtained with a commercial kit incorporating an indirect ELISA that measures antibody to the rabies glycoprotein. There was a high correlation (r = 0.79) between the two tests. The blocking ELISA provides a single test suitable for the rapid detection of antibodies against rabies virus in the sera of any animal species and for that reason is particularly apt for epidemiological investigations in regions where species diversity is important, as in southern Africa.

  2. Immunoreactive insulin in diabetes mellitus patient sera detected by ultrasensitive ELISA with thio-NAD cycling.

    PubMed

    Ito, Etsuro; Kaneda, Mugiho; Kodama, Hiromi; Morikawa, Mika; Tai, Momoko; Aoki, Kana; Watabe, Satoshi; Nakaishi, Kazunari; Hashida, Seiichi; Tada, Satoshi; Kuroda, Noriyuki; Imachi, Hitomi; Murao, Koji; Yamashita, Masakane; Yoshimura, Teruki; Miura, Toshiaki

    2015-12-01

    To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 μL of serum was required for testing, with a limit of detection (LOD) for the assay of 10(-16) moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.

  3. Development of a blocking ELISA for detection of antibodies against avian hepatitis E virus.

    PubMed

    Liu, Baoyuan; Zhao, Qin; Sun, Yani; Wang, Xinjie; Zhao, Jinan; Du, Taofeng; Wang, Chengbao; Xiao, Shuqi; Mu, Yang; Zhang, Gaiping; Luo, Jianxun; Hsu, Walter H; Zhou, En-Min

    2014-08-01

    A blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of immunoglobulin G antibodies against avian hepatitis E virus (HEV). In the bELISA, the coating antigen was a truncated protein containing C-terminal 268-amino acid region of ORF2 from an avian HEV strain isolated in China (CaHEV) and blocking antibody was a monoclonal antibody (mAb) 1H5 recognizing the epitope within amino acids 384-414 in the C-terminal 268-amino acid region. The concentration of blocking mAb 1H5 was determined as that yielded an OD450nm value of 1.0 for binding to the coating antigen and the antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 20.7% at the mean percent inhibition plus 3 standard deviations was determined by testing 265 negative sera. The bELISA had a sensitivity of 98.3% by testing 116 positive sera from chickens infected experimentally with CaHEV and had no cross-reaction with other anti-avian virus antibodies. The compliance rates of the bELISA with indirect ELISA and Western blot were 83.7% and 93.3%, respectively, by testing 300 field chicken sera. These results suggested that the bELISA developed in this study can be used for detection of antibodies against avian HEV and showed high reproducibility compared with indirect ELISA and Western blot methods.

  4. Development and application of an indirect ELISA for detection of antibodies against avian hepatitis E virus.

    PubMed

    Zhao, Qin; Sun, Yani; Zhao, Jinan; Hu, Shoubin; Zhao, Feifei; Chen, Fuyong; Clavijo, Alfonso; Zhou, En-Min; Xiao, Yihong

    2013-01-01

    An indirect enzyme-linked immunosorbent assay (iELISA) that could detect immunoglobulin G antibodies against avian hepatitis E virus (HEV) was developed. This assay employs a truncated C-terminal 268-amino acid recombinant ORF2 protein from an avian HEV genotype 3 strain isolated in China (CaHEV) as the coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 0.368 at OD(450nm) was determined by testing 120 positive and 200 negative chicken sera for avian HEV antibodies using the two-graph receiver operating characteristic (TG-ROC) analysis. This iELISA has a sensitivity of 96.1% and a specificity of 95.8%. The overall agreement between the iELISA and a corresponding Western blot was 97%. The iELISA was used to evaluate the seroprevalence of avian HEV in poultry farms in the Shandong province. The avian HEV seropositive rate of 35.9% was determined by testing 1871 serum samples that were collected from 10 chicken flocks ranged from 10 to 60 weeks of age. The iELISA that was developed in this study can be used for detection of immunoglobulin G antibodies against avian HEV.

  5. Rapid and sensitive detection of Staphylococcus species in milk by ELISA based on monodisperse magnetic particles.

    PubMed

    Yazdankhah, S P; Hellemann, A L; Rønningen, K; Olsen, E

    1998-04-30

    Staphylococcus spp. are the single most important cause of mastitis in dairy cows. Aiming for a rapid and sensitive method for detection of staphylococci, a magnetic bead-based enzyme-linked immunosorbent assay (ELISA) employing MAb for the detection of staphylococci in milk was developed. Pretreatment of the sample with lysostaphin increased the sensitivity of the assay. The method is specific for most of staphylococci which cause mastitis in bovines, detects between 10(4)-10(5) organisms per ml and takes three hours. Thus, the principle for staphylococci ELISA based on monodisperse magnetic beads is a way for rapid detection of staphylococci in milk and possibly also for detecting staphylococci by direct testing of other clinical specimens. The system may be automated.

  6. 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)

  7. 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...

  8. 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.

  9. Evaluation of the ELISA test for detection of Entamoeba histolytica in feces.

    PubMed

    Merino, E; Glender, W; del Muro, R; Ortiz-Ortiz, L

    1990-01-01

    The clinical utility of an ELISA test with monoclonal antibodies to detect antigen of Entamoeba histolytica in feces was evaluated in 150 patients with gastrointestinal symptoms. Each subject was examined by rectosigmoidoscopy with rectal smear and/or a triple stool search for ova-bacteria-parasite (OBP); in addition, one stool sample was collected for the ELISA test. All the tests were independent and double blind. E. histolytica was detected by OBP and/or rectosigmoidoscopy in 66 patients; 61 patients had other parasites; and in 23, no parasites were identified. Of all patients, 116 were positive for the ELISA test. Of these, E. histolytica was identified in 52. In 47, other parasites were identified and in 17, no parasites were found. The ELISA test with a monoclonal antibody against E. histolytica antigen showed higher sensitivity than the standard diagnostic methods: the ability to detect the presence of E. histolytica antigen regardless of the destruction of the parasite or of the error due to misidentification of the parasite resulting from faulty preparation of the samples.

  10. 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.

  11. Development of a monoclonal antibody blocking-ELISA for detection of antibodies against Maedi-Visna virus.

    PubMed

    Fevereiro, M; Barros, S; Fagulha, T

    1999-08-01

    A monoclonal antibody (MAb) blocking ELISA (Blck-ELISA) was developed to detect antibodies against Maedi-Visna virus (MVV) in sheep sera. The assay employs a MAb directed against the envelope protein p90 of the virus in a sandwich blocking procedure. To determine whether the MAb was a potential antibody for developing a Blck-ELISA, a collection of three hundred sera obtained from several sheep flocks known to be infected with MVV were used to examine the sensitivity of the Blck-ELISA. A total of 50 serum samples originating from a flock free of MVV were tested to assess the specificity of the assay. The results were compared with a commercial indirect ELISA (I-ELISA) and samples giving a conflicting or doubtful result were tested by immunoblot. The Blck-ELISA proved to be specific, sensitive and it showed high reproducibility and low variability. PMID:10488767

  12. 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

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

    PubMed

    Weng, Xuan; Gaur, Gautam; Neethirajan, Suresh

    2016-06-07

    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.

  14. 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

  15. 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

  16. 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.

  17. A rapid antigen capture ELISA for the detection of orthopox viruses.

    PubMed

    Johann, S; Czerny, C P

    1993-10-01

    A genus-specific antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection orthopox viruses (OPV) was established, testing various combinations of monoclonal antibodies (MAbs) and rabbit hyper-immune sera. The most sensitive assay was based on capturing antigen with a non-neutralizing MAb against a highly conserved antigenic site of the OPV fusion protein. Bound antigen was detected by a mixture of the two broadly-reactive rabbit anti-monkey-pox- and rabbit anti-vaccinia virus hyper-immune sera. The reaction was visualized by the corresponding anti-species-horseradish-peroxidase- (HRP) conjugate and 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. This system was able to identify all OPV-species and isolates which were investigated. The ELISA demonstrated complete agreement with conventional procedures undertaken for qualitative and quantitative OPV-detection. In parallel, 16 defined OPV-strains were analysed by quantitative electron microscopy (calculation of the number of physical particles; PN) and infectivity titration (TCID50/ml) on tissue cultures. When ELISA data were compared with infectivity titers, the detection limits in the ELISA varied in a wide range of 10(2)-10(4) TCID50/ml., depending on different replicative potencies of the individual virus strains. However, when PN was the basis for calculation, a minimum of 10(5) physical virus particles/ml could be detected. Gradient-purified virus was detectable up to a protein content of 15 ng/ml. Maximum optical densities in the sample (OD450nm) reached 2.0, while mock infected tissue cultures of negative samples offered corresponding values of < 0.01.

  18. 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

  19. 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).

  20. Development of ELISA for detection of mercury based on specific monoclonal antibodies against mercury-chelate.

    PubMed

    Zhang, Yi; Li, Xiaobing; Liu, Guowen; Wang, Zhe; Kong, Tao; Tang, Jiajia; Zhag, Peng; Yang, Wei; Li, Dongna; Liu, Lei; Xie, Guanghong; Wang, Jianguo

    2011-12-01

    Immunoassays for heavy metals offer an alternative approach to traditional techniques for detection of mercury. In this study, a mercury-chelate was prepared with 1-(4-aminobenzyl) ethylenediamine-N,N,N',N'-tetraacetic acid (aminobenzyl-EDTA). The resulting complex was linked to keyhole limpet hemocyanin (KLH) or bovine serum albumin via the amino group and used as the immunizing antigen or detection antigen, respectively. BALB/c mice were immunized with KLH-aminobenzyl-EDTA-Hg and spleen cells from BALB/C mice were fused with Sp2/0 cells. One cell line (5F7) produced monoclonal antibodies with preferential selectivity and sensitivity for aminobenzyl-EDTA-Hg. This cell line had an affinity constant of 4.31 × 10(9) L/mol and its cross-reactivity (CR) with other metals was <2%. The antibody was used for competitive indirect ELISA (CI-ELISA) for Hg(2+) measurements. The detection range was 0.087-790.4 μg/L and the lower limit of detection was 0.042 μg/L. The concentrations of mercury in environmental water samples obtained by CI-ELISA correlated well with graphite furnace atomic absorption spectrometry (GFAAS), and the mean recovery was 88.82% to 104.64%. These results indicate that this method could be used for monitoring mercury of water.

  1. Establishment of hepatitis A detection antibodies set BRR batch 3 for antigen content determination by ELISA.

    PubMed

    Morgeaux, S; Manniam, I; Variot, P; Daas, A; Costanzo, A

    2015-01-01

    The current batch of the European Pharmacopoeia (Ph. Eur.) Biological Reference Reagents (BRRs) used for the in vitro potency assay of hepatitis A vaccines (HAV) by ELISA (enzymelinked immunosorbent assay) was established in 2012 for use in conjunction with Ph. Eur. general chapter 2.7.14 Assay of hepatitis A vaccine. It is composed of a coating reagent and a set of detection antibodies. As stocks of the latter are running low, the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised a collaborative study to qualify replacement batches. The candidate BRR antibodies (primary monoclonal antibody and labelled secondary antibody) were prepared under appropriate conditions from starting materials similar to those used for the current batches. The new batches of antibodies were tested alongside previous batches of BRRs to ensure continuity, and the results confirmed that they were suitable for use in the potency assay of hepatitis A vaccines by ELISA using the standard method referenced in Ph. Eur. general chapter 2.7.14 at the same final concentrations as the previous batches, i.e. 1:500 for the primary monoclonal antibody and 1:400 for the secondary conjugated antibody. The outcome of the study allowed their establishment by the Ph. Eur. Commission in March 2015 as anti-hepatitis A virus primary detection antibody BRR batch 3 and conjugated secondary detection antibody BRR batch 3 respectively. They are available from the EDQM as hepatitis A vaccine ELISA detection antibodies set BRR batch 3.

  2. 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

  3. 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.

  4. 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.

  5. Use of monoclonal antibodies in blocking ELISA detection of transmissible gastroenteritis virus in faeces of piglets.

    PubMed

    Rodák, L; Smíd, B; Nevoránková, Z; Valícek, L; Smítalová, R

    2005-04-01

    Monoclonal antibodies (mAb) to the transmissible gastroenteritis virus (TGEV) nucleoprotein (N) and membrane protein (M) were prepared and used for the comparative assessment of three blocking ELISA variants to detect TGEV. The competitive blocking ELISA format showed the highest sensitivity, allowing detection of 10(3) TCID50 TGEV/ml in culture medium. Ninety-nine porcine field faecal samples obtained from 37 herds affected with diarrhoea were examined, and various TGEV levels were found in nine samples from six herds. However, only in three samples were significant TGEV concentrations demonstrated. The relationship between incidence of TGEV gastroenteritis and the spread of porcine respiratory coronavirus infection in pig farms is discussed.

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

    PubMed

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

    2014-01-21

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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Development of monoclonal antibody-based sandwich ELISA for detection of dextran.

    PubMed

    Wang, Sheng-Yu; Li, Zhe; Wang, Xian-Jiang; Lv, Sha; Yang, Yun; Zeng, Lian-Qiang; Luo, Fang-Hong; Yan, Jiang-Hua; Liang, Da-Feng

    2014-10-01

    Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.

  12. 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.

  13. Development of ELISA based detection system for lethal toxin of Clostridium sordellii

    PubMed Central

    Arya, Preetika; Ponmariappan, S.; Singh, Lokendra; Kumar, Om

    2013-01-01

    Background & objectives: Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii. Methods: The catalytic domain (1.6kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund's complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin. Results: The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody. Interpretation & conclusions: An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples. PMID:23852299

  14. Development and application of an indirect ELISA for the detection of antibodies to novel duck reovirus.

    PubMed

    Yun, Tao; Chen, Haipeng; Yu, Bin; Zhang, Cun; Chen, Liu; Ni, Zheng; Hua, Jionggang; Ye, Weicheng

    2015-08-01

    A novel duck reovirus (N-DRV) disease emerged in China in 2000 and it has become an epidemic genotype. A test for detection of virus-specific antibodies in serum samples would be useful for epidemiological investigations. Currently, Currently, serological assays for N-DRV diagnosis are not available. A test for detection of virus-specific antibodies in serum samples would be useful for epidemiological investigations. In this study, a highly sensitive and specific indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to N-DRV was developed. The outer capsid (σC) of N-DRV was cloned and expressed in Escherichia coli as a coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration. Furthermore, the specificity of σC-ELISA assay was confirmed by cross checking with other duck viral pathogens. In comparison with the western blot, the sensitivity and specificity of the σC-ELISA was 92.6% and 88.9%, respectively, and agreement of two tests was excellent with κ value of 0.786 (p < 0.05). A serological survey was performed using the assay on serum samples from different age and species of duck flocks in the Zhejiang and Jiangsu Province, China. The seropositive rate of the 1209 serum samples was 57.7%. In conclusion, the developed σC-ELISA assay is a very specific and sensitive test that will be useful for large-scale serological survey in N-DRV infection and monitoring antibodies titers against N-DRV.

  15. Taenia solium cysticercosis in a village in northern Viet Nam: seroprevalence study using an ELISA for detecting circulating antigen.

    PubMed

    Erhart, Annette; Dorny, Pierre; Van De, Nguyen; Vien, Ha Viet; Thach, Dang Cam; Toan, Nguyen Duy; Cong, Le Dinh; Geerts, Stanny; Speybroeck, Niko; Berkvens, Dirk; Brandt, Jef

    2002-01-01

    An enzyme-linked immunosorbent assay (ELISA) for detecting circulating Taenia solium antigen was evaluated in Viet Nam; 12 of 210 people gave a positive result, including 5 persons with epilepsy. Cysticercosis was confirmed in 9 persons. Agreement between the ELISA, computerized tomography scanning and biopsy examination was high. PMID:12174775

  16. Biotin-Avidin ELISA Detection of Grapevine Fanleaf Virus in the Vector Nematode Xiphinema index.

    PubMed

    Esmenjaud, D; Walter, B; Minot, J C; Voisin, R; Cornuet, P

    1993-09-01

    The value of biotin-avidin (B-A) ELISA for the detection of grapevine fanleaf virus (GFLV) in Xiphinema was estimated with field populations and greenhouse subpopulations. Samples consisted of increasing numbers of adults ranging from 1 to 64 in multiples of two. Tests with virus-free X. index populations reared on grapevine and fig plants as negative controls did not reveal a noticeable effect of the host plant. ELISA absorbances of virus-free X. index samples were greater than corresponding absorbances of X. pachtaicum samples. Differences occurred between two X. index field populations from GFLV-infected grapevines in Champagne and Languedoc. In most tests, 1-, 2-, 4-, and 8-nematode samples of virus-free and virus-infected populations, respectively, could not be separated. Consequently, B-A ELISA was not a reliable method for GFLV detection in samples of less than 10 X. index adults, but comparison of the absorbances obtained with increasing numbers may allow differentiation of the viral infectious potential of several populations.

  17. Recombinant phosphoprotein based single serum dilution ELISA for rapid serological detection of Newcastle disease virus.

    PubMed

    Das, Moushumee; Kumar, Sachin

    2015-12-01

    Newcastle disease virus (NDV) is the causative agent of a highly contagious disease in avian species. All strains of NDV belong to avian paramyxovirus serotype-1. The disease is endemic in different parts of the world and vaccination is the only way to protect birds from NDV infection. The virus non-structural phosphoprotein (P) is the second most abundant protein and a major modulator of viral replication. Although P protein shows lesser evolutionary divergence among NDV isolates, it is known to be highly divergent among different avian paramyxovirus serotypes. In the present study, a recombinant P protein based single serum dilution ELISA was developed which showed better sensitivity, specificity and accuracy as compared to conventional methods for NDV detection. The recombinant P protein based ELISA could be an alternative to existing diagnostics against NDV infection in chickens.

  18. Development of sandwich ELISA for detection and quantification of invertebrate major allergen tropomyosin by a monoclonal antibody.

    PubMed

    Zhang, Hong; Lu, Ying; Ushio, Hideki; Shiomi, Kazuo

    2014-05-01

    Muscle protein tropomyosins of invertebrates are major allergens responsible for wide spread allergic reactions against invertebrates such as shellfish and insects. In order to develop a sandwich enzyme-linked immunoadsorbent assay (ELISA) for detection and quantification of the invertebrate pan-allergen tropomyosin, a specific monoclonal antibody (MAb), CE7B2, was produced. We have successfully established a sandwich ELISA for measuring invertebrate tropomyosin concentrations in food and food materials. The sandwich ELISA system using the MAb CE7B2 is a useful tool to detect and quantify levels of tropomyosin in food. The method is also helpful to detect mite and cockroach tropomyosins, the important indoor allergens.

  19. 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.

  20. Verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking ELISA methods.

    PubMed

    Rodák, L; Smíd, B; Nevoránková, Z; Smítalová, R; Valícek, L

    2004-05-01

    Monoclonal antibodies to group A rotavirus Vp6 protein were prepared and used for verification of three blocking enzyme-linked immunosorbent assay (ELISA) modifications to detect rotavirus A. Selected competitive blocking ELISA (CB-ELISA) and electron microscopy (EM) were used for examination of 194 field faecal samples of piglets affected with diarrhoea. Rotavirus was detected in 43 samples (22.2%) by CB-ELISA method, whereas in 26 (13.4%) samples by EM examination. However, of 26 samples positive by EM, rotavirus A was detected by CB-ELISA in 19 (73.1%) samples; indicating the share of group A rotavirus in all cases of gastroenteritis caused by rotavirus. The sensitivity and specificity of the CB-ELISA was verified both by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV) in each analysis and by comparative examination of samples with the commercial ELISA kit. The CB-ELISA sensitivity was positively affected by examination of samples in the presence of chelating agent.

  1. 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.

  2. Indirect competitive ELISA based on monoclonal antibody for the detection of 5-hydroxymethyl-2-furfural in milk, compared with HPLC.

    PubMed

    Guan, Yongguang; Wu, Xinlan; Meng, Hecheng

    2013-08-01

    In this study, a method for rapid detection of 5-hydroxymethyl-2-furfural (HMF) was investigated. Monoclonal antibody (anti-HMF) was prepared and evaluated by an indirect competitive ELISA (ic-ELISA) format. The optimized standard curve was y=-0.2097x+1.0432 [where x is the logarithm (base 10) of the values of the HMF concentration and y is the absorbance of ic-ELISA results tested at 490 nm] and the linear detection range was 0.008 to 32.768 mg/L. The percentage of cross-reactivity of HMF with 5 major furfural derivatives was less than 2.92%. Finally, the established ic-ELISA format was used to test HMF in milk, and compared with the result obtained by HPLC, which produced an error of about 0.3%. Based on the data in this experiment, we concluded that the established ic-ELISA format was reliable with a high specificity.

  3. Development and application of ELISA for the detection of IgG antibodies to lymphocytic choriomeningitis virus.

    PubMed

    Lapošová, K; Lukáčiková, Ľ; Ovečková, I; Pastoreková, S; Rosocha, J; Kuba, D; Beňa, Ľ; Tomášková, J

    2016-06-01

    Lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen, which can cause severe illnesses in humans. The most vulnerable are the human foetus and immunosuppressed individuals. Since there is no commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of anti-LCMV antibodies in human sera, we developed a sandwich ELISA method detecting anti-nucleoprotein IgG antibodies, using a specific monoclonal anti-nucleoprotein antibody and cells persistently infected with LCMV strain MX as antigen. In the present study we show standardization of this ELISA protocol, determination of its clinical specificity and sensitivity and its application on 30 clinical samples from multiorgan donors. Comparison of these results to the indirect immunofluorescence antibody test (IFA) demonstrates that ELISA is more sensitive. The developed ELISA assay provides a fast, simple and efficient tool for the clinical detection of anti-nucleoprotein antibodies in human sera. PMID:27265463

  4. 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.

  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. Development of an ELISA for the detection of bromoxynil in water.

    PubMed

    Cao, Yongsong; Lu, Yitong; Long, Shengyou; Hong, Jingbo; Sheng, Guoqing

    2005-01-01

    For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the nitrile herbicide bromoxynil, the polyclonal antibodies raised against 2,6-dibromo-4-cyano-phenoxyacetic acid (hapten) conjugated to bovine serum albumin (BSA) by the N-hydroxysuccinimide-activated ester method. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 77 days after the primary immunization. Antiserum A2 was applied to the residual analysis of some water samples, under optimized ELISA condition, the quantitative working range was from 10 to 500 ppb with a limit of detection of 5 ppb. Cross-reactivity to structurally similar agrochemicals and related chemicals was determined. The antiserum showed little cross-reactivity with 2,6-dibromophenol and bromoxynil octanoate ester which have a dibromophenol group as common structure, but showed no cross-reactivity with other herbicides. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween20 in the assay buffer. These four kinds of water samples were fortified with bromoxynil at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution, the mean recovery was 102.3%, and the mean coefficient of variation was 5.96%. The proposed ELISA turned out to be a powerful tool for monitoring of residual bromoxynil in water samples at trace level.

  7. Comparison of an ELISA assay for the detection of adhesive/invasive Neospora caninum tachyzoites.

    PubMed

    Pereira, Luiz Miguel; Yatsuda, Ana Patrícia

    2014-03-01

    Neospora caninum belongs to the phylum Apicomplexa, the causative agent of neosporosis, which leads to economic impacts on cattle production. A common feature among apicomplexan parasites is the invasive process driven mostly by the parasite. As a first evaluation of candidate molecules that play a possible role by interfering in this invasive process, the in vitro invasion assay is a fast and direct way to screen future agonists or antagonists. This work involved the development of a new cell culture ELISA and transient β-galactosidase activity applied to the semi-quantitative detection of N. caninum in Vero cell culture. Cell culture ELISA is based on histochemistry and immunology, resulting in a colorimetric reaction. The β-galactosidase activity was obtained by the transient transfection of the lacZ gene under control of RPS13 promoter of N. caninum. These methods were used to evaluate the effects of temperature (37°C and 85°C) on the invasion and adhesion of tachyzoites. The three tested methods (real time PCR, β-galactosidase activity and ELISA) showed a similar pattern, indicating that different methods may be complementary. PMID:24728359

  8. 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

  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

    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

  10. 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

  11. 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.

  12. Development of ELISA for the detection of transgenic vegetative insecticidal protein in GM crops/produce.

    PubMed

    Kumar, R

    2012-01-11

    In the process of the development of insect-resistant genetically modified (GM) crops and also to evaluate the consistency in the expression of toxin under field conditions, immunological assays are commonly being used. An immunoassay was developed to support the labelling of vegetative insecticidal protein (Vip3A)-based GM produce. The developed ELISA for the measurement of Vip3A is a triple antibody sandwich procedure utilising a polyclonal capture antibody (mouse anti-Vip3A) and a polyclonal detection antibody (rabbit anti-Vip3A) followed by use of a third HRP-conjugated anti-species antibody (goat anti-rabbit IgG). The limit of detection limit of the ELISA assay was 16 ng ml(-1) with a linear quantification range from approximately 31 to 500 ng ml(-1) of Vip3A protein. Furthermore, the assay was in-house validated with GM brinjal samples. The assay was specific, sensitive and reproducible, which can be helpful to detect and track down the spread of unapproved and intentionally/unintentionally released GM produce harbouring Vip protein.

  13. 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

  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. Development and evaluation of an immunomagnetic separation-ELISA for the detection of Alicyclobacillus spp. in apple juice.

    PubMed

    Wang, Zhouli; Yue, Tianli; Yuan, Yahong; Cai, Rui; Niu, Chen; Guo, Caixia

    2013-08-16

    The immunomagnetic separation (IMS) technique was used in combination with an enzyme-linked immunosorbent assay (ELISA) procedure to shorten the total analysis time and improve the sensitivity for the detection of Alicyclobacillus spp. in apple juice samples. The specificity of IMS-ELISA for twenty strains of Alicyclobacillus spp. and eighteen strains of non-Alicyclobacillus spp. was determined and there was little cross-reaction with non-Alicyclobacillus strains. Artificially contaminated apple juice with different concentrations of Alicyclobacillus acidoterrestris was detected by IMS-ELISA, and the detection limit of the assay in apple juice was 10(3)CFU/mL. Furthermore, the sample inoculated with 1CFU/mL of A. acidoterrestris could be detected as positive after incubation for 24h. The IMS-ELISA described, allows for the identification of suspect positive samples within 3h of testing versus 3-5days required by standard culture methods while significantly reducing the materials and labor required for the detection of Alicyclobacillus spp. in apple juice samples. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS-ELISA for 102 naturally contaminated apple juice samples were 91.3%, 96.02% and 95.09%, respectively. These results demonstrated that the newly proposed IMS-ELISA procedure can be a potentially useful analytical method for the detection of Alicyclobacillus spp. in apple juice.

  16. 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

  17. Detectability of lupine seeds by ELISA and PCR may be strongly influenced by potential differences between cultivars.

    PubMed

    Röder, Martin; Kleiner, Kornelia; Sachs, Andrea; Keil, Nicole; Holzhauser, Thomas

    2013-06-26

    Accurate methods for allergen detection are needed for the verification of allergen labeling and the avoidance of hidden allergens. But systematic data on the influence of different cultivars of allergenic crop species on their detectability in enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are lacking. As one example, seeds of 14 different cultivars of lupine (Lupinus albus, Lupinus angustifolius, Lupinus luteus) were investigated for total protein according to a Kjeldahl method, and for their relative quantitative detectability in three commercial lupine-specific ELISA tests and four lupine-specific PCR methods. Total Kjeldahl nitrogen allowed an accurate quantification of total protein. Relative differences in quantitative response between cultivars of 390-5050% and 480-13,600% were observed between ELISA kits and PCR methods, respectively. Hence, quantitative results of selected ELISA and PCR methods may be strongly influenced by the examined lupine cultivar.

  18. Detection of dermcidin for sweat identification by real-time RT-PCR and ELISA.

    PubMed

    Sakurada, Koichi; Akutsu, Tomoko; Fukushima, Hisayo; Watanabe, Ken; Yoshino, Mineo

    2010-01-30

    We evaluated the performance of real-time RT-PCR and ELISA assays for detection of dermcidin (DCD) in sweat and body-fluid stains. DCD, a small antibiotic peptide secreted into human sweat, was detected by real-time RT-PCR in 7-day-old stains containing as small as 10 microL of sweat, and the assay showed high specificity when testing 7-day-old stains containing 30 microL of other body-fluid. ELISA using anti-human dermcidin mouse monoclonal antibody detected DCD sweat diluted up to approximately 10,000-fold and could specifically detect DCD in 10 microL of body-fluid stains. The performance of the two assays was tested during winter on samples that simulated forensic case samples: an undershirt and a sock worn for 20 h, a handkerchief used to wipe the brow several times within 12h, a cap and a cotton glove worn for 4h, and a white robe worn at intervals for 2 years. The result showed that the former assay detected DCD in all sites of the undershirt examined (armpit, back, and breast), and the latter gave a relatively high OD value in the armpit among the three sites. For the socks, although the latter assay gave very high OD values in both the center and toe of the foot sole, the former could not detect DCD in both of them. These results indicate that highly damp conditions, such as inside a shoe, might promote the degradation of mRNA in samples such as socks. In the other case samples, sweat was adequately detected by both assays. This study is the first demonstration of the use of real-time RT-PCR to sensitively identify sweat among body-fluid stains, and it confirmed that dermcidin was an excellent marker for sweat identification. In addition, the usefulness of ELISA was also verified. Positive sweat identification using these assays is expected to assist forensic practice.

  19. 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

  20. 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

  1. Detection of Bovine IgG Isotypes in a PPA-ELISA for Johne's Disease Diagnosis in Infected Herds

    PubMed Central

    Fernández, Bárbara; Gilardoni, Liliana Rosa; Jolly, Ana; Colavecchia, Silvia Beatriz; Paolicchi, Fernando Alberto; Mundo, Silvia Leonor

    2012-01-01

    Johne's Disease or Paratuberculosis is a chronic granulomatous enteritis disease affecting ruminants. Detection of subclinically infected animals is difficult, hampering the control of this disease. The aim of this work was to evaluate the performance of detection of IgG isotypes in a PPA-ELISA to improve the recognition of cattle naturally infected with Map in different stages. A total of 108 animals from Tuberculosis-free herds were grouped as follows: exposed (n = 30), subclinically infected (n = 26), clinically infected (n = 14), and healthy controls (n = 38). Receiver-operating characteristic (ROC) curves of isotypes/PPA-ELISAs were constructed and areas under the curves were compared to evaluate the performance of each test. Our study demonstrated that the conventional PPA-ELISA (detecting IgG) is the best to identify clinically infected animals with high sensitivity (92.9%) and specificity (100%). Meanwhile, IgG2/PPA-ELISA improved the number of subclinically infected cattle detected as compared with conventional IgG/PPA-ELISA (53.8 versus 23.1%). In addition, it had the maximum sensitivity (65.0%, taking into account all Map-infected cattle). In conclusion, the combination of IgG and IgG2/PPA-ELISAs may improve the identification of Map-infected cattle in different stages of disease. The usefulness of IgG2 detection in serological tests for Johne's Disease diagnosis should be further evaluated. PMID:22792511

  2. 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

  3. 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

  4. Bioassay Development for Ultrasensitive Detection of Influenza A Nucleoprotein Using Digital ELISA.

    PubMed

    Leirs, Karen; Tewari Kumar, Phalguni; Decrop, Deborah; Pérez-Ruiz, Elena; Leblebici, Pelin; Van Kelst, Bram; Compernolle, Griet; Meeuws, Hanne; Van Wesenbeeck, Liesbeth; Lagatie, Ole; Stuyver, Lieven; Gils, Ann; Lammertyn, Jeroen; Spasic, Dragana

    2016-09-01

    Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection. PMID:27487722

  5. Report of a joint DMRQC/Organon field trial to detect hepatitis A IgM by ELISA.

    PubMed

    Supran, E M; Craske, J; Hart, R J; Kurtz, J B; Parry, J V; Skidmore, S J; Gardner, P S

    1983-10-01

    The results of a field trial of a joint DMRQC/Organon ELISA kit for the detection of hepatitis A IgM antibody are reported. The participating laboratories were asked to use the kit to test a panel of 360 specimens consisting of duplicate coded samples of 180 sera. The panel was also tested by MACRIA in the Virus Reference Laboratory, Colindale. The ELISA was shown to be specific and sensitive giving good discrimination between acute and late convalescent hepatitis A sera. It was proposed that the same cut-off control as is used in the RIA (equivalent to 10 RIA units) should be adopted for the ELISA also.

  6. 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

  7. Detection of influenza A antibodies in avian serum samples by ELISA.

    PubMed

    Chappell, Len; Killian, Mary Lea; Spackman, Erica

    2014-01-01

    ELISA assays are a fast and relatively inexpensive way to screen sera for antibodies to avian influenza virus. Commercial ELISA kits are available, and although they are more expensive, they provide a ready-to-use assay with good quality control. Various sample types can be processed for ELISA: serum, plasma, egg yolk, blood collected on filter paper. Quality samples are critical to accurate results. The basics of AIV antibody ELISA, sample processing, results interpretation, and troubleshooting are discussed. PMID:24899428

  8. Zinc reduces the detection of cocaine, methamphetamine, and THC by ELISA urine testing.

    PubMed

    Venkatratnam, Abhishek; Lents, Nathan H

    2011-07-01

    Federal workplace drug testing was initiated during the late 1980s. Since then, numerous methods have been employed to subvert these drug tests, adulteration of urine samples being the most common. A wide variety of adulterants has been reported to date along with suitable methods of their detection. Recently, websites have claimed that zinc sulfate can be an effective adulterant to bypass drug testing. Herein, these claims are investigated using standard drug detection kits and urine samples adulterated with zinc. Drug-free urine samples were fortified with different amounts methamphetamines and benzoylecgonine, to which zinc sulfate was added to study its effect. Urine samples from acute marijuana smokers were also obtained in order to study the effects of zinc supplements on THC drug testing. All urine drug testing was performed using ELISA detection kits manufactured by Immunalysis. Both zinc sulfate and zinc supplements are effective in interfering with the detection of all three drugs by Immunalysis drug detection kits. Also, no suitable method could be established to detect zinc in urine samples. Zinc can be an effective adulterant in urine for some illicit drugs that are commonly screened under routine drug testing.

  9. Development and evaluation of a VP3-ELISA for the detection of goose and Muscovy duck parvovirus antibodies.

    PubMed

    Zhang, Yun; Li, Yongfeng; Liu, Ming; Zhang, Dabing; Guo, Dongchun; Liu, Chunguo; Zhi, Haidong; Wang, Xiaomei; Li, Gang; Li, Na; Liu, Shiguo; Xiang, Wenhua; Tong, Guangzhi

    2010-02-01

    The VP3-encoding gene of goose parvovirus (GPV) Ep22 strain was cloned and expressed in Escherichia coli. The GPV VP3-encoding gene was 1605 bp in length, and it encoded a 534 amino acid protein with a predicted molecular mass of 59.9 kDa. The VP3 fusion protein expressed in E. coli was detected by goose and Muscovy duck anti-parvovirus polyclonal sera. In addition, an ELISA (VP3-ELISA) using the VP3 protein as the coating antigen for the detection of antibodies to GPV in geese and antibodies to Muscovy duck parvovirus (MDPV) in Muscovy ducks was developed. Compared to the virus neutralization test, the specificity and sensitivity of the VP3-ELISA was 90.2% and 95.2% for goose sera and 91.8% and 96.7% for Muscovy duck sera, respectively. The VP3-ELISA did not react with the anti-sera to other goose or duck pathogens, indicating that this protein is specific for the reorganization of goose or duck anti-parvovirus antibodies. Cross-reactivity between immunoglobulin G antibodies from geese and Muscovy ducks was also tested, and the results reflected the phylogenetic distance between these two birds when using the ELISA. In conclusion, the VP3-ELISA is a sensitive and specific method for detecting antibodies against GPV or MDPV.

  10. Precision, limit of detection and range of quantitation in competitive ELISA.

    PubMed

    Hayashi, Yuzuru; Matsuda, Rieko; Maitani, Tamio; Imai, Kazuhiro; Nishimura, Waka; Ito, Katsutoshi; Maeda, Masako

    2004-03-01

    This paper develops a mathematical model for describing the within-plate variation as the RSD (relative standard deviation) of absorbance measurements in a wide concentration range in competitive ELISA and proposes a method for determining the limit of detection (LOD) and range of quantitation (ROQ). The ELISA for 17 alpha-hydroxyprogesterone is taken as an example. The theoretical RSD description involves analyte concentration as an independent variable and error sources as parameters which concern the pipetting and absorbance measurement. Our model can dispense with repeated experiments of real samples, but the error parameters should be determined experimentally. The theory is in good agreement with the experiments. The most influential error sources at low and high sample concentrations are shown to be the pipetting of a viscous solution of antiserum and the absorbance inherent to the wells of a plate, respectively. The LOD and ROQ are defined as the concentration with 30% RSD and the region with <10% RSD, respectively, and are found in the theoretical plot of the RSD of concentration estimates vs concentration.

  11. Development of ELISA and Colloidal Gold-PAb Conjugate-Based Immunochromatographic Assay for Detection of Abrin-a.

    PubMed

    Xu, Chuang; Li, Xiaobing; Liu, Guowen; Xu, Chuchu; Xia, Cheng; Wu, Ling; Zhang, Hongyou; Yang, Wei

    2015-10-01

    When abrin-a was combined with several polyclonal antibodies (PAb), the detection limit could be increased. In this way, a monoclonal antibody (capture) and polyclonal antibody (detection) sandwich enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-PAb conjugate-based immunochromatographic assay for detection of abrin-a were developed. The ELISA had a detection limit of 3.9 ng/mL for abrin-a in standard solution and 7.8 ng/mL in soybean milk, and was more sensitive than polyclonal antibody (capture) and monoclonal antibody (detection) ELISA, which had a detection limit of 15.6 ng/mL. The test strip had a detection range of 50 to 500 ng/mL for abrin-a and a detection limit in standard solution or soybean milk samples of 50 ng/mL. However, the test strip had a reduced detection capability compared with a colloidal gold-monoclonal antibody conjugate-based immunochromatographic assay test strip, which had a lower detection limit of 10 ng/mL. The developed ELISAs and test strip show the specificity towards abrin-a and have no cross-reactivity towards abrin-b, -c, -d, ricin, or the agglutinins from either castor beans or rosary peas. PMID:26492622

  12. 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

  13. 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.

  14. 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

  15. 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.

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

    PubMed Central

    Dai, Hua; Xu, Zheng-zhong; Wang, Meiling; Chen, Jun-hua; Chen, Xiang; Pan, Zhi-ming; Jiao, Xin-an

    2016-01-01

    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

  17. Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

    PubMed Central

    Nakatsuma, Akira; Kaneda, Mugiho; Kodama, Hiromi; Morikawa, Mika; Watabe, Satoshi; Nakaishi, Kazunari; Yamashita, Masakane; Yoshimura, Teruki; Miura, Toshiaki; Ninomiya, Masaki; Ito, Etsuro

    2015-01-01

    To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA. PMID:26098695

  18. Detection limit estimated from slope of calibration curve: an application to competitive ELISA.

    PubMed

    Hayashi, Yuzuru; Matsuda, Rieko; Ito, Katsutoshi; Nishimura, Waka; Imai, Kazuhiro; Maeda, Masako

    2005-02-01

    This paper theoretically derives a general rule that while the slope of the semi-logarithmic plot (Y vs. log X) of a calibration curve varies depending on analyte concentration, X, the slope takes a specific value at the detection limit (L(D)). This rule holds good irrespective of the shape of the calibration curve (linear or non-linear) and in this paper, is applied to competitive ELISA (enzyme linked immunosorbent assay). The following relationship is deduced: slope of log-dose B/B0 at L(D) = [relative standard deviation (RSD) of blank responses] / 0.13. The L(D) obtained from the above-mentioned slope corresponds to the dose at which the RSD of dose estimates is 0.3 (= 30%). A commercial kit for 17alpha-hydroxyprogesterone is taken as an example.

  19. Characterisation of an ELISA detecting immunoglobulin G to Mycobacterium avium subsp. paratuberculosis in bovine colostrum.

    PubMed

    Zervens, Lisa Marie-Louise; Nielsen, Søren Saxmose; Jungersen, Gregers

    2013-09-01

    Although colostrum has been used to detect specific immunoglobulin (Ig) G to Mycobacterium avium subsp. paratuberculosis (MAP) in cattle, confounding, non-specific reactions can be a problem. The objectives of this study were to determine the proportion of non-specific ELISA reactions in samples of colostrum taken between 0 and 4 days-in-milk (DIM), and to assess the probability of an animal testing positive for MAP specific IgG over this time-period. Non-specific reactions were found in 3/365 (0.8%) of samples. The odds of an animal testing positive on day of calving were 130 times higher than at 4 DIM. The findings suggest colostral samples may have enhanced diagnostic potential over milk samples in determining if cattle have been exposed to or infected with MAP. PMID:23611487

  20. [Indirect ultramicroElisa for the detection of total antibodies against cytomegaloviruses in human blood].

    PubMed

    Laferte, J; Marrero, M; Alvarez, M; Jomarron, L; Garcia, S; Vazquez, S; Morier, L; Ulacia, M; Melchor, A

    1992-01-01

    We have standardized an indirect ultramicro ELISA assay for detecting antibodies to human Cytomegalovirus (CMV) using human serum samples (UMELISA CMV). The optimal concentration of coating antigen (30 ug/ml), serum dilution (1:40) and anti-human conjugate working dilution (1:1500), were determined by a check board titration method. The UMELISA CMV was compared with the latex agglutination test for antibodies to CMV (Dupont de Nemours) and with an indirect immunofluorescent method. The results have showed the high coincidence, sensitivity and especificity of the proposed assay regarding the two methods compared with, and supporting its use either for a blood donors screening or in the serological diagnosis of this infection by paired serum samples.

  1. The detection of rotavirus specific antibody in colostrum and milk by ELISA.

    PubMed

    Ellens, D J; de Leeuw, P W; Straver, P J

    1978-01-01

    The blocking method of ELISA for the detection and titration of rotavirus-specific antibody in colostrum is described. The results obtained were positively correlated with those of a neutralizing antibody test. On one farm colostrum samples were obtained over a period of 18 months. No relationship was found between the titer of colostrum obtained shortly after calving, and the development of rotavirus-associated diarrhoea in calves. On a second farm only samples obtained during the calving season were tested. Within this restricted period high colostral antibody titers appeared to reduce the incidence of diarrhoea among calves and to delay the onset of rotavirus excretion in the faeces. These results are discussed in relation to the rapid decline in antibody content of colostrum after calving.

  2. Preliminary evaluation of the ELISA as a tool for the detection of rotaviruses in activated sewage sludge.

    PubMed

    Agbalika, F; Wullenweber, M; Prévot, J

    1985-05-01

    Samples of activated sludge were taken in three different treatment plants near Nancy (France) and in West Berlin (F.R.G.), and were investigated for their content of rotaviruses. After elution with beef extract (0.25%, pH 7.3) and ultrasonication, the samples were concentrated by protamine flocculation and, in a few cases, in parallel by ultracentrifugation. A commercially available ELISA test kit (Rotazyme, Abbott Diagnostics) was routinely used for rotavirus detection. Although both the Rotazyme and a laboratory-designed ELISA gave positive results for the suldge concentrates, confirmation with a blocking test and electron microscope (EM) was negative in all cases so far investigated. Therefore, the exclusive use of ELISA cannot be recommended for similar investigations as it may lead to false positive results. In this light, an additional verification step with neutralizing antibodies should be included by the ELISA kits' manufacturers to enable a quick confirmation of the user's results.

  3. 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

  4. 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.

  5. 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

  6. The merits of designed ELISA avidity kit in detection of Toxoplasma gondii IgG antibody in laboratory conditions

    PubMed Central

    Sadraie, Javid; Bahadory, Ehsan Shariat; Marsusi, Vajihe

    2013-01-01

    Background: Toxoplasmosis is a parasitic disease which may cause some laboratory symptoms in infected individuals. One of the main ways to transmit this organism is placenta to the fetus pathway. If this transmission occurs in the 3rd month of pregnancy, the abortion, central nerve system and ocular disorder will happen. Because of this issue, the precise technique for the detection of Toxoplasma antibodies such as immunoglobulin G (IgG) and immunoglobulin M IgM are important, as they contain ELISA and ELISA avidity. Materials and Methods: In this survey, the main samples are serum and amniotic fluid that were collected from 48 pregnant women infected with Toxoplasma gondii in Shariaty hospital. This survey is attempted to design ELISA avidity kit in Tarbiat Modates University. Results: The results from this survey show that, in these total pregnant women the infection by T. gondii has occurred and many of them are infected currently. Conclusions: In the simple ELISA technique, the only antibody that can be detected precisely is IgM; however, using this technique the IgG antibody can also be detected. In this new technique or ELISA avidity, in addition to detection of IgG antibody against T. gondii, the month of transmission of Toxoplasma is also interpreted. PMID:23961440

  7. 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.

  8. Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA.

    PubMed

    Platteau, C; Cucu, T; De Meulenaer, B; Devreese, B; De Loose, M; Taverniers, I

    2011-02-01

    Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used. PMID:21253985

  9. 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

  10. Detection of double-stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides.

    PubMed

    Aramburu, J; Navas-Castillo, J; Moreno, P; Cambra, M

    1991-06-01

    An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by several serological techniques. DsRNA was readily detected by indirect ELISA (ELISA-I) and dot immunobinding assay (DIA). Addition of the antigen to poly-L-lysine-precoated plates and blocking with uncreamed milk powder allowed detection levels of 100 pg.ml-1 In-Cn by ELISA-I. Concentrations as low as 1 ng.ml-1 were detected by DIA using polyvinyliden difluoride (PVDF) membranes. Detection capacity with nitrocellulose membranes was 1000 times lower than with PVDF. ELISA-I and DIA enabled detection of dsRNA in enriched fractions from cucumber mosaic virus (CMV)- and citrus tristeza virus (CTV)-infected plants and from virus-infected Penicillium chrysogenum mycelium. These techniques showed similar or higher sensitivity for detection of dsRNA than separation by polyacrylamide gel electrophoresis and silver staining. PMID:1939501

  11. 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).

  12. 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

  13. Influence of baking time and matrix effects on the detection of milk allergens in cookie model food system by ELISA.

    PubMed

    Monaci, Linda; Brohée, Marcel; Tregoat, Virginie; van Hengel, Arjon

    2011-07-15

    Milk allergens are common allergens occurring in foods, therefore raising concern in allergic consumers. Enzyme-linked immunosorbent assay (ELISA) is, to date, the method of choice for the detection of food allergens by the food industry although, the performance of ELISA might be compromised when severe food processing techniques are applied to allergen-containing foods. In this paper we investigated the influence of baking time on the detection of milk allergens by using commercial ELISA kits. Baked cookies were chosen as a model food system and experiments were set up to study the impact of spiking a matrix food either before, or after the baking process. Results revealed clear analytical differences between both spiking methods, which stress the importance of choosing appropriate spiking methodologies for method validation purposes. Finally, since the narrow dynamic range of quantification of ELISA implies that dilution of samples is required, the impact of sample dilution on the quantitative results was investigated. All parameters investigated were shown to impact milk allergen detection by means of ELISA.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. [Comparison of the indirect immunofluorescent (IFAT), ELISA test and the comercial Chagatek test for anti-Trypanosoma cruzi antibodies detection].

    PubMed

    Enciso, Clara; Montilla, Marleny; Santacruz, María M; Nicholls, Rubén Santiago; Rodríguez, Adriana; Mercado, Marcela; Puerta, Concepción

    2004-03-01

    Chagas disease is a public health problem in Colombia, particularly in the eastern region. Because of human migration from rural areas to urban centers, the possibility of transfusional transmission becomes increasingly important. However the risk can be minimized by a careful screening of blood donors by means of serological tests. Colombian blood banks use comercial, foreign serological tests for screening for T. cruzi infection. The purpose of the current study was to compare the IFAT and ELISA tests (both use antigen obtained from Colombian strains) with the comercially available Chagatek tests. Sera of blood donors were classified in two groups on the basis of the IFAT: group I, 15 positive patients and group II, 14 negative patients. Sera from each group were tested by the ELISA and Chagatek tests. The ELISA test detected 100% of the patients as positive in group I and 7% (1/14) of patients as positive in group II. The Chagatek test detected 93% (14/15) of the patients as positive in group I and 50% (7/14) in group II. The kappa index for concordance between the ELISA and IFAT tests was 0.93 (95% C.I.: 0.80-1.00); between IFAT and Chagatek 0.43 (95% C.I.: 0.26-0.62), and between ELISA and Chagatek 0.49 (95% C.I.: 0.31-0.67). These results highlighted the importance of using autochtonous Colombian strains as antigens in screening tests for blood donors.

  19. 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%.

  20. 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

  1. Detection by ELISA of circulating anti-blood fluke (Carettacola, Hapalotrema, and Learedius) immunoglobulins in Hawaiian green turtles (Chelonia mydas).

    PubMed

    Graczyk, T K; Aguirre, A A; Balazs, G H

    1995-06-01

    An enzyme-linked immunosorbent assay (ELISA) is described utilizing the surface glycocalyx crude antigen of adult blood trematodes Learedius learedi, Hapalotrema dorsopora, and Carettacola hawaiiensis, for the detection of circulating antibodies (Ab) in Hawaiian green turtles (Chelonia mydas) naturally infected with the parasites and with or without green turtle fibropapillomatosis (GTFP). A concentration of 10.0 micrograms/ml of antigen was optimal in terms of test specificity and sensitivity. A direct ELISA with anti-reptilian/amphibian phosphatase-labeled IgG identified C. mydas Ab at a dilution of 1/12,800. Utilizing indirect ELISA, it was possible to detect Ab to blood flukes at a dilution of 1/3,200. The gross lesions and histopathology were typical for cardiovascular spirorchidiasis in C. mydas. No significant relationship was found between the size of turtles and the degree of GTFP severity. Forty-seven of 59 (80%) samples, from 5 sites, gave a positive ELISA reaction; 6 of the 47 (13%) specimens gave significantly (P < 0.001) higher absorbance values, with 5 from the same location. All 12 (20%) ELISA-negative turtles originated from 1 site, and the absorbance values of the 41 animals from this location were significantly lower (P < 0.015) when compared with the other 4 sites. The proposed assay is fast, has the feature of visual scoring, and can be used for determination of exposure of C. mydas to the spirorchid trematodes.

  2. 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.

  3. Evaluation of an indirect ELISA for detection of antibodies in bulk milk against bluetongue virus infections in the Netherlands.

    PubMed

    Mars, M H; van Maanen, C; Vellema, P; Kramps, J A; van Rijn, P A

    2010-12-15

    After the introduction of bluetongue virus serotype 8 (BTV-8) in western Europe in 2006, an indirect ELISA for detection of serogroup-specific antibodies against BTV in serum samples was validated for individual milk samples by the Central Veterinary Institute and the Animal Health Service in the Netherlands (Kramps et al., 2008). In order to develop a cost-effective monitoring tool, we now have evaluated this ELISA also for use in bulk milk. Therefore, bulk milk samples and individual milk samples were collected from 92 herds in the affected southern region in the Netherlands in 2007, before the start of the vaccination campaign. In addition, bulk milk samples collected from 88 herds before the bluetongue introduction in 2006 ("historically negative" samples) have been tested. With these results ROC analyses were performed and herd specificity and herd sensitivity of the bulk milk ELISA were estimated. All "historically negative" bulk milk samples were negative in the ELISA, with a mean S/P ratio of 10 ± 0.8%. The herd sensitivity and herd specificity of the ELISA in bulk milk samples depend on the cut-off that is chosen. In order to detect a within-herd-prevalence of 1%, the optimal cut-off S/P ratio 13% was found. A few herds with one or two milk-positive animals would then be missed. The specificity will be 100%. A within-herd-prevalence of 10% can be detected with 100% sensitivity at a cut-off S/P ratio of 96%. In conclusion, the indirect ELISA in bulk milk samples is a very specific and sensitive test which can be implemented in monitoring and surveillance systems in unvaccinated populations.

  4. Rapid and sensitive detection of rare cancer cells by the coupling of immunomagnetic nanoparticle separation with ELISA analysis

    PubMed Central

    Cheng, Hao-Yuan; Lai, Lee-Jene; Ko, Fu-Hsiang

    2012-01-01

    This study presents a rapid and sensitive method for detecting cancer cells occurring at low concentration. The method involves the simultaneous detection of two biomarkers of T helper cancer cells. One biomarker conjugates with immunofunctionalized magnetic nanoparticles (MNPs), enabling the separation of the T helper cells from a mixed population of cells. The other biomarker is used for detection during enzyme-linked immunosorbent assay (ELISA) analysis. The specific T helper cells can be quantified according to their ELISA absorbance values following magnetic separation. The experimental results demonstrate that immunofunctionalized MNPs can function as magnetic sensors and separate specific T helper cells from a mixed population with high efficiency and high specificity. Coupled with the ELISA technique, the immunofunctionalized MNPs can simultaneously detect rare cells. Results indicated increasing absorbance with increasing T cell number (from 10 to 106). The total detection time was less than 15 minutes, even at a low T cell count. The advantages of the proposed method for detecting specific cells at low concentration include ease of preparation, low cost, rapid detection, and high sensitivity. The proposed system can be adopted to detect circulating tumor cells in early tumor stages for diagnostic or prognostic purposes. PMID:22787392

  5. 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.

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

    PubMed

    Jenko, Kathryn L; 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(-1) (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg mL(-1) (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg mL(-1) (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 cross-reactivity 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. An indirect ELISA for detection of Theileria sergenti antibodies in water buffalo using a recombinant major piroplasm surface protein.

    PubMed

    Wang, L X; Zhao, J H; He, L; Liu, Q; Zhou, D N; Zhou, Y Q; Zhao, J L

    2010-06-24

    In this study we investigated the prevalence and enzootic potential of Theileria spp. in water buffalo in the Hubei province in China. An indirect ELISA based on a recombinant major piroplasma surface protein was developed. The complete ORF of the 33-kDa major piroplasma surface protein (p33) was obtained from Theileria sergenti genomic DNA by PCR, cloned into the pET-28(a) vector and expressed in E. coli as a His-fusion protein. Then the recombinant p33 (rp33) was purified and used as the antigen to develop an iELISA. Specificity test showed that there was no cross-reaction with Babesia orientalis, Schistosoma japonicum, Anaplasma marginale and Toxoplasma gondii. 178 water buffaloes raised in different locations in Hubei province in China were detected by this iELISA, all samples were also examined by PCR and microscopy at the same time. The iELISA result showed a higher positive rate (27.5%) than PCR (22.5%) and microscopy (12.9%). This result indicated that the iELISA is a suitable method for the diagnosis of T. sergenti infection and could be used in serological surveys to map out the prevalence of the disease.

  13. Rapid diagnosis of echovirus type 33 meningitis by specific IgM detection using an enzyme linked immunosorbent assay (ELISA).

    PubMed

    Chomel, J J; Thouvenot, D; Fayol, V; Aymard, M

    1985-01-01

    During an outbreak of meningitis in France (in the Lyon area), from June to October 1982, serum and stool samples were collected from 227 patients. An enzyme-linked immunosorbent assay (ELISA) for titrating IgG and IgM antibodies anti-echovirus type 33 was developed and compared with the virus isolation technique, and with the titration of neutralizing antibodies. In 39 patients excreting echovirus 33 in faeces, the ELISA test allowed a positive serodiagnosis in 85% of the cases by detection of specific IgM (64% of the cases) and by seroconversion (21%). Compared with the neutralization (Nt) test, ELISA was found to be more sensitive. The antibody titres in ELISA were over 50 times higher and detected earlier than the neutralizing antibodies. This early immune response allowed a rapid diagnosis by specific IgM detection in the acute sera collected within 8 days after the appearance of the clinical symptoms in more than 50% of the 97 patients examined, whereas the Nt test allowed a positive serodiagnosis in only 32% of the patients. The use of a caesium chloride purified antigen insured the specificity of the reactions.

  14. Concise estimate of the expected number of detections for stellar-mass binary black holes by eLISA

    NASA Astrophysics Data System (ADS)

    Kyutoku, Koutarou; Seto, Naoki

    2016-10-01

    We study prospects for detecting extragalactic binary black holes similar to GW150914 by evolved Laser Interferometer Space Antenna (eLISA). We find that the majority of detected binary black holes will not merge within reasonable observation periods of eLISA in any configuration. While long-arm detectors are highly desired for promoting multiband gravitational-wave astronomy by increasing the detections of merging binaries, the number of total detections can be increased also by improving the acceleration noise. A monochromatic approximation works well to derive semiquantitative features of observational prospects for non-merging binaries with clearly indicating the parameter dependence. Our estimate also suggests that the number of galaxies in the error volume is so small that the host galaxy may be determined uniquely with high confidence.

  15. 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.

  16. Implementation of Microfluidic Sandwich ELISA for Superior Detection of Plant Pathogens

    PubMed Central

    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

  17. 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

  18. 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.

  19. Field evaluation of an indirect ELISA for detection of brucellosis in lowland Bolivia.

    PubMed

    Kerby, P J; Quiroga, J L; McGrane, J J; Stagg, D A

    1997-05-01

    Bovine brucellosis exists endemically at an estimated prevalence of 10% in the developing dairy industry of Santa Cruz in tropical Bolivia. This paper describes field testing of an FAO/IAEA indirect ELISA for brucellosis, as a possible replacement confirmatory test for the complement fixation test (CFT). The ELISA and CFT were compared on sera from 3 cattle populations: a non-vaccinated negative population, an S19-vaccinated negative population, and a brucellosis-positive population of unknown vaccination status. The CFT and ELISA showed excellent specificities of 100% and 98% respectively against the negative non-vaccinated group. The CFT maintained a specificity of 98% against the S19-vaccinated negative group, but ELISA specificity fell to 83% using a cut-off of 20% of positive control, and 94% using a cut-off of 40% of positive control. Against sera from the positive population, the ELISA gave many more positive reactions than the CFT, probably a combination of both higher sensitivity and lower specificity. It is concluded that as Santa Cruz is entering a phase of brucellosis control rather than eradication, the extra sensitivity of the ELISA is not valuable enough to risk a higher level of false positive reactions, especially as S19 vaccination is being increasingly used.

  20. Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus.

    PubMed

    Ding, Meng-Die; Wang, Hong-Ning; Cao, Hai-Peng; Fan, Wen-Qiao; Ma, Bing-Cun; Xu, Peng-Wei; Zhang, An-Yun; Yang, Xin

    2015-01-01

    An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.

  1. Development of a solid-phase competition ELISA to detect antibodies against newly emerged Duck Tembusu virus.

    PubMed

    Fu, Yuguang; Ji, Yanhong; Liu, Bin; Dafallah, Rihab Muhammad; Zhu, Qiyun

    2015-11-01

    Duck Tembusu virus (DTMUV) was first isolated in Northern China in 2010, and is an emerging viral pathogen that is now rapidly spreading to most Chinese provinces causing a severe disease and economic losses for the duck breeding industry. The virus can affect ducks, chickens and geese, but currently there are no commercial ELISA methods for detecting antibodies against DTMUV from different species, or for assessing antibody titers in infected birds. In the present study, a solid-phase competition ELISA for this purpose was developed. The sensitivity and specificity of this test were 97.6% and 100%, respectively, when the percent inhibition (PI) was set at 15% using a serum dilution of 1:16. No cross-reaction with the sera against other avian viral pathogens was found in the study and the ELISA results generated for duck and chicken sera from the field were totally consistent with those of the classical virus neutralization assay. Therefore, this solid-phase competition ELISA represents a sensitive, specific and quantitative tool for the serological detection of Tembusu virus in different birds to facilitate the prevention and control of this disease.

  2. Establishment and application of SPA-co-operated ELISA for detection of anti-HCV-IgM.

    PubMed

    Li, F H; Guo, L S; Yu, Z Q; Wang, Y K; Qi, J Y; Yuan, X W; Hao, L J

    1993-01-01

    A staphylococcus aureus protein A co-operated ELISA (SPA-ELISA) for the detection of anti-HCV-IgM has been established using HCV antigenic polypeptide, SPA-bearing germs and horseradish peroxidase labelled anti-human IgM. The specificity of SPA-ELISA has been confirmed by some substitution tests, blocking tests and destroying test with 2-mercaptoethanol. The results showed that the rate of anti-HCV-IgG in a group of patients with acute hepatitis and there were significant difference in anti-HCV-IgM was higher than that of anti-HCV-IgM detected rates between patients with acute hepatitis and those with chronic hepatitis (32.26%, P < 0.01). On the other hand, the positive rates of anti-HCV-IgM were 53.66% and 63.41% in transfusion associated hepatitis, 38.10% and 42.86% in sporadic hepatitis, 6.11% and 16.33% in people who have had active social activities, 40.00% and 10.00% in a group of blood donors respectively. Furthermore, taking into account the characteristics of HCV polypeptide used, its easiness of manipulation, and elimination of the interference of anti-HCV-IgG in sera, the new SPA-ELISA is believed to be of practical value in clinical and epidemiological studies of hepatitis C. PMID:7512150

  3. 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

  4. 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

  5. Indirect ELISA and indirect immunofluorescent antibody assay for detecting the antibody against murine norovirus S7 in mice.

    PubMed

    Kitagawa, Yota; Tohya, Yukinobu; Ike, Fumio; Kajita, Ayako; Park, Sang-Jin; Ishii, Yoshiyuki; Kyuwa, Shigeru; Yoshikawa, Yasuhiro

    2010-01-01

    To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.

  6. Diagnostic accuracy study of a factor VIII ELISA for detection of factor VIII antibodies in congenital and acquired haemophilia A.

    PubMed

    Batty, Paul; Moore, Gary W; Platton, Sean; Maloney, James C; Palmer, Ben; Bowles, Louise; Pasi, K John; Rangarajan, Savita; Hart, Daniel P

    2015-10-01

    Antibody formation to factor VIII (FVIII) remains the greatest clinical and diagnostic challenge to the haemophilia-treating physician. Current guidance for testing for inhibitory FVIII antibodies (inhibitors) recommends the functional Nijmegen-Bethesda assay (NBA). A FVIII ELISA offers a complementary, immunological approach for FVIII antibody testing. It was the aim of this study to retrospectively evaluate the performance of a FVIII ELISA (index) for detection of FVIII antibodies, compared with the NBA (reference). All samples sent for routine FVIII antibody testing at two haemophilia Comprehensive Care Centres, were tested in parallel using the NBA and a solid-phase, indirect FVIII ELISA kit (Immucor). A total of 497 samples from 239 patients (severe haemophilia A=140, non-severe haemophilia A=85, acquired haemophilia A=14) were available for analysis. Sixty-three samples tested positive by the NBA (prevalence 12.7%, 95% confidence interval [CI], 9.9-15.9 %), with a median inhibitor titre of 1.2 BU/ml (range 0.7-978.0). The FVIII ELISA demonstrated a specificity of 94.0% (95%CI, 91.3-96.0), sensitivity of 77.8% (95%CI, 65.5-87.3), negative predictive value of 96.7% (95%CI, 94.5-98.2), positive predictive value 65.3% (95%CI, 53.5-76.0), negative likelihood ratio 0.2 (95%CI, 0.1-0.4), positive likelihood ratio 13.0 (95%CI, 8.7-19.3) and a diagnostic odds ratio of 54.9 (95%CI, 27.0-112.0). Strong positive correlation (r=0.77, p<0.001) was seen between the results of the NBA (log adjusted) and FVIII ELISA optical density. In conclusion, FVIII ELISA offers a simple, specific, surveillance method enabling batch testing of non-urgent samples for the presence of FVIII antibodies.

  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. Detection of Hypoderma actaeon infestation in Cervus elaphus with ELISA and western blotting.

    PubMed

    Domínguez, Julia; Panadero, Rosario; de la Fuente-López, Concepción

    2010-07-01

    We used enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) to evaluate the reactivity of first-stage larval extracts of Hypoderma lineatum with antibodies in sera from 76 red deer from an endemic area for Hypoderma actaeon. Antibodies in sera from deer infested with H. actaeon recognized hypodermin C from H. lineatum in both ELISA and WB assays. ELISA values were correlated with the epidemiology of the fly infestation in the area where the deer sera were collected. There was no clear relationship between anti-hypodermin C antibody levels and the age of the animals or the number of Hypoderma grubs found at necropsy in the hides of the deer. Our results suggest that first-instar antigens of H. lineatum can be used to diagnose natural infestations by H. actaeon by indicating the presence of first-stage larvae, which can help to accurately describe H. actaeon epidemiology. PMID:20688700

  9. Detection of Hypoderma actaeon infestation in Cervus elaphus with ELISA and western blotting.

    PubMed

    Domínguez, Julia; Panadero, Rosario; de la Fuente-López, Concepción

    2010-07-01

    We used enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) to evaluate the reactivity of first-stage larval extracts of Hypoderma lineatum with antibodies in sera from 76 red deer from an endemic area for Hypoderma actaeon. Antibodies in sera from deer infested with H. actaeon recognized hypodermin C from H. lineatum in both ELISA and WB assays. ELISA values were correlated with the epidemiology of the fly infestation in the area where the deer sera were collected. There was no clear relationship between anti-hypodermin C antibody levels and the age of the animals or the number of Hypoderma grubs found at necropsy in the hides of the deer. Our results suggest that first-instar antigens of H. lineatum can be used to diagnose natural infestations by H. actaeon by indicating the presence of first-stage larvae, which can help to accurately describe H. actaeon epidemiology.

  10. 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

  11. Development of a sensitive ELISA for the detection of casein-containing fining agents in red and white wines.

    PubMed

    Deckwart, Marina; Carstens, Carsten; Webber-Witt, Manuella; Schäfer, Volker; Eichhorn, Lisa; Kang, Sophie; Fischer, Markus; Brockow, Knut; Christmann, Monika; Paschke-Kratzin, Angelika

    2014-07-16

    Fining of wine with proteinogenic fining agents such as casein from cow's milk is a traditional and commonly used technique all over the world. Casein and other proteins from cow's milk are well-known food allergens, which pose a risk for allergic consumers. Temporary regulations exempting the labeling of milk and products thereof in wine expired. Since July 1, 2012, these fining agents have to be declared on the wine label under Regulation (EU) No. 579/2012 in conjunction to article 120g of Regulation (EU) No. 1234/2007 if exceeding the threshold of 0.25 mg/L allergenic protein. The aim of the presented study was to develop sensitive ELISA methods for the detection of casein in white and red wines and to investigate the risk of allergenic residues in fined wines. In this context it was shown that the used substance for calibration is highly relevant. Casein wine fining agents of different commercial producers were investigated by LDS-PAGE and immunoblot. In addition to casein, they contain other milk proteins, which are potentially allergic and therefore have to be incorporated in the development of antibodies for an ELISA method to be set up. An indirect ELISA for the investigation of white wine was developed. The LOD is 0.1 mg/L. For red wine the LOD is 0.2 mg/L in an indirect sandwich ELISA setup. The LOD of the indirect sandwich ELISA for white wine depends on the calibration standard. It is 0.1 mg/L for the fining agent casein and 0.01 mg/L for casein from a chemical trader. It is also shown that the use of different technological procedures during winemaking leads to no detectable amounts of casein in various wine samples.

  12. [Comparative evaluation of culture techniques and ELISA test in detection of Helicobacter pylori infection].

    PubMed

    Sito, E; Karczewska, E; Szczeba, J; Kamiński, K; Oleksy, J; Nowak-Sadzikowska, J; Maj, A

    1994-01-01

    The paper evaluates the correlation between culture tested for Helicobacter pylori from biopsy material obtained from patients diagnosed with stomach ulcer, duodenal ulcer and gastritis, and the level of specific IgG antibodies measured by the ELISA test. As compared with culture, the ELISA test yielded a higher percentage of positive assays. Antibody titer was particularly raised in patients with duodenal ulcer, which accounted for 83% of cases. The highest frequency of positive cultures (50%) was also obtained in this group of patients. Essentially, the results are in agreement with findings reported by other authors.

  13. [A sensitive enzyme-linked immunosorbent assay (ELISA) for the serological detection of antibodies against the European hog cholera virus].

    PubMed

    Schagemann, G; Greiser-Wilke, I; Liess, B; Moennig, V

    1991-02-01

    An ELISA for the detection of antibodies against hog cholera virus (HCV) was developed. The HCV-specific glycoprotein gp53 served as diagnostic antigen after immobilization using a monoclonal capture antibody. Due to the higher affinity of HCV-specific antibodies to the viral gp53, sera cross reacting with bovine viral diarrhea (BVD) virus were discriminated by the slope of the titration curves.

  14. COMPARISON OF ELISAS FOR DETECTING VITELLOGENIN IN THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

    EPA Science Inventory

    Measurement of vitellogenin (VTG) concentrations in the fathead minnow is currently being evaluated and considered for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp, was recently evaluate...

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. Development of an indirect competitive ELISA for detection of Campylobacter jejuni subsp.jejuni O:23 in foods.

    PubMed

    Hochel, I; Viochna, D; Skvor, J; Musil, M

    2004-01-01

    An indirect enzyme immunoassay for rapid detection of Campylobacter jejuni subsp. jejuni 0:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 microg/mL, and 8 microg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/microL, limit of quantification being 480 CFU/microL. By testing 5 chromogens, viz. 1,2-benzenediamine, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3',5,5'-tetramethylbenzidine, bi(4,4'-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3',5,5'-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain of C. sputorum subsp. sputorum (21.5 %) and with G+ bacterium Micrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according to CSN ISO 10272 and commercially available Singlepath Campylobacter GLISA-Rapid Test.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. Recombinant UL30 antigen-based single serum dilution ELISA for detection of duck viral enteritis.

    PubMed

    Aravind, S; Patil, B R; Dey, Sohini; Mohan, C Madhan

    2012-11-01

    A recombinant UL30 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure specific antibody in the sera of ducks against duck enteritis virus (DEV). The partial UL30 gene of DEV was cloned, expressed, purified and tested for its diagnostic use by designing a single serum dilution enzyme linked immuno-sorbent assay (ELISA). A total of 226 duck sera samples were tested using the assay. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived which demonstrated this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be specific, sensitive and accurate as compared to the virus neutralization test with a specificity, sensitivity and accuracy being 96%, 95% and 95% respectively.

  8. Indirect Haemagglutination Test in Comparison with ELISA for Detection of Antibodies against Invasive Amoebiasis

    PubMed Central

    Dhanalakshmi, Sankaramoorthy; Meenachi, Chidambaram

    2016-01-01

    Introduction Diagnosis of amoebiasis is based on combination of tests like microscopy, imaging, serology and molecular methods. In absence of molecular techniques, serology can be used as an alternative aid. Various serological techniques were reported with different sensitivity and specificity. The diagnostic efficiency of these assays mainly depends on the characteristics of antigen that is being used and various conditions of performance. Aim To evaluate the efficiency of recombinant calcium binding domain containing protein by Indirect Haemagglutination Assay (IHA) against a commercial ELISA among amoebic liver abscess cases and control group. Materials and Methods The study was carried out during the period of 2011-2015 and blood samples were collected from suspected amoebiasis cases who were attending the clinics of Medicine and Paediatrics department, JIPMER. A total of 200 sera samples which included 100 Amoebic Liver Abscess (ALA), 50 cases of other parasitic infections and liver diseases and 50 presumed healthy controls were examined by IHA and commercial ELISA. In brief, chick cells were stabilized by Double Aldehyde Sensitization (DAS) method. Optimum Sensitizing Dose (OSD) of the antigen was determined. The test was performed in a U-bottomed microtiter plate with recombinant amoebic antigen (12.5μg/ml), incubated at Room Temperature (RT) for 2 hours. RIDASCREEN Entamoeba IgG ELISA kit which is commercially available was used to evaluate the samples as per manufacturer’s instruction. Results The overall sensitivity and specificity of the IHA was 62% and 96%, respectively when compared to ELISA having sensitivity and specificity of 69% and 90%, respectively. The positive predictive value of the IHA was 91% while negative predictive value was 79%. Similarly, the positive predictive value of the ELISA was 87% while negative predictive value was 74%. Conclusion As serology heavily suffers due to lack of a standardised test system employing the native

  9. Iodine-Mediated Etching of Gold Nanorods for Plasmonic ELISA Based on Colorimetric Detection of Alkaline Phosphatase.

    PubMed

    Zhang, Zhiyang; Chen, Zhaopeng; Wang, Shasha; Cheng, Fangbin; Chen, Lingxin

    2015-12-23

    Here, we propose a plasmonic enzyme-linked immunosorbent assay (ELISA) based on highly sensitive colorimetric detection of alkaline phosphatase (ALP), which is achieved by iodine-mediated etching of gold nanorods (AuNRs). Once the sandwich-type immunocomplex is formed, the ALP bound on the polystyrene microwells will hydrolyze ascorbic acid 2-phosphate into ascorbic acid. Subsequently, iodate is reduced to iodine, a moderate oxidant, which etches AuNRs from rod to sphere in shape. The shape change of AuNRs leads to a blue-shift of longitudinal localized surface plasmon resonance. As a result, the solution of AuNRs changes from blue to red. Benefiting from the highly sensitive detection of ALP, the proposed plasmonic ELISA has achieved an ultralow detection limit (100 pg/mL) for human immunoglobulin G (IgG). Importantly, the visual detection limit (3.0 ng/mL) allows the rapid differential diagnosis with the naked eye. The further detection of human IgG in fetal bovine serum indicates its applicability to the determination of low abundance protein in complex biological samples.

  10. A sensitive phage-based capture ELISA for sub-femtomolar detection of protein variants directly from biological samples.

    PubMed

    Williams, Stephanie; Schulz, Philip; Sierks, Michael R

    2015-01-01

    To determine the role of proteins, and in particular protein variants, in human health, it may often be necessary to quantitatively determine the concentration of a specific protein variant present in complex biological samples such as blood, cerebral spinal fluid (CSF), or tissue. Many protein variants are present only at trace levels and therefore a simple assay with very high sensitivity and reliability would greatly facilitate correlation of the presence of particular protein variants with the progression of specific diseases. We have developed a simple phage based capture ELISA system that enables femtomolar or better detection of individual protein variants directly from complex biological samples. The protocol utilizes a capture reagent that selectively recognizes a unique epitope of the protein variant and a phage based detection reagent that binds to a second epitope present in all forms of the target protein. The phage based detection reagent is essentially a self-assembling nanoparticle consisting of several thousand coat proteins that can each be labeled to amplify the detection signal by several orders of magnitude. Here we demonstrate that we can achieve subfemtomolar detection of individual protein variants that have been implicated in neurodegenerative disease directly from complex tissue homogenates and sera. The ELISA system should facilitate identification of disease specific protein variants or other compounds even when present at trace amounts in samples including blood, CSF, saliva and urine. PMID:25203940

  11. A sensitive and specific ELISA using a monoclonal capture antibody for detection of Tamm-Horsfall urinary glycoprotein in serum.

    PubMed

    Hunt, J S; Peach, R J; Brünisholz, M C; Lynn, K L; McGiven, A R

    1986-07-11

    An enzyme-linked immunosorbent assay (ELISA) has been established using Nunc polystyrene immunoplates coated with a monoclonal antibody to human Tamm-Horsfall urinary glycoprotein (THP) to detect and measure THP in human serum. Optimal reaction conditions for both the monoclonal capture antibody and the affinity-purified rabbit anti-human THP second antibody were established to produce standard curves which showed linearity between 20-90 ng/ml with a sensitivity of 2-3 ng/ml. The plate-to-plate standard curve mean coefficient of variation (CV) was 5.9 +/- 2.9% on assays performed on the same day while day to day mean CV was 13.3 +/- 2.4%. The specificity of the ELISA was demonstrated by inhibition of binding after preincubation of both urinary THP standards and serum with monoclonal anti-THP antibody. Sera from 195 blood donors tested by the ELISA had a mean concentration of THP antigenic determinants of 260 +/- 105 ng/ml. Results from three control sera run on all plates used in the survey showed mean CV less than 7.6% while no binding was observed with sera from an anephric patient.

  12. Detecting diclofenac in livestock carcasses in India with an ELISA: a tool to prevent widespread vulture poisoning.

    PubMed

    Saini, Mohini; Taggart, Mark A; Knopp, Dietmar; Upreti, Suchitra; Swarup, Devendra; Das, Asit; Gupta, Praveen K; Niessner, Reinhard; Prakash, Vibhu; Mateo, Rafael; Cuthbert, Richard J

    2012-01-01

    Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), has caused catastrophic vulture declines across the Indian sub-continent. Here, an indirect ELISA is used to detect and quantify diclofenac in 1251 liver samples from livestock carcasses collected across India between August 2007 and June 2008, one to two years after a ban on diclofenac manufacture and distribution for veterinary use was implemented. The ELISAs applicability was authenticated with independent data obtained using LC-ESI/MS. Of 1251 samples, 1150 (91.9%) were negative for diclofenac using both methods, and 60 (4.8%) were positive at 10-4348 and 10-4441 μg kg(-1) when analysed by ELISA and LC-ESI/MS, respectively. The residue level relationship in the 60 positive samples was highly significant (p < 0.001, r(2) = 0.644). Data suggest that this immunological assay could be used not only for cost effective sample screening, but also for residue level semi-quantification.

  13. A sensitive epitope-blocking ELISA for the detection of Chikungunya virus-specific antibodies in patients.

    PubMed

    Goh, Lucas Y H; Kam, Yiu-Wing; Metz, Stefan W; Hobson-Peters, Jody; Prow, Natalie A; McCarthy, Suzi; Smith, David W; Pijlman, Gorben P; Ng, Lisa F P; Hall, Roy A

    2015-09-15

    Chikungunya fever (CHIKF) has re-emerged as an arboviral disease that mimics clinical symptoms of other diseases such as dengue, malaria, as well as other alphavirus-related illnesses leading to problems with definitive diagnosis of the infection. Herein we describe the development and evaluation of a sensitive epitope-blocking ELISA (EB-ELISA) capable of specifically detecting anti-chikungunya virus (CHIKV) antibodies in clinical samples. The assay uses a monoclonal antibody (mAb) that binds an epitope on the E2 protein of CHIKV and does not exhibit cross-reactivity to other related alphaviruses. We also demonstrated the use of recombinant CHIK virus-like particles (VLPs) as a safe alternative antigen to infectious virions in the assay. Based on testing of 60 serum samples from patients in the acute or convalescent phase of CHIKV infection, the EB-ELISA provided us with 100% sensitivity, and exhibited 98.5% specificity when Ross River virus (RRV)- or Barmah Forest virus (BFV)-immune serum samples were included. This assay meets the public health demands of a rapid, robust, sensitive and specific, yet simple assay for specifically diagnosing CHIK-infections in humans.

  14. Evaluation of recombinant Bhlp29.7 as an ELISA antigen for detecting pig herds with swine dysentery.

    PubMed

    La, Tom; Phillips, Nyree D; Hampson, David J

    2009-01-01

    Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera (n=1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.

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

    PubMed Central

    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

  16. 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.

  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.

  18. 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

  19. 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

  20. Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry.

    PubMed

    McCoy, Leslie F; Scholl, Peter F; Sutcliffe, Anne E; Kieszak, Stephanie M; Powers, Carissa D; Rogers, Helen S; Gong, Yun Yun; Groopman, John D; Wild, Christopher P; Schleicher, Rosemary L

    2008-07-01

    Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B(1). In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

  1. 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

  2. Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA.

    PubMed

    Sellek, Ricela; Jimenez, Oscar; Aizpurua, Carmen; Fernandez-Frutos, Begoña; De Leon, Patricia; Camacho, Maite; Fernandez-Moreira, Daniel; Ybarra, Carmen; Carlos Cabria, Juan

    2008-03-01

    The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4 gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.

  3. 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

  4. Accuracy of ELISA detection methods for gluten and reference materials: a realistic assessment.

    PubMed

    Diaz-Amigo, Carmen; Popping, Bert

    2013-06-19

    The determination of prolamins by ELISA and subsequent conversion of the resulting concentration to gluten content in food appears to be a comparatively simple and straightforward process with which many laboratories have years-long experience. At the end of the process, a value of gluten, expressed in mg/kg or ppm, is obtained. This value often is the basis for the decision if a product can be labeled gluten-free or not. On the basis of currently available scientific information, the accuracy of the obtained values with commonly used commercial ELISA kits has to be questioned. Although recently several multilaboratory studies have been conducted in an attempt to emphasize and ensure the accuracy of the results, data suggest that it was the precision of these assays, not the accuracy, that was confirmed because some of the underlying assumptions for calculating the gluten content lack scientific data support as well as appropriate reference materials for comparison. This paper discusses the issues of gluten determination and quantification with respect to antibody specificity, extraction procedures, reference materials, and their commutability.

  5. 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

  6. 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

  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. 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

  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. 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.

  12. 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

  13. Patents for ELISA tests to detect antibiotic residues in foods of animal origin.

    PubMed

    Reig, Milagro; Toldrá, Fidel

    2011-05-01

    The use of antibiotics in the European Union was banned since the 1(st) of January 2006. At present, and even though such ban, residues of drugs used in animal therapy or, illegally, as growth promoters might be found in food. Such residues may cause adverse health effects on consumers like allergic reactions in sensitive individuals develop transmissible antimicrobial resistance in food microflora, and have technological implications in the manufacture of fermented products. This is the reason why to check food for the presence of antibiotics residues is still necessary. ELISA test constitutes an attractive screening technique due to its simplicity, large number of samples per kit and possibility of automation. The aim of this manuscript is to provide a review of the numerous patents and applications for the analysis of antibiotic residues in foods. PMID:21428870

  14. Patents for ELISA tests to detect antibiotic residues in foods of animal origin.

    PubMed

    Reig, Milagro; Toldrá, Fidel

    2011-05-01

    The use of antibiotics in the European Union was banned since the 1(st) of January 2006. At present, and even though such ban, residues of drugs used in animal therapy or, illegally, as growth promoters might be found in food. Such residues may cause adverse health effects on consumers like allergic reactions in sensitive individuals develop transmissible antimicrobial resistance in food microflora, and have technological implications in the manufacture of fermented products. This is the reason why to check food for the presence of antibiotics residues is still necessary. ELISA test constitutes an attractive screening technique due to its simplicity, large number of samples per kit and possibility of automation. The aim of this manuscript is to provide a review of the numerous patents and applications for the analysis of antibiotic residues in foods.

  15. 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.

  16. 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.

  17. A sensitive sandwich ELISA for the detection of trace amounts of cashew (Anacardium occidentale L.) nut in foods.

    PubMed

    Wei, Yanhong; Sathe, Shridhar K; Teuber, Suzanne S; Roux, Kenneth H

    2003-05-21

    Trace amounts of cashew nut protein can provoke severe allergic reactions in sensitive patients. Consequently, commercial food processors and regulatory agencies must be vigilant to prevent cashew nut cross-contamination among foods and ensure proper labeling. Toward this end, we have developed a sandwich enzyme-linked immunosorbent (ELISA) to detect the predominant cashew protein fraction (anacardein or cashew major protein, CMP) that can be extracted in aqueous buffer from food matrixes. Protein G-purified goat antiwhole cashew extract IgG and rabbit anti-CMP IgG were used as capture and secondary antibodies, respectively. Immunoadsorption against several nut and seed proteins significantly minimized the inherent cross-reactivity of these reagents. Food samples spiked with cashew flour and CMP were extracted and tested in a sandwich ELISA where standard curves were based on reactivity with CMP. The assay was optimized to detect as little as 20 ng/mL (0.02 ppm) of CMP and was successfully used to quantify CMP, and thus cashew, in various food matrixes.

  18. 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.

  19. Evaluation of a French ELISA for the detection of Salmonella Enteritidis and Salmonella Typhimurium in flocks of laying and breeding hens.

    PubMed

    Jouy, E; Proux, K; Humbert, F; Rose, V; Lalande, F; Houdayer, C; Picault, J-P; Salvat, G

    2005-09-30

    In France, the regular and compulsory detection of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in flocks of breeding and laying hens is based on bacteriological examination of environmental swabs and faeces samples. The aim of this study was to compare this bacteriological examination with a serological method (ELISA) developed in our laboratory. This ELISA was first evaluated by use of artificially infected hens. During these experimental infection studies, several groups of hens were inoculated with SE, ST, different vaccines and different Salmonella serovars to calculate the experimental parameters of our ELISA. Then, in a field study, 43 flocks were followed monthly using two bacteriological samples (environmental swab and pool of faeces) and 20 serological samples (sera or yolks). Twenty-seven flocks without SE or ST gave a negative serological response throughout their surveillance. Among the 10 various serovars different from SE and ST isolated in this study, S. Heidelberg, S. Agona and S. Hadar gave seropositive results in seven flocks. Consequently, this ELISA was not specific of SE and ST as it detected serovars sharing or not common antigens with SE and ST. Seropositive results were also obtained each month for two flocks where no Salmonella could be isolated. Finally, in seven flocks found infected with SE or ST, the positive ELISA results appeared later than the bacteriological detection. Therefore, for the detection of chicken flocks recently infected with SE or ST, bacteriological examination currently used in France seems to be more appropriate than this ELISA.

  20. 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.

  1. 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

  2. Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes.

    PubMed

    Novo, Pedro; Prazeres, Duarte Miguel França; Chu, Virginia; Conde, João Pedro

    2011-12-01

    Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated. PMID:22012414

  3. The use of a sandwich ELISA for the detection of staphylococcal enterotoxin A in foods from outbreaks of food poisoning.

    PubMed Central

    Wieneke, A. A.; Gilbert, R. J.

    1985-01-01

    Foods from outbreaks of food poisoning were examined for the presence of staphylococcal enterotoxin A (SEA) by a sandwich ELISA using microtitre trays as the solid phase and SEA antibodies raised in sheep. The presence of SEA was confirmed by neutralization tests. The toxin was detected in 12 of 15 foods from separate outbreaks of staphylococcal food poisoning; all 15 foods contained a strain of Staphylococcus aureus which produced SEA. For most foods a simple extraction procedure without a concentration step was sufficient to detect the toxin. The method was semi-quantitative and recoveries of SEA added to control foods varied from 30 to 80%. The foods from outbreaks contained between 1 and 10 micrograms of SEA/100 g. SEA was not found in foods from 21 outbreaks in which an SEA-producing strain of Staph. aureus was not isolated. PMID:3926871

  4. The effect of heat treatment on the detection of peanut allergens as determined by ELISA and real-time PCR.

    PubMed

    Scaravelli, Elena; Brohée, Marcel; Marchelli, Rosangela; van Hengel, Arjon J

    2009-09-01

    Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitised individuals. The detection of peanut traces in food products is therefore of prime importance. Peanut traces which can be (unintentionally) present in food products have usually undergone one or more processing steps like roasting and baking. Therefore, methods designed to detect such traces have to be capable of detecting heat-treated peanuts. Commonly used methodologies designed to detect peanut traces in food products are enzyme-linked immunosorbent assays (ELISAs) that detect peanut-specific proteins, and polymerase-chain-reaction (PCR)-based methods targeting peanut-specific DNA. A comparative analysis of such methods was performed and the impact of heat treatment on peanut kernels as well as the impact on a peanut-containing food matrix are investigated. Our results show that heat treatments have a detrimental effect on the detection of peanut with either type of method and that both types of methods are affected in a similar manner.

  5. Utility of slot-blot-ELISA as a new, fast, and sensitive immunoassay for detection of carcinoembryonic antigen in the urine samples of patients with various gastrointestinal malignancies.

    PubMed

    El-Masry, Samir; El-Sayed, Ibrahim H; Lotfy, Mahmoud; Mahmoud, Lamiaa; El-Naggar, Mohamed

    2007-01-01

    Carcinoembryonic antigen (CEA) is the most widely used clinical tumor marker. CEA immunoassay has found acceptance as a diagnostic adjunct in clinical diagnosis of gastrointestinal tumors (GIT). Several immunoassays have been established for detection of CEA in plasma, serum, tissue, feces, and urine of cancer patients using polyclonal or monoclonal antibodies raised against CEA. Some of these assays display both high sensitivity and specificity for the detection of CEA. However, these assays require special and highly expensive equipment and the procedures require long periods for their completion. In the present study, we established a Slot-Blot Enzyme Linked Immunosorbent Assay (SB-ELISA), based on anti-CEA monoclonal antibody (CEA-mAb), as a new, simple, fast, cheap, and non-invasive immunodiagnostic technique for detection of CEA in the urine of GIT patients. Urine and serum samples were collected from 248 GIT patients (58 with pancreatic cancer, 20 with hepatoma, 23 with ampullary carcinoma, 15 with hilar cholangiocarcinoma, 28 with gastric cancer, 14 with esophageal cancer, and 90 with colorectal cancer). Moreover, urine and serum samples were collected from 50 healthy individuals to serve as negative controls. The traditional ELISA technique was used for determination of CEA in the sera of GIT patients using anti-CEA monoclonal antibody. A comparison between the results of both techniques (ELISA and SB-ELISA) was carried out. The traditional ELISA detected CEA in the sera of 154 out of 248 GIT patients with a sensitivity of 59.8%, 51.7% positive predictive value (PPV) and 75.37% negative predictive value (NPV). In addition, it identified 15 false positive cases out of 50 healthy individuals with a specificity of 70%. The urinary CEA was identified by a Western blotting technique and CEA-mAb at a molecular mass of 180 Kda. The developed SB-ELISA showed higher sensitivity, specificity, PPV, and NPV (70.1%, 78%, 62.4%, and 82.13%, respectively) for detection

  6. An ELISA for detecting anti-Plasmodium spp. antibodies in African black-footed penguins (Spheniscus demersus).

    PubMed

    Graczyk, T K; Cranfield, M R; Skjoldager, M L; Shaw, M L

    1994-02-01

    An enzyme-linked immunosorbent assay (ELISA) utilizing 3 Plasmodium falciparum antigens, R32tet32, P.F.R27, and crude red blood cell extract, was developed for the detection of circulating anti-Plasmodium relictum or anti-Plasmodium elongatum antibodies in sera from naturally infected adult African black-footed penguins (Spheniscus demersus) at The Baltimore Zoo, Maryland. A concentration of 2.0 micrograms/ml of each antigen was optimal in terms of specificity, sensitivity, and test speed. It was possible to detect anti-Plasmodium spp. antibodies at a dilution of 10(-4.11). Low absorbance values (less than 0.050) of nonspecific background were observed. The binding efficacy of anti-penguin IgG coupled to alkaline phosphatase to antibodies in the penguin sera was significantly higher than the binding efficacy of anti-chicken IgG. All penguins, bled in the winter time, in controlled mosquito-free conditions had anti-Plasmodium spp. antibodies reactive with P. falciparum antigens. The penguins showed age-dependent variation in antibody levels. There was a decrease in antibody titration units that was significantly correlated with the number of outdoor exposure years experienced by the birds, despite the season-comparable epizootiologic conditions in their summer open-air habitat. We concluded that the decrease of anti-malarial antibodies could be explained by an antibody-mediated equilibrium of immunity in naturally immunized birds harboring endothelial-stage parasites. The ELISA described is sensitive, and it requires a minimal amount of equipment to collect the blood samples. The assay can be used for detecting and monitoring levels of anti-Plasmodium spp. antibodies in selected groups of penguins.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. [The standardization of an ultramicro ELISA assay for the detection of IgG antibodies to the respiratory syncytial virus].

    PubMed

    Savón, C; Laferté, J; Goyenechea, A; Valdivia, A; Morier, L; Tejeiro, Y

    1996-01-01

    An ultramicro ELISA assay of double antibody for the detection of IgG antibodies to the respiratory syncytial virus (RSV) was standardized. It was used a RVS antiprotein F monoclonal antibody produced by the Genetic Engineering and Biotechnology Center (GEBC) in Havana. The use of this antibody allowed to include crude antigenic preparations instead of purified fractions, which caused a significant reduction of the reactivity obtained with the antigen control. The assay conditions were determined by crossed titration. It was obtained a sensitivity of 97.2%, a coincidence of 91%, and a specificity of 83.3% of the UMELISA as regards the complement fixation. The results may be qualitatively expressed or by antibody titres using only one serum dilution (1:40) and a pattern curve.

  13. 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

  14. Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine.

    PubMed

    Miller, Eleanor I; Torrance, Hazel J; Oliver, John S

    2006-03-01

    The purpose of this study was to validate the Immunalysis Buprenorphine Microplate enzyme-linked immunosorbent assay (ELISA) for the detection of buprenorphine in urine samples. Sixty-nine urine samples were obtained from volunteers on the Subutex treatment program and from routine samples submitted to the laboratory for buprenorphine testing. For ELISA analysis, samples were diluted 1:10 with K(2)HPO(4) (0.1M, pH 7.0). The limit of detection was calculated as 0.5 ng/mL buprenorphine. The intra-assay and interday precision was 3.8% (n = 10) and 8.6% (n = 50) respectively at 1 ng/mL buprenorphine. At a low concentration of norbuprenorphine (1 ng/mL), the immunoassay demonstrated a cross-reactivity of 78%. A higher cross-reactivity of 116% was observed at a higher concentration of norbuprenorphine (10 ng/mL). Dihydrocodeine, codeine, tramadol, morphine, propoxyphene, methadone, and EDDP were tested at concentrations of 10 ng/mL and 10,000 ng/mL and demonstrated no cross-reactivity with the assay. For liquid chromatography-tandem mass spectrometry (LC-MS-MS), deuterated internal standard mixture, 1M acetate buffer (pH 5.0), and b-glucuronidase were added to the standards and samples, which were then incubated for 3 h at 60 degrees C. After incubation, 3 mL K(2)HPO(4) (0.1M, pH 6.0) was added and the pH altered to pH 6.0 using 1M KOH. Buprenorphine and norbuprenorphine were subsequently extracted by solid-phase. Twenty-one samples were confirmed positive and 48 samples were confirmed negative by LC-MS-MS. Using a cut-off value of 0.5 ng/mL buprenorphine, the immunoassay demonstrated a sensitivity and specificity of 100%. PMID:16620543

  15. Development of a monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants (PPR) virus.

    PubMed

    Singh, R P; Sreenivasa, B P; Dhar, P; Shah, L C; Bandyopadhyay, S K

    2004-01-14

    Peste des petits ruminants (PPR) is an acute febrile, viral, disease of small ruminants with great economic importance. A competitive-ELISA (c-ELISA) test was developed for detection of antibodies to PPR virus in the sera samples of goats and sheep. The test uses monoclonal antibody to a neutralizing epitope of haemagglutinin protein of the virus. Based on the distribution of known negative sera samples (n=933) in respect of PPR virus antibodies in the test, a cut-off value was set as 38%. This value was the result of mean of negative population added with two times the standard deviations. A total of 1668 sera samples from goat and sheep and 32 sera from cattle were screened by c-ELISA and virus neutralization test (VNT). Efficacy of c-ELISA compared very well with VNT having high relative specificity (98.4%) and sensitivity (92.4%). The sensitivity of c-ELISA for PPR sero-surveillance could further be increased (95.4%), if the target population is non-vaccinated. c-ELISA test correlated well with VNT (r=0.845) for end-point titration of PPR virus antibody in 64 goat sera samples. It could clearly separate infected population from uninfected in field sera. Using c-ELISA test paired sera samples from 13 goats provided a clear diagnosis of PPR virus infection. Furthermore, antibodies to PPR virus could be successfully detected during 1 year after vaccination in four goats inoculated with an experimental PPR vaccine. Findings suggest that the c-ELISA test developed can easily replace VNT for sero-surveillance, sero-monitoring, diagnosis from paired sera samples and end-point titration of PPR virus antibodies.

  16. Detection of anti-drug antibodies using a bridging ELISA compared with radioimmunoassay in adalimumab-treated rheumatoid arthritis patients with random drug levels

    PubMed Central

    Jani, Meghna; Isaacs, John D.; Morgan, Ann W.; Wilson, Anthony G.; Plant, Darren; Hyrich, Kimme L.; Chinoy, Hector

    2016-01-01

    Objective. To determine the concordance between RIA and bridging ELISA at detecting anti-drug antibodies (ADAbs) in the context of random adalimumab levels and investigate the additional clinical utility of detecting ADAbs in RA patients who test ADAb positive by RIA and negative by ELISA. Methods. ADAb levels were determined using RIA and bridging ELISA in 63 adalimumab-treated RA patients (159 samples). Immunogenicity concordance was determined using receiver operating characteristic curves. To determine the additional clinical value provided by a positive RIA in the presence of negative ELISA, association between treatment response (ΔDAS28), adalimumab drug levels and ADAbs was evaluated longitudinally using generalized estimating equation. Results. Of the 60 RIA+ samples (n = 31 patients), 19 (n = 10 patients) were also ELISA+, corresponding to 31.7% of samples. Area under the curve for detecting ADAbs using ELISA (compared with RIA) using receiver operating characteristic curves was 0.65 (95% CI: 0.59, 0.71); this increased to 0.91 (95% CI: 0.81, 0.99) if ADAbs were ⩾100 AU/ml using RIA. In RIA+/ELISA− patients, adalimumab levels were associated with ΔDAS28 over 12 months [regression coefficient: 0.098 (95% CI: 0.043, 0.15), P < 0.0001] and while ADAbs were significantly associated with drug level, they were not directly associated with ΔDAS28 over 12 months [β coefficient: 0.00083 (95% CI: −0.0038 to 0.0054), P = 0.72]. Conclusion. ADAbs were detected using ELISA more frequently when present in high titres as measured by RIA. In RIA+/ELISA− patients, only drug levels were significantly associated with treatment response. Although ADAbs were not independently associated with treatment response, they may be helpful in determining the aetiology of low drug levels. PMID:27565176

  17. Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant toxin for detection of antibodies against Pasteurella multocida toxin.

    PubMed

    Takada-Iwao, Asuka; Uto, Takehiko; Mukai, Tetsuya; Okada, Munenori; Futo, Satoshi; Shibata, Isao

    2007-06-01

    To facilitate the control of progressive atrophic rhinitis (PAR) of swine caused by toxigenic Pasteurella multocida, an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (NT) have recently been developed to detect antibodies against the P. multocida dermonecrotic toxin (PmDNT). However, the NT is a cumbersome and time-consuming technique. To overcome these drawbacks, we developed an indirect ELISA, using recombinant PmDNT expressed in Escherichia coli, for the detection of antibodies to PmDNT in serum samples from pigs. The practical usefulness of this ELISA was compared with the NT using serum samples obtained from experimentally infected and naturally infected pigs. In the pigs experimentally inoculated with vaccine including PmDNT toxoid, the ELISA and neutralization antibodies were detected at almost the same time, and a good correlation was demonstrated between both tests (P<0.01, R(2)=0.807). Therefore, the ELISA can be used to evaluate the immune reaction of pigs after vaccination with P. multocida toxoid. In a survey conducted on a field herd with a history of clinical AR, the seropositivity by ELISA in pigs of age 4.5-6 months was increased even though the NT was negative, and the correlation was low between the results obtained with the two tests (P<0.01, R(2)=0.38). Therefore, the results indicated that this ELISA might be a useful alternative to the NT currently used to detect the antibody to PmDNT after vaccination or infection with P. multocida. PMID:17611352

  18. 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

  19. 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.

  20. Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA

    PubMed Central

    Emmerich, Petra; Mika, Angela; Schmitz, Herbert

    2013-01-01

    Background Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination. Methods In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens. Results Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified. Conclusions The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value

  1. 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

  2. Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows.

    PubMed

    Kramps, Johannes A; van Maanen, Kees; Mars, Maria H; Popma, Johan K; van Rijn, Piet A

    2008-07-27

    A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.

  3. 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

  4. 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

  5. Development of an indirect ELISA and immunocapture rt-PCR for Lily virus detection.

    PubMed

    Kim, Jin Ha; Yoo, Ha Na; Bae, Eun Hye; Jung, Yong-Tae

    2012-12-01

    Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.

  6. 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

  7. [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.

  8. Bulk tank milk ELISA for detection of antibodies to Mycobacterium avium subsp. paratuberculosis: Correlation between repeated tests and within-herd antibody-prevalence.

    PubMed

    Nielsen, Søren Saxmose; Toft, Nils

    2014-01-01

    Detection of bulk tank milk (BTM) antibodies using ELISA (BTM-ELISA) may constitute an inexpensive test for surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds provided that the test is accurate and consistent. The objectives of this study were to determine: (a) the correlation between repeated BTM reactions; and (b) the association between the BTM antibody ELISA-level and the within-herd prevalence of antibody-positive cows. Eight BTM samples per herd and approximately four milk samples per lactating cow per herd were collected from each of 108 Danish Holstein herds over a period of one year. All samples were tested using a commercial indirect ELISA for detection of MAP specific antibodies. The individual cow's results were dichotomised and used to estimate the within-herd antibody prevalence at each test-date. These prevalences were then combined with the ELISA reading on the BTM test-date closest to the cow-level test-date. A mixed-effect analysis of covariance with autoregressive type 1 correlation structure was carried out using the log-transformed BTM-ELISA results as outcome. This model was used to assess the correlation between repeated tests with and without correction for within-herd antibody prevalence. The repeated BTM-recordings were highly correlated with a correlation of 0.80 between samples collected 1.5 months apart. The within-herd antibody prevalence significantly influenced this estimate (p<0.0001), which dropped to 0.60 when corrected for the within-herd antibody prevalence. Although the test-results were relatively consistent and correlated with the within-herd prevalence, the magnitude of the test-values makes it difficult to use the BTM-ELISA for surveillance of MAP infections in practice.

  9. 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

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

    PubMed

    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.

  11. 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

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

    PubMed

    Woldehiwet, Z; Yavari, C

    2012-01-01

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

  13. 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.

  14. 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

  15. 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

  16. Development of an indirect ELISA for detection of E. Coli antibodies in cow serum using a recombinant OmpT as antigen.

    PubMed

    Tong, Chunyu; Wu, Zhijun; Yu, Liquan; Fan, Ziyao; Chen, Liang; Hu, Rui; Ma, Jinzhu; Song, Baifen; Zhu, Zhanbo; Cui, Yudong

    2014-01-01

    The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection of E. coli in cattle. OmpT, a highly conserved protease in all E. coli strains, was successfully expressed in E. coli XL-1-Blue strain with PET32a vector. Molecular weight of recombinant protein was identified by analyzing SDS-PAGE and the immunogenicity of OmpT was confirmed by Western Blotting. The recombinant OmpT was then employed as capture antigen in the ELISA. The antigen concentration and serum dilution were determined using a checkerboard titration. Results showed that the optimal concentration of coated antigen was 1 μg/ml at a serum dilution of 1:640 and the cut-off value of the assay was 0.335. In addition, the cross-reactivity assay showed that the OmpT was E. coli specific and the reproducibility experiments displayed good repeatability of the assay. Three hundred and forty cattle serum samples were tested by rOmpT-ELISA and sera coagulation tests. The ELISA has showed relative sensitivity of 100% and specificity of 96.47%. Results of these experiments indicated that the rOmpT-ELISA is a simple, rapid, and convenient method for detection the infection of E. coli with different serotype strains.

  17. Performance Evaluation of an Automated ELISA System for Alzheimer's Disease Detection in Clinical Routine.

    PubMed

    Chiasserini, Davide; Biscetti, Leonardo; Farotti, Lucia; Eusebi, Paolo; Salvadori, Nicola; Lisetti, Viviana; Baschieri, Francesca; Chipi, Elena; Frattini, Giulia; Stoops, Erik; Vanderstichele, Hugo; Calabresi, Paolo; Parnetti, Lucilla

    2016-07-22

    The variability of Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers undermines their full-fledged introduction into routine diagnostics and clinical trials. Automation may help to increase precision and decrease operator errors, eventually improving the diagnostic performance. Here we evaluated three new CSF immunoassays, EUROIMMUNtrademark amyloid-β 1-40 (Aβ1-40), amyloid-β 1-42 (Aβ1-42), and total tau (t-tau), in combination with automated analysis of the samples. The CSF biomarkers were measured in a cohort consisting of AD patients (n = 28), mild cognitive impairment (MCI, n = 77), and neurological controls (OND, n = 35). MCI patients were evaluated yearly and cognitive functions were assessed by Mini-Mental State Examination. The patients clinically diagnosed with AD and MCI were classified according to the CSF biomarkers profile following NIA-AA criteria and the Erlangen score. Technical evaluation of the immunoassays was performed together with the calculation of their diagnostic performance. Furthermore, the results for EUROIMMUN Aβ1-42 and t-tau were compared to standard immunoassay methods (INNOTESTtrademark). EUROIMMUN assays for Aβ1-42 and t-tau correlated with INNOTEST (r = 0.83, p < 0.001 for both) and allowed a similar interpretation of the CSF profiles. The Aβ1-42/Aβ1-40 ratio measured with EUROIMMUN was the best parameter for AD detection and improved the diagnostic accuracy of Aβ1-42 (area under the curve = 0.93). In MCI patients, the Aβ1-42/Aβ1-40 ratio was associated with cognitive decline and clinical progression to AD.The diagnostic performance of the EUROIMMUN assays with automation is comparable to other currently used methods. The variability of the method and the value of the Aβ1-42/Aβ1-40 ratio in AD diagnosis need to be validated in large multi-center studies. PMID:27447425

  18. Diagnostic performance of an indirect enzyme-linked immunosorbent assay (ELISA) to detect bovine leukemia virus antibodies in bulk-tank milk samples.

    PubMed

    Nekouei, Omid; Durocher, Jean; Keefe, Greg

    2016-07-01

    This study assessed the diagnostic performance of a commercial ELISA for detecting bovine leukemia virus antibodies in bulk-tank milk samples from eastern Canada. Sensitivity and specificity of the test were estimated at 97.2% and 100%, respectively. The test was recommended as a cost-efficient tool for large-scale screening programs.

  19. 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

  20. A multi-species indirect ELISA for detection of non-structural protein 3ABC specific antibodies to foot-and-mouth disease virus.

    PubMed

    Hosamani, M; Basagoudanavar, S H; Tamil Selvan, R P; Das, Varsha; Ngangom, Pravina; Sreenivasa, B P; Hegde, Raveendra; Venkataramanan, R

    2015-04-01

    Reliable diagnostic tests that are able to distinguish infected from vaccinated animals are a critical component of regional control programs for foot-and-mouth disease (FMD) in the affected countries. Non-structural protein (NSP) serology based on the 3ABC protein has been widely used for this purpose, and several kits are commercially available worldwide. This report presents the development of a 3ABC-antigen-based indirect ELISA, employing a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Recombinant 3ABC protein was expressed in insect cells and purified using affinity column chromatography. Using this protein, an indirect ELISA was developed and validated for the detection of NSP antibodies in serum samples collected from animals with different status of FMD. Diagnostic sensitivity and specificity were found to be 95.8 (95 % CI: 92.8-97.8) and 97.45 % (95 % CI: 94.8-99.0), respectively. The in-house ELISA compared well with the commercially available prioCHECK FMDV NS-FMD kit, with a high agreement between the tests, as determined by the kappa coefficient, which was 0.87. The in-house ELISA showed higher sensitivity for detecting vaccinated and subsequently infected animals, when compared to the reference test. Both of the tests were able to detect NSP antibodies as early as 7-8 days after experimental infection.

  1. 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

  2. Detection of serum antibody response in patients infected with one strain of Campylobacter jejuni with a DIG-ELISA method.

    PubMed

    Melby, K; Tønjum, T; Skjørten, F

    1986-12-01

    Paired sera from 10 patients and a convalescence sample from one patient suffering from campylobacteriosis were analysed for IgG, IgM and IgA antibodies against the infective organism (Campylobacter jejuni PEN 0:6,7) with a DIG-ELISA system. Either formalinized, ethanol-inactivated or heat-inactivated preparations of the infecting organism were used as antigens. Cross reactivity was tested with human sera having agglutinating antibodies against Yersinia enterocolitica (N = 6) or Salmonella typhi or S. parathyphi b (N = 7). All patients displayed IgA and IgM levels in the convalescence sample above that found in healthy blood donors (N = 55). Using the ethanol-inactivated or formalinized preparations more than 90% of the convalenscence sera showed IgG levels above that found in blood donors whereas the heat-inactivated preparation detected 73% IgG positives in the same group of sera. Serum from one patient infected with S. parathyphi b was positive in the test. This finding was interpreted as most likely due to a double infection. The study suggests that serum IgA may be a valuable marker for infection with this microorganism. Both the formalinized and the ethanol-inactivated preparations showed presence of flagella in contrast to the heat-inactivated preparation.

  3. DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA.

    PubMed

    Guo, Qi; Han, Jiao-Jiao; Shan, Shan; Liu, Dao-Feng; Wu, Song-Song; Xiong, Yong-Hua; Lai, Wei-Hua

    2016-12-15

    This study reported on a novel sandwich enzyme linked immunosorbent assay (ELISA) for the sensitive determination of Escherichia coli O157:H7 (E. coli O157:H7) by using DNA-based hybridization chain reaction (HCR) and biotin-streptavidin signal amplification. The anti-E. coli O157:H7 polyclonal antibody (pAb) was immobilized in the ELISA wells. The anti-E. coli O157:H7 monoclonal antibody (mAb) and initiator strand (DNA1) were labeled on gold nanoparticle (AuNP) to form a mAb-AuNP-DNA1 complex. In the presence of the target E. coli O157:H7, the sandwiched immunocomplex, which is pAb-E. coli O157:H7-mAb-AuNP-DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA (dsDNA) that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase-streptavidin and substrate/chromogen solution. Under optimal conditions, E. coli O157:H7 could be detected in the range of 5×10(2) CFU/mL to 1×10(7) CFU/mL; the limit of detection was 1.08×10(2) CFU/mL in pure culture. The LOD of the novel ELISA was 185 times lower than that of traditional ELISA. The proposed method is considerably specific and can be applied in the detection of whole milk samples inoculated with E. coli O157:H7. The coefficient of variation of in pure culture and in whole milk was 0.99-5.88% and 0.76-5.38%, respectively. This method offers a promising application in the detection of low concentrations of food-borne pathogens.

  4. DNA-based hybridization chain reaction and biotin-streptavidin signal amplification for sensitive detection of Escherichia coli O157:H7 through ELISA.

    PubMed

    Guo, Qi; Han, Jiao-Jiao; Shan, Shan; Liu, Dao-Feng; Wu, Song-Song; Xiong, Yong-Hua; Lai, Wei-Hua

    2016-12-15

    This study reported on a novel sandwich enzyme linked immunosorbent assay (ELISA) for the sensitive determination of Escherichia coli O157:H7 (E. coli O157:H7) by using DNA-based hybridization chain reaction (HCR) and biotin-streptavidin signal amplification. The anti-E. coli O157:H7 polyclonal antibody (pAb) was immobilized in the ELISA wells. The anti-E. coli O157:H7 monoclonal antibody (mAb) and initiator strand (DNA1) were labeled on gold nanoparticle (AuNP) to form a mAb-AuNP-DNA1 complex. In the presence of the target E. coli O157:H7, the sandwiched immunocomplex, which is pAb-E. coli O157:H7-mAb-AuNP-DNA1, could be formed. Two types of biotinylated hairpin were subsequently added in the ELISA well. A nicked double-stranded DNA (dsDNA) that contained abundant biotins was formed after HCR. Detection was performed after adding horseradish peroxidase-streptavidin and substrate/chromogen solution. Under optimal conditions, E. coli O157:H7 could be detected in the range of 5×10(2) CFU/mL to 1×10(7) CFU/mL; the limit of detection was 1.08×10(2) CFU/mL in pure culture. The LOD of the novel ELISA was 185 times lower than that of traditional ELISA. The proposed method is considerably specific and can be applied in the detection of whole milk samples inoculated with E. coli O157:H7. The coefficient of variation of in pure culture and in whole milk was 0.99-5.88% and 0.76-5.38%, respectively. This method offers a promising application in the detection of low concentrations of food-borne pathogens. PMID:27498326

  5. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of Campylobacter jejuni antibodies, and comparison with a complement fixation test (CFT).

    PubMed

    Oosterom, J; den Uyl, C H; Bänffer, J R; Lauwers, S; Huisman, J; Busschbach, A E; Poelma, F G; Bellemans, R

    1985-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of total anti-Campylobacter immunoglobulins in human sera. In this assay disintegrated Campylobacter bacteria were used as the antigen. Absorption tests including other possibly enteropathogenic bacterial species showed that the ELISA system displayed a high immunological specificity for Campylobacter. Using this ELISA it was found that in about 80% of Campylobacter patients these Campylobacter antibodies are produced to almost maximal levels within 8 days after onset of disease, and that they may persist for at least 4 months. Indeed, Campylobacter antibodies were demonstrated at low levels in a large number of control sera. However, accepting an antibody titre of 1:640 as indicative of Campylobacter infection, the statistical sensitivity of the ELISA system was 77% and the specificity 95%. In an epidemiological survey a high association was demonstrated between the severity of Campylobacter-related symptoms and antibody titre values. Assessment of Campylobacter antibody titres by means of this ELISA and by a complement fixation test in 92 sera from index patients and contacts with and without symptoms showed a high association of results.

  6. Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in Yeast Pichia pastoris and its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle.

    PubMed

    Behera, Sthita Pragnya; Mishra, Niranjan; Nema, Ram Kumar; Pandey, Pooja Dubey; Kalaiyarasu, Semmannan; Rajukumar, Katherukamem; Prakash, Anil

    2015-01-01

    The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.

  7. 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

  8. 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

  9. 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

  10. Intra and inter-laboratory reproducibility of a monoclonal antibody cocktail based ELISA for detection of allergen specific IgE in dogs: proficiency monitoring of macELISA in six laboratories.

    PubMed

    Lee, Kenneth W; Blankenship, Karen D; McCurry, Zachary M; McKinney, Brennan; Ruffner, Rick; Esch, Robert E; Tambone, Cecilia; Faas, Rebecca; Hermes, Darren; Brazis, Pilar; Drouet, Laurent

    2012-08-15

    The purpose of this study was to evaluate the reproducibility of results yielded using a monoclonal antibody based ELISA for detection of allergen specific IgE when run in six separate affiliated laboratories. On two separate occasions, duplicate samples of 15 different sera pools were independently evaluated by each laboratory in a single blinded fashion. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.2% (range 2.6-18.2%), while the average intra-laboratory inter-assay variance was 12.1% (range 8.0-17.1%). The overall inter-assay inter-laboratory variance was consistent among laboratories and averaged 15.6% (range 15.1-16.6%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose-response profiles observed in each of the laboratories were indistinguishable. Considering positive/negative results, inter-assay inter-laboratory concordance of results exceeded 95%. Correlation of OD values between and among all laboratories was strong (r>0.9, p<0.001). Correlation of OD values between the two separate evaluations was also high for all allergens except olive, which was attributed to lot-to-lot differences of allergen coated wells. Collectively, the results demonstrated that the monoclonal antibody based ELISA for measuring allergen specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory, but between laboratories using the same monoclonal-based ELISA.

  11. Detection of antibodies against foot-and-mouth disease virus using a liquid-phase blocking sandwich ELISA (LPBE) with a bioengineered 3D protein.

    PubMed

    O'Donnell, V K; Boyle, D B; Sproat, K; Fondevila, N A; Forman, A; Schudel, A A; Smitsaart, E N

    1996-04-01

    A liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA-3D) was developed to detect specific antibodies to the 3D protein in sera from foot-and-mouth disease (FMD) virus (FMDV)-infected animals. The assay uses a nonstructural 3D recombinant protein and two polyclonal antisera, one for capture (bovine) and the other for detector (guinea pig). The specificity of the assay was demonstrated by negative results with 101 sera of cattle from the FMD-free zone in Argentina and with bovine and porcine sera raised against various RNA and DNA viruses. The ELISA-3D was able to detect antibodies in cattle after natural or experimental infection with FMDV of A, O, or C types as early as 5 days postinfection and at later stages in persistently infected animals. Comparison of the results with those obtained with the routinely used agar gel immunodiffusion test and a previously described ELISA, both employing a partially purified virus-infection-associated antigen, shows that the ELISA-3D is highly sensitive and specific and gives reproducible results. Its use as a tool for monitoring viral activity and for certification of FMDV-free animals is recommended. PMID:8744733

  12. Cloning, expression and purification of duck hepatitis B virus (DHBV) core protein and its use in the development of an indirect ELISA for serologic detection of DHBV infection.

    PubMed

    Liu, Qiang; Jia, Renyong; Wang, Mingshu; Huang, Juan; Zhu, Dekang; Chen, Shun; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue; Cheng, Anchun

    2014-05-01

    Infecting ducks with duck hepatitis B virus (DHBV) is widely accepted as a relevant model for studying aspects of human HBV infection. However, efficient and sensitive diagnostic methods for the various infection models are limited. In order to provide a more simple and convenient method for serologic diagnosis, we improved the production of recombinant DHBV viral capsid protein (core protein) and then used it to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting anti-DHBc antibodies (DHBcAg ELISA) in DHBV-infected ducks. Given the positive/negative cut-off value, the maximum dilution of duck sera in which anti-DHBc antibodies could be detected was 1:12,800. In addition, the DHBcAg ELISA displayed no cross reactivity with duck antisera against duck circovirus (DuCV), duck plague virus (DPV), duck hepatitis virus (DHV), duck swollen head septicemia virus (DSHSV), avian influenza virus (AIV), Riemerella anatipestifer, Salmonella anatum, or Escherichia coli. Furthermore, the coefficients of variation (CVs) of inter-assay and intra-assay experiments were both below than 10 %. When compared to PCR for accuracy on clinical samples from cases of suspected DHBV infection, the DHBcAg showed 95.45 % coincidence with PCR. In conclusion, recombinant DHBc was readily produced and used to establish a simple DHBcAg ELISA that provided a highly specific and sensitive method for analysis of clinical samples.

  13. Expression and self-assembly of virus-like particles from two genotypes of marine vesiviruses and development of an ELISA for the detection of antibodies

    PubMed Central

    McClenahan, Shasta D.; Bok, Karin; Sosnovtsev, Stanislav V.; Neill, John D.; Burek, Kathy A.; Beckmen, Kimberlee B.; Smith, Alvin W.; Green, Kim Y.; Romero, Carlos H.

    2009-01-01

    Sequences encoding the major and minor capsid proteins (VP1 and VP2) from two marine vesivirus isolates (Steller sea lion viruses V810 and V1415) were engineered for expression of virus-like particles (VLPs) in the baculovirus system. The resulting VLPs were morphologically similar to native vesivirus virions. Purified VLPs were probed in immunoblots with pooled antisera specific for nine San Miguel sea lion virus (SMSV) types, and a predominant protein of approximately 60 kDa was detected. An enzyme linked immunosorbent assay (ELISA) for the detection of antibodies was developed in which the VLPs served as antigen. The VLPs were adsorbed to the wells of a microplate, and the specificity of the ELISA was established with hyperimmune sera raised against 24 serotypes of the genus Vesivirus. The ELISA was used to screen for the presence of vesivirus specific antibodies in the sera of free-ranging Steller sea lions. The ELISA results demonstrated that Steller sea lions that inhabit the Pacific Ocean waters of southeast Alaska are widely exposed to antigenically-related marine vesiviruses, while no previous exposure could be demonstrated using VLP antigens in 17 Steller sea lions from the Aleutian Islands. The broad reactivity of these VLPs and their non-infectious nature will facilitate global sero-epidemiological studies aimed at determining the incidence and prevalence of marine vesiviruses in mammals that inhabit the Pacific and Atlantic oceans as well as susceptible terrestrial animals. PMID:19913368

  14. Detection of antibodies against foot-and-mouth disease virus using a liquid-phase blocking sandwich ELISA (LPBE) with a bioengineered 3D protein.

    PubMed

    O'Donnell, V K; Boyle, D B; Sproat, K; Fondevila, N A; Forman, A; Schudel, A A; Smitsaart, E N

    1996-04-01

    A liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA-3D) was developed to detect specific antibodies to the 3D protein in sera from foot-and-mouth disease (FMD) virus (FMDV)-infected animals. The assay uses a nonstructural 3D recombinant protein and two polyclonal antisera, one for capture (bovine) and the other for detector (guinea pig). The specificity of the assay was demonstrated by negative results with 101 sera of cattle from the FMD-free zone in Argentina and with bovine and porcine sera raised against various RNA and DNA viruses. The ELISA-3D was able to detect antibodies in cattle after natural or experimental infection with FMDV of A, O, or C types as early as 5 days postinfection and at later stages in persistently infected animals. Comparison of the results with those obtained with the routinely used agar gel immunodiffusion test and a previously described ELISA, both employing a partially purified virus-infection-associated antigen, shows that the ELISA-3D is highly sensitive and specific and gives reproducible results. Its use as a tool for monitoring viral activity and for certification of FMDV-free animals is recommended.

  15. An ELISA technique to detect IgG antibody to the early herpes simplex virus type 2 (HSV-2) antigen AG-4 in HSV-2 patients.

    PubMed

    Evans, L; Arsenakis, M; Sheppard, M; May, J T

    1983-05-01

    An ELISA system, based on urease activity was used for the detection and titration of IgG to the immediate early AG-4 antigen in sera from HSV-2 patients. It detected low levels of IgG to the AG-4 antigen in 32% of patients' sera known to contain complement fixing antibody to the antigen. Furthermore, the sensitivity of the ELISA system was 2- to 10-fold higher than the complement-fixation system depending on when the sera was taken from the HSV-2 patients. The system also allowed the easy detection and quantitation of AG-4 antigen production when various HSV-1 X HSV-2 intertypic recombinant viruses were used to infect BHK-21 cells.

  16. Development of a double antibody sandwich ELISA for West Nile virus detection using monoclonal antibodies against non-structural protein 1.

    PubMed

    Ding, Xi-Xia; Li, Xiao-Feng; Deng, Yong-Qiang; Guo, Yong-Hui; Hao, Wei; Che, Xiao-Yan; Qin, Cheng-Feng; Fu, Ning

    2014-01-01

    The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans. PMID:25303282

  17. Development of a double antibody sandwich ELISA for West Nile virus detection using monoclonal antibodies against non-structural protein 1.

    PubMed

    Ding, Xi-Xia; Li, Xiao-Feng; Deng, Yong-Qiang; Guo, Yong-Hui; Hao, Wei; Che, Xiao-Yan; Qin, Cheng-Feng; Fu, Ning

    2014-01-01

    The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans.

  18. 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

  19. An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes.

    PubMed

    Starke-Buzetti, W A; Machado, R Z; Zocoller-Seno, M C

    2001-05-01

    Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation.

  20. The efficacy of a commercial ELISA as an alternative to virus neutralisation test for the detection of antibodies to EAV.

    PubMed

    Duthie, S; Mills, H; Burr, P

    2008-03-01

    Infection with equine arteritis virus is a notifiable disease with sporadic occurrence in the UK. As stallions may harbour the virus after infection, horses are screened for exposure by serological testing prior to breeding. The virus neutralisation test is considered the 'gold standard' serological screening test, but it is time-consuming and labour intensive; consequently there is a move towards more rapid screening methodology. In this study, a commercially available EVA antibody ELISA is assessed. The ELISA performed poorly with a specificity [corrected] of 26% and a sensitivity [corrected] of 96% in the samples analysed. It was concluded that this ELISA would be of little value for reducing sample turnaround time. The study emphasises the need for in-house validation of commercially available kits.

  1. Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA

    PubMed Central

    Hsu, Min-Yen; Hung, Yu-Chien; Hwang, De-Kuang; Lin, Shang-Chi; Lin, Keng-Hung; Wang, Chun-Yuan; Choi, Hin-Yeung; Wang, Yu-Ping; Cheng, Chao-Min

    2016-01-01

    Intraocular vascular endothelial growth factor (VEGF) levels play an important role in the pathogenesis of blindness-related diseases, such as age-related macular degeneration (AMD). Here, we aimed to develop a paper-based enzyme-linked immunosorbent assay (P-ELISA) to analyze the suppression of aqueous VEGF concentrations following intravitreal injection (IVI) of anti-VEGF antibody (bevacizumab or ranibizumab). A total of 25 eyes with wet AMD, one with myopic neovascularization, and one with polypoidal choroidal vasculopathy were enrolled in this study. The limit of detection using P-ELISA was 0.03 pg/mL. Forty-six consecutive samples of aqueous humor were acquired. From all samples, 66.67% (10/15) achieved complete VEGF suppression (below the detection limit) within 5 weeks of receiving IVI of anti-VEGF antibody. Only 13.33% of samples (2/15) achieved complete VEGF suppression 5 weeks after receiving treatment. In some patients, elevated VEGF was still detected 5 weeks after receipt of anti-VEGF antibody, and all samples (10/10) were found to have elevated VEGF levels 49 days after treatment. Thus, we suggest that monthly IVI of anti-VEGF antibody may be required to ensure durable VEGF inhibition. Ultrasensitive P-ELISA can detect elevated VEGF at an earlier time point and may facilitate decision-making regarding appropriate treatment strategies. PMID:27725716

  2. Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein as antigen.

    PubMed

    Dea, S; Wilson, L; Therrien, D; Cornaglia, E

    2000-06-01

    The 15 kDa nucleocapsid (N) protein is the most abundant protein of the porcine reproductive and respiratory syndrome virus (PRRSV), and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. In this study, complementary DNA corresponding to the entire N gene of the IAF-Klop strain of PRRSV was cloned into the pGEX-4T-1 vector, and the N protein was expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein. The resulting GST-N recombinant fusion protein was purified by affinity chromatography and used as antigen for serological testing by indirect enzyme-linked immunosorbent assay (ELISA). Two anti-N specific monoclonal antibodies (MAbs) (IAF-K8 and IAF-2B4), obtained following fusion experiments with spleen cells of BAlb/c mice that were immunized with the purified virus, were used in a competitive assay to increase the specificity of the ELISA. Both MAbs were found to be directed against highly conserved conformational epitopes of North American isolates of PRRSV. Optimal concentration of GST-N protein was determined by checkerboard titration, using hyperimmune pig antiserum to the homologous PRRSV strain, and corresponded to a range of 0.1-0.5 microg protein per well. When tested on 95 sera from pigs that were experimentally infected with the IAF-Klop strain, the competitive ELISA (K8-ELISA) was capable of detecting anti-PRRSV antibodies in 86.7% (65/75) and 92.6% (63/68) of pig sera known to be seropositive by indirect immunofluorescence (antibody titers >16) and a currently used commercial ELISA (HerdCheck(R); Idexx), with specificity values of 100 and 96.2%, respectively. When tested on clinical samples (542 sera) from 28 positive and 28 negative pig herds, the K8-ELISA performed in a similar way to HerdCheck(R) and immunofluorescence (IF) tests as shown by kappa values of 0.762 and 0.803. The sensitivity and specificity of K8-ELISA were 100% on a

  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. Development and evaluation of a Luminex multiplex serology assay to detect antibodies to bovine herpes virus 1, parainfluenza 3 virus, bovine viral diarrhoea virus, and bovine respiratory syncytial virus, with comparison to existing ELISA detection methods.

    PubMed

    Anderson, Steve; Wakeley, Phil; Wibberley, Guy; Webster, Kath; Sawyer, Jason

    2011-03-01

    Detection of circulating antibodies to bovine herpes virus 1 (BHV-1), parainfluenza 3 virus (PI3V), bovine viral diarrhoea virus (BVDV) and bovine respiratory syncytial virus (BRSV) using ELISA is widely used for veterinary diagnostics and surveillance. In this paper, the potential of a multiplex serology test based on Luminex technology, where all antibodies are simultaneously detected in a single assay was investigated. The performance of "in-house" separate ELISAs which use relatively crude lysates of cultured virus as capture antigens, was compared to the multiplex assay where the same antigens were covalently bound to the fluorescent beads used in the Luminex platform. A panel of field serum samples was tested by the multiplex assay in parallel with the separate routine ELISAs to provide a comparison between tests. The BHV-1 and PI3V components of the multiplex test showed similar sensitivities and specificities to the separate "in-house" ELISAs. The performance of the BVDV and BRSV components was less successful and was attributed to relatively low signal strength for these antigens, leading to higher assay variability and a reduced ability to distinguish positive and negative samples compared to the "in-house" ELISAs. The results illustrated that antigens commonly used successfully in ELISAs cannot always be transferred for use in alternative assay systems. The use of recombinant BVDV E2 protein was investigated and was shown to lead to an appreciable increase in signal strength compared to the use of crude BVDV antigen in the Luminex system.

  5. [Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

    PubMed

    Dzagurova, T K; Solopova, O N; Sveshnikov, P G; Korotina, N A; Balovneva, M V; Leonovich, O A; Varlamov, N E; Malkin, G A; Sotskova, S E; Tkachenko, E A

    2013-01-01

    The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

  6. A simple heat dissociation method increases significantly the ELISA detection sensitivity of the nonstructural-1 glycoprotein in patients infected with DENV type-4.

    PubMed

    Lima, Monique da Rocha Queiroz; Nogueira, Rita Maria Ribeiro; Filippis, Ana Maria Bispo de; Nunes, Priscila Conrado Guerra; Sousa, Carla Santos de; Silva, Manoela Heringer da; Santos, Flavia Barreto dos

    2014-08-01

    The secreted form of the dengue virus (DENV) nonstructural-1 (NS1) glycoprotein has been shown to be useful for the diagnosis of DENV infections in patients' serum samples. In a number of studies, the sensitivity of the commercially available DENV NS1 glycoprotein detection assays was higher against some DENV serotypes (DENV-1>DENV-3>DENV-2=DENV-4) than others and were also lower using patients' serum samples with secondary versus primary DENV infections. In this study, 471 DENV-4 positive acute phase patients' serum samples were selected from a large panel collected in Brazil from March 2011 to October 2012 by RT-PCR and/or virus isolation followed by serotype determination. The sera from primary (n=228) and secondary (n=238) DENV-4 infections were identified using IgM and IgG capture ELISAs. The sensitivity of a commercial DENV NS1 glycoprotein detection ELISA was then assessed when these serum samples were not pre-treated or pre-treated by acid or heat dissociation prior to being tested. Acid and heat dissociation of patients' serum samples with primary and secondary DENV-4 infections increased significantly the sensitivity of the DENV NS1 glycoprotein detection ELISA from 54.4% to 77.2% (p<0.05) and 82% (p<0.05) and from 39.1% to 63.9% (p<0.05) and 73.1% (p<0.05), respectively. Treatment of DENV infected patients' serum samples using simple and rapid heat dissociation step (100°C for 5min) was, therefore, shown to be very useful for increasing the sensitivity of the DENV NS1 glycoprotein detection ELISA using serum samples from either primary or secondary DENV infected patients.

  7. 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.

  8. 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

  9. Development and Evaluation of an in-House IgM-Capture ELISA for the Detection of Chikungunya and Its Application to a Dengue Outbreak Situation in Kenya in 2013.

    PubMed

    Wasonga, Caroline; Inoue, Shingo; Kimotho, James; Morita, Kouichi; Ongus, Juliette; Sang, Rosemary; Musila, Lillian

    2015-01-01

    Chikungunya (CHIK) is a mosquito-borne viral disease. In the 2004 CHIK outbreak in Kenya, diagnosis was delayed because of the lack of accurate diagnostics. Therefore, this study aimed to develop and evaluate an in-house IgM-capture enzyme linked immunosorbent assay (ELISA) (in-house ELISA) for the detection of chikungunya virus (CHIKV) infections. Anti-CHIKV antibodies were raised in rabbits, purified and conjugated to horseradish peroxidase. These anti-CHIKV antibodies and cell-culture derived antigen were used to develop the ELISA. To validate the in-house ELISA, 148 patient sera from the 2005 Comoros CHIK outbreak were tested with centers for disease control and prevention (CDC) IgM-capture ELISA (CDC ELISA) and focus reduction neutralization test (FRNT) as reference assays. The in-house ELISA had a sensitivity of 97.6% and specificity of 81.3% compared to the CDC ELISA and a sensitivity of 91.1% and specificity of 96.7% compared to FRNT. Furthermore, 254 clinically suspected dengue patient samples from Eastern Kenya, collected in 2013, were tested for CHIKV IgM using the in-house ELISA. Out of the 254 samples, 26 (10.2%) were IgM positive, and of these 26 samples, 17 were further analyzed by FRNT and 14 (82.4%) were positive. The in-house ELISA was able to diagnose CHIKV infection among suspected dengue cases in the 2013 outbreak.

  10. 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.

  11. An ELISA Lab-on-a-Chip (ELISA-LOC).

    PubMed

    Rasooly, Avraham; Bruck, Hugh A; Kostov, Yordan

    2013-01-01

    Laminated object manufacturing (LOM) technology using polymer sheets is an easy and affordable method for rapid prototyping of Lab-on-a-Chip (LOC) systems. It has recently been used to fabricate a miniature 96 sample ELISA lab-on-a-chip (ELISA-LOC) by integrating the washing step directly into an ELISA plate. LOM has been shown to be capable of creating complex 3D microfluidics through the assembly of a stack of polymer sheets with features generated by laser micromachining and by bonding the sheets together with adhesive. A six layer ELISA-LOC was fabricated with an acrylic (poly(methyl methacrylate) (PMMA)) core and five polycarbonate layers micromachined by a CO(2) laser with simple microfluidic features including a miniature 96-well sample plate. Immunological assays can be carried out in several configurations (1 × 96 wells, 2 × 48 wells, or 4 × 24 wells). The system includes three main functional elements: (1) a reagent loading fluidics module, (2) an assay and detection wells plate, and (3) a reagent removal fluidics module. The ELISA-LOC system combines several biosensing elements: (1) carbon nanotube (CNT) technology to enhance primary antibody immobilization, (2) sensitive ECL (electrochemiluminescence) detection, and (3) a charge-coupled device (CCD) detector for measuring the light signal generated by ECL. Using a sandwich ELISA assay, the system detected Staphylococcal enterotoxin B (SEB) at concentrations as low as 0.1 ng/ml, a detection level similar to that reported for conventional ELISA. ELISA-LOC can be operated by a syringe and does not require power for operation. This simple point-of-care (POC) system is useful for carrying out various immunological assays and other complex medical assays without the laboratory required for conventional ELISA, and therefore may be more useful for global healthcare delivery. PMID:23329460

  12. Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine*

    PubMed Central

    Wu, Jian-xiang; Zhang, Shao-en; Zhou, Xue-ping

    2010-01-01

    A protein conjugate of streptomycin (streptomycin-bovine serum albumin (BSA) conjugate) was prepared and used as immunogen to produce monoclonal antibodies (MAb). One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentration (IC50) value of 4.65 ng/ml and the IC20 value of 0.21 ng/ml in phosphate buffered saline (PBS). At optimum conditions, an indirect competitive enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based immunochromatographic assay (CGIA) were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/ml were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum detection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples. PMID:20043352

  13. 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.

  14. 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.

  15. An evaluation study of enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 for detection of antibody against Bartonella bacilliformis infection among the Peruvian population.

    PubMed

    Angkasekwinai, Nasikarn; Atkins, Erin H; Romero, Sofia; Grieco, John; Chao, Chien Chung; Ching, Wei Mei

    2014-04-01

    Reliable laboratory testing is of great importance to detect Bartonella bacilliformis infection. We evaluated the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 (rPap31) for the detection of antibodies against B. bacilliformis as compared with immunofluorescent assay (IFA). Of the 302 sera collected between 1997 and 2000 among an at-risk Peruvian population, 103 and 34 samples tested positive for IFA-immunoglobulin G (IgG) and IFA-IgM, respectively. By using Youden's index, the cutoff values of ELISA-IgG at 0.915 gave a sensitivity of 84.5% and specificity of 94%. The cutoff values of ELISA-IgM at 0.634 gave a sensitivity of 88.2% and specificity of 85.1%. Using latent class analysis, estimates of sensitivity and specificity of almost all the assays were slightly higher than those of a conventional method of calculation. The test is proved beneficial for discriminating between infected and non-infected individuals with the advantage of low-cost and high-throughput capability.

  16. 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

  17. An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein.

    PubMed

    Encinas, P; Gomez-Casado, E; Estepa, A; Coll, J M

    2011-09-01

    An enzyme linked immunosorbent assay (ELISA) method to study serum antibodies to viral haemorrhagic septicemia virus (VHSV) was designed by using recombinant fragments of their G protein. By using this fragment-ELISA, we describe the binding of antibodies against recombinant G fragments of 45-445 amino acids present in VHSV-hyperimmunized trout sera. Fragments were designed by taking into account their tridimensional pH-dependent structure and functional domains. Sera were obtained from hyperimmunized trout following 4-5 intraperitoneal injections of VHSV antigens by using Freund's or saponin adjuvants. Sera from different hyperimmunized trout differed quantitatively rather than qualitatively in their recognition of solid-phase frg11 (56-110), frg12 (65-109), frg13 (97-167), frg14 (141-214), frg15 (65-250), frg16 (252-450) and G (G21-465) by Western blot and ELISA. However, titres were higher when using frg11, frg15 or frg16, rather than G21-465, suggesting higher accessibility to G epitopes. Further knowledge of the antigenicity of the G protein of rhabdoviruses by using fragments might be used to improve current vaccines. On the other hand, they might be used to dissect the trout antibody response to VHSV infections, to complement in vitro neutralizing assays, and/or to quantitate anti-VHSV antibodies in VHSV-infected/vaccinated trout, other fish and/or other body fluids such as mucus.

  18. Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry.

    PubMed

    Pradhan, Sunil K; Kamble, Nitin M; Pillai, Aravind S; Gaikwad, Satish S; Khulape, Sagar A; Reddy, M R; Mohan, C Madhan; Kataria, Jag Mohan; Dey, Sohini

    2014-12-01

    Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

  19. Antibody-capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients.

    PubMed

    Bundo, K; Igarashi, A

    1985-05-01

    Immunoglobulin M (IgM) antibody titers in paired sera from 19 encephalitis and 44 dengue hemorrhagic fever (DHF) patients in Thailand and 42 Japanese encephalitis (JE) patients in Japan were measured by the antibody capture ELISA and applied to distinguish JE virus infection from dengue virus infection. Titer distribution and the ratio of the titers against JE and dengue antigens led to the following diagnostic criteria. The specimens can be considered as positive with JE when IgM-ELISA titer showed over 200 against JE and 4-fold or more higher than titers against any types of dengue antigens. The specimens can be considered as positive with dengue infection when IgM ELISA titer showed over 200 against one of the 4 types of dengue antigens and 4-fold or more higher than against JE antigen. Based on these criteria, 41 of 42 patients in Japan and 11 of 19 encephalitis patients in Thailand could be diagnosed as having JE virus infection while 2 of 19 encephalitis patients in Thailand and 26 of 44 DHF patients in Thailand could be diagnosed as having dengue virus infections.

  20. Limits of diagnostic accuracy of anti-hepatitis C virus antibodies detection by ELISA and immunoblot assay.

    PubMed

    Suslov, Anatoly P; Kuzin, Stanislav N; Golosova, Tatiana V; Shalunova, Nina V; Malyshev, Nikolai A; Sadikova, Natalia V; Vavilova, Lubov M; Somova, Anna V; Musina, Elena E; Ivanova, Maria V; Kipor, Tatiana T; Timonin, Igor M; Kuzina, Lubov E; Godkov, Mihail A; Bajenov, Alexei I; Nesterenko, Vladimir G

    2002-07-01

    When human sera samples are tested for anti-hepatitis C virus (HCV) antibodies using different ELISA kits as well as immunoblot assay kits discrepant results often occur. As a result the diagnostics of HCV infection in such sera remains unclear. The purpose of this investigation is to define the limits of HCV serodiagnostics. Overall 7 different test kits of domestic and foreign manufacturers were used for the sampled sera testing. Preliminary comparative study, using seroconversion panels PHV905, PHV907, PHV908 was performed and reference kit was chosen (Murex anti-HCV version 4) as the most sensitive kit on the base of this study results. Overall 1640 sera samples have been screened using different anti-HCV ELISA kits and 667 of them gave discrepant results in at least two kits. These sera were then tested using three anti-HCV ELISA kits (first set of 377 samples) or four anti-HCV ELISA kits (second set of 290 samples) at the conditions of reference laboratory. In the first set 17.2% samples remained discrepant and in the second set - 13.4%. "Discrepant" sera were further tested in RIBA 3.0 and INNO-LIA immunoblot confirmatory assays, but approximately 5-7% of them remained undetermined after all the tests. For the samples with signal-to-cutoff ratio higher than 3.0 high rate of result consistency by reference, ELISA routing and INNO-LIA immunoblot assay was observed. On the other hand the results of tests 27 "problematic" sera in RIBA 3.0 and INNO-LIA were consistent only in 55.5% cases. Analysis of the antigen spectrum reactive with antibodies in "problematic" sera, demonstrated predominance of Core, NS3 and NS4 antigens for sera, positive in RIBA 3.0 and Core and NS3 antigens for sera, positive in INNO-LIA. To overcome the problem of undetermined sera, methods based on other principles, as well as alternative criteria of HCV infection diagnostics are discussed.

  1. 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

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

    PubMed Central

    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

  3. Detection of avian leukosis virus antigens by the ELISA and its use for detecting infectious virus after cultivation of samples and partial characterization of specific pathogen-free chicken lines maintained in this laboratory.

    PubMed

    Tsukamoto, K; Hihara, H; Kono, Y

    1991-06-01

    An enzyme-linked immunosorbent assay (ELISA) for detecting avian leukosis virus (ALV) antigens was developed with rabbit anti-ALV serum. The ELISA detected purified ALV of subgroups A and B at a concentration of 0.4 ng/well and about 10(3) infectious units/well estimated by a resistance-inducing factor (RIF) test, and antigens in culture fluids from chicken embryo fibroblasts infected with subgroups A, B or E of ALV. These results showed that common antigens among the subgroups were detected by the ELISA. When virus titration was performed, virus infectivity could be determined by the ELISA within 7 days after cultivation. The titer was similar to that obtained by the RIF test on 19 days after 3 subcultures. These results indicate that the ALV-isolation test by the ELISA was superior to the RIF test in rapidity and applicability to large-scale field trials. Four specific pathogen-free (SPF) chicken lines maintained in this laboratory were examined for endogenous ALV antigens by the ELISA. Sera from laying hens had considerably high absorbance (A) values, whereas albumen samples showed low A values except for some samples (7/40 hens). Although most of sera from 1-day-old SPF chicks showed lower A values than those from laying hens, some sera showed A values as high as those from viremic chicks in 2 lines. Endogenous ALV was isolated from sera from laying hens (6/40) and their albumens (4/7) with high A values. Two SPF chicken lines were found to produce endogenous virus at a high frequency.

  4. Application of a recombinant Fab fragment from a phage display library for sensitive detection of a target antigen by an inhibition ELISA system.

    PubMed

    Itoh, K; Suzuki, K; Ishiwata, S; Tezuka, T; Mizugaki, M; Suzuki, T

    1999-02-01

    We have found that the recombinant Fab (rFab) produced by phage display system was detectable for a target antigen more sensitive than the parental monoclonal antibody (MoAb). The Fab phage display library was constructed from hybridoma cells producing APU-6 MoAb specific for a modified nucleoside, pseudouridine that have been studied as a urinary marker for malignancy. Fab-displayed phage clones were screened by a direct ELISA, and the single positive clone was finally obtained. Although the reaction pattern of rFab against pseudouridine and uridine was almost identical to that of MoAb, detection sensitivity of rFab was approximately 30 times higher than that of MoAb. Since the sensitivity of rFab was almost identical to that of Fab fragment prepared by papain digestion of MoAb, the increased sensitivity is considered to be the nature of Fab fragment. The sensitivity of established assay system was sufficient for quantitative determination of serum pseudouridine levels in healthy individuals and cancer patients. This procedure may be applicable for improvement of detection sensitivity of a MoAb-based inhibition ELISA system for drugs or low molecular weight compounds.

  5. Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods.

    PubMed

    Stephan, Oliver; Vieths, Stefan

    2004-06-16

    Hidden allergens in food products are, especially for peanut-allergic consumers, a serious problem because even low amounts (approximately 200 microg) of peanut can elicit allergic reactions. Undeclared peanut traces can be found in processed food products, because contaminations with peanut during production processes are frequent. To minimize the risk of such cross-contaminations, it is necessary to develop sensitive analytical methods for the detection of hidden allergens in foods. For this approach we developed two peanut-specific assays based on the detection of peanut protein by specific antibodies (sandwich ELISA) and by the detection of peanut-specific DNA (part of the coding region of Ara h 2) by a real-time PCR. Both tests did not show any cross-reactivity with 22 common food ingredients (cereals, nuts, legumes), and the limit of detection is <10 ppm peanut in processed foods. Thirty-three random samples of food products were tested for the presence of peanut to compare both assay types with each other and to evaluate the percentage of foods on the German market that are contaminated with peanut traces. We found that four products (13.3%) without peanut in the list of ingredients contained peanut protein in a range from 1 to 74 ppm peanut protein and that the results of both tests correlated well. The real-time PCR was able to detect one more positive sample than the sandwich ELISA. In conclusion, both assays are sensitive and specific tools for the detection of hidden allergens in processed foods.

  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.

  7. Serologic detection of bluetongue virus infection of black-tailed deer: comparison of serum neutralization, agar gel immunodiffusion, and competitive ELISA assays.

    PubMed

    Patton, J F; Work, T M; Jessup, D A; Hietala, S K; Oliver, M N; Maclachlan, N J

    1994-01-01

    Three adult black-tailed deer (Odocoileus hemionus columbianus) and four fawns were inoculated with bluetongue virus (BTV) serotype 10 or 17, or epizootic hemorrhagic disease virus (EHDV) serotype 1. Animals were bled at irregular intervals thereafter and the presence of virus-specific antibodies in serum determined by agar gel immunodiffusion (AGID), serum neutralization (SN) and competitive enzyme-linked immunosorbent assay (C-ELISA) tests. Serum antibodies to BTV were detected in all three tests for 692 days after inoculation (DAI) of adult deer, but both the SN and AGID tests gave either erroneous or misleading results. Serum from one deer was negative by the AGID test at 409 DAI with BTV-10 but was positive at 248 and 692 DAI; also one adult and one fawn had antibodies by the SN test to serotypes of BTV with which they were not inoculated. The AGID test for EHDV had false positive results with some sera from animals inoculated only with BTV, and it consistently had false negative results with serum samples collected from an EHDV-inoculated deer at 140 DAI and thereafter. The C-ELISA was the most useful test for the detection of antibodies to BTV because it rapidly gave quantitative and accurate results.

  8. Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin.

    PubMed

    Paudel, Madan Kumar; Putalun, Waraporn; Sritularak, Boonchoo; Morinaga, Osamu; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-09-01

    A colloidal gold conjugated anti-baicalin monoclonal antibody (anti-BA MAb) was prepared and used in an immunochromatographic assay (ICA) for BA in Scutellariae Radix and Kampo medicines. This competitive ICA uses an anti-BA MAb which shows a high specificity for BA and baicalein. Its advantages include a short assay time (15 min), no dependence on any instrumental systems, and it can detect BA in plant materials and Kampo medicines. The limit of detection for the ICA was found to be around 0.6 μg mL(-1)of baicalin. Moreover, the usefulness of the combination of indirect competitive ELISA and the ICA using anti-BA MAb as a quality control method was confirmed for analysis of BA in Scutellariae Radix and Kampo medicines with a sufficient sensitivity (200 ng mL(-1) to 2 μg mL(-1)), obtainable in an easy and timely manner.

  9. 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

  10. Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study

    PubMed Central

    Kashyap, Rajpal S; Rajan, Anju N; Ramteke, Sonali S; Agrawal, Vijay S; Kelkar, Sanjivani S; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F

    2007-01-01

    Background Diagnosis of tuberculosis (TB) remains problematic despite many new advanced diagnostic methods. A reliable and rapid diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnoses of TB. In the present study we describe a prospective evaluation for demonstrating Antigen (Ag) 85 complex in the sera from TB patients. Methods Indirect ELISA, employing monoclonal antibodies (mAb) against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in sera from TB patients. Serum samples were obtained from 197 different groups of patients: confirmed TB {n = 24}, clinically diagnosed TB {n = 104}, disease controls {n = 49} and healthy controls {n = 20}. Receiver operating curve (ROC) was used to calculate the cut off value and comparison between TB and non-TB groups were done by the chi-square test. Results The indirect ELISA method, using an mAb against Ag 85 complex, yielded 82% sensitivity (95% confidence interval [CI] 67 to 93%) and 86% specificity (95% CI, 57 to 98%) for the diagnosis of TB. The serum positivities for Ag 85 complex in cases of confirmed and clinically diagnosed TB patients were 96% (23/24) and 79% (82/104) respectively, while the positivity for patients in the non-tuberculosis group was 14% (10/69). Conclusion The detection of Ag 85 complex in sera from TB patients by indirect ELISA using mAb against purified Ag 85 complex gives a reliable diagnosis and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity. PMID:17620147

  11. The increasing prevalence of Endemic Typhus in Kuala Lumpur and an evaluation of a diagnostic ELISA dot test for the detection of antibodies to Rickettsia typhi.

    PubMed

    Sekhar, W Y; Devi, S

    2000-05-01

    A seroepidemiology study was done in response to the recent increase of Endemic Typhus cases diagnosed at University Hospital. The serosurvey was based on doctors' request for the Weil Felix (WF) or the Indirect Immunoperoxidase (IIP) test in Pyrexia of Unknown Origin (PUO) patients for the years 1991 to 1997. Over the 7 years, we found that the incidence of Endemic typhus is increasing with gender (male:female = 2:1), age (20-40 years) and race distribution (Indians > Malay > Chinese) that reflects socioeconomic circumstances. A commercially available ELISA dot assay [INDX (E2R3) Dip-S-Ticks], for the detection of antibodies against R. typhi was compared with the indirect immunoperoxidase test (IIP). The ELISA assay was done against 219 IIP tested sera. The Dip-S-Ticks was found to be comparable to the IIP with a sensitivity of 91.7% and specificity of 92.8% at cut-off titres of > 1:80 IIP.

  12. Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

    PubMed

    Wang, Qian; Ju, Huanyu; Li, Yanwei; Jing, Zhiqiang; Guo, Lu; Zhao, Yu; Ma, Bo; Gao, Mingchun; Zhang, Wenlong; Wang, Junwei

    2014-12-01

    An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV.

  13. Application of an ELISA-type screen printed electrode-based potentiometric assay to the detection of Cryptosporidium parvum oocysts.

    PubMed

    Laczka, Olivier; Skillman, Lucy; Ditcham, William G; Hamdorf, Brenton; Wong, Danny K Y; Bergquist, Peter; Sunna, Anwar

    2013-11-01

    We report a novel electrochemical method for the rapid detection of the parasitic protozoan, Cryptosporidium parvum. An antibody-based capture format was transferred onto screen-printed electrodes and the presence of horseradish peroxidase-labelled antibodies binding to the oocysts was potentiometrically detected. This method allowed the detection of 5 × 10(2)Cryptosporidium oocysts per mL in 60 min.

  14. Development and validation of an ELISA kit (YF MAC-HD) to detect IgM to yellow fever virus.

    PubMed

    Basile, Alison Jane; Goodman, Christin; Horiuchi, Kalanthe; Laven, Janeen; Panella, Amanda J; Kosoy, Olga; Lanciotti, Robert S; Johnson, Barbara W

    2015-12-01

    Yellow fever virus (YFV) is endemic in tropical and sub-tropical regions of the world, with around 180,000 human infections a year occurring in Africa. Serologic testing is the chief laboratory diagnostic means of identifying an outbreak and to inform the decision to commence a vaccination campaign. The World Health Organization disseminates the reagents for YFV testing to African reference laboratories, and the US Centers for Disease Control and Prevention (CDC) is charged with producing and providing these reagents. The CDC M-antibody capture ELISA is a 2-day test, requiring titration of reagents when new lots are received, which leads to inconsistency in testing and wastage of material. Here we describe the development of a kit-based assay (YF MAC-HD) based upon the CDC method, that is completed in approximately 3.5h, with equivocal samples being reflexed to an overnight protocol. The kit exhibits >90% accuracy when compared to the 2-day test. The kits were designed for use with a minimum of equipment and are stored at 4°C, removing the need for freezing capacity. This kit is capable of tolerating temporary sub-optimal storage conditions which will ease shipping or power outage concerns, and a shelf life of >6 months was demonstrated with no deterioration in accuracy. All reagents necessary to run the YF MAC-HD are included in the kit and are single-use, with 8 or 24 sample options per kit. Field trials are envisioned for the near future, which will enable refinement of the method. The use of the YF MAC-HD is anticipated to reduce materials wastage, and improve the quality and consistency of YFV serologic testing in endemic areas.

  15. Development and validation of an ELISA kit (YF MAC-HD) to detect IgM to yellow fever virus.

    PubMed

    Basile, Alison Jane; Goodman, Christin; Horiuchi, Kalanthe; Laven, Janeen; Panella, Amanda J; Kosoy, Olga; Lanciotti, Robert S; Johnson, Barbara W

    2015-12-01

    Yellow fever virus (YFV) is endemic in tropical and sub-tropical regions of the world, with around 180,000 human infections a year occurring in Africa. Serologic testing is the chief laboratory diagnostic means of identifying an outbreak and to inform the decision to commence a vaccination campaign. The World Health Organization disseminates the reagents for YFV testing to African reference laboratories, and the US Centers for Disease Control and Prevention (CDC) is charged with producing and providing these reagents. The CDC M-antibody capture ELISA is a 2-day test, requiring titration of reagents when new lots are received, which leads to inconsistency in testing and wastage of material. Here we describe the development of a kit-based assay (YF MAC-HD) based upon the CDC method, that is completed in approximately 3.5h, with equivocal samples being reflexed to an overnight protocol. The kit exhibits >90% accuracy when compared to the 2-day test. The kits were designed for use with a minimum of equipment and are stored at 4°C, removing the need for freezing capacity. This kit is capable of tolerating temporary sub-optimal storage conditions which will ease shipping or power outage concerns, and a shelf life of >6 months was demonstrated with no deterioration in accuracy. All reagents necessary to run the YF MAC-HD are included in the kit and are single-use, with 8 or 24 sample options per kit. Field trials are envisioned for the near future, which will enable refinement of the method. The use of the YF MAC-HD is anticipated to reduce materials wastage, and improve the quality and consistency of YFV serologic testing in endemic areas. PMID:26342907

  16. [Design of a curve model for the detection of human immunoglobulin G against herpes simplex virus in ultramicroELISA from a single serum dilution].

    PubMed

    Angeles Ribas, M A; Otero, A; Alvarez, M; Marrero, M; Vázquez, S

    1994-01-01

    The use of a curve model in the application of a ultramicroELISA technique for the detection of human immunoglobulin G against herpes simplex virus is reported. Based on end point titration (linear regression) of 100 initial sera, 51 of these, with r2 > 0.98, were selected, and 4 curve models were elaborated to relate the natural fluorescence logarithm for 4 dilutions with the previously determined natural end point titration logarithm. The curve corresponding to the 1:40 (r2 = 0.9645) was selected to evaluate 39 additional serum samples whose approximate titration was known through the graphic design of the 4 dilutions. An 89% coincidence was found regarding the latter. Divergence of the other 11% was due to the lack of accuracy of the graphic method in the points near the cutline.

  17. Development and use of a biotinylated 3ABC recombinant protein in a solid-phase competitive ELISA for the detection of antibodies against foot-and-mouth disease virus.

    PubMed

    Clavijo, Alfonso; Zhou, En-Min; Hole, Kate; Galic, Boris; Kitching, Paul

    2004-09-15

    A biotinylated 3ABC recombinant protein was developed and used in a competitive ELISA (cELISA) to detect foot-and-mouth disease virus (FMDV) antibodies in cattle, sheep and pigs. In this report, we describe the cloning and expression of 3ABC protein in Escherichia coli cells as fusion protein with 6xHis and biotin. This cELISA uses streptavidin to capture bacterially expressed and in vivo biotinylated 3ABC antigen. The antigen capture strategy provides a simple and reliable method, which does not require purification of recombinant antigen before the serological assay. An hyperimmune guinea pig antiserum produced against purified 6xHis-3ABC was used as competitor in the test. The potential use of this cELISA for the identification of antibodies induced by FMD virus infection from those induced by vaccination is discussed. PMID:15288965

  18. An ELISA-on-a-chip biosensor system coupled with immunomagnetic separation for the detection of Vibrio parahaemolyticus within a single working day.

    PubMed

    Seo, Sung-Min; Cho, Il-Hoon; Jeon, Jin-Woo; Cho, Hyun-Kyu; Oh, Eun-Gyoung; Yu, Hong-Sik; Shin, Soon-Bum; Lee, Hee-Jung; Paek, Se-Hwan

    2010-08-01

    In this study, we constructed a rapid detection system for a foodborne pathogen, Vibrio parahaemolyticus, by using enzyme-linked immunosorbent assay (ELISA)-on-a-chip (EOC) biosensor technology to minimize the risk of infection by the microorganism. The EOC results showed a detection capability of approximately 6.2x10(5) cells per ml, which was significantly higher than that of the conventional rapid test kit. However, this high level of sensitivity required cultivation of the pathogen prior to analysis, which typically exceeded a day. To shorten the test period, we combined the EOC technology with immunomagnetic separation (IMS), which could enhance the sensitivity of the biosensor. IMS was carried out with magnetic particles coated with a monoclonal antibody specific to the microbe. To test the performance of the IMS-EOC method, fish intestine samples were prepared by artificially inoculating less than 1 or 5 CFU/10 g, allowing for enrichment over predetermined times, and analyzing the sample by using the EOC sensor after concentrating the culture 86-fold via IMS. Using this approach, the bacterium was detected after (at most) 9 h, which approximately corresponds to standard working hours. Thus, the IMS-EOC method allowed for the rapid detection of V. parahaemolyticus, which is responsible for foodborne diseases, and this method could be used for early isolation of contaminated foods before distribution.

  19. Use of monoclonal antibodies in an ELISA to detect IgM class antibodies specific for Toxoplasma gondii.

    PubMed Central

    Balfour, A H; Harford, J P; Goodall, M

    1987-01-01

    Two monoclonal antibodies CH6 and C1E3 were used in an antibody class capture assay for the detection of IgM antibodies specific for Toxoplasma gondii. CH6 was used on the solid phase to capture human IgM. After a Toxoplasma gondii antigen had been added, specifically bound material was detected using C1E3 coupled to horseradish peroxidase. The assay was compared with an established system using polyclonal antisera at both the capture and antigen detection stages. A good correlation was found, with 97.3% (125 of 128) of sera giving the same classification in both assays. Three sera were positive only in the polyclonal system. No false positive results were found when 118 negative sera were examined. The two monoclonal antibodies provide a viable alternative to the use of polyclonal sera at the capture and antigen detection stages in the antibody class capture assay for the measurement of specific IgM against T gondii. PMID:3654986

  20. Rapid detection of S. mutans surface antigen I/II using a sensitive monoclonal anti-Ag I/II antibody by ELISA.

    PubMed

    Kim, Mi-Ah; Jeon, Hyun-Soon; Shin, Se-Young; Baik, Byeong-Ju; Yang, Yeon-Mi; Lee, Kyung-Yeol; Kim, Jae-Gon

    2013-10-01

    The cell-surface protein antigen I/II (Ag I/II) is expressed in oral streptococci, which are known as the causative agent of a number of diseases including dental caries, endocarditis, gingivitis, and periodontal disease. Consequently, monoclonal antibodies (MAb) capable of recognizing the streptococcal Ag I/II protein could be a useful tool for the diagnosis and cure of these diseases. In this study, a previously generated monoclonal anti-Ag I/II antibody, ckAg I/II, was used to detect a small amount of Streptococcus mutans (S. mutans) surface antigen Ag I/II. The ckAg I/II was proved to be very sensitive and able to detect as little as 1 ng of recombinant Ag I/II protein within 5 min and Ag I/II in saliva within 10 min, as well as native Ag I/II in 20 μL of culture supernatant by ELISA. These results suggest that ckAg I/II can be used as a fast and efficient diagnostic tool to detect Ag I/II. PMID:24111865

  1. Comparison of six commercial ELISA kits for their specificity and sensitivity in detecting different major peanut allergens.

    PubMed

    Jayasena, Shyamali; Smits, Mieke; Fiechter, Daniëlle; de Jong, Aard; Nordlee, Julie; Baumert, Joe; Taylor, Steve L; Pieters, Raymond H; Koppelman, Stef J

    2015-02-18

    Six commercial peanut enzyme-linked immunosorbent assay kits were assessed for their ability to recover peanut from the standard reference material 2387 peanut butter and also for their specificity in detecting four major peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6. The percentage recovery of peanut from peanut butter differed across different kits as well as at different sample concentrations. The highest recovery was observed with the Romer and R-Biopharm kits, while four other kits were found to underestimate the protein content of the reference peanut butter samples. Five of the kits were most sensitive in detecting Ara h 3 followed by Ara h 1, while hardly recognizing Ara h 2 and Ara h 6. The other kit showed the highest sensitivity to Ara h 2 and Ara h 6, while Ara h 1 and Ara h 3 were poorly recognized. Although Ara h 2 and Ara h 6 are known to be heat stable and more potent allergens, antisera specific to any of these four peanut proteins/allergens may serve as good markers for the detection of peanut residues.

  2. Comparison of six commercial ELISA kits for their specificity and sensitivity in detecting different major peanut allergens.

    PubMed

    Jayasena, Shyamali; Smits, Mieke; Fiechter, Daniëlle; de Jong, Aard; Nordlee, Julie; Baumert, Joe; Taylor, Steve L; Pieters, Raymond H; Koppelman, Stef J

    2015-02-18

    Six commercial peanut enzyme-linked immunosorbent assay kits were assessed for their ability to recover peanut from the standard reference material 2387 peanut butter and also for their specificity in detecting four major peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6. The percentage recovery of peanut from peanut butter differed across different kits as well as at different sample concentrations. The highest recovery was observed with the Romer and R-Biopharm kits, while four other kits were found to underestimate the protein content of the reference peanut butter samples. Five of the kits were most sensitive in detecting Ara h 3 followed by Ara h 1, while hardly recognizing Ara h 2 and Ara h 6. The other kit showed the highest sensitivity to Ara h 2 and Ara h 6, while Ara h 1 and Ara h 3 were poorly recognized. Although Ara h 2 and Ara h 6 are known to be heat stable and more potent allergens, antisera specific to any of these four peanut proteins/allergens may serve as good markers for the detection of peanut residues. PMID:25651402

  3. Use of Monoclonal Antibodies in an ELISA for Detecting an Invasive Pest Insect, Liriomyza trifolii (Diptera: Agromyzidae).

    PubMed

    Zhao, W C; Shang, H W; Guo, W; Xu, D; Huang, T Y; Zhu, L X

    2015-04-01

    A monoclonal antibody was prepared by the hybridoma technology. It reacted only with the protein of Liriomyza trifolii (Burgess) and not with that of Chromatomyia horticola Goureau or Liriomyza sativae Blanchard in indirect enzyme-linked immunosorbent assay. It was effective even after being diluted more than 8.192×10(6)-fold. The detection sensitivity of the antibody was 31.3 µg/ml under controlled conditions. Positive reaction was achieved with all laboratory-reared L. trifolii samples, including larvae, pupae, and adults. An indirect enzyme-linked immunosorbent assay system was successfully established to detect L. trifolii in the field. This antibody was successfully used to determine the L. trifolii collected in different locations, from different host plants, and in different seasons. More than 50% of leafminers collected on Brassica chinensis var chinensis, Apium graveolens (Miller) Persoon, Vigna unguiculata (L.) Walpers, Phaseolus vulgaris L., Lactuca sativa L., and Chrysanthemum coronarium (L.) Cassini ex Spach were L. trifolii, indicating that those six plant species might be the preference host plants of L. trifolii. Population of L. trifolii peaked in September, October, or November in Hangzhou, Zhejiang. These results suggest a great potential of using this McAb for precisely identifying L. trifolii and monitoring the population dynamics of L. trifolii in the field. PMID:26470159

  4. A new ELISA kit which uses a combination of Plasmodium falciparum extract and recombinant Plasmodium vivax antigens as an alternative to IFAT for detection of malaria antibodies

    PubMed Central

    Doderer, Cecile; Heschung, Aurelie; Guntz, Phillippe; Cazenave, Jean-Pierre; Hansmann, Yves; Senegas, Alexandre; Pfaff, Alexander W; Abdelrahman, Tamer; Candolfi, Ermanno

    2007-01-01

    Background The methods most commonly used to measure malarial antibody titres are the Indirect Fluorescence Antibody Test (IFAT), regarded as the gold standard, and the Enzyme-Linked ImmunoSorbent Assay (ELISA). The objective here was to assess the diagnostic performance, i.e. the sensitivity and specificity, of a new malaria antibody ELISA kit in comparison to IFAT. This new ELISA kit, the ELISA malaria antibody test (DiaMed), uses a combination of crude soluble Plasmodium falciparum extract and recombinant Plasmodium vivax antigens. Methods Two groups were used: 95 samples from malaria patients to assess the clinical sensitivity and 2,152 samples from blood donors, who had not been exposed to malaria, to assess the clinical specificity. Results The DiaMed ELISA test kit had a clinical sensitivity of 84.2% and a clinical specificity of 99.6% as compared with 70.5% and 99.6% respectively, using the IFAT method. The ELISA method was more sensitive than the IFAT method for P. vivax infections (75% vs. 25%). However, in 923 malaria risk donors the analytical sensitivity of the ELISA test was 40% and its specificity 98.3%, performances impaired by large numbers of equivocal results non-concordant between ELISA and IFAT. When the overall analytical performances of ELISA was compared to IFAT, the ELISA efficiency J index was 0.84 versus 0.71 for IFAT. Overall analytical sensitivity was 93.1% and the analytical specificity 96.7%. Overall agreement between the two methods reached 0.97 with a reliability k index of 0.64. Conclusion The DiaMed ELISA test kit shows a good correlation with IFAT for analytical and clinical parameters. It may be an interesting method to replace the IFAT especially in blood banks, but further extensive investigations are needed to examine the analytical performance of the assay, especially in a blood bank setting. PMID:17313669

  5. Evaluation of the solid phase competition ELISA for detecting antibodies against the six foot-and-mouth disease virus non-O serotypes.

    PubMed

    Li, Yanmin; Swabey, Kate G; Gibson, Debi; Keel, Phil J; Hamblin, Pip; Wilsden, Ginette; Corteyn, Mandy; Ferris, Nigel P

    2012-08-01

    The solid-phase competition ELISA (SPCE) has been evaluated in both screening and titration assay formats for detecting antibodies against foot-and-mouth disease virus (FMDV) for the six non-O serotypes A, C, SAT 1, SAT 2, SAT 3 and Asia 1. Cut-off values were determined as a percentage inhibition of 40 for the SAT serotypes and 50 for serotypes A, C and Asia 1, which gave rise to specificity values ranging from 99.41% to 99.9% for the different serotypes. The relative sensitivity between the SPCE and LPBE/virus neutralisation test was 100%/109%. Antiserum titres derived by the SPCE for samples of serotypes O, A(22) and Asia 1 were more than 11, 1 and 5 times of those determined by virus neutralisation test, respectively. This study indicated that the non-type O SPCEs have sufficient sensitivities and specificities for use as serological diagnostic tests for the qualitative and quantitative detection of antibodies against FMDV.

  6. Detection of Campylobacter antibodies in sheep sera by a Dot-ELISA using acid extracts from c. fetus ssp. fetus and c. jejuni strains and comparison with a complement fixation test.

    PubMed

    Gürtürk, K; Ekin, I H; Aksakal, A; Solmaz, H

    2002-04-01

    In this study, a dot-enzyme-linked immunosorbent assay (Dot-ELISA) was evaluated in comparison with a complement fixation test (CFT) for the detection of Campylobacter antibodies in sheep sera. Acid glycine extracts (AGE) of both Campylobacter fetus ssp. fetus and Campylobacter jejuni strains that had been isolated from the gall-bladder of slaughtered sheep was used as antigen in both tests. A total of 153 sheep sera from aborted (74) and slaughtered (79) sheep were examined by both Dot-ELISA and CFT. Twenty-two sera showed anti-complementary activity were not suitable for CFT. Of the 22 sera showing anti-complementary activity, two sera were found to be positive in Dot-ELISA. Eighty-eight (67.2%) of the remaining 131 sera were negative by both Dot-ELISA and CFT using AGE of both Campylobacter strains whereas 43 sera (32.8%) gave different reaction patterns in Dot-ELISA and CFT with the extracts of both Campylobacter strains. Twelve sera were positive by both tests using AGE of C. fetus ssp. fetus but CFT failed to detect antibodies in nine of these sera when AGE of C. jejuni was used. Twelve sera were positive by both tests only when AGE of C. fetus ssp. fetus was used. Eleven sera were positive only by CFT. Seven of these reacted only with the AGE of C. fetus ssp. fetus and four sera were positive by using AGE of both Campylobacter strains. The remaining eight sera were found to be positive only by dot-immunobinding assay either with the AGE of both Campylobacter strains or with the AGE of one of the Campylobacter strains. It is concluded that Dot-ELISA using AGE from C. fetus ssp. fetus could be employed for the detection of Campylobacter antibodies in sheep sera and the additional use of AGE from C. jejuni as antigen appeared not to be profitable for this purpose.

  7. Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera.

    PubMed

    Saenger, Thorsten; Braukmann, Achim; Vordenbäumen, Stefan; Altendorfer, Irina; Bleck, Ellen; Hochwallner, Heidrun; Valenta, Rudolf; Schneider, Matthias; Jose, Joachim

    2014-08-01

    The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method - in particular no need for time-consuming and expensive antigen production and purification - the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies. PMID:24747146

  8. Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera

    PubMed Central

    Saenger, Thorsten; Braukmann, Achim; Vordenbäumen, Stefan; Altendorfer, Irina; Bleck, Ellen; Hochwallner, Heidrun; Valenta, Rudolf; Schneider, Matthias; Jose, Joachim

    2015-01-01

    The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method – in particular no need for time-consuming and expensive antigen production and purification – the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies. PMID:24747146

  9. Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera.

    PubMed

    Saenger, Thorsten; Braukmann, Achim; Vordenbäumen, Stefan; Altendorfer, Irina; Bleck, Ellen; Hochwallner, Heidrun; Valenta, Rudolf; Schneider, Matthias; Jose, Joachim

    2014-08-01

    The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method - in particular no need for time-consuming and expensive antigen production and purification - the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies.

  10. Rapid detection of immunity against bacteria in Asian honeybee and Western honeybee with quantification of royalisin in the hemolymphe by fast ELISA.

    PubMed

    Shen, Li-Rong; Dilireba, Shatar; Zhou, Wen-Xiu; Wang, Yi-Ran; Li, Mei-Lu; Zhai, Liang

    2014-09-24

    Royalisin from royal jelly (RJ) is a valuable peptide both for the prevention of honeybee diseases and for RJ preservation. ELISA for fast determination of royalisin content in hemolymphe (RCH) of honeybees with polyclonal antibody against recombinant royalisin from Asian honeybee was established. Assay on RCHs of health samples from Asian honeybee and Western honeybee showed the former (7.06 μg/mL) was significantly higher than that of the latter (5.64 μg/mL, p < 0.01). Moreover, relative to the non infection, the RCHs of Asian honeybees at 24 and 48 h post infection of Eschericha coli were higher than those of Western honeybees by 32.90% and 29.66%, respectively. Evidence revealed that Asian honeybee possesses higher innate immunity and immune response against bacteria in relation to the Western honeybee. The method will be a potential tool for detection of resistant levels to pathogens in honeybees and for quantification of royalisin in RJ products. PMID:25183454

  11. Rapid detection of immunity against bacteria in Asian honeybee and Western honeybee with quantification of royalisin in the hemolymphe by fast ELISA.

    PubMed

    Shen, Li-Rong; Dilireba, Shatar; Zhou, Wen-Xiu; Wang, Yi-Ran; Li, Mei-Lu; Zhai, Liang

    2014-09-24

    Royalisin from royal jelly (RJ) is a valuable peptide both for the prevention of honeybee diseases and for RJ preservation. ELISA for fast determination of royalisin content in hemolymphe (RCH) of honeybees with polyclonal antibody against recombinant royalisin from Asian honeybee was established. Assay on RCHs of health samples from Asian honeybee and Western honeybee showed the former (7.06 μg/mL) was significantly higher than that of the latter (5.64 μg/mL, p < 0.01). Moreover, relative to the non infection, the RCHs of Asian honeybees at 24 and 48 h post infection of Eschericha coli were higher than those of Western honeybees by 32.90% and 29.66%, respectively. Evidence revealed that Asian honeybee possesses higher innate immunity and immune response against bacteria in relation to the Western honeybee. The method will be a potential tool for detection of resistant levels to pathogens in honeybees and for quantification of royalisin in RJ products.

  12. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

    PubMed

    Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

    2016-01-01

    The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.

  13. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

    PubMed

    Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

    2016-01-01

    The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings. PMID:26930597

  14. Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves

    PubMed Central

    Feng, Xia; Ma, Jun-Wu; Sun, Shi-Qi; Guo, Hui-Chen; Yang, Ya-Min; Jin, Ye; Zhou, Guang-Qing; He, Ji-Jun; Guo, Jian-Hong; Qi, Shu-yun; Lin, Mi; Cai, Hu; Liu, Xiang-Tao

    2016-01-01

    The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings. PMID:26930597

  15. Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures.

    PubMed

    Waters, Sinéad M; Doyle, Sean; Murphy, Richard A; Power, Ronan F G

    2005-12-01

    Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora. This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)-horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction. PMID:15949857

  16. Development and evaluation of two truncated recombinant NP antigen-based indirect ELISAs for detection of bovine parainfluenza virus type 3 antibodies in cattle.

    PubMed

    Yang, Yong; Wang, Feng-Xue; Sun, Na; Cao, Li; Zhang, Shu-Qin; Zhu, Hong-Wei; Guo, Li; Cheng, Shi-Peng; Wen, Yong-Jun

    2015-09-15

    Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory pathogens in both young and adult cattle. Nucleocapsid protein (NP) is the most abundant viral protein and the main regulator of virus replication and transcription. In this study, amino acid sequence data of BPIV3 NP was used to identify potential linear epitopic regions, which were subsequently used to design truncated recombinant NP antigens. The amino-terminal region (aa 9-157, NP-N) and the carboxy-terminal region (aa 391-500, NP-C) were selected, and these two truncated recombinant BPIV3 NP proteins were expressed in Escherichia coli based on the results of prediction studies. Furthermore, Enzyme-Linked Immunosorbent Assays (ELISAs) were established using the truncated recombinant BPIV3-N proteins as antigens, and 154 clinical samples were used to evaluate the newly established ELISA systems in comparison with a virus neutralisation test (VNT) as a reference. The results showed that a high coincidence rate was observed for the data that were obtained by the two methods. The sensitivity of NP-N ELISA and NP-C ELISA were 98.4% and 94.6%, respectively, and the specificity of both ELISAs was 100% with reference to the VNTs. Our data indicated that both ends of NP have high immunogenicity during BPIV3 infection and that they were good targets for serodiagnosis. The ELISAs based on the two truncated proteins were especially suitable for use in large-scale epidemiological investigations.

  17. The Dot Blot ELISA.

    ERIC Educational Resources Information Center

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  18. IgE ELISA using antisera derived from epsilon chain antigenic peptides detects allergen-specific IgE in allergic horses.

    PubMed

    Kalina, Warren V; Pettigrew, Howard D; Gershwin, Laurel J

    2003-05-12

    Equine disease with an allergic etiology is common. Environmental antigens most often implicated as allergens in horses include molds, dusty hay, grass pollen, hay dust mites, and insect saliva. Although intradermal testing with allergen is a useful diagnostic tool for some species, skin testing frequently produces false positive results in horses. Allergen deprivation as a diagnostic tool is often impossible and at best it is ineffective at diagnosing the specific allergic reactivity. Synthesis of IgE after exposure to allergen is the instigator of the allergic process. While IgE exerts its effect after binding strongly to mast cell Fc receptors, the presence of free IgE in the serum can be used to quantify and determine the allergen specificity of the allergic disease. A lack of widely available reagents for detection of equine IgE has limited this approach in horses. We have used the nucleotide sequence of equine IgE to prepare a peptide-based immunogen to elicit equine epsilon chain-specific antisera. Selection of peptides was based on antigenic attributes of the deduced amino acid sequence of the equine epsilon chain. Six peptides were selected for conjugation to carrier molecules and rabbit immunization. Of these, one peptide elicited antisera that was successfully used in enzyme linked immunosorbant assay (ELISA) to screen horse serum from 64 allergic horses for allergen-specific IgE. Twenty-four of the 64 horses showed positive reactivity to one or more of the following allergens: grass, grain mill dust, mosquito, and horsefly. This study demonstrates the usefulness of peptide-based immunogens for development of antisera to rare or difficult to purify antigens such as IgE. Resultant antisera has great usefulness in diagnostic assays for equine allergy and as a research tool. PMID:12730014

  19. Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

    PubMed

    Singh, Harpal; Shimojima, Masayuki; Fukushi, Shuetsu; Le Van, An; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

  20. Increased sensitivity of 3D-Well enzyme-linked immunosorbent assay (ELISA) for infectious disease detection using 3D-printing fabrication technology.

    PubMed

    Singh, Harpal; Shimojima, Masayuki; Fukushi, Shuetsu; Le Van, An; Sugamata, Masami; Yang, Ming

    2015-01-01

    Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines. PMID:26406036

  1. [Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents].

    PubMed

    Polat, Ceylan; Karataş, Ahmet; Sözen, Mustafa; Matur, Ferhat; Abacıoğlu, Hakan; Öktem, Mehmet Ali

    2016-04-01

    Hantaviruses infect humans via inhalation of viral particles in infected rodents' secretions such as saliva, urine and faeces or via direct contact with infected rodents. The rodent species that are known as the carriers of Dobrava (DOBV), Puumala (PUUV), Saaremaa (SAAV), Tula (TULV) and Seoul (SEOV) viruses are found in our country. The presence of specific antibodies against hantaviruses have been demonstrated in rodents collected from Black Sea and Aegean Regions of Turkey in 2004 for the first time. The first hantavirus-related hemorrhagic fever with renal syndrome (HFRS) cases were reported in Black Sea region in 2009. The determination of the hantavirus prevalence in wild life and rodent populations in the field is crucial for the information about hantavirus-related cases and to clarify the state of risk. There is no commercial product optimized for the screening of rodent serum samples in terms of HFRS agents like DOBV and PUUV that are widely seen in Eurasia as well as Turkey. In this study, the antigens belonging to the commercial enzyme-linked immunoassay (ELISA) and immunoblot tests that are produced for the screening of human sera were used for the development of antibody screening tests against hantavirus in rodent sera and were optimized. The most appropriate serum and conjugate dilutions were determined for the optimization of ELISA (Anti-Hantavirus Pool ELISA; Euroimmun, Germany) and immunoblot (Euroline Anti-Hanta Profile 1 strips; Euroimmun, Germany) methods. Optimized ELISA method was used for the screening and optimized immunoblot method was used for the confirmation. A total of 84 wild rodent sera that belonged to Apodemus and Microtus species were evaluated with this procedure and the cut-off value, sensitivity and specificity of optimized ELISA method were determined. For the optimization of ELISA 1/50, 1/100 and 1/200 serum dilutions and 1/10.000, 1/20.000 and 1/40.000 conjugate dilutions were tested. For the optimization of immunoblot, 1

  2. [Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents].

    PubMed

    Polat, Ceylan; Karataş, Ahmet; Sözen, Mustafa; Matur, Ferhat; Abacıoğlu, Hakan; Öktem, Mehmet Ali

    2016-04-01

    Hantaviruses infect humans via inhalation of viral particles in infected rodents' secretions such as saliva, urine and faeces or via direct contact with infected rodents. The rodent species that are known as the carriers of Dobrava (DOBV), Puumala (PUUV), Saaremaa (SAAV), Tula (TULV) and Seoul (SEOV) viruses are found in our country. The presence of specific antibodies against hantaviruses have been demonstrated in rodents collected from Black Sea and Aegean Regions of Turkey in 2004 for the first time. The first hantavirus-related hemorrhagic fever with renal syndrome (HFRS) cases were reported in Black Sea region in 2009. The determination of the hantavirus prevalence in wild life and rodent populations in the field is crucial for the information about hantavirus-related cases and to clarify the state of risk. There is no commercial product optimized for the screening of rodent serum samples in terms of HFRS agents like DOBV and PUUV that are widely seen in Eurasia as well as Turkey. In this study, the antigens belonging to the commercial enzyme-linked immunoassay (ELISA) and immunoblot tests that are produced for the screening of human sera were used for the development of antibody screening tests against hantavirus in rodent sera and were optimized. The most appropriate serum and conjugate dilutions were determined for the optimization of ELISA (Anti-Hantavirus Pool ELISA; Euroimmun, Germany) and immunoblot (Euroline Anti-Hanta Profile 1 strips; Euroimmun, Germany) methods. Optimized ELISA method was used for the screening and optimized immunoblot method was used for the confirmation. A total of 84 wild rodent sera that belonged to Apodemus and Microtus species were evaluated with this procedure and the cut-off value, sensitivity and specificity of optimized ELISA method were determined. For the optimization of ELISA 1/50, 1/100 and 1/200 serum dilutions and 1/10.000, 1/20.000 and 1/40.000 conjugate dilutions were tested. For the optimization of immunoblot, 1

  3. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

    PubMed

    Cao, Biyun; He, Guangzhao; Yang, Hong; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-10-15

    Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist illegally used as feed additives for growth promotion. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of PA in tissue and feed samples was developed and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). By reduction of nitryl group to amino group, the PA derivative was synthesized and coupled to carrier proteins with diazobenzidine method. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All antisera displayed high sensitivity with IC50 values lower than 0.48 ng mL(-1). The most sensitive ELISA was established with IC50 and limit of detection (LOD) values of 0.049 ng mL(-1) and 0.003 ng mL(-1), respectively. The cross-reactivity (CR) values of the antisera with three frequently used β-adrenergic agonists (clenbuterol, salbutamol and ractopamine) were lesser than 0.39%; there was no CR of the antisera with other six compounds including two structurally related substances (isoproterenol, phenylephrine). To investigate the accuracy and precision of the assay, swine kidney, liver, meat and feed samples were fortified with PA at different content and analyzed by ELISA. Acceptable recovery rates of 92.2-113.7% and intra-assay coefficients of variation of 3.8-10.9% (n=3) were achieved. Seven spiked samples were simultaneously analyzed by ELISA and LC-MS/MS. There was a high correlation coefficient of 0.9956 (n=7) between the two methods. The proposed ELISA proven to be a feasible quantitative/screening method for PA analysis in tissue and feed samples with the properties of high sensitivity and specificity, high sample throughput and low expensive.

  4. Detection of human leptospirosis as a cause of acute fever by capture ELISA using a Leptospira interrogans serovar Copenhageni (M20) derived antigen

    PubMed Central

    2013-01-01

    Background Leptospirosis is a potentially lethal zoonosis mainly affecting low-resource tropical countries, including Peru and its neighbouring countries. Timely diagnosis of leptospirosis is critical but may be challenging in the regions where it is most prevalent. The serodiagnostic gold standard microagglutination test (MAT) may be technically prohibitive. Our objective in this study was to assess the sensitivity, specificity, and predictive value of an IgM antibody capture enzyme-linked immunoassay (MAC-ELISA) derived from the M20 strain of Leptospira interrogans serovar Copenhageni (M20) by comparison to MAT, which was used as the gold standard method of diagnosis. Methods Acute and convalescent sera from participants participating in a passive febrile surveillance study in multiple regions of Peru were tested by both IgM MAC-ELISA and MAT. The sensitivity, specificity, positive and negative predictive value (PPV, NPV) of the MAC-ELISA assay for acute, convalescent and paired sera by comparison to MAT were calculated. Results The sensitivity, specificity, PPV and NPV of the MAC-ELISA assay for acute sera were 92.3%, 56.0%, 35.3% and 96.6% respectively. For convalescent sera, the sensitivity, specificity, PPV and NPV of the MAC-ELISA assay were 93.3%, 51.5%, 63.6% and 89.5% respectively. For paired sera, the sensitivity, specificity, PPV and NPV of the MAC-ELISA assay were 93.6%, 37.5%, 59.2%, 85.7% respectively. Conclusions The M20 MAC-ELISA assay performed with a high sensitivity and low specificity in the acute phase of illness. Sensitivity was similar as compared with MAT in the convalescent phase and specificity remained low. Paired sera were the most sensitive but least specific by comparison to MAT serodiagnosis. NPV for acute, convalescent and paired sera was high. The limited specificity and high sensitivity of the MAC-ELISA IgM suggests that it would be most valuable to exclude leptospirosis in low-resource regions that lack immediate access to

  5. Taguchi optimisation of ELISA procedures.

    PubMed

    Jeney, C; Dobay, O; Lengyel, A; Adám, E; Nász, I

    1999-03-01

    We propose a new method in the field of ELISA optimization using an experimental design called the Taguchi method. This can be used to compare the net effects of different conditions which can be both qualitative and quantitative in nature. The method reduces the effects of the interactions of the optimized variables making it possible to access the optimum conditions even in cases where there are large interactions between the variables of the assay. Furthermore, the proposed special assignment of factors makes it possible to calculate the biochemical parameters of the ELISA procedure carried out under optimum conditions. Thus, the calibration curve, the sensitivity of the optimum assay, the intra-assay and inter-assay variability can be estimated. The method is fast, accessing the results in one step, compared to the traditional, time-consuming 'one-step-at-a-time' method. We exemplify the procedure with a method to optimize the detection of ScFv (single chain fragment of variable) phages by ELISA. All the necessary calculations can be carried out by a spreadsheet program without any special statistical knowledge. PMID:10089092

  6. Development of an ELISA to detect circulating anti-asparaginase antibodies in dogs with lymphoid neoplasia treated with Escherichia coli l-asparaginase.

    PubMed

    Kidd, J A; Ross, P; Buntzman, A S; Hess, P R

    2015-06-01

    Resistance to Escherichia coli l-asparaginase in canine lymphoma occurs frequently with repeated administration, a phenomenon often attributed, without substantiation, to the induction of neutralizing antibodies. To test the hypothesis that treated dogs develop antibodies against the drug, we created an enzyme-linked immunosorbent assay (ELISA) to measure plasma anti-asparaginase immunoglobulin G responses. Using samples from dogs that had received multiple doses, specific reactivity against l-asparaginase was demonstrated, while naïve patients' samples were negative. The optimized ELISA appeared sensitive, with endpoint titers >1 600 000 in positive control dogs. Intra- and inter-assay coefficients of variation were 3.6 and 14.5%. The assay was supported by the observation that ELISA-positive plasma could immunoprecipitate asparaginase activity. When clinical patients were evaluated, 3/10 dogs developed titers after a single injection; with repeated administration, 4/7 dogs were positive. l-asparaginase antibodies showed reduced binding to the PEGylated drug formulation. The ELISA should prove useful in investigating the potential correlation of antibody responses with resistance.

  7. Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk

    PubMed Central

    Nia, Yacine; Rodriguez, Mélanie; Zeleny, Reinhard; Herbin, Sabine; Auvray, Frédéric; Fiebig, Uwe; Avondet, Marc-André; Munoz, Amalia; Hennekinne, Jacques-Antoine

    2016-01-01

    The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness. PMID:27649244

  8. Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk.

    PubMed

    Nia, Yacine; Rodriguez, Mélanie; Zeleny, Reinhard; Herbin, Sabine; Auvray, Frédéric; Fiebig, Uwe; Avondet, Marc-André; Munoz, Amalia; Hennekinne, Jacques-Antoine

    2016-01-01

    The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness. PMID:27649244

  9. Development of a new method for the determination of residues of the neonictinoid insecticide imidacloprid in juvenile Chinook (Oncorhynchus tyshawytscha) using ELISA detection

    USGS Publications Warehouse

    Frew, John A.; Grue, Christian E.

    2012-01-01

    The neonicotinoid insecticide imidacloprid (IMI) has been proposed as an alternative to carbaryl for controlling indigenous burrowing shrimp on commercial oyster beds in Willapa Bay and Grays Harbor, Washington. A focus of concern over the use of this insecticide in an aquatic environment is the potential for adverse effects from exposure to non-target species residing in the Bay, such as juvenile Chinook (Oncorhynchus tshawytscha) and cutthroat trout (O. clarki). Federal registration and State permiting approval for the use of IMI will require confirmation that the compound does not adversely impact these salmonids following field applications. This will necessitate an environmental monitoring program for evaluating exposure in salmonids following the treatment of beds. Quantification of IMI residues in tissue can be used for determining salmonid exposure to the insecticide. Refinement of an existing protocol using liquid-chromatography mass spectrometry (LC-MS) detection would provide the low limits of quantification, given the relatively small tissue sample sizes, necessary for determining exposure in individual fish. Such an approach would not be viable for the environmental monitoring effort in Willapa Bay and Grays Harbor due to the high costs associated with running multiple analyses, however. A new sample preparation protocol was developed for use with a commercially available enzyme-linked immunosorbent assay (ELISA) for the quantification of IMI, thereby providing a low-cost alternative to LC-MS for environmental monitoring in Willapa Bay and Grays Harbor. Extraction of the analyte from the salmonid brain tissue was achieved by Dounce homogenization in 4.0 mL of 20.0 mM Triton X-100, followed by a 6 h incubation at 50–55 °C. Centrifugal ultrafiltration and reversed phase solid phase extraction were used for sample cleanup. The limit of quantification for an average 77.0 mg whole brain sample was calculated at 18.2 μg kg-1 (ppb) with an average

  10. Validation of an indirect ELISA to detect antibodies against BoHV-1 in bovine and guinea-pig serum samples using ISO/IEC 17025 standards.

    PubMed

    Parreño, Viviana; Romera, S Alejandra; Makek, Lucia; Rodriguez, Daniela; Malacari, Darío; Maidana, Silvina; Compaired, Diego; Combessies, Gustavo; Vena, María Marta; Garaicoechea, Lorena; Wigdorovitz, Andrés; Marangunich, Laura; Fernandez, Fernando

    2010-10-01

    Two ELISAs to quantify antibodies to BoHV-1 in the sera of cattle and immunized guinea pigs were developed and validated using ISO/IEC 17025 standards. The cut-off value of the assay was established at 20% positivity of a high positive control for screening of cattle. Using this threshold, the assay properly classified the OIE bovine reference sera EU1, EU2 and EU3. For vaccine potency testing, a cut-off of 40% was selected for both species. The reliability of the assays, given by their diagnostic sensitivity and specificity, using the threshold of 40% was 89.7% and 100%, respectively, for bovines and 94.9% and 100% for guinea pigs, respectively. There was almost perfect agreement between the ELISA and virus neutralization results. In addition, after vaccination, there was a good correlation between the neutralizing and ELISA antibody titers of the serum from the same bovine or guinea pig, sampled at 60 and 30 days post-vaccination, respectively (R(bovine)=0.88, R(guinea pig)=0.92; p<0.0001). A similar correlation was observed when analyzing the mean antibody titers of groups of vaccinated animals (R(bovine)=0.95 and R(guinea pig)=0.97; p<0.0001), indicating the relevance of the ELISAs for batch to batch vaccine potency testing in the target species and in the laboratory animal model. The intermediate precision of the assays expressed as the relative coefficient of variation (CV) of the positive control assayed over a 3-year period in the same laboratory was 22.2% for bovines and 23.1% for guinea pigs. The reproducibility of both techniques obtained in inter-laboratory assays was CV=12.4% for bovines and CV approximately 0 for guinea pigs, which met the requirements of the OIE (CV<30%). The validated ELISAs represent important methods for vaccine potency testing and for controlling BoHV-1 infections. PMID:20655331

  11. Development of ELISA for diagnosis of allergy to Dermatophagoides farinae.

    PubMed

    Ho, T M; Radha, K; Shahnaz, M; Singaram, S P

    1993-09-01

    An indirect enzyme linked immunosorbent assay (ELISA) was developed for the diagnosis of allergy to a house dust mite, Dermatophagoides farinae. The efficacy of the ELISA was then evaluated against a prick test using a commercial allergen. Eighty five suspected allergic rhinitis patients from the Otorhinolaryngology Department, Kuala Lumpur General Hospital, were tested with the ELISA and prick test. Prick test and ELISA results were positive in 84.7% and 80.0% of the patients respectively. The ELISA was found to have 87.5% sensitivity, 61.5% specificity, 92.6% positive predictive value, 47.1% negative predictive value, 7.4% false positive and 52.9% false negative. There was total agreement between the prick test and ELISA for prick test grades of 3+ and 4+. It is concluded that the ELISA is a useful assay for detection of individuals who are highly sensitive to D. farinae.

  12. Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

    PubMed

    Alam, Muhammad Azhar; Maqbool, Azhar; Nazir, Muhammad Mudasser; Lateef, Muhammad; Khan, Muhammad Sarwar; Ahmed, Atif Nisar; Ziaullah, M; Lindsay, David S

    2015-04-01

    Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P < 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirty-nine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple

  13. Assessment of the repeatability and border-plate effects of the B158/B60 enzyme-linked-immunosorbent assay for the detection of circulating antigens (Ag-ELISA) of Taenia saginata.

    PubMed

    Jansen, Famke; Dorny, Pierre; Berkvens, Dirk; Van Hul, Anke; Van den Broeck, Nick; Makay, Caroline; Praet, Nicolas; Gabriël, Sarah

    2016-08-30

    The monoclonal antibody-based circulating antigen detecting ELISA (B158/B60 Ag-ELISA) has been used elaborately in several studies for the diagnosis of human, bovine and porcine cysticercosis. Interpretation of test results requires a good knowledge of the test characteristics, including the repeatability and the effect of the borders of the ELISA plates. Repeatability was tested for 4 antigen-negative and 5 antigen-positive reference bovine serum samples by calculating the Percentage Coefficient of Variation (%CV) within and between plates, within and between runs, overall, for two batches of monoclonal antibodies and by 2 laboratory technicians. All CV values obtained were below 20% (except one: 24.45%), which indicates a good repeatability and a negligible technician error. The value of 24.45% for indicating the variability between batches of monoclonal antibodies for one positive sample is still acceptable for repeatability measures. Border effects were determined by calculating the %CV values between the inner and outer wells of one plate for 2 positive serum samples. Variability is a little more present in the outer wells but this effect is very small and no significant border effect was found.

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

    PubMed

    Odashima, Newton Satoru; Takayanagui, Osvaldo Massaiti; Figueiredo, José Fernando de Castro

    2002-06-01

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

  15. Assessment of the repeatability and border-plate effects of the B158/B60 enzyme-linked-immunosorbent assay for the detection of circulating antigens (Ag-ELISA) of Taenia saginata.

    PubMed

    Jansen, Famke; Dorny, Pierre; Berkvens, Dirk; Van Hul, Anke; Van den Broeck, Nick; Makay, Caroline; Praet, Nicolas; Gabriël, Sarah

    2016-08-30

    The monoclonal antibody-based circulating antigen detecting ELISA (B158/B60 Ag-ELISA) has been used elaborately in several studies for the diagnosis of human, bovine and porcine cysticercosis. Interpretation of test results requires a good knowledge of the test characteristics, including the repeatability and the effect of the borders of the ELISA plates. Repeatability was tested for 4 antigen-negative and 5 antigen-positive reference bovine serum samples by calculating the Percentage Coefficient of Variation (%CV) within and between plates, within and between runs, overall, for two batches of monoclonal antibodies and by 2 laboratory technicians. All CV values obtained were below 20% (except one: 24.45%), which indicates a good repeatability and a negligible technician error. The value of 24.45% for indicating the variability between batches of monoclonal antibodies for one positive sample is still acceptable for repeatability measures. Border effects were determined by calculating the %CV values between the inner and outer wells of one plate for 2 positive serum samples. Variability is a little more present in the outer wells but this effect is very small and no significant border effect was found. PMID:27523940

  16. Evaluation of the diagnostic accuracy of the modified agglutination test (MAT) and an indirect ELISA for the detection of serum antibodies against Toxoplasma gondii in sheep through Bayesian approaches.

    PubMed

    Mainar-Jaime, R C; Barberán, M

    2007-09-01

    The diagnostic accuracies of the modified agglutination test (MAT) and indirect ELISA test for the detection of serum antibodies against Toxoplasma gondii in sheep were evaluated through Bayesian approaches on two populations of sheep created from three different groups of animals (T. gondii-aborted ewes, colostrums-deprived newborn lambs, and ewe-lambs and adult ewes with unknown T. gondii infection status). Tests showed a high degree of agreement (kappa statistic = 0.93; 95% confidence interval = 0.87, 0.98) and a significant specificity (Sp) correlation (gamma(Sp) = 0.26; 95% credibility interval = 0.017, 0.61). When prior information was used for all unknown parameters the posterior medians for the sensitivity (Se) and Sp of the MAT and ELISA were, respectively, 92.6% (95% credibility interval = 85.2, 96.9), 95.5% (89.9, 98.7), 90.5% (83.4, 95.6), and 97.8% (94.2, 99.5). These estimates remained similar when uninformative priors were included. The Se estimates of the MAT and ELISA were higher than those obtained on pigs in other study using the same approach (Se = 80.6% and Sp = 89.5% for the MAT, and Se = 71.5% and Sp = 85.5% for the ELISA [Georgiadis, M.P., Wesley, O.J., Gardner, I.A., Singh, R., 2003. Correlation-adjusted estimation of sensitivity and specificity of two diagnostic tests. Appl. Stat. 52, 63-78]. This finding supported the believe that test performances may vary when applied on different animal species. Thus, if these tests are planned to be used on animal species other than sheep or pigs, their diagnostic accuracy should be re-assessed to prevent biased inferences from their results.

  17. Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

    PubMed

    Shan, Zhifu; Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2016-07-01

    Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds. PMID:27565564

  18. Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

    PubMed

    Shan, Zhifu; Yamasaki, Takeshi; Suzuki, Akio; Hasebe, Rie; Horiuchi, Motohiro

    2016-07-01

    Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.

  19. Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

    PubMed

    Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

    2016-09-01

    There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA.

  20. Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

    PubMed

    Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

    2016-09-01

    There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. PMID:27180038

  1. Long-term RNA persistence of porcine rubulavirus (PorPV-LPMV) after an outbreak of a natural infection: the detection of viral mRNA in sentinel pigs suggests viral transmission.

    PubMed

    Cuevas-Romero, S; Hernández-Baumgarten, E; Kennedy, S; Hernández-Jáuregui, P; Berg, M; Moreno-López, J

    2014-08-01

    The persistence of porcine rubulavirus (PorPV-LPMV) in five pigs that had survived an outbreak of a natural infection was determined. After the resolution of the outbreak, each animal was housed in an isolation pen together with one sentinel pig. Approximately every 2 months thereafter one group of animals was euthanized and tissue samples taken for virological and serological analysis. Infectious virus was not isolated from any samples; antibodies to PorPV-LPMV were detected in convalescent pigs by virus neutralisation test and blocking ELISA but not in sentinel pigs. PorPV-LPMV mRNA of the nucleoprotein (NP) and phosphoprotein (P) genes was detected by a nested polymerase chain reaction (nPCR) in samples of trigeminal and optic nerves, cervical spinal cord, tonsils, salivary gland, lung and pancreas from convalescent pigs. mRNA was also detected in the midbrain, corpus callosum, or olfactory bulb in four out of five pigs by nRT-PCR, this result was confirmed by the sequencing of a 260bp PCR product of P gene region. The highest average viral copies/μg of total RNA occurred in the olfactory bulb and pancreas tissues of convalescent pigs and midbrain, tonsil and pancreas of sentinel pigs housed with the convalescent pigs. Satellitosis and gliosis of the midbrain, olfactory bulb, corpus callosum, medulla oblongata or choroid plexus were microscopically observed in four convalescent pigs. The control pig remained negative in all tests. The results indicate that PorPV-LPMV mRNA persists and induces a durable humoral immune response in pigs that have recovered from a natural infection. After a possible reactivation of the virus, it was transmitted to sentinel pigs in contact with the convalescent pigs.

  2. Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep showing pathology of Johne's disease.

    PubMed

    Sonawane, Ganesh G; Tripathi, Bhupendra N

    2013-12-01

    A quantitative real-time PCR (qPCR) assay employing IS900 gene specific primers of Mycobacterium avium subsp. parartuberculosis (MAP) was compared with conventional PCR, bacterial culture and enzyme-linked immunosorbent assay in 38 sheep showing granulomatous enteritis and lymphadenitis with and without demonstration of acid-fast bacilli (AFB). The lesions were classified as multibacillary (MB) (n = 23), which had diffuse granulomatous lesions with abundant AFB, and paucibacillary (PB) (n = 15), which had focal or multifocal granulomatous lesions with few or no AFB. In the multibacillary group (MB), IS900 PCR detected 19 (82.6%), and qPCR detected all 23 (100%) sheep positive for MAP in the intestine and lymph node tissues. In the paucibacillary group (PB), IS900 PCR detected 2 (13.3%), and qPCR detected all 15 (100%) sheep positive for MAP in tissues. When results of both groups were taken together, IS900 PCR detected 21(55.2%), and qPCR detected all 38 (100%) animals positive for MAP genome either in the intestine or lymph node tissues. On Herrold egg yolk medium, tissues of 14 (60.9%) MB and 5 (33.3%) PB sheep were found to be positive for MAP. Out of 27 sheep (PB = 8, MB = 19) tested by an ELISA, 21 (77.7%) were found to be positive for MAP antibody, of which 25% (2/8) and 100% (19/19) sheep were from PB and MB sheep, respectively. Based on the results of the present study, it was concluded that qPCR was a highly sensitive test in comparison to conventional PCR, ELISA and bacterial culture for the diagnosis of paratuberculosis on infected tissues especially from paucibacillary sheep.

  3. Antigen characterization from second instars of oestrid bot flies for the detection of anti-Cephenemyia stimulator antibodies by ELISA in roe deer (Capreolus capreolus).

    PubMed

    Arias, M S; Pajares, G; Paz-Silva, A; Díez-Baños, N; Suárez, J L; Díez-Baños, P; Sánchez-Andrade, R; Morrondo, P

    2014-08-01

    A study to determine the most appropriate antigen for use in the serodiagnosis of Cephenemyia (Diptera: Oestridae) infestation in roe deer (Capreolus capreolus) was carried out using immunoenzymatic tests. Serum samples from 43 roe deer from northern Spain were obtained post-mortem and corresponding numbers of bot fly larvae established. Three antigen complexes were tested, including Cephenemyia stimulator Clark excretory/secretory antigens (CsES), C. stimulator somatic antigens (CsSA) and Oestrus ovis L. (Diptera: Oestridae) excretory/secretory antigens (OoES). In addition, the composition of each antigen was analysed using an electrophoresis system. Cephenemyia stimulator larvae were found in 25% of roe deer; the mean intensity of infection was 24.3 larvae per infested animal. In the antigen analysis, CsSA showed four exclusive bands of molecular weight (17-19, 62, 65 and 67-70 kDa). A positive correlation between immunoglobulin G (IgG) values and total number of larvae was found with CsES and CsSA. The highest sensitivity value, negative predictive value and negative likelihood ratio were obtained using CsES. The highest specificity value, positive likelihood ratio and kappa value were achieved with CsSA. The predictive values of the enzyme-linked immunosorbent assay (ELISA) using CsES and CsSA reached statistical significance and seroprevalence values were 26-44%. The use of ELISA with CsES and CsSA seems promising in the non-invasive diagnosis of Cephenemyia infestation in roe deer. PMID:25171610

  4. Antigen characterization from second instars of oestrid bot flies for the detection of anti-Cephenemyia stimulator antibodies by ELISA in roe deer (Capreolus capreolus).

    PubMed

    Arias, M S; Pajares, G; Paz-Silva, A; Díez-Baños, N; Suárez, J L; Díez-Baños, P; Sánchez-Andrade, R; Morrondo, P

    2014-08-01

    A study to determine the most appropriate antigen for use in the serodiagnosis of Cephenemyia (Diptera: Oestridae) infestation in roe deer (Capreolus capreolus) was carried out using immunoenzymatic tests. Serum samples from 43 roe deer from northern Spain were obtained post-mortem and corresponding numbers of bot fly larvae established. Three antigen complexes were tested, including Cephenemyia stimulator Clark excretory/secretory antigens (CsES), C. stimulator somatic antigens (CsSA) and Oestrus ovis L. (Diptera: Oestridae) excretory/secretory antigens (OoES). In addition, the composition of each antigen was analysed using an electrophoresis system. Cephenemyia stimulator larvae were found in 25% of roe deer; the mean intensity of infection was 24.3 larvae per infested animal. In the antigen analysis, CsSA showed four exclusive bands of molecular weight (17-19, 62, 65 and 67-70 kDa). A positive correlation between immunoglobulin G (IgG) values and total number of larvae was found with CsES and CsSA. The highest sensitivity value, negative predictive value and negative likelihood ratio were obtained using CsES. The highest specificity value, positive likelihood ratio and kappa value were achieved with CsSA. The predictive values of the enzyme-linked immunosorbent assay (ELISA) using CsES and CsSA reached statistical significance and seroprevalence values were 26-44%. The use of ELISA with CsES and CsSA seems promising in the non-invasive diagnosis of Cephenemyia infestation in roe deer.

  5. Informative prior distributions for ELISA analyses.

    PubMed

    Klauenberg, Katy; Walzel, Monika; Ebert, Bernd; Elster, Clemens

    2015-07-01

    Immunoassays are capable of measuring very small concentrations of substances in solutions and have an immense range of application. Enzyme-linked immunosorbent assay (ELISA) tests in particular can detect the presence of an infection, of drugs, or hormones (as in the home pregnancy test). Inference of an unknown concentration via ELISA usually involves a non-linear heteroscedastic regression and subsequent prediction, which can be carried out in a Bayesian framework. For such a Bayesian inference, we are developing informative prior distributions based on extensive historical ELISA tests as well as theoretical considerations. One consideration regards the quality of the immunoassay leading to two practical requirements for the applicability of the priors. Simulations show that the additional prior information can lead to inferences which are robust to reasonable perturbations of the model and changes in the design of the data. On real data, the applicability is demonstrated across different laboratories, for different analytes and laboratory equipment as well as for previous and current ELISAs with sigmoid regression function. Consistency checks on real data (similar to cross-validation) underpin the adequacy of the suggested priors. Altogether, the new priors may improve concentration estimation for ELISAs that fulfill certain design conditions, by extending the range of the analyses, decreasing the uncertainty, or giving more robust estimates. Future use of these priors is straightforward because explicit, closed-form expressions are provided. This work encourages development and application of informative, yet general, prior distributions for other types of immunoassays.

  6. Rapid Immuno-Chromatographic Assay for the Detection of Antibodies to HIV Compare with Elisa among Voluntary and Replacement Blood Donor of Mymensingh Medical College Hospital.

    PubMed

    Chakrabarty, P; Rudra, S; Hossain, M A; Begum, S A; Mirza, T T; Rudra, M

    2015-04-01

    Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from voluntary and replacement blood donors & HIV-infected patients (positive samples from BSMMU, Dhaka). Five rapid HIV assays: Determine™ HIV-1/2 (Inverness Medical), SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), First Response HIV Card 1-2.0 (PMC Medical India Pvt Ltd.), HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc) and Uni-Gold™ HIV-1/2 (Biotech) were evaluated between 1st February to 30th June, 2013 using 400 whole blood samples from voluntary and replacement blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics). Only 01 sample including ten positive samples from BSMMU were confirmed HIV-1 antibody positive, while 399 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold™ was 100% (95% CI; 99.1-100) while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2-99.9) and 97.7% (95% CI; 95.7-98.9) respectively, which increased to 100% (95% CI; 99.1-100) on repeat testing. The initial specificity of the Uni-Gold™ assay was 100% (95% CI; 99.6-100) while specificities were 99.6% (95% CI; 99-99.9), 99.4% (95% CI; 98.8-99.7), 99.6% (95% CI; 99-99.9) and 99.8% (95% CI; 99.3-99.9) for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was concordantly false positive in Uni-Gold™, Determine and SD Bioline assays. An alternative confirmatory HIV testing strategy based on initial testing on either SD Bioline or Determine assays followed by testing of reactive

  7. Sensitivity improvement of a sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen isoforms in human serum using electrochemical impedance spectroscopy and an ordered and hierarchically organized interfacial supramolecular architecture.

    PubMed

    Gutiérrez-Zúñiga, Gabriela Guadalupe; Hernández-López, José Luis

    2016-01-01

    A gold millielectrode (GME) functionalized with a mixed (16-MHA + EG3SH) self-assembled monolayer (SAM) was used to fabricate an indirect enzyme-linked immunosorbent assay (ELISA) immunosensor for the sensitive detection of prostate-specific antigen (PSA), a prostate cancer (PCa) biomarker, in human serum samples. To address and minimize the issue of non-specific protein adsorption, an organic matrix (amine-PEG3-biotin/avidin) was assembled on the previously functionalized electrode surface to build up an ordered and hierarchically organized interfacial supramolecular architecture: Au/16-MHA/EG3SH/amine-PEG3-biotin/avidin. The electrode was then exposed to serum samples at different concentrations of a sandwich-type immunocomplex molecule ((Btn)Ab-AgPSA-(HRP)Ab), and its interfacial properties were characterized using electrochemical impedance spectroscopy (EIS). Calibration curves for polarization resistance (RP) and capacitance (1/C) vs. total and free PSA concentrations were obtained and their analytical quality parameters were determined. This approach was compared with results obtained from a commercially available ELISA immunosensor. The results obtained in this work showed that the proposed immunosensor can be successfully applied to analyze serum samples of patients representative of the Mexican population.

  8. Sensitivity improvement of a sandwich-type ELISA immunosensor for the detection of different prostate-specific antigen isoforms in human serum using electrochemical impedance spectroscopy and an ordered and hierarchically organized interfacial supramolecular architecture.

    PubMed

    Gutiérrez-Zúñiga, Gabriela Guadalupe; Hernández-López, José Luis

    2016-01-01

    A gold millielectrode (GME) functionalized with a mixed (16-MHA + EG3SH) self-assembled monolayer (SAM) was used to fabricate an indirect enzyme-linked immunosorbent assay (ELISA) immunosensor for the sensitive detection of prostate-specific antigen (PSA), a prostate cancer (PCa) biomarker, in human serum samples. To address and minimize the issue of non-specific protein adsorption, an organic matrix (amine-PEG3-biotin/avidin) was assembled on the previously functionalized electrode surface to build up an ordered and hierarchically organized interfacial supramolecular architecture: Au/16-MHA/EG3SH/amine-PEG3-biotin/avidin. The electrode was then exposed to serum samples at different concentrations of a sandwich-type immunocomplex molecule ((Btn)Ab-AgPSA-(HRP)Ab), and its interfacial properties were characterized using electrochemical impedance spectroscopy (EIS). Calibration curves for polarization resistance (RP) and capacitance (1/C) vs. total and free PSA concentrations were obtained and their analytical quality parameters were determined. This approach was compared with results obtained from a commercially available ELISA immunosensor. The results obtained in this work showed that the proposed immunosensor can be successfully applied to analyze serum samples of patients representative of the Mexican population. PMID:26703258

  9. High-Throughput Analysis of Serum Antigens Using Sandwich ELISAs on Microarrays

    SciTech Connect

    Servoss, Shannon; Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

    2009-05-11

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection and validation of disease biomarkers. ELISA microarrays are capable of simultaneous detection of many proteins using a small sample volume. Although there are many potential pitfalls to the use of ELISA microarrays, these can be avoided by careful planning of experiments. In this chapter we describe a high-throughput protocol for processing ELISA microarrays that will result in reliable and reproducible data.

  10. Gray level entropy matrix is a superior predictor than multiplex ELISA in the detection of reactive stroma and metastatic potential of high-grade prostatic adenocarcinoma.

    PubMed

    Jia, Xiaopeng; Sun, Yanan; Wang, Baozhi

    2014-12-01

    Recent reports have indicated that not only the primary glandular tissue but also the surrounding stromal tissue plays an active role in the progression of carcinoma. Such is true for cancer tissues arising in the prostate. However, the precise role of stromal tissue in benign prostatic hyperplasia (BPH) and prostate adenocarcinoma is not well described. We undertook this current investigation to examine the changes in orientation of the extracellular matrix and correlate with prostatic cancer progression. We used a novel form of image analysis called gray level entropy matrix (GLEM) texture analysis to evaluate morphometric changes in stromal tissues. We used normal prostatic tissue obtained from cadaveric specimen and compared with BPH, prostatic intraepithelium neoplastic, hormone responsive prostatic adenocarcinoma and castration-resistant prostatic adenocarcinoma tissues. GLEM showed higher entropy in disease-resistant prostatic tissues, compared with benign forms of all spectra of pathologically diagnosed prostatic tissues (P < 0.05, ANOVA, between groups). Higher entropy is reflective of the disorganized morphological organization of the stroma, possibly reflecting the reactive matrix. In contrast, ELISA revealed that although individually correlated with the progressive stages of benign and carcinomatous prostatic tissues and trend correlation between groups, intergroup comparisons failed to arrive at statistical significance of comparisons between markers of neovasculogenesis, vascular endothelial growth factor, epithelial-mesenchymal transition (beta1-integrin, E-cadherin, MMP3) and osteogenic metastasis (RANKL and osteoprotegerin). The results of our study demonstrate the potential of GLEM entropy of gray level pixel in providing quasiquantitative estimate of a reactive stroma in advance stages of prostatic adenocarcinoma and thus can be routinely used in clinical decision making.

  11. ELISA versus Conventional Methods of Diagnosing Endemic Brucellosis

    PubMed Central

    Mantur, Basappa; Parande, Aisha; Amarnath, Satish; Patil, Giridhar; Walvekar, Ravindra; Desai, Arun; Parande, Mahantesh; Shinde, Rupali; Chandrashekar, Masiyappa; Patil, Satish

    2010-01-01

    The diagnostic value of enzyme-linked immunosorbent assay (ELISA) was evaluated when blood specimens of 92 patients suspected of brucellosis underwent the ELISA (IgM and IgG), standard tube agglutination (SAT), and 2-mercaptoethanol (2-ME) tests and blood cultures; 38 sera from non-brucellosis patients and 34 sera from blood donors were also subjected to ELISA, SAT, and 2-ME tests. SAT was able to pinpoint only 23 (25%), whereas ELISA confirmed the etiology in 56 (60.9%; P < 0.001) patients with brucellosis, including 31 culture-confirmed cases. The sensitivity and specificity of ELISA were 100% and 71.31%, respectively. Because they were confirmed by ELISA, the diagnosis could never be excluded with SAT in 33 cases. ELISA has been found to be more sensitive in acute (28% higher sensitivity; P < 0.02) and chronic (55% higher sensitivity; P < 0.01) cases. For accurate diagnosis in suspected brucellosis cases detection, we recommend both ELISA IgM and IgG tests. ELISA IgG and 2-ME tests seem to be promising tools in judging prognosis. PMID:20682874

  12. Variation in E**rns viral glycoprotein associated with failure of immunohistochemistry and commercial antigen capture ELISA to detect a field strain of bovine viral diarrhea virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea virus (BVDV) effects cattle populations causing clinical signs that range from subclinical immunosuppression to severe reproductive and respiratory problems. Detection and removal of persistently infected (PI) calves is the single most important factor for control and eradicat...

  13. Immunoglobulin detection in wild birds: Effectiveness of three secondary anti-avian IgY antibodies in direct ELISAs in 41 avian species

    USGS Publications Warehouse

    Fassbinder-Orth, Carol A.; Wilcoxen, Travis E.; Tran, Tiffany; Boughton, Raoul K.; Fair, Jeanne M.; Hofmeister, Erik K.; Grindstaff, Jennifer L.; Owen, Jen C.

    2016-01-01

    4.This study indicates that the anti-passerine IgY secondary antibody can effectively be used in immunological assays to detect passerine IgY for species in most passerine families and is preferred over anti-chicken and anti-bird secondary antibodies for the majority of passerine species. This anti-passerine antibody will allow for more accurate detection and quantification of IgY in more wild bird species than was possible with previously available secondary antibodies.

  14. Development of a simple gel permeation clean-up procedure coupled to a rapid disequilibrium enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I dye in spices and sauces.

    PubMed

    Oplatowska, Michalina; Stevenson, Paul J; Schulz, Claudia; Hartig, Lutz; Elliott, Christopher T

    2011-09-01

    Sudan dyes have been found to be added to chilli and chilli products for illegal colour enhancement purposes. Due to the possible carcinogenic effect, they are not authorized to be used in food in the European Union or the USA. However, over the last few years, many products imported from Asian and African countries have been reported via the Rapid Alert System for Food and Feed in the European Union to be contaminated with these dyes. In order to provide fast screening method for the detection of Sudan I (SI), which is the most widely abused member of Sudan dyes family, a unique (20 min without sample preparation) direct disequilibrium enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on polyclonal antibodies highly specific to SI. A novel, simple gel permeation chromatography clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The assay was validated according to the Commission Decision 2002/657/EC criteria. The detection capability was determined to be 15 ng g(-1) in sauces and 50 ng g(-1) in spices. The recoveries found ranged from 81% to 116% and inter- and intra-assay coefficients of variation from 6% to 20%. The assay was used to screen a range of products (85 samples) collected from different retail sources within and outside the European Union. Three samples were found to contain high amounts (1,649, 722 and 1,461 ng g(-1)) of SI by ELISA. These results were confirmed by liquid chromatography-tandem mass spectrometry method. The innovative procedure allows for the fast, sensitive and high throughput screening of different foodstuffs for the presence of the illegal colorant SI.

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

    PubMed

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

    1994-01-01

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

  16. Preliminary evaluation on the efficiency of the kit Platelia Dengue NS1 Ag-ELISA to detect dengue virus in dried Aedes aegypti: a potential tool to improve dengue surveillance

    PubMed Central

    2014-01-01

    Background Surveillance is a critical component of any dengue prevention and control programme. Herein, we investigate the efficiency of the commercial kit Platelia Dengue NS1 Ag-ELISA to detect dengue virus (DENV) antigens in Aedes aegypti mosquitoes infected under laboratory conditions. Methods Under insectary conditions, four to five day-old mosquitoes were orally challenged with DENV-2 titer of 3.6 x 105 PFU equivalent/ml, incubated for 14 days and then killed. At ten time-points following mosquito death (0, 6, 12, 24, 72, 96, 120, 144 and 168 h), i.e., during a one-week period, dried mosquitoes were comparatively tested for the detection of the NS1 antigen with other methods of detection, such as qRT-PCR and virus isolation in C6/36 cells. Results We first observed that the NS1 antigen was more effective in detecting DENV-2 in Ae. aegypti between 12 and 72 h after mosquito death when compared with qRT-PCR. A second round involved comparing the sensitivity of detection of the NS1 antigen and virus isolation in C6/36 cells. The NS1 antigen was also more effective than virus isolation, detecting DENV-2 at all time-points, i.e., up to 168 h after mosquito death. Meanwhile, virus isolation was successful up to 96 h after Ae. aegypti death, but the number of positive samples per time period presented a tendency to decline progressively over time. From the 43 samples positive by the virus isolation technique, 38 (88.4%) were also positive by the NS1 test. Conclusion Taken together, these results are the first to indicate that the NS1 antigen might be an interesting complementary tool to improve dengue surveillance through DENV detection in dried Ae. aegypti females. PMID:24690324

  17. Development of slide ELISA (SELISA) for detection of four poultry viral pathogens by direct heat fixation of viruses on glass slides.

    PubMed

    Desingu, P A; Singh, S D; Dhama, K; Kumar, O R Vinodh; Singh, R; Singh, R K

    2014-12-01

    The development of an easy and simpler method of slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of four economically important poultry viruses viz., Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) and egg drop syndrome 76 virus (EDS 76) and the use of SELISA for semi quantitation of NDV are described. The positive signals for viral aggregates were detected under light microscope. This is the first report regarding the development of SELISA based on heat fixation for the diagnosis of viral pathogens. PMID:25218174

  18. Diagnostic accuracy of ELISA methods as an alternative screening test to indirect immunofluorescence for the detection of antinuclear antibodies. Evaluation of five commercial kits.

    PubMed

    Tonuttia, Elio; Bassetti, Danila; Piazza, Anna; Visentini, Daniela; Poletto, Monica; Bassetto, Franca; Caciagli, Patrizio; Villalta, Danilo; Tozzoli, Renato; Bizzaro, Nicola

    2004-03-01

    Detection of antinuclear antibodies (ANA) is a fundamental laboratory test for diagnosing systemic autoimmune diseases. Currently, the method of choice is indirect immunofluorescence (IIF) on a HEp-2 cell substrate. The goal of this study was to evaluate the diagnostic accuracy of five commercially available enzyme immunoassay (EIA) kits for ANA detection and to verify the possibility of using them as an alternative to the IIF method. The study involved 1513 patients, 315 of whom were diagnosed with a systemic autoimmune disease and 1198 in whom an autoimmune disorder was excluded. For all sera, ANA detection was performed via IIF and with five different EIA kits. The results were evaluated in relation to clinical diagnosis and the presence of possible specific autoantibodies (anti-ENA or anti-dsDNA); lastly, they were compared with the results obtained using ANA-IIF as the method of reference. The positive rate of the ANA-IIF test in subjects with systemic autoimmune diseases was 92%, whereas in the five ANA-EIA kits there was broad diversity in terms of response, with positive rates ranging from 74 to 94%. All the EIA kits correctly detected the presence of antibodies (anti-dsDNA, anti-RNP, anti-Ro/SSA) responsible for homogeneous and speckled fluorescence pattern, but at the same time they showed substantial inaccuracy with the nucleolar pattern, with a mean sensitivity of approximately 50% in this case. Instead, there was a large kit-to-kit difference in terms of identification of anti-Scl70 and centromere patterns, for which sensitivities ranged between 45 and 91%, and between 49 and 100%, respectively. The results of the study demonstrate that the commercially available ANA-EIA kits show different levels of sensitivity and specificity. Some of them have a diagnostic accuracy that is comparable and, in some cases, even higher than the IIF method. Consequently, these could be used as an alternative screening test to IIE. However, others do not ensure acceptable

  19. Development of slide ELISA (SELISA) for detection of four poultry viral pathogens by direct heat fixation of viruses on glass slides.

    PubMed

    Desingu, P A; Singh, S D; Dhama, K; Kumar, O R Vinodh; Singh, R; Singh, R K

    2014-12-01

    The development of an easy and simpler method of slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of four economically important poultry viruses viz., Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) and egg drop syndrome 76 virus (EDS 76) and the use of SELISA for semi quantitation of NDV are described. The positive signals for viral aggregates were detected under light microscope. This is the first report regarding the development of SELISA based on heat fixation for the diagnosis of viral pathogens.

  20. Enzyme-linked immunosorbent assay (ELISA) for detection of sulfur-rich protein (SRP) in soybeans (Glycine max L.) and certain other edible plant seeds.

    PubMed

    Monaghan, Erin K; Venkatachalam, Mahesh; Seavy, Margaret; Beyer, Kirsten; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

    2008-02-13

    As a result of methionine deficiency, legume proteins are considered to be incomplete, and therefore there is a need to explore ways to improve legume protein amino acid balance. Using rabbit anti-soybean sulfur-rich protein (SRP) polyclonal antibodies (pAb), sensitive immunoassays (nanogram sensitivity) were developed. The immunoassays detected SRP in all soybean seeds and soybean-based commercial samples examined. In addition, the presence of pAb cross-reactive proteins was detected in certain dry beans and oilseeds. The cross-reactive proteins were isolated using purified IgG-based immunoaffinity column chromatography. Biochemical analyses including N-terminal amino acid sequencing and amino acid composition indicated that the cross-reactive proteins were comparable to soybean SRP. The cross-reactive proteins contained methionine (1.6-2.4 residues/100 residues) and cysteine (2.4-3.6 residues/100 residues), which satisfies the FAO/WHO recommended pattern for sulfur amino acids in both adults and children (2-5 years old). The results suggest the presence of constitutive SRPs in several dry beans and oilseeds. PMID:18189355

  1. Detection of protective antibody titers against feline panleukopenia virus, feline herpesvirus-1, and feline calicivirus in shelter cats using a point-of-care ELISA.

    PubMed

    Digangi, Brian A; Gray, Lauren K; Levy, Julie K; Dubovi, Edward J; Tucker, Sylvia J

    2011-12-01

    Serum antibody titers are a useful measurement of protection against infection (feline panleukopenia virus [FPV]) or clinical disease (feline herpesvirus-1 [FHV] and feline calicivirus [FCV]), and their determination has been recommended as part of disease outbreak management in animal shelters. The objective of this study was to determine the sensitivity, specificity, and inter-observer and inter-assay agreement of two semi-quantitative point-of-care assays for the detection of protective antibody titers (PAT) against FPV, FHV and FCV in shelter cats. Low sensitivity for FPV antibodies (28%) rendered a canine point-of-care assay inappropriate for use in cats. The feline point-of-care assay also had low sensitivity (49%) and low negative predictive value (74%) for FPV PAT detection, but was highly accurate in the assessment of FHV and FCV PAT. Improvements in accuracy and repeatability of FPV PAT determination could make this tool a valuable component of a disease outbreak response in animal shelters. PMID:21885311

  2. Analysis of fenbendazole residues in bovine milk by ELISA.

    PubMed

    Brandon, David L; Bates, Anne H; Binder, Ronald G; Montague, William C; Whitehand, Linda C; Barker, Steven A

    2002-10-01

    Fenbendazole residues in bovine milk were analyzed by ELISAs using two monoclonal antibodies. One monoclonal antibody (MAb 587) bound the major benzimidazole anthelmintic drugs, including fenbendazole, oxfendazole, and fenbendazole sulfone. The other (MAb 591) was more specific for fenbendazole, with 13% cross-reactivity with the sulfone and no significant binding to the sulfoxide metabolite. The limit of detection of the ELISA method in the milk matrix was 7 ppb for MAb 587 and 3 ppb for MAb 591. Fenbendazole was administered in feed, drench, and paste form to three groups of dairy cattle. Milk was collected immediately before dosing and then every 12 h for 5 days. The ELISA indicated that residue levels varied widely among individual cows in each group. Fenbendazole levels peaked at approximately 12-24 h and declined rapidly thereafter. Metabolites were detected at much higher levels than the parent compound, peaked at approximately 24-36 h, and declined gradually. Residue levels were undetectable by 72 h. The ELISA data correlated well with the total residues determined by chromatographic analysis, but the use of the two separate ELISAs did not afford an advantage over ELISA with the single, broadly reactive MAb 587. The ELISA method could be used to flag high-residue samples in on-site monitoring of fenbendazole in milk and is a potential tool for studying drug pharmacokinetics.

  3. Ultrasensitive ELISA using enzyme-loaded nanospherical brushes as labels.

    PubMed

    Qu, Zhenyuan; Xu, Hong; Xu, Ping; Chen, Kaimin; Mu, Rong; Fu, Jianping; Gu, Hongchen

    2014-10-01

    Improving the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is of utmost importance for meeting the demand of early disease diagnosis. Herein we report an ultrasensitive ELISA system using horseradish peroxidase (HRP)-loaded nanospherical poly(acrylic acid) brushes (SPAABs) as labels. HRP was covalently immobilized in SPAABs with high capacity and activity via an efficient "chemical conjugation after electrostatic entrapment" (CCEE) process, thus endowing SPAABs with high amplification capability as labels. The periphery of SPAAB-HRP was further utilized to bind a layer of antibody with high density for efficient capture of analytes owing to the three-dimensional architecture of SPAABs. Using human chorionic gonadotrophin (hCG) as a model analyte, the SPAAB-amplified system drastically boosted the detection limit of ELISA to 0.012 mIU mL(-1), a 267-fold improvement as compared to conventional ELISA systems.

  4. Enzyme-linked immunosorbent assay (ELISA) for the detection of use of the synthetic cannabinoid agonists UR-144 and XLR-11 in human urine.

    PubMed

    Mohr, Amanda L A; Ofsa, Bill; Keil, Alyssa Marie; Simon, John R; McMullin, Matthew; Logan, Barry K

    2014-09-01

    Ongoing changes in the synthetic cannabinoid drug market create the need for relevant targeted immunoassays for rapid screening of biological samples. We describe the validation and performance characteristics of an enzyme-linked immunosorbent assay designed to detect use of one of the most prevalent synthetic cannabinoids in urine, UR-144, by targeting its pentanoic acid metabolite. Fluorinated UR-144 (XLR-11) has been demonstrated to metabolize to this common product. The assay has significant cross-reactivity with UR-144-5-OH, UR-144-4-OH and XLR-11-4-OH metabolites, but <10% cross-reactivity with the parent compounds, and no measurable cross-reactivity with other synthetic cannabinoids and their metabolites at concentrations of <1,000 ng/mL. The assay's cutoff is 5 ng/mL relative to the pentanoic acid metabolite of UR-144, which is used as the calibrator. The method was validated with 90 positive and negative control urine samples for UR-144, XLR-11 and its metabolites tested versus liquid chromatography-tandem mass spectrometry. The accuracy, sensitivity and specificity were determined to be 100% for the assay at the specified cutoff. PMID:24908262

  5. Optimising and Evaluating the Characteristics of a Multiple Antigen ELISA for Detection of Mycobacterium bovis Infection in a Badger Vaccine Field Trial

    PubMed Central

    Aznar, Inma; Frankena, Klaas; More, Simon J.; Whelan, Clare; Martin, Wayne; Gormley, Eamonn; Corner, Leigh A. L.; Murphy, Denise; De Jong, Mart C. M.

    2014-01-01

    A long-term research programme has been underway in Ireland to evaluate the usefulness of badger vaccination as part of the national bTB (bovine tuberculosis) control strategy. This culminated in a field trial which commenced in county Kilkenny in 2009 to determine the effects of badger vaccination on Mycobacterium bovis transmission in badgers under field conditions. In the present study, we sought to optimise the characteristics of a multiplex chemiluminescent assay for detection of M. bovis infection in live badgers. Our goal was to maximise specificity, and therefore statistical power, during evaluation of the badger vaccine trial data. In addition, we also aimed to explore the effects of vaccination on test characteristics. For the test optimisation, we ran a stepwise logistic regression with analytical weights on the converted Relative Light Units (RLU) obtained from testing blood samples from 215 badgers captured as part of culling operations by the national Department of Agriculture, Food and the Marine (DAFM). The optimised test was applied to two other datasets obtained from two captive badger studies (Study 1 and Study 2), and the sensitivity and specificity of the test was attained separately for vaccinated and non-vaccinated badgers. During optimisation, test sensitivity was maximised (30.77%), while retaining specificity at 99.99%. When the optimised test was then applied to the captive badger studies data, we observed that test characteristics did not vary greatly between vaccinated and non-vaccinated badgers. However, a different time lag between infection and a positive test result was observed in vaccinated and non-vaccinated badgers. We propose that the optimized multiplex immunoassay be used to analyse the vaccine trial data. In relation to the difference in the time lag observed for vaccinated and non-vaccinated badgers, we also present a strategy to enable the test to be used during trial evaluation. PMID:24983473

  6. [A simple ELISA method for the detection of HBsAg: Organon Teknika HBsAg Uniform II screening and confirmation test. Comparative study using the HBsAg Hapanostika method. A multicenter study].

    PubMed

    Pár, A; Mihály, I; Kömives, K

    1994-09-25

    An one-step enzyme-linked immunoabsorbent method, named as HBsAg Uniform II has been described for the detection of serum HBsAg, and a comparison was made with a widely used ELISA technique HBsAg Hepanostika test, to evaluate sensitivity, specificity and reproducibility of the method. A total of 531 serum samples from patients with liver disease and with renal failure, as well as 1065 samples from blood donors have been investigated. While the sensitivity of Uniform II vs. Hepanostika was 99.5% vs. 72.7%, the specificity was 99.2% of both methods. The positive predictive values did not differ (99.5% vs. 99.2%), however, the negative predictive values were 99.2% vs. 71.7%, respectively, in favour of Uniform II test. The Uniform II confirmatory test confirmed the positive HBsAg results in 94%, this rate was 74% using Hepanostika system. The new method proved to be a simple, quick, reliable test, which can be useful as a valuable tool in both the clinical diagnosis and blood donor screening.

  7. [The systematization of the enzyme-linked immunosorbent assay (=ELISA)].

    PubMed

    Gränzer, W

    1989-06-01

    Using the example of the detectability of immunological methods the study shows the considerable influence exerted by this detectability on the quantitative and qualitative parameters and the necessity of a systematization of the methods. This systematization already starts with the product labeling: A sufficient description of antibodies has to contain five qualities. The profitability of the experimental research is improved by optimized product and method systematization. ELISA is presented in a systematized form as a modular system optimizing the comparability of these methods. In order to detect the gain of information transparency obtained by systematization, an ELISA, described in the literature, is presented in the original and in the systematized form.

  8. Status of eLISA

    NASA Astrophysics Data System (ADS)

    Stebbins, Robin T.; Jennrich, O.; Consortium, eLISA

    2013-01-01

    Scientists from the member states of the European Space Agency (ESA) that proposed the New Gravitational Wave Observatory (NGO) are organizing the eLISA Consortium to propose for the next large mission opportunity. The Evolved Laser Interferometer Space Antenna (eLISA) concept is derived from the well studied LISA concept for a space-based, gravitational-wave mission. eLISA will use the technology being developed in the LISA Pathfinder mission in a two-arm version that achieves much of the science of LISA. eLISA will: (1) survey very many compact stellar-mass binaries and study the structure of the Milky Way; (2) discover black holes formed at early epochs that grew over cosmic time to generate the supermassive black holes present in most galactic nuclei today; (3) trace the interaction of galaxy growth and massive black hole growth over the entire history of galaxy formation; (4) explore the populations of stellar-mass compact objects in galactic nuclei and their dynamics; (5) observe highly relativistic coalescences of black hole binaries, and provide exceptionally strong tests of the predictions of General Relativity; and (6) probe new physics and cosmology with gravitational waves, and search for unforeseen sources of gravitational waves. The key parameters of the design concept and a quantitative assessment of the science performance are given. NASA and the U.S. research community may be able to participate, and possibly extend the capabilities of the mission.

  9. [Immunoenzyme (ELISA) diagnosis of spring viremia in carp].

    PubMed

    Rodák, L; Pospísil, Z; Tománek, J; Veselý, T; Hampl, J

    1986-08-01

    The enzymoimmunologic (ELISA) method was used to diagnose spring viremia in carp. The specific antibodies to Rhabdovirus carpio in the fish sera were detected by this method. Reaction specificity was tested by the use of viral and control antigens, by examination of positive and negative sera and by serum-free control incubation. Applying the ELISA method, the antiviral antibodies can be detected after infection or vaccination: this method is therefore suitable for thorough studies and when choosing the appropriate treatments and preventive measures.

  10. [E.L.I.S.A. in human coccidioidomycosis].

    PubMed

    Tiraboschi, I N; Marticorena, B; Negroni, R

    1991-01-01

    An E.L.I.S.A. test for antibody detection, with an exo-antigen of Coccidioides immitis was standardized in 67 humans sera diluted in 1/1000, 1/2000, 1/4000 and 1/8000. Eighteen sera from mycologically proved cases of coccidioidomycosis were studied: 5 were negative and 13 were positive in some dilutions. 3/26 sera of healthy persons who presented positive skin tests with coccidioidin were positive and the other 23 sera did not have positive reactions. None of the 15 sera of healthy human exhibited positive E.L.I.S.A. Serum samples of 8 patients suffering other deep mycosis were studied, 4 of them presented cross-reactions in E.L.I.S.A. tests. E.L.I.S.A. test seems to be a useful serologic technique for antibody detection in anticomplementary serum samples or when a low concentration of antibodies should be detected. As it is very sensitive, cross-reactions with other mycoses are frequent, thus the use other more specific serologic technique together E.L.I.S.A. is recommended.

  11. Application of ELISA in determining the fate of tetracyclines in land-applied livestock wastes.

    PubMed

    Aga, Diana S; Goldfish, Randall; Kulshrestha, Pankaj

    2003-06-01

    The potential use of a class-specific enzyme-linked immunosorbent assay (ELISA) in studying the occurrence and fate of tetracyclines in the environment was evaluated. Several manure samples collected from hog lagoons and cattle feedlots were screened for the presence of tetracycline residues using ELISA. The levels varied from less than the detection limit (0.5 parts per billion) to 200 parts per million. The degradation of tetracyclines in soil-applied manure was followed using ELISA to measure the decline in tetracycline concentrations. Low levels of tetracyclines remained detectable in soil for up to 28 days. The ELISA procedure also proved useful in determining the leaching potential of tetracyclines in undisturbed soil columns and in the analysis of total tetracyclines in manure, soil, and water. Based on the cross-reactivity of the antibodies employed, this ELISA method can be an important screening tool for the presence of other tetracycline compounds, such as chlortetracycline and oxytetracycline. The ELISA method also detects the epimers of tetracyclines and the corresponding dehydration by-products, anhydrotetracyclines. Analysis of selected manure extracts by liquid chromatography with mass spectrometry (LC-MS) showed lower concentrations of total tetracyclines compared to the values obtained by ELISA, indicating the presence of other structurally related compounds or transformation products of tetracyclines being detected by ELISA in the samples. Because analysis of manure and soil samples by LC-MS requires extensive clean-up procedures, ELISA provides an alternative method for conducting environmental fate and transport studies of antibiotics.

  12. Calculation of the ELISA's cut-off based on the change-point analysis method for detection of Trypanosoma cruzi infection in Bolivian dogs in the absence of controls.

    PubMed

    Lardeux, Frédéric; Torrico, Gino; Aliaga, Claudia

    2016-07-01

    In ELISAs, sera of individuals infected by Trypanosoma cruzi show absorbance values above a cut-off value. The cut-off is generally computed by means of formulas that need absorbance readings of negative (and sometimes positive) controls, which are included in the titer plates amongst the unknown samples. When no controls are available, other techniques should be employed such as change-point analysis. The method was applied to Bolivian dog sera processed by ELISA to diagnose T. cruzi infection. In each titer plate, the change-point analysis estimated a step point which correctly discriminated among known positive and known negative sera, unlike some of the six usual cut-off formulas tested. To analyse the ELISAs results, the change-point method was as good as the usual cut-off formula of the form "mean + 3 standard deviation of negative controls". Change-point analysis is therefore an efficient alternative method to analyse ELISA absorbance values when no controls are available. PMID:27384081

  13. Efficacy demonstration of tetanus vaccines by double antigen ELISA.

    PubMed

    Rosskopf, U; Noeske, K; Werner, E

    2005-09-01

    This paper describes a double antigen ELISA (DAE) for rapid, specific and reliable assessment of the antitetanus immune status of horses and sheep. Compared with the indirect ELISA, the double antigen ELISA has the advantage of species-independent testing of sera. Thanks to its test design, it is more specific since the detected antibodies are forced to bind tetanus toxoid twice. In addition, it is very sensitive to tetanus antibodies, enabling the detection of low antibody titres, in range which is relevant for the assessment of the protective status (tetanus toxin neutralising antibodies). The detection limit of the DAE for tetanus antibodies is in the order of 10(-4) EU/ml. A comparison of in vitro results of individual sera with in vivo titres showed that horse sera with titres of 0.04 and 0.05 EU/ml in the DAE showed titres of > 0.05 IU and 0.034 IU/ml respectively during in vivo testing thus indicating good agreement. For tested sheep sera which were rated > 0.05 IU/ml in vivo, the corresponding titre in the DAE was 0.24 EU/ml. Clear tetanus antitoxin establishment of protective ELISA limits requires further comparative examination of sera with low titres (< 1.0 EU/ml) in the double antigen ELISA and the toxin neutralisation test. With the double antigen ELISA, efficacy can be determined for marketing authorisation procedures of tetanus vaccines ad us. vet. As a consequence, the toxin neutralisation test (still being the standard method of choice for quantifying tetanus toxin neutralising antitoxin titres) could be replaced, since it requires too great a number of animals per test and involves considerable suffering for the animals. The test described here reduces the use of mice and guinea pigs within vaccine efficacy testing. In addition, it involves less exposure of the laboratory personnel to toxin.

  14. Enhanced ELISA based on carboxymethylated dextran coatings.

    PubMed

    Liberelle, Benoît; Fortier, Charles; De Crescenzo, Gregory

    2014-01-01

    In a "sandwich" enzyme-linked immunosorbent assay (ELISA) designed to detect an antigen in a complex protein mixture, the antigen is usually captured via an antibody adsorbed to the wells of a microplate. Plate preparation for standard assay involves a passive adsorption of capture antibodies followed by the incubation of blocking agents. Here, we describe a new strategy that replaces these two time-consuming adsorption steps (up to 15 h) by a unique step corresponding to the covalent grafting of the capture antibody on a carboxymethylated dextran (CMD) layer, a single step completed in 15 min. Taking advantage of the CMD low-fouling properties, blocking agent-free buffer solutions can be used as diluent in the improved approach.

  15. Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA

    PubMed Central

    Parameswaran, N.; O'Handley, RM.; Grigg, ME.; Fenwick, SG.; Thompson, RCA.

    2009-01-01

    Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7-20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a κ coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay. PMID:19567231

  16. Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA.

    PubMed

    Parameswaran, N; O'Handley, R M; Grigg, M E; Fenwick, S G; Thompson, R C A

    2009-06-01

    Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7-20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a kappa coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay.

  17. Application of PCR-ELISA in Molecular Diagnosis

    PubMed Central

    Sue, Mei Jean; Yeap, Swee Keong; Omar, Abdul Rahman; Tan, Sheau Wei

    2014-01-01

    Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality. PMID:24971343

  18. Microalbuminuria measurements by two in-house ELISA methods.

    PubMed

    Ng, L C; Teng, L C; Ng, M L; Sazali, B S; Khalid, B A

    2000-12-01

    Detection of microalbuminuria is important in the management of diabetic patients since it is predictive of development of proteinuria and nephropathy. Two sensitive and specific in-house ELISAs for microalbuminuria were established and validated. One of the ELISAs was based on antigen coating while the other employed antibody coating. Recovery and linearity experiments gave acceptable results of 100 +/- 10%, while precision results were <10% for intra-assay and <12% for inter-assay coefficients of variation (CVs). The standard curve ranged from 10-625 ug/l, equivalent to 0.2-12.5 mg/l for urine samples diluted 1:20 fold. When the antibody coated ELISA was compared to antigen coated ELISA, a correlation of r=0.996 was obtained. When compared to commercial kits, the in-house ELISAs gave good correlations of r=0.961 versus the Boehringer Mannheim Micral Test strips and r=0.940 versus Ames Microalb Turbidimetry. The normal microalbumin reference ranges determined for 12h, first morning and random urine samples were 0.7-5.3 mg, 0.1-10.2 mg/l and 0.8-26.1 mg/l respectively. The normal albumin excretion rate (AER) was 1.0-7.3 ug/min while untimed urine samples gave results of 0.1-0.9 and 0.2-1.6 mg/mmol after dividing by creatinine concentrations. The ELISAs were used to detect microalbuminuria in 338 random urine samples from diabetic patients. A high percentage 47.9% was found to be positive for microalbuminuria and 18.0% had macroalbuminuria >25 mg/mmol. Thus screening for microalbuminuria together with creatinine measurements using random urine samples can be used for management of diabetic patients. PMID:16329538

  19. Microalbuminuria measurements by two in-house ELISA methods.

    PubMed

    Ng, L C; Teng, L C; Ng, M L; Sazali, B S; Khalid, B A

    2000-12-01

    Detection of microalbuminuria is important in the management of diabetic patients since it is predictive of development of proteinuria and nephropathy. Two sensitive and specific in-house ELISAs for microalbuminuria were established and validated. One of the ELISAs was based on antigen coating while the other employed antibody coating. Recovery and linearity experiments gave acceptable results of 100 +/- 10%, while precision results were <10% for intra-assay and <12% for inter-assay coefficients of variation (CVs). The standard curve ranged from 10-625 ug/l, equivalent to 0.2-12.5 mg/l for urine samples diluted 1:20 fold. When the antibody coated ELISA was compared to antigen coated ELISA, a correlation of r=0.996 was obtained. When compared to commercial kits, the in-house ELISAs gave good correlations of r=0.961 versus the Boehringer Mannheim Micral Test strips and r=0.940 versus Ames Microalb Turbidimetry. The normal microalbumin reference ranges determined for 12h, first morning and random urine samples were 0.7-5.3 mg, 0.1-10.2 mg/l and 0.8-26.1 mg/l respectively. The normal albumin excretion rate (AER) was 1.0-7.3 ug/min while untimed urine samples gave results of 0.1-0.9 and 0.2-1.6 mg/mmol after dividing by creatinine concentrations. The ELISAs were used to detect microalbuminuria in 338 random urine samples from diabetic patients. A high percentage 47.9% was found to be positive for microalbuminuria and 18.0% had macroalbuminuria >25 mg/mmol. Thus screening for microalbuminuria together with creatinine measurements using random urine samples can be used for management of diabetic patients.

  20. eLISA eccentricity measurements as tracers of binary black hole formation

    NASA Astrophysics Data System (ADS)

    Nishizawa, Atsushi; Berti, Emanuele; Klein, Antoine; Sesana, Alberto

    2016-09-01

    Up to hundreds of black hole binaries individually resolvable by eLISA will coalesce in the Advanced LIGO and Virgo band within 10 yr, allowing for multiband gravitational wave observations. Binaries formed via dynamical interactions in dense star clusters are expected to have eccentricities e0˜10-3-10-1 at the frequencies f0=10-2 Hz where eLISA is most sensitive, while binaries formed in the field should have negligible eccentricity in both frequency bands. We estimate that eLISA should always be able to detect a nonzero e0 whenever e0≳10-2; if e0˜10-3, eLISA should detect nonzero eccentricity for a fraction ˜90 % (˜25 %) of binaries when the observation time is Tobs=5 (2) yr, respectively. Therefore eLISA observations of black hole binaries have the potential to distinguish between field and cluster formation scenarios.

  1. Assessment of an ELISA Laboratory Exercise

    ERIC Educational Resources Information Center

    Robinson, David L.; Lau, Joann M.

    2012-01-01

    The enzyme-linked immunosorbent assay (ELISA) is a powerful immunological technique for quantifying small amounts of compounds and has been used in research and clinical settings for years. Although there are laboratory exercises developed to introduce the ELISA technique to students, their ability to promote student learning has not been…

  2. A Simple ELISA Exercise for Undergraduate Biology.

    ERIC Educational Resources Information Center

    Baker, William P.; Moore, Cathy R.

    Understanding of immunological techniques such as the Enzyme Linked Immuno Sorbent Assay (ELISA) is an important part of instructional units in human health, developmental biology, microbiology, and biotechnology. This paper describes a simple ELISA exercise for undergraduate biology that effectively simulates the technique using a paper model.…

  3. A Protein Microarray ELISA for Screening Biological Fluids

    SciTech Connect

    Varnum, Susan M.; Woodbury, Ronald L.; Zangar, Richard C.

    2004-02-01

    Protein microarrays permit the simultaneous measurement of many proteins in a small sample volume and therefore provide an attractive approach for the quantitative measurement of proteins in biological fluids, including serum. This chapter describes a microarray ELISA assay. Capture antibodies are immobilized onto a glass surface, the covalently attached antibodies bind a specific antigen from a sample overlaying the array. A second, biotinylated antibody that recognizes the same antigen as the first antibody but at a different epitope is then used for detection. Detection is based upon an enzymatic signal enhancement method known as tyramide signal amplification (TSA). By coupling a microarray-ELISA format with the signal amplification of tyramide deposition, the assay sensitivity is as low as sub-pg/ml.

  4. ELISA-BASE: An Integrated Bioinformatics Tool for Analyzing and Tracking ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Collett, James L.; Seurynck-Servoss, Shannon L.; Daly, Don S.; Zangar, Richard C.

    2009-06-15

    ELISA-BASE is an open-source database for capturing, organizing and analyzing protein enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Soft-ware Environment (BASE) database system, which was developed for DNA microarrays. In order to make BASE suitable for protein microarray experiments, we developed several plugins for importing and analyzing quantitative ELISA microarray data. Most notably, our Protein Microarray Analysis Tool (ProMAT) for processing quantita-tive ELISA data is now available as a plugin to the database.

  5. Multicenter comparative study of a new ELISA, PLATELIA RABIES II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people.

    PubMed

    Feyssaguet, M; Dacheux, L; Audry, L; Compoint, A; Morize, J L; Blanchard, I; Bourhy, H

    2007-03-01

    The envelope glycoprotein G of rabies virus induces the production of neutralising antibodies, which are important in protection against rabies. Therefore, titration of anti-envelope glycoprotein antibodies is a good indicator of the degree of immunity in people during anti-rabies treatment or after vaccination. According to the World Health Organization (WHO) guidelines, a booster vaccine dose should be given if the rabies antibody titre falls below 0.5 IU/ml. Titration of anti-rabies antibodies is also useful for plasma centers in the preparation and standardization of human anti-rabies gamma-globulins for therapeutic use and to a lesser extent for the diagnosis of rabies in human sera and cerebrospinal fluid (CSF). This paper presents a new enzyme-linked immunosorbent assay (ELISA), PLATELIA RABIES II, developed for rabies envelope glycoprotein antibody detection or titration and its comparison to the current reference method (RFFIT). The data collected during validation of the test in a multicenter study are analysed to give a sound overall knowledge of the capabilities of the PLATELIA RABIES II, for instance specificity, linearity, accuracy, precision, detection limit and quantitation limit. To this aim, human serum samples from a total of 1348 vaccinated or non-vaccinated people were tested in parallel using the new ELISA and the RFFIT for the presence of anti-rabies antibodies. Data generated indicate a linear relationship across the range of titration between the two methods. The sensitivity reaches 98.6% and the specificity 99.4%. This study indicates that this new ELISA test is as sensitive and specific as the current standardized reference method. The method is simple, safe, rapid and can be considered as a useful alternative to the neutralisation test.

  6. Simultaneous measurement of multiple ear proteins with multiplex ELISA assays.

    PubMed

    Trune, Dennis R; Larrain, Barbara E; Hausman, Frances A; Kempton, J Beth; MacArthur, Carol J

    2011-05-01

    A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 h following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys Biosciences, Aushon Biosystems SearchLight (both microplate-based), MILLIPLEX MAP Sample (bead-based), and a RayBiotech, Inc (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokines related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1β, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative

  7. The efficacy of ELISA commercial kits for the screening of equine infectious anemia virus infection.

    PubMed

    Alvarez, Irene; Cipolini, Fabiana; Wigdorovitz, Andrés; Trono, Karina; Barrandeguy, Maria E

    2015-01-01

    The most used and reliable indicator of Equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100% diagnostic sensitivity, and 92.3-94.3% diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV infected animals, allowing to identify a higher number of positive horse cases. Thus, ELISA assays can be useful tools in EIA control and eradication.

  8. Investigation of enrofloxacin residues in broiler tissues using ELISA and LC-MS/MS.

    PubMed

    Panzenhagen, Pedro Henrique N; Aguiar, Waldemir S; Gouvêa, Raquel; de Oliveira, Andréa M G; Barreto, Fabiano; Pereira, Virgínia L A; Aquino, Maria Helena C

    2016-01-01

    This study investigated the efficiency of an enrofloxacin ELISA test kit to detect the presence of enrofloxacin residues in broiler tissues compared with LC-MS/MS. Broiler tissues from 72 samples consisting of 60 breast muscle, six pools of livers (500 g each) and six pools of kidneys (500 g each) were obtained from six different slaughterhouses. Breast muscle from 10 carcasses and pools of livers and kidneys from approximately 200 carcasses of the same flock were collected from each slaughterhouse. ELISA and HPLC were used to identify and quantify the contamination of the samples with enrofloxacin. A total of 72% of the analysed samples contained enrofloxacin residues detected by the ELISA and 22.2% were detected by LC-MS/MS. The mean values of enrofloxacin contamination found in chicken breast by ELISA and HPLC were 8.63 and 12.25 μg kg(-1), respectively. None of the samples exceeded the maximum limit of 100 μg kg(-1) by both methods set by the European Union as well as the Brazilian Agriculture Ministry. All positive samples for enrofloxacin residues detected by LC-MS/MS were also positive by ELISA. These data confirm the efficiency of the ELISA test, and suggest its use as a screening method for enrofloxacin residues in poultry tissues due to its quick results, low price and ease of applicability.

  9. [Use of immunoenzyme (ELISA) diagnosis of Aujeszky's disease in pigs during the recovery period].

    PubMed

    Rodák, L; Smíd, B; Valícek, L; Jurák, E; Veselý, T

    1986-10-01

    Samples of blood and blood serums of pigs were examined for the presence of antibodies to the Aujeszky's disease virus. The virus-neutralizing (VN) test and the enzymoimmunologic (ELISA) method were used for this examination. As indicated by comparison of the average titres of antiviral antibodies determined by both methods, the ELISA method is 60 to 600 times more sensitive than the VN test. The high sensitivity of the ELISA method enabled to detect antiviral antibodies even in samples considered as negative after VN-testing. The method has been used with success for the sanitation of three swine stocks where the Aujeszky's disease was eradicated without interruption of operation.

  10. Evaluation of a commercially available ELISA kit as a tool to determine BTEX in groundwater.

    PubMed

    Francioni, E; Fillmann, G; Hamacher, C; Wagener, A De Luca R; Depledge, M H; Readman, J W; Meniconi, M De Fátima Guadalupe

    2003-06-01

    The reliability of enzyme-linked immunosorbent assay (ELISA) tests as a screening technique to address groundwater contamination was tested in an area following leakage of gasoline from a petrol station. Immunoassay data of benzene, toluene, ethylbenzene, and o-, m- and p-xylene (BTEX) were compared with results obtained using capillary gas chromatographic analysis. Detection limits were of 20 microg l(-1) for ELISA and 0.3 microg l(-1) for gas chromatography with flame ionization and photoionization detectors (GC-FID/PID) determination. Despite an observed overestimation of BTEX concentrations as given by ELISA, the tests responded reliably to different levels of contamination.

  11. The Single Intradermal Cervical Comparative Test Interferes with Johne's Disease ELISA Diagnostics.

    PubMed

    Kennedy, Aideen E; Da Silva, Ana T; Byrne, Noel; Govender, Rodney; MacSharry, John; O'Mahony, Jim; Sayers, Riona G

    2014-01-01

    Enzyme-linked immunosorbent assays (ELISA) of milk and serum samples are a routinely used method of screening herds for Mycobacterium avium subspecies paratuberculosis (MAP). Infection with MAP causes granulomatous enteritis of ruminants known as Johne's disease (JD). The sensitivity (Se) and specificity (Sp) of MAP ELISAs leads to difficulties in the identification of both infected and infectious animals. Interference with MAP ELISA Se and Sp has been reported in MAP seronegative cows following administration of purified protein derivative (PPD) as part of intradermal testing for bovine tuberculosis (bTB). The aim of this study is to examine the impact of the single intradermal cervical comparative test (SICCT) for bTB, on both serum and milk MAP ELISA tests, in a herd containing both seropositive and seronegative cows pre-SICCT. A secondary objective is to provide appropriate timing of JD ELISA tests in relation to the SICCT. A herd of 139 cows were serum and milk sampled pre- and post-SICCT administration. Prior to SICCT, 6% of the herd tested seropositive for MAP using milk ELISA, with 8% positive on serum. ID Screen Paratuberculosis Indirect Screening Test (ID Vet) was used to screen the herd. Within 14 days of PPD administration, a significant increase in the prevalence of seropositive cows was recorded. Identical prevalence's were recorded with both test matrices (39%). ELISA values remained significantly higher until day 43 post-SICCT in milk (P = 0.850), and day 71 in serum (P = 0.602). If the "new" positives detected post-bTB testing are deemed false positives due to generation of cross-reacting antibodies by administration of PPD, milk would appear a more suitable sample for JD ELISA testing within 2 months of SICCT. In summary, sampling for JD utilizing milk ELISA should be avoided in the 43-day period following PPD administration, with serum ELISA sampling avoided for an additional 28 days.

  12. Development of indirect ELISAs for differential serodiagnosis of classical and highly pathogenic porcine reproductive and respiratory syndrome virus.

    PubMed

    Xiao, Y H; Wang, T T; Zhao, Q; Wang, C B; Lv, J H; Nie, L; Gao, J M; Ma, X C; Hsu, W H; Zhou, E M

    2014-08-01

    The objective of this study was to develop two indirect enzyme-linked immunosorbent assays (iELISAs) for detection of serum antibodies against classical vaccine strain of porcine reproductive and respiratory syndrome virus (PRRSV) and highly pathogenic PRRSV (HP-PRRSV). To detect the common antibodies against classical and HP-PRRSV, the coating antigen used in the iELISA (designated iELISA-180) was the antigen of Nsp2-180, the 180aa at amino terminal of Nsp2. To detect the different antibodies against classical and HP-PRRSV, the coating antigen in the second iELISA (designated iELISA-D29) was Nsp2-D29, the deleted 29aa in Nsp2 of HP-PRRSV. The antigen concentration and serum dilutions were optimized using a draughtboard titration. The cut-off values of 0.361 at OD(450nm) for the iELISA-180 and 0.27 at OD(450nm) for the iELISA-D29 were determined by testing a panel of 120 classical PRRSV positive and 198 PRRSV negative pig serum samples, which generated the specificity of 97.1% and 96.7%, the sensitivity of 96.9% and 96.3% for iELISA-180 and iELISA-D29, respectively. The agreements between the Western blot and iELISA-180 and iELISA-D29 were 98%, 96.7%, respectively. The developed iELISAs can be used to differentiate serologically HP-PRRSV from the vaccinated or classical PRRSV in clinical serum samples.

  13. Development of a near-infrared fluorescence ELISA method using tyramide signal amplification.

    PubMed

    Gong, Haibiao; Cradduck, Mark; Cheung, Lael; Olive, D Michael

    2012-07-01

    In this study, we applied tyramide signal amplification (TSA) to fluorescence enzyme-linked immunosorbent assay (ELISA) employing horseradish peroxidase (HRP) as the detection enzyme. When used with a human epidermal growth factor ELISA kit, the TSA method led to a >100-fold increase in fluorescence signal intensity in comparison to an unamplified method. It also showed wider dynamic range and better sensitivity compared to a conventional method using tetramethylbenzidine as the HRP substrate. PMID:22490466

  14. Use of a commercial ELISA for the detection of measles-specific immunoglobulin G (IgG) in dried blood spots collected from children living in low-resource settings.

    PubMed

    Colson, K Ellicott; Potter, Alan; Conde-Glez, Carlos; Hernandez, Bernardo; Ríos-Zertuche, Diego; Zúñiga-Brenes, Paola; Iriarte, Emma; Mokdad, Ali H

    2015-09-01

    Seroepidemiological monitoring of population immunity to vaccine-preventable diseases is critical to prevent future outbreaks. Dried blood spots (DBS), drops of capillary blood dried on filter paper, are an affordable, minimally invasive alternative to venipuncture for collecting blood in field settings. However, few proven methods exist to analyze DBS for the presence of protective antibodies. This study validates a novel technique for measuring measles-specific immunoglobulin G (IgG) in capillary DBS using a commercial ELISA. The predictive performance of a new method for analyzing DBS was tested by comparing matched serum and DBS samples from 50 children. The accuracy, precision, and reliability of the procedure were evaluated, and the optimal cut points to classify positive and negative samples were determined. The method was then applied to 1,588 DBS collected during a large survey of children in Mexico and Nicaragua. Measles-specific IgG in serum samples were 62% negative, 10% equivocal, and 28% positive. In comparisons with matched serum, DBS results were 100% sensitive and 96 · 8% specific, and agreed in 46 of 50 (92%) cases. The inter-assay and intra-assay coefficients of variation from kit-provided controls were greater than desired (24.8% and 8.4%, respectively). However, in predictive simulations the average misclassification was only 3.9%. Procedures were found to be acceptable to surveyors and participants. Analyzing DBS collected in low-resources settings is a feasible and accurate means of measuring population immunity to measles and should be used to generate objective measures of health status and health system performance. PMID:25988945

  15. A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

    PubMed Central

    Hjelmevoll, Stig Ove; Olsen, Merethe Elise; Sollid, Johanna U. Ericson; Haaheim, Håkon; Unemo, Magnus; Skogen, Vegard

    2006-01-01

    Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection. PMID:17065426

  16. Diagnosis of theileria equi infections in horses in the Azores using cELISA and nested PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Equine piroplasmosis is a tick-borne disease of equids that is often caused by the parasite Theileria equi. We applied competitive ELISA (cELISA) and nested PCR diagnostic methods to detect this parasite in horses by screening 162 samples from mainland Portugal where the parasite is endemic, and 143...

  17. Comparison of GD2 Binding Capture ELISA Assays for Anti-GD2-Antibodies Using GD2-Coated Plates and a GD2-Expressing Cell-Based ELISA

    PubMed Central

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Mitra, Gautam

    2011-01-01

    Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2’-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment. PMID:21893062

  18. Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

    PubMed Central

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

    2013-01-01

    Background Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. Aim The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. Results A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. Conclusions Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms. PMID:23509607

  19. The design of wavelength selector for full-automatic ELISA analyzer

    NASA Astrophysics Data System (ADS)

    Bao, Yan; Dong, Mingli; Zhu, Lianqing; Chang, Haitao; Li, Hong

    2010-12-01

    In recent years, ELISA technology has been developed rapidly and full-automatic ELISA analyzer, which is of significant practical value is widely used in the diagnosis of many diseases, such as, bacteria and viruses. Optical detection system is the hard core of fully automated ELISA analyzer and the key part of system is high-precision wavelength selector. The authors in this paper present the design of wavelength selector composed of light source, optical circuit plan, color filters and the module of signal acquisition. A control system for stepper motor which is used to choose the suitable color filters based on the microcontroller 8051 is introduced. From the results of experiment test, it can be seen that the wavelength selector is sufficient to meet the requirements of the full-automatic ELISA analyzer.

  20. The design of wavelength selector for full-automatic ELISA analyzer

    NASA Astrophysics Data System (ADS)

    Bao, Yan; Dong, Mingli; Zhu, Lianqing; Chang, Haitao; Li, Hong

    2011-05-01

    In recent years, ELISA technology has been developed rapidly and full-automatic ELISA analyzer, which is of significant practical value is widely used in the diagnosis of many diseases, such as, bacteria and viruses. Optical detection system is the hard core of fully automated ELISA analyzer and the key part of system is high-precision wavelength selector. The authors in this paper present the design of wavelength selector composed of light source, optical circuit plan, color filters and the module of signal acquisition. A control system for stepper motor which is used to choose the suitable color filters based on the microcontroller 8051 is introduced. From the results of experiment test, it can be seen that the wavelength selector is sufficient to meet the requirements of the full-automatic ELISA analyzer.

  1. Synthetic Peptide-Based ELISA and ELISpot Assay for Identifying Autoantibody Epitopes.

    PubMed

    Pozsgay, Judit; Szarka, Eszter; Huber, Krisztina; Babos, Fruzsina; Magyar, Anna; Hudecz, Ferenc; Sarmay, Gabriella

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is an invaluable diagnostic tool to detect serum autoantibody binding to target antigen. To map the autoantigenic epitope(s), overlapping synthetic peptides covering the total sequence of a protein antigen are used. A large set of peptides synthesized on the crown of pins can be tested by Multipin ELISA for fast screening. Next, to validate the results, the candidate epitope peptides are resynthesized by solid-phase synthesis, coupled to ELISA plate directly, or in a biotinylated form, bound to neutravidin-coated surface and the binding of autoantibodies from patients' sera is tested by indirect ELISA. Further, selected epitope peptides can be applied in enzyme-linked immunospot assay to distinguish individual, citrullinated peptide-specific autoreactive B cells in a pre-stimulated culture of patients' lymphocytes. PMID:26490479

  2. Development of an automated on-chip bead-based ELISA platform

    PubMed Central

    Campbell, Jennifer; Pollock, Nira; Sharon, Andre; Sauer-Budge, Alexis F.

    2015-01-01

    We present a lab-on-a-chip and associated instrument for heterogeneous enzyme-linked immunosorbent assay (ELISA)-based detection of proteins from liquid samples. The system performs all necessary ELISA steps (starting from antigen incubation) in a quarter of the time required for corresponding plate-based protocols. We have previously described the instrument, which automates fluidic control via remote valve switching and detects fluorescence from reacted substrate, for use in a molecular diagnostics application. The ELISA chip reported here utilizes a high surface area bead bed to enhance capture efficiency and increase the dynamic range of the assay as compared to a standard plate-based ELISA. Its functionality is demonstrated using human IL-10 as a model antigen, but theoretically any sandwich ELISA could be ported onto this “open source platform.” We show that our automated on-chip assays have greater sensitivities than the corresponding standard manual plate-based ELISAs, and that single samples can be assayed in a fraction of the time. PMID:26523155

  3. Yersinia enterocolitica serodiagnosis: a dual role of specific IgA. Evaluation of microagglutination and ELISA.

    PubMed

    Bitzan, M; Häck, H J; Mauff, G

    1987-12-01

    The microagglutination technique for the detection of antibodies against Y. enterocolitica, serovars 3 and 9 (corresponding to O-groups I and V), was compared with the conventional tube agglutination. An immunoglobulin class specific, indirect ELISA (polyvalent immunoglobulin, IgG, IgM, and IgA) was established employing as antigens formalinized whole bacteria ("OH"-antigens) and LPS preparations (hot phenol-water extraction). ELISA titers and net absorbancy (ELISA-"units") of single serum dilutions were in good agreement; the same was true for ELISA and agglutination results. Specificity (against healthy controls) and sensitivity of both serologic techniques were comparable. Cross-reacting antibodies against serovars 3 and 9 could be identified in the ELISA. Correct serovar-specific diagnosis was possible in 95% with a single assay (polyvalent Ig assay with LPS-antigen). The sensitivity of the LPS-ELISA was superior to the "OH" antigen assay after infections by serovar 3 strains, and antibodies were detected with LPS preparations for a longer period following reconvalescence. Specific IgA, due to its rapid decrease during reconvalescence, on one hand impresses as a valuable marker for the differentiation of recent disease from uncomplicated past infections, while persistence of IgA appears to be associated with Yersinia-induced arthritis. Persisting IgM but rarely IgA titers were characteristically found in patients with prolonged enteric yersiniosis.

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

    PubMed

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

    2008-04-17

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

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  6. eLISA: Astrophysics and cosmology in the millihertz regime

    NASA Astrophysics Data System (ADS)

    Amaro-Seoane, Pau; Aoudia, Sofiane; Babak, Stanislav; Binétruy, Pierre; Berti, Emanuele; Bohé, Alejandro; Caprini, Chiara; Colpi, Monica; Cornish, Neil J.; Danzmann, Karsten; Dufaux, Jean-François; Gair, Jonathan; Hinder, Ian; Jennrich, Oliver; Jetzer, Philippe; Klein, Antoine; Lang, Ryan N.; Lobo, Alberto; Littenberg, Tyson; McWilliams, Sean T.; Nelemans, Gijs; Petiteau, Antoine; Porter, Edward K.; Schutz, Bernard F.; Sesana, Alberto; Stebbins, Robin; Sumner, Tim; Vallisneri, Michele; Vitale, Stefano; Volonteri, Marta; Ward, Henry; Wardell, Barry

    2013-05-01

    This document introduces the exciting and fundamentally new science and astronomy that the European New Gravitational Wave Observatory (NGO) mission (derived from the previous LISA proposal) will deliver. The mission (which we will refer to by its informal name ``eLISA'') will survey for the first time the low-frequency gravitational wave band (about 0:1 mHz to 1 Hz), with sufficient sensitivity to detect interesting individual astrophysical sources out to z = 15. The measurements described here will address the basic scientific goals that have been captured in ESA's ``New Gravitational Wave Observatory Science Requirements Document''; they are presented here so that the wider scientific community can have access to them. The eLISA mission will discover and study a variety of cosmic events and systems with high sensitivity: coalescences of massive black holes binaries, brought together by galaxy mergers; mergers of earlier, less-massive black holes during the epoch of hierarchical galaxy and black-hole growth; stellar-mass black holes and compact stars in orbits just skimming the horizons of massive black holes in galactic nuclei of the present era; extremely compact white dwarf binaries in our Galaxy, a rich source of information about binary evolution and about future Type Ia supernovae; and possibly most interesting of all, the uncertain and unpredicted sources, for example relics of inflation and of the symmetry-breaking epoch directly after the Big Bang. eLISA's measurements will allow detailed studies of these signals with high signal-to-noise ratio, addressing most of the key scientific questions raised by ESA's Cosmic Vision programme in the areas of astrophysics and cosmology. They will also provide stringent tests of general relativity in the strong-field dynamical regime, which cannot be probed in any other way. This document not only describes the science but also gives an overview on the mission design and orbits. LISA's heritage in the eLISA design

  7. Specificity of a Polyclonal Fecal Elastase ELISA for CELA3

    PubMed Central

    Budde, Christoph; Lerch, Markus M.

    2016-01-01

    Introduction Elastase is a proteolytic pancreatic enzyme that passes through the gastrointestinal tract undergoing only limited degradation. ELISA tests to determine stool elastase concentrations have therefore been developed for the diagnosis of exocrine pancreatic insufficiency. Five different isoforms of pancreatic elastase (CELA1, CELA2A, CELA2B, CELA3A, CELA3B) are encoded in the human genome. We have investigated three different polyclonal antisera that are used in a commercial fecal elastase ELISA to determine their specificity for different pancreatic elastase isoforms. Material and Methods Different polyclonal rabbit antisera against human elastase peptides (BIOSERV Diagnostics GmbH, Germany) were tested by Western blot analysis of human pancreatic juice, in HEK-293 cells expressing Elastase constructs, and in the protein content of porcine pancreatin, used for treatment of exocrine pancreatic insufficiency. Results In human pancreatic juice the polyclonal antisera detected proteins at the corresponding size of human pancreatic elastase isoforms (~29kDa). Transiently expressed GFP fusion protein of elastase isoform CELA3A (CELA3A-GFP), but not CELA2A (CELA2A-GFP) could be precipitated from HEK-293 cell lysates with the elastase antisera. We detected no cross-reactivity with elastases in the porcine pancreatic extracts (pancreatin) used for enzyme replacement therapy. Conclusion The polyclonal antisera used in a commercial fecal elastase ELISA are specific for the human pancreatic elastase isoform CELA3 and do not cross-react with elastase contained in pig pancreatin. While pancreatic elastase 1 (CELA1) is not expressed in the adult human pancreas, possible differences between the other isoforms regarding their cellular expression, pathophysiological role and relevance in exocrine pancreatic insufficiency deserve further investigation. PMID:27459204

  8. Development of an antibody capture ELISA using inactivated Ebola Zaire Makona virus.

    PubMed

    Krähling, Verena; Becker, Dirk; Rohde, Cornelius; Eickmann, Markus; Eroğlu, Yonca; Herwig, Astrid; Kerber, Romy; Kowalski, Katharina; Vergara-Alert, Júlia; Becker, Stephan

    2016-04-01

    The 2014 Zaire Ebola virus (ZEBOV) outbreak in West Africa represents an international public health concern. Highly sensitive and precise diagnostic tools are needed. In the present study, we developed a ZEBOV-specific enzyme-linked immunosorbent assay (ELISA) using inactivated ZEBOV isolate Makona from March 2014. Mock antigen was used to address nonspecific binding. Specificity, reproducibility and precision were determined to measure assay performance. The ZEBOV ELISA proved to be specific (96 %), reproducible and precise (Intra-assay CV 8 %, Inter-assay CV 18 %). Using the human monoclonal antibody KZ52, we showed that the ELISA was able to detect conformation-specific antibodies. Monitoring antibody development in 29 PCR-positive EBOV disease (EVD) patients revealed seroconversion in all cases. In addition, the ELISA was used to detect ZEBOV glycoprotein (GP)-specific antibodies in a vaccinated volunteer from day 14 until 5 years post-vaccination with a VSV-ZEBOV candidate vaccine. The results demonstrate the high reproducibility, specificity and sensitivity of this newly developed ELISA, which is suitable for the detection of specific antibody responses directed against different ZEBOV proteins in EVD patients and against the ZEBOV surface glycoprotein GP in vaccinated individuals. PMID:26475282

  9. Development of antigen capture ELISA for the quantification of EIAV p26 protein.

    PubMed

    Hu, Zhe; Chang, Hao; Ge, Man; Lin, Yuezhi; Wang, Xuefeng; Guo, Wei; Wang, Xiaojun

    2014-11-01

    An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9-62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3-8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3-8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R (2) =0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium.

  10. Development of an antibody capture ELISA using inactivated Ebola Zaire Makona virus.

    PubMed

    Krähling, Verena; Becker, Dirk; Rohde, Cornelius; Eickmann, Markus; Eroğlu, Yonca; Herwig, Astrid; Kerber, Romy; Kowalski, Katharina; Vergara-Alert, Júlia; Becker, Stephan

    2016-04-01

    The 2014 Zaire Ebola virus (ZEBOV) outbreak in West Africa represents an international public health concern. Highly sensitive and precise diagnostic tools are needed. In the present study, we developed a ZEBOV-specific enzyme-linked immunosorbent assay (ELISA) using inactivated ZEBOV isolate Makona from March 2014. Mock antigen was used to address nonspecific binding. Specificity, reproducibility and precision were determined to measure assay performance. The ZEBOV ELISA proved to be specific (96 %), reproducible and precise (Intra-assay CV 8 %, Inter-assay CV 18 %). Using the human monoclonal antibody KZ52, we showed that the ELISA was able to detect conformation-specific antibodies. Monitoring antibody development in 29 PCR-positive EBOV disease (EVD) patients revealed seroconversion in all cases. In addition, the ELISA was used to detect ZEBOV glycoprotein (GP)-specific antibodies in a vaccinated volunteer from day 14 until 5 years post-vaccination with a VSV-ZEBOV candidate vaccine. The results demonstrate the high reproducibility, specificity and sensitivity of this newly developed ELISA, which is suitable for the detection of specific antibody responses directed against different ZEBOV proteins in EVD patients and against the ZEBOV surface glycoprotein GP in vaccinated individuals.

  11. Multicolor ELISA based on alkaline phosphatase-triggered growth of Au nanorods.

    PubMed

    Li, Yanyan; Ma, Xiaoming; Xu, Zhengming; Liu, Meihua; Lin, Zhenyu; Qiu, Bin; Guo, Longhua; Chen, Guonan

    2016-05-10

    Seed-mediated synthesis of gold nanorods (AuNRs) has been widely used for diverse applications in the past decade. In this work, this synthetic process is demonstrated for multicolor biosensing for the first time. Our investigation reveals that ascorbic acid acts as a key factor to mediate the growth of AuNRs. This phenomenon is incorporated into the alkaline phosphatase (ALP)-enzyme-linked immunosorbent assay (ELISA) system based on the fact that ALP can catalyze the conversion of ascorbic acid-phosphate into ascorbic acid with high efficiency. This allows us to develop a multicolor ELISA approach for sensitive detection of disease biomarkers with the naked eye. We show the proof-of-concept multicolor ELISA for the detection of prostate-specific antigen (PSA) in human serum. The results show that different colors are presented in response to different concentrations of PSA, and a detection limit of 3 × 10(-15) g mL(-1) in human serum was achieved. The proposed multicolor ELISA could be a good supplement to conventional ELISA for POC diagnostics. PMID:27050384

  12. Quantitative Analysis of Protein-DNA Interaction by qDPI-ELISA.

    PubMed

    Fischer, Stefan M; Böser, Alexander; Hirsch, Jan P; Wanke, Dierk

    2016-01-01

    The specific binding of DNA-binding proteins to their cognate DNA motifs is a crucial step for gene expression control and chromatin organization in vivo. The development of methods for the identification of in vivo binding regions by, e.g. chromatin immunoprecipitation (ChIP) or DNA adenine methyltransferase identification (Dam-ID) added an additional level of qualitative information for data mining in systems biology or applications in synthetic biology. In this respect, the in vivo techniques outpaced methods for thorough characterization of protein-DNA interaction and, especially, of the binding motifs at single base-pair resolution. The elucidation of DNA-binding capacities of proteins is frequently done with methods such as yeast one-hybrid, electrophoretic mobility shift assay (EMSA) or systematic evolution of ligands by exponential enrichment (SELEX) that provide only qualitative binding information and are not suited for automation or high-throughput screening of several DNA motifs. Here, we describe the quantitative DNA-protein-Interaction-ELISA (qDPI-ELISA) protocol, which makes use of fluorescent fusion proteins and, hence, is faster and easier to handle than the classical DPI-ELISA. Although every DPI-ELISA experiment delivers quantitative information, the qDPI-ELISA has an increased consistency, as it does not depend on immunological detection. We demonstrate the high comparability between probes and different protein extracts in qDPI-ELISA experiments. PMID:27557760

  13. Validation of an ELISA for the quantitation of lanoteplase, a novel plasminogen activator.

    PubMed

    Stouffer, B; Habte, S; Vachharajani, N; Tay, L

    1999-11-01

    An ELISA was developed and validated for the quantitation of lanoteplase in human citrated plasma. The ELISA employed a monoclonal anti-lanoteplase antibody absorbed onto 96-well microtiter plates to capture lanoteplase in citrated human plasma samples containing PPACK, a protease inhibitor. The captured lanoteplase was detected using a biotinylated rabbit anti-lanoteplase polyclonal antibody. The standard curve range in human plasma for the ELISA was 7-100 ng/ml. Assessment of individual standard curve variability indicated reproducible responses with r2 values of > or = 0.985. The accuracy (% DEV) and precision (%RSD) estimates for the ELISA based on the predicted values from quality control (QC) samples were within 7.3% and 11%, respectively. Cross-reactivity with t-PA was determined to be less than 11% by ELISA. The stability of lanoteplase was established in human citrated PPACK plasma for 24 hours at 4 degrees C, for 2 months at -20 degrees C, for 22 months at -70 degrees C, three weeks at room temperature, and through four freeze/thaw cycles. To quantify lanoteplase plasminogen activator (PA) activity, a commercially available chromogenic activity assay was also validated. This method and its application is described briefly here. The lanoteplase ELISA as well as the commercial activity method were successfully employed to evaluate the pharmacokinetic parameters of lanoteplase in support of clinical Phase II/III studies. PMID:10595857

  14. ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

    PubMed

    Tong, Qing-He; Tao, Tao; Xie, Li-Qi; Lu, Hao-Jie

    2016-06-15

    Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs.

  15. Quantitative estimation of Newcastle disease virus antibody levels in chickens and turkeys by ELISA.

    PubMed

    Adair, B M; McNulty, M S; Todd, D; Connor, T J; Burns, K

    1989-01-01

    A quantitative single-well ELISA for estimation of Newcastle disease (ND) virus antibodies in chickens and turkeys was developed using purified antigen from PMV-1/Chicken/Ulster 2C/71. The test was standardised using sera from 20-week-old chickens or 20- 30-week-old turkeys. Absorbance values for negative sera in chickens increased with the age of the birds but overall was lower than the cut-off for the test. ND haemagglutination inhibition (HI) positive field sera were always positive by ELISA and the mean was significantly higher than that of the negative population. Standard antisera to four of seven of the other PMV serotypes (including PMV-3) gave positive reactions in ELISA and three were also positive at low level by PMV-1 HI test. Absorbance values remained negative in turkeys given two inoculations of PMV-3 vaccine in spite of good PMV-3 HI responses. Doubling dilutions of chicken and turkey sera were tested by ELISA and end-point titres calculated. Standard curves relating ELISA titre and absorbance of each sample at 1/100 dilution were constructed and used to determine titres of test samples from single well absorbance values. A significant positive correlation between ELISA titre and HI titre for chicken and turkey sera was demonstrated. Sensitivity of the test was investigated using birds experimentally infected with PMV-1/Chicken/Ulster 2C/71 or a pigeon PMV-1 isolate. Seroconversion was detected at the same time by ELISA and HI. In experiments to estimate the ELISA titre required to protect birds against virulent ND, five groups of chickens were vaccinated twice with one of four commercial ND vaccines (three inactivated; one live) on two occasions and challenged with a virulent ND strain (PMV-1/ Chicken/Antrim/73). Two of the groups vaccinated with inactivated vaccines were protected against challenge. In another group also given inactivated vaccine, clinical signs were seen in one bird and ELISA proved a better indicator of immune status than HI. In

  16. Comparison of a commercial dengue IgM capture ELISA with dengue antigen focus reduction microneutralization test and the Centers for Disease Control dengue IgM capture-ELISA.

    PubMed

    Welch, Ryan J; Chang, Gwong-Jen J; Litwin, Christine M

    2014-01-01

    Dengue (DENV) infection is caused by an arbovirus that is a member of the family Flaviviridae, genus Flavivirus. The diagnosis of acute dengue infection using clinical signs and symptoms can be difficult since the manifestations cannot be readily differentiated from other infections. Therefore the diagnosis of acute dengue infection depends upon laboratory assays. Dengue virus ELISAs have been designed for the detection of IgM and IgG antibodies in addition to nonstructural 1 (NS1) antigens. The InBios IgM Dengue ELISA was compared to the Antigen Focus Reduction Microneutralization Test (FRμNT90) and Centers for Disease Control Dengue IgM Capture-ELISA (CDC MAC-ELISA). The calculated sensitivity, specificity and agreement of the InBios ELISA compared to FRμNT90 and CDC MAC-ELISA was 88.7% (C.I. 81.4-93.7), 93.1% (C.I. 89.1-95.8) and 91.5% (C.I. 86.3-95.0), respectively. In summary the InBios IgM Dengue ELISA sensitivity and specificity is comparable to other commercially available IgM Capture-ELISAs.

  17. A Double-Sandwich ELISA for Identification of Monoclonal Antibodies Suitable for Sandwich Immunoassays.

    PubMed

    Stanker, Larry H; Hnasko, Robert M

    2015-01-01

    The sandwich immunoassay (sELISA) is an invaluable technique for concentrating, detecting, and quantifying target antigens. The two critical components required are a capture antibody and a detection antibody, each binding a different epitope on the target antigen. The specific antibodies incorporated into the test define most of the performance parameters of any subsequent immunoassay regardless of the assay format: traditional ELISA, lateral-flow immunoassay, various bead-based assays, antibody-based biosensors, or the reporting label. Here we describe an approach for identifying monoclonal antibodies (mAbs) suitable for use as capture antibodies and detector antibodies in a sELISA targeting bacterial protein toxins. The approach was designed for early identification of monoclonal antibodies (mAbs), in the initial hybridoma screen.

  18. Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1

    PubMed Central

    Li, Meng; Zhang, Bicheng; Chen, Kangming; He, Kongwang

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC’s intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples. PMID:27603508

  19. Diagnostic parameters of serological ELISA for invasive amoebiasis, using antigens preserved without enzymatic inhibitors.

    PubMed

    Flores, María S; Carrillo, Perla; Tamez, Eva; Rangel, Roberto; Rodríguez, Elba G; Maldonado, María G; Isibasi, Armando; Galán, Luis

    2016-02-01

    Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemagglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis. PMID:26684288

  20. Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.

    PubMed

    Zhang, Xuehan; Li, Meng; Zhang, Bicheng; Chen, Kangming; He, Kongwang

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC's intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples. PMID:27603508

  1. Diagnostic parameters of serological ELISA for invasive amoebiasis, using antigens preserved without enzymatic inhibitors.

    PubMed

    Flores, María S; Carrillo, Perla; Tamez, Eva; Rangel, Roberto; Rodríguez, Elba G; Maldonado, María G; Isibasi, Armando; Galán, Luis

    2016-02-01

    Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemagglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis.

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

    PubMed

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

    2014-10-21

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

  3. Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes

    PubMed Central

    Liu, Chengqian; Skaldin, Maksym; Wu, Chengxiang; Lu, Yuanan; Zavialov, Andrey V.

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens. PMID:27510152

  4. Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes.

    PubMed

    Liu, Chengqian; Skaldin, Maksym; Wu, Chengxiang; Lu, Yuanan; Zavialov, Andrey V

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens. PMID:27510152

  5. DLISA: A DNAzyme-Based ELISA for Protein Enzyme-Free Immunoassay of Multiple Analytes.

    PubMed

    Hu, Rong; Liu, Tao; Zhang, Xiao-Bing; Yang, Yunhui; Chen, Tao; Wu, Cuichen; Liu, Yuan; Zhu, Guizhi; Huan, Shuangyan; Fu, Ting; Tan, Weihong

    2015-08-01

    A DNAzyme-based ELISA, termed DLISA, was developed as a novel protein enzyme-free, triply amplified platform, combining a catalytic and molecular beacon (CAMB) system with a cation exchange reaction for ultrasensitive multiplex fluorescent immunosorbent assay. Classical ELISA, which employs protein enzymes as biocatalysts to afford amplified signals, suffers from poor stability caused by the irreversible denaturation of these enzymes under harsh conditions, such as heat and acidity. Compared with proteins, nucleic acids are more stable and adaptable, and they can be easily produced using a commercial DNA synthesizer. Moreover, the catalytic and cleavage activities of DNAzyme can be achieved in solution; thus, no enzyme immobilization is needed for detection. Taken together, these attributes suggest that a DNAzyme-based ELISA detection approach will be more robust than current ELISA assays. Importantly, the proposed triply amplified DLISA immunoassay method shows ultrasensitive detection of such targets as human IgG with a detection limit of 2 fg/mL (3 × 10(-17) M), which is well within the range of many important disease biomarkers. DLISA can also be used to construct a sensing array for simultaneous multiplexed detection. With these merits, this high-throughput, stable, simple, sensitive, and low-cost multiplex fluorescence immunoassay shows promise for applications in clinical diagnosis.

  6. An expression of within-plate uncertainty in sandwich ELISA.

    PubMed

    Hayashi, Yuzuru; Matsuda, Rieko; Maitani, Tamio; Ito, Katsutoshi; Nishimura, Waka; Imai, Kazuhiro; Maeda, Masako

    2004-09-21

    This paper puts forward a method to describe an equation of the within-plate uncertainty (relative standard deviation (R.S.D.) of measurements) as a function of analyte concentration in sandwich enzyme-linked immunosorbent assay (ELISA). A kit for thyroid stimulating hormone is taken as an example. The pipetting procedures of analyte solution and chromogen-substrate solution and absorbance inherent to the wells of a microplate are identified as major error sources and their variability is included as parameters in the uncertainty equation. These parameters can be determined by the experiments with distilled water. The theoretical R.S.D. is shown to be in good agreement with the results of the repeated experiments using the real samples. Since the theory gives a continuous plot of R.S.D. against concentration, the uncertainty structure of the ELISA kit can be recognized over a wide concentration range and the detection limit and quantitation range can easily be determined on the plot.

  7. Development of a microplate reader compatible microfluidic chip for ELISA.

    PubMed

    Hou, Fenghua; Zhang, Qin; Yang, Jianping; Li, Xinchun; Yang, Xiujuan; Wang, Shuping; Cheng, Zhiyi

    2012-08-01

    We report a novel microfluidic device use for sandwich enzyme-linked immunoassay assay (ELISA). The related procedures including the introduction of reagents, dilution and distribution of samples, as well as immobilization of enzyme can be readily carried out on a poly (dimethylsiloxane) (PDMS) chip. Particularly, this microfluidic chip comprising of two distinct parallel units, and has an identical dimension as a conventional microtiter plate, which offers access to the directly quantitative detection by the microplate reader. Gradient-concentration reacting solutions at six different concentrations level generated by the microfluidic channel network are simultaneously transported to 24 reaction chambers to form enzymatic products. Alkaline phosphatase (ALP), 4-methylumbelliferyl phosphate (4-MUP) and KH(2)PO(4) are used as enzyme-substrate-inhibitor model, to demonstrate the utility of the developed microchip-based enzyme inhibitor assay. Various conditions such as the surface treatment of chip channels, fluids velocities, substrate concentration, and buffer pH are investigated. The present microfluidic device for ELISA holds several advantages, for instance frugal usage of samples and reagents, less of operating time, favorably integrated configuration, ease of manipulation, and could be explored to a variety of high throughput drug screening. PMID:22526682

  8. Survey of Field Soils for Cercospora beticola by PCR and ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We surveyed several fields in the Lower Yellowstone River Valley (Eastern Montana and Western North Dakota) for Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet. We used both PCR based technique and ELISA for detection of C. beticola in soil. Soils were sampled from severa...

  9. Survey of field soils for cercospora beticola by PCR and ELISA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We surveyed several fields in the Lower Yellowstone River Valley (Eastern Montana and Western North Dakota) for Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet. We used both PCR based technique and ELISA for detection of C. beticola in soil. Soils were sampled from severa...

  10. An overview of recent developments and current status of gluten ELISA methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ELISA methods for detecting and quantitating allergens have been around for some time and they are continuously improved. In this context, the development of gluten methods is no exception. Around the turn of the millennium, doubts were raised whether the existing “Skerritt-ELISA” would meet the 20 ...

  11. Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA.

    PubMed

    González, I; García, T; Fernández, A; Sanz, B; Hernández, P E; Martín, R

    1999-02-01

    Direct enumeration of Escherichia coli from oysters was achieved using a polymerase chain reaction (PCR) amplification of the lamB gene coupled with an enzyme-linked immunosorbent assay (ELISA). Amplified PCR products generated using a digoxigenin-labelled primer were heat denatured before being quantified by an ELISA. A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments that were detected with a peroxidase antidigoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying oyster samples containing E. coli in the range 10-10(5) cfu g-1.

  12. [ELISA method for the determination of factor VII antigen].

    PubMed

    Jorquera, J I; Aznar, J A; Monteagudo, J; Montoro, J M; Casaña, P; Pascual, I; Bañuls, E; Curats, R; Llopis, F

    1989-12-01

    The low plasma concentration of clotting factor VII makes it difficult to assay its antigenic fraction by the conventional methods of precipitation with specific antigens. Simple and peroxidase-conjugated antisera are currently available from commercial sources, thus allowing one to determine F VII:Ag by enzyme immunoassay. An ELISA method has been developed in this laboratory which provides sensitivity limits about 0.1% of the plasma concentration of F VII and correlates significantly with its functional activity (r = 0.603, n = 44, p less than 0.001). This technique can be highly helpful in characterising molecular variants of F VII, as well as in detecting acquired deficiencies of this factor.

  13. Determination of PCBs in fish using enzyme-linked immunosorbent assay (ELISA)

    USGS Publications Warehouse

    Lasrado, J.A.; Santerre, C.R.; Zajicek, J.L.; Stahl, J.R.; Tillitt, D.E.; Deardorff, D.

    2003-01-01

    Polychlorinated biphenyls (PCBs) were determined in fish tissue using an enzyme-linked immunosorbent assay (ELISA). Standard curves for Aroclor 1248, 1254, and 1260 in catfish tissue were developed with ranges from 0.05 to 0.5 ppm and 0.5 to 5.0 ppm. Wild fish were initially analyzed using gas chromatography/electron-capture detection (GC/ECD) and those having residues within the standard curve ranges were analyzed with ELISA. Results obtained using ELISA and GC/ECD were not significantly different (p < 0.05) from 0.05 to 0.5 ppm. From 0.5 to 5.0 ppm, the standard curve for Aroclor 1254 was the best predictor of total PCB in wild fish samples.

  14. Low-frequency Gravitational-wave Science with eLISA

    NASA Astrophysics Data System (ADS)

    Vallisneri, Michele; eLISA Science Study Team

    2013-01-01

    I review the expected science performance of eLISA, the proposed European-led gravitational-wave detector that may be selected as ESA "L2" Cosmic Vision mission. eLISA will survey the low-frequency gravitational-wave sky (from 0.1 mHz to 1 Hz), detecting and characterizing a broad variety of systems and events throughout the Universe, including the coalescences of massive black holes brought together by galaxy mergers; the inspirals of stellar-mass black holes and compact stars into central galactic black holes; several millions of ultracompact binaries, both detached and mass transferring, in the Galaxy; and possibly unforeseen sources such as the relic gravitational-wave radiation from the early Universe. eLISA's high signal-to-noise measurements will provide new insight into the structure and history of the Universe, and they will test general relativity in its strong-field dynamical regime.

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

    PubMed

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

    2003-09-01

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

  16. Short communication: ELISA system for screening of bovine mastitis caused by Prototheca zopfii.

    PubMed

    Kano, Rui; Sato, Ayano; Sobukawa, Hideto; Sato, Yuko; Ito, Takaaki; Suzuki, Kazuyuki; Hasegawa, Atsuhiko; Kamata, Hiroshi

    2016-08-01

    Prototheca zopfii is an achlorophyllic alga that causes bovine mastitis, resulting in a reduction in milk production and the secretion of thin, watery milk with white flakes. This study evaluated the use of an ELISA system for distinguishing cows with mastitis due to P. zopfii genotype 2 from healthy cows and cows with chronic candidal mastitis. We also investigated the transitional changes of specific antibody titers in healthy cows injected with inactivated P. zopfii genotype 2 cells. The ELISA system exhibited the highest sensitivity (94%) and specificity (100%) for chronic protothecal mastitis when the positive cutoff value was set at 43.4 ELISA units. Anti-protothecal IgG titers were positive in all cows after they were inoculated with inactivated P. zopfii genotype 2 cells. These results indicated that ELISA detection of anti-protothecal IgG in serum provided specificity and sensitivity sufficient for diagnosing protothecal mastitis. Thus, an ELISA system incorporating this specific antiserum is expected to be valuable for definitive field-based diagnosis of bovine mastitis due to P. zopfii genotype 2. PMID:27265167

  17. Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5.

    PubMed

    Dummer, Luana A; Araujo, Itauá L; Campos, Fabrício S; da Rosa, Matheus C; Finger, Paula F; de Oliveira, Patricia D; Conceição, Fabricio R; Fischer, Geferson; Roehe, Paulo M; Leite, Fábio P L

    2016-01-01

    Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.

  18. Development of a species-specific coproantigen ELISA for human Taenia solium taeniasis.

    PubMed

    Guezala, Maria-Claudia; Rodriguez, Silvia; Zamora, Humberto; Garcia, Hector H; Gonzalez, Armando E; Tembo, Alice; Allan, James C; Craig, Philip S

    2009-09-01

    Taenia solium causes human neurocysticercosis and is endemic in underdeveloped countries where backyard pig keeping is common. Microscopic fecal diagnostic methods for human T. solium taeniasis are not very sensitive, and Taenia saginata and Taenia solium eggs are indistinguishable under the light microscope. Coproantigen (CoAg) ELISA methods are very sensitive, but currently only genus (Taenia) specific. This paper describes the development of a highly species-specific coproantigen ELISA test to detect T. solium intestinal taeniasis. Sensitivity was maintained using a capture antibody of rabbit IgG against T. solium adult whole worm somatic extract, whereas species specificity was achieved by utilization of an enzyme-conjugated rabbit IgG against T. solium adult excretory-secretory (ES) antigen. A known panel of positive and negative human fecal samples was tested with this hybrid sandwich ELISA. The ELISA test gave 100% specificity and 96.4% sensitivity for T. solium tapeworm carriers (N = 28), with a J index of 0.96. This simple ELISA incorporating anti-adult somatic and anti-adult ES antibodies provides the first potentially species-specific coproantigen test for human T. solium taeniasis.

  19. Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5

    PubMed Central

    Campos, Fabrício S.; da Rosa, Matheus C.; Finger, Paula F.; de Oliveira, Patricia D.; Conceição, Fabricio R.; Fischer, Geferson; Roehe, Paulo M.; Leite, Fábio P. L.

    2016-01-01

    Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5. PMID:26866923

  20. Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed Indirect ELISA: Practical considerations

    PubMed Central

    Sharma, K. K.; Kshirsagar, D. P.; Kalyani, I. H.; Patel, D. R.; Vihol, P. D.; Patel, J. M.

    2015-01-01

    Aim: The work was conducted to diagnose peste des petits ruminants (PPR) outbreak through an in house developed indirect ELISA (thereafter referred as iELISA) its comparison with other available diagnostic tests and description of practical considerations in its development, utility and limitations. Materials and Methods: An outbreak resembled to PPR occurred in two different places of southern Gujarat viz. Vapi and Navsari, affecting 622 animals, including both goat (n = 476) and sheep (n = 146). Animals displayed the typical signs of PPR at Vapi; however diarrhea was the inconsistent feature in animals of Navsari. The affection caused morbidity of 100% and mortality were 73.68% (n = 392/532) and 56.67% (n = 51/90) in Vapi and Navsari outbreaks, respectively. Relevant ante mortem and post mortem samples were collected from representative animals. At the outset of the epidemic no kit was available with us, so agar gel immunodiffusion (AGID) was carried out and a commercial ELISA (cELISA) kit was ordered for making diagnosis through antibody demonstration. Meanwhile, an iELISA was developed in house using PPR vaccine as antigen and protein G conjugated HRPO antibody as detector. Histopathology and results of sandwich ELISA were also used to diagnose PPR virus (PPRV) in the outbreak. Results: The iELISA developed had detected PPRV antibodies in 22/24 samples (91.66%). Significant difference was observed in disease sensitivity pattern of two species by Chi-square test. While AGID failed to detect antibodies in any sample. Results were reconfirmed by comparing with commercially available cELISA kit. Conclusion: PPR is an economically important disease and for the rapid diagnosis of PPR the in house developed antibody capture iELISA can be a suitable cost effective alternative. PMID:27047112

  1. Quantitation of rat lacrimal secretion: a novel sandwich ELISA with high sensitivity.

    PubMed

    Sanghi, S; Kumar, R; Walton, S; Laurie, G W

    2000-05-01

    Modulation of lacrimal acinar cell tear secretion may involve multiple factors acting both in subtle and pronounced ways. Functional screens of recombinant protein products arising from gene array technologies, or protein fractions derived from lacrimal conditioned media or extracellular matrix, will require a highly sensitive assay capable of monitoring tear protein secretion by small replicate cultures. To improve significantly on current methods, a rat- and mouse-specific sandwich ELISA was developed. For this purpose, chickens and rabbits were immunized with serum-free secretion media from carbachol and VIP-stimulated rat lacrimal acinar cell cultures. Immune sera were characterized by ELISA, Western blotting and immunohistochemistry, and subsequently optimized for use in a sandwich ELISA. Both antisera detected a wide range of different rat tear proteins, and immunostained only the secretory granule-rich juxtalumenal region in sections of rat lacrimal gland. Chicken, but not rabbit, antiserum cross-reacted with rabbit and human tears. In sandwich ELISA, capture with purified chicken immunoglobulin fraction and detection with rabbit antiserum detected as little as 1 ng ml-1 tear protein in 10,000-fold diluted rat secretion medium--a level of sensitivity 8000 times greater than the rat tear peroxidase assay. Such specificity and sensitivity greatly reduce the quantity of media needed for assay, and makes feasible functional screens for scarce factors that may influence lacrimal secretory processes, and in turn possibly play a role in human lacrimal insufficiency syndromes.

  2. Standardization of a PCR-ELISA in serum samples: diagnosis of active parvovirus B19 infection.

    PubMed

    Zerbini, M; Gallinella, G; Manaresi, E; Musiani, M; Gentilomi, G; Venturoli, S

    1999-10-01

    To standardize a PCR assay for the detection of parvovirus B19 DNA in serum samples three different sample treatments were evaluated on the basis of the efficiency of recovery, reproducibility, convenience of sample handling, and presence of PCR inhibitors. Moreover, the presence of an internal standard competitor as the working reagent at one defined concentration in a competitive PCR-ELISA has been suggested as a valid tool to standardize and validate the assay. The results indicated that serum sample treatment by rapid heating fulfilled the criteria for a routine practice in the diagnostic laboratory. Titration experiments carried out to define the optimal amount of the internal standard competitor to use in PCR-ELISA showed that at 2 x10(2) competitor copies, any amplification interferences between target and competitor sequences were avoided. The internal standard competitor in a competitive PCR-ELISA allows the detection of false-negative results due to PCR inhibitors in the samples or large amounts of target DNA. Heating treatment and competitive PCR-ELISA for the detection of parvovirus B19 DNA were applied to the testing of 347 serum samples, which were submitted to the laboratory for B19 investigation. Of the 34 serum samples that were positive for B19 DNA, 15 were from adult patients and 19 from pediatric subjects. B19 infection was associated with haematological disorders, nonimmunological foetal hydrops, atypical rash, arthropathies, hepatic dysfunction, nonspecific symptoms, and congenital infections.

  3. Dispensing an enzyme-conjugated solution into an ELISA plate by adapting ink-jet printers.

    PubMed

    Lonini, Luca; Accoto, Dino; Petroni, Silvia; Guglielmelli, Eugenio

    2008-04-24

    The rapid and precise delivery of small volumes of bio-fluids (from picoliters to nanoliters) is a key feature of modern bioanalytical assays. Commercial ink-jet printers are low-cost systems which enable the dispensing of tiny droplets at a rate which may exceed 10(4) Hz per nozzle. Currently, the main ejection technologies are piezoelectric and bubble-jet. We adapted two commercial printers, respectively a piezoelectric and a bubble-jet one, for the deposition of immunoglobulins into an ELISA plate. The objective was to perform a comparative evaluation of the two classes of ink-jet technologies in terms of required hardware modifications and possible damage on the dispensed molecules. The hardware of the two printers was modified to dispense an enzyme conjugate solution, containing polyclonal rabbit anti-human IgG labelled with HRP in 7 wells of an ELISA plate. Moreover, the ELISA assay was used to assess the functional activity of the biomolecules after ejection. ELISA is a common and well-assessed technique to detect the presence of particular antigens or antibodies in a sample. We employed an ELISA diagnostic kit for the qualitative screening of anti-ENA antibodies to verify the ability of the dispensed immunoglobulins to bind the primary antibodies in the wells. Experimental tests showed that the dispensing of immunoglobulins using the piezoelectric printer does not cause any detectable difference on the outcome of the ELISA test if compared to manual dispensing using micropipettes. On the contrary, the thermal printhead was not able to reliably dispense the bio-fluid, which may mean that a surfactant is required to modify the wetting properties of the liquid.

  4. Dot-ELISA in diagnosis of schistosomiasis.

    PubMed

    Madwar, M A; Hassan, M M

    1989-12-01

    One microliter of S. mansoni egg antigen was dotted directly on the nitrocellulose paper sheet acting as the adsorbent surface (9 dots/paper). The sera of 25 Egyptian patients and 15 healthy persons (2 microliters of each) were dotted over the antigen dots, then 2 ml of each of the blocking, washing, HRP-conjugated IgG and DAB adding procedures, were added over the nitrocellulose paper in the petri-dish at room temperature. An intact brown circle (by naked-eye) indicates a positive in Dot-ELISA. There is an insignificant dot colour intensities in different clinical stages of S. mansoni infected Egyptians whereas, a direct relation was obtained between egg count and the colour intensity of the dots. The test had 100% sensitivity and 86% specificity thus it appears to be useful for both laboratory and field studies. PMID:2794577

  5. eLISA and the Gravitational Universe

    NASA Astrophysics Data System (ADS)

    Danzmann, Karsten

    2015-08-01

    The last century has seen enormous progress in our understanding of the Universe. We know the life cycles of stars, the structure of galaxies, the remnants of the big bang, and have a general understanding of how the Universe evolved. We have come remarkably far using electromagnetic radiation as our tool for observing the Universe. However, gravity is the engine behind many of the processes in the Universe, and much of its action is dark. Opening a gravitational window on the Universe will let us go further than any alternative. Gravity has its own messenger: Gravitational waves, ripples in the fabric of spacetime. They travel essentially undisturbed and let us peer deep into the formation of the first seed black holes, exploring redshifts as large as z ~ 20, prior to the epoch of cosmic re-ionisation. Exquisite and unprecedented measurements of black hole masses and spins will make it possible to trace the history of black holes across all stages of galaxy evolution, and at the same time constrain any deviation from the Kerr metric of General Relativity. eLISA will be the first ever mission to study the entire Universe with gravitational waves. eLISA is an all-sky monitor and will offer a wide view of a dynamic cosmos using gravitational waves as new and unique messengers to unveil The Gravitational Universe. It provides the closest ever view of the early processes at TeV energies, has guaranteed sources in the form of verification binaries in the Milky Way, and can probe the entire Universe, from its smallest scales around singularities and black holes, all the way to cosmological dimensions.

  6. Analysis of High-Throughput ELISA Microarray Data

    SciTech Connect

    White, Amanda M.; Daly, Don S.; Zangar, Richard C.

    2011-02-23

    Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

  7. Enzyme-linked immunosorbant assay (ELISA) of size-selected crotalid venom antigens by Wyeth's polyvalent antivenom.

    PubMed

    Schaeffer, R C; Randall, H; Resk, J; Carlson, R W

    1988-01-01

    The binding of Antivenom (Crotalidae) Polyvalent to fractions from crude venoms of eight crotalid and one viperid snake, obtained by high performance size-exclusion chromatography, was determined with an indirect enzyme-linked immunosorbent assay (ELISA). Most of the large (greater than 30,000 mol. wt) molecular mass crotalid venom fractions were associated with high (greater than 0.7 absorbance units) ELISA values. Similarly, the medium (13,000-30,000 mol. wt) and small (less than 14,000 mol. wt) molecular mass crotalid venom fractions were coincident with moderate (0.3-0.7 absorbance units) and low (less than 0.3 absorbance units) ELISA levels. Some variability in this pattern was seen with individual venom fractions. A distinctly different pattern of ELISA values were observed with two rattlesnake venoms: the South American (Crotalus durissus terrificus) and Mojave desert (Crotalus scutulatus scutulatus) rattlesnakes. The elution profile from these venoms showed a progression of low to moderate ELISA values within the large molecular mass fractions. This pattern was followed by a decline to low ELISA values throughout the remainder of the elution profile. When saw scaled viper (Echis carinatus leucogaster) venom fractions were tested, only background ELISA values were detected with antivenom. Similarly, background ELISA values were associated with the small molecular mass fractions of all venoms tested. In addition, the elution position for the basic peptides of southern Pacific (Crotalus viridis helleri) and timber (Crotalus h. horridus) rattlesnake venoms showed minimal ELISA values. These data support the view that except for the venom of C. durissus terrificus and C. s. scutulatus, most antivenom antibodies bind large (greater than 30,000 mol. wt) venom fractions. Thus, antivenom contains minimal levels of antibodies to the basic peptides in these venoms. PMID:3347932

  8. [Cryptosporidiosis in Senegalese children: prevalence study and use of ELISA serologic diagnosis].

    PubMed

    Faye, B; Dieng, T; Tine, R C; Diouf, L; Sylla, K; Ndiaye, M; Sow, D; Ndiaye, J L; Ndiaye, D; Ndiaye, M; Badiane, A S; Seck, M C; Dieng, Y; Faye, O; Ndir, O; Gaye, O

    2013-10-01

    This is a prospective, descriptive and analytic study conducted from July 2011 to September 2011 at the Children National Hospital Albert Royer of Dakar and at the Vélingara Health District. It was focused on children under 15 without reference to HIV status. For each child, a sample of stool was examined by the Ziehl-Neelsen modified staining and by ELISA using the "Cryptosporidium Antigen Detection Microwell ELISA kit" designed to detect Cryptosporidium spp antigens. The aim of our study was to determine the prevalence of cryptosporidiosis in rural and hospital areas and to measure the performance of the ELISA kit that we used. Out of the 375 stool examinations performed with the Ziehl-Neelsen modified staining, 17 had revealed the presence of Cryptosporidium spp (4.53%). The prevalence in rural areas was 2% while the hospital prevalence was 7.4%, of which 1.8% (1/57) were from urban areas and 9.8% (12/122) from suburban areas. No positive case was observed in children over 10 years. By ELISA, 23 positives cases were reported corresponding to a prevalence of 6.13% (1.8% in children living in urban areas, 13.1% in children from suburban areas and 3%living in rural areas).The correlation of this assay with the Ziehl-Neelsen modified staining, considered as the reference method, found that this assay had a sensitivity of 58.82% and a high specificity reaching 96.37%. The positive predictive value (PPV) was 43.4% while the negative predictive value was 98%. Cryptosporidiosis is a significant cause of parasitic infection among children in Senegal. Antigen detection of Cryptosporidium spp by ELISA in stool can be a complementary tool in the diagnosis of cryptosporidiosis.

  9. LISA Pathfinder and eLISA news

    NASA Technical Reports Server (NTRS)

    Thorpe, James Ira; Mueller, Guido

    2014-01-01

    Two important gatherings of the space-based gravitational-wave detector community were held in Zurich, Switzerland this past March. The first was a meeting of the Science Working Team for LISA Pathfinder (LPF), a dedicated technology demonstrator mission for a future LISA-like gravitational wave observatory. LPF is entering an extremely exciting phase with launch less than 15 months away. All flight components for both the European science payload, known as the LISA Technology Package (LTP), and the NASA science payload, known as the Space Technology 7 Disturbance Reduction System (ST7-DRS), have been delivered and are undergoing integration. The final flight component for the spacecraft bus, a cold-gas thruster based on the successful GAIA design, will be delivered later this year. Current focus is on completing integration of the science payload (see Figures 1 and 2) and preparation for operations and data analysis. After a launch in Summer 2015, LPF will take approximately 90 days to reach its operational orbit around the Earth-Sun Lagrange point (L1), where it will begin science operations. After 90 days of LTP operations followed by 90 days of DRS operations, LPF will have completed its prime mission of paving the way for a space-based observatory of gravitational waves in the milliHertz band. Immediately following the meeting of the LPF team, the eLISA consortium held its third progress meeting. The consortium (www.elisascience.org) is the organizing body of the European space-based gravitational-wave community, and it was responsible for the "The Gravitational Universe" whitepaper that resulted in the November 2013 election of a gravitational-wave science theme for ESA's Cosmic Visions L3 opportunity. In preparation for an L3 mission concept call, which is expected later this decade, and for launch in the mid 2030s, the eLISA consortium members are coordinating technology development and mission study activities which will build on the LPF results. The final

  10. Development and comparison of two competitive ELISAs for estimation of cotinine in human exposed to environmental tobacco smoke.

    PubMed

    Lei, Yajing; Zhang, Qian; Fang, Lizheng; Akash, Muhammad Sajid Hamid; Rehman, Kanwal; Liu, Zhiming; Shi, Weixing; Chen, Shuqing

    2014-10-01

    We simultaneously set up two competitive (direct and indirect) enzyme-linked immunosorbent assays (ELISAs) based on the same antibody for estimation of cotinine (COT) in pregnant women especially and population generally exposed to environmental tobacco smoke. The results show that the limits of detection (LODs) for direct competitive ELISA and indirect competitive ELISA were 0.04 μgL(-1) and 0.1 μgL(-1), respectively. Direct competitive ELISA was found to be more sensitive than indirect competitive ELISA. Thereafter, we applied our direct competitive ELISA for the detection of COT from urinary samples taken from 450 volunteers from the Zhejiang Province of China. COT was detected in 100% of participants with concentration ranging from LOD to 5358.0 μgL(-1). The GM and 95th percentile concentration of COT in pregnant women were 6.3 μgL(-1) and 57.2 μgL(-1), respectively. Males had statistically higher COT concentrations than females (P < 0.0001), active smokers had statistically higher COT concentrations than non-smokers (P < 0.0001), whereas, non-pregnant women were found to have higher COT concentration than pregnant women. We conclude that our developed direct competitive ELISA is useful for detecting the COT in urinary concentration of human. The human urinary data obtained in this study indicated that common people generally and pregnant women especially were highly exposed to COT. Further studies are needed to focus on the sources of exposure, potential health effects and risk assessment of exposure to COT.

  11. Studies of Clusters and Biomolecules in ELISA

    NASA Astrophysics Data System (ADS)

    Andersen, J. U.; Andersen, L. H.; Hvelplund, P.; Lapierre, A.; M Ø ller, S. P.; Nielsen, S. B.; Pedersen, U. V.; Tomita, S.

    Application of the electrostatic ion storage ring ELISA to studies of clusters and biomolecules is discussed. Ions injected from a plasma source or a sputter source are hot, and at short times the yield of neutrals is usually dominated by decay of metastable ions. We have demonstrated that the decay function is close to a 1/t dependence when the internal energy of the ions is conserved, i.e., when photon emission can be ignored. Deviations from a 1/t distribution therefore gives information about the radiative lifetime or, for larger systems, about the intensity of the emitted radiation. Systematic measurements have been carried out for fullerene anions CN-, for even values of N from 36 to 96, to test a classical dielectric model. Recently we have installed an electrospray ion source with a Paul trap for bunching, which can be used to inject biomolecular ions from solution, and the first experiments on laser spectroscopy of biomolecules have been carried out. Also lifetimes of excited states have been measured for stored biomolecular ions excited by a laser pulse.

  12. AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

    EPA Science Inventory

    An ELISA assay for heme oxygenase (HO-l )

    Abstract

    A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

  13. A micro ELISA for the diagnosis of cerebral cisticercosis.

    PubMed

    Nunez, R; Munoz, A; Nunez, C; Gomez, B

    1989-01-01

    A micro ELISA for cysticercosis in the central nervous system was evaluated in 24 patients and compared with indirect hemagglutination. CSF ELISA results were positive in 17 of 20 patients (sensitivity 85%), whereas CSF indirect hemagglutination was positive in only 14 patients (sensitivity 70%).

  14. Multi-Laboratory Validation of Estrone (E1) ELISA Methods

    EPA Science Inventory

    This project is a round-robin evaluation of commercially available Enzyme-Linked Immunosorbent Assay (ELISA) technology to quantitatively or qualitatively measure the hormone estrone (E1) in combined animal feeding operation (CAFO) receiving streams. ELISA is meant to be a simpl...

  15. Differential Diagnosis of Japanese Encephalitis Virus Infections with the Inbios JE Detect™ and DEN Detect™ MAC-ELISA Kits

    PubMed Central

    Johnson, Barbara W.; Goodman, Christin H.; Jee, Youngmee; Featherstone, David A.

    2016-01-01

    Japanese encephalitis virus (JEV) is the leading cause of pediatric viral neurological disease in Asia. The JEV-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) in cerebrospinal fluid (CSF) and serum is the recommended method of laboratory diagnosis, but specificity of JEV MAC-ELISA can be low due to cross-reactivity. To increase the specificity of the commercially available JE Detect™ MAC-ELISA (JE Detect), a differential testing algorithm was developed in which samples tested by JE Detect with positive results were subsequently tested by the DEN Detect™ MAC-ELISA (DEN Detect) kit, and results of both tests were used to make the final interpretation. The testing algorithm was evaluated with a reference panel of serum and CSF samples submitted for confirmatory testing. In serum, the false Japanese encephalitis (JE) positive rate was reduced, but sequential testing in CSF resulted in reduced JE specificity, as true JEV+ CSF samples had positive results by both JE Detect and DEN Detect and were classified as JE− (dengue virus [DENV]+). Differential diagnosis of JE by sequential testing with JE Detect and DEN Detect increased specificity for JE in serum, but more data with CSF is needed to make a final determination on the usefulness of this testing algorithm for CSF. PMID:26856911

  16. Applicability of ELISA-based Determination of Pesticides for Groundwater Quality Monitoring

    NASA Astrophysics Data System (ADS)

    Tsuchihara, Takeo; Yoshimoto, Shuhei; Ishida, Satoshi; Imaizumi, Masayuki

    The principals and procedures of ELISA (Enzyme-linked Immunosorbent Assay)-based determination of pesticides (Fenitrothion) in environmental samples were reviewed, and the applicability of the ELISA method for groundwater quality monitoring were validated through the experimental tracer tests in soil columns and the field test in Okinoerabu Island. The test results showed that the ELISA method could be useful not only for screening but also for quantitative analysis of pesticides. In the experimental tracer tests in soil columns, the retardation of pesticides leaching compared with conservative tracers were observed. In the field test, the contamination of the pesticide was detected in groundwater samples in Okinoerabu Island, even though the targeted pesticide was considered to be applied to the upland field 4 months ago. In order to investigate the transport and fate of pesticides in groundwater taking into account retardation from the field to groundwater table and the residue in groundwater, continuous observations of pesticides in groundwater are in a strong need, and the ELISA method is applicable to the long-term quality groundwater monitoring.

  17. Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis.

    PubMed

    Rhodes, Shelley G; Gruffydd-Jones, Tim; Gunn-Moore, Danièlle; Jahans, Keith

    2008-08-15

    There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-gamma responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.

  18. Science with the space-based interferometer eLISA: Supermassive black hole binaries

    NASA Astrophysics Data System (ADS)

    Klein, Antoine; Barausse, Enrico; Sesana, Alberto; Petiteau, Antoine; Berti, Emanuele; Babak, Stanislav; Gair, Jonathan; Aoudia, Sofiane; Hinder, Ian; Ohme, Frank; Wardell, Barry

    2016-01-01

    We compare the science capabilities of different eLISA mission designs, including four-link (two-arm) and six-link (three-arm) configurations with different arm lengths, low-frequency noise sensitivities and mission durations. For each of these configurations we consider a few representative massive black hole formation scenarios. These scenarios are chosen to explore two physical mechanisms that greatly affect eLISA rates, namely (i) black hole seeding, and (ii) the delays between the merger of two galaxies and the merger of the black holes hosted by those galaxies. We assess the eLISA parameter estimation accuracy using a Fisher matrix analysis with spin-precessing, inspiral-only waveforms. We quantify the information present in the merger and ringdown by rescaling the inspiral-only Fisher matrix estimates using the signal-to-noise ratio from nonprecessing inspiral-merger-ringdown phenomenological waveforms, and from a reduced set of precessing numerical relativity/post-Newtonian hybrid waveforms. We find that all of the eLISA configurations considered in our study should detect some massive black hole binaries. However, configurations with six links and better low-frequency noise will provide much more information on the origin of black holes at high redshifts and on their accretion history, and they may allow the identification of electromagnetic counterparts to massive black hole mergers.

  19. Serodiagnosis of neurocysticercosis in patients with epileptic seizure using ELISA and immunoblot assay.

    PubMed

    Ishida, Maria M I; Peralta, Regina Helena S; Livramento, José A; Hoshino-Shimizu, Sumie; Peralta, José M; Vaz, Adelaide J

    2006-01-01

    Sera from 88 patients from Santa Catarina and São Paulo states of Brazil, with epileptic seizures who underwent cerebral computed tomography (CT) were analyzed for the detection of antibodies to T. solium cysticercus by ELISA and Immunoblot (IB) with the following antigens: Taenia solium cysticercus total saline (Tso), Taenia crassiceps cysticercus vesicular fluid (Tcra-vf) and T. crassiceps cysticercus glycoproteins (Tcra-gp). ELISA carried out with Tso, Tcra-vf and Tcra-gp antigens showed 95%, 90% and 80% sensitivities, respectively, and 68%, 85% and 93% specificities, respectively. In the epileptic patients group, ELISA positivity was 30%, 51% and 35% with Tso, Tcra-vf and Tcra-gp antigens respectively. Considering the IB as the confirmatory test, the positivity was 16% (14/88) in the epileptic patients total group and 22% (12/54) in the epileptic patients with positive CT and signals of cysticercosis. We found a significant statistical correlation among ELISA or IB results and the phase of the disease when any antigens were used (p < 0.05). We emphasize the need to introduce in the laboratory routine the search for neurocysticercosis (NC) in patients presenting with epileptic seizures because of the high risk of acquiring NC in our region and its potential cause of epilepsy.

  20. A rapid method for estimating the binding of ligands to ELISA microwells.

    PubMed

    Steinitz, M; Baraz, L

    2000-04-21

    This report presents a rapid and simple assay for estimating to what extent the surface of