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PubMed1983

Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA).

The biotin/avidin system was incorporated into the enzyme-linked immunosorbent assay (ELISA) technique to increase the sensitivity of the standard ELISA for the detection of mouse antibody to hepatitis B surface antigen ((anti-HBs and HBsAg, respectively). Two biotin/avidin ELISA designs were studied. In both assays, 96-well polystyrene plates were coated with HBsAg, post-coated with 0.5% gelatin and incubated with dilutions of mouse anti-HBs. In the biotin/avidin (BA) ELISA, reagents were added to antibody reacted wells in the following sequence: biotinylated goat anti-mouse IgG (b-GAMG), avidin-alkaline phosphatase (Av-AP) and substrate. The order of reactants after mouse antibody in the biotin/avidin/biotin (BAB) ELISA was b-GAMG, avidin, biotinylated alkaline phosphatase (b-AP) and substrate. The sensitivities of BA ELISA, BAB ELISA and a standard ELISA using a glutaraldehyde conjugated goat anti-mouse enzyme were compared to AUSAB (a commercial radioimmunoassay) using a panel of 23 mouse anti-HBs sera. All 3 ELISAs were more sensitive than AUSAB; the standard ELISA, BAB ELISA and BA ELISA were respectively 50, 1173 and 4134 times more sensitive than AUSAB for detection of mouse anti-HBs activity.

Animals↗
PubMed1997

An improved ELISA for the detection of serum antibodies directed against classical swine fever virus.

The complex-trapping-blocking (CTB) ELISA for detection of antibodies against classical swine fever virus (CSFV) using two monoclonal antibodies (mAbs) directed against envelope glycoprotein E2, has been improved using recombinant CSFV E2-antigen. The newly developed Ceditest ELISA for CSFV-Ab is a modification of the CTB-ELISA as described by Wensvoort et al. (1988) and Bloemraad et al. (1993). The old CTB-ELISA format comprised of a two-step, single-dilution test which had to be performed by hand at 37 degrees C. The Ceditest ELISA is a one-step, single-dilution test which can be performed by hand, or automated at room temperature. A set of 505 pig sera was tested for CSFV antibodies in the CTB-ELISA, in the Ceditest ELISA and in the neutralizing peroxidase-linked assay (NPLA). Concordance between the two ELISAs was 96.7%. Sensitivity and specificity of the Ceditest ELISA were 99% and 99% compared to the CTB-ELISA. The Ceditest ELISA discriminates between antibodies directed against bovine viral diarrhea virus (BVDV) and CSFV. The discrimination between antibodies directed against some Border disease virus (BDV) strains and CSFV is as inconsistent as with the NPLA. We conclude that the Ceditest ELISA for CSFV-Ab is a promising tool for the fast and simple screening of large numbers of pig sera for detection of antibodies directed against CSFV.

Animals↗
PubMed1998

Highly sensitive and specific ELISA with monoclonal antibody capture to measure Dermatophagoides farinae 1-specific IgE.

BACKGROUND: Dermatophagoides farinae (Der f) is a major allergen in Der f extract. Measurement of Der f I-specific IgE has not been commonly used, because of the difficulty in obtaining large amounts of purified Der f 1. OBJECTIVES: To improve the diagnosis of dust mite allergy, we wanted to develop an ELISA to measure Der f 1-specific IgE in which purified Der f 1 is not required. METHODS: Using a monoclonal antibody (mAb) against Der f 1, a mAb-capture ELISA was developed. Microplates coated with the mAb were sequentially incubated with crude Der f extract, serum samples, goat anti-human IgE, and conjugated rabbit anti-goat IgG. A conventional ELISA using purified Der f 1 as capture (Der f 1-ELISA) was developed to compare it with the mAb-capture ELISA. RESULTS: There was a significant correlation between the two ELISAs (r = 0.89, P < .001) with a sensitivity of 0.8 U/mL. Both the assays were inhibited by the crude Der f extract in a dose-dependent manner. Using the two ELISAs, serum Der f 1-specific IgE was evaluated in 12 allergic patients with positive skin tests to Der f extract, 14 allergic patients with negative skin tests to Der f extract, and 15 healthy subjects. The mean Der f 1-IgE and the positivity rate of Der f I-IgE were higher in the mite-allergic group and lower in the healthy group when the mAb-ELISA was used (P < .01) than when the Der f 1-ELISA was used (P < .05). CONCLUSIONS: Der f 1-specific IgE can be measured by mAb-capture ELISA using crude Der f extract. The mAb-ELISA is more sensitive and specific than the conventional Der f 1-capture ELISA in diagnosing dust mite allergy.

Adolescent↗
PubMed2004

Development of a neutralizing monoclonal antibody-based blocking ELISA for detection of equine herpesvirus 1 antibodies.

A single-dilution, sensitive and specific monoclonal antibody-based blocking enzyme-linked immunosorbent assay (B-ELISA) was developed as an alternative to the cumbersome virus neutralization test (VNT) for detection of equine herpesvirus-1 (EHV-1) antibodies. Neutralizing monoclonal antibodies (1H6 and 9C6) raised against EHV-1 (Hisar-90-7 strain) and sera from 70 horses (30 known negative and 40 known positive for EHV-1 antibodies by VNT) were used for standardization of the B-ELISA. Using a single serum dilution of 1:250 in B-ELISA, 100% specificity was obtained with both monoclonal antibodies (Mabs) in comparison to VNT. Similarly, the sensitivity of the B-ELISA was 92.5% and 100% with 1H6 and 9C6 Mabs, respectively. A very high correlation coefficient (r = 0.85) was observed between B-ELISA and VNT that was significant at the p < 0.01 level. B-ELISA detected a more than 3-fold rise in antibody titres in paired serum samples collected from mares aborting owing to EHV-1 infection. Mab 9C6 was chosen for testing 231 field sera from apparently healthy vaccinated and non-vaccinated horses from organized breeding farms belonging to 11 Indian states, and from Bhutan, by B-ELISA and VNT. There was very good agreement between the results obtained by both VNT and B-ELISA (K = 0.9438). Of 231 field sera, 144 samples were negative for EHV- 1 antibodies by both VNT and B-ELISA and 81 were positive by both tests. Two samples negative by VNT were found positive in B-ELISA. On the other hand, four weakly positive samples in VNT (VN antibody titre 0.9 1.2 log10) were negative in B-ELISA. The Mab (9C6)-based B-ELISA was found to be a suitable alternative to VNT for screening large numbers of field sera and enabled confirmatory EHV-1 serodiagnosis.

Animals↗
PubMed2006

Sandwich ELISAs for soluble immunoglobulin superfamily receptor translocation-associated 2 (IRTA2)/FcRH5 (CD307) proteins in human sera.

BACKGROUND: The immunoglobulin superfamily receptor translocation-associated 2 (IRTA2) gene encodes both membrane and secreted forms of the B-cell differentiation antigen (also identified as FcRH5). The membrane form is highly expressed on the surface of hairy cell leukemia (HCL) cells from patients. This study aimed to develop immunoassays for soluble IRTA2/FcRH5 proteins in human serum. METHODS: Two sandwich ELISAs for soluble IRTA2/FcRH5 were designed using two pairs of monoclonal antibodies specific to four different epitopes on IRTA2/FcRH5. RESULTS: In both ELISAs, the lower limit of quantitation for soluble IRTA2/FcRH5 in human serum was 30 ng/mL. The analytical recovery of 0.3-2.1 ng/mL of IRTA2/FcRH5-Fc used as the standard, from a 100-fold dilution of IRTA2/FcRH5-free sera, was 94-106% for ELISA #1 and 89-97% for ELISA #2. Between-assay coefficients of variance were 7.7-17.6% for ELISA #1 and 7.7-32.7% for ELISA #2. Both ELISAs detected soluble IRTA2/FcRH5 protein in sera from normal donors (median 169 ng/mL in ELISA #1 and 146 ng/mL in ELISA #2, n=123) without correlations to gender or age. A marked increase in soluble IRTA2/FcRH5 levels was observed in samples from patients with HCL (medians 719 ng/mL in ELISA #1 and 754 ng/mL in ELISA #2, n=44). CONCLUSIONS: These ELISAs may be useful for monitoring soluble IRTA2/FcRH5 in HCL and other B-cell malignancies.

Adolescent↗
PubMedDate not supplied

[Standardization of ELISA IgM and IgA for immunodiagnosis of human trichinosis].

An ELISA test for trichinosis using as antigen a larvae soluble fraction from Trichinella spiralis was carried out for the detection of IgM and IgA specific antibodies in 45 serum samples from patients confirmed or suspected to have trichinosis by strong clinical and epidemiological evidences. All the patients had positive serology detected by precipitin test, bentonite floculation test, indirect hemagglutination test and ELISA IgG test. The cut-off value was determined using two criteria. Criterion A was determined in each plate, using three positive controls and two negative ones; the average of the negative controls and the weakest positive control, multiplied by a 1.2 factor was, considered the cut-off value. Criterion B was determined using the average plus three standard deviations from 64 apparently healthy persons serum samples. In both cases, three serum dilutions (1:10, 1:100 and 1:500) were used. The sensitivity of ELISA IgM was 100.0, 93.3 and 82.2% using serum dilutions of 1:10, 1:100 and 1:500 respectively (criterion A) and 100.0, 97.8 and 95.6% for the same dilutions (criterion B), whereas the values for ELISA IgA were: 100.0, 91.1 and 86.7% (criterion A) and 100.0, 100.0 and 91.1% (criterion B). In order to find out the specificity of ELISA IgM and ELISA IgA, additional 118 serum samples from individuals with other parasitoses, such as cysticercosis (18) hydatidosis (39), fascioliasis (12), toxocariasis (30), Chagas' disease (12) and individuals with non-specific eosinophilia (7), were also tested. ELISA IgM presented a specificity of 92.3, 93.4 and 97.3% (criterion A) and 96.2, 97.8 and 97.8% (criterion B) whereas the results for ELISA IgA were 97.8, 98.9 and 99.4% (criterion A) and 98.4% for the 1:10 and 1:100 dilutions and 100.0% for the 1:500 dilution (criterion B). The positive predictive values of ELISA IgM were 76.3, 77.8 and 88.1% (criterion A) and 86.5, 91.7 and 91.5% (criterion B) whereas the negative ones were 100.0, 98.3 and 95.7% (criterion A) and 100.0, 99.4 and 98.9% (criterion B). The positive predictive values of ELISA IgA were 91.8, 95.3 and 97.5% (criterion A) and 93.8, 93.8 and 100.0% (criterion B) whereas the negatives ones were: 100.0, 97.8 and 96.8% (criterion A) and 100.0, 100.0 and 97.8% (criterion B). The use of ELISA IgM and ELISA IgA in the immunodiagnosis of trichinosis is discussed.

Adult↗
PubMed2001

Performance of two ELISAs for antifilaggrin autoantibodies, using either affinity purified or deiminated recombinant human filaggrin, in the diagnosis of rheumatoid arthritis.

OBJECTIVE: To develop a standardisable enzyme linked immunosorbent assay (ELISA), using human filaggrin, for detection of antifilaggrin autoantibodies in rheumatoid arthritis (RA). To compare the diagnostic performance of the ELISA with those of reference tests: "antikeratin antibodies" ("AKA"), and antibodies to human epidermis filaggrin detected by immunoblotting (AhFA-IB). METHODS: Two ELISAs were developed using either affinity purified neutral-acidic human epidermis filaggrin (AhFA-ELISA-pur) or a recombinant human filaggrin deiminated in vitro (AhFA-ELISA-rec) as immunosorbent. Antifilaggrin autoantibodies were assayed in 714 serum samples from patients with well characterised rheumatic diseases, including 241 RA and 473 other rheumatic diseases, using the two ELISAs. "AKA" and AhFA-IB tests were carried out in the same series of patients. The diagnostic performance of the four tests was compared and their relationships analysed. RESULTS: The titres of "AKA", AhFA-IB, and the AhFA-ELISAs correlated strongly with each other. The diagnostic sensitivity of the AhFA-ELISA-rec, which was better than that of AhFA-ELISA-pur, was 0.52 for a specificity of 0.95. This performance was similar to those of "AKA" or AhFA-IB. However, combining AhFA-ELISA-rec with AhFA-IB led to a diagnostic sensitivity of 0.55 for a specificity of 0.99. CONCLUSION: A simple and easily standardisable ELISA for detection of antifilaggrin autoantibodies was developed and validated on a large series of patients using a citrullinated recombinant human filaggrin. The diagnostic performance of the test was similar to that of the "AKA" and AhFA-IB. Nevertheless, combining the AhFA-ELISA-rec with one of the other tests clearly enhanced the performance.

Adolescent↗
PubMed1992

Two-site sandwich ELISA for detection of Plasmodium vivax blood stage antigens using monoclonal and polyclonal antibodies.

Two systems of sandwich enzyme-linked immunosorbent assay (ELISA), a two-site monoclonal antibody sandwich ELISA MAb-MAb sandwich ELISA) and a two site polyclonal-monoclonal antibody sandwich ELISA (PAb-MAb sandwich ELISA) for the detection of Plasmodium vivax antigens were developed. The assays showed good correlation with the level of parasitemia when tested against serially diluted P. vivax parasites (r = 0.937, and 0.997 for MAb-MAb and PAb-MAb sandwich ELISA, respectively), with the ability to detect as few as 6.68 parasites/10(6) erythrocytes and 2.69 parasites/10(3) erythrocytes, respectively. The MAb-MAb sandwich ELISA was specific, since it was positive only with P. vivax-infected erythrocytes from vivax malaria patients and negative when erythrocytes from 34 healthy individuals and 30 falciparum malaria cases were tested. In contrast, cross-reaction was found in the PAb-MAb sandwich ELISA when the plates were coated with polyclonal IgG and tested against the serially diluted P. falciparum SO strain antigen prepared from in vitro cultures. Comparison between the two systems of two-site sandwich ELISA showed that the MAb-MAb sandwich ELISA was superior to the PAb-MAb sandwich ELISA: (1) it gave a higher sensitivity when tested with serially diluted P. vivax antigen preparations from vivax malaria patients; (2) it gave a higher specificity when tested with the SO strain of P. falciparum from in vitro cultures, (3) it gave a lower absorbance value when tested with erythrocytes from healthy individuals. All 281 cases of vivax malaria already proven by microscopic examination were positive by MAb-MAb sandwich ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Anti-HIV positivity in Thailand: the usefulness of another ELISA test kit and Western blot as confirmatory tests.

Sera from 47 individuals repeatedly reactive in one screening ELISA system (designated as ELISA-A) for antibodies against human immunodeficiency virus (HIV) were evaluated by a second ELISA system (designated as ELISA-B) as well as by the Western blot technique. Both ELISA systems and the Western blot were positive in all of the 14 patients with clinical diagnoses of AIDS and AIDS-related persistent generalized lymphadenopathy (PGL). Of the 7 asymptomatic gays whose sera were repeatedly reactive in ELISA-A, 5 were also reactive in ELISA-B and these were the ones with positive Western blot tests. Eight and 17 ELISA-A reactive individuals were uncovered during a survey of 2,699 female prostitutes and 15,210 potential workers for Saudi Arabia respectively. All of these 25 individuals were ELISA-B and Western blot negative, an indication of false-positive reactivity with ELISA-A. Our studies indicate that the prevalence of HIV infection among the general Thai population is still low, and that the specificity of two ELISA test kits for anti-HIV may differ considerably. We concluded that evaluation of test kits should include studies in tropical countries where ecological conditions, climate and background endemic disease patterns are different than in the countries producing the diagnostic systems. Such studies are needed to identify the most sensitive and specific kits for worldwide application. We did discover that concordant positivity of two different ELISA test kits served as a reliable and inexpensive confirmatory test for anti-HIV.

AIDS-Related Complex↗
PubMedDate not supplied

High rate of p53 overexpression in head and neck carcinomas detected with a refined ELISA.

p53 mutations are amongst the most prevalent alterations in human cancer. Overexpression of p53 is usually caused by mutation and is observed in a high percentage of squamous cell carcinomas of the head and neck (SCCHN). Fifty two fresh samples of SCCHN were examined for p53 overexpression and 23 tumor-free tonsils served as controls. Using the monoclonal antibody Pab 1801 a refined ELISA technique was employed. In contrast to established ELISA procedures tumor tissue was pulverized at -80 degrees C prior to the actual ELISA (ELISA I). Comparative p53 detection was carried out by immunohistochemistry (IHC) and a commercially available ELISA kit (ELISA II). The modified ELISA I revealed p53 overexpression in 48 tumor samples (92%). p53 detection was obtained in 26 cases (50%) with ELISA II and with IHC 39 stained positive for p53 (75%). All of the controls were negative for p53 with ELISA and IHC. The p53 staining in IHC showed a significant correlation with the grading of the tumor. The ELISA results of the p53 overexpression did not show any correlation with tumor size or stage and rate of metastasis or other clinical parameters. This ELISA represents a more sensitive detection method of p53 than any other technique so far. It improves on former ELISA and IHC results on p53 overexpression in squamous cell carcinoma of the head and neck and underlines the involvement and the importance of the p53 tumor suppressor protein in carcinogenesis of head and neck cancer.

Adult↗
PubMed1997

Recombinant 52 kDa Ro(SSA) ELISA detects autoantibodies in Sjögren's syndrome sera that go undetected by conventional serologic assays.

OBJECTIVE: To determine the utility of a recombinant 52 kDa Ro(SSA) ELISA for detecting Ro autoantibodies in Sjögren's syndrome (SS) sera. METHODS: Several different groups of SS sera previously tested for Ro and La(SSB) autoantibodies in clinical diagnostic labs were tested by ELISA with a recombinant human 52 kDa Ro fusion protein. RESULTS: Five of 18 primary SS sera (28%) that had undetectable Ro and La autoantibodies by conventional immunodiffusion (ID) or ELISA in clinical diagnostic laboratories had significant reactivity with a recombinant 52 kDa Ro (r52) ELISA. On repeat testing these 5 sera were again negative for Ro and La antibodies by ELISA with purified 60 kDa Ro and La antigens, but 3 of these sera were reactive with r52 by immunoblot, and immunoprecipitated a 52 kDa protein from human cell extracts. Twelve of 12 primary SS sera that had detectable Ro autoantibodies by ID also reacted with the r52 ELISA, whereas none of 11 normal sera and only one of 27 ID-defined Ro negative systemic lupus erythematosus sera did. Eleven of 28 sera from patients with suspected SS were Ro positive by ID and 60 kDa Ro ELISA. All 11 were also Ro positive by the r52 ELISA. Two of the 28 suspected SS sera were Ro positive by the r52 Ro ELISA, but were Ro and La negative by ID and 60 kDa Ro and La ELISA. CONCLUSION: Anti-52 kDa Ro autoantibodies are frequently present in primary SS sera, but may go undetected by commonly used Ro serologic assays. Our r52 ELISA is more sensitive in detecting Ro antibodies in SS than conventional ID and 60 kDA Ro ELISA.

Antibody Specificity↗
PubMed2003

Establishment of ELISA on 384-well microplate for AFP, CEA, CA 19-9, CA 15-3, CA 125, and PSA-ACT: higher sensitivity and lower reagent cost.

The 384-well microplate contains four times as many wells as the regular 96-well microplate. Establishing enzyme linked immunosorbent assay (ELISA) on 384-well microplate should lead to savings in reagents and specimens. To determine that ELISAs on 384-well microplate have acceptable assay precision, ELISAs for tumor markers, including AFP, PSA-ACT, CEA, CA 125, CA 15-3, and CA 19-9, were compared to the same ELISAs established on 96-well microplate. We found that ELISAs established on 384-well microplate had similar sensitivity and covered similar concentration ranges as ELISAs on 96-well microplate. All within-day and day-to-day precisions for the 384-ELISA had %CV less than 10%. Compared to ELISA on 96-well microplate, 384-ELISA used less reagents, less specimen, and exhibited approximately a two-fold increase in sensitivity. Overall cost of the 384-384-ELISA was also greatly reduced. Our results suggest that 384-ELISA is suitable for use in routine clinical laboratories.

Biomarkers, Tumor↗
PubMed1994

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks.

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N'-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.

Animals↗
PubMed1988

Dot-ELISA, a potential immunoassay for the detection of Plasmodium falciparum antibodies.

The dot enzyme-linked immunosorbent assay (dot-ELISA) was compared to the indirect immunofluorescent assay (IIFA) and radioimmunoassay (RIA) for the detection of malaria antibodies. Of 281 sera tested by dot-ELISA, 220 were from Ethiopia, 11 from the Sudan, and 50 from Egypt. A close correlation between the dot-ELISA and RIA results was observed in 92% of the 220 Ethiopian cases. Of the remainder, 6% gave positive RIA and negative dot-ELISA results, and 2% gave positive dot-ELISA and negative RIA results. Antibody titres determined by dot-ELISA and RIA were positively correlated in 10 of the 11 Sudanese cases tested by direct microscopical examination. The eleventh case was positive by dot-ELISA at 1:1000 dilution, but negative by RIA and direct examination. With the 50 Egyptian sera, the dot-ELISA results showed close correspondence to the IIFA results, but the dot-ELISA was 20-40 fold more sensitive than the IIFA. To test specificity, 62 samples from patients with 11 different diseases and conditions were examined by dot-ELISA. No malaria antibodies were detected in any of these or in sera from healthy controls. Dot-ELISA is a potentially useful method for sero-epidemiological studies of malaria.

Animals↗
PubMed1987

Use of enzyme amplification in an ELISA to increase sensitivity of detection of barley yellow dwarf virus in oats and in individual vector aphids.

A new technique of alkaline phosphatase amplification in an ELISA (amplified ELISA) was used to increase the sensitivity of detection of barley yellow dwarf virus (BYDV) from oat plant sap and in individual vector aphids. Amplified ELISA differs from conventional direct double antibody sandwich ELISA (DAS-ELISA) in the enzyme substrate reaction. The bound enzyme-labelled antibody catalyzes the conversion of NADP to NAD which is then used in a secondary enzyme-mediated cyclic reaction producing a red-coloured end product. Amplified ELISA was compared with DAS-ELISA for the detection of BYDV and each assay was done with both monoclonal and polyclonal antibody reagents. Both types of antibodies detected BYDV from oat sap and amplified ELISA increased the sensitivity of detection sufficiently to allow a diagnostic test to be completed in less than 2 h using microtitre plates precoated with antibodies. However, in the amplified ELISA using polyclonal antibodies the absorbance values obtained with the healthy oat sap samples were much greater than those obtained in the DAS-ELISA, or with the monoclonal antibodies, and were too large to be acceptable for reliable diagnostic tests. Both monoclonal and polyclonal antibodies were used successfully to detect BYDV in individual virus-carrying Rhopalosiphum padi by amplified ELISA and there was little nonspecific background reaction in the control samples with either of the antibodies.

Alkaline Phosphatase↗
PubMed2006

Development and evaluation of a rapid IgM capture ELISA (JEV-Chex) for the diagnosis of Japanese encephalitis.

BACKGROUND: The IgM capture ELISA has been the most widely used diagnostic method for Japanese encephalitis. However, the lack of availability of validated commercial kits as well as the short shelf life of the kit reagents has limited the use of this technique to very few centres in Asia. OBJECTIVES: Development and evaluation of a rapid IgM capture ELISA (JEV Chex) in comparison to the conventional IgM capture ELISA. Produce key reagents such as cell culture derived JEV antigen and biotinylated monoclonal antibody which are stable at room temperature. STUDY DESIGN: The conventional IgM capture ELISA was modified to reduce the total assay time and two key reagents used in the assay JEV antigen and biotinylated anti-JEV monoclonal antibodies were rendered stable at room temperature using a special procedure. A multi-centric evaluation of this rapid ELISA was carried out using well characterized stored CSF and serum samples. Long term stability of the key reagents was also assessed over a period of 6 months. RESULTS: The rapid IgM capture ELISA developed by us showed complete concordance with the results obtained using the conventional ELISA at all the three centres where it was evaluated. In addition, the stability studies carried out with the inactivated cell culture antigen and the biotinylated monoclonal antibodies stored at room temperature for a period of 180 days revealed that both these reagents yielded consistent optical density values in the ELISA. CONCLUSIONS: The rapid ELISA format of the IgM capture ELISA (JEV-Chex) developed by us as well as the stability of reagents achieved by us in this study is what renders this rapid IgM capture ELISA very robust and user friendly since reagents can be stored at 4 degrees C by peripheral labs.

Aedes↗
PubMed2006

Peptide ELISA for measuring antibodies to N-protein of porcine reproductive and respiratory syndrome virus.

Indirect and competition ELISAs with synthetic peptides were used to characterize the epitopes of the N-protein of porcine reproductive and respiratory syndrome virus (PRRSV) that are recognized by a battery of monoclonal antibodies (mAbs) and by antibodies from infected pigs. Four linear epitopes recognized by mAbs have been identified in the most hydrophilic segment of the N-protein (AA25-57). Similarly, at least four linear epitopes in this segment are immunogenic in PRRSV-infected pigs, but only one corresponds to an epitope recognized by one of the mAbs (AA36-45). Antibody formation to these epitopes varied greatly between individual pigs. Most infected pigs generated antibodies that bound to both peptides and HerdChek plates, which are commonly used in the sero-diagnosis of PRRSV infections, but the time course of formation of peptide binding antibodies and antibodies that react with HerdChek plates differed greatly between pigs. This suggests that, although the peptide and HerdChek ELISAs may detect antibodies to some of the same epitopes, they also seem to detect antibodies to epitopes that are uniquely expressed by one and not the other. Some mAbs fail to bind to HerdChek ELISA plates and this is also the case for certain pig antibodies. Peptide ELISA results identified four herds in which most or all pigs possessed N-protein peptide binding antibodies, even though they were HerdChek ELISA sero-negative and exhibited no other signs of PRRSV infection. Thus PRRSV infections may be more widespread than presently realized involving strains that cause asymptomatic infections. The peptide ELISA is useful as an adjunct to the HerdChek ELISA or it could replace it since only two serum samples among 450 tested were HerdChek ELISA positive but peptide ELISA negative. The peptide ELISA is also considerably cheaper than the HerdChek ELISA, more flexible and can provide information on the epitope specificity of the reacting antibodies.

Amino Acid Sequence↗
PubMed2000

Detection of Trypanosoma congolense antibodies with indirect ELISAs using antigen-precoated microtitre plates.

The study reports the performance of four indirect enzyme-linked immunosorbent assays (ELISAs) for antibody (AB) detection using microtitre plates which were precoated with native or heat/detergent denatured antigens (AGs) from Trypanosoma congolense (T.c.) and T. vivax (T.v.), and stored for between 1 to 206 days at +37 degrees C. Bovine serum samples were obtained by sequential bleeding of 3-months old T.c.-infected bulls and their uninfected cohorts, as well as by a single bleeding of uninfected adult cattle. The first day of AB detection, and observations on samples after this (defined as estimated ELISA sensitivity), depended on the cut-off value in the specific ELISAs. Cut-off values from pre- and early post-infection samples of individual animals demonstrated a seroconversion in all ELISAs on average after 10-15 days post-infection (dpi). The AB detection was delayed in the T.c. native and denatured AG-based ELISAs using cut-off points from uninfected cohort cattle (16.5 dpi, 19.3 dpi) and the adult cattle population (22.1 dpi, 25.0 dpi). The T.v. AG-based ELISAs however lacked crossreactiviy to T.c. ABs. The estimated sensitivity of each T.c. AG-based ELISA was above 96% throughout, but significantly lower for the T.c. native AG-based ELISA (91.1%) when the adult cattle derived cut-off point was used (p<0.01). The sensitivity of the phase contrast buffy coat technique was similar to the T.c. AG-based ELISAs, but significantly lower when the T.c. denatured AG-based ELISA was used at the adult cattle derived cut-off point (p<0.05). The implications of the results and future research aspects on ELISAs to detect trypanosomal ABs and AGs are discussed.

Animals↗