Comparison of five commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis

Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of five commercially available ELISA kits with the help of 65 serum specimens which were repetitively tested for evaluation of the kits. The specimens contained 20 paired serum samples from patients with clinical pertussis, 15 samples were from children vaccinated with a diphtheria-tetanus-acellular pertussis vaccine, seven specimens were taken from an interlaboratory comparison of ELISAs, and there were three reference preparations from the Food and Drug Administration's (FDA's) Laboratory of Pertussis and from our laboratory. Reference values were obtained from the FDA or from results obtained with an in-house ELISA. Commercial ELISAs were compared with respect to their reproducibility and variability, their ability to detect significant titer rises in paired serum samples, their ability to detect an immune response after vaccination, and the comparability of semiquantitative and quantitative results. Reproducibility was generally good (>89%), intra-assay variation ranged from 2.4 to 28.7%, and indeterminate results were recorded in up to 18.5% of all specimens. Most kits correctly identified the antibody response to an acellular pertussis vaccine. None of the commercial kits identified all cases of pertussis correctly, and the sensitivity ranged between 60 and 95%. All five commercial ELISAs showed great discrepancies when comparing semiquantitative results and contained obviously different antigen preparations. Our data suggest that the five commercial ELISAs tested here need further improvement and standardization.     

Comparison of the enzyme-linked immunosorbent assay (elisa), haemagglutination inhibition test and agar gel precipitation test for detection of antibodies to avian infectious bronchitis virus

The immune response after vaccination with infectious bronchitis virus (IBV) under field conditions was measured by the ELISA, haemagglu-tination-inhibition (HI) and agar-gel precipitin (AGP), tests. Vaccinations were performed in three flocks and one experimental group via the drinking water with the vaccine strains H 120 and H 52. In each flock 40 random serum samples were taken every 2 weeks and tested individually. In the experimental group blood samples were collected every week from each of the 10 chickens. The primary vaccination with H 120 resulted in a rapid increase of antibody titre as detected by ELISA followed by a slow decrease over the next few weeks. By the HI and AGP tests no antibody responses could be seen after this primary vaccination. Revaccination with the H 52 strain provoked a further increase in ELISA titres. In the experimental group, and in flock W, a similar increase occurred by the HI test and precipitating antibodies appeared. The formation of HI antibodies in flock T (nipple waterers) was somewhat retarded and precipitating antibodies were just detectable. In flock F revaccination did not result in the immediate production of HI and AGP antibodies. However, 6 weeks after revaccination a significant rise in ELISA, HI and AGP antibodies was observed, probably as the result of a field infection. It was demonstrated that, based on the higher sensitivity, the ELISA test is more suitable than HI and AGP to monitor antibody responses to vaccination against infectious bronchitis. Strain specificity in the HI test is discussed as a reason for its failure to detect antibodies after primary vaccination with the highly attenuated vaccine strain H 120.     

Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis

Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.     

Evaluation of two recombinant Maedi-visna virus proteins for use in an enzyme-linked immunosorbent assay for the detection of serum antibodies to ovine lentiviruses.

Defined segments of the gag polyprotein and transmembrane envelope glycoprotein from Maedi-visna virus were expressed as glutathione S-transferase fusion proteins in Escherichia coli and evaluated singly and in combination for use in an enzyme-linked immunosorbent assay (ELISA). Two hundred sixty field serum specimens from 15 sheep flocks were tested in parallel with recombinant and whole-virus antigens, and the relative sensitivities and specificities of the recombinant antigens were calculated. When the recombinant gag and transmembrane proteins were used in combination, a sensitivity of 97.4% and a specificity of 99.4% relative to whole-virus antigen were observed, indicating the utility of these proteins in diagnostic testing.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Junin virus in rodents

Junin virus is the etiological agent of Argentine hemorrhagic fever, a serious rodent-borne disease. An enzyme-linked immunosorbent assay (ELISA) to detect Junin virus IgG antibodies in rodents was evaluated using sera from 27 Calomys musculinus and five Calomys laucha, inoculated experimentally with a live attenuated strain of this arenavirus. The test performance was compared against an indirect immunofluorescence assay (IFA). The ELISA had a sensitivity and specificity of 100% and a reproducibility of 87.9% for samples with titers above the selected cut-off value. IFA had lower sensitivity (53%) with the same specificity. The ELISA results were similar, whether carried out on whole blood or serum samples, thus eliminating the need for serum separation. A high correlation (K=0.86) between ELISA and IFA results was obtained from 1011 wild sigmodontine and murine rodents collected within and outside of the Argentine hemorrhagic fever endemic area. These results indicate that Junin virus IgG ELISA is the most suitable assay for detection of Junin virus antibodies in rodent samples.     

Comparison of the enzyme-linked immunosorbent assay and double immunodiffusion test for the detection and quantitation of antibodies in farmer's lung disease

An ELISA for the detection of antibodies to the thermophilic actinomycetes associated with farmer's lung disease (FLD) has been described and compared with the standard double immunodiffusion (DID) assay. Parallel determinations of antibody were made with both test systems on sera from 10I0 patients being tested for FLD, as well as on sera from 133 additional subjects previously diagnosed with FLD or assessed as antibody-positive farmers. The ELISA was more sensitive than DID in detecting antibody to the panel of antigens used and also in detecting patients with symptons consistent with FLD. The ELISA was also useful in quantitating antibody in previously diagnosed FLD. When compared with farmers with antibody to the thermophilic actinomycetes and no clinical illness, FLD patients had a higher mean titer as a group, which was not statistically significant.     

Comparison of seven commercial enzyme-linked immunosorbent assays for the detection of anti-diphtheria toxin antibodies

Determination of immune status of patients to diphtheria toxin is based mainly on the results of commercially available ELISA kits. The aim of the present study was to compare the results obtained by ELISAs from seven different manufacturers: Mikrogen, Immunolab, Sekisui Virotech, NovaTec, VirionSerion, IBL International and Euroimmun. All assays were performed according to the manufacturers’ instructions. The concentrations of the anti-diphtheria toxin antibodies in 72 serum samples were calculated on the basis of curves constructed from standards supplied by manufacturers and the new reference material—International Standard for Diphtheria Antitoxin (10/262). The repeatability and reproducibility of all the ELISA kits tested were good. Number of sera with concentrations of the anti-diphtheria toxin antibodies below the WHO-recommended level of protection (0.1 IU/ml) were dependent on the ELISA used: Mikrogen, 20/72 samples (27.7 %); Immunolab, 11/72 samples (15.3 %); Sekisui Virotech, 0/72 samples (0 %); NovaTec 18/72 samples (25.0 %); Serion 12/72 samples (16.7 %); IBL International, 7/72 samples (9.7 %); and Euroimmun, 17/72 samples (23.6 %). However, the results obtained in particular ELISAs, with the exception of Sekisui Virotech, were much more consistent when the concentrations of the anti-diphtheria toxin antibodies in 72 sera measured by using curves constructed from the International Standard 10/262. The data obtained clearly demonstrated that manufacturer-dependent differences between anti-diphtheria IgG ELISA kits exist. The differences in recommendations accepted by the individual manufacturers together with differences shown in our studies in sensitivity greatly affect the clinical interpretation of results.     

Relative sensitivity and specificity of agar gel immunodiffusion,enzyme immunosorbent assay,and immunoblotting for detection of anti-bovine leukemia virus antibodies in cattle

Three serologic methods for the detection of antibodies to bovine leukemia virus (BLV) were compared using the sera of 140 dairy cows. A widely used commercial agarose immunodiffusion screening assay and a commercial antibody capture enzyme immunosorbent assay were compared for sensitivity and specificity with immunoblotting as the standard. The immunoblot utilized the same antigen preparations that were provided in the commercial kits. The agarose immunodiffusion and the enzyme immunosorbent assay were comparable in the number of positive animals detected. However, the commercial screening kits failed to detect 39% (agarose immunodiffusion) and 35% (immunosorbent assay), respectively, of the animals determined serologically positive by immunoblot. These findings corroborate those of some other groups and emphasize the need for more sensitive tests to identify BLV positive cattle for culling or separation in order to create BLV-free herds.     

Epitope-blocking enzyme-linked immunosorbent assays for detection of west nile virus antibodies in domestic mammals

We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.     

Comparison of three enzyme-linked immunosorbent assays suitable for the detection of antibodies to rotaviruses in epidemiological studies

Three enzyme-linked immunosorbent assays which use commercially available reagents are presented as alternatives to the complement fixation procedure for large-scale detection of rotavirus antibodies. Comparison of results in 75 sera tested by indirect, competition and blocking ELISA and by complement fixation demonstrated that the ELISA techniques were rapid, easy to perform and 26 to 29 times more sensitive than complement fixation; furthermore the ELISA techniques permitted extrapolation of an approximate titer from a single dilution test. Although it could detect class-specific antibodies, the indirect assay was less sensitive, specific and reproducible than competition and blocking assays; furthermore the titer extrapolated from a single dilution test in the indirect assay had a lower correlation with the end-point titer than in the other two methods. Competition ELISA was easier to perform whereas blocking ELISA had better reproducibility and serotype studies were possible with it using faecal rotavirus isolates. The respective ELISA technique can be recommended for use in seroepidemiologic surveys according to the study needs.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to bovine leukemia virus in serum and milk.

The results of examination of 375 bovine serum and 150 bovine milk samples for detection of bovine leukemia virus infection by the immunodiffusion technique were compared with those of an enzyme-linked immunosorbent assay (ELISA). It was concluded that the ELISA is another useful method for the detection of antibodies to bovine leukemia virus in serum and milk. The ELISA provides a quantitative result and has the advantage of being more sensitive and less time-consuming than the conventional immunodiffusion technique.     

Comparison of complement fixation with two enzyme-linked immunosorbent assays for the detection of antibodies to respiratory viral antigens

We compared complement fixation (CF) for the measurement of antibodies against influenza A, influenza B, respiratory syncytial virus (RSV), human adenovirus, and parainfluenza viruses 1, 2, and 3 (para-1, para-2, and para-3) with 2 enzyme-linked immunosorbent assays (ELISA kits, A and B). The IgG ELISA kits compared very well with each other except for the influenza A and B IgG ELISAs. The IgG ELISAs, in general, did not agree with CF In contrast, the IgM ELISAs compared well with CF and each other except for the consensus parainfluenza panel from ELISA B. The poor agreement of the IgG ELISAs with the CF test can be explained by the increased sensitivity of the ELISAs and differences between CF antigens and the ELISA antigens. The influenza A and influenza B ELISA antigens differed between both kits, which may explain their poor agreement. The ELISA is a suitable replacement for CF, providing greater sensitivity, isotype specificity, and ease of use.     

Correlation of haemagglutination inhibition and enzyme-linked immunosorbent assays for antibodies to Newcastle disease virus

Antibody responses of commercial broiler chickens vaccinated for Newcastle disease were assayed by haemagglutination inhibition and enzyme-linked immunosorbent assay, and the results from the two methods compared. Three-hundred-and-nineteen chickens from 37 farms were assayed. The correlation coefficient was 0.85. Analysis of individual slopes of data from the 37 farms indicated that at least eight chickens from a farm must be sampled to achieve an acceptable degree of parallelism.     

Evaluation of a dipstick enzyme-linked immunosorbent assay for detection of antibodies to dengue virus.

Accurate serological confirmation of dengue (DEN) infection is difficult, because simple reliable assays for the detection of DEN antibodies are not available. To address this problem, a dipstick enzyme-linked immunosorbent assay (ELISA) was evaluated. The dipstick contained dots of serially diluted DEN 2 antigen. To detect immunoglobulin G (IgG), the dipstick was processed through four reaction cuvettes containing test serum, enhancer, enzyme-conjugated anti-human IgG and IgM antibody, and substrate. Total assay time was 45 min. To detect IgM, the serum was passed through a protein G device to remove IgG. The dipstick was then processed as before, except that the incubation times were longer and enzyme-conjugated anti-human IgM was used. The total assay time was 3 h. The dipstick ELISA results were compared with results from microplate ELISA. The IgG dipstick ELISA showed a sensitivity of 95.2% and a specificity of 100% compared to an IgG microplate ELISA with serum samples from 125 individuals living in an area in which DEN is endemic. In tests with 75 serum samples from patients with clinically suspected acute DEN infections, the IgM dipstick ELISA showed a sensitivity of 97.9% and specificity of 100% compared to those of an IgM antibody capture microplate ELISA. These results showed that the dipstick ELISA was a sensitive and specific test for the detection of either DEN IgM or IgG in human serum. The dipstick ELISA was also shown to be useful for detecting seroconversions to DEN IgM or IgG in paired serum samples from 20 patients with virus isolation-confirmed acute DEN infections.     

An enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibodies to porcine parvovirus (PPV). Antisera to PPV were raised in pigs, for which PPV grown on PK15 cells was used for primary intranasal inoculations, and PPV cultured on autologous kidney cells for booster immunisations. A competition ELISA was developed, based on the principle of a double antibody sandwich assay, using immunoglobulin fractions prepared from these sera. The ELISA was compared with a haemagglutination inhibition (HI) test. The tests were equally sensitive for detecting antibodies early after infection and for detecting a significant increase in antibody titre between paired sera. A high correlation was found between antibody titres of field sera measured by the two tests (r = 0.91). We conclude that ELISA is preferable to the HI test, because it is labour-saving and can be standardised better and automated.     

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to west nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WNV or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.1112G potentially discriminated between WNV and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2B2 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.67G, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.     

Development of an enzyme-linked immunosorbent assay for detection of antibodies to Babesia bigemina in cattle

Monoclonal antibodies, directed against a 58-kDa Babesia bigemina merozoite antigen that reacted strongly with immune sera from experimentally and naturally infected cattle in Western blots, were used to develop a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). As based on the testing of 70 antibody-positive sera from experimentally infected cattle and 166 antibody-negative sera collected in non-endemic areas of Australia, the sensitivity and specificity of the ELISA were 95.7% and 97.0%, respectively. In sequential sera collected from six calves during the course of experimental B. bigemina infections the ELISA detected seroconversion at about 10 days post-inoculation. The specificity of the ELISA was not affected by the presence of antibodies to B. bovis, Anaplasma marginale or Theileria buffeli. In 42 sera from cattle experimentally infected with B. bovis but negative for B. bigemina the specificity of the ELISA was 95.2%. The use of a competitive-inhibition ELISA format detecting only antibody directed against a single epitope on the 58-kDa antigen appears to have overcome many of the specificity problems that have plagued serological tests for B. bigemina in the past. The test should be useful for epidemiology studies, particularly in areas where B. bovis and B. bigemina have overlapping distributions. Received: 28 December 1997 / Accepted: 10 February 1998     

Development of an enzyme-linked immunosorbent assay for detection of IgE antibodies specific to Dermatophagoides pteronyssinus

Allergy to Dermatophagoides pteronyssinus was determined in 61 rhinitis patients using prick test (PT), enzyme-immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). A total of 43 patients tested positive with PT. Forty six patients were positive when tested with EIA and ELISA. With PT as standard test, EIA was found to have 83.7% sensitivity and 44.4% specificity; ELISA had 81.4% sensitivity and 38.9% specificity. There was a linear relationship between absorbance values obtained by EIA and ELISA. The performance time was 8 hours, 24 hours and 30 minutes for ELISA, EIA and PT respectively. The cost per test for ELISA, EIA and PT was US$ 0.20, US$ 5.20 and US$ 0.14 respectively. It was concluded that ELISA was more cost-effective than EIA should be used to supplement PT for a more complete diagnosis of allergy.     

Comparison of enzyme-linked immunosorbent and indirect hemagglutination assays for determining anthrax antibodies.

An enzyme-linked immunosorbent assay has been established to measure anthrax antibody titers. The protective antigen component of anthrax toxin was used as the capture antigen. Two types of conjugates (protein A-horseradish peroxidase and anti-human immunoglobulin G plus immunoglobulin A plus immunoglobulin M-horseradish peroxidase) were tested. Results from enzyme-linked immunosorbent assay testing were compared with those from indirect hemagglutination titers on serum from vaccinees. The overall trend of enzyme-linked immunosorbent assay and indirect hemagglutination titers was significant. The enzyme-linked immunosorbent assay offered speed, precision, and reduced cost per test.