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 two commercial enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen

Two commercial enzyme-linked immunosorbent assays (ELISA) for detection of herpes simplex virus (HSV) antigen in direct clinical specimens were compared with cell culture in primary rabbit kidney and MRC-5. With a total of 238 specimens, the sensitivity and specificity of the Dako ELISA method were determined to be 70.1% and 82.4%, respectively, and 60.4% and 88.2%, respectively, for the Ortho Antigen ELISA.     

Performance and reliability of five commercial enzyme-linked immunosorbent assay kits in screening for anti-human immunodeficiency virus antibody in high-risk subjects.

Anti-human immunodeficiency virus enzyme-linked immunosorbent assay kits marketed by Electro-Nucleonics Inc. (ENI), Genetic Systems Corp. (GSC), Organon Teknika Inc. (OTI), Ortho Diagnostic Systems Inc. (ODSI), and Wellcome Diagnostics (WD) were evaluated by using 289 randomly selected serum samples from a high-risk population and 53 serum samples likely to produce false-positive results. The radioimmunoprecipitation assay was used as the reference test. Sensitivities ranged from 96.51% (ODSI, WD) to 97.67% (ENI, GSC, OTI). Sera showing antibodies to viral glycoproteins only produced the false-negative results. Specificities ranged from 99.6% (ENI, GSC, ODSI, OTI) to 100% (WD). False-positive results were obtained with sera from patients with autoimmune disease or Epstein-Barr virus infection. Only results from GSC and OTI kits were distributed in two compact clusters well segregated on either side of the cutoff point. ODSI and GSC kits had the best intralot reproducibility. The GSC kit had the best interlot reproducibility. Cutoff values for ODSI and GSC kits were the least variable. Intraplate repeatability was good for all kits. Sample localization was not an important source of variability. Our results do not point out one outstanding kit among the five evaluated. However, the GSC kit showed the best overall results.     

Evaluation and diagnostic usefulness of domestic and imported enzyme-linked immunosorbent assays for detection of human immunodeficiency virus type 1 antibody in India

Diagnosis of human immunodeficiency virus (HIV) infection is important for patient management and prevention of new infections. The number of test kits available for the detection of HIV antibodies is unprecedented. In order to identify appropriate test kits, we evaluated a variety of commercial kits manufactured abroad as well as in India. The plasma and serum specimens (n=264) were collected from individuals attending the Voluntary Counseling and Testing Centre at the YRG Centre for AIDS and Education. The specimens were used to evaluate six commercially available HIV test kits: Enzaids HIV 1+2, HIV-CheX, Murex HIV-1.2.0, Genscreen HIV 1/2 version 2, Vironostika HIV Uni-Form II Ag/Ab, and CombAids RS Advantage. High sensitivities and specificities (>or=99%) were observed for the Enzaids, Murex, Vironostika, and CombAids assays. HIV-CheX showed the highest number of false-positive and false-negative results. The Genscreen test also gave many false positives. The study indicated that the Enzaids, Murex, and Vironostika enzyme-linked immunosorbent assay kits and the CombAids RS Advantage rapid assay could be used to achieve acceptable results for the detection of HIV antibodies. A combination of two tests is recommended to optimize the efficiency of HIV antibody testing algorithms, especially when evaluation with an HIV Western blot confirmatory test is not possible.     

Comparison of two enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen.

Two enzyme-linked immunosorbent assays (ELISAs) for herpes simplex virus (HSV) detection were compared with culture in a prospective, blinded study with 153 patients with suspected recurrent oral or genital HSV. A subset of 15 of these subjects were studied daily until symptom resolution during a single episode of recurrent HSV. Direct-site specimens were collected and either placed in viral transport media (for Ortho ELISA and fresh inoculation into primary rabbit kidney cells) or frozen in ELISA collection media (DuPont). One hundred eighty-six culture-ELISA comparisons were analyzed. On the basis of culture positivity, the DuPont and Ortho ELISAs differed substantially with regard to sensitivity (93 versus 35%) but had similar specificities (95 versus 100%) and positive (85 versus 100%) and negative (98 versus 85%) predictive values. There were seven DuPont ELISA-positive, culture-negative samples which were confirmed positive for HSV by blocking antibody test (revised specificity, 100%; positive predictive value, 100%). Six of these discrepant samples were from previously culture-positive subjects. These results demonstrate that currently available ELISA kits vary substantially as to their sensitivities in detecting HSV antigen from direct-site specimens. In addition, antigen detection, by ELISA technology, is not always synonymous with state of viral infectivity as judged by tissue culture cytopathic effect.     

Comparison of competitive and indirect enzyme-linked immunosorbent assays for detection of bluetongue virus antibodies in serum and whole blood.

An indirect (I) enzyme-linked immunosorbent assay (ELISA) and a competitive (C) ELISA, using a group-specific monoclonal antibody against bluetongue virus (BTV), are described for the detection of antibodies to BTV in cattle and sheep sera. The performance of these assays in detecting anti-BTV antibody in sequential serum samples and eluates from whole blood (WB) dried on filter paper from three calves and four sheep experimentally infected with type 10 BTV was evaluated. The C-ELISA was superior to the I-ELISA in the detection of anti-BTV antibody in the sera and WB samples from both cattle and sheep early after infection with BTV. BTV antibodies were demonstrable by C-ELISA in all the bovine and ovine sera and WB eluates by 9 days postinfection; whereas the I-ELISA results for sheep sera and WB eluates were similar, anti-BTV antibody was not detected in bovine serum and WB eluates until 26 and 14 days postinfection, respectively. While both ELISAs proved reliable, under the present test conditions involving detection of early postinfection reactions of experimentally infected animals, the C-ELISA was always as sensitive or more sensitive than the standard agar gel immunodiffusion test, the modified complement fixation test, and the plaque neutralization tests in the detection of anti-BTV antibodies. Unlike observations with the immunodiffusion test, no reaction was seen between BTV antigen and bovine epizootic hemorrhagic disease virus antiserum in either ELISA. The results suggest that either ELISA may be suitable for routine diagnostic testing and may have the potential to replace other tests for detection of anti-BTV group-specific antibodies and that the C-ELISA may have the most potential.     

Comparison of six serological assays for human immunodeficiency virus antibody detection in developing countries.

Three commercially available assays for the detection of human immunodeficiency virus (HIV) antibodies-Vironostika enzyme immunoassay (EIA), Wellcozyme competitive EIA, and JLC Allaman indirect immunofluorescence assay--were tested on 300 serum samples from African subjects with and without HIV-related conditions. Two experimental assays both rapid and simple to perform (Biotech dip stick and Cambridge Bioscience latex agglutination) were also evaluated on the same serum samples. The results were compared with those of a commercial Western blot (WB) (immunoblot) assay from Biotech, used as the reference technique. All assays were tested in the laboratory of the AIDS Project in Kigali, Rwanda. Calculated specificity ranged from 90.8% (dip stick) to 98.6% (Vironostika EIA, Wellcozyme competitive EIA, and Cambridge Bioscience latex agglutination). Sensitivity ranged from 95.2% (Cambridge Bioscience latex agglutination) to 98.0% (Vironstika EIA) and JLC indirect immunofluorescence assay). However, the sensitivity of the latex agglutination test improved to 98.6% after the prozone effect was controlled for by serial twofold dilution of latex agglutination-negative, WB-positive samples. In situations with a high prevalence of HIV infection, any one of these tests can be regarded as an alternative to the more expensive, time-consuming, and difficult WB assay.     

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.     

Reaction pattern of human anti-Mycoplasma pneumoniae antibodies in enzyme-linked immunosorbent assays and immunoblotting.

In serological diagnosis of Mycoplasma pneumoniae disease the frequently used complement fixation test is based on a cross-reacting glycolipid. Recently enzyme-linked immunoassays have been developed to overcome this lack of specificity. To study the involvement of the various proteins and the influence of age on the level of antiprotein antibodies present, we investigated by enzyme-linked immunoassays (immunoglobulin M [IgM] and IgG) and immunoblotting the sera of healthy persons of different age groups as well as sera of patients (including paired sera) with M. pneumoniae infection. In sera of children with nonrespiratory diseases and in healthy blood donors the IgM antibodies rose during the first 2 years of life to a relatively constant background level (optical density at 405 nm of 0.15 to 0.21). In contrast IgG remained low up to the seventh year and then increased to moderate levels (optical density of 0.15). The blotting patterns showed few IgM bands in the age group of 20 to 30 years. IgG blots revealed, up to 7 years, only very few reactions with a 168-kilodalton protein, but in higher age groups a considerable number of reactions with proteins of 193, 168, 84, 69, and 56 kilodaltons were detected. In the sera of patients, positive IgM blots were most numerous in the third week, whereas the number of IgG blots increased up to the fourth and the fifth week. At this time all sera contained antibodies against the 168-kilodalton protein, which is identical with the adhesin of M. pneumoniae. In a patient with acute infection, who had a high preinfection IgG level, no IgM response developed. The data indicate a relatively high background of antibodies against M. pneumoniae proteins in older age groups, suggesting a requirement for paired sera. Furthermore, reinfections of adults may occur without a concomitant IgM response.     

Molecular specificity of two commercial enzyme linked immunosorbent assays for human immunodeficiency virus antigens.

Only "fair" agreement has been shown between the Abbott and DuPont enzyme linked immunosorbent assays when used for the detection of human immunodeficiency virus (HIV) antigen in serum samples from asymptomatic HIV antibody positive homosexual men. To investigate the discrepancies between the two ELISA results, further experiments were performed. The rabbit detector antibody solutions of both tests were western blotted and showed that the DuPont test was specific for p24; the Abbott detector antibody had bands for p18, p41-43, gp120 as well as p24. By using dilutions of a known amount of HIV antigen, the Abbott test could detect 20 pg/ml p24; the DuPont test could detect 30 pg/ml p24. The DuPont test was also more sensitive than the Abbott test at detecting a synthetic 104mer peptide of p24. Within the 104mer sequence two regions (294-318, 334-348 amino acids) inhibited the binding of the DuPont detector antibody, but no blocking was observed with the Abbott antibody. Although the Abbott test was slightly more sensitive at detecting HIV protein than the DuPont test, the major difference between the tests was in the molecular specificity, in that the Abbott test detected proteins other than p24. This may not be important for detecting antigen in cell culture, but it may affect the detection of antigenaemia in patients' sera.     

Comparison of five commercial western blot kits for detection of human immunodeficiency virus 1

Twenty-two human immunodeficiency virus 1 (HIV-1) enzyme immunoassay (EIA) reactive and two non-reactive patient specimens were analyzed using five commercially available HIV-1 Western blot kits. The percentage of HIV-1 bands detected by each kit was recorded. The differences between pairs of kits were not found to be statistically significant at the 0.05 level. All EIA reactive specimens were reconfirmed as reactive by each Western blot kit tested.     

Detection of human calicivirus antigen and antibody by enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect human calicivirus (HCV) antigen and antibody to HCV. The ELISAs were specific for HCV and as sensitive as a previously developed radioimmunoassay. These ELISAs were used to search for evidence of HCV infection in the United States, where HCV gastroenteritis has rarely been reported. One hundred sixty-three stool samples collected from children hospitalized with diarrhea were examined; one sample was positive in the ELISA. Typical calicivirus particles were found in this stool sample, and these particles reacted with a hyperimmune guinea pig anti-HCV serum by immune electron microscopy. The age-related acquisition of antibody to HCV in hospitalized infants and children (from birth to 19 years old) without gastroenteritis and in healthy adults was also evaluated. The pattern of acquisition of antibody to HCV was similar to that for group A rotaviruses, namely, beginning in infancy and becoming 100% by the age of 4 years. These data suggest that HCV is associated with infantile gastroenteritis in the United States, that infections with HCV are common, and that many infections with HCV (Sapporo strain) may not require hospitalization.     

Agreement of rubella IgG antibody measured in serum and dried blood spots using two commercial enzyme-linked immunosorbent assays

Cases of congenital rubella are now rare in the United Kingdom. However, in certain areas such as London, where a significant proportion of pregnant women has been born abroad and uptake of trivalent measles-mumps-rubella (MMR) vaccination is low, the risk of a rubella outbreak remains. Prior to carrying out a seroprevalence study using rubella IgG antibody in newborn dried blood spots as an indirect marker of maternal immunity, rubella IgG antibody concentrations in serum and dried blood spot samples were investigated. Anonymous paired serum-dried blood spot samples left over from occupational health screening were tested for rubella IgG antibody by two commercially available enzyme-linked immunosorbent assays (ELISAs) (Dade Behring, Marburg, Germany, and Diesse, Siena, Italy). Agreement between serum samples and dried blood spot samples was high for both assays. There were no significant differences in antibody concentrations in paired samples, as 67 of 73 samples tested with the Diesse ELISA (91.8%), and 76 out of 79 samples tested with the Dade Behring ELISA (96.2%) were within two standard deviations of the mean difference. Commercial ELISAs are an appropriate test for seroprevalence surveys based on rubella IgG in dried blood spot samples.     

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.     

Development of quality control procedures for the human immunodeficiency virus type 1 antibody enzyme-linked immunosorbent assay.

A standardized pool of human sera that was positive for human immunodeficiency virus type 1 (HIV-1) antibody was developed. This positive control serum was used to analyze test differences among eight laboratories, among the HIV-1 antibody test kits of three different manufacturers, among different lots of the same test kit, and among pipetting devices and techniques. The standardized pool of human sera was tested 327 times by the different laboratories. In terms of positive tests, a reproducibility of 99.69% was achieved; however, significant test variance among laboratories, among test kit lots, and among pipetting devices and techniques could be demonstrated if the tests were compared on the basis of the net positive optical density (OD) value. This value was calculated by subtracting the cutoff OD value (i.e., the value below which an OD value was considered negative for HIV-1 antibody) from the observed OD value of the standardized pool of human sera. The results obtained suggest that this strategy can be used for proficiency testing, for monitoring the quality of HIV-1 antibody enzyme-linked immunosorbent assay reagents, and for evaluating pipetting devices and techniques.     

Comparison of Track XI fluorometric immunoassay with Bio-EnzaBead enzyme-linked immunosorbent assay for detection of serum antibody to mouse hepatitis virus.

The Track XI system (Microbiological Associates, Bethesda, Md.) was compared with the Bio-EnzaBead assay (Organon Teknika, Durham, N.C.) for the detection of antibody to mouse hepatitis virus (MHV). Strain A/J mice were inoculated intranasally with MHV type 3. Sera were collected at 1, 2, 4, and 9 weeks postinoculation and tested. Individual serum samples were retested twice by each method. The results suggested that the Track XI system was more sensitive and reliable than the Bio-EnzaBead assay in detecting antibody to MHV type 3 in individual serum samples from A/J mice.     

Comparison of enzyme-linked immunosorbent assay with enzyme-linked fluorescence assay with automated readers for detection of rubella virus antibody and herpes simplex virus.

The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available.     

Comparison of three enzyme-linked immunosorbent assays and a direct fluorescent-antibody test for detection of respiratory syncytial virus antigen.

We prospectively evaluated three enzyme immunoassays (EIAs) and a direct fluorescent-antibody (DFA) test for respiratory syncytial virus detection. Of 90 specimens, 79% gave the same results in all four tests (30 positive and 41 negative) and 97% were in agreement in three of the four assays. The agreement between the direct fluorescent-antibody test and each enzyme immunoassay was greater than or equal to 86%.