Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus,diphtheria, and Haemophilus influenzae Type b

We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.     

Comparison of three enzyme-linked immunosorbent assays for detection of immunoglobulin g antibodies to tetanus toxoid with reference standards and the impact on clinical practice

Accurate determination of the concentrations of immunoglobulin G (IgG) antibody to tetanus toxoid is important in order to evaluate the immunogenicity of tetanus toxoid vaccines, determine immune competence in individual patients, and measure the prevalence of immunity in populations. The performance of three commercially available enzyme-linked immunosorbent assays (ELISAs) for IgG antibodies to tetanus toxoid were evaluated. Serially diluted NIBSC 76/589 and TE-3 human tetanus IgG immunoglobulin international reference standards were analyzed in quadruplicate using ELISAs manufactured by The Binding Site, Inc. (VaccZyme); Scimedx; and Euroimmun. In addition, IgG antibodies to tetanus toxoid were measured in 83 deidentified serum specimens using each manufacturer's ELISA. Each ELISA provided linear results when evaluated with the reference preparations. The Binding Site ELISA provided results that closely corresponded to the reference preparations (y = 1.09x − 0.08), whereas the Scimedx ELISA gave results that were consistently lower (y = 0.21x − 0.07) and the Euroimmun ELISA gave results that were consistently higher (y = 1.5x + 0.30) than the reference preparation concentrations. Using the recommended cutoff for each ELISA (<0.10 IU/ml), the overall agreement of all of the ELISA methods was 78%. Three of eighty-three (3.6%) human serum samples demonstrated inadequate immunity with all three assays. The Binding Site ELISA yielded nonprotective antibody concentrations in only these 3 samples, whereas 19 samples (22.9%) according to the Scimedx ELISA and 6 samples (7.2%) according to the Euroimmun ELISA demonstrated nonprotective concentrations. The performance characteristics of ELISAs for tetanus immunoglobulin titers were manufacturer dependent, and the differences translated into important disparities in reported results.     

Antibody response to pertussis toxin in patients with clinical pertussis measured by enzyme-linked immunosorbent assay

Serum antibody response to pertussis toxin was measured by enzyme-linked immunosorbent assay in 172 patients with clinical symptoms typical of whooping cough. The diagnosis was verified by culture in 100 patients. Serum antibodies were either not detectable or present only at low levels in sera obtained in the early stage of disease. Significant changes in serum levels of IgG, IgM and/or IgA were demonstrated in 143 patients (83 %). The lack of comparable increases in most of the other patients may be due to inappropriate timing of serum collection. Thus, detection of antibodies against pertussis toxin in paired serum samples can be used for serological diagnosis of pertussis. However, the presence of IgM and/or IgA in a single serum sample does not confirm a diagnosis of pertussis, since such antibodies were found in healthy adults as well as in patients two years after the disease. High levels of these antibodies are, however, suggestive of on-going or recent disease.     

An enzyme-linked immunosorbent assay (ELISA) for the determination of diphtheria toxin antibodies.

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of diphtheria toxin antibodies is described. When the ELISA technique was compared to a single radial immunodiffusion assay the results correlated well but the ELISA technique was ten thousand times more sensitive. It was also at least ten times as sensitive as the in vivo rabbit skin test.     

Normalized enzyme-linked immunosorbent assay for determining immunoglobulin G antibodies to cytomegalovirus

A normalized enzyme-linked immunosorbent assay for the determination of immunoglobulin G antibodies to cytomegalovirus is described. The rapid assay involves three 30-min incubations and permits the quantitation of antibody levels with a single-specimen dilution in conjunction with a reference antibody preparation. The results obtained with the normalized enzyme-linked immunosorbent assay correlated closely with the results of complement fixation titrations and another commercially available enzyme-linked immunosorbent assay. The specificity of the procedure was further demonstrated by viral absorption, using cytomegalovirus from two different sources and other viral antigen preparations, including rubella and influenza. The reproducibility of the normalized test results is good and allows for greater uniformity of reporting on a day-to-day basis, as well as between laboratories.     

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.     

Ability of enzyme-linked immunosorbent assays to detect early immunoglobulin G antibodies toToxoplasma gondii

Two ELISA procedures, one using sonicated antigen coated with carbonate buffer and the other formalin fixed trophozoites with dry coating, differ in their ability to detect early antibodies in toxoplasmosis. In order to identify factors responsible for this difference, seven ELISA systems differing from each other in antigen used and/or coating procedure were compared. Both fixation of the trophozoites with formalin and air-drying of the antigen in the microtiterplate were important factors determining the ability of the assay to detect IgG antibodies in the early stage of infection. Differences in the results of the two ELISA procedures can be used to distinguish between the acute and chronic stages of infection.     

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.     

Interlaboratory evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to Toxoplasma gondii.

One hundred and fifteen serum samples from healthy laboratory personnel and 50 consecutive samples from 19 patients with anamnestic clinical signs of toxoplasmosis were assayed by four laboratories for the presence of immunoglobulin M antibodies to Toxoplasma gondii by an indirect enzyme-linked immunosorbent assay (ELISA), an antibody capture assay with peroxidase-labeled toxoplasma antigen, and an immunoblotting assay. In addition, a commercially available antibody capture ELISA was used. Highly significant correlation coefficients were obtained between the four laboratories and the commercial test. The indirect ELISA and antibody capture ELISA showed equal sensitivity in detection of immunoglobulin M antibodies to toxoplasma in early-stage serum samples. However, in this study, the antibody capture assay discriminated better between serum samples obtained at early or late stages of toxoplasma infection.     

Development and validation of a multiplex immunoassay for the simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus

To increase testing of vaccine induced humoral immunity in immune surveillance studies and vaccine trials, a rapid and simple microsphere-based multiplex assay (pentaplex) was developed for the quantitation of IgG serum antibodies directed against the Bordetella pertussis antigens: Pertussis Toxin (Ptx), Filamentous hemagglutinin (FHA), Pertactin (Prn) and to Diphtheria toxin and Tetanus toxin. All individual antigens were covalently linked to carboxylated microspheres. The method was validated with different serum panels (n=60-78 samples). With the Multiplex Immunoassay (MIA) no evidence for bead interference between monoplex and pentaplex was found. The specificity of the method was shown by a heterologous inhibition of <16% and homologous inhibition of >92%. The pentaplex MIA appeared sensitive with lower limits of quantitation (LLOQ) well below those for ELISA (enzyme-linked immuno-sorbant assay). Assay reproducibility was high with intra-assay variability less than 10% and inter-assay variability below 14%. The reproducibility of the bead conjugation was good and beads could be stored up to at least 6 months without quality reduction. Importantly, the correlation of the pentaplex MIA with the individual ELISAs was excellent, R>0.98 for the Pertussis antigens and R=0.95 for Diphtheria and R=0.98 for Tetanus. Serum IgG antibodies to B. pertussis, Diphtheria and Tetanus can be measured easily, specific and reproducible using the pentaplex MIA. The pentaplex MIA shares features of the ELISA with the additional advantages of high sample throughput and small sample volumes and antigen required.     

Fast, antigen-saving multiplex immunoassay to determine levels and avidity of mouse serum antibodies to pertussis, diphtheria, and tetanus antigens

To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra- and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.     

Detection of immunoglobulin M antibodies toTreponema pallidum in a modified enzyme-linked immunosorbent assay

The indirect enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin M (IgM) antibodies toTreponema pallidum in sera of syphilitic patients is complicated by false positive reactions due to the interference of IgM rheumatoid factor (IgM-RF) activity and the presence of treponemal IgG antibodies. Another source of error producing false negative results is the competition between treponemal IgG and IgM antibodies for the binding sites on the antigen. To avoid these complications in the indirectTreponema pallidum-specific IgM-ELISA, total IgG was immunoprecipitated from sera of syphilitic patients prior to the assay. The IgM-RF from non-precipitated sera reacted in an IgM-RF-ELISA and in theTreponema pallidum-IgM-specific ELISA with identical titers. After precipitation of total IgG no reactíon of the IgM-RF in the assay could be demonstrated. Competition between IgG and IgM antibodies can be prevented almost completely by the precipitation procedure. The sensitivity and specificity of theTreponema pallidum-specific IgM-ELISA after immunoprecipitation of total serum IgG were shown to be higher than 97 percent.     

Detection of antichlamydial immunoglobulin G and M antibodies by enzyme-linked immunosorbent assay

Chlamydia trachomatis causes a wide range of infections in adults and conjunctivitis and pneumonia in neonates. The complement fixation test for chlamydial antibody is broadly reactive, but possesses low sensitivity, whereas the microimmunofluorescence test is highly sensitive, but technically difficult to perform. A simple, rapid enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of immunoglobulin G (IgG) and IgM antibodies to C. trachomatis. Wells of microtiter plates were coated with Renografin-purified elementary bodies (serotype L2) grown in cycloheximide-treated McCoy cells, and serum antibody was detected with peroxidase-labeled goat antihuman IgG and IgM antibody. Of 41 sera tested from patients with lymphogranuloma venereum, pelvic inflammatory disease, cervicitis, or urethritis there was a 90 and 63% correlation of positive results for IgG and IgM, respectively, by microimmunofluorescence and ELISA. Of the positive correlates, ELISA titers were up to 128 times higher than microimmunofluorescence titers for IgG and IgM. The ELISA detected no false-positive results, but missed two positive results for IgG. Both of these sera were reactive against serotypes C and J, suggesting that the ELISA with LGV L2 antigen may not measure antibodies to serotypes within the C serogroup. The IgM ELISA detected 7 negative and 4 positive results not detected by the microimmunofluorescence test. Of four paired sera examined by ELISA, three showed a fourfold rise in IgG antibody titer, and one showed a twofold rise. Further evaluation of this ELISA will be required to determine how useful it will be in seroepidemiological studies and as a diagnostic tool.     

Detection of immunoglobulin G antibodies to cytomegalovirus antigens by antibody capture enzyme-linked immunosorbent assay

An antibody capture enzyme-linked immunosorbent assay (ELISA) that uses horseradish-peroxidase-labeled antigen for the detection of immunoglobulin G (IgG) antibodies to cytomegalovirus (CMV) is described. A microtiter plate was coated with anti-human IgG and consecutively incubated with serum specimens, enzyme-labeled CMV antigen made from CMV-infected cell nuclei, and substrate. The CMV IgG antibody content was determined spectrophotometrically and expressed as absorbance. Furthermore, to reveal any nonspecific reactions, all sera were tested against an enzyme-labeled control antigen made from uninfected cell nuclei. The problem with nonspecific reactions was small and was circumvented by the addition of unlabeled control antigen to the conjugates. For epidemiological studies the test was not as sensitive as other serological tests. On the other hand, the IgG antibody capture ELISA was highly sensitive for detecting the serological antibody response in patients with primary and recurrent CMV infections. Thus, one positive serum remained positive at a serum dilution of 1:10(7). The specificity of the test was shown by a blocking experiment and by testing 126 complement fixation-positive sera, of which 97% were positive. There was a rather good correlation between the complement fixation test and the IgG antibody capture ELISA (rs = 0.79, P less than 0.001). The test is especially useful when tests for CMV antibodies of the IgM, IgA, and IgE classes are run by similar antibody capture ELISAs, since the same procedure and conjugate are used.     

Detection of immunoglobulin M antibodies to hepatitis A virus by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay for the detection of immunoglobulin M (IgM) antibodies to hepatitis A virus is described. The test uses the principle of binding of IgM antibodies to anti-IgM-coated microtiter plates to determine whether the IgM antibodies attached have specificities for hepatitis A virus. In three patients with hepatitis type A followed up to 12 months, IgM antibodies to hepatitis A virus could be demonstrated from the onset of illness and during the following 2 to 3 months. When acute-phase sera from 48 patients with acute hepatitis were tested, IgM antibodies to hepatitis A virus could only be demonstrated in 18 patients previously classified as type A, whereas 30 patients with type B and non-A non-B hepatitis were negative. IgM antibodies to hepatitis A virus could not be demonstrated in 108 normal sera nor in 55 sera containing rheumatoid factor. These results indicate that the enzyme-linked immunosorbent assay for IgM antibodies to hepatitis A virus is useful in the serodiagnosis of acute hepatitis type A on a single serum sample taken during the acute phase of illness.     

Microtiter enzyme-linked immunosorbent assay for immunoglobulin g cholera antitoxin in humans: sensitivity and specificity

Serum samples were obtained from 92 informed, community volunteers before and 10, 21, and 28 days after they ingested 10(3) to 10(6)Vibrio cholerae of Inaba or Ogawa serotype and classical or El Tor biotype as part of a cholera vaccine development program. Pre- and postchallenge sera were examined for neutralizing antibody to cholera toxin by the rabbit skin permeability factor and adrenal cell techniques. Immunoglobulin G-binding antibodies to cholera toxin were quantitated by enzyme-linked immunosorbent assay (ELISA) in serum diluted 1:200. The results obtained in these cholera volunteers were compared with a negative control population comprising 30 people who ingested enteropathogenic Escherichia coli or E. coli which produced heat-stable but not heat-labile enterotoxin. Although all three antitoxin assays correlated closely with each other in both groups of volunteers, ELISA was more sensitive than either neutralization assay in detecting both subclinical and overt cholera infections. Seroconversion was demonstrated by ELISA in 58 of 66 (88%) volunteers who excreted V. cholerae, including 50 of 54 (93%) with clinical cholera, compared with 47 of 66 (71%) and 52 of 66 (79%) by the rabbit skin permeability factor and adrenal cell techniques, respectively. Although ELISA does not measure the toxin-neutralizing activity of antibodies directly, it provides a practical alternative to the rabbit skin permeability factor and adrenal cell assays.     

Determination of tetanus antibodies by a double-antigen enzyme-linked immunosorbent assay in individuals of various age groups

In this study, tetanus immunity was determined in 549 randomly chosen individuals of various age groups in Ankara, Turkey. Antibody levels in sera of the individuals were measured using a double-antigen enzyme-linked immunosorbent assay. Overall, 66.5% (95%CI, 62.4–70.4) of the population studied was found to have basic protection (0.01 IU/ml) against tetanus. Protective levels of tetanus antibodies declined progressively with age. The rate of protection in children and adolescents (aged <20 years) exceeded 90%, while only 16.3% (95%CI, 8.9–26.2) of those over 60 years of age were protected. Females over 60 years of age were less immune than males of the same age group (p=0.034). Although the rates of protection in children and adolescents are regarded as satisfactory, the rates among adults are low. Preventive measures against tetanus should therefore focus on scheduled booster immunization for adults as well as children.     

Comparison of Western immunobloting to an enzyme-linked immunosorbent assay for the determination of anti-Bordetella pertussis antibodies

During Bordetella pertussis infection, it has been established that an increase of anti-pertussis toxin (PT) and anti-filamentous hemagglutinin (FHA) antibodies occurs. Immunoblots from two manufacturers using FHA and PT antigens were compared with an enzyme-linked immunosorbent assay (ELISA) that used both FHA and PT. One manufacturer used two concentrations of PT bands for the IgG immunoblot, calibrated to the World Health Organization standard for PT in international units (IU/ml), 100 IU/ml (PT-100) and 8 IU/ml (PT). The second immunoblot kit measured antibodies to a single calibrated PT band. Both kits measured IgA antibodies, and one additionally measured IgM antibodies. Two of 41 (5%) ELISA IgM positives were confirmed positive by IgM immunoblotting, suggesting poor specificity of the IgM ELISA. The agreements of the IgG and IgA immunoblots with the ELISA ranged from 72.5% to 85.3%, with only 38 to 51% of IgA positives confirmed by immunoblotting and only 61 to 68% of IgG positives confirmed by immunoblotting. The two immunoblots correlated well with each other, with 91.7% and 94.3% agreement for IgG and IgA, respectively. When the FHA band was used with the PT band as the criterion for positivity, significant differences existed in specificity compared to the ELISA (IgG, 84.1% versus 33.3%; IgA, 82.4% versus 71.0%). When the positive IgA immunoblots (evidence of natural recent infection) were compared to the positive PT-100 IgG immunoblots (evidence of recent infection or vaccination), the PT-100 blot showed a 71% sensitivity in detecting natural recent infection. B. pertussis immunoblots, alone or in combination with ELISAs, can aid in the diagnosis of B. pertussis infection.     

Quantitation of rabbit immunoglobulin G antibodies to Salmonella minnesota Re by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed which allowed the measurement of rabbit immunoglobulin G antibodies directed to Salmonella minnesota Re glycolipid. Efficient adsorption of the antigen to polystyrene could only be effected provided it had been previously dialyzed against 0.2 M EDTA (pH 7.0) and subsequently treated with 0.2% sodium deoxycholate (15 min at 56 degrees C) in 0.05 M diethanolamine buffer (pH 9.6). The method is by far more sensitive than quantitative precipitation in the determination of IgG antibodies. Inhibition by glycolipid of binding S. minnesota Re antibodies to immobilized glycolipid attests to the specificity of the assay and permits the detection of as little as 100 ng of glycolipid.