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.     

磁微粒化学发光法与酶联免疫吸附法测定抗中性粒细胞胞浆抗体的结果比较

目的:比较分析磁微粒化学发光法与酶联免疫吸附法测定抗PR3 抗体、抗MPO 抗体的检测结果。方法:分别采用磁微粒化学发光法(A 方法)和酶联免疫吸附法(B 方法)对166 例自身免疫病患者血清、50 例健康者血清中抗PR3 抗体、抗MPO 抗体进行定量检测,对检测结果进行统计分析。结果:A 方法测定高、中、低质控血清的批内和批间重复性优于B方法,A、B 方法测定质控血清的准确度均符合要求;A、B 方法测定抗PR3 抗体、抗MPO 抗体临床样本的线性相关系数r 分别为0.987 8,0.989 6;A、B 方法的检测结果采用kappa 分析,kappa 系数分别为0.897 和0.882。结论:磁微粒化学发光法(A 方法)测定抗PR3 抗体、抗MPO 抗体优于酶联免疫吸附法(B 方法),更符合临床应用要求。     

Comparison of an enzyme-linked immunosorbent assay with radioimmunoassay for the measurement of pneumococcal capsular polysaccharide antibodies

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure antibodies against pneumococcal polysaccharides of the IgA, IgG and IgM isotypes. Antibodies against pneumococcal polysaccharide types 1, 3, 6A, 8 and 9N were measured by ELISA and radioimmunoassay. Similar antibody responses were observed when comparing both assays. The study included 39 persons at high risk of developing pneumococcal infection and 13 healthy adults. Within 1 month after immunization IgM was the principle isotype. After 1 month, IgG was the principle isotype. Very low levels of IgA were detected in the post-immunization serum. The ELISA procedure described can be used to study the immune response to pneumococcal vaccines.     

Competitive enzyme-linked immunosorbent assay for Treponema pallidum antibodies

A competitive enzyme-linked Treponema pallidum immunosorbent assay (CETPIA) was compared with the standard serological tests for syphilis. Of 3081 serum samples submitted, 2883 gave negative results in the CETPIA and the routine screening tests. Positive results were obtained in the CETPIA and in one or more of the specific treponemal tests with 115 samples. Discrepancies in the results of the CETPIA and standard serological tests were found with 83 serum samples, most of these were attributed to biological false positive reactions in the Venereal Disease Research Laboratory (VDRL) test. CETPIA may have a role in the serological diagnosis of syphilis.     

Detection of antibodies to Mycoplasma pulmonis by an enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay, which entails the use of antigen-coated tubes and enzyme-labeled anti-immunoglobulins, was applied for the detection of antibodies against Mycoplasma pulmonis in mice. A lysate of M. pulmonis was used as the antigen, and anti-mycoplasmal antibodies of the different immunoglobulin classes were detected by class-specific anti-immunoglobulin labeled with alkaline phosphatase. The optimal conditions for the test were determined, the specificity was evaluated, and the assay was compared with other procedures for detection of mycoplasmal infection. The enzyme-linked immunosorbent assay was found to be a specific, highly sensitive, and reliable procedure for detecting anti-mycoplasmal antibodies in mice.     

Measurement of antibodies to influenza virus neuraminidase by an enzyme-linked immunosorbent assay

The contribution of influenza A neuraminidase antibodies to the reaction with whole virus in an enzyme-linked immunosorbent assay (ELISA) was assessed by specific absorption of rabbit hyperimmune sera. Although measurable and independent, the effect of neuraminidase antibodies was less than that of hemagglutinin antibodies. Recombinants with an irrelevant hemagglutinin were used successfully as antigens in an ELISA test for measuring neuraminidase antibodies in rabbit hyperimmune sera, but a low cross-reaction between N1 and N2 subtypes was observed. However, for the measurement of N2 antibody rises in human sera, ELISA was highly specific and compared favorably with two other methods, neuraminidase inhibition and single radial hemolysis.     

An enzyme-linked immunosorbent assay for the determination of the IgA subclass distribution of antigen-specific antibodies

An ELISA for the determination of the IgA subclass distribution of antigen-specific antibodies was developed using commercially available monoclonal anti-IgA1 anti-IgA2 subclass antibodies. Furthermore an anti-A2m allotype-specific antibody was included in the study. The specificity and sensitivity of the monoclonal anti-immunoglobulin antibodies used was analyzed using sera from normal and IgA class- or subclass-deficient individuals (with or without homozygous C alpha 1 subclass gene deletions). Human IgA1 and IgA2 hybridoma antibodies were also used. In this particular assay, only two out of four tested anti-IgA1 and two out of three tested anti-IgA2 antibodies proved to be specific for their corresponding IgA subclass. The anti-A2m(2) monoclonal antibody was shown to be specific for the corresponding allotype. These ELISA methods may facilitate further work on the regulation of IgA subclass production in man.     

Anti-endometrial antibodies in women measured by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed tomeasure anti-endometrial antibody concentrations in the serumof women with endometriosis. Pooled cytosolic protein extractsfrom the endometrial gland cells of 10 women were used as anantigen source. Serum samples were obtained from women withendometriosis before (n = 51) and after 6 months treatment withdanazol or nafarelin (n = 30). Control sera came from womenwith a normal pelvis at laparoscopy, performed for sterilization(n = 23) or the investigation of pain and/or infertility (n= 22), 13 women with Rokitansky syndrome, and 10 umbilical cordbloods and adult males. There were no significant differencesin serum anti-endometrial antibody concentrations before andafter treatment, or between women with endometriosis and withoutendometriosis. Concentrations were lower in male and cord bloodserum than in female's serum (P < 0.0001). We conclude thatthe ELISA is not a useful diagnostic tool for endometriosisunless more specific antigens can be isolated.     

Comparison of Western blot and enzyme-linked immunosorbent assay for diagnosis of Lyme borreliosis

The usefulness of Western blot in the serological diagnosis of Lyme borreliosis was evaluated compared with an ELISA using a whole cell sonicate antigen. Fifty-three of 68 (78 %) patients with neuroborreliosis had positive IgM and/or IgG immunoblots and 40 of 68 (59 %) had positive IgM and/or IgG ELISA titers in serum. Eight of 44 (18 %) controls with meningitis/encephalitis of non-borrelia etiology had positive IgM and/or IgG immunoblots and 4 of 44 (9 %) had positive IgM and/or IgG ELISA titers in serum. Western blot was more sensitive than ELISA, the difference being most pronounced in sera from patients with neurological disease for four weeks or less. Both patients and controls lived in an area endemic for Lyme borreliosis and some ELISA negative but Western blot positive controls were thought to have been previously exposed toBorrelia burgdorferi. However, the specificity for current disease was not improved by Western blot. In conclusion, Western blot does not seem to be the method of choice for screening purposes in a routine laboratory but can be used as a complement to ELISA for serodiagnosis in patients with disease of short duration.     

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.     

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.     

Quantitative determination of human IgG antibodies to the peptide subunit determinant of peptidoglycan by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative measurement of IgG antibodies to the immunodominant R-D-Ala-D-Ala-OH determinant of peptidoglycan. Synthetic peptides R-D-Ala-D-Ala-OH, revealing structural analogy with the C-terminal sequence of the antigenic determinant H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 of peptidoglycan, were coupled covalently to albumin via their amino groups. The resulting peptidyl proteins were employed as an antigen in an ELISA for the specific detection of human IgG antibodies against the C-terminal R-D-Ala-D-Ala-OH moiety of H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2. Antigenic specificity was proved by comparing the high binding to albumin-(D-Ala-D-Ala-D-Ala-OH)9 with a lack of binding to albumin-(L-Ala-L-Ala-L-Ala-OH)13 and by appropriate inhibition studies of the ELISA. IgG, totally free from IgA and IgM, was isolated from reference serum 004, and the particular specificity was entirely found in this fraction. Quantification of the ELISA was effected by affinity chromatography. Isolated IgG was applied to an affinity column of Sepharose-[albumin-(D-Ala-D-Ala-D-Ala-OH)9]n, unbound IgG was eluted with phosphate-buffered saline and specific IgG against the C-terminal R-D-Ala-D-Ala-OH moiety of H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 was eluted with 6 M guanidinium chloride.     

Production of subgroup-specific monoclonal antibodies against human rotaviruses and their application to an enzyme-linked immunosorbent assay for subgroup determination

Nonneutralizing monoclonal antibodies were prepared against two strains, S2 and YO, of human rotaviruses isolated in cell culture. S2-37 and YO-5 antibodies had subgroup I and subgroup II specificities, respectively. The remaining antibodies (S2-65, YO-71, YO-89, and YO-156) reacted commonly with all the rotaviruses examined. All of the monoclonal antibodies agglutinated exclusively single-shelled particles and immunoprecipitated 42,000-dalton protein, a major component of inner capsid. Using the three monoclonal antibodies (S2-37, YO-5, and YO-156), an enzyme-linked immunosorbent assay was developed for detecting and subgrouping human rotavirus isolates.     

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.     

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.     

A highly sensitive enzyme-linked immunosorbent assay for idiotype-bearing antibodies

A sensitive and specific immunoenzyme assay (ELISA) for quantitation of total and cross-reactive idiotype-bearing (CRI) anti-ABA antibodies is described. Total anti-ABA antibodies are directly assessed in ABA-BGG coated polyvinyl wells with enzyme-labelled rabbit anti-mouse immunoglobulins. By interpolation on a standard curve absorbance values give the concentration of anti-ABA antibodies with a sensitivity of 30 ng/ml. CRI+ antibodies are quantitated by inhibition of enzyme-labelled monoclonal CRI+ antibody binding to solid-phase coated rabbit anti-CRI immunoglobulins. The concentration of CRI+ antibodies, evaluated by interpolation on a standard inhibition curve, can be measured at the level of 10 ng/ml. This highly sensitive, rapid, specific and reproducible assay is easily used, with minor modifications, to detect specific antibodies in any idiotype system.     

Specific enzyme-linked immunosorbent assay for the detection of antibodies to the human spumavirus

Recombinant plasmid clones were constructed harbouring the central domains of the outer membrane protein and the transmembrane protein of the env gene of human spumaretrovirus (HSRV). The corresponding fusion proteins were expressed in E. coli, purified and used subsequently to produce antibodies against the HSRV env proteins in rabbits. The authenticity of the bacterially produced domain of the HSRV env proteins was shown by radioimmunoprecipitation of the viral env glycoprotein from HSRV-infected human cells with rabbit antibodies raised against the recombinant antigens. The recombinant viral antigens were used to establish a sensitive and spumavirus-specific enzyme-linked immunosorbent assay (ELISA). This anti HSRV antibody ELISA makes it possible to screen human sera for the presence of spumavirus infections.     

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.     

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.