Automated microparticle enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii.

Fully automated microparticle enzyme immunoassays (MEIA) for the IMx immunoassay analyser were developed to detect IgG and IgM antibodies to Toxoplasma gondii. The IgG MEIA results are expressed in International Units (IU) of IgG antibody interpolated from a six point calibration curve covering the range from 0 to 300 IU/ml. Reproducible results were obtained from a calibration curve stored in the instrument for at least one month. The qualitative IgM MEIA expresses results as an index using a single calibrator included in each run. The Toxo IgG MEIA and Toxo IgM MEIA were in 98% and 97% agreement, respectively, with the reference assays used. Twenty four sera can be completely processed in about 35 minutes.     

Evaluation of the Captia enzyme immunoassays for detection of immunoglobulins G and M to Treponema pallidum in syphilis.

Two new enzyme-linked immunosorbent assays (ELISA), one for the measurement of immunoglobulin G (IgG) (Captia Syphilis-G) and one for the measurement of IgM (Captia Syphilis-M), were evaluated for detecting antibodies to Treponema pallidum. Serum samples from 169 patients, 96 with various stages of untreated syphilis, 63 with treated syphilis, and 10 who were noninfected, were investigated. All sera were also examined by traditional treponemal and cardiolipin tests and by the fluorescent treponemal antibody absorption (FTA-ABS) test for 19S(IgM). The overall sensitivity of Captia Syphilis-G was 98.3%. The IgG ELISA was very sensitive (100%) in all stages of untreated syphilis, except in primary syphilis (82%). In all diagnostic groups of syphilis, the reactivity of Captia Syphilis-M was similar to that of the 19S(IgM) FTA-ABS test, except in reinfections, in which the IgM capture ELISA was less sensitive. False-positive IgM capture ELISA results were not found in the 10 neonates born to mothers adequately treated for syphilis. However, of six serum samples containing rheumatoid factor, two were reactive in the Captia Syphilis-M test but not in the 19S(IgM) FTA-ABS test. This indicated that the specificity of the IgM capture ELISA was not absolute. All serum samples from treated patients were reactive in the IgG ELISA, but only 15 samples were reactive in the IgM capture ELISA, which appeared to be as effective as the 19S(IgM) FTA-ABS test in monitoring the effect of treatment. Simultaneous measurement of IgG and IgM antibodies for T. pallidum by the Captia immunoassays appears to be an efficient and simple method for confirming the diagnosis of syphilis as well as for indicating whether active disease is present.     

Detection of immunoglobulins G and M to rubella virus by time-resolved immunofluorometry.

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.     

Evaluation of rubella IgM enzyme immunoassays.

BACKGROUND: Rubella virus generally causes a mild fever, rash illness similar in clinical presentation to infections by other viruses including measles and parvovirus B19. Rubella infections in pregnant women in the first trimester carry a high risk of congenital rubella syndrome (CRS) which can result in severe congenital defects in the infants. The goal of rubella immunization programs is therefore to eliminate CRS. The primary test for the laboratory confirmation of rubella is IgM serology. It is therefore important to evaluate currently available commercial rubella IgM immunoassays to ensure high quality rubella diagnostic testing. STUDY DESIGN: In this study, we compared the performance of seven commercial rubella IgM enzyme immunoassays (EIA) (Meddens, Denka Seiken, Behring, Wampole, Captia, Sigma and Abbott Axsym) using well-defined panels of sera from rubella and non-rubella/rash-illness cases. RESULTS: The Meddens, Denka Seiken, Behring and Wampole rubella IgM EIAs all performed similarly for sensitivity (range of 74.1-76.8%) and specificity (range of 93.9-96.1%). Relative to the other assays, the Axsym had a higher sensitivity (78.9%) but lower specificity (86.5%). The Captia assay had the lowest overall sensitivity (66.4%), while the Sigma assay had a lower specificity (85.6%) in relation to the other assays. CONCLUSIONS: In conclusion, the Meddens, Denka Seiken, Behring and Wampole rubella IgM EIAs are comparable in their overall performance with respect to sensitivity and specificity.     

Serological detection of HBeAg and anti-HBe using automated microparticle enzyme immunoassays.

Fully automated microparticle enzyme immunoassays (EIA) were developed for the detection of HBeAg (IMx HBe) and antibodies against HBeAg (IMx anti-HBe), respectively. Specimens from blood donors, diagnostic and hospital patients and individuals with a variety of infectious and immune diseases were tested both in house and at four clinical sites. The overall agreement between IMx HBe and Abbott HBe RIA/EIA was 99.7% (2985 of 2994) and between IMx anti-HBe and anti-HBe RIA/EIA was 95.8% (2330 of 2432). Almost all anti-HBe discordant specimens (94.1%, 96 of 102) were reactive by IMx anti-HBe but negative by anti-HBe RIA/EIA. off anti-HBe discordant specimens were also reactive for anti-HBc. The IMx anti-HBe assay was 2- to 4-fold more sensitive than the current RIA as determined by serial dilution of anti-HBe reactive specimens. The ability of these IMx assays to detect HBeAg and anti-HBe in 199 HBsAg reactive specimens was also evaluated. 43.7% (87 of 199) and 66.3% (132 of 199) specimens were reactive for HBeAg and anti-HBe by IMx, respectively. Only one specimen was negative for both IMx assays compared to 14 (7.0%) non-reactive for both HBe and anti-HBe RIA. There were 24 specimens (12.1%) positive for both HBeAg and anti-HBe by IMx compared to 1 (0.5%) positive by the corresponding RIAs. This increased detectability of anti-HBe in HBsAg carriers using IMx anti-HBe may result from increased sensitivity for 'free' anti-HBe and/or increased ability to detect anti-HBe in immune complex. IMx anti-HBe also detected more reactives among volunteer blood donor specimens reactive for anti-HBc but negative for HBsAg (55.5%, 86 of 155), compared to RIA (38.7%, 60 of 155). IMx anti-HBe may be useful in confirming prior exposure to HBV in blood screened positive by Corzyme.     

Comparison of four enzyme immunoassays for detection of immunoglobulin M antibodies against rubella virus.

We evaluated four tests for the detection of rubella virus-specific immunoglobulin M antibodies. Primarily, consecutive serum samples were tested by two different assays. Selected panels of sera from patients with proven or likely recent rubella and false-positive and true-negative results in the two primary assays were further tested with two recently developed, fully automated techniques. The four tests were comparable in overall accuracy, but their dynamic ranges may differ considerably. Ways to optimize the predictive values are discussed. We conclude that automated assays may be used without causing significant changes in diagnostic accuracy or distortions in notifications of the incidence of rubella compared with the use of established tools.     

Detection of rubella-specific immunoglobulin G: comparison of the enzyme-linked immunosorbent assay and an automated microparticle enzyme immunoassay (IMx).

An automated microparticle enzyme immunoassay (IMx Rubella IgG Antibody Assay; Abbott Laboratories, North Chicago, Ill.) was compared with a conventional enzyme-linked immunosorbent assay (ELISA) for detection of rubella-specific immunoglobulin G (IgG) in 400 consecutive antenatal patients. There was complete agreement between the two tests in this population, which had a positivity rate of 99% for rubella-specific IgG antibodies. The performance of the IMx was also evaluated at the cutoff zone by assaying 64 selected antenatal serum samples with low or negative rubella antibody titers as determined by ELISA. Overall, the IMx was found to be a specific, sensitive assay for the detection of rubella-specific IgG and is virtually fully automated for easy performance.     

Evaluation of two enzyme immunoassays for detection of immunoglobulin G antibodies to mumps virus

To determine suitability for national serosurveys, we compared two commercial enzyme-linked immunosorbent assays (ELISAs) for mumps antibody, Enzygnost Anti-Parotitis-Virus/IgG (which uses a whole-virus antigen) and Microimmune Mumps IgG Screen ELISA (which uses a recombinant nucleoprotein antigen), by testing 1,915 opportunistically collected sera submitted to diagnostic laboratories across Australia in 1997 to 1998. The proportion of positive results increased with age in both ELISAs but was significantly higher with the Microimmune than with the Enzygnost ELISA overall (88% versus 63%; P < 0.01) and in all age groups. However, the proportion of equivocal results was significantly higher with the Enzygnost than with the Microimmune ELISA (9% versus 4%; P < 0.01). Of the 572 sera with discrepant or equivocal results, 508 had sufficient sample remaining to perform the neutralization test (NT). A proportion with concordant results in both ELISAs were also tested by the NT. For sera with discrepant results, there was significantly better agreement between the NT and Microimmune than between the NT and Enzygnost (310/444 [70%] versus 135/348 [39%]; P < 0.01). Of 64 sera with equivocal Microimmune results, 45 (70%) were positive in the NT compared with 140 of 160 (88%) equivocal Enzygnost results (P < 0.01). Compared with the NT, the Microimmune ELISA is more sensitive (96% versus 80%) but apparently less specific (36% versus 85%) than the Enzygnost ELISA. However, this is likely to be due to the generally lower sensitivity of the NT, since the Microimmune results reflect expected seroprevalence, based on vaccine uptake in the age groups studied. We conclude that the Microimmune ELISA is a more appropriate assay than the Enzygnost ELISA for estimation of mumps seroprevalence.     

Indirect enzyme-linked immunosorbent assay for immunoglobulin G and four immunoassays for immunoglobulin M to Toxoplasma gondii in a series of heart transplant recipients.

Toxoplasma gondii infections in heart transplant recipients were monitored by indirect enzyme-linked immunosorbent assay for immunoglobulin G (ELISA-IgG), indirect ELISA-IgM in serum IgM fractions, antibody capture ELISA-IgM, IgM-immunosorbent agglutination assay (ISAGA), and IgM immunoblotting. Basic immunosuppression consisted of cyclosporine and low-dose steroids. Before transplantation, 26 of 43 recipients showed serological evidence of infection. In serum samples from 15 (35%) recipients, specific antibodies were not detected. Approximately 50% of the heart donors, were toxoplasma seropositive. Eight of the fifteen seronegative recipients received hearts from toxoplasma-seropositive donors. In four of the eight recipients, seroconversion could be demonstrated with all tests used. In three of these four patients, clinical disease developed. One patient with strong serological evidence of toxoplasmosis died, but toxoplasma parasites and antigens were not detected at autopsy. In two patients, toxoplasma cysts were found in cardiac biopsies. Seroconversion was not prevented by the use of spiramycin prophylaxis in two recipients. Reactivations of latent infections or reinfections were detected by indirect ELISA in six (23%) seropositive recipients, but symptoms and signs of active T. gondii infection were not seen. Seroconversion and reactivation of infection were readily found by a combined use of indirect ELISA-IgG and ELISA-IgM and antibody capture ELISA-IgM. Discrepancies in results could be examined by immunoblotting. IgM-ISAGA retained stable positive values longer than IgM-ELISAs did. Cyclosporine treatment did not hamper detection of seroconversion but could cause antibody levels to remain relatively low in primary infections. Seronegative recipients should receive antitoxoplasma treatment on seroconversion.     

Selective reactivity of antibodies to human immunoglobulins G, M, and A with rubella virus proteins

Proteins of purified rubella virus were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with human sera and immunoglobulin class heavy-chain-specific peroxidase conjugates. The levels of rubella antibodies in these sera were predetermined by the radial hemolysis test, the density gradient centrifugation method for immunoglobulin M (IgM) antibodies, and IgG-, IgM-, and IgA-specific enzyme immunoassays. In immunoblotting, rubella-specific IgG antibodies reacted with both envelope glycoproteins (E1 and E2) and the capsid protein (C). In contrast, rubella IgM antibodies reacted predominantly with E1, whereas the specific reactivity of IgA antibodies was directed mainly to the capsid protein. Purified IgM rheumatoid factor added to IgG-positive, IgM-negative serum did not give false-positive reactivity in the immunoblotting test as it did in solid-phase enzyme immunoassays. The immunoglobulin class-specific reactivities with the different viral proteins are expected to have diagnostic applications.     

Diagnosis of Colorado tick fever virus infection by enzyme immunoassays for immunoglobulin M and G antibodies.

An immunoglobulin M (IgM) capture enzyme immunoassay technique was adapted for the detection of antibody to Colorado tick fever virus in sera from 84 individuals for whom diagnosis had been confirmed by virus isolation or neutralization test. Titers were compared with those for IgG and neutralizing antibodies in these Colorado tick fever cases. IgM antibody titers were higher than neutralizing antibody titers, but neither appeared until 1 to 2 weeks after the onset of illness. Neutralizing antibodies were detected earlier than IgM antibodies, and both were detected with greater frequency than IgG antibodies. Late-convalescent-phase sera contained both neutralizing and IgG antibodies, but IgM was all but undetectable by 2 months after onset. Although the neutralization test may remain the serological test of choice, the enzyme immunoassay for IgM antibody offers a simple and more rapid method of serodiagnosis; the enzyme immunoassay is, however, less sensitive than the neutralization test. Furthermore, because there was a sharp decline in IgM antibody after 45 days, the presence of IgM antibody in a single serum sample provides a basis for the presumptive serodiagnosis of recent Colorado tick fever virus infection.     

Comparison of novel synthetic peptide-based DETECT-RUBELLA enzyme immunoassays with Enzygnost and IMx for detection of rubella-specific immunoglobulin G.

Enzyme immunoassays (EIAs) using synthetic peptides SP-E1 and SP-E1E2 (DETECT-RUBELLA [Bio-Chem]) were compared with two viral lysate-based EIAs (Enzygnost [Behring] and IMx [Abbott]) for the detection of rubella virus-specific immunoglobulin G antibodies. Sensitivities of 94.7, 100, 98.6, and 100% and specificities of 100, 97.4, 100, and 73.7% were found for the SP-E1, SP-E1E2, Enzygnost, and IMx EIAs, respectively.     

Evaluation of Toxoplasma gondii immunoglobulin G (IgG) and IgM assays incorporating the newVidia analyzer system

The new Vidia system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles, which allows a fast measurement of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM levels. The analytical performances of the Vidia Toxo IgG and IgM assays were compared with those of the automated Vidas, AxSYM, and Liaison Toxo IgG and IgM assays. The comparative evaluation was performed utilizing 204 frozen sera belonging to 166 subjects and 201 fresh sera collected from 198 subjects. For the Vidia Toxo IgG system, the sensitivities were 100% in both the retrospective and prospective studies, and specificities were 98.39% in the retrospective study and 100% in the prospective study, respectively. The sensitivities of the other three Toxo IgG assays were 100%, and the specificities ranged from 96.77% to 100%. For the Vidia Toxo IgM assay, the sensitivity and specificity were 100% in both the retrospective and prospective studies. The overall sensitivities and specificities of the other three Toxo IgM assays ranged from 80% to 100% and from 99.44% to 100%, respectively. In our study, the Vidia system revealed excellent sensitivity (100% for both IgG and IgM assays) and good specificity (99.25% for IgG and 100% for IgM assays).     

Serological diagnosis of Toxoplasma gondii infections by rapid separation of serum immunoglobulins M and G with CM Bio-Gel A.

A simple and rapid method has been developed for the separation of serum immunoglobulin M (IgM) and IgG. CM Bio-Gel A chromatography was used in the technique, which resulted in an IgM-rich fraction containing 31% of the original serum IgM and less than 2% of the serum IgG. The procedure was used to detect masked IgM antibodies in patients suspected of having Toxoplasma gondii infections.     

Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection

The fully automated IMx immunoassay analyzer was used to develop a system for the detection of IgG and IgM antibodies to rubella virus for immune status screening and diagnosis of primary infections. Reagents and assay protocol software were developed using rubella virus sensitized microparticles as the solid phase to capture specific antibodies from serum samples. Anti-human IgG or IgM antibody coupled to alkaline phosphatase enzyme followed by methylumbelliferyl phosphate substrate was used to detect the presence or absence of antibodies specific to the antigens on the solid phase. To evaluate the efficacy of the IMx rubella IgG assay, immune status screening was performed with a clinical patient population of 501 sera. When compared to an IgG specific enzyme immunoassay and passive hemagglutination assay the agreement was greater than 99%. The IMx rubella IgM assay was utilized to determine the presence of rubella specific IgM antibodies in 462 sera. These results were compared to IgM specific enzyme immunoassay results and also demonstrated greater than 99% agreement. Seroconversion following rubella vaccination of susceptible individuals was demonstrated by IgG and IgM antibody responses as early as two weeks postvaccination. In addition to automation, the IMx system offers rapid assay times and calibration curve storage without sacrificing clinical efficacy.     

Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM.     

Evaluation of five enzyme immunoassays for detection of immunoglobulin M antibodies to Epstein-Barr virus viral capsid antigens.

Five enzyme immunoassays for the detection of Epstein-Barr virus immunoglobulin M were evaluated versus indirect immunofluorescence assays with 128 serum specimens. The sensitivities and specificities, respectively, of these kits were as follows: Gull, 92 and 100%; Incstar, 53 and 100%; Ortho, 89 and 100%; Sigma, 78 and 86%; and BioWhittaker, 94 and 70%. Indeterminate results were produced by 2, 2, 0, 8, and 14 specimens with the Gull, Incstar, Ortho, Sigma, and BioWhittaker tests, respectively. The Gull and Sigma tests had the best day-to-day reproducibilities, and the Gull test was the easiest to use. No correlation between immunofluorescence titers and enzyme immunoassay values was observed.     

Use of enzyme immunoassays and the latex agglutination test to measure the temporal appearance of immunoglobulin G and M antibodies after natural infection or immunization with rubella virus

The time course of appearance of antibodies after infection with rubella virus was determined with an immunoglobulin G (IgG) detection enzyme-linked immunosorbent assay, a latex agglutination test, and an IgM detection enzyme-linked immunosorbent assay. In six naturally infected rubella patients and 26 vaccinees, antibodies measured by either the IgG enzyme-linked immunosorbent assay or the latex agglutination test generally appeared in parallel with those detected by the hemagglutination inhibition test. By 28 days after inoculation of live virus vaccine and by 2 days postonset of clinical rubella symptoms caused by natural infection, antibodies were found by the two tests for all individuals. A commercially available enzyme-linked immunosorbent assay kit was used to detect rubella-specific IgM. After natural infection, IgM appeared earlier than IgG, and although IgM titers decreased rapidly postinfection, in four of five patients antibodies were still detectable 40 to 43 days after the onset of clinical symptoms. After vaccine-induced infection, rubella-specific IgM was lower in titer than after natural infection and was detected in only three of seven vaccinees 70 days post-immunization.     

An enzyme immunoassay for immunoglobulin M antibodies to Toxoplasma gondii which is not affected by rheumatoid factor or immunoglobulin G antibodies.

An enzyme-linked immunosorbent assay (ELISA) for total antibodies to Toxoplasma gondii was modified to measure specific immunoglobulin M (IgM) antibodies. The assay requires three incubation periods totaling 2 h and enzyme-labeled-heavy-chain-specific antibodies to human IgM. The objective read-out in absorbance was normalized to percent of a standardized positive control for interpretations. No difference was observed between the assay results with or without previous absorption of the samples by Staphylococcus aureus protein A to remove most of the IgG antibodies. Addition of serum containing very high levels of IgG antibodies to another containing both IgG and IgM antibodies did not change the IgM assay values for the latter. None of the 22 sera containing high levels of IgM rheumatoid factor (RF) gave positive ELISA IgM results, even though 8 of them also had high levels of IgG toxoplasma antibodies. Mixtures of sera containing high concentrations of RF with sera having high levels of IgG toxoplasma antibodies also failed to show any false-positive reactions in the IgM toxoplasma assay. Thus, this ELISA for T. gondii IgM antibodies was not affected by IgG toxoplasma antibodies and RF.