Solid-phase enzyme immunoassay for immunoglobulin M antibodies to cytomegalovirus.

A sensitive, solid-phase enzyme immunoassay for the detection of immunoglobulin M antibodies to cytomegalovirus is described. The results of the enzyme immunoassay correlated well with those obtained by an indirect immunofluorescence method. Horseradish peroxidase proved to be a more sensitive label than alkaline phosphatase. Nonspecific reactions, occurring with commercially available cytomegalovirus antigens, could be avoided by using a nuclear antigen prepared from sonically disrupted nuclei of cytomegalovirus-infected cells.     

Evaluation of a commercial enzyme immunoassay for detection ofMycoplasma pneumoniae specific immunoglobulin G antibodies

A commercial enzyme immunoassay (Platelia Mycoplasma) infections was evaluated and found not to be suitable for the purpose. More than 80 % of healthy persons and patients with non-Mycoplasma pneumoniae respiratory infection, all with a negativeMycoplasma pneumoniae complement fixation test, had a positive EIA. Paired sera did not show the positive correlation between a rise in complement fixation titre and the EIA ratio reported by the manufacturer.     

Rapid double-sandwich enzyme-linked immunosorbent assay for detection of human immunoglobulin M anti-Toxoplasma gondii antibodies.

The double-sandwich enzyme-linked immunosorbent assay has been compared with the indirect fluorescence assay for the detection of immunoglobulin M antibodies against Toxoplasma gondii in humans. Incubation times have been shortened, permitting the test to be completed within 2 h. The double-sandwich enzyme-linked immunosorbent assay is confirmed to be more sensitive and more specific than the immunofluorescence assay.     

Evaluation of Abbott CMV-M enzyme immunoassay for detection of cytomegalovirus immunoglobulin M antibody.

The Abbott CMV-M enzyme immunoassay (EIA) for the qualitative determination of immunoglobulin M (IgM) antibody to cytomegalovirus in human serum was compared with the indirect fluorescent-antibody (IFA) test on 338 human serum specimens. Discordant specimens were evaluated by IFA following isolation of IgM fractions. Discordant specimens remaining after IFA testing were evaluated by an IgM-specific EIA (CYTOMEGELISA M; M.A. Bioproducts). After resolution of discordant specimens, the CMV-M EIA was 94.7% sensitive and 99.1% specific.     

Comparison of immunofluorescence and enzyme immunoassay for detection of measles-specific immunoglobulin M antibody.

During a measles outbreak, 283 serum specimens from 221 suspected cases of measles were tested by immunofluorescence and enzyme immunoassay for the presence of measles-specific immunoglobulin M (IgM) antibodies by using commercially available reagents. There was 97% agreement between the two assays; thus, the choice of the method for diagnostic testing is a matter of convenience and experience. In all 62 cases of measles from which a single blood sample was available, measles IgM-specific antibodies were detectable by both methods. Fifty percent of the 62 cases were positive within 3 days after onset of the rash. This increased to 91% 10 days after onset of the rash.     

Diagnosis of acute toxoplasmosis by an enzyme immunoassay for specific immunoglobulin m antibodies

A recently developed enzyme-linked immunosorbent assay for detection of immunoglobulin M (IgM) class antibodies to Toxoplasma gondii was evaluated with respect to specificity and sensitivity. By using an antibody capture principle and F(ab')2 conjugates, interference of rheumatoid factors was absent. No cross-reactions with anti-toxoplasma IgG occurred, and no interference with antinuclear antibodies was found. A large-scale study with about 1,500 clinical specimens revealed a 100% specificity. By testing 79 sera from patients with acute-phase acquired toxoplasmosis, sensitivity was found to be 97%. In routine clinical practice, the IgM-enzyme-linked immunosorbent assay proved to be a more sensitive tool for diagnosis than the immunofluorescent-antibody test. The course of IgM-enzyme-linked immunosorbent assay antibodies in acute patients was studied; IgM reached peak levels within 1 month after onset of illness, and could be demonstrated up to an average of 8 months after onset.     

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.     

Evaluation of a new enzyme immunoassay based on recombinant Rubella virus-like particles for detection of immunoglobulin M antibodies to Rubella virus.

A new enzyme immunoassay for the detection of specific antibodies to rubella virus was evaluated at two different sites. This assay, the Roche Cobas Core Rubella IgM EIA recomb, uses a recombinant rubella virus-like particle and is based upon the immunoglobulin M (IgM) capture principle. It was compared to the Abbott IMx Rubella IgM test and to the Sorin ETI-RUBEK-M reverse test. The relative clinical specificities were 99.30% for the Roche test, 98.26% for the Abbott test, and 100% for the Sorin test. The relative clinical sensitivities were 100, 93.87, and 82.65%, respectively. In the case of most primary infections, IgM antibodies could be detected immediately at the onset of the disease and for up to 7 weeks. In the case of vaccinations, they could be detected between 3 and 12 weeks after vaccination.     

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.     

Enzyme immunoassay for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae.

An enzyme immunoassay (EIA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complement fixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infections of known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoid factor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combined measurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgM antibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificity was 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We found no false positives among serum samples from patients with non-M. pneumoniae respiratory infection of known etiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA results became positive earlier in the course of illness than the MPCF titer. While children and teenagers responded predominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% of cases), probably because of reinfection. We conclude that this EIA is a good alternative to the combined MPCF and cold agglutinin tests in the diagnosis of M. pneumoniae infection.     

Fluoroimmunoassay for detection of rubella-specific immunoglobulin M: comparison with indirect enzyme immunoassay and mu-chain capture.

The performance of a commercially-available method of fluoroimmunoassay (Rubella M FIAX, International Diagnostic Technology, Santa Clara, Calif.), designed for the detection of rubella-specific immunoglobulin M, was tested with 137 selected sera, including 52 from cases of primary rubella, 29 from healthy pregnant women, 21 containing rheumatoid factor, and 35 from cases of infectious mononucleosis (IM) caused by Epstein-Barr virus. The results were compared with those obtained by commercial indirect enzyme immunoassay (EIA) and EIA anti-mu chain capture tests. The fluoroimmunoassay technique showed a satisfactory level of sensitivity, but low values had to be interpreted with caution as false-positive results were detected with sera with rheumatoid factor and from IM cases, even after preliminary treatment of sera with the anti-human immunoglobulin G antisera provided in the kit. On the other hand, no false-positive results in the analysis of IM sera were seen in the EIA anti-mu chain capture method. Because of its sensitivity and specificity, we recommend the use of the latter technique for the diagnosis of primary rubella.     

The detection of measles specific immunoglobulin M antibodies using biotinylated antigens

The methods of reverse type enzymeimmunoassay (EIAs) with biotinylated antigens were used to determine IgM antibodies to measles virus in human sera. These antigens, either purified measles virus antigen or lysate type measles-vero antigen with lysate vero control antigen, were used in the two separate IgM-tests. Paired sera from 15 measles patients as well as 456 sera from patients with viral infections other than measles, with mycoplasma pneumoniae infections, from rheumatoid arthritis patients and blood donors, were assayed in a dilution of 1:200. Both the test systems detected all the 30 serum specimens from the measles patients as measles IgM positive, but the sera of all the other groups proved to be measles IgM negative. These tests developed for measles specific IgM antibodies, avoiding the interference of IgM-class rheumatoid factor, offer valuable tools for routine virus serology.     

Detection of immunoglobulin M antibodies to hepatitis E virus by class capture enzyme immunoassay

The measurement of antibodies to hepatitis E virus (anti-HEV) has been essential for understanding the epidemiology of hepatitis E. Studies to determine the prevalence of HEV infections require a reliable serologic assay that is sensitive and specific. It is also important to distinguish the acute from the convalescent phase of an infection; this usually requires the detection of the immunoglobulin M (IgM) class of antibody. Few enzyme immunoassays (EIAs) that measure IgM anti-HEV have been described, and most have utilized the sandwich method. The present study describes an EIA that detects IgM anti-HEV by antibody class capture methodology. The assay was validated by using serum and/or plasma panels from experimentally infected nonhuman primates. It was used to demonstrate an anamnestic response and the reappearance of IgM anti-HEV in a chimpanzee experimentally challenged with HEV at two different times 45 months apart. The class capture method was more sensitive than the sandwich EIA when used to test clinical samples from two hepatitis E epidemics in Pakistan; it also had the advantage of distinguishing IgM anti-HEV in the presence of high titers of IgG anti-HEV.     

Enzyme immunoassay for detection of human immunodeficiency virus-specific immunoglobulin A antibodies.

Early diagnosis of human immunodeficiency virus (HIV) infection may be difficult in adults with acute or recent HIV infection and in infants with perinatally acquired HIV. Detection of HIV-specific immunoglobulin A (IgA) antibodies in infant serum by Western blot (immunoblot) has been suggested as a reliable method to identify HIV-infected infants, especially those over the age of 6 months, and as an adjunct to diagnosis of acute HIV infection in adults. We developed a simple enzyme immunoassay for detection of HIV-specific IgA, using standard commercially available reagents. Enzyme immunoassay was comparable to Western blot for detection of HIV-specific IgA in sera from adults (n = 216), older children (n = 49), and infants born to HIV-infected mothers (n = 65). Specificity was 100% and sensitivity ranged from 80 to 92%. IgA-enzyme immunoassay is a simple, highly sensitive method for detection of HIV-specific IgA antibodies and is easily adapted to the standard clinical laboratory.     

Microparticle enzyme immunoassay for determination of immunoglobulin G antibodies to human cytomegalovirus.

A microparticle enzyme immunoassay (LEIA) that uses passive latex agglutination was used to detect cytomegalovirus immunoglobulin G antibodies in 495 serum samples. LEIA was in excellent concordance with latex agglutination (97%) and an enzyme-linked immunosorbent assay (98.3%). The sensitivity and specificity of LEIA compared with those of the enzyme-linked immunosorbent assay were 100 and 92.5%, respectively.     

Reverse enzyme immunoassay for the determination of Dermatophagoides pteronyssinus IgE antibodies

Sera from patients with suspected mite sensitivity were assayed using both the radioallergosorbent test (RAST) and a reverse enzyme immunoassay for IgE antibodies (REIA). Of the 133 sera studied, the RAST gave positive results in 48 cases and the REIA in 59. Negative results for both assays were obtained in 72 of the sera. The immunoenzymatic assay described here, using micro-plates as solid phase, has proven to be IgE- and antigen-specific. The allergen conjugate has a prolonged shelf life and the method is easy to perform. The sensitivity of the REIA for Dermatophagoides pteronyssinus IgE antibodies is limited by the small amount of IgE bound by the well surface (less than 100 ng). However, we think that the method offers a reliable alternative for the diagnosis of D. pteronyssinus-allergic patients.     

Evaluation of a commercial measles virus immunoglobulin M enzyme immunoassay.

Paired serum samples from 93 patients suspected of having measles were assayed for measles virus-specific immunoglobulin M (IgM) antibodies by an enzyme immunoassay (EIA), and the results were compared with results from a complement fixation assay and an EIA for measles virus IgG. By using significant serologic rises as the standard for comparison, the IgM EIA assay had a sensitivity of 85.7%, a specificity of 81.3%, a positive predictive value of 95.7%, and a negative predictive value of 54.2%. This assay can be expected to perform well in outbreak situations.     

Detection by enzyme immunoassay of serum immunoglobulin G antibodies that recognize specific Cryptosporidium parvum antigens

Human infection with Cryptosporidium parvum usually elicits characteristic immunoglobulin G (IgG), IgA, and IgM antibody responses against two sporozoite surface antigens with apparent molecular masses of approximately 27 and 17 kDa. We have determined that these two antigens are actually complex families of related antigens. We have developed two new enzyme-linked immunosorbent assays (ELISAs) for the detection and quantitation of serum IgG antibodies against both antigens. The assays utilize a recombinant form of the 27-kDa antigen and a partially purified native fraction isolated from sonicated whole oocysts that contains 17-kDa antigen. An immunoblot assay previously developed in our laboratory served as the reference, or "gold standard," seroassay for the assessment of the new ELISAs. Positive responses with the recombinant-27-kDa-antigen ELISA were correlated with the immunoblot results for the 27-kDa antigen, with a sensitivity and specificity of 90 and 92%, respectively. Similarly, positive responses with the partially purified native-17-kDa-antigen ELISA correlated with the immunoblot results for the 17-kDa antigen, with a sensitivity and specificity of 90 and 94%, respectively. For both ELISAs the median IgG antibody levels for serum sets collected during outbreaks of waterborne C. parvum infection were at least 2.5-fold higher than the levels determined for a nonoutbreak set. Using the immunoblot as the "gold standard," the new ELISAs were more specific and, in the case of the 27-kDa-antigen ELISA, more sensitive than the crude oocyst antigen ELISA currently in use. These assays will be useful in future epidemiologic studies.