Analysis of eight commercial enzyme immunoassay tests for detection of antibodies to Mycoplasma pneumoniae in human serum

Mycoplasma pneumoniae is an important etiologic agent of primary atypical pneumonia in children and adults. The diagnosis of M. pneumoniae infection is commonly confirmed through serologic testing. In this study, we used paired sera from 51 patients (all with confirmed M. pneumoniae infection and positive complement fixation [CF] titers) to compare the results of eight enzyme immunoassays (EIAs) available commercially in the United States. We compared two single-use EIAs and six plate-type EIAs. Results from acute-phase sera ranged from only 7 (14%) positive by ImmunoWELL (GenBio) immunoglobulin M (IgM) EIA to 23 (45%) positive by Zeus IgG EIA. When both the acute-phase and convalescent-phase serum samples were examined, positive results ranged from 20 (39%) by the ImmunoWELL (GenBio) IgM assay to 45 (88%) positive by the Remel IgG-IgM EIA. In this study, the single-use EIAs by Remel and Meridian were more reliable than were the plate-type EIAs. Among the plate-type EIAs, the Zeus and DiaSorin assays (which detect antibodies to protein antigens) were more sensitive than the ImmunoWELL assay (which detects antibodies to glycolipid antigens). In general, IgG EIAs on convalescent-phase sera were more concordant with one another than were IgM EIAs with one another. Scatter plot analysis of convalescent-phase sera showed that, as the CF titer dropped, the IgM assays identified fewer positive convalescent-phase sera. In contrast, the IgG assays provided fairly consistent positive results for convalescent-phase sera with CF titers of 64 and above. Results of individual tests and overall limitations of serodiagnostics for M. pneumoniae infections are discussed.     

A sandwich enzyme immunoassay for the detection of human IgG antibodies to dog allergens

A double-sandwich enzyme immunoassay (DSEIA) was developed for the detection of human IgG antibodies to dog dander and hair (DH) allergens. Since DH allergens are immunogenic in rabbits, the gamma-globulin fraction of rabbit antiserum to DH (RGG-a-DH; 10 micrograms/ml) was used for coating of polystyrene microtiter plates. Dog allergens (1 microgram/ml of DH extract) were bound to RGG-a-DH. Binding of human IgG Ab to nonimmune RGG (10 micrograms/ml)--used as a background control--was substracted from the DH specific one and nonspecific binding was further eliminated by using 0.5% of normal rabbit serum in the dilution buffer. Sera were diluted 1:10 in this buffer. Specificity of the assay was shown by absorption experiments: protein A, anti-IgE discs (Phadebas PRIST) and dog or birch (e5, t3; Phadebas RAST) allergen discs were used to remove total and allergen specific IgG and IgE, respectively. The results were expressed as net absorbances or as a percentage of a reference serum. A significant correlation (r = 0.65, p less than 0.001) between IgG (DSEIA) and IgE (RAST) antibodies to dog allergens was observed in 40 untreated dog allergic subjects. The DSEIA was found to be more sensitive than conventional ELISA in detecting IgG Ab in 15 asthmatic children during hyposensitization: a significant rise was observed in 14 compared to 12 with ELISA, while no significant increases were observed in the placebo-treated group.     

A solid-phase enzyme immunoassay for the detection of tetanus toxin using human and murine monoclonal antibodies

Three human and three murine monoclonal antibodies were tested for their reactivity to tetanus toxin and toxoid and used to establish an enzyme immunoassay specific for tetanus toxin. The dissociation constants of the monoclonal antibodies were between 3.91 x 10(-9) and 8.48 x 10(-12). Two human monoclonal antibodies recognized conformation determinants on the toxin, whereas the others reacted to the heavy chain. Only a combination of antibodies of the two species allowed the development of an enzyme immunoassay for the detection of tetanus toxin with a lower detection limit of 1.2 micrograms/l.     

An enzyme labelled nuclear antigen immunoassay for detection of cytomegalovirus IgM antibodies in human serum: specific and non-specific reactions

A mu-capture enzyme linked immunosorbent assay was developed for detection of IgM antibody to cytomegalovirus (CMV). Virus-specific IgM was detected using horseradish peroxidase labelled nuclear CMV antigen (CMV-ELA). False-positive reactions caused by Paul-Bunnell-Davidsohn (PBD) positive sera and antinuclear antibody (ANA) positive sera were identified in a combination assay employing enzyme labelled nuclear control antigen (CO-ELA) in parallel to the CMV-ELA. Four of five PBD positive and 30 of 31 ANA positive sera reactive with the CMV-ELA were identified as false positive reactions in the combined ELA-assay. The reactivity in PBD-positive sera could not be explained by antigenic cross reactivity between CMV and Epstein-Barr virus, and the results further suggested that different cell specified components of the CMV-ELA were responsible for the reactivity of PBD-positive as compared to ANA-positive sera. One of 314 healthy blood donors, 12 of 12 patients with primary CMV infection, and 11 of 15 patients with secondary CMV infection had detectable CMV IgM antibodies. Comparison of different CMV-ELAs revealed that pronounced differences in specificity as well as sensitivity may exist.     

Two-site column enzyme immunoassay for neuron-specific enolase (NSE) in human serum using monoclonal antibodies

A new method for the rapid determination of neuron-specific gamma-enolase (NSE), gamma-subunit of alpha gamma- and gamma gamma-enolase in human serum was developed by employing monoclonal antibodies for the separation method. The assay system consists of 0.1 ml Sepharose 4B column with immobilized rabbit anti-mouse IgG antibodies for the separation of bound label, Fab' fragments of rabbit anti-bovine gamma gamma-enolase IgG labeled with beta-D-galactosidase from Escherichia coli, and F(ab')2 fragments of two mouse monoclonal antibodies to gamma gamma-enolase. Serum samples or standard NSE solutions were incubated at 30 degrees C with the monoclonal antibody fragments. 10 min later, the galactosidase-labeled antibody fragments were added to the mixture, and incubated at 30 degrees C for 30 min. Then the reaction mixture was applied to a micro-column of Sepharose 4B with immobilized anti-mouse IgG antibodies. From the galactosidase activity bound in the column, NSE concentration in the samples could be estimated within 2 h. The minimum detection limit of the assay system was 30 pg/tube, being sufficiently sensitive for the assay of serum NSE with a satisfactory precision. Serum concentrations of NSE determined by the present method correlated well with that by the colorimetric solid-phase immunoassay method.     

A mu-capture immunoassay for detection of human herpes virus-6 (HHV-6) IgM antibodies in human serum.

BACKGROUND: Human herpes virus-6 (HHV-6) was first isolated in 1986. It has been shown to cause exanthema subitum and has been associated with various other diseases. HHV-6 infection is widespread, and more than 90% of the population have antibodies against HHV-6 at the age of 2 years. Once acquired, the virus remains latent in the body. This makes it difficult to draw any conclusions about a causal relationship between the demonstration of HHV-6 and a specific disease. OBJECTIVES: This work was to develop a mu-capture HHV-6 IgM enzyme linked immuno sorbent assay (ELISA) for use in routine diagnosis and for wide scale patient population analysis. STUDY DESIGN: A mu-capture HHV-6 IgM ELISA was established. A total of 682 sera consisting of 585 sera from Danish blood donors and 97 sera from patients with autoimmune antibodies were analysed in the HHV-6 IGM ELISA. One hundred and ninety-two sera had earlier been analysed for total HHV-6 antibody content in a competitive ELISA, 94 sera were analysed for cytomegalovirus (CMV) IgM and 57 sera for Epstein Barr virus (EBV) antibodies, using different ELISA assays. The results for 12 primary infections with HHV-6 are also reported. RESULTS: A HHV-6 IgM optical density (OD)-ratio was calculated according to a constant positive control. An empirical cut off of 0.5 HHV-6 IgM OD-ratio was chosen (with regard to the 10 HHV-6 seroconverters), which resulted in a specificity of 97.5% of the HHV-6 IgM ELISA. Two of the three donor sera with HHV-6 IgM OD-ratios more than 1.05 had total HHV-6 antibody titers significantly above the group with IgM OD-ratios below 0.7 consisting with HHV-6 reactivation. There was no cross reactions to EBV or CMV IgM positive sera. CONCLUSION: The HHV-6 IgM ELISA seems valid to diagnose primary HHV-6 infection in particular in combination with the HHV-6 total antibody assay.     

Competitive enzyme immunoassay for detection of complement-fixing antibodies in diagnostic virology.

A competitive enzyme immunoassay was developed to detect serum complement-fixing antibodies in virus diseases. This assay utilized conglutinin-covered plastic beads as the solid phase to detect specific antibody-antigen complexes that competed for complement with a probe complex comprised of Escherichia coli beta-galactosidase and its specific antibody. Binding to the solid phase is C3bi mediated, and when specific antibody-antigen complexes are not present the probe is bound and an enzymatic reaction ensues. This type of competitive assay was introduced in the field of immunopathology for investigating circulating immune complexes by Manca et al. (Clin. Immunol. Immunopathol. 16:131-141, 1980). The assay gave satisfactory results in terms of specificity, reproducibility, and handling, enabling laboratories to obtain results in 5 h. The sensitivity of the method coincided with that of the complement fixation test. However, this technique offers several advantages over conventional complement fixation because it requires less time, is easier to perform, and gives more reliable quantitative results. The data obtained indicated that this competition assay offers a feasible alternative to conventional complement fixation tests and should be useful in routine diagnostic applications and in seroepidemiological surveys.     

A fluorescent enzyme immunoassay for Salmonella detection

A double antibody sandwich immunoassay (EIA) was developed for the detection of Salmonella. The assay utilizes a beta-galactosidase-murine myeloma monoclonal antibody (M467) conjugate prepared with the heterobifunctional coupling reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and uses 4-methyl umbelliferyl beta-D-galactoside as a fluorogenic substrate for the enzyme. The EIA is sensitive and rapid, and compared favorably with the conventional cultural technique in the analysis of 60 feed samples naturally contaminated with Salmonella. Proteins or natural contaminants from the sample did not interfere in the assay.     

A sensitive two-site sandwich enzyme immunoassay for human angiotensin converting enzyme utilizing monoclonal antibodies.

A sensitive enzyme immunoassay was developed for human angiotensin converting enzyme. Monoclonal antibodies specific for two unique converting enzyme epitopes were utilized to develop a two-site sandwich enzyme immunoassay. Alkaline phosphatase conjugated to the detecting antibody hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) to NAD. Subsequently, NAD is cycled between its reduced and oxidized forms by an alcohol dehydrogenase/diaphorase catalyzed redox cycle. Each cycle converts iodonitrotetrazolium violet to a highly colored formazan which is quantitated. With this assay, as little as 94 pg/ml of native converting enzyme is detectable without interference from either therapeutic or endogenous converting enzyme inhibitors.     

A biotin-streptavidin enzyme immunoassay for detection of antibodies to porcine granulosa cell antigens.

A colorimetric solid-phase enzyme immunoassay has been developed which quantifies antibodies to porcine granulosa cell membrane antigens in rabbits immunized with porcine granulosa cells. A cell-free, particulate membrane preparation of porcine granulosa cells was used as coating antigen. A biotinylated second antibody in conjunction with a streptavidin-beta-galactosidase conjugate was utilized to amplify reactivity. The enzyme beta-galactosidase was used due to high background obtained using peroxidase, presumably due to endogenous peroxidase activity of the tissue. Sigmoidal serum dilution curves were obtained with immune rabbit sera indicating that absorbance was related to the concentration of antibodies. Assay activity was reduced by preincubation of immune serum with granulosa cell membranes. Sera from ovariectomized or pre-immune rabbits did not yield any specific binding in the assay. This assay has potential applicability for quantifying antiovarian and antigranulosa cell antibodies in women suspected of having autoimmune premature ovarian failure.     

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.     

Reverse enzyme immunoassay for detection of specific anti-Toxoplasma immunoglobulin M antibodies.

A reverse enzyme immunoassay (R-EIA) is described, in which polystyrene muplates are sensitized with anti-immunoglobulin M (IgM) (mu chain) antibodies and then sequentially allowed to react with patient's serum, peroxidase-labeled Toxoplasma gondii soluble antigen, and substrate. Measurement of activity of the solid-phase bound enzyme conjugate was done by colorimetric reading of the final developed color and kinetically by the initial rate of color development. This R-EIA allowed full resolution between absorbance values of a group of 36 sera which presented positive results in the Toxoplasma IgM immunofluorescence test and the remaining groups, which consisted of 39 normal individuals, 22 rheumatoid factor-positive sera, 8 Waldenstrom's macroglobulinemic sera, 3 infectious mononucleosis samples, and 6 high-titered IgG anti-T. gondii sera. No interference of rheumatoid factor IgM or inhibition by high-titered specific IgG was detected, even in the false IgM immunofluorescence-positive rheumatoid factor samples. Likewise, false-negative IgM immunofluorescence samples gave positive R-EIA even without adsorption with Staphylococcus aureus protein A. The possibility of direct tagging of the antigen with the enzyme eliminates the need for using antigen and anti-antigen conjugates as separate layers, therefore eliminating one step in the assay.     

A quantitative immunoassay using chicken antibodies for detection of native and recombinant alpha-amidating enzyme.

A sensitive competitive ELISA has been developed for the detection and quantitation of native and recombinant alpha-amidating enzyme. Chickens immunized with purified enzyme (75 kDa) isolated from a rat medullary thyroid carcinoma, produced IgY antibodies specific for the native enzyme. The assay is defined by a standard curve with a linear range of 0.78-12.5 ng/ml in phosphate-buffered saline, and a limit of sensitivity for detection of the enzyme of 0.20 ng/ml. The immunoassay is capable of detecting enzyme from both tumor derived sources, and from cells genetically engineered to secrete the enzyme into tissue culture medium containing up to 10% fetal calf serum.     

Rapid dot enzyme immunoassay for the detection of antibodies to cytomegalovirus.

Cytomegalovirus (CMV) antigen was coated onto a white opaque plastic card as small dots inside circles marked in the microtiter plate well pattern. The card with antigen dots could be cut according to the number of test samples to be assayed. Small drops of undiluted serum samples, goat antibodies to human immunoglobulin G labeled with alkaline phosphatase, and finally substrate (5-bromo-4-chloro-3-indolyl phosphate) were sequentially added to the antigen spots and incubated in the open air at room temperature for 5 min each. The antigen dots showed blue color for sera with immunoglobulin G antibodies to cytomegalovirus but no color for those without. The developed antigen dots could be rinsed with water and kept as permanent records. For the assay of a large number of serum samples, a modified procedure with serum diluted 1:10 and longer first two incubations (20 min each) was found to be more comfortable to perform. The results of this assay for 123 undiluted and 256 diluted serum samples revealed very good correlations with those obtained by a commercially available test kit for immunoglobulin G antibodies to cytomegalovirus with 97 and 99% agreement, respectively. This dot test was very reproducible and required no instrumentation. The reagents, including coated antigen dots, are stable at room temperature for at least 2 months and are ready for use.     

Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies

The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed. We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively. Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result.     

Evaluation of an enzyme immunoassay technique for detection of antibodies against Treponema pallidum

In the present study, the performance of an enzyme-linked immunosorbent assay (ELISA) technique (Eti-syphilis-G and Eti-syphilis-M; DiaSorin) for detection of Treponema pallidum immunoglobulin M (IgM) and IgG antibodies for the laboratory diagnosis of syphilis was evaluated. Four hundred forty-one samples were studied. The sensitivity and specificity of the ELISA were 100 and 93%, respectively, compared with the results of a microhemagglutination assay for Treponema pallidum (MHA-TP) and 99.4 and 100%, respectively, compared with the results of the fluorescent treponemal antibody absorption (FTA-Abs) test. The results of the ELISA technique were concordant with those of MHA-TP for 98% of the samples tested, while the rate of concordance with the FTA-Abs test was 99.5%. The sensitivities of the rapid plasma reagin (RPR) test, MHA-TP, and the ELISA in the different phases of syphilis compared with the results of the FTA-Abs test were 92, 88, and 100%, respectively, for patients with primary syphilis; 100% for all tests evaluated for patients with secondary syphilis; 97.2, 99.4, and 100%, respectively, for patients with latent syphilis; and 57.9, 92.6, and 97.9%, respectively, for patients with past treated syphilis. The RPR test was reactive with 12 samples that were negative by all the specific tests. IgM antibodies were most frequently detected by the ELISA for IgM antibodies (32.8%) than by the FTA-Abs for IgM antibodies (28.4%). Detection of these antibodies by the FTA-Abs test and the ELISA for IgM antibodies decreased with the stage of disease (72 and 88%, respectively, for patients with primary syphilis to 17 and 19%, respectively, for patients with early latent syphilis). The high sensitivity and specificity of this ELISA technique during all stages of syphilis, together with the fact that it is a simple, objective, and easily automated method, lead us to believe that it could be used as a screening test for syphilis.     

Sensitive two-site sandwich enzyme immunoassay with antipeptide antibodies for detection of viral antigens

Two-site sandwich ELISAs with antipeptide antibodies were developed for measuring the amounts of polyoma virus medium T and SV40 virus large T antigen in extracts from virus infected and transformed cells. In order to exclude crossreactive proteins, two antipeptide antibodies directed against different regions of the antigen were used in series. Both antigenic determinants have to be recognized for the assay to score positive. A positive signal was obtained with as little as 10 μ1 of polyoma virus infected cell lysate and 100 μl of extract prepared from medium T antigen-transformed FR18-1 cells which were shown to contain 37 and 15 pg of medium T antigen, respectively. This two-site sandwich ELISA is a sensitive tool to detect novel or closely related proteins.     

An enzyme immunoassay for detection of IgA class antibodies against hepatitis delta virus

A sensitive and reproducible enzyme-linked immunoassay (ELISA) for IgA class antibodies against the Delta antigen (HDAg) is described. Specificity of the assay was demonstrated by the absence of binding to an unrelated antigen or to uncoated plates and the finding that binding to HDAg was independent of total IgA concentrations in sera. Positive results were obtained with sera from 11 of 14 patients with chronic Delta virus infection (seropositive for HBsAg and IgM anti-HDAg, negative for IgM anti-HBc) at serum dilutions of up to 1:10(6). Sera from four normal healthy individuals and from 25 patients with chronic hepatitis B or other liver disorders who had no evidence of exposure to HDV were all negative in the assay.     

Solid-phase enzyme immunoassay for Chlamydial antibodies.

An enzyme immunoassay (EIA) for chlamydial immunoglobulin G antibodies was developed by using microtiter wells coated with partially purified reticulate bodies of Chlamydia trachomatis serotype L2, grown in McCoy cells, and uninfected McCoy cells as a control. Duplicate testing of a single serum dilution, 1:500, was found to be sufficient. A good correlation between positive reactions was observed in a comparative study of 421 patient sera with the EIA and an inclusion immunofluorescence test. A good correlation between positive reactions was also observed in a comparative study of 140 patient sera with EIA and microimmunofluorescence tests in which chlamydial elementary or reticulate bodies were used as antigens. Sera of 77 healthy control individuals with low titers in inclusion immunofluorescence or complement fixation tests gave negative results in the EIA. Immunoblotting experiments showed that the major antigenic component in the EIA antigen was a protein with an Mr of 39,000.