Use of enzyme-linked immunosorbent assay to detect antibodies to staphylococcal protein A in the rabbit

An ELISA technique is described which employs both solid phase and labelled free SpA. The technique allows the detection of anti-SpA antibodies in the rabbit. It is not suited for the same purpose with human sera, because human immunoglobulins are not saturated by non-specific interaction with solid phase SpA.     

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.     

Hemagglutinin-specific enzyme-linked immunosorbent assay for antibodies to influenza A and B viruses.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies present in human serum or nasal washes directed against influenza A or B hemagglutinin glycoproteins. The assay was modified to measure the immunoglobulin isotype specificity of the anti-hemagglutinin response in serum and nasal secretions. In the postinfection sera anti-hemagglutinin of the immunoglobulin G isotype was predominant, whereas in nasal secretions the antibody was predominantly immunoglobulin A. The antibody response detected by the ELISA manifested hemagglutinin subgroup specificity. In addition, there was a good correlation between the ELISA antibody titer and the hemagglutination-inhibition or neutralizing antibody titer. The ELISA was more sensitive than the hemagglutination-inhibition assay, and the range of antibody titers measurable by ELISA in human serum was from less than 1:20 for children who had never experienced influenza infection to 1:400,000 for adults convalescing from a secondary infection. With more sensitive tests to detect antibody to the influenza hemagglutinin it should be possible to determine the relative contribution of local and systemic immunity to resistance to influenza virus infection.     

A micro enzyme-linked immunosorbent assay for insulin antibodies in serum

A solid-phase micro enzyme-linked immunosorbent assay for the measurement of insulin antibodies in serum is described and its performance compared with that of an established radiobinding assay. Interassay precision in the ELISA was 10% or less at widely spaced points on the dilution curves for human, porcine and bovine insulins. Specificity was demonstrated by substituting purified human gamma-globulin for the test serum and glucagon for the insulin. The influence on ELISA of endogenous insulin in the test serum was examined by measuring antibody binding before and after extraction of the insulin. The correlation between results from extracted and unextracted sera was 0.96 and the fit ideal: y = 1.00x + 0.38%. The correlation between the results of measuring insulin antibody in 256 diabetic sera by the 2 assays was r = 0.74, P less than 0.001 (human insulin) and r = 0.71, P less than 0.001 (porcine insulin). ELISA is cheap and simple to perform. We believe it may prove to be a practical alternative to radioassay in both the routine detection and investigative research of insulin antibodies.     

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 alphaviruses by enzyme-linked immunosorbent assay.

Cell culture-derived antigens detected antibodies to alphaviruses in human sera with the enzyme-linked immunosorbent assay technique. Results correlated with those from hemagglutination inhibition and neutralization tests.     

Rapid serotyping of infectious pancreatic necrosis virus by one-step enzyme-linked immunosorbent assay using monoclonal antibodies.

A one-step ELISA has been developed for detection and serotyping of infectious pancreatic necrosis virus (IPNV) in infected cell cultures using monoclonal antibodies (mAb) raised against strains representing the Sp, Ab and VR 299 serotypes of IPNV. This assay uses a serotype-specific mAb as capture and a mAb directed against a common epitope as detector antibody. Avidin-peroxidase was employed for amplification. The assay was performed in 90 min by simultaneous incubation of the samples, the biotin labelled mAb and the avidin-peroxidase, and detected 37 ng/ml of purified virus. Serotyping of 34 isolates from different areas of Europe, Asia and America showed a total concordance with the results obtained by the neutralization assay.     

A highly sensitive and specific enzyme-linked immunosorbent assay of antibodies to hepatitis C virus

In this study, a 178 amino acids long portion of the hepatitis C virus (HCV) core gene was cloned, sequenced, expressed in Escherichia coli, and purified. The resulting antigen (C178) was tested with human sera enzyme-linked immunosorbent assay (ELISA) in order to assess its ability to diagnose HCV. It was shown by ELISA that 92% of the patients sera, diagnosed previously by a 3rd generation enzyme immunoassay (EIA) as HCV-positive, had antibodies against the C178 antigen. This antigen gave no false positive results when tested with anti-HCV-negative sera.     

Combined use of enzyme-linked immunosorbent assay and flow cytometry to detect antibodies to Trypanosoma cruzi in domestic canines in Texas

Canines may be sentinels and/or reservoirs for human Trypanosoma cruzi exposures. This study adapted a method originally designed for human diagnostics to detect serum immunoglobulin G to T. cruzi in canines. The method combined an enzyme-linked immunosorbent assay (ELISA) for screening and flow cytometry detection of anti-live trypomastigote antibodies (ALTA) for confirmation. The assays were optimized by using known positive and negative control canine sera, and cutoff values were established. The ELISA and ALTA assay easily distinguished between reactive (positive controls) and nonreactive (negative controls) sera and were used to test sera collected in a cross-sectional seroprevalence survey of 356 domestic canines from Harris County, Tex., and the surrounding area. Fifty-three (14.9%) of 356 asymptomatic canines in the survey were positive by ELISA, and 5 (1.4%) were confirmed positive with the ALTA assay, with an additional 4 (1.1%) canines classified as "suspect positive." Thus, the overall prevalence of T. cruzi antibodies in this population was 2.6%. This is the first U.S. study to use the combination of ELISA and ALTA to detect serum antibodies to T. cruzi and the first report of the prevalence of T. cruzi infection in domestic canines in the Houston, Tex. (Harris County), region. Our results demonstrate that the combination of ELISA and ALTA has been successfully adapted for use in testing canines for serological evidence of T. cruzi infection. Seroprevalence survey results suggest that T. cruzi antibody-positive domestic canines in the peridomestic setting are present in the Houston, Tex., region and further suggest that T. cruzi is enzootic in the region.     

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 for antibodies to native and denatured DNA.

A modification of the enzyme-linked immunosorbent assay (ELISA) is described, that permits determination of antibodies to native DNA (nDNA). The same approach can be used to measure antibodies to denatured DNA (dDNA). Poor binding of nDNA to the polystyrene solid phase has presented difficulties in using the ELISA method for assaying anti-nDNA activity (Engvall, 1976), but we find that precoating of the solid phase with protamine sulfate circumvents this problem. Assays for anti-dDNA are also enhanced by the use of protamine sulfate coated tubes. We have used the ELISA method to assay 15 SLE and 27 non-SLE sera for anti-nDNA and anti-dDNA activity. The results are compared with those obtained using the GF/A glass fiber filter assay, previously described by Lewis et al. (1973).     

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.     

Detection of rotavirus-specific IgG antibodies by immunoperoxidase assay and enzyme-linked immunosorbent assay

An indirect immunoperoxidase assay (IPA) has been developed for determination of IgG antibodies to rotavirus. The technique employed as antigen, SA-11 infected MA 104 cells, which were air-dried on glass slides and acetone-fixed. In parallel, rota-specific IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA). Specific IgG antibodies to rotavirus were determined in sera of healthy children and in sera of patients suffering from gastroenteritis. A good correlation (r = 0.92) and (r = 0.98) for healthy children and patients, respectively, was found between IPA and ELISA techniques. The IPA technique is rapid and simple and positive results, because of the intensive staining, are easily read by low-power light microscope. The potential application of IPA and ELISA methods in serodiagnosis of rotavirus infections is discussed.     

An enzyme-linked immunosorbent assay for measuring anti-sheep erythrocyte antibodies

A simple ELISA assay for detecting murine anti-SRBC antibodies of IgG class was developed and the variation of the results according to different experimental conditions was investigated. Erythrocytes were left to settle in flexible plastic microtiter plates, after which they were fixed with glutaraldehyde and the remaining binding sites in the plates saturated with ovalbumin. Serum or monoclonal IgG antibodies were then allowed to react with the erythrocytes. Protein A coupled to alkaline phosphatase caused a color change in the subsequently added enzyme substrate. The results proved to be of good reproducibility, specificity and sensitivity. The assay can be used for measuring IgG concentration, estimating antibody avidity and number of antigenic determinants on the SRBC, as well as screening IgG anti-SRBC hybridomas. The precision of concentration estimates was very good when standard curves were used.     

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.     

A reverse-type sandwich enzyme-linked immunosorbent assay for detecting antibodies to Borna disease virus.

To investigate whether there is an epidemiological correlation between Borna disease virus (BDV) infection and human neuropsychiatric diseases, we established a reverse-type sandwich enzyme-linked immunosorbent assay (RS-ELISA) for detecting specific antibodies to BDV. In this assay, microplate wells were coated dispersely with BDV p40 antigen, followed by the addition of test samples at a low dilution and then the biotinylated p40. A preformed complex of streptavidin and horseradish peroxidase-conjugated biotin and an enzyme substrate were used to measure the captured biotinylated p40. Theoretically, RS-ELISA should specifically detect anti-BDV antibodies without nonspecific signals; such signals possibly occur in conventional serological assays. Additionally, the RS-ELISA could be applied under the same protocols to test samples from a variety of animals. By using anti-BDV rat and rabbit sera, the assay was standardized so that it had high specificity and sensitivity. When we used the RS-ELISA to determine the presence of anti-BDV antibodies in plasma from 70 patients with chronic schizophrenia as well as 40 healthy individuals in the Tokyo area of Japan, no plasma sample was found to possess specific antibodies to BDV p40, indicating no association between BDV infection and the disease in our testing population. A negative reaction was also shown for the sera that had previously been judged to be seropositive for BDV by an immunofluorescence or immunoblot test. These findings suggested that false-positive cases of infection due to nonspecific reactions may be included in previous seroepidemiological information with regard to BDV.     

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.     

Monoclonal antibodies to porcine tumor necrosis factor alpha: development of an enzyme-linked immunosorbent assay.

Five hybridomas (4F4, 14H1, 9B4, 6E10, and 8G7) secreting antibodies to porcine tumor necrosis factor alpha (PTNF-alpha) were obtained from one fusion. Four of the 5 monoclonal antibodies (Mab) recognized recombinant PTNF-alpha (rPTNF-alpha) on western blot and were able to neutralize both rPTNF-alpha and native (released by porcine macrophages) PTNF (nPTNF-alpha, only 4F4, 14H1, and 9B4 tested for the neutralization of nPTNF-alpha) in vitro. A sandwich enzyme-linked immunosorbent assay (ELISA) for PTNF-alpha was developed using Mab 4F4 and purified rabbit polyclonal antibodies against PTNF-alpha. The test detected PTNF-alpha concentrations as low as 400 pg/ml and did not cross react with native porcine TNF-beta, recombinant human TNF-alpha, recombinant mouse TNF-beta or native mouse TNF-alpha. The Mabs and the ELISA should be useful for assessing PTNF-alpha levels in swine serum during disease processes and possibly for alleviation of toxic effects of TNF-alpha.     

Simple standardized enzyme-linked immunosorbent assay for human antibodies to Entamoeba histolytica.

A simple solid-phase enzyme-linked immunosorbent assay procedure for the detection of human antibodies to Entamoeba histolytica was developed which showed a high degree of correlation with the agar gel diffusion, counterelectrophoresis, and indirect hemagglutination methods, as well as with clinical data. The enzyme-linked immunosorbent assay is rapid (1 h 15 min, total incubation time), and the reported values are referenced to a positive control so that they correlate with levels of antibody sufficient to be detected by the gel diffusion methods. The enzyme-linked immunosorbent assay is highly reproducible, specific, and sensitive; it can be used qualitatively or quantitatively.