Determination of IgA antibodies to human cytomegalovirus by enzyme-linked immunosorbent assay (ELISA)

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies cytomegalovirus (CMV). The antigen consisted of a sonicated extract of CMV infected human embryo cells. The tested sera were absorbed with staphylococcus aureus (strain Cowan 1) before analysis. Rabbit antihuman IgA peroxidase conjugate was used to detect human IgA bound to viral antigen. In parallel, Igm and IgG antibodies to CMV were studied by ELISA and by the immunoperoxidase antibody to membrane antigen (IPAMA) technique, respectively. CMV IgA was detected in high titers by ELISA in eight of nine CMV patients. The maximal IgA titers were generally lower than the IgM titers detected by ELISA. No CMV IgA antibodies (titer less than 20) were detected by ELISA in 57 control sera (healthy adults, hospitalized patients with various other diseases), paired sera of five patients with acute herpes simplex, infection, two patients with Epstein-Barr infections, one patients with varicella, and one with zoster infections. The potential application of the ELISA CMV IgA technique in serodiagnosis of CMV infections is discussed.     

Detection by ELISA of IgA and IgM antibodies in secretion and IgM antibodies in serum in primary lower respiratory syncytial virus infection

Twenty-six infants and children with primary lower RS virus infection, diagnosed by the detection of RS virus in nasopharyngeal secretion (NPS) by use of immunofluorescent antibody (FA) technique, were studied with respect to the presence of IgA and IgM antibodies. Samples of NPS and serum obtained during the first 3-4 months following the beginning of illness, were investigated. Employing a reverse ELISA technique, we found IgM antibodies in the acute, but not during the convalescent, phase of illness in NPS from 20 of the patients and in serum from 21 of the patients. The majority of the IgM antibody conversions observed occurred in NPS as well as in serum on days 5-8 following the illness. RS virus IgA antibodies, also detected by a reverse ELISA technique, were demonstrated in NPS in 22 of the patients, with antibody conversions being found in 19 of the patients on days 5-8 following the beginning of the illness. Two patients still had IgA antibodies in NPS approximately 3 months FSOI. By comparison, RS virus was detected in acute-phase NPS by double-antibody sandwich ELISA in 25 of the 26 patients investigated.     

Detection by ELISA of IgG-subclass-specific antibodies in primary respiratory syncytial (RS) virus infections

The IgG-subclass specific antibody response was investigated in primary RS-virus infections in infants and small children by using an ELISA with monoclonal antibodies against the four human IgG subclasses. When 78 serum samples obtained from 21 patients during the first 3-4 mo following the onset of illness were studied, only IgG-1 and IgG-3 antibodies could be detected. Both of these subclass specific antibodies appeared during the second week following the beginning of illness. Approximately 4 wk after the onset of illness, IgG-1 antibodies reached maximum levels, which persisted during the following months. Approximately 3 wk after the onset of illness, IgG-3 antibodies reached maximum levels, which decreased during the following months. Both IgG-1 and IgG-3 antibodies showed neutralizing capacity.     

Determination of human IgG and IgM class antibodies to West Nile virus by enzyme linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed and used for the detection of IgG and IgM antibodies to West Nile virus in human sera. Thirteen paired sera of clinical cases and 24 control sera taken randomly from a blood bank repository were tested. The sera were reacted in microtiter plates coated with PEG-treated WNV antigen. IgG or IgM antibodies were quantitated by the use of alkaline-phosphatase-conjugated anti-human IgG or IgM antibodies. Of the 24 randomly collected serum samples, 7 were positive in the IgG-ELISA test. One positive by the IgM-ELISA was found to contain rheumatoid factor. In 12 of 13 paired sera of clinical cases, IgM as well as IgG antibodies were detected in the second serum sample taken about 3 wk after the onset of clinical signs. The IgM positive sera were screened for rheumatoid factor (RF) on IgG-coated plates. None of them contained RF. Antibody titers obtained by ELISA showed a good correlation with titers obtained by hemagglutination inhibition, complement fixation, and neutralization tests. The ELISA tests for detection of IgM and IgG antibodies to WNV therefore can replace the other serological methods for epidemiological surveillance and diagnostic purposes.     

The evaluation of a quantitative enzyme-linked immunosorbent assay (ELISA) for anti-Aspergillus fumigatus IgG

A standard enzyme-linked immunosorbent assay method for anti-Aspergillus fumigatus IgG in human serum was modified to produce a quantitative assay. The resulting assay was reproducible and capable of separating individual precipitin line groups and provided a means of monitoring the variation in antibody levels over long periods in patients with pulmonary aspergillosis.     

Evaluation of an enzyme-linked immunosorbent assay that measures rhinovirus-specific antibodies in human sera and nasal secretions

Rhinovirus-specific antibodies have traditionally been detected by their ability to neutralise the homologous rhinovirus serotype in tissue culture. Recently, however, we have described an enzyme-linked immunosorbent assay that detects rhinovirus-specific antibodies in sera and nasal secretions [Barclay and Al-Nakib, 1987]. Here we describe an evaluation of the ELISA in a study involving 71 adult volunteers inoculated intranasally with human rhinovirus type 2 (HRV-2). Pre- and post-inoculation serum samples and pre-inoculation nasal washings were tested for the presence of HRV-2-specific antibodies by ELISA. Such antibodies were associated with protection against infection when present locally in nasal secretions, but when also present in the serum they were associated with protection against both infection and the development of illness. The antibody concentrations showed strong correlation with each other and with that of antibodies detected by the neutralisation test. Following HRV-2 infection, rises in HRV-2-specific IgA in sera detected by ELISA occurred more frequently than rises in neutralising antibody. These results suggest that the ELISA is a sensitive and reliable indicator of recent infection, as well as a predictor of homologous immune status.     

Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA)

Competitive-inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM are described. These assays can be readily performed with commercial antisera and a recently developed method for purifying human IgA and IgM with high yield. The assays described are specific, with undetectable (<0.5%) cross-reactivity between the immunoglobulin classes in all systems, except with purified IgM, which cross-reacted to 1.9% with the IgG enzyme immunoassay.

Minimal detectable concentrations of 2.5±0.8 ng/ml for IgG 4.2±0.9 ng/ml for IgA and 7.2±1.4 ng/ml for IgM were recorded, indicating that these assays are particularly sensitive. There is little within-assay variation (mean coefficient of variation = 3.9%), although the between-assay variation was substantially greater (mean coefficient of variation = 23.5%). These assay systems appear to be particularly suited to the measurement of immunoglobulin production by lymphocytes in culture. In such studies the assay must be specific, sensitive and be capable of discriminating between levels of immunoglobulin produced in response to various experimental treatments.     

Enzyme-linked immunosorbent assay for subclass distribution of human IgG and IgA antigen-specific antibodies

In this study we describe an ELISA using monoclonal antibodies to IgG 1, 2, 3, 4, IgA1 and IgA2 for determining the subclass distribution of human-specific antibodies. No cross-reactivity of the subclass-specific reagents under the conditions used was observed. The sensitivity was 0.5 ng/ml for IgG1, 3, 4; 1.5 ng/ml for IgG2 and 50 ng/ml for IgA1 and IgA2. The reproducibility as described by the coefficient of variation calculated on repeated runs was 8-26% if the data were obtained by relating the absorbance values to a positive serum run in the assay, 17-58% when relating the OD figures to those of a standard myeloma plate. The method may be considered semiquantitative with high sensitivity and specificity, easy to handle and with small day-to-day variation. The assay has been applied to a number of antigens of protein and polysaccharide nature.     

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.     

Simplified enzyme-linked immunosorbent assay for specific antibodies to respiratory syncytial virus.

A simplified and reliable enzyme-linked immunosorbent assay (ELISA) was applied to the detection of serum antibodies against respiratory syncytial virus (RSV). RSV-infected cells were fixed and dried on 96-well microtiter plates and kept at 4 degrees C. The titers of reference sera were determined by endpoint dilution. A linear relation was found between the titers and the logarithm of absorbance values of sera diluted to 1:1,000 (r = 0.93, P less than 0.001). Measurement of RSV antibodies was done by using a single serum dilution (1:1,000) in conjunction with a standard curve. A strong correlation was found between complement fixation and ELISA results (r = 0.89, P less than 0.001). In addition, the ELISA method exhibited higher titers and a greater sensitivity than did complement fixation, although the applicability of the assay is limited with positive serum samples of low titer.     

Enzyme-linked immunosorbent assay for IgG antibodies to Epstein-Barr virus-associated early antigens and viral capsid antigen

An enzyme-linked immunosorbent assay has been developed for the detection of IgG antibodies to Epstein-Barr virus-associated early antigens and late antigens including the viral capsid antigen. The antibody titers of human sera determined in this way correlate well with those by indirect immunofluorescence. ELISA was more sensitive than the IF method. The assays described may be used for rapid and sensitive diagnosis of EBV-related diseases. In addition, the ELISA will be useful for the determination of antibody titers to isolated EBV-associated antigens, e.g., purified components of the EA complex.     

Enzyme-linked immunosorbent assay (ELISA) for mumps virus antibodies

An ELISA for the detection of mumps virus-specific IgG and IgM antibodies was developed. Three different antigen preparations were compared. Equally good results were obtained with virus concentrated by ultracentrifugation and with virus that was further purified by sucrose gradient centrifugation. Crude infected allantoic fluid was unsuitable for use as antigen. The variability and reproducibility of the tests were satisfactory. When the ELISA was compared to conventional serological methods, a good correlation of IgG absorbance values with complement fixation (CF) antibody titers was found (r = 0.574), the ELISA being more sensitive in detecting antibodies in acute-phase sera. For the determination of immunity, ELISA IgG values were compared with results obtained in hemagglutination-inhibition (HI) and hemolysis-in-gel (HIG) tests. Again there was a good correlation with both tests (r_HI = 0.528, r_HIG = 0.667). The ELISA was more sensitive than the HI and HIG test for the detection of low levels of antibodies.     

Use of the enzyme-linked immunosorbent assay for measurement of allergen-specific antibodies

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for allergen-specific IgG antibodies is described. Various solid-phase supports (microtiter plates), coating procedures, binding kinetics, and presentation of allergen in the assay were investigated. Using optimal conditions the indirect ELISA, in which the allergen is coated onto the well, was capable of detecting 2.4 ng/ml specific IgG antibodies to bee venom phospholipase A2(PLA2). The sandwich ELISA, in which the allergen was immobilized via specific antibody precoated onto the well, detected 0.24 ng/ml IgG antibodies to PLA2.     

Persistence of serum IgA antibodies to herpes simplex, varicella-zoster, cytomegalovirus, and rubella virus detected by enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays were used to detect IgG and IgA antibodies to herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and rubella virus in sera from 68 adult female gynaecological patients. Of the patients who had virus-specific IgG antibodies, the proportion who also had virus-specific IgA was 98% for HSV, 75% for VZV, 73% for rubella virus, and 42% for CMV. IgA antibodies to all four viruses were only found when specific IgG antibodies were also detected in the serum. These results suggest that virus-specific IgA may persist for several years; possible explanations for this are discussed.     

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay (ELISA) respiratory syncytial virus antigen detection kit (Ortho Diagnostics, Inc., Raritan, N.J.) was compared with virus culture and with the indirect fluorescent antibody test (FAT) by using fresh nasal washings from children with suspected respiratory syncytial virus infection. Both uncentrifuged nasal washings and pellets from centrifuged split specimens were examined by ELISA. The ELISA was considered positive when the optical density was greater than the mean background optical density plus 0.15. Specimens positive by ELISA but negative by culture and FAT were reexamined in an ELISA blocking assay. Of 337 uncentrifuged specimens, 124 (37%) were positive by culture, 107 (32%) were positive by FAT, and 166 (49%) were positive by ELISA. Blocking assays showed that 21 of 30 (70%) of the seemingly false-positive ELISA tests were, in fact, true-positives and that the cultures and FATs were falsely negative. A patient specimen was considered positive when it was positive by virus culture, FAT, or blocking assay. The sensitivity, specificity, and positive predictive value of the ELISA test were 88, 94, and 95%, respectively. Centrifugation of nasal washings raised the sensitivity from 88 to 92% but reduced the specificity from 94 to 81%. We conclude that the Ortho ELISA test of uncentrifuged nasal washings is more sensitive than virus culture or indirect FAT and is highly specific.     

Enzyme-linked immunosorbent assay (ELISA) for detecting antisperm antibodies in mice with testicular autoimmunity

We developed an ELISA for measuring antisperm antibodies in the mouse by using serum samples obtained from mice immunized with murine testicular antigens in complete Freund's adjuvant (CFA) as well as from mice rendered vasectomized. Sperm antigens used were syngeneic epididymal spermatozoa and two types of soluble, murine testicular antigens prepared in our laboratory. This study deals with a) the sequential changes of antisperm antibody levels following immunization; b) determination of immunoglobulin classes of these antibodies; c) a correlation between the absorbance values and the endpoint titers of antisperm antibodies; and d) comparison of endpoint titers of antisperm antibodies detected by ELISA with those by immunoperoxidase staining method in immune and nonimmune sera. It is suggested that serum dilution as high as 1/800 or more is required for detecting antibody titers of immune sera, because nonimmune mouse sera reveal a definite, although low, level of absorbance value at a serum dilution of 1/400 or less.     

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.     

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses

A human monoclonal anti-HBsAg antibody (IgGl, λ) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgGl antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay, indirect immunofluorescence, and virus isolation: a comparative study

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.     

Rapid enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against

A rapid ELISA was developed for the detection of specific IgG against Micropolyspora faeni and Thermoactinomyces vulgaris and compared with counterimmunoelectrophoresis (CIE) in twenty-seven patients with suspected farmer's lung disease (FLD). Seventeen patients had precipitins to M. faeni or T. vulgaris or both, and ten had no precipitins. The optimum conditions for each step in the ELISA were determined: pre-equilibration of reagents at 37⁰C and vigorous agitation during incubation enabled the total test time required for the procedure to he reduced to 4 hr. A serum dilution of 10-fold produced good differentiation between CIE-positive and -negative sera. Little correlation was seen between CIE and ELISA for either M. faeni or T. vulgaris antigens in tests with sera from patients with precipitins: high readings were often recorded in ELISA where no precipitins had been detected with the same antigen. Positive- negative discrimination of unknown sera in ELISA was achieved through the inclusion of CIE-positive and -negative reference sera in each test run. Thirteen CIE-positive sera were classed as positive in ELISA with the M. faeni antigen while eight of thirteen CIE-positive sera were positive in ELISA with the T. vulgaris antigen. For both antigens, four CIE-negative sera were recorded as positive in ELISA.