Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody to hepatitis B core antigen.

An enzyme-linked immunosorbent assay for detection of specific immunoglobulin M (IgM) antibodies against the core antigen of the hepatitis B virus (anti-HBc IgM) is described. The interference of IgM rheumatoid factor was evaluated quantitatively. In the anti-HBc IgM test, the rheumatoid factor gave false-positive results when the concentration exceeded 20 IU/ml. The rheumatoid-positive sera were disclosed by a control and retested for anti-HBc IgM after absorption of rheumatoid factor with latex particles aggregated with human IgG. In five of seven selected patients with acute hepatitis B followed to biochemical and clinical recovery, anti-HBc IgM was present transiently until antibodies against hepatitis B surface antigen (anti-HBs) appeared. Two patients had persistent anti-HBc IgM during the follow-up period. Four patients with hepatitis B surface antigenemia and progression to chronic liver disease did not clear their anti-HBc IgM in the period of observation (11 to 24 months). Anti-HBc IgM could not be demonstrated in 223 of 225 Danish blood donors. The two donors found positive for anti-HBc IgM also had anti-HBs. Twenty patients with acute A or non-A non-B hepatitis were negative for anti-HBc IgM. The enzyme-linked immunosorbent assay for anti-HBc IgM described here has a high specificity and sensitivity. The diagnostic relevance needs further evaluation, including quantitation of anti-HBc IgM, but the results presented indicate that anti-HBc IgM may be helpful in differentiating between prior and recent or ongoing hepatitis B infection.     

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.     

Determination of immunoglobulin M antibodies for hepatitis B core antigen with a capture enzyme immunoassay and biotin-labeled core antigen produced in Escherichia coli.

A new capture enzyme immunoassay for the determination of immunoglobulin M (IgM) antibodies against hepatitis B core antigen (HBcAg) is described. Core antigen produced in Escherichia coli was labeled with biotin and subsequently detected by an avidin-biotin-peroxidase complex. The biotin-labeled core antigen was effective at concentrations as low as 20 ng/ml. Of 561 serum samples from different groups of patients that were tested, 465 samples were negative for other hepatitis B virus markers and also for anti-HBcAg IgM. Sera from the early stages of hepatitis B infection had high levels of anti-HBcAg IgM, and a clear correlation with the acuteness of the disease was observed in 45 follow-up sera from 23 patients with acute or recent hepatitis B. Sera from 21 patients with past hepatitis B were all negative for anti-HBcAg IgM. Twenty serum samples from chronic carriers of hepatitis B surface antigen showed slightly elevated antibody levels for anti-HBcAg IgM. Ten sera which were positive for anti-HBcAg IgG antibodies and had high levels of rheumatoid factor were negative for anti-HBcAg IgM.     

Development of an enzyme-linked immunosorbent assay for immunoglobulin M antibodies against measles virus

Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).     

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.     

Demonstration of immunoglobulin M class antibodies to Toxoplasma gondii antigenic component p35000 by enzyme-linked antigen immunosorbent assay

On the basis that 89% of 48 acute-phase toxoplasmosis patients showed immunoglobulin M (IgM) class antibodies to the 35,000-molecular-weight antigenic component (p35000) of Toxoplasma gondii, as demonstrated by IgM immunoblotting, the antigen was purified by sucrose gradient centrifugation and enzyme labeled for use in an enzyme-linked antigen immunosorbent assay (ELA) for the demonstration of IgM class antibodies to the p35000 component. The ELA showed a specificity of 96% with 139 serum specimens at a serum dilution of only 1:5. The test serologically detected 73 symptomatic acute-phase toxoplasmosis patients; 64 were positive in the 19S IgM indirect immunofluorescent-antibody test, and 9 were negative, although they showed IgM antibodies to p35000, as demonstrated by IgM immunoblotting. Also, the ELA turned out to be independent of IgM rheumatoid factors in six acute-phase toxoplasmosis serum specimens.     

Use of immunoglobulin M antibody to hepatitis B core antigen in diagnosis of viral hepatitis.

To determine the diagnostic value of hepatitis B core (HBc)-specific immunoglobulin M (IgM) antibody (anti-HBc IgM), the sera of six patients with known recent acute viral hepatitis B were examined for the presence of anti-HBc periodically for up to 21 months from the onset of the attack by a sensitive radioimmunoassay technique (CORAB, Abbott Laboratories). It was found that anti-HBc IgM was detectable for approximately 17 months from the onset of the illness. Hence the finding of anti-HBc IgM suggests infection by hepatitis B virus, probably within the preceding 1 to 2 years. A high level of anti-HBc IgM in the acute-phase serum of an individual with viral hepatitis is indicative of recent hepatitis B virus etiology; one patient, however, showed a low titer of anti-HBc IgM in the acute-phase serum sample, which remained virtually unchanged 15 months from onset. The diagnostic use of this serologaical marker is illustrated in 25 patients with viral hepatitis, in whose acute-phase sera anti-HBc was found.     

Diagnosis of invasive amebiasis by enzyme-linked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies

Saliva from subjects with amebic liver abscess (ALA), acute amebic colitis, asymptomatic infection with Entamoeba histolytica or Entamoeba dispar, and uninfected controls was tested by enzyme-linked immunosorbent assay (ELISA) for the presence of E. histolytica galactose-inhibitable lectin antigen and salivary immunoglobulin (IgG) antibodies to a recombinant cysteine-rich lectin-derived protein (LC3). Salivary lectin antigen was found in 65.8% of subjects with acute colitis, compared to 22.2% of those convalescent from ALA, 10.0% with asymptomatic E. histolytica infection, 9.8% with E. dispar infection, and 2.6% of controls (subjects from the United States and study patients with nonamebic diarrhea) (P < 0.001 for each compared to values for subjects with colitis). Salivary anti-LC3 IgG antibodies were found in 92% of ALA patients regardless of duration of illness and in 83.3% of colitis patients who were symptomatic for at least 7 days (P < 0.001 compared to other study groups). Serum anti-LC3 IgG antibodies were detected in 56.3% of subjects with acute colitis, 100% of subjects with ALA or prolonged colitis, 45% of subjects with asymptomatic E. histolytica infection, 32.3% of subjects with E. dispar infection, and 23.4% of diarrhea controls. In comparison to ELISA for serum anti-LC3 IgG antibodies, the salivary lectin antigen assay is a more sensitive and specific test for acute amebic colitis. Detection of salivary anti-LC3 IgG antibodies by ELISA is an effective means for the diagnosis of ALA and prolonged cases of amebic colitis.     

Solid-phase enzyme-linked immunosorbent assay for detection of hepatitis A-specific immunoglobulin M.

A solid-phase enzyme linked immunosorbent assay was developed for the detection of immunoglobulin M antibody to hepatitis A virus. The system was capable of detecting hepatitis A-specific immunoglobulin M in a single dilution of serum and appears to be a reliable and rapid means of establishing a diagnosis of hepatitis A infection. Specific immunoglobulin M was only detected in patients with serologically confirmed hepatitis A and not in patients with other forms of hepatitis, chronic liver disease, or autoimmune disease. In patients with hepatitis A, specific immunoglobulin M was usually detectable for 6 weeks after the onset of dark urine, and the longest period for which it was present in any patient was 115 days. This enzyme-linked immunosorbent assay is rapid, simple to perform, and does not require complicated equipment. Provided adequate supplies of purified reagents can be obtained, this enzyme-linked immunosorbent assay procedure is likely to simplify hepatitis A serology, because the same antibody-coated plates can be utilized to detect hepatitis A virus, anti-hepatitis A virus, and hepatitis A-specific immunoglobulin M.     

Detection of antichlamydial immunoglobulin G and M antibodies by enzyme-linked immunosorbent assay

Chlamydia trachomatis causes a wide range of infections in adults and conjunctivitis and pneumonia in neonates. The complement fixation test for chlamydial antibody is broadly reactive, but possesses low sensitivity, whereas the microimmunofluorescence test is highly sensitive, but technically difficult to perform. A simple, rapid enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of immunoglobulin G (IgG) and IgM antibodies to C. trachomatis. Wells of microtiter plates were coated with Renografin-purified elementary bodies (serotype L2) grown in cycloheximide-treated McCoy cells, and serum antibody was detected with peroxidase-labeled goat antihuman IgG and IgM antibody. Of 41 sera tested from patients with lymphogranuloma venereum, pelvic inflammatory disease, cervicitis, or urethritis there was a 90 and 63% correlation of positive results for IgG and IgM, respectively, by microimmunofluorescence and ELISA. Of the positive correlates, ELISA titers were up to 128 times higher than microimmunofluorescence titers for IgG and IgM. The ELISA detected no false-positive results, but missed two positive results for IgG. Both of these sera were reactive against serotypes C and J, suggesting that the ELISA with LGV L2 antigen may not measure antibodies to serotypes within the C serogroup. The IgM ELISA detected 7 negative and 4 positive results not detected by the microimmunofluorescence test. Of four paired sera examined by ELISA, three showed a fourfold rise in IgG antibody titer, and one showed a twofold rise. Further evaluation of this ELISA will be required to determine how useful it will be in seroepidemiological studies and as a diagnostic tool.     

The use of monoclonal antibodies specific for seal immunoglobulins in an enzyme-linked immunosorbent assay to detect canine distemper virus-specific immunoglobulin in seal plasma samples

A method is described for the measurement of antigen-specific immunoglobulin in seal pup plasmas. Four monoclonal antibodies (H1a, H13a, H24b and H49a) raised against grey seal (Halichoerus grypus) immunoglobulin were used in an ELISA procedure. Levels of canine distemper virus (CDV) specific macroglobulin (IgM like protein) were found to peak approximately 10 days after the first vaccination. Levels of other smaller CDV-specific immunoglobulins (IgG like protein) also increased after vaccination Using immunoblotting the CDV specific IgG-like protein reacted with a CDV protein, having a molecular weight of approximately 75 kDa.     

Detection of immunoglobulin M antibodies toTreponema pallidum in a modified enzyme-linked immunosorbent assay

The indirect enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin M (IgM) antibodies toTreponema pallidum in sera of syphilitic patients is complicated by false positive reactions due to the interference of IgM rheumatoid factor (IgM-RF) activity and the presence of treponemal IgG antibodies. Another source of error producing false negative results is the competition between treponemal IgG and IgM antibodies for the binding sites on the antigen. To avoid these complications in the indirectTreponema pallidum-specific IgM-ELISA, total IgG was immunoprecipitated from sera of syphilitic patients prior to the assay. The IgM-RF from non-precipitated sera reacted in an IgM-RF-ELISA and in theTreponema pallidum-IgM-specific ELISA with identical titers. After precipitation of total IgG no reactíon of the IgM-RF in the assay could be demonstrated. Competition between IgG and IgM antibodies can be prevented almost completely by the precipitation procedure. The sensitivity and specificity of theTreponema pallidum-specific IgM-ELISA after immunoprecipitation of total serum IgG were shown to be higher than 97 percent.     

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.     

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.     

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.     

Interlaboratory evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to Toxoplasma gondii.

One hundred and fifteen serum samples from healthy laboratory personnel and 50 consecutive samples from 19 patients with anamnestic clinical signs of toxoplasmosis were assayed by four laboratories for the presence of immunoglobulin M antibodies to Toxoplasma gondii by an indirect enzyme-linked immunosorbent assay (ELISA), an antibody capture assay with peroxidase-labeled toxoplasma antigen, and an immunoblotting assay. In addition, a commercially available antibody capture ELISA was used. Highly significant correlation coefficients were obtained between the four laboratories and the commercial test. The indirect ELISA and antibody capture ELISA showed equal sensitivity in detection of immunoglobulin M antibodies to toxoplasma in early-stage serum samples. However, in this study, the antibody capture assay discriminated better between serum samples obtained at early or late stages of toxoplasma infection.     

Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay

Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus.     

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.     

Direct enzyme-linked immunosorbent assay that uses peroxidase-labeled antigen for determination of immunoglobulin M antibody to cytomegalovirus.

A direct enzyme-linked immunosorbent assay was developed for the measurement of immunoglobulin M (IgM) antibody to cytomegalovirus (CMV). Wells of microtiter plates were coated with anti-human IgM. Each patient's serum was added at a dilution of 1:100, and IgM from the serum was allowed to react with anti-human IgM. The amount of CMV-specific IgM antibody bound was determined by measuring the intensity of color change after the addition of peroxidase-labeled CMV antigen and substrate. Nuclei of infected cells served as an antigen source. CMV IgM could be detected only in IgM fractions of sera from patients with a recent CMV infection. Rheumatoid factor did not cause false-positive results. No cross-reactions were observed when paired sera from 22 patients with herpes simplex or varicella and single sera from 12 patients with suspected infectious mononucleosis were tested by the direct enzyme-linked immunosorbent assay. Each of 17 patients with a seroconversion for CMV antibody showed CMV-specific IgM antibody. In six of these patients the antibody was detected in the initial serum. The direct enzyme-linked immunosorbent assay for CMV IgM is a specific and sensitive test for the diagnosis of recent CMV infections and possesses distinct advantages over indirect tests.     

Evaluation of a synthetic-peptide enzyme-linked immunosorbent assay for immunoglobulin M to human parvovirus B19.

A synthetic peptide corresponding to a part of the virus protein 1-virus protein 2 overlapping region of human parvovirus B19 was used in an indirect enzyme-linked immunosorbent assay. Antibodies of the immunoglobulin (Ig) M class were measured in serum samples from patients with erythema infectiosum and controls. In comparison with an IgM assay using native B19 viral antigen, the peptide antigen assay was 92% sensitive and 87% specific. B19 IgM reactivities were seen in a limited number of children with other viral diseases. Specific IgM reactivities to short synthetic viral peptides have previously been reported only with Epstein-Barr virus. Since other sources of viral antigen are limited, the peptide antigen assay may be a useful alternative for the diagnosis of B19-associated disease in human beings.