Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.     

Competitive inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM

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.     

Comparison of four-layer radioimmunoassay and electron microscopy for detection of human rotavirus.

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.     

Chronological evolution of IgM, IgA, IgG and neutralisation antibodies after infection with SARS-associated coronavirus

Abstract Serum levels of IgG, IgM and IgA against severe acute respiratory distress syndrome (SARS)-associated coronavirus (SARS-CoV) were detected serially with the use of immunofluorescent antibody assays in 30 patients with SARS. Seroconversion for IgG (mean 10 days) occurred simultaneously, or 1 day earlier, than that for IgM and IgA (mean 11 days for both). IgG could be detected as early as 4 days after the onset of illness. The earliest time at which these three antibodies reached peak levels was similar (mean 15 days). A high IgG level (1:800) could persist for > 3 months. The kinetics of neutralisation antibodies obtained with 100x the tissue culture infective dose (TCID50) of the SARS-CoV TW1 strain in five patients with SARS nearly paralleled those for IgG. There were no significant differences in the kinetics of the IgG, IgM and IgA responses between patients with or without underlying medical disease, steroid or intravenous immunoglobulin therapy, or mechanical ventilation.     

Measurement of antirotavirus IgM/IgA/IgG responses in the serum samples of Indian children following rotavirus diarrhoea and their mothers

Rotavirus specific, serum IgM/IgA/IgG levels among hospitalized children and their respective mothers were determined. Children were grouped as having rotavirus diarrhoea (RVD) and non-rotavirus diarrhoea (NRVD) on the basis of fecal excretion measured by ELISA and RT-PCR. Although IgM seropositivity was observed among children of both the groups, it was significantly higher in the acute as well as convalescent phase serum samples (P < 0.05 for both) of RVD group. Five out of ten acute sera from the NRVD group were positive for IgM and seven showed IgA/IgG seroconversion indicating rotavirus infection among these children in the past. It was noted that, three out of 24 mothers' sera from RVD group, showed presence of IgM in the serum collected during convalescence of their children. The observation suggests, subclinical rotavirus infection among mothers probably contacted from their children. This is supported by the seroconversion for IgA/IgG among these three mothers. Such a phenomenon was not noticed among the mothers from NRVD group. In general, IgA positivity did not vary significantly among the children from both the groups. IgA seropositivity was significantly higher (P < 0.001) from children of RVD group as compared to healthy group of children following rotavirus infection. From RVD group, all the child patients and 12 mothers out of 24 (50%) showed IgA/IgG seroconversion. None of the mothers from NRVD group showed seroconversion. Serum samples of healthy children and adults, showed IgM positivity at equal level (10%), but a significant difference (P < 0.01) was observed in IgA positivity. In conclusion, subclinical transmission of rotavirus infection from children to their mothers may occur. Seroconversion alone cannot be considered as a marker of rotavirus diarrhoea in children. Moreover, about 40-50% of subjects lacked rotavirus specific IgA at protective levels, making them susceptible to rotavirus infection.     

Kinetics of antigen-specific IgM/IgG/IgA antibody responses during Zika virus natural infection in two patients

Understanding of kinetics of antibody responses is crucial for developing rapid serological tests and studying the mechanisms of Zika virus (ZIKV) infection. Most of the serological diagnostic assays previously published are based on either IgM or IgG titer, little is known on the level of IgA antibody in saliva and urine. In this study, we investigated the kinetics of IgM/IgG/IgA antibody responses in serum, saliva, and urine obtained from two ZIKV infected individuals from as early as the second day of onset of symptoms to as long as 2 years postinfection. Other than detecting robust early IgM response, long lasting IgG response, we discovered strong early IgA response specific for ZIKV in saliva in both patients. This unique observation provides a novel strategy and scientific basis for the development of noninvasive rapid tests for ZIKV infection.     

New bacterial absorption method for determination of hepatitis A IgM and IgA antibodies

Antibodies against hepatitis A virus (anti-HAV) can be determined by a commercially available radioimmunoassay (RIA) (HavabTM, Abbott). To discriminate between recent and past hepatitis A infection this RIA was used in combination with absorption with protein A-containing staphylococci. However, nonabsorbable anti-HAV was repeatedly detected in late-convalescent sera using this methods. The nature of these antibodies was studied in serum samples from 12 such patients. In all patients, the late-convalescent sera contained no IgM class anti-HAV as judged by sucrose density gradient centrifugation. The restricted specificity of staphylococcal protein A explains the lack of absorption. Some recently described streptococcal strains capable of binding all IgG subclasses (including IgG3) as well as both IgA subclasses were, therefore, added to the staphylococci. Absorption studies using these strains indicated that the previously nonabsorbable anti-HAV in these 12 patients was mainly of the IgA class. A bacterial mixture including IgA-binding streptococci seems preferable to routine determination of IgM anti-HAV in acute hepatitis A diagnosis. The results also indicate that IgA anti-HAV in serum can persist for more than two years after a hepatitis A infection.     

A user's guide to the indirect solid-phase radioimmunoassay for the detection of cytomegalovirus-specific IgM antibodies

In this review full laboratory details are given of a solid-phase indirect radioimmunoassay for the detection of specific IgM antibodies against cytomegalovirus. Practical advice is given on readily available commercial sources of reagents, a simple iodination procedure, the rapid dilution of sera under test and calculation of the results with a computer program available from the authors. Problems encountered with the assay are also detailed such as interference by rheumatoid factor, deterioration of the radiolabel and high background binding found with some sera. If these problems are avoided by the methods given in the text then the radioimmunoassay has been shown to give results of 100% specificity with 89% sensitivity for detecting congenital infection and 92% sensitivity for identifying primary cytomegalovirus infection in pregnant women.     

Solid-phase radioimmunoassay of herpes simplex virus IgG and IgM antibodies.

A solid-phase radioimmunoassay for detection of herpes simplex virus-specific IgG and IgM antibodies in human serum specimens is presented. Virus antigen is adsorbed on polystyrene balls and antibodies which attach to the antigen are detected by 125I-labeled antihuman-gamma or antihuman-mu immunoglobulins. A total of 76 specimens have been tested. The appearance of virus-specific IgG and IgM antibodies in primary herpetic infections was readily demonstrated. When serum samples from patients with past exposure to herpes simplex virus were tested, endpoint titers of virus-specific IgG antibodies were found to be 8 to 2048 times higher than titers determined by a complement fixation test. Apparent cross reactivity with varicella-zoster virus was observed in the present radioimmunoassay.     

Solid-phase microtiter radioimmunoassay for detection of the Norwalk strain of acute nonbacterial,epidemic gastroenteritis virus and its antibodies

The development of microtiter solid-phase radioimmunoassays for the detection of Norwalk antigen and its antibody is described. The tests are simple to perform and are sensitive and specific. The test for antigen can be used on crude stool filtrates and suspensions. Both tests are at least as sensitive as immune electron microscopy and more sensitive than immune adherence assay.     

Passive immunizations of suckling mice and infants with bovine colostrum containing antibodies to human rotavirus.

After immunizing 8-month pregnant Holstein cows with the human rotavirus MO strain, cow colostrum containing neutralizing antibody to four different serotypes of human rotavirus, designated Rota colostrum, was obtained. Oral inoculation of human rotavirus MO strain into 5-day-old BALB/c mice causes gastroenteritis characterized by diarrhea. Using this small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of Rota colostrum. Rota colostrum completely protected against rotavirus infection, but purified IgG and IgA obtained from Rota colostrum were unable to protect against infection. After grouping randomly 20 infants from a baby care center, 10 infants received 20 ml of Rota colostrum per day for 2 weeks and 10 control infants did not. Rotavirus-associated diarrhea developed in 7 of 10 infants in the control group. None of the three infants in the every day recipient group of Rota colostrum had such symptoms, and one of three infants in the every other day recipient group developed rotavirus-induced diarrhea. All four infants who received Rota colostrum after symptoms appeared developed diarrhea. Oral administration of Rota colostrum seems to be an effective and safe means of preventing diarrhea caused by human rotavirus infection.     

Rescue of IgM,IgG, and IgA production in common varied immunodeficiency by T cell-independent stimulation with Epstein-Barr virus

We previously defined three categories of B-cell defects in common varied immunodeficiency (CVI): failure to produce IgG and IgA in response to T cell-dependent (TD) stimulation byStaphylococcus bacteria (Sac) plus pokeweed mitogen or B-cell inducing factor (BIF), failure to produce any immunoglobulin, and failure of Sac-induced proflieration and differentiation. The present study includes the responses of 22 CVI patients to T cell-independent (TI) stimulation by Epstein-Barr virus (EBV). In the majority of patients, EBV-stimulated B cells showed normal proliferation and IgM production. In addition, IgG and IgA production was in the range of that for EBV-stimulated normal cells in many patients. Among 11 patients with no TD production of immunoglobulin of any isotype, two showed normal IgM secretion in response to EBV and five others had significant but subnormal responses. Four patients never had humoral responses despite repeated testing and removal of potentially suppressing T cells and monocytes. Concanavalin A stimulation of the T cells from all the patients tested resulted in the production of B-cell inducing factor at higher levels than for normal donor T cells, as assayed on normal Sac-stimulated B cells. These results show that many cases of B-cell defects in CVI patients involving TD production of IgM, switching to TD production of IgG and IgA, and mitogen responses to Sac are not absolute defects. The B cells will respond normally to some stimuli.     

Rotavirus fecal IgA antibody response in adults challenged with human rotavirus

Our studies of rotavirus challenge in adult volunteers enabled us to evaluate the relationship of pre-existing antirotavirus fecal IgA antibody to infection and illness and to investigate the local response to this infection. No relationship could be found between the pre-existing levels of fecal antirotavirus IgA antibody and protection from infection or illness. A greater than six-fold increase in the level of antibody was seen in 16/19 infected volunteers with determinable increases but in 0/15 controls who received less than the minimal infectious dose of rotavirus. Antibody levels increased rapidly in infected volunteers and were consistent with an anamnestic response. Two of seven volunteers who received an infectious dose of rotavirus but were considered uninfected on the basis of other laboratory methods had greater than or equal to six-fold increases of fecal antibody and one of these experienced symptoms compatible with a rotavirus infection. This finding indicates that an increase in fecal antibody may be a reliable indicator of rotavirus infection even in the absence of detectable shedding or seroconversion.     

Serum or breast milk immunoglobulins mask the self‐reactivity of human natural IgG antibodies

B cells producing IgG antibodies specific to a variety of self‐ or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein‐destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab’)₂ fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self‐reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.     

Rapid detection of rotavirus in stool by latex agglutination: comparison with radioimmunoassay and electron microscopy and clinical evaluation of the test

A latex agglutination test (LX) using antisera prepared against Nebraska calf diarrhea virus (NCDV) is described for the detection of rotavirus in stool of children with acute gastroenteritis. The test was compared with electron microscopy (EM) and radioimmunoassay (RIA) with 100 stools positive or negative for rotavirus. Out of 53 stools positive in RIA or EM, 49 were positive in LX and 4 were negative. Two specimens negative in EM and RIA were falsely positive in LX. The method was also tested in two clinical series with 115 stools from 101 children. Altogether 67/115 stools were positive in RIA, and 62/115 in LX. Out of 7 stools with contradictory results, 6 were negative in LX but positive in RIA, and 1 was positive in LX but negative in RIA. The results indicate that the LX is suitable for rapid screening of rotavirus gastroenteritis in clinical practice.     

Induction and persistence of local rotavirus antibodies in relation to serum antibodies

The induction and persistence of local rotavirus antibodies, including stool IgA, jejunal IgA, and jejunal neutralizing antibody, were evaluated in 14 adult volunteers infected with the CJN strain of human rotavirus. In addition, the relationships between local rotavirus IgA and serum rotavirus IgA, IgG, and neutralizing antibody were determined. Both stool and serum rotavirus IgA appeared to have similar kinetics. Both antibodies peaked by days 14-17 after inoculation in all subjects, then decreased rapidly. By days 26-28, titers had fallen to 13% and 30% of their respective peaks. Serum rotavirus IgG peaked somewhat later, occurring in five subjects on days 26-28. Serum neutralizing antibody peaked on days 26-28 in all but three subjects. Both serum IgG and neutralizing antibodies also declined more slowly than rotavirus IgA. Although all antibody concentrations had decreased to only a fraction of their peak responses by days 270-365 after rotavirus inoculation they remained higher than baseline levels. For example, stool rotavirus IgA concentrations were 13.5-fold higher than baseline, while jejunal rotavirus IgA and neutralizing antibody were 8.9- and 4.3-fold above baseline, respectively. Similarly, serum antibodies remained 3.7- to 11.2-fold higher than baseline at 270-365 days after rotavirus inoculation. These studies imply that serum rotavirus IgA is a good indicator of local antibody responses. Furthermore, although both serum and local antibody titers peaked within 2-4 weeks after infection, these antibodies persisted at above baseline concentrations for at least 9-12 months after infection.     

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.     

Faecal and pharyngeal shedding of rotavirus and rotavirus IgA in children with diarrhoea

In 70 children 0-4 years of age with acute diarrhoea, the shedding of rotavirus and the excretion of rotavirus-specific IgA antibody in the stools were examined throughout the period of clinical symptoms. Quantitative detection of rotavirus and IgA was performed by an ELISA technique. The maximal rotavirus shedding was found between the second and fifth day and the maximal excretion of IgA antibody about the seventh day, which marked the clinical recovery of most children. Throat swabs were examined for both virus particles and specific IgA antibody to try to document the respiratory spread of rotavirus infection. Rotavirus antigen could not be demonstrated in the throat swabs, but specific IgA antibody was detected at levels comparable to the faecal specimens obtained at clinical recovery. The observations indicate that the presence of rotavirus secretory IgA limits the duration of diarrhoea and plays a major role in the intestinal resistance to infection.     

Solid-phase radioimmunoassay determination of virus-specific IgM antibody levels in a follow-up of patients with naturally acquired measles infections

The question of the exact disappearance time or possible persistence of measles-specific IgM antibodies after naturally acquired measles virus infections was studied with a sensitive solid-phase radioimmunoassay (RIA) method. A total of 30 patients were analyzed with follow-up times varying from 4.5 to 8 months; all were measles IgM positive in the first serum specimen obtained after the onset of rash. In 29 of 30 patients, the measles IgM declined to undetectable levels by approximately 90 days. The remaining patient developed postmeasles encephalitis, however, and was found to have a prolonged measles IgM antibody response. For comparison, the measles-specific IgG response was also studied and was found to develop only slightly later than the IgM response, with levels then remaining high and stable up to 8 months later. Although apparent measles IgM antibodies were found in 1 of 64 nonmatched adult controls, they were due to the presence of high levels of IgM-class rheumatoid factor. The data presented indicate that measles IgM antibodies begin to decline soon after the onset of rash and reach negative levels 1 to 3 months later; in complicated infections, however, measles IgM antibody synthesis may not terminate normally.     

The protective capacity of immune sera in experimental mouse salmonellosis is mainly due to IgM antibodies

Passive immunization was used to protect mice against a general infection caused by Salmonella typhimurium and our purpose was to compare the protective capacity of different immunoglobulin isotypes (classes and subclasses). Three antisera were studied, one pool of mouse serum against the envelope of rough bacteria, and two rabbit sera against smooth bacteria. Three different methods were used to separate isotypes. The consistent finding was that only IgM antibodies protected efficiently. A unit of IgG antibodies had an effect that was 1/50th of the IgM effect or less. This effect could have been due to a contamination by IgM. IgA appears to be non-protective like IgG. In two of the antisera a considerable proportion of protective antibodies were against a defined antigenic determinant (anti-0–4,5 or anti-0–9). IgG antibodies of these sera measured by the solid phase assay were also predominantly anti-0–4,5 or anti-0–9, respectively. This argues that the failure of IgG antibodies to protect cannot be explained by assuming that unlike IgM antibodies they are directed against “non-protective” determinants. We conclude that the observed difference between the protective capacities of IgM and IgG antibodies is due to C-region differences between the μ- and γ-chains.