Class and subclass distribution of hantavirus-specific serum antibodies at different times after the onset of nephropathia epidemica

Sera from Dutch and Belgium individuals who suffered from nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), were tested for the distribution of classes and subclasses of Hantavirus (HV)-specific antibodies at different times after the onset of the disease, with class- and subclass-specific Ig capture enzyme-linked immunosorbent assays (ELISAs). In the acute, early convalescent, and convalescent phases, predominantly specific IgA, IgM, and IgGS antibodies were detected. Specific IgG2 antibodies were only detected at low levels in the early convalescent and convalescent phases. In the late convalescent phase specific IgG1 and IgGS antibodies were found, whereas in the late post convalescent phase only specific IgG1 antibodies proved to be present. Specific IgG4 antibodies were not detected in any of the respective phases. These data show that the simultaneous determination of classes and subclasses of HV specific serum antibodies allows the estimation of the time elapsed after the onset of NE. © 1994 Wiley-Liss, Inc.     

A critical study of the use of staphylococci containing protein A for separation of IgG and IgM antibodies

This study was to determine the best conditions for using staphylococci bearing protein A to separate IgG from IgM. The validity of the technique was evaluated for detection of IgM with antimicrobial activity and for typing monoclonal IgM. The results indicate that separation of IgG and IgM is not entirely satisfactory in normal sera and worse in hyperglobulinemic sera. The detection and titration of IgM antimicrobial antibodies (rubella and hepatitis B core (HBc) specific IgM) was unreliable because IgG was only partially absorbed by staphylococcal cells, while a significant portion of IgM was bound. The use of higher concentrations of staphylococci did not improve the results because the more IgG was absorbed, the more IgM was also bound. It is shown that with anti-HBc specific IgM the risk of misinterpretation is very high with a sensitive radioimmunoassay technique allowing detection of trace amounts of nonabsorbed IgG. In contrast staphylococcal protein A proved useful in typing monoclonal IgM.     

Detection of IgM antibodies to delta antigen after coinfection and superinfection with the delta virus

A sensitive microtitre radioimmunoassay was developed for detection of IgM antibodies to delta antigen. The assay was based on the selective binding of IgM from test sera to antihuman IgM (u-chain specific) fixed to wells of a microtitre plate, and utilized delta antigen extracted from the liver of an experimentally infected chimpanzee. This test proved to be useful in distinguishing between coinfection and superinfection with the hepatitis delta virus (HDV). Transient anti-delta IgM responses were observed in patients coinfected with HDV, while prolonged elevated IgM levels were found in HBsAg carriers with chronic liver disease superinfected with HDV. Two distinct serological patterns were observed in both coinfection and superinfection. In coinfection, only 50% of patients with detectable anti-delta IgM went on to develop a long-lasting antibody response. Following superinfection with HDV either stationary or fluctuating levels of IgM antibody were demonstrated. In patients with fluctuating antibody levels, the presence or absence of IgM antibody related to the level of viral replication.     

Significance of maternal and infant serum antibodies to hepatitis B core antigen in hepatitis B virus infection of infancy

The significance of IgM and IgG class antibodies to hepatitis B virus (HBV) core component (anti-HBc) was investigated in a study of maternal-fetal HBV transmission. An IgM anti-HBc response was lacking in the majority (49/53) of HBV-infected infants. This antibody thus cannot be used as an indicator of transplacental infection. However, most infants who became HBsAg positive during the first 6 months of life acquire infection in the perinatal period rather than transplacentally. Passively transferred maternal IgG anti-HBc in the infant and additional IgM anti-HBc positively in the carrier mother have no modulating influence on HBV infection of infants born to HBV carrier women.     

Isotypic analysis of grass-pollen-specific antibodies in human plasma. III. Relationship to autoantibodies to IgE

Since it has been shown that autoanti-IgE may be mistaken for antiallergen antibodies, thus appearing as pseudo-allergen-specific antibodies, it is crucial to separate true- from pseudo-allergen-specific antibodies and to determine to what extent autoanti-IgE appeared as pseudo-allergen-specific antibodies. For this purpose, human Ig pools were affinity-purified successively on a grass-pollen column and then on an antihuman-IgE column. IgG1–4, IgA, and IgM antibodies that were eluted from the grass-pollen column separated into pseudo- (∼30–40%) and true-allergen-specific antibodies that were coretained and not coretained, respectively, with the IgE on the anti-IgE column. Levels of autoanti-IgE were determined in individual plasma samples by surface plasmon resonance and statistically compared to the concentrations of allergen-specific antibodies obtained previously in the same plasma samples. A positive correlation between IgM autoanti-IgE levels and grass-pollen-"specific" IgM concentrations ( P < 0.0002), and negative correlations between IgA autoanti-IgE and both IgE anti-grass pollen and IgG2 autoanti-IgE levels ( P < 0.03, in both cases) were observed for the first time. This supports the contentions that: (1) autoanti-IgE antibodies appeared as pseudo-grass-pollen-specific antibodies, (2) they hid IgE antibodies when the latter were measured, and (3) they compete with one another in binding IgE. Lastly, a model of large Ig complexes is discussed.     

Elisa test system with monoclonal antibodies to human IgG4 for the detection of allergen-specific antibodies in pollinosis

G. N. Gabrichevskii Moscow Research Institute of Epidemiology and Microbiology. Institute. of Experimental Cardiology, All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. I. Vorob'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 11, pp. 574–577, November, 1989.     

Serum immunoglobulin G subclass responses in different phases of hepatitis E virus infection

To investigate the specific immunoglobulin (Ig) G subclass responses in patients with hepatitis E virus (HEV) infection, an open reading frame 2 (ORF2) protein based enzyme‐linked immunosorbant assay was used to measure antibody levels in sera obtained at different phases of infection. Sera were collected at 2–31 days and at 6 months after the onset of symptoms corresponding to the acute (n = 48, 100% IgM‐positive) and convalescent (n = 17/48, 53% IgM‐positive) phases of infection, respectively. IgM‐negative sera from 61 individuals infected at least ≥6 months ago (prior exposure) were also tested. IgG1, IgG2, IgG3, and IgG4 antibodies were detected in 100%, 6%, 56%, and 4% of acute phase sera, respectively, and in 100%, 0%, 0%, and 65% of convalescent phase sera, respectively. IgG1 antibody levels were significantly higher than those of the other detectable subclasses of IgG in the acute and convalescent sera (P < 0.05). The IgG3 antibodies in six acute phase patients were replaced by IgG4 antibodies in the convalescent phase of infection. Patients with prior exposure to HEV had low total IgG antibody titers and decreased IgG1 seropositivity compared with those in the acute and convalescent phases. IgG1 was the only major subclass of antibody to be detected in all the three phases of infection. Other than IgG1 antibodies, the subclass antibody response was restricted to IgG3 and IgG4 antibodies in the acute and convalescent phases of infection, respectively. J. Med. Virol. 85:828–832, 2013. © 2013 Wiley Periodicals, Inc.     

Persistence of neutralizing antibodies a year after SARS-CoV-2 infection in humans

Most subjects develop antibodies to SARS-CoV-2 following infection. In order to estimate the duration of immunity induced by SARS-CoV-2 it is important to understand for how long antibodies persist after infection in humans. Here, we assessed the persistence of serum antibodies following WT SARS-CoV-2 infection at 8 and 13 months after diagnosis in 367 individuals. The SARS-CoV-2 spike IgG (S-IgG) and nucleoprotein IgG (N-IgG) concentrations and the proportion of subjects with neutralizing antibodies (NAb) were assessed. Moreover, the NAb titers among a smaller subset of participants (n = 78) against a WT virus (B) and variants of concern (VOCs): Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) were determined. We found that NAb against the WT virus persisted in 89% and S-IgG in 97% of subjects for at least 13 months after infection. Only 36% had N-IgG by 13 months. The mean S-IgG concentrations declined from 8 to 13 months by less than one third; N-IgG concentrations declined by two-thirds. Subjects with severe infection had markedly higher IgG and NAb levels and are expected to remain seropositive for longer. Significantly lower NAb titers against the variants compared to the WT virus, especially after a mild disease, suggests reduced protection against VOCs.     

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.     

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 development of monoclonal antibodies to respiratory syncytial virus and their use in diagnosis by indirect immunofluorescence

Twelve clones of murine hybridoma cells secreting antibody specific for respiratory syncytial (RS) virus were classified into four groups on the basis of their pattern of staining of unfixed RS virus-infected HEp-2 cells in an indirect immunofluorescence test. Three of the groups reacted with virus antigens present on the membrane of the cells, whilst the fourth group failed to stain most live cells, suggesting specificity for an antigen expressed internally. Representative monoclonals from the membrane antigen staining groups immunoprecipitated the 86K glycoprotein (G), 50K plus 19K glycoprotein (F1,2) and a 23K non-glycosylated protein (VP23). A representative monoclonal from the fourth group that appeared to stain an internally expressed protein immunoprecipitated the virion 34K phospho-protein (P). All four monoclonals stained acetone-fixed tissue culture cells infected with either the Long strain of RS virus or with strains isolated in Newcastle during the 1965, 1972, and 1983 winter epidemics. The anti-fusion protein antibody stained acetone-fixed cells from all of 26 nasopharyngeal secretions from infants with RS virus infection. The anti-G glycoprotein antibody and the anti-VP23 antibody stained cells from secretions poorly or not at all, whilst the anti-P protein antibody stained cells in half the secretions tested but reacted with only a small proportion of cells in comparison with the anti-F or polyclonal antibodies. A pool of all four monoclonals produced more intense staining than the anti-F monoclonal alone and gave a more clearly defined staining reaction than the polyclonal antiserum used for routine diagnosis in over half the secretions. These results indicate that monoclonal antibodies will be of value in the diagnosis of RS virus by indirect immunofluorescence if care is taken in the selection of a suitable pool.     

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.     

Immunoglobulin responses to echovirus type 11 by enzyme linked immunosorbent assay: Single-serum diagnosis of acute infection by specific IgM antibody

An indirect solid-phase enzyme-linked immunosorbent assay (ELISA) for the determination of specific IgM and IgG antibodies to echovirus type 11 in a single dilution of serum was developed using partially purified echovirus type 11 bound to microplates. Whole serum was used for IgG antibody but prior to assaying for IgM antibody interfering IgG was removed by ion exchange chromatography. The ELISA for echovirus type 11 IgG antibody was a more sensitive, rapid, technically easier and less costly alternative to the neutralisation test. With the IgG ELISA 12 of 132 sera (10.6%) known to contain enterovirus antibodies other than echovirus type 11 were positive but it could not be determined to what extent this was due to the greater sensitivity of the ELISA or cross-reactions. The IgM ELISA was even more sensitive than the IgG ELISA with acute sera, and showed a reactivity in 4 of 36 sera (11.1%) with no detectable echovirus type 11 neutralising antibodies. Echovirus type 11 IgM antibody was detected in all sera collected after the first week of infection and up to 30 days after infection. However, it was only detected in 58% of sera collected during the first week after onset thus limiting its use for rapid diagnosis. The echovirus type 11 IgM ELISA appears to have considerable laboratory diagnostic potential when a rising antibody level cannot be demonstrated in paired sera or when virus is not cultured.     

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1.

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314-330 (EP3) covering the central conserved sequence of this domain. Anti-hepatitis C virus-positive blood donors were screened for anti-EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA-confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti-EP3-producing heterohybridoma cell lines: Ul/F30 and Ul/F31 produced IgM-kappa, whereas Ul/F32 and Ul/F33 secreted the isotypes IgG1-lambda and IgG1-kappa, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies Ul/F32 and Ul/F33 have dissociation constants to the peptide of approximately 10(-9) M, binding to recombinant E1 protein expressed in COS-7 cells was different. Only Ul/F33 detected envelope protein of approximately 24-35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.     

Production of monoclonal antibodies to gluten proteins and their use in developing tests for gluten quality

There is a need for rapid, simple tests to give added assurance in gluten quality evaluation which address the needs of plant breeders and the milling and baking industry. As part of fundamental research into gluten protein structure‐function relationships, a library of monoclonal antibodies (MAbs) has been developed to various gluten protein fractions, including both high and low molecular weight subunits of glutenin. The difficulties presented by working with insoluble, heterogeneous gluten proteins in raising MAbs of the requisite specificity are discussed. Several of the MAbs have been used to develop rapid immunoassays which are capable of analyzing a single sample in 10 min, and these have been applied to the quantification of total glutenin proteins, and high and low molecular weight subunits of glutenin in flour samples, illustrating the potential of immunotechnology for monitoring flour quality.     

Monoclonal antibodies to human alpha-foetoprotein: analysis of the behaviour of three different antibodies

Three mouse monoclonal antibodies directed to different epitopes on human alpha-foetoprotein have been produced. Two are of IgG1 subclass and the third is IgA. The polyethylene glycol-dependent immunoprecipitation system, designed for conventional antisera, had to be adapted before reproducible results could be obtained with the reagents. In this adapted system, as well as in a radioimmunoassay using solid-phase second antibody, a mixture of the 3 monoclonal antibodies exhibits cooperativity. However, the sensitivity of the radioimmunoassay with pooled monoclonals is not good as that of conventional antiserum. Low affinity monoclonal antibodies have been used for immunopurification of the antigen, whilst the high affinity one is useful for antigen quantitation in a labelled antibody-dependent system which requires absolute antibody specificity.     

Quantification of IgA and IgG and specificities of antibodies to viral proteins in parotid saliva at different stages of HIV-1 infection

Paired sera and parotid saliva from 75 HIV-1-infected patients, divided in three equal groups with CD4⁺ cell counts > 500, 200–500 and < 200/mm³, respectively, were analysed for IgG, IgA and secretory IgA (sIgA) concentrations and for IgG and IgA antibody directed to HIV-1. Twenty-nine age-matched HIV⁻ subjects were used as controls. In serum the concentrations of immunoglobulins were significantly increased in HIV-infected subjects compared with controls, and a progressive increase of IgA and sIgA was noticed while the CD4⁺ cell count decreased. In contrast, concentrations of IgA and sIgA were not different in parotid saliva between the four subject groups. By an ELISA test directed towards HIV-1 proteins, 73 of the 75 serum specimens from the HIV-infected subjects (97%) and 43 of the corresponding saliva (57%) were found positive for specific IgA antibodies to HIV-1, with an even distribution among the three groups of patients. By Western blotting multiple specificities of IgA to HIV-1 proteins were not frequently found in patients. By contrast, in spite of an IgG concentration in saliva about 100 times lower than that of IgA, reactivities were significantly higher for IgG than for IgA antibodies, especially to env and to pol HIV-1 products. Altogether, these data suggest that the regulation of IgA production in HIV-infected subjects is independent in serum and in parotid saliva. This imbalance of IgA/IgG antibodies to HIV-1 at the mucosal level appears to be a specific feature of HIV-1 infection, and may raise important issues in terms of local protection after immunization.     

Persistence of antibodies to West Nile virus nonstructural protein 5

BACKGROUND: Because IgM antibody against West Nile virus (WNV) pre-membrane/envelope (preM/E) recombinant protein may persist for more than 1 year, an assay distinguishing recent from past WNV infection would be useful. Published findings for a single patient suggest that the presence of antibody against WNV nonstructural protein 5 (NS5) indicates recent infection. OBJECTIVES: To compare the persistence of WNV NS5 antibodies and preM/E IgM using plasma samples from blood donors who were viremic at the time of donation. STUDY DESIGN: Follow-up plasma samples from 35 viremic donors were tested for WNV NS5 antibodies using a microsphere immunoassay, and compared to WNV preM/E IgM antibodies determined on the same samples using an enzyme-linked immunosorbent assay (ELISA). RESULTS: At 90+/-14 days of follow-up, 20/26 donors (77%) were positive for NS5 antibodies; 6/25 (24%) were positive at 180+/-27 days, and 3/23 (13%) were positive at 365+/-55 days. The comparable values for preM/E IgM antibodies were 77%, 32% and 17%, respectively. CONCLUSION: Persistence of WNV NS5 antibody in plasma is similar to that of preM/E IgM antibody. WNV NS5 antibody cannot be used to distinguish recent from past WNV infection.     

Serum antibodies to Pseudomonas aeruginosa outer-membrane proteins and iron-regulated membrane proteins at different stages of chronic cystic fibrosis lung infection

Serum samples collected over periods up to 15 years from nine patients with cystic fibrosis (CF) were investigated by immunoblotting and crossed immuno-electrophoresis (CIE) for antibodies to Pseudomonas aeruginosa outer-membrane proteins (OMPs). The earliest antibody response to OMPs was directed against proteins G, H1 and I. Detection by immunoblotting sometimes preceded the CIE response; the appearance of antibodies to the other major OMPs was co-incident with an increase in CIE precipitins. Isolation of the mucoid form of P. aeruginosa was associated with a rapid increase in both precipitin numbers and antibodies detected by immunoblotting. Antibodies to iron-regulated OMPs could be detected in all the serum samples that showed eight or more CIE precipitins but their presence became pronounced only in the advanced stages of disease. The clinical strain used in this study and other isolates from CF patients showed several atypical OMPs, perhaps as a consequence of antibiotic therapy or related to the serum sensitivity of mucoid P. aeruginosa. Their expression in vivo was confirmed by detecting antibodies to them in patients' serum.