Serum levels of IgA, IgM, IgD and IgE in IgG paraproteinaemias

The dependence of serum levels of polyclonal IgA, IgM, IgD and IgE on the amount of IgG paraprotein was studied in a series of 58 IgG paraproteinaemias. A significant correlation was found for IgA (r = -0.4782, p = 0.0007), for IgM (r = -0.3296, p = 0.0237) and for IgD (r = -0.3589, p = 0.0143) in the whole series of IgG paraproteinaemias and in the subgroup of myeloma paraproteinaemias (n = 34) for IgM (r = -0.4276, p = 0.0207) and for IgD (r = -0.4384, p = 0.0196).     

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.     

In vivo and in vitro IgE isotype switching in human B lymphocytes: Evidence for a predominantly direct IgM to IgE class switch program

Molecular analysis of circular excision products and composite genomic switch regions has demonstrated that in mice, immunoglobulin (Ig) isotype switching from IgM to IgE often proceeds sequentially via IgG1. Based on analysis of Ig production in cell cultures, it has been suggested that human B cells may switch to IgE via IgG4, whereas limited molecular data from in vitro switched B cells suggest a direct IgM to IgE switch program. To obtain a quantitative assessment of direct versus sequential IgE switching in humans, we have analyzed the nucleotide sequences of 29 composite Sμ/S? switch regions from freshly isolated human B lymphocytes from patients with atopic dermatitis and from B lymphocytes induced to switch to IgE synthesis in vitro. The data show that in these B cells IgE isotype switching progressed directly from IgM to IgE. We conclude that, in contrast to the murine IgM/IgE switch program, the IgM to IgE switch in B lymphocytes from patients with atopic dermatitis as well as in vitro stimulated B cells from healthy donors preferentially proceeds via direct Sμ to S? switch recombination.     

Interleukin-3-treated non-B, non-T cells switch activated B cells to IgG1/IgE synthesis.

The switch of activated B cells to IgE synthesis is an interleukin (IL)-3-dependent process. It is currently thought that specific T cells activated by antigen presented in the context of class II major histocompatibility complex are the major source of IL-4. Recently it has been demonstrated that a splenic non-T non-B cell population (termed NBNT) has the capacity to produce IL-4 following IgE and IgG receptor cross-linkage. In this study we demonstrate that IL-4 producing NBNT cells can induce the switch of lipopolysaccharide-activated B cells to the synthesis of IgG1 and IgE antibodies. Furthermore, it was found that not only IgE receptor cross-linkage but IL-3 was able to stimulate NBNT cells to produce IL-4 and induce the switch of B cells to IgE synthesis. NBNT cells derived from the spleen and bone marrow of SCID mice were able to produce IL-4 on exposure to IL-3. This suggested that the ability of IL-3 to stimulate IL-4 production was not dependent on prior exposure of the NBNT cells to antibody complexes in vivo. Taken together these findings represent the first observation that enough IL-4 is produced by NBNT cells to actually influence a B cell IgG/Ig response. The findings also clearly demonstrate that B cells do not need high concentrations of IL-4 to be directed to switch to IgG1 and IgE synthesis.     

Antisera to human IgE can also recognize IgA and IgM molecules as produced by selected B lymphoblastoid cell lines.

A series of polyclonal and monoclonal anti-human IgE sera reacted with a panel of 11 selected IgA and IgM producing B lymphoblastoid cell lines (BLCLs) in cytoplasmic fluorescence, ELISA and immunoprecipitation assays. The reactions were discordant between the BLCLs and the individual antisera. The panel of 11 BLCLs was selected from over 500 BLCLs by an ELISA spot assay using a polyclonal anti-human IgE serum. The BLCLs produced exclusively IgM or IgA, both with either kappa or lambda light chains as shown by immunoprecipitation/PAGE and mRNA analyses. The data indicate that IgE antisera may recognize IgE-like epitopes on the variable parts of some IgM or IgA molecules. These reactivities of IgE antisera may lead to false positive results of serum IgE determinations. Selected BLCLs are useful targets for exposing such reactivities in IgE antisera.     

Multiplexed serum measurement of IgG, IgA, IgM, and IgE antibody responses to therapeutic biologicals

Analytical methods characterizing the immunogenicity of therapeutic proteins are useful for monitoring, characterizing and predicting reactions to biopharmaceuticals. A multiplexed assay capable of isotyping the specific IgG, IgA, IgM and IgE (IgGAME) antibody responses against a biotherapeutic was demonstrated in a hyper-immunized cynomolgus monkey, over a 15-month period. The quantitative range of the antibody measurements was determined to be 15 ng/ml to 50 ng/ml in 10% serum. By the use of any biotinylated or fluorescently tagged therapeutic as a detector, this multiplexed isotyping assay can be broadly applied to human and non-human primate IgG, IgA, IgM and IgE immunogenicity studies.     

Inhibition of T cell-dependent human B cell proliferation and B cell differentiation by polyspecific monomeric IgG.

A commercially available polyspecific, monomeric IgG preparation suitable for intravenous administration (IgSRK; Sandoglobulin) can inhibit pokeweed mitogen (PWM)-induced proliferation of peripheral blood mononuclear cells (PBMC) by a small, but statistically significant, amount compared to control cultures. Such inhibition could not be demonstrated when PBMC were stimulated with the T cell mitogen phytohaemagglutinin. Surface phenotype analysis of the PWM-stimulated cells indicated that in IgSRK-containing cultures, the proportion of B cells was decreased and the proportion of T cells was increased compared to control cultures. This alteration in T:B ratio was not due to antigenic modulation of B or T cell markers from their surfaces. In addition, IgSRK inhibited the proliferation of T cell-depleted PBMC cultures stimulated by B cell proliferation factors (BCPF) but not by fixed protein A-bearing Staphylococcus aureus strain Cowan I. The capacity to inhibit B cell proliferation was independent of and distinct from its capacity to inhibit B cell differentiation, since IgSRK inhibited the differentiation of a B cell differentiation factor (BCDF)-sensitive line by BCDF (which contains no BCPF activity). IgSRK inhibited PWM-induced generation of cytoplasmic Ig+ cells but had no effect on Ig secretion from mature Ig-secreting cells. Taken together, these findings suggest that IgSRK (which contains the IgG fraction from pooled plasma from 2,000 healthy donors) can inhibit T cell-dependent or T cell factor-dependent B cell proliferation and B cell differentiation.     

Low zone tolerance: a possible defect in the switch from IgM to IgG production

Specific antibody production during tolerogenic treatment with bacteriophage fd precedes the manifestation of low zone tolerance. The antibodies produced are sensitive to treatment with dithiothreitol, thus, most likely IgM antibodies. Upon challenge, this initial immune response is not amplified. Repeated injections of subimmunogenic doses of antigen affect the classes of antibodies produced upon challenge in different ways. IgG production is already suppressed with doses which are without influence on the production of IgM. It is suggested that low zone tolerance affects the switch from IgM to IgG.     

Interleukin 2 induces T cell-dependent IgM production in human B cells

Conditioned medium, obtained from mononuclear cells after activation with concanavalin A and phorbol myristate, strongly stimulated the in vitro production of IgM by human mononuclear cells. Partial purification by gel filtration showed that this activity co-eluted with interleukin 2 (IL 2). Via removal by adsorption and further purification by chromatofocusing techniques, it was demonstrated that the factor in this conditioned medium, responsible for the IgM production, was in fact IL 2. Experiments with purified IL 2 from the human IL 2-producing Jurkat cell line as well as recombinant IL 2 confirmed its capacity to induce IgM production. In the absence of T cells, IL 2 could not activate Ig synthesis, suggesting an indirect effect of IL 2 in the induction of the helper signals for B cells. Evidence is presented that partially purified conditioned medium contained another factor, distinct from IL 2, with the capacity to differentiate human B lymphocytes in the absence of T cells into IgM-producing cells.     

Fc receptors for IgG, IgM and IgE on human leukaemic lymphocytes.

Fc receptors for IgG, IgM, IgE and the cell surface immunoglobulins (SIg) were analysed on lymphocytes from seventeen patients with chronic lymphatic leukaemia (CLL), one with lympho-sarcoma cell leukaemia (LSL), two each with hairy cell leukaemia (HCL), acute lymphatic leukaemia (ALL) and Sézary syndrome. Fc receptors for IgG and IgM were detected by rosette formation with ox erythrocytes (E_O) sensitized with rabbit IgG (E_OA_G) and IgM (E_OA_M) anti-E_O antibodies, respectively. Fc receptors for IgE were analysed either with E_O coated with glutaraldehyde coupled complexes consisting of rabbit Fab'' fragments of anti-E_O antibodies and Fc fragments of an IgE myeloma protein (E_OA_E), or with aldehyde fixed E_O to which IgE was adsorbed. SIg of classes IgM, IgD, IgG and κ and λ light chain type were detected with E_O coated with complexes consisting of Fab''-anti-E_O and purified F(ab'')₂ fragments of specific goat antibodies.Lymphocytes of all patients with CLL, LSL and HCL had Fc receptors for IgG (65±15% E_OA_G⁺, normal 22·0±5·8%). Ten patients had significant numbers of cells with IgM Fc receptors (37±22% E_OA_M⁺, normal 1·2±1·5%) which were detected without overnight culturing of the lymphocytes. Lymphocytes of four patients (two CLL, one LSL, one HCL) had Fc receptors for IgE (22–88% E_OA_E⁺, normal 1·8±0·7%). The cells of three of these four patients were also E_OA_M⁺. The high numbers of rosetting cells indicated that individual lymphocytes must have carried more than one class of Fc receptors. The lymphocytes of the ALL and Sézary syndrome patients had few Fc receptor positive cells.Of the seventeen patients with CLL, twelve were SIgM⁺ and/or SIgD⁺, only four κ⁺ or λ⁺ and one had no SIg. The cells of the LSL and one of the HCL patients were SIgM⁺ and SIgD⁺, whilst the cells of the other HCL patient were SIgG, λ⁺. None of the other patients had more than 10% SIgG⁺ cells. The ALL and Sézary syndrome patients had low numbers of SIg⁺ and Fc receptor positive cells.These data indicate that lymphocytes of patients with B-cell leukaemias can carry different classes of Fc receptors simultaneously; the different classes are found in a decreasing frequency of IgG>IgM>IgE.     

Antitrichomonas IgG,IgM, IgA,and IgG subclass responses in human intravaginal trichomoniasis

Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is a major nonviral sexually transmitted disease. Clinical spectrum varies from an asymptomatic state to mild, moderate, or severe symptoms. However, the exact factors leading to the variations in symptoms have not been well elucidated. Host’s immune response to the parasite may be playing a role in varied symptomatology. The present study reports antitrichomonas IgM, IgA, IgG and its subclasses in doubling dilutions of serum and diluted vaginal washes of six T. vaginalis-infected symptomatic and four T. vaginalis-infected asymptomatic women and uninfected controls by enzyme-linked immunosorbent assay (ELISA). No significant difference was observed in serum IgG ELISA absorbance values from symptomatic compared to asymptomatic subjects (p > 0.05) while a significant difference (p < 0.05) was noted in serum IgM in all the tested dilutions and IgA up to a dilution of 400. This is the first report of the detection of specific IgG subclass response in T. vaginalis-infected female patients, and quantitative analysis of the antibody responses indicated that the production of local IgG particularly IgG1 in vaginal secretions may be playing a significant role in establishing symptomatic infection. The interesting observation of the present study is that the specific IgM was detected in 2 (33.3%) symptomatic and T. vaginalis-infected patients in ≥800 dilutions and in 1 (16.6%) up to 200 dilutions in serum, while it was not detectable in the vaginal secretions of symptomatic patients or in the serum and vaginal secretions of asymptomatic T. vaginalis-infected patients.     

Kinetics of specific IgA, IgD, IgE, IgG, and IgM antibody responses in rubella

Rubella-specific IgD and IgE antibodies were determined with a solid-phase enzyme immunoassay using enzyme-labeled heavy-chain specific anti-immuno-globulins, and the antibody responses in rubella infection were compared to IgM, IgA, and IgG antibodies. IgD and IgE antibodies increased rapidly after the onset of infection, remained at a high level for at least 2 months, and declined slightly by 6 months. In comparison, the IgM antibodies decreased more rapidly, whereas the IgG antibodies persisted longer at a steady level. By 6 months the mean levels of the different antibodies had declined from their maximal mean levels as follows: IgM, 52%; IgA, 42%; IgE, 35%; IgD, 29%; and IgG, 8%. Thus IgD and IgE antibodies, in spite of their known short half lives, persisted longer than IgM and IgA antibodies, which limits their diagnostic value. The IgA antibody responses were found too variable to substitute for IgM antibody determination in diagnosis of a recent rubella virus infection from a single serum specimen. Comparison of maternal and cord blood sera indicated that, in addition to IgG antibodies, rubella-specific IgD antibodies were found to cross the placenta.     

IgA, IgG, IgM, and IgE levels in normal, healthy, non-atopic Israeli children

IgA, IgG, IgM, and IgE levels in healthy, non-atopic, Israeli-born children aged 20 days to 16 years were analyzed and showed similar age-related values and dynamics as those of white populations found in other countries. No significant effect of sex of the individual or ethnic origin of the parents was found on the IgE values at different ages. This may indicate that total IgE levels are strongly influenced by environmental factors. Establishing tolerance limits at 97.5, 95, 75, 25 and 5th percentiles and the geometric mean provides the practitioner with more complete reference values. The use of multivariate control charts with tolerance limits from normal IgA, IgG, IgM, and IgE levels is described and is offered as an additional tool for the diagnosis of an allergic individual.     

Characterization of grass pollen-specific IgE, IgA, IgM classes and IgG subclasses in allergic patients

Water-soluble grass pollen extracts (Dactylis glomerata) were separated by isoelectric focusing in a wide pH range (2-11) in agarose gel. After focusing, two successive gel prints were taken. The first one, on an ordinary nitrocellulose filter during 10 s, enabled the visualization of separated components of the pollen, after india ink staining. The second one, on a cyanogen bromide-activated nitrocellulose filter obtained after a 10-min transfer and saturation step, was incubated overnight with patient sera, and the specific antibodies bound to antigens or allergens were detected. The screening of different patient sera showed great variability in the antigen spectra. There was no obvious relationships between IgM and IgA antigen spectra as compared to IgE. Some association between IgE and IgG4 subclass was observed.     

Interleukin-9 potentiates the interleukin-4-induced immunoglobulin (IgG,IgM and IgE) production by normal human B lymphocytes

IgE production by normal peripheral blood lymphocytes (PBL) is known to be triggered upon stimulation by interleukin (IL)-4. In the present study we showed that IL-9, another T cell-derived cytokine, markedly potentiated IgE production induced by suboptimal doses of IL-4, whereas no effect of IL-9 was observed in the absence of IL-4. The potentiating effect of IL-9 appeared to be associated with the increased frequency of IgE-producing cells, as revealed by a specific ELISA-spot assay. Under the same experimental conditions, IL-9 also enhanced the IL-4-induced IgG production but did not elicit IgM production. However, IL-9 did not amplify the IL-4-dependent expression of membrane-bound and soluble low affinity receptor for IgE (CD23). IL-4-induced IgE production was also potentiated by IL-6 but not by tumor necrosis factor-α and IL-β The possibility that the activity of IL-9 was mediated by IL-6 released from accessory cells was excluded by the observations that monocyte depletion did not abolish the effect of IL-9 and that IL-9 was still active on fluorescence assisted cell sorted CD20⁺ B lymphocytes co-cultured with irradiated murine EL4 cells. In addition, IL-9 was shown to potentiate the IL-4-induced IgG and IgM production by normal human B lymphocytes preactivated with Staphylococcus aureus Cowan strain. Taken together, these data suggest that IL-9 plays a regulatory role in the IL-4-dependent immunoglobulin production.     

Immature B cells in fetal development and immunodeficiency: studies of IgM, IgG, IgA and IgD production in vitro using Epstein-Barr virus activation

Epstein-Barr virus has been used as a B cell mitogen to explore the parallels between the B cells found in patients with hypogammaglobulinemia and the immature B cells in fetal tissues. Peripheral blood lymphocytes from 29 cases of late onset hypogammaglobulinemia (common variable immunodeficiency) and from 10 cases of X-linked hypogammaglobulinemia were depleted of T lymphocytes and stimulated with virus in vitro. Immunoglobulin production was measured over a 4-week culture period using inhibition radioimmunoassays for IgM, IgG, IgA and IgD. The results were compared with those seen with fetal liver cells, cord blood lymphocytes and adult lymphocytes. Virus-stimulated cells from fetal sources produced small amounts of IgG and IgA relative to IgM, the ratio of IgM to IgG in the second week being in all cases greater than 10. Similar patterns were seen in 25/29 cases of late onset hypogammaglobulinemia, and in the eight cases of X-linked hypogammaglobulinemia that responded in vitro. In contrast, the ratio of IgM to IgG was always less than 8 in cultures of normal adult peripheral blood or bone marrow lymphocytes, and also in cultures from four cases of hypogammaglobulinemia known independently to have abnormal circulating suppressor cells. Eight cases of selective IgA deficiency showed reduced IgA production; six of these showed a normal ratio of IgM to IgG production. Thus, the B lymphocytes which circulate in many patients with hypogammaglobulinemia are functionally immature.