The significance of blood levels of IgM,IgA, IgG and IgG subclasses in Sudanese visceral leishmaniasis patients

We developed an ELISA test using leishmania antigenic extracts to detect antigen-specific antibody responses, including subclass and isotype analysis, in visceral leishmaniasis (VL) patients from the Sudan. A total of 92 parasitologically proven patients were compared with cutaneous leishmaniasis, schistosomiasis, malaria, onchocerciasis and tuberculosis patients, as well as with healthy endemic and non-endemic controls. Some VL patients were examined before and after chemotherapy. VL patients showed significantly higher IgG responses compared with all other groups (93·4% sensitivity, 93·7% specificity), and higher (but not significantly) IgM responses. All groups showed low IgA levels. All groups showed low IgA levels. All IgG subclasses, IgG1, 2, 3, and 4, showed higher levels in patients than all other groups, with IgG1 and IgG3 levels being significantly reduced following treatment. The rank order for specificity and sensitivity for IgG subclasses was IgG3 > IgG I> IgG2> IgG4.     

Distribution of IgG subclasses in antimonial unresponsive Indian kala-azar patients

Sodium antimony gluconate (SAG) is the mainstay of treatment for visceral leishmaniasis (VL) or kala-azar. In view of the increasing incidence of refractoriness to SAG in India, we compared the levels of parasite-specific IgG and IgG subclasses in 20 longitudinally followed up kala-azar patients. In both SAG-responsive (n = 10) and unresponsive patients (n = 10), the levels of total IgG, IgG1, IgG2, IgG3 and IgG4 were increased, the rank order being IgG1 > IgG2 > IgG3 = IgG4. Following treatment, a significant decrease in total IgG and the four subclasses occurred in the SAG-responsive group, whereas in the SAG-unresponsive group these levels were unchanged or slightly increased. Therefore, monitoring of IgG1 and IgG2 levels in Indian kala-azar patients is a good serologic alternative to monitoring the disease status.     

Allotype-associated differences in concentrations of human IgG subclasses

The concentrations of seven immunoglobulin isotypes (IgA, IgE, IgM, IgG1, IgG2, IgG3, and IgG4) were measured in the sera of 207 Finnish blood donors, and they were allotyped with anti-Gm antibodies: anti-f, anti-a, anti-x, and anti-n. The above population could be divided into 12 phenotypes, and significant differences in isotype concentrations between different phenotypes were observed. They are best explained by postulating that the following alleles of different loci are associated with a high concentration of the product of the locus: a(x)-IgG1, n-IgG2, b-IgG3, and perhaps 4b-IgG4. The following concentration differences between the low and the high homozygotes were found: IgG1, 1.2-fold; IgG2, 1.5-fold; and IgG3, 2.6-fold. No significant allotype-associated differences in the concentrations of IgA, IgM, or IgE could be detected.     

Chemokines in onchocerciasis patients after a single dose of ivermectin

Ivermectin treatment will effectively diminish microfilariae (Mf) of Onchocerca volvulus in the skin of patients, but therapy is associated with adverse host inflammatory responses. To investigate the association of proinflammatory chemokines with the intensity of infection and clinical adverse reactions, chemokine serum levels were measured in patients following ivermectin treatment (100 microg/kg, 150 microg/kg or 200 microg/kg) or placebo. The density of O. volvulus Mf per mg skin decreased by 85%, 97%, 97% and 90% at day 3, at month 3, month 6 and at 1 year post-ivermectin. The cutaneous T cell-attracting chemokine (CTACK/CCL27) was found highly elevated in onchocerciasis patients compared to infection-free European controls (P = 0.0004) and it did not change following ivermectin or placebo to 1 year post-therapy. The chemokine RANTES/CCL5 (regulated on activated and normally T cell-expressed) was similarly high in onchocerciasis patients and infection-free European controls; the RANTES/CCL5 levels did not change following treatment until 6 months post-therapy but were slightly elevated at 1 year post-therapy (P < 0.02). In contrast, the Th2-type chemoattractants, thymus and activation regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), were activated at 3 days post-ivermectin (P < 0.0001) to return to pretreatment or lower levels thereafter. The Th1-type chemoattractants, macrophage inflammatory protein (MIP)-1alpha/CCL3 and MIP-1beta/CCL4 were low before ivermectin treatment, but following clearance of microfilariae of O. volvulus their levels increased from 6 months post-therapy onwards (for both at 12 months post-therapy, P < 0.0001). The adverse reaction scores (RS) in treated patients increased significantly on day 3 (P < 0.02) while it remained unchanged in those who received placebo (P = 0.22); RS interacted with the microfilarial density (P = 0.01), but not with the dose of ivermectin or with the serum levels of MIP-1alpha/CCL3, MIP-1beta/CCL4, TARC/CCL17, MDC/CCL22 and CTACK/CCL27. Our observations suggest that following ivermectin, macrophages as well as memory Th2-type lymphocytes and B cells, attracted and activated by MDC/CCL22, TARC/CCL17 and CTACK/CCL27, may contribute to dermal immune responses and O. volvulus Mf killing and clearance. The transient changes of TARC/CCL17 and MDC/CCL22 were not associated with clinical adverse responses, and the later rise of MIP-1alpha/CCL3 and MIP-1beta/CCL4 showed a reactivation of Type 1 immune responses associated with persistent low levels of O. volvulus microfilariae and an expiring O. volvulus infection.     

Chromatographic fractionation of rhesus monkey (Macaca mulatta) IgG subclasses using DEAE cellulose and protein A-Sepharose

Rhesus monkey serum was applied to a protein A-Sepharose column at pH 7.3 IgG was not detectable in the unbound effluent. Bound protein was eluted with a pH gradient of citrate-phosphate buffer. Two major elution peaks reproducibly resulted at pH 4.85–4.9 and pH 4.35–4.43, respectively, with only slight variability among individuals. Since the immunoelectrophoretic mobility of IgG in the peak at pH 4.85–4.9 was slower than that of the peak at pH 4.35–4.43, serum was first fractionated by ion exchange chromatography into 2 fractions: the first did not bind to DEAE cellulose in 0.01 M phosphate buffer pH 7.8, the second did bind and was eluted by an NaCl gradient. Each DEAE fraction was then further fractionated on protein A-Sepharose. IgG in the first DEAE fraction bound to protein A at pH 7.3 and eluted in a single peak at pH 4.72–5.0. IgG in the second DEAE fraction also bound to protein A, but eluted in 2 peaks at pH 4.65–5.1, and 4.25–4.6, respectively. All 3 IgG fractions eluted in the same position from an A5m column, had immunodiffusion reactions of identity with anti-human γ chain and were composed of similar heavy and light chains judged by SDS polyacrylamide gel electrophoresis. However, IgG eluting from protein A at the low pH (4.25–4.6) had a reaction of partial identity with other IgG fractions when tested by immunodiffusion against rabbit anti-rhesus γ chain, suggestive of an antigenically different subclass. Analysis of rabbit antisera prepared against the 3 IgG fractions confirmed the occurrence of at least 3 antigenically distinct rhesus monkey IgG subclasses. These 3 subclasses have been provisionally designated IgG I, IgG II and IgG III.     

The functional affinities of antibodies of different IgG subclasses to dietary antigens in mothers and their babies

The quantity and functional affinities of IgG1 and IgG4 antibodies to the dietary antigens casein and ovalbumin were measured in unselccted mothers and their 1-year-old infants. In these infants, the titre of IgG antibodies to both antigens was highest in the IgG1 subclass, while in their mothers the litre of IgG1 and IgG4 antibodies to these foods was similar. High affinity IgG4 responses to both casein and ovalbumin were frequently found in mothers, whilst IgG1 responses, particularly to ovalbumin. were of 1ow functional affinity. By contrast, in the 1-year-old infants, the functional affinity of IgG1 antibody to ovalbumin was substantially higher than in their mothers (Student's paired t-test, P < 0.001). indicating that higher affinity IgG1 antibody was produced on first exposure to ovalbumin rather than following chronic exposure. The effect of antigenic load on affinity maturation was further investigated by comparing the affinity of IgG1 antibody to casein in bottle, mixed and breast fed infants. Bottle fed infants had significantly higher-affinity IgG1 antibodies to casein compared with breast or mixed fed infants (Student's unpaired t-test. P < 0.01 and 0.02), suggesting that antigen exposure via the gut was able to drive the affinity maturation process. In studying the immune response it is clear that account must be taken of the affinity as well as of the litre of the antibody produced.     

尿IgG亚类的分离纯化和鉴定

目的： 建立一种简便快捷的分离纯化肾病患者尿IgG亚类的方法。 方法： 收集的肾病患者24h尿液经硫酸铵盐析、DEAE-cellulose 离子交换层析和葡萄球菌A蛋白（SPA ）亲和层析等3步分离纯化，即可得到IgG₁、IgG₂、IgG₃、IgG₄ 4种亚类，最后用Western blotting和SDS－PAGE 电泳的方法鉴定样品及其纯度。 结果： SPA亲和层析洗脱曲线显示有4个独立的洗脱峰， Western blotting和SDS－PAGE方法鉴定表明提纯的样品为高纯度的IgG₁、IgG₂、IgG₃、IgG₄。 结论： 本方法通过3步即可一次性将样品中的4种不同的IgG亚类全部分离纯化，且纯度高，简便快捷，效果良好。     

Predominance of IgG1 and IgG4 subclasses of anti-neutrophil cytoplasmic autoantibodies (ANCA) in patients with Wegener's granulomatosis and clinically related disorders.

In view of the supposed hypersensitivity, the elevated levels of IgE, and the occurrence of eosinophilia reported in Wegener's granulomatosis and related conditions, we studied the IgG subclass distribution of ANCA directed against a 29-kD serine protease and myeloperoxidase (MPO) in 41 untreated ANCA-positive patients with several forms of active vasculitis and/or glomerulonephritis. We found that both 29-kD ANCA and MPO ANCA were predominantly of the IgG1 and IgG4 subclass in all groups of patients. The additional presence of IgG3 subclass was associated with renal involvement. We compared the subclass distribution of ANCA with that of total IgG subclass levels, and with the IgG subclass distribution of antibodies to cytomegalovirus (CMV) as a persistent endogenous antigen and antibodies to tetanus toxoid (TT) as an exogenous recall antigen. Total levels of IgG4 were elevated in the majority of the patients together with elevated IgG1 levels. Antibodies to CMV and TT, however, had the same subclass distribution as found in normals and did not show enhanced IgG4 expression. ANCA belong predominantly to the IgG1 and IgG4 subclass, which may suggest that the production of ANCA is related to recurrent exposition to the antigen(s) involved, possibly as part of a hypersensitivity reaction.     

IgG subclasses in human chronic schistosomiasis: over-production of schistosome-specific and non-specific IgG4

IgG subclasses were determined quantitatively in sera from 63 Egyptian men who were infected with Schistosoma mansoni. Total and antigen-specific IgG was measured pre- and post-treatment. Total IgG subclass antibodies were determined by immunoradiometric assay (IRMA) using monoclonal antibodies (MoAbs). The anti-worm and anti-egg specific S. mansoni IgG subclass antibodies were quantitatively measured by ELISA using specific MoAbs and standards obtained by affinity chromatography. Our data show that total IgG of the patients was elevated in the range of two to three times above normal. The magnitude of increase differed markedly among the four subclasses of IgG. The IgG1, IgG2 and IgG3 concentrations were approximately two to four times higher than normal, whereas the IgG4 concentrations was 20 times normal (9000 mg/l). IgG1 and IgG4 tended to dominate the IgG subclass distribution of anti-soluble worm antigen preparation (SWAP) antibodies followed by IgG2 and IgG3. On the other hand, IgG1 and IgG2 dominated the IgG subclass distribution of anti-soluble egg antigen (SEA) antibodies. As with IgG1, IgG2 and IgG3, most IgG4 was non-specific. The role of IgG subclasses in the pathogenesis of schistosomiasis is not clear. However, the high concentration of IgG4 might act as IgE blocking antibody, possibly as anti-idiotypes that may play a role in down-regulation of the immune system when it is challenged with an excess of antigen.     

The significance of IgG subclasses and mannan-binding lectin (MBL) for susceptibility to infection in apparently healthy adults with IgA deficiency.

The aim of this study was to investigate the significance of IgG subclasses and MBL for susceptibility to infection in association with IgA deficiency. The study population consisted of 139 apparently healthy adult blood donors with IgA deficiency and normal serum levels of IgG and IgM, and an increased susceptibility to infection demonstrated at a population level. Additionally, 216 controls matched for age and sex were investigated. IgG4 deficiency was more common and the mean level of IgG4 lower in persons with IgA deficiency than in the controls. No significant associations could be demonstrated between overt IgG subclass deficiencies and increased susceptibility to infection. However, when the mean concentrations of IgG subclasses were analysed with regard to medical history, that of IgG1 was lower in persons who reported recurrent viral respiratory infections, that of IgG3 in persons who had episodes of severe infection in their history, and that of IgG4 in persons who had recurrent mild respiratory infections, compared with those who had no particular history of infections. In contrast, MBL deficiency-alone or combined with that of the IgG subclass-was not associated with increased susceptibility to infection in persons with IgA deficiency. The results indicate that the proneness to infections observed in a population of otherwise healthy persons with IgA deficiency can only for a small part be accounted for by concomitant deficiencies of IgG subclasses. Contrary to expectations, no synergism between the deficiencies of IgA and MBL could be demonstrated.     

Preparation of F(ab')2 fragments from mouse IgG of various subclasses

We have investigated the effects of peptic digestion on mouse monoclonal immunoglobulins of each subclass of IgG. F(ab')2 fragments were obtained in good yield from IgG1, IgG2a and IgG3 proteins. The relative rates of digestion were IgG3 greater than IgG2a greater than IgG1. Variations in rate of digestion were noted for individual monoclonal IgG3 proteins. Five different IgG2b proteins were degraded very rapidly without the detectable formation of antigen-binding F(ab')2 fragments. One IgG1 protein out of four tested was also rapidly degraded.     

Fine specificity and subclasses of IgG anti-actin autoantibodies differ in health and disease

Current opinions suggest that autoantibodies occurring in autoimmune diseases are generated by B-cells which primarily produce polyspecific natural autoantibodies, through either polyclonal activation or specific antigen selection of these B-cells. In this study, we compared the immunological properties (polyspecificity, fine specificity and IgG subclasses) between natural anti-actin antibodies (N-AAA) and disease-associated AAA (D-AAA). IgG AAA from sera of healthy donors, patients with autoimmune hepatitis type 1 (AIH-1) and patients with primary biliary cirrhosis (PBC) were affinity-purified on actin immunoadsorbent and tested initially for polyspecificity against various cytoskeleton proteins by enzyme-linked immunosorbent assay (ELISA). Fine specificity was studied by Western blotting using proteolytic peptides of actin and by ELISA using synthetic 12 mer peptides, spanning the 221-377 aa sequence of actin. Results showed that both N-AAA and D-AAA are polyspecific. Nevertheless, D-AAA from both diseases showed a specific reactivity pattern as compared to N-AAA, against the 16 kDa C-terminal (229-377 aa) proteolytic peptide of actin and more specifically against the P36 synthetic peptide (351-362 aa). Quantitation of AAA IgG subclasses revealed that IgG1 and IgG3 were specifically increased in D-AAA from AIH-1 and PBC, respectively, as compared to N-AAA. We conclude that D-AAA are differentiated from N-AAA in terms of fine specificity and IgG subclasses, probably through specific antigen selection of B-cells primarily producing N-AAA.     

The predominance of IgG1 and IgG3 subclass antisperm antibodies in infertile patients with serum antisperm antibodies.

Mouse monoclonal antibodies (MAb) specific for each of the four human IgG subclasses and immunofluorescence flow cytometry were used to evaluate the subclass of the IgG antibody response to sperm in serum samples from 13 men and 6 women with a high titer (greater than 1:15,625) of IgG antisperm antibodies (ASA] determined by an indirect immunobead test. Five sera without ASA were also studied as a control. All 19 (100%) of the ASA-positive sera contained immunoglobulin (Ig)G ASA of the IgG1 and IgG3 subclasses. A 1:1 correlation was observed between the presence of IgG1 and IgG3 ASA. IgG2 was essentially undetectable, while IgG4 reactivity, although less intense than IgG1 and IgG3, was more prominent in the sera from the five vasectomized men. The ability of the IgG1 and IgG3 ASA-positive sera to deposit complement (C) on sperm was demonstrated by the concomitant binding to antibody-laden sperm of polyclonal antibodies to the membrane attack complex (C5b-9) of C. Both C-fixing and non-C-fixing ASA-positive sera were found to possess IgG1 and IgG3 antisperm antibodies. The predominance of IgG1 and IgG3 subclasses suggested a T-cell dependent immune response to sperm antigens.     

Evaluation of Onchocerca volvulus-specific IgG4 subclass serology as an index of onchocerciasis transmission potential of three Gabonese villages

The major objective of this study was to evaluate the usefulness of IgG4 ELISA and Western blot analysis, using a crude extract of Onchocerca volvulus adult worms as antigens, for diagnosing onchocerciasis in a Gabonese paediatric population with mixed filarial infections. The subjects had loaisis, streptocercosis or mansonellosis in addition to onchocerciasis. Control sera from loaisis or mansonellosis subjects residing outside the endemic zone were used to provide the cut-off point for positive results. The IgG4 ELISA had a specificity of 96% but a lower sensitivity of 78·7%. It detected 25 onchocerciasis cases out of 65 individuals who were negative on parasitological examination. Furthermore, the ELISA provided a more accurate picture of onchocerciasis transmission in a village with very low skin microfilartal load. A 27·5-kD antigen was identified on Western blots as a marker of onchocerciasis. The paediatric population provided a reliable window for assessing the parasitologic and serologic parameters in the three villages with disparate levels of onchocerciasis transmission.     

IgG isotype and isotype specificity of murine monoclonal IgG rheumatoid factors

Immune complexes of lipopolysaccharide (LPS) with homologous IgG antibody induces rheumatoid factor (RF) predominantly of the IgG class in normal mice, while LPS alone induces mostly IgM RF directed to homologous IgG1. In this study, IgG monoclonal RFs (mRF) were prepared from hybridomas derived from spleen cells of BALB/c mice which were immunized with complexes of TNP-LPS with anti-TNP mouse IgG and their specificity to mouse IgG subclasses was assessed by analysing dissociation kinetics of the ligands due to RF-specific and non-specific interactions. Of the 19 IgG mRFs (11 IgG1, five IgG2a, one IgG2b and two IgG3 types) tested, 14 were directed to either IgG3 or IgG2b or both, while only one exhibited a significant binding capacity to IgG1. Other mRFs, although reactive to rabbit IgG, exhibited little homophilic activity. None of these mRFs reacted strongly with their own isotypes. The results suggest that the IgG RF producing cells are not direct progenies of the IgG1-directed IgM RF-producing cells but may have developed via a rigorous selection process to eliminate clones that produce self-reactive RF.     

Anti-neutrophil cytoplasm antibody IgG subclasses in Wegener's granulomatosis: a possible pathogenic role for the IgG4 subclass

A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.     

IgG subclass distribution of thyroglobulin antibodies in patients with thyroid disease

The IgG subclass distribution of thyroglobulin antibodies (TgAb) has been studied in Hashimoto and Graves’ patients by several investigators with conflicting results, in part explainable by methodological problems. We have recently developed a quantitative ELISA to measure in absolute terms the serum concentration of TgAb subclasses. The aim of the present study was to apply this method in a large series of patients with autoimmune as well as, for the first time, non-autoimmune thyroid diseases. We examined 28 patients with Hashimoto's thyroiditis, 30 with Graves’ disease, 21 with thyroid carcinoma and 18 with non-toxic goitre, all selected for the presence of TgAbs. The results indicated that TgAbs in thyroid diseases were not restricted to any particular isotype, but comprised all four IgG subclasses. IgG1 was represented similarly in the four groups. The same was true for IgG3, even though its contribution to the total antibody content was very small. IgG4 was the dominant subclass in patients with Graves’ disease, thyroid carcinoma and non-toxic goitre, probably reflecting a prolonged antigenic challenge. In Hashimoto's thyroiditis IgG2 was dominant, possibly because T helper lymphocytes infiltrating the thyroid are typically Th1 type.     

Determination of IgG subclasses of immunoglobulin preparations for intravenous injection and the problems involved

The IgG subclasses of immunoglobulin for intravenous injection (IVIg) were investigated after various different types of treatment. The level of IgG2 was relatively low in preparations treated with pepsin, but by changing the clone producing anti-IgG2 antibody, the level approached that of normal human serum. Though undetectable in sulfonated immunoglobulin preparations, IgG3 was detected after reoxidation. When determining the IgG subclasses of IVIg, it must be kept in mind that the apparent value may subsequently become lower due to the depressed reaction of the antibody.