Quantitation of Gm Allotypes

A method for quantitation of Gm allotypes is described. Alternative Gm allotypes of the three IgG subclasses, IgG1, IgG2 and IgG3, were investigated for the six most common Caucasian Gm phenotypes, Quantitation of Glm(a), Glm(f) of IgG1, G2m(n) of IgG2 and G3m(b) of IgG3 was performed with specific monoclonal antisera and purified myeloma proteins of different Gm allotypes. Mean±SD are given as percentage of a normal serum pool and in g/1 for the Gm allotypes Glm(a), Glm(f), G2m(n) and G3m(b), For homozygous individuals the G2m(",") values are equal to the IgG2 levels and the G3m(g,g) values equal to the IgG3 levels. For heterozygous individuals the value for G2m(") is calculated as IgG2 minus G2m(n) and for G3m(g) as IgG3 minus G3m(b), Homozygous individuals have about double the amounts of the Gm allotype compared with heterozygous individuals. The gene activity of heterozygous individuals is given by quotients, mean±SD for G1 m(a)/G1 m(f) of IgG 1, G2m(n)/G2m(") of IgG2 and G3m(b)/G3m(g) of IgG3 in different Gm phenotypes. Heterozygous individuals on all three IgG subclass loci have at least six different qualities of IgG molecules compared with three for homozygous individuals.     

Lack of the G2m(n) Allotype in IgG Subclass Deficiency, in IgG2 Deficiency Together with Lack of G1m(a) and G3m(g), and in IgG3 Deficiency Together with Lack of G1m(f) and G3m(b)

Lack of G2m(n) was demonstrated in both IgG2-deficient and IgG3-deficient Caucasian patients. Lack of G2m(n) or G2m(",") was found together with homozygosity for both G1m and G3m allotypes as the dominant finding, i.e. for IgG2-deficient patients together with G1m (f,f) and G3m(b,b), constituting the Gm(f,",b) phenotype, and for IgG3-deficient patients together with G1m(a,a) and G3m(g,g), constituting the Gm(a,",g) phenotype. The group with IgG2 deficiency and the selected patients with the Gm(f,",b) phenotype expressed characteristically very low or undetectable IgG4, significantly increased IgG3, and normal IgG1. The group with IgG3 deficiency and the selected patients with the phenotype Gm(a,",g) expressed instead normal IgG4 and nearly normal IgG2 and IgG1 levels. The lack of G2m(n) together with lack of one or the other of the alternative G1m genes and corresponding G3m genes give different IgG2 levels and different IgG subclass patterns. The frequency of G1m allotypes and corresponding G3m allotypes also deviated significantly when the IgG2 deficiency and IgG3 deficiency groups were compared with each other. Most IgG subclass-deficient patients are homozygous in the Gm system and lack genetic variants in the three IgG subclasses, IgG1, IgG2, and IgG3.     

Immunoglobulin allotypes and IgG subclass antibody response to Pseudomonas aeruginosa antigens in chronically infected cystic fibrosis patients.

Chronic Pseudomonas aeruginosa lung infection is the leading cause of death in patients with cystic fibrosis (CF). Poor prognosis correlates with a high number of anti-pseudomonas precipitins and with high levels of IgG2 and IgG3 anti-pseudomonas antibodies. Reports of several highly significant associations between certain Gm (genetic markers of IgG on human chromosome 14) and Km (k-type light chain determinants on chromosome 2) phenotypes and immune responsiveness to various antigens suggest that allotype-linked immune response genes do exist in man. Furthermore correlation between Gm types and IgG subclass levels has been reported. A group of 143 CF patients were investigated (31 non-infected and 112 chronic infected). The IgG subclass antibodies to three different P. aeruginosa antigens (P. aeruginosa standard antigen (St-Ag), alginate and LPS) were determined. Immunoglobulin allotypes were determined by haemagglutination inhibition. Samples were typed for G1m(1,2,3, and 17), G2m(23), G3m(5,21), and Km(1,3). Statistical analysis of our data demonstrate that IgG3 anti-pseudomonas antibody levels and Gm markers are related. IgG3 antibody levels to all investigated P. aeruginosa antigens are significantly higher in sera homozygous for Gm(3;5), somewhat lower in heterozygous sera, and significantly lower in sera homozygous for Gm(1,2,17;21). We suggest that genetic differences between the patients may explain the present differences in subclass patterns.     

Linkage of IgA deficiency to Gm allotypes; the influence of Gm allotypes on IgA-IgG subclass deficiency

IgA deficiency (IgAD) is the most common immunodeficiency, characterized by an arrest in B cell differentiation. It has a sporadic occurrence or variable inheritance pattern, and is also linked to the HLA genes. IgA deficiency is sometimes associated with IgG subclass deficiency. In this study the Gm allotypes, as genetic characteristics of the IgG1, IgG2 and IgG3, were analysed in 83 Caucasian IgAD individuals. Half of the patients presented with IgG4 < 0·01 g/l compared with 5% (P < 0·001) in a healthy population. Three of the 83 had significantly low IgG2 and four had significantly low IgG3 levels. Gm allotype frequencies in IgAD deviated compared with a normal population. Of the 83 patients, 44 (53%) showed homozygous G2m(“,”) expression on the IgG2 locus (33% in controls, P < 0·01). In IgAD the Gm(a, g) haplotype was more frequent (43%) compared with controls (31%, P < 0·01). The Gm homozygous phenotype Gm(a', g/a', g) was most common, found in 20 of 83 patients (24%, P < 0·05) compared with controls (14%). On the other hand the Gm(f,n,b) haplotype of IgAD was rare (28%) compared with controls (45%, P < 0·001). The low IgG4, < 0·01 g/l, found in 50% of the patients, was even more frequent (56–69%) among the G2m(“,”) phenotypes. IgG subclass levels were given for different Gm phenotypes of the IgAD group and compared with controls. Significantly low IgG4 was revealed in the Gm(a,“,g/a,”,g) phenotype (P < 0·01) and significantly low IgG2 in the Gm(a,“,g/f,”,b) phenotype (P < 0·01). The Gm(a,“,g/f,”,b) phenotype contained the three patients found with IgG2 levels < - 2 s.d., and the four patients with IgG3 levels < -2 s.d. were present among those with the homozygous Gm(a,“,g/a,”,g) phenotype; both phenotypes with G2m(“,”) on the IgG2 locus. The ‘compensatory’ increase of IgG was significant for both IgG1 and IgG3 in all Gm phenotypes, but in the Gm(a,“,g/f,”,b). Thus, the susceptibility of IgAD with the additional IgG antibody deficiencies, down-regulated IgG4 and IgG2/IgG3, is associated with Gm allotypes, especially the homozygous G2m(“,”) expression on the IgG2 locus.     

Serum Gm Allotype Development During Childhood

Gm allotypes are genetic variants of the immunoglobulin heavy G chains (IGHG) of IgG molecules, coded from chromosome 14q32, characterized by differences in amino acid epitopes of the constant heavy G chains and inherited in the Mendelian manner. Gm allotypes have influence on IgG subclass levels, and serum Gm allotype levels have been given for different Gm genotypes in adults. Four hundred and thirty healthy children, aged 1-15 years, were examined for serum Gm allotypes and IgG subclasses from the six most common Gm genotypes and different age groups were measured using competitive enzyme-linked immunosorbant assay and radial immunodiffusion methods. Quantities (in g/l) of G1m(a) and G1m(f) of IgG1, G2m(n) and G2m(-n) of IgG2 and G3m(g), and G3m(b) of IgG3 are given. Different maturation rates of the alternative Gm allotypes within IgG1, IgG2 and IgG3 were shown. G2m(n) development was strikingly retarded compared with G2m(-n) from the gamma2 locus. This was found comparing IgG2 levels from homozygous G2m(-n-n) and G2m(nn) individuals, but was also seen in heterozygous G2m(n-n) genotypes. From the gamma1 locus G1m(f) levels dominated significantly, but inconstantly, over G1m(a) levels in heterozygous G1m(af) individuals. In homozygous G1m genotypes, G1m(aa) compared with G1m(ff) of the same age, one or the other dominated, sometimes significantly. Serum levels of G3m(b) from the gamma3 locus of homozygous G3m(bb) individuals were increased significantly compared with G3m(g) levels of homozygous G3m(gg) individuals, in ages over 3 years. However, in heterozygous G3m(gb) individuals G3m(b) dominance was not evident. There is a relatively rapid development of G1m(f) molecules and a retarded development of G2m(n) in the Gm(f;n;b) haplotype. In comparison, G1m(a) is retarded and G2m(-n) is enhanced in the Gm(a;-n;g) haplotype. The retarded serum G2m(n) development is comparable with serum IgA development during childhood. Different maturation rates of Gm allotypes within the same IgG subclass provide further explanation for the variation of the antibody response during childhood. Quantitative Gm allotype determinations give information of the activity from IGHG genes. The genetic variation constitutes an additional basis for evaluation of IgG antibodies in different diseases in childhood.     

The Cγ3 Homology Region in Human IgG Subclasses and Allotypes

Amino acid composition, end-group analysis, and antigenic analysis have been performed on isolated pFc' fragments (Cγ3 homology region) from the major allotypic variants of human IgG. A comparison of the amino acid composition of Gm(a+x+) and Gm(a+x−) pFc' fragments showed less serine and more glycine in the Gm(a+x+) proteins. Two IgG2 pFc' fragments were shown to resemble IgG1 Gm(f) pFc' fragments in their amino acid compositions, except that there was more threonine and less alanine in the IgG2 fragments. This was also observed in two IgG3 Gm(g) proteins and may be the basis for the expression of a non b antigen. IgG3 fragments of both Gm(g) and Gm(b) allotypes showed six amino acid differences from the IgG1 subclass. There was more arginine, serine, and isoleucine and less histidine, lysine, and valine in both allotypes. In addition, analysis of a single Gm(b) pFc' fragment showed differences from the Gm(g) proteins; there was more phenylalanine, alanine, and proline and less tyrosine, threonine, and, possibly, aspartic acid.     

Immunoglobulin haplotypes of two population groups in Iran

Genetic markers of IgG and IgA were investigated in two population groups from Iran. The Gm-Am haplotypes found were mainly those prevalent in Caucasians, with a low frequency of Asiatic haplotypes. Twenty samples had phenotypes that led to the assumption of rare haplotypes. The main ones were: Gm(z;n;b)a2m(1) and Gm(za;n;g)A2m(2). The first haplotype differs from the common haplotypes because G1m(z) is present instead of G1m(f), and the second because it has G2m(n) and A2m(2) in combination with G1m(za) and G3m(g).     

Enzyme linked immunosorbent assay (ELISA) for human IgG (Gm) allotype determination

An inhibition enzyme-linked immunosorbent assay (inhibition-ELISA) was developed for the quantitative determination of human IgG (Gm) allotypes using rabbit anti-Gm antisera, alkaline-phosphatase-conjugated goat anti-rabbit IgG and, as the calibrant, purified human myeloma proteins possessing the relevant Gm allotype. The assay is reproducible and can detect as little as 10 ng/ml of G1m(a), G2m(n) or G3m(st), and 100 ng/ml of G1m(f) or G3m(g). Using this assay, the "gene dosage effect" and "allelic balance" in healthy Japanese were studied.     

Direct antiglobulin reactions in Gambian children with P. falciparum malaria. III. Expression of IgG subclass determinants and genetic markers and association with anaemia.

The IgG subclass and Gm allotype distribution of red cell-bound IgG molecules from Gambian children with past or present falciparum malaria has been determined. The results show that the antibody is polyclonal with some predominance of the IgG2 and IgG4 subclasses. A restriction towards IgG2 antibodies may indicate specificity for a schizont-derived antigen which is carbohydrate in nature. Not all the allotypes normally carried by the G3m(b) allele could be demonstrated on IgG3-sensitized cells, a finding which remains unexplained. Expression of G3m(10), (11) and (14) allotypes of the IgG3 molecule was noted. Sensitization of red cells with IgG1 molecules correlated with the presence of anaemia but red cells sensitized with IgG2, IgG3 or IgG4 usually came from children with haematological findings within the normal range for that population. The implications of the results are discussed with reference to the few reports on the subclass and Gm allotype of malaria-specific IgG.     

Imbalanced switch of the IGHG (immunoglobulin constant heavy G chain) Gm(bfn) genes in atopic childhood asthma

BACKGROUND: The IGHG genes on chromosome 14q32, 5'micron delta gamma3 gamma1alpha1 gamma2 gamma4 epsilon alpha2 3', as studied by Gm allotypes, are involved in the inheritance of atopy. The 5'micron delta b f alpha1 n gamma4 epsilon alpha2 3', Gm(bfn) haplotype of the genetic B1-cell variant has been found to be associated with the atopic phenotype of children with bronchial asthma. METHODS: An indirect competitive enzyme-linked immunosorbent assay for quantitation in serum of the alternative serum Gm allotypes from the gamma3-, gamma1-, and gamma2 loci and radial immunodiffusion for quantitation of IgG subclasses were used. Children with the genetic B1-cell variants B1/B1 (= Gm[bfn/bfn]), B1/B2 (=Gm[bfn/bf-n]), and B1/B4 (=Gm[bfn/ga-n]) and bronchial asthma were investigated and compared to healthy children of the same age and B-cell type. RESULTS: The three groups with B1/B1, B1/B2, and B1/B4 cells exhibited increased IgE. In both homozygous and heterozygous B1 or Gm(bfn), the serum G1m(f) levels from gamma1 loci were significantly downregulated to 75% of normal, while G2m(n) from gamma2 loci were significantly upregulated to about double the normal level. In heterozygous patients with additional B2 or B4 cells, the G2m(-n) levels from gamma2 loci were instead downregulated. G1m(a) from gamma1 of B4 cells was also downregulated. CONCLUSIONS: Children with atopic bronchial asthma demonstrated an imbalanced class switch in rearrangement of the genes for IgG. The activity of G1m(f) from the gamma1 locus was downregulated, but G2m(n) from gamma2 was upregulated together with the closely situated epsilon locus downstream of the IGH genes. Low levels of G1m(f), Glm(a), and G2m(-n) indicated a low pressure of infections. The imbalanced activation of the IGH genes in more hygienic environments might be one explanation of the increased prevalence of atopy in children in recent decades.     

The Importance of G1m and 2 Allotypes for the IgG2 Antibody Levels and Avidity Against Pneumococcal Polysaccharide Type 1 Within Mono- and Dizygotic Twin-Pairs

Eighty-two mono-or dizygotic Caucasian twins vaccinated with a 23-valent pneumococcal vaccine, who had previously had their IgG2 antibody levels to pneumococcus type 1 determined before and after vaccination, were included in this study. Their IgG2 antibody levels were related to their G1m and G2m allotypes/phenotypes and their Gm amounts.
Eight different Gm phenotypes were found and characteristically IgG2 antibody levels were related to them. G2m (n) homozygotic twins had significantly higher IgG2 levels than heterozygotic twins who had significantly higher levels than G2m (-n) homozygotic twins (P <0.05). The G1m allotype, on the other hand was without influence on the IgG2 levels and so were the Gm amounts among G2m (n) heterozygotic twins. The IgG2 antibody avidities were not related to Gm allotypes but significantly correlated to IgG2 levels (P = 0.05). Finally, a highly significant intra-pair correlation was found for avidity in the monozygotic twins supporting a genetic regulation of avidity (P <0.002).
These results may explain our earlier findings that IgG2 antibody levels after pneumococcal vaccination are significantly more closely correlated within mono-compared to dizygotic twins.     

Preparation of IgG subclass allotypes from polyclonal IgG

Two allotypes have been identified for each of the IgG subclasses IgG1, IgG2 and IgG3. These allotypes are referred to as G1m(a) and G1m(f), G2m(n) and G2m(-n), and G3m(g) and G3m(b). Using a pool of normal human serum and a combination of preparative electrophoresis, DEAE ion-exchange and protein A-Sepharose chromatography, it was possible to separate G1m(f) from G1m(a), G2m(-n) from G2m(n) and G3m(g) from G3m(b). Purification of G2m(-n) molecules is of special interest as no genetic marker has been found to identify this allotype.     

A SEARCH FOR ASSOCIATION BETWEEN IgD CONCENTRATIONS AND IMMUNOGLOBULIN ALLOTYPES IN 936 SERA FROM A GENETIC ISOLATE IN NEWFOUNDLAND

Immunoglobulin allotype (Gm) data has been analysed against immunoglobulin D (IgD) concentrations in a population study in Newfoundland. There was no significant difference between the distribution of IgD concentrations in people homozygous for the alleles Glm(f) and G3m(b) when compared with people homozygous for the alleles G1m(a) and G3m(g). These findings, involving 573 homozygous individuals as opposed to ninety-eight in an earlier study on a New York population, do not confirm the earlier findings. Thus a genetic influence on IgD concentration by Gm genes or genes closely linked to them is not universally demonstrable by typing for these four markers and by using the Mancini technique for measuring IgD concentration.     

Correlation between atopy and Gm allotypes

In 50 consecutive atopic Caucasian patients with increased IgE greater than 600 kU/l, the phenotypic Gm allotype constellation deviated from that to be expected, with significantly increased frequency of patients with the phenotype Gm(f,n,b). There was an increased frequency of the G2m(n) allotype, more frequent in patients with IgE greater than 1,000 kU/l, and in patients with IgG4 greater than 1 g/l. In patients with IgE greater than 1,000 kU/l the phenotype Gm(a,f,n,b) was significantly increased and in patients with IgG4 greater than 1 g/l the phenotype Gm(f,n,b) was significantly increased. Those atopic patients with increased IgE and increased IgG4, according to earlier studies known to have the most severe forms of the disease, were thus mainly found to have the m(f,n,b) phenotype.     

Assay restriction profiles of three monoclonal antibodies recognizing the G3m(u) allotype. Development of an allotype specific assay

Three monoclonal antibodies raised against a purified human IgG3 paraprotein were found to exhibit a restriction profile for IgG3/G3m(u) and pan-IgG specificity which was dependent on the assay system. When adapted to an IgG3 subclass capture ELISA, all three McAbs discriminated between paraproteins expressing G3m(u) and antithetical markers G3m(st). One of the antibodies (PNF69C) was selected and conditions were optimised for Gm typing purposes. Using this system G3m(u) could be detected on captured IgG3 derived from human sera. This system may prove useful in the elucidation of Gm allotype profiles.     

Quantification of IgG subclasses in sera of normal adults and healthy children between 4 and 12 years of age.

The concentration of the four subclasses of IgG was determined in sera of normal adults and healthy children between 4 and 12 years of age, using the radial immunodiffusion technique. A relation between the concentration of IgG subclasses and Gm type was studied in adults. No influence of Gm type on IgG1 concentration could be shown, except that the group of Gm(fb) individuals had a higher level than the others. The mean concentration of IgG2 was higher in sera positive for Gm(n) than in those lacking this genetic marker. High IgG3 concentrations corresponded to the presence of Gm(b). No clearcut evidence was obtained for a relation between IgG4 concentration and Gm factors, although in general Gm(n) positive individuals had higher and Gm (zag) positive individuals lower concentrations of this subclass in their serum. Quantification of IgG subclasses in sera from healthy children of different ages revealed that the amount of IgG2 rises slowly with age, having not yet reached the adult level at the age of 12 years. This also holds for IgG4, although in a lesser degree. No significant differences from the adult level were found for the concentrations of IgG1 and IgG3.     

Monoclonal antibodies against IgG allotypes G1m(z), G1m(a), G1m(f), G3m(b1/u) and G3m(g1): their usefulness in HAI and capture ELISA

Monoclonal antibodies (McAbs) were produced against the IgG allotypes G1m(z), G1m(a), G1m(f), G3m(b1/u) and G3m(g1). Four out of the six McAbs described in this paper showed in the haemagglutination assay cross-reactivity with some or all IgG-coated cell samples. In the haemagglutination inhibition assay, all six McAbs are useful as typing reagent for the above allotypes. In this assay, two of the McAbs show two different specificities, which depend on the Ig-coated cell sample used. Five McAbs are useful for allotyping in a capture ELISA. The results with four of these are promising for the development of a quantitative determination of Gm allotypes.     

Qualitative and quantitative studies on IgG2 globulins in individual human sera with an antiserum capable of differentiating between Gm(n+) and Gm(n-) proteins

A monkey antiserum was obtained by immunization with Gm(n+) IgG2 proteins. After it had been rendered specific for the IgG2 subclass, it was shown to contain antibodies of two specificities, i.e. anti-γ2 and anti-Gm(n). Precipitation and haemagglutination experiments, using unabsorbed antiserum and antiserum absorbed with Gm(n+) or Gm(n−) proteins, made it clear that the anti-Gm(n) was unable to form precipitates with Gm(n+) proteins without the assistance of anti-γ2 antibodies. Results obtained in radial immunodiffusion with serum samples of known Gm genotype showed that the antiserum differentiates three types of individuals: homozygous Gm(n− /n−), heterozygous Gm(n+ /n−) and homozygous Gm(n+ /n+).The amount of IgG2 present in serum samples from seventy-four individuals of variable Gm types was determined by comparison with a standard serum. It was found that the mean level of female sera (4·2 mg/ml) was higher than that of male sera (3·3 mg/ml). It was further shown that the IgG2 level was correlated with Gm(n). The total IgG2 content of individuals possessing the Gm(n) marker was higher than that of individuals of otherwise identical Gm type but lacking this factor. In individuals heterozygous for Gm(n) the level of Gm(n+) protein was significantly higher than that of Gm(n−) protein.     

Quantitative and qualitative investigations of serum IgG subclasses in immunodeficiency diseases.

Determinations of IgG subclasses were made by electroimmunoassay and crossed immunoelectrophoresis, and Gm markers were typed in sera from seventeen patients with well-defined immunodeficiency diseases. Certain IgG subclass and Gm patterns were recognized in various diseases: IgG2 deficiency and homozygosity of Gm (4,5) in the cartilage-hair-hypoplasia syndrome, in the ataxia telangiectasia syndrome and in selective IgG subclass deficiency; and IgG3 deficiency and homozygosity of Gm(1,-5) in the Wiskott-Aldrich syndrome. The findings suggest a common structural or regulator gene defect in some immunodeficiency diseases. In IgA deficiencies, the levels of IgG1 were raised. In patients with IgG subclass deficiencies there was sometimes a compensatory increase of the remaining IgG subclasses, with a preponderance of IgG1 and IgG3. The increased Ig1 showed restricted heterogeneity with only an increase of the electrophoretically cathodal part. This part contained both kappa and lambda chaings. IgG subclass deficiency indicates treatment with gammaglobulin even if the serum levels of IgG are normal or increased.     

The Reaction of Rheumatoid Anti-Gm Antibodies with Native and Aggregated Gm-Negative IgG

Rheumatoid anti-Gm(a) antibodies were shown to react with native Gm(a-) IgG. This reaction takes place with the C_H3 domain (pFc' fragment) of IgG and is not due to anti-isotype specificities contaminating the test system. The reactivity of the Gm(a-) IgG is greatly increased by heat aggregation, and this increase is clearly related to the amount of aggregates formed. Rheumatoid anti-Gm(a) reacts with aggregated IgG1. IgG2. and IgG3 Gm(g+) proteins but not with IgG3 Gm(b+) or IgG4 proteins. Thus there is a clear specificity in the reactions with aggregated IgG of different subclasses. Quantitative hemagglutination inhibition studies in the AutoAnalyzer suggested that the antigenic determinants of native and aggregated Gm(a+) or Gm(a-) IgG involved in the reaction with rheumatoid anti-Gm(a) were very similar. Furthermore, immunosorbent studies showed that the saint population of rheumatoid anti-Gm(a) antibodies is reacting with the native and aggregated Gm(a+) and Gm(a-) IgG Analogous findings were obtained using rheumatoid anti-Gm(b¹) and anti-Gm(g).