One allele [G2m(n)] of the human IgG2 locus is more productive than the other

Earlier investigators have shown that a high serum concentration of IgG2 and a high IgG2 antibody response to several polysaccharide antigens is associated with the allotype G2m(n) of the IgG2 subclass. We now show that the total IgG2 concentrations are significantly higher in G2m(n) than in G2m(-n) homozygotes (difference of means was 1.26-fold, p = 0.002). Furthermore, in G2m(n) heterozygotes, the G2m(n) allele contributed more than the G2m(-n) allele to specific IgG2 antibody responses and to the total IgG2.     

Rapid immunoassays for the measurement of immunoglobulin G subclass concentration in immunoglobulin preparations and human serum.

Enzyme immunoassays (EIA) capable of determining total IgG1, IgG2, IgG3 and IgG4 subclass concentrations in human serum preparations have been developed. Subclass-specific monoclonal antibodies (mAbs) are bound to polyacrylamide bead-conjugated anti-mouse immunoglobulin antibodies. Bound immunoglobulins are detected with a peroxidase-conjugated anti-IgG antibody or a biotin-conjugated anti-IgG antibody followed by peroxidase streptavidin. The standard curves were found to be linear in the regions 16.0-2.0 micrograms/ml for IgG1, 4.0-0.5 micrograms/ml for IgG2, 0.4-0.06 micrograms/ml for IgG3 and 0.25-0.05 micrograms/ml for IgG4. Coefficient of variation (CV) values range from 0.32-7.32% for IgG1, 0.66-4.85% for IgG2, 1.62-6.85% for IgG3 and 0.05-6.47% for IgG4 standard curves. The inter-assay variability for the control human serum samples was 9.6% for IgG1, 6.7% for IgG2, 9.5% for IgG3 and 6.8% for IgG4.     

Monoclonal antibody-based immunoenzymetric assays for quantification of human IgG and its four subclasses

A panel of 5 immunoenzymetric assays (IEMAs) has been developed for quantification of the total and four subclasses of immunoglobulin G (IgG) in human serum using IUIS/WHO documented monoclonal antibodies (MoAb). Human IgG specific MoAb was adsorbed to microtiter plates and used to capture IgG from serum. Peroxidase conjugated forms of polyclonal mouse anti-human IgG Fc or a mixture of 4 MoAb (anti-kappa, anti-lambda, anti-IgG Fc PAN and anti-IgG Fd PAN) were used as detection antibodies. Use of monoclonal antibody in chromatographically purified form was required for acceptable assay sensitivity (S) and working ranges (WR). All 5 IEMAs displayed good precision (intra-assay %CV less than 5%, inter-assay %CV less than 12%) and parallelism (inter-dilutional CV less than 20%). Both HP6069 or HP6070 (anti-IgG1 Fc) worked well alone or together as capture antibodies in the IgG1 IEMA: WR = 20-1250 ng/ml, S = 15 ng/ml. HP6002 (anti-gG2 Fc) alone or in combination with HP6014 (anti-IgG2 Fd) produced an IgG2 IEMA with a WR of 5-200 ng/ml and S of 5 ng/ml. HP6047 (anti-IgG3 hinge) alone generated a sensitive IgG3 IEMA with a narrow working range: WR = 2-50 ng/ml, S = 1.6 ng/ml. Both HP6025 and HP6023 (anti-IgG4 Fc) worked equally well alone and together to produce a useful IgG4 IEMA: WR = 8-250 ng/ml, S = 7.8 ng/ml. HP6017 (anti-IgG PAN Fc) was combined in an equal molar ratio with HP6046 (anti-IgG PAN Fd) to produce a total IgG PAN IEMA with a WR of 5-530 ng/ml and a sensitivity of 5 ng/ml. All 5 IEMAs fulfilled requirements for robust clinical immunoassays that permit the quantitation of human IgG and its 4 subclasses.     

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 measurement of IgG anti-IgG antibody in rheumatoid arthritis

Serum IgG anti-IgG antibody was measured in patients with rheumatoid arthritis and osteo-arthritis and in healthy young adults. The antibody was isolated by immunoadsorption and evaluated by titration with latex particles. After establishing the method's specificity for IgG anti-IgG, its reproducibility and a means for converting titer to IgG concentration, IgG anti-IgG was found in greatest amounts in rheumatoid arthritis when compared with two other groups. The concentration of IgG anti-IgG, however, was consistently lower in our study than in studies by other workers. This discrepancy appears to be related to our procedure, namely dilution of serum prior to exposure to immunoadsorption, the latter tending to dissociate immune complexes (removal of IgG antigen from IgG anti-IgG antibody). The eluate obtained in this manner contains antibody IgG freed of IgG antigen, as manifested by mainly 7 S IgG, rather than 7 S IgG admixed with complexes of higher sedimentation coefficients. The method reported in this article, therefore, appears to measure IgG anti-IgG antibody with a greater degree of accuracy than previously reported techniques.     

Demonstration of Electrophoretic Heterogeneity of Serum β2-Microglobulin in Systemic Lupus Erythematosus and Rheumatoid Arthritis: Evidence against Autoantibodies to β2-Microglobulin

A sensitive crossed radioimmunoelectrophoretic method (CRIE), originally developed to study lymphocyte-associated beta 2-microglobulin (beta 2m), was applied in the study of serum beta 2m in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). In six of seven patients with SLE and nineteen of twenty-seven patients with RA a considerable electrophoretic heterogeneity of serum beta 2m was found. In addition to the normally seen symmetric beta 2m precipitate, a beta 2m precipitate exhibiting complete immunochemical identity was found in the alpha-electrophoretic region. Binding of isolated 125I-labelled beta 2m to the abnormal precipitate was demonstrated in crossed immunoelectrophoresis. After gel filtration of sera exhibiting the above-mentioned beta 2m binding, all beta 2m was eluted in low molecular weight fractions corresponding to free beta 2m. By application of appropriate antisera and a glycoprotein-binding lectin in intermediate gels in CRIE, it was shown that the possible beta 2m-binding ligand is not an antibody, not a major constituent of normal human serum, and not unmodified HLA alloantigen. The abnormality was not restricted to patients with high disease activity but was found more frequently and was more pronounced (mean score 1.6 arbitrary units against 0.57 arbitrary units, P less than 0.01) in such patients. Thus our data exclude the possibility that autoantibodies to beta 2m were present in serum from patients with SLE and RA.     

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.     

Presence of IgG4 on the membrane of human basophils. Histamine release is induced by monoclonal antibodies directed against the Fab but not the Fc region of the IgG4 molecule

We have studied the possible role of human IgG4 as an anaphylactic antibody. For that purpose, we have determined the induction of histamine release (HR) from human basophils by anti-IgE and anti-IgG4 monoclonal antibodies (MoAbs) recognizing different epitopes located at the Fc and Fab regions of the IgG4 molecule. The results show that anti-IgG4 (Fab) MoAb was able to induce HR in 93% of donors tested, with no differences between atopics and non-atopics. That HR is calcium dependent and is accompanied by the synthesis and release of leukotriene C4. In contrast, no HR could be induced by anti-IgG4(Fc) MoAbs in any individual, even in the presence of D2O or after a second challenge with a polyclonal goat anti-mouse IgG antibody. The results obtained suggest the presence of IgG4 on the basophil membrane and that the epitope recognized by the anti-IgG4 (Fc) MoAbs is probably hidden in cell-bound IgG4. This was demonstrated by immunofluorescence techniques: IgG4 bound to the basophil membrane could be detected with anti-IgG4(Fab) but not with anti-IgG4(Fc) MoAbs. In addition, we found that nine donors were unresponsive to an anti-IgE stimulus, while they released histamine efficiently after challenge with anti-IgG4(Fab), suggesting the existence of different receptors for both immunoglobulins.     

Effect of rheumatoid factor and anti-immunoglobulin G antibodies on complement-mediated lysis of herpes simplex virus-infected human fibroblasts.

The effect of various anti-immunoglobulin G (IgG) antibodies on the complement-mediated lysis of herpes simplex virus-infected human fibroblasts was determined. IgM rheumatoid factor, a naturally occurring anti-human Fc, inhibited lysis, whereas rabbit anti-human IgG serum potentiated immune cytolysis. We attempted to explain this disparity by determining the effect various classess of anti-IgG's with differing specificities had on complement-mediated lysis. Inhibition of cytolysis occurred with IgM anti-Fc and all of the IgG antiglobulins (anti-IgG, Fab, and Fc). In contrast, IgM anti-Fab enhanced lysis. IgM anti-IgG suppressed immune cytolysis when high concentrations of antiviral serum were incubated with the virus-infected cell, but augmented lysis when low concentrations of anti-herpes simplex virus antibody were exposed to the fibroblasts. The experiments indicated that whether a particular antiglobulin potentiates or inhibits lysis depends on the concentration of antibody bound to the target cells as well as the class and specificity of the antiglobulin exposed to the antibody-coated cell.     

Three new alleles of IGHG2 and their prevalence in Danish Caucasians,Mozambican Blacks and Japanese

The human IGHG2 gene locus is polymorphic, encoding two known allotypes of IgG2: G2m(n-) and G2m(n+). The allele prevalence varies greatly between different ethnic groups and individual genotypes correlate with the level of plasma IgG2 and with antibody responses to certain polysaccharide antigens. In this study, we present three new alleles of IGHG2 (IGHG2*03, 04, and 05), and a complete sequence specific PCR typing system allowing discrimination between the different allotypes of IgG2. A hitherto unknown allotype, which we name G2m(ny), is encoded by IGHG2*04 and differs from G2m(n-) by asparagine rather than serine in CH1 residue 75 and by phenylalanine rather than leucine in CH1 residue 76 (EU numbering 192 and 193). The polymorphic residues are probably surface exposed near the hinge region. The same residues are also found in IgG1, IgG3, and IgG4, and G2m(ny) is therefore an isoallotype that probably arises by gene conversion within the heavy chain locus. The IGHG2*04 allele is present among Danish Caucasians with a low prevalence (2.5%), but was not found in Japanese or Mozambicans. The two other new alleles (IGHG2*03 and IGHG2*05) both encode the G2m(n-) allotype. The IGHG2*03 allele encodes most of the IgG2 of the G2m(n-) allotype in Danish Caucasians.     

Antibody quantitation using an immunoadsorbent and the unlabeled antibody enzyme method

Measurement of specific immunoadsorbent-bound antibodies has been accomplished by the unlabeled antibody enzyme method (Sternberger et al., 1970). Sepharose-4B containing specific antigen (or ligand) is treated with diluted specific immune serum (primary serum), such as 1 ml of serum diluted 6000–20,000 fold, followed by antiserum against immunoglobulin G (IgG) of the primary serum and then by peroxidase-antiperoxidase (antigen-antibody) complex (PAP) derived from the same species as the primary serum. Radiolabeled primary antibody and anti-IgG have confirmed the stoichiometry of the reaction. The immunoadsorbent binds the antibody of interest quantitatively and to equal extent after 15 min or 48 h. The enzymatic activity of the PAP complex followed a direct linear relationship to its concentration indicating the stability of binding in the PAP complex. A direct relation between the enzymatic activity measured when both the primary antiserum and the anti-IgG are used allows for quantitation of the antibody level of the primary serum.     

Association between Km1 immunoglobulin allotype and pulmonary tuberculosis in Indonesians

Allotypes were determined in 121 cases of smear-positive pulmonary tuberculosis and 33 healthy controls from Indonesia. It was found that the occurrence of Km1 was significantly lower in patients than in controls (p = 0.011), and that phenotypes lacking G1m (17) and G3m(21) as well as Km1 occurred much more frequently among patients than controls (p = 0.0025). Some evidence for an allotypic influence on the antibody response to mycobacterial antigens was found. A lack of G1m(17) or G3m(21) was associated with increased antibody levels in the IgG2 subclass in control subjects and a lack of Km1 with decreased antibody levels in the IgG4 subclass in patients.     

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.     

The choice of methods for immunoglobulin IgG purification: Yield and purity of antibody activity

Six methods for the purification of immunoglobulin G (IgG) from serum were compared, using rabbit antiserum to Bacillus anthracis spores as a model. Antibody activity was monitored by a solid-phase immunoradiometric assay (IRMA). Salt precipitation/ion exchange chromatography and ethanol precipitation both resulted in IgG of high purity but there was considerable inactivation of antibody. Salt precipitation/affinity chromatography gave poor yields of antibody. PEG precipitation and gel filtration of Sephacryl S-300 gave moderate yields and purity of IgG, with little evidence of antibody inactivation. Salt precipitation was marginally more destructive than the last 2 methods, but is recommended for routine use on grounds of its simplicity. Should IgG prepared by salt precipitation prove inadequate for particular applications, gel filtration is recommended since it allows the balance of yield and purity to be altered at will.     

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.     

Immunoglobulin G and M composition of naturally occurring antibody to type III group B streptococci.

Human sera were examined by an enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgM antibodies to purified type III polysaccharide of group B streptococci. The antigen-binding capacity of a reference human serum was determined by a radioimmunoassay, and the total antibody content was determined by quantitative precipitation. The serum was then depleted of IgM and IgA to determine the effect on the antigen-binding capacity. Duplicate samples of 81 sera were tested by the enzyme-linked assay in comparison with reference standard serum. Although levels of IgG antibody were greater in subjects who had carried type III streptococci during pregnancy, concentrations of this antibody were generally low. Only 2 of 28 sera (7%) from parturient subjects and 7 of 25 sera (28%) from adult volunteers contained greater than or equal to 1 microgram of IgG antibody per ml; the mean levels were 0.13 and 0.53 micrograms/ml, respectively. In contrast, 19 of 28 maternal sera (68%) and 22 of 25 (88%) volunteer adult sera contained greater than or equal to 1 microgram/ml of IgM antibody; mean levels were 1.33 and 1.54 micrograms/ml, respectively. The cord serum levels of IgG antibody were almost identical to maternal serum concentrations, whereas IgM antibody was essentially undetected.     

Determination of antibodies to TT virus (TTV) and application to blood donors and patients with post-transfusion non-A to G hepatitis in Japan

Recently, a nonenveloped single-stranded DNA virus named TT virus (TTV) has been reported in association with non-A to G post-transfusion as well as sporadic acute and chronic liver disease. A method was developed for the detection of antibody to TTV (anti-TTV) by means of immune precipitation and detection of TTV DNA by the polymerase chain reaction. The test serum was incubated with TTV, recovered from feces of a carrier, and after incubation, the formed immune complexes were precipitated with goat antiserum to human IgG. TTV DNA was sought for by the polymerase chain reaction in both precipitate and supernatant. The detection of TTV DNA in the precipitate, but not in the supernatant, was considered to represent anti-TTV in the test serum. Of the 44 healthy blood donors in Japan, anti-TTV was detected in one of the six (17%) with TTV DNA and 11 of the 38 (29%) without TTV DNA. In the two patients with post-transfusion non-A to G hepatitis, free anti-TTV developed as they cleared TTV in serum. Anti-TTV complexed with TTV in serum, detectable by precipitating sera with goat anti-human IgG and testing for TTV DNA, elicited while the patients had elevated alanine transaminase levels. The determination of anti-TTV would be useful for detecting resolved infection in surveys for exposure to TTV in the general population, and for establishing the mechanism of liver injury associated with TTV infection.     

The proportion of two b locus allotypic determinants in rabbit antisera raised against pneumococcal polysaccharide SSS III antigen

An agar gel radial diffusion technique has been devised to examine the amount of IgG and relative amounts of As 4 and As 5 in the sera of twenty heterozygous As/4,5 rabbits immunized with Type III pneumococci. Serial absorption of the sera with the polysaccharide SSS III antigen before reaction with anti-IgG and anti-allotypic sera was used to show how much specific IgG antibody was present in each serum and the distribution of this antibody between molecules bearing one or other or neither of the allotypic determinants.

The results indicate that anti-SSS III antibody generally occurred as As 4 rather than As 5 molecules although in some animals this preference was reversed. Non-allotypic (b-minus) molecules contributed significantly to the antibody response.

     

Effect of Gm allotypes on IgG2 antibody responses and IgG2 concentrations in children and adults

Earlier studies have suggested that in adults the n-positive allele of the human IgG2 gene is more productive than the n-negative allele. This superiority was seen to be manifested in IgG2 antibody responses to polysaccharides, in the higher serum concentration of total IgG2 in the n/n than in -/- individuals, and in the higher concentration of n-positive than n-negative IgG2 in heterozygotes. The present study shows that in 1- or 2-year-old children, the concentration of IgG2 was independent of allotype G2m(n), and both alleles of a heterozygote contributed an average of one-half of the total IgG2. On the other hand, the superiority of the n-positive allele was also seen in young children in IgG2 antibody responses induced by the Haemophilus influenzae type b polysaccharide (Hib). The effect of allotype n on antibody responses was evident only when the immunogen was the Hib polysaccharide. When the immunogen was a conjugate of Hib and diphtheria toxoid, the IgG2 antibody responses of n-positive and n-negative vaccinated individuals were almost equal, both in adults and in children.     

The surface expression of a tumor-associated antigen on human kappa myeloma cells

The monoclonal antibody K-1-21 defines an antigen, KMA (kappa myeloma antigen) on the surface of human kappa myeloma cells. K-1-21 also recognizes human kappa light chains in free form but not when covalently bonded to heavy chains. To examine the relationship between KMA and this determinant on free kappa chains, the surface expression of KMA was examined on the IgG, kappa myeloma line LICR LON/HMy2 (HMy2). No patching or capping was observed in the presence of K-1-21 alone, but KMA could be capped if the cells were incubated with K-1-21 followed by fluorescein isothiocyanate-conjugated sheep F(ab')2 anti-mouse immunoglobulin. Capping was not affected by the inhibitors calcium ionophore or dibucaine. When IgG molecules were removed from the cell surface by capping with anti-IgG antiserum both KMA and free kappa light chains could still be detected with K-1-21 and a polyvalent anti-kappa antiserum, respectively. By contrast, after removal of all surface kappa chains with the polyvalent anti-kappa serum, no staining was observed with K-1-21 indicating that KMA may be an epitope on free kappa chains inserted in the membrane of kappa myeloma cells but absent from normal cells. KMA cell surface expression varied with the stage of the cell cycle. Flow cytometric analysis of K-1-21-stained HMy2 cells from either continuous cultures or from elutriated fractions enriched for various cell cycle phases showed that, within the cycling population, cells in G2 + M expressed KMA at a higher frequency and density than did cells in G1.