Monoclonal anti-human IgG antibodies for quantitation of allergen-specific IgG in human sera

Eight monoclonal anti-human IgG antibodies were fully characterized and evaluated as possible reagents in solid phase radioimmunoassay for quantitating allergen-specific IgG antibody. Four monoclonal antibodies (HG24D, HG2-14, HG2-18, and HG2-25) recognize CH2 domain of human IgG and bind to human IgG fixed to microtiter plate with high affinities. These monoclonal antibodies were more suitable than polyclonal rabbit anti-human IgG antibody in Phadebas RAST for honey bee venom-specific IgG antibody. Nonspecific binding was much lower, and the slopes of standard curves were much steeper. In contrast to polyclonal antibody, the standard curve was hardly influenced by human serum IgG in sample diluent. These advantages of monoclonal antibodies that recognize CH2 domain of human IgG made it possible to quantitate egg white- and Dermatophagoides pteronyssinus-specific IgG antibodies with use of allergen disks prepared for IgE RAST. This property allows a single system to be used for measurement of IgG and IgE antibodies against clinically relevant allergens.     

Specific IgG antibodies in sera in patients with penicillin allergy

The role of IgG antibodies in inducing or modifying allergic reaction has not been sufficiently clarified. The objective of this investigation is to elucidate the relationship between IgG antibodies and penicillin allergy, between IgG and IgE antibodies in allergic patients. Enzyme-linked immunosorbent assay and Radioallergosorbent test were used to examine eight kinds of specific IgG and IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO) and phenoxomethylpenicilloyl (PVO), and minor antigenic determinants: benzylpenicillanyl (BPA), ampicillanyl (APA), amoxicillanyl (AXA) and phenoxomethylpenicillany (PVA), in the sera of 249 patients with penicillin allergy. Except BPA-IgG, seven kinds of antigenic determinants IgG antibodies levels were significantly higher than that of control group (P < 0.05). Positive rates of specific IgG and IgE were 47.0 and 57.8%, while positive rate of IgE and IgG together was 77.9%. The positive rate of IgG antibodies to major antigenic determinants (42.2%) was significantly higher than that of minor antigenic determinants (8.8%) (P < 0.05). The positive rate of IgG antibodies of patients with typical clinical symptoms after penicillin administration when skin tests were negative was significantly higher than that of patients with positive skin test (P < 0.01). There were no differences between the IgG positive rates to three kinds of determinants and that of all of eight kinds. The study indicates that IgG may be important in penicillin allergy with negative skin test and IgG antibodies to major antigenic determinants probably play a more important role in the process of allergic reaction. This project was supported by the Science Foundation for Distinguished Young Scholars of Henan Province (No. 0312002100) and the Nature Science Foundation of Henan Province (No. 0211040100).     

IgG2 deficiency in a healthy blood donor. Concomitant lack of IgG2, IgA and IgE immunoglobulins and specific anti-carbohydrate antibodies

The autologous and allogeneic mixed lymphocyte reactions were measured using peripheral blood cells from 13 patients with hairy cell leukaemia (HCL), six of whom had been splenectomized when first studied. T cells from five patients responded to autologous stimulation, and one of these had received splenectomy. Significant alloreactivity was observed in nine patients, of whom three were splenectomized. An absent autologous reaction was associated with ratios of OKT4:OKT8 positive cells less than 1.5, and such ratios were more often observed after splenectomy. Non-T cells from 10 of 11 patients stimulated allogeneic normal T cells. The amounts of HLA-DR antigens on hairy cells were similar to those on normal peripheral blood non-T cells. These data suggest that the defects of response in HCL reflect abnormalities at the responder T cell level. Such defects may contribute to the defective host defence frequently observed in HCL.     

IgG antibodies against polyisocyanates in car painters

A group of thirty car painters exposed to vapours and aerosols of paint containing prepolymer and monomer of hexametylene diisocyanate (HDI) was investigated. Specific antibodies against monomer HDI and prepolymerized HDI were analysed with RAST (IgE) and ELISA (IgG) assays after conjugation of the haptens with human serum albumin. There was no significant increase of serum IgG antibodies against HDI monomer, nor of specific IgE antibodies against HDI monomer or prepolymer. Specific IgG antibodies against prepolymerized HDI were significantly increased, as compared with non-exposed referents (medians 0.11 vs 0.03 absorbance (A)). Six car painters were found to have specific IgG antibodies of subclass 4 against HDI prepolymer, four also against HDI monomer. This shows an association between exposure and specific IgG antibodies. Thirteen subjects had suffered symptoms of rhinitis and/or conjunctivitis, and ten had symptoms from the bronchi (two asthma). There was no significant association between symptoms and levels of specific antibodies. Most of the symptoms were slight and unspecific, probably due to irritative effects of the exposure.     

Immunofluorescent IgG and IgM antibodies in human sera by enterovirus infections

IgG and IgM antibodies to selected enterovirus serotypes (P2, CB 1-5, E2, 4, 20), to prototype collection strains and to fresh isolates were examined by immunofluorescence in paired sera of 11 children and 4 adults in whom enterovirus had been isolated, or a rise of specific antibodies had been proved by neutralization test. One six-month- and one seven-month-old child had antibodies of both classes to the isolated virus strain and to poliovirus only. Children from eight month onwards and adults had antibodies to the majority of enterovirus serotypes tested. Among all enteroviruses tested, heterologous reactions were observed not only with IgG but also with IgM class antibodies.     

The subclasses of human IgG antibodies against tetanus toxoid.

The subclass of IgG antibodies against tetanus present in the serum of thirty-five human individuals, who received an injection with tetanus toxoid, was determined. Six successive serum samples were obtained from twenty-five normal individuals (laboratory personnel) 0, 3, 7, 14, 28 days and 2-3 months after the injection with tetanus toxoid had been given. Another ten serum samples were obtained from ten persons with a positive IgE-RAST, taken 2 weeks after the injection. Antibodies were determined with a quantitative immunofluorescence method known as the defined antigen substrate spheres (DASS) system. The normal individuals in whose serum a clearly positive IgG binding was found (nineteen) showed activity in all four subclasses. The binding activity in all individuals reached a maximum between 2 and 4 weeks after the injection. The antibody activity in the serum of four individuals whose serum gave weak IgG binding was confined to IgG1. Two individuals did not show any IgG binding activity at all. In the ten persons with a positive IgE-RAST and three of the normal individuals, who also had a positive IgE-RAST, the distribution of the antibodies over the subclasses was the same as in the others.     

Antiidiotypic antibodies against anti-DNA antibodies in sera of families of lupus patients

We searched for antiidiotypes directed against anti-DNA in sera of healthy family members of lupus patients. Controls were healthy individuals without a personal or family history of lupus. No significant differences were noted between the family members' and the control group's sera with respect to binding to DNA or to non-anti-DNA F(ab)₂ fragments. Family members' sera had higher binding to anti-DNA F(ab)₂ and to normal IgG F(ab)₂ fragments (P<0.01). Sera of the family members had significantly higher binding to anti-DNA F(ab)₂ than to normal IgG F(ab)₂ fragments (P<0.0036). Inhibition experiments have shown that the antiidiotype is directed against the framework determinants and not against the antigen binding sites of the idiotype. The anti-idiotypic antibodies were directed against cross-reactive anti-DNA idiotypes and were not restricted to the idiotypes of the lupus proband. Age, sex, and blood relationship to the lupus patient did not influence the presence of antiidiotypes in the family members. The possible role of environmental factors in the induction of antiidiotypes and the role of the latter in regulating anti-DNA antibodies are discussed.     

Patients with myasthenia gravis and thymoma have in their sera IgG autoantibodies against titin.

Patients with myasthenia gravis (MG) and thymoma have in their sera antibodies which react with non-receptor antigens from striated muscle. The purpose of this investigation was to characterize the antigen(s). Polypeptides in homogenates from rat skeletal muscle were separated by SDS-PAGE and trans-blotted to nitrocellulose. Sera from six patients with MG and thymoma stained a large (molecular weight greater than 500 kD) polypeptide, while no staining was observed with sera from 20 non-thymoma MG patients. Titin is one of the large (greater than 500 kD) polypeptides of striated muscle and the antibody containing MG sera have antibodies that bind to titin in a preparation of myofibrillary proteins from rabbit skeletal muscle. The staining pattern is identical to that obtained with antiserum to titin, showing that the antigen has the same electrophoretic mobility as titin. Antibodies from the sera of the patients with MG and thymoma, affinity-purified on the large polypeptide, reacted with skeletal muscle sections in a cross-striational pattern, near the A/I band junction but within the I band, corresponding to the localization of one of the epitopes of titin. Our findings therefore indicate that the muscle antibodies found in the sera from some MG patients with thymoma are directed against titin.     

Preparation of antibodies against subclasses of human IgG

Several species were immunized with IgG myeloma proteins to prepare subclass-specific antisera. Rabbits produced easily antibodies against IgG₁ and IgG₃. Antibodies against IgG₂ and IgG₄ were easily raised in sheep. The antisera were tested by precipitation and agglutination techniques.     

Human antibodies against bovine immunoglobulin M in IgA deficient sera

Analysis of the sera of seventeen patients with a selective deficiency of IgA revealed that 24% gave a precipitin reaction on gel diffusion with normal bovine serum and milk. None of 100 normal adults and only one of approximately 500 patients with various diseases without an IgA deficiency had precipitins when studied by this same technique. Analysis of the nature of this reaction revealed that an IgG type antibody present in the human sera was reacting with the Fc fragment of the IgM present in bovine milk and serum.

An immunologically identical reaction occurred with goat and sheep, and reactions of partial identity with horse and pig sera. No reactions were found with sera from other species including man. In two of the IgA deficient sera additional but much weaker precipitin lines were noted with cow milk. These data are consistent with the hypothesis that oral immunization with the highly antigenic macroglobulin present in cow milk was facilitated by the mucosal antibody defect accompanying the IgA deficiency.

     

Lipid A in anaerobic bacteria.

The ability of lipid A preparations from strains of Bacteroides, Fusobacterium, and Veillonella to inhibit the lipid A-anti-lipid A reaction in an enzyme-linked immunosorbent assay was tested. Anti-lipid A serum was prepared with lipid A from Salmonella minnesota R595, and lipid A from Escherichia coli EH100 was used as control antigen. Preparations from three of four different species of Bacteroides were unable to inhibit the anti-lipid A activity, whereas lipid A preparations from Fusobacterium and Veillonella strains inhibited 50% of the activity at 1 to 141 micrograms. One of the Bacteroides strains, Bacteroides oralis, showed a very weak inhibiting activity at the highest concentration used. The results confirm that Bacteroides species have a unique lipopolysaccharide structure, in contrast to other anaerobic genera which have a lipopolysaccharide structure similar to that of the Enterobacteriaceae.