Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides.

Fourteen synthetic peptides of 15 amino acid residues length, overlapping by five residues and spanning the entire sequence of the major allergen Der p II from the house dust mite Dermatophagoides pteronyssinus were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope mapping studies using human IgE antisera. These antibodies bound predominantly to the peptide comprising residues 65-78, the binding of which was inhibited by native Der p II. In addition these antisera bound, to a lesser extent, to the peptide that comprised residues 1-15, which binding was not inhibited by native Der p II. Thus, we found one sequential epitope for a number of IgE sera.

ELISA procedures for the measurement of IgG subclass antibodies to bacterial antigens.

We have developed enzyme-linked immunosorbent assays (ELISA) of IgG subclass antibodies against whole bacteria and bacterial antigens using enzyme-labelled mouse monoclonal antibodies. The properties of different anti-subclass antibodies were compared. In sera from 18 healthy adults we measured the IgG subclass distribution of specific antibodies against Staphylococcus aureus and Haemophilus influenzae b and against distinct bacterial components: pneumococcal capsular polysaccharides, dextran and tetanus toxoid. We found that antibodies against protein (tetanus toxoid) were mainly IgG1, with some contribution of IgG4 and IgG2. Antibodies against polysaccharides (pneumococcal PS and dextran) and whole bacteria were restricted mainly to IgG1 and IgG2.

The determination of the complement components C1q, C4 and C3 in serum and cerebrospinal fluid by radioimmunoassay.

Non-competitive 2-site radioimmunoassays (RIA) for the determination of the complement proteins C1q, C4 and C3 in cerebrospinal fluid (CSF) are described. The quantitative results of the RIAs were the same as those obtained by other assay methods: radial immunodiffusion and turbidimetry and, in the case of C4, the haemolytic assay. The concentrations of the complement proteins in paired CSF and serum samples from a group of 60 patients were measured, as well as those of albumin and IgG. The ratios (concentration in CSF)/(concentration in serum) of the complement proteins correlated poorly with that of albumin. In contrast, the ratio of IgG was significantly correlated with that of albumin. The ratios of the complement proteins were higher than might be expected on the basis of their molecular masses. This suggests that these proteins may be synthesized within the normal central nervous system.

Heterogeneity in the IgE binding to a peptide derived from the house dust mite allergen Der p II indicates that the IgE response is highly polyclonal.

The fine specificity of IgE antibody binding to peptide 65-78 of the house dust mite major allergen Der p II was examined by comparison with binding to two peptides in which the cysteines corresponding to cys73 and cys78 in Der p II were substituted by serines and methionines. Differences in binding behavior indicated that at least three different subpopulations of IgE antibodies bound to peptide 65-78. Even at the level of such a small fragment the IgE response in individual donors proved to be polyclonal.

Complement activation by polyclonal immunoglobulin G1 and G2 antibodies against Staphylococcus aureus, Haemophilus influenzae type b, and tetanus toxoid.

To obtain information on effector functions of human immunoglobulin G2 (IgG2), we have measured the complement-activating properties of polyclonal IgG subclass antibodies against bacterial antigens. IgG1 and IgG2 were purified from serum samples from five healthy individuals, and complement activation was measured with different bacterial antigens. We used Staphylococcus aureus Wood 46 (STAW), which is a common antigen, Haemophilus influenzae type b (Hib), which is a common pathogenic microorganism in children, and formaldehyde-inactivated tetanus toxin (TT). Bacteria were incubated with antibodies and then incubated with sera from agammaglobulinemic patients as a complement source, and C3c deposition was measured by enzyme-linked immunosorbent assay. We found that anti-STAW IgG2 activated complement to a level similar to that of anti-STAW IgG1. Anti-Hib IgG1 complement activation was as much as seven times higher than that of anti-Hib IgG2 in four individuals. In one individual, anti-Hib IgG2 was more effective in complement activation than anti-Hib IgG1. Anti-TT antibodies showed patterns similar to those of anti-Hib. Our results indicate that IgG2 antibodies may contribute significantly to antibacterial defense. Also, individual differences in antibody effector functions should be taken into account when evaluating the immune status of patients and during early phase 1 studies of new vaccines.