Populations Of Antibody Molecules Research Articles

Antibodies to the strain-specific and cross-reactive determinants of the haemagglutinin of influenza H3N2 viruses. 1. Preparation of antibodies and in vitro studies.

The serological analysis of antibodies to the haemagglutinin (HA) of influenza A viruses of the Hong Kong (H3N2) subtype is described, using haemagglutination-inhibition, immuno-double-diffusion and single-radial-diffusion techniques. By cross-absorption of antisera to purified HA antigens, different populations of antibody molecules were obtained, which are designated strain-specific and cross-reactive and characterized in terms of their antigenic specificities for HA antigens of the homologous and antigenically variant H3N2 viruses. A narrowly strain-specific population of antibodies (SS"HK) was obtained as the residual antibody in antiserum to A/Hong Kong/1/68 HA after absorption with the closely related A/England/42/72 virus, whilst a contrasting broadly cross-reactive population (CR'HK) was obtained by absorption of the anti-A/Hong Kong/1/68 HA serum with the more distantly related strain A/Victoria/3/75 and eluting the cross-reactive antibodies from the absorbing virus. Similarly, specific and cross-reactive antibodies were derived from antiserum to A/Victoria/3/75 HA antigen by absorption with A/Hong Kong/1/68 virus. Single-radial-diffusion tests were performed, involving sequential application of different antibody preparations in the same wells in immunoplates containing intact virus particles. The cross-reactive and strain-specific antibodies differed in their property of mutual interference of attachment ot antigen. The results suggested that the cross-reactive antigenic determinants on the HA subunit may be located closer to the distal end of the molecule than the strain-specific determinants. Further tests employing single-radial-diffusion showed that there are more cross-reactive than strain-specific sites available for antibody in the intact virus particle. The strain-specific antibodies also gave higher haemagglutination-inhibition titres per microgram IgG than the cross-reactive antibodies.

Antibody responses to antigenic determinants of influenza virus hemagglutinin. I. Thymus dependence of antibody formation and thymus independence of immunological memory.

Using immunodiffusion methods it has been shown that purified hemagglutinin (HA) extracted from two related strains of influenza A viruses (A/PR8/34 and A/FM1/47) have two distinct antigenic determinants, or groups of determinants. One determinant is cross-reactive while the other is strain-specific. Antisera raised in normal mice against HA were shown to contain two populations of antibody molecules, each directed against one of the determinants. Immunization of thymus-deprived (TXBM) mice showed a strong thymus dependence of antibody formation to HA. However, the thymus dependence of antibody formation against the cross-reactive determinant could be overcome by repeated inoculations of HA in TXBM mice, indicating a different handling of two portions of the same molecule by the immunological system. Strong, secondary-type responses to the strain-specific determinant were observed in primed thymus-deprived mice after reconstitution with virgin thymus cells, showing that specific immunological memory was elicited by this determinant despite the absence of detectable antibody secretion. These findings are interpreted as examples of immunological recognition and memory mediated by B lymphocytes and discussed in terms of mechanisms of T and B lymphocyte co-operation. It is suggested that the helper effect of T lymphocytes is exerted at a late stage in the differentiation of specific populations of B cells into antibody-secreting cells.

Concentration of Cross-Reacting Idiotypic Specificities in Unrelated Mouse Immunoglobulins

Abstract Myeloma proteins and populations of antibody molecules from individual donors possess unique antigenic (idiotypic) determinants (1–3). Gell and Kelus (4) first proposed that a given idiotypic specificity is characteristic of a clone of cells, i.e., of cells descended from a common precursor. It has been suggested, however, that certain idiotypic specificities might actually reflect the presence of unique antigenic determinants in certain subgroups of polypeptide chains, selected by the antigen (5). If this were the case nonspecific immunoglobulin populations would be expected to exhibit various idiotypic specificities in low concentrations. The present experiments define upper limits for the concentrations of idiotypic specificities of mouse antihapten antibodies in normal IgG and in mouse antibodies of unrelated specificity. The antibodies used to elicit antiidiotypic (anti-D)2 antibodies were prepared in strain A/J mice by immunization with keyhole limpet hemocyanin (KLH)-p-azophenylarsonate or KLH-p-azobenzoate (6). Anti-D antibodies directed to the antihapten antibodies were prepared and were quantified by an indirect precipitation method, as described elsewhere (6).

Development of a Quantitative Immunological Assay for the Study of Spore Coat Synthesis and Morphogenesis

A quantitative assay employing (125)I-labeled antibody has been developed for Bacillus cereus T spore coat protein. Populations of antibody molecules with various affinities for inner or outer coat can be prepared by selective adsorption to and elution from different coat preparations. Adsorption to and elution from intact spores results in an antibody preparation at least 15 times more reactive to outer coat. This antibody is useful for measuring the time and extent of spore coat maturation, i.e., outer spore coat formation. Rifampin inhibits the increase in content of this coat antigen.

Evidence for Ammonium and Guanidinium Groups in the Combining Sites of Anti-p-azobenzenearsonate Antibodies— Separation of Two Different Populations of Antibody Molecules

Abstract The effect of modification of amino and guanidinium groups of rabbit antibodies directed against the negatively charged p-azobenzenearsonate hapten (anti-Rp antibodies) was studied. Purified antibodies from either a single rabbit or from serum pooled from several rabbits lost at least 85 to 90% of their combining sites when 100% of their amino groups were selectively modified with maleic anhydride and 75 to 80% of their guanidinium groups were modified by subsequent treatment with glyoxal. However, they lost 50% or less of their combining sites when only the amino groups were modified. The loss of combining sites could be prevented by the presence of specific hapten (p-nitrobenzenearsonate) during the modification reactions. These results suggest that some anti-Rp antibodies have combining sites which contain guanidinium groups, while others have combining sites which contain amino groups. It was possible to separate these antibody molecules into two such populations. Maleylated anti-Rp antibody molecules were separated into two fractions with a specific solid immunoadsorbent. Approximately half of the maleylated antibodies remained in the solution because they had lost their combining sites as a result of maleylation of an amino group in the combining sites. It was possible to recover completely the antibody activity by removing most of the maleyl groups by acid hydrolysis. These antibodies did not contain glyoxal-reactive groups in their combining sites because treatment with glyoxal either before or after hydrolysis of the maleyl groups resulted in no loss of antibody combining sites. The second fraction of the maleylated antibodies, those which were adsorbed on the solid immunoadsorbent, was eluted and treated again with maleic anhydride, and their combining sites were found to be 100% effective. This showed that there were no amino groups in the combining sites of these antibodies. When these antibodies were treated subsequently with glyoxal, 75% of their combining sites were lost, showing the presence of glyoxal-reactive groups in these sites. Modification with both maleic anhydride and glyoxal of antibodies directed against a positively charged (p-azophenyltrimethylammonium) hapten and a neutral (3-azopyridine) hapten did not result in any losses of combining sites, showing that these antibodies did not contain amino- or glyoxalreactive groups in their combining sites. This investigation showed the presence and separation of at least two distinct populations of anti-Rp antibody molecules —those with combining sites containing guanidinium or other glyoxal-reactive groups and no amino groups, and those with combining sites containing amino groups and no other glyoxal-reactive groups. Amino acid analyses of the glyoxal-modified antibodies showed that arginyl residues had been irreversibly modified. No evidence was found that any other residues had been modified. However, the possibility still exists that other residues were modified, but that the modification was reversed during hydrolysis of the antibodies. Nevertheless, the immunochemical evidence is that the important group altered was a guanidinium group, since only antibodies directed against negatively charged haptens are affected while antibodies directed against neutral and positively charged haptens are not affected.

SPECIFIC FRACTIONATION OF HUMAN ANTIDEXTRAN ANTIBODIES. II. ASSAY OF HUMAN ANTIDEXTRAN SERA AND SPECIFICALLY FRACTIONATED PURIFIED ANTIBODIES BY MICROCOMPLEMENT FIXATION AND COMPLEMENT FIXATION INHIBITION TECHNIQUES.

Human antidextran of one individual, absorbed specifically on sephadex, was fractionated into two populations of antibody molecules by successive elution with oligosaccharides of the isomaltose series of increasing size. The purified antibody fractions and some whole antidextran sera were found to fix complement with dextrans of molecular weight of 195,000 and above. It could be demonstrated by quantitative microcomplement fixation inhibition assays that the antibody eluted with isomaltotriose had a higher affinity for smaller oligosaccharides relative to isomaltohexaose, indicating a high content of antibody molecules with smaller combining sites, while with the second fraction, eluted with isomaltohexaose, the small haptens were very poor inhibitors and the larger oligosaccharides inhibited readily, presumably due to a higher proportion of molecules with larger combining site size. Assays of similarly prepared fractions, obtained from earlier bleedings of the same individual (1), with inhibition of complement fixation were in good agreement with those obtained by inhibition of precipitation. The two purified antidextran fractions were shown to differ with respect to their complement-fixing capacity. The fraction with molecules with smaller size-combining sites fixed only about half as much complement per unit antibody N as did the fraction containing largely molecules with larger combining sites suggesting that the strength of complement fixation is affected by the strength of the antigen-antibody interaction.