Studies on Antigen-binding Activity of Macroglobulin Antibody Subunits and Their Enzymatic Fragments

Abstract

Abstract To gain insight into reasons for the presence of only five combining sites in γM antibodies, studies were undertaken to determine the number of antigen-binding sites on γM subunits and on Fabµ fragments of a γM rheumatoid factor anti-γ-globulin from a patient with mixed cryoglobulins. The rheumatoid factor contained both κ and λ light chains and reacted with human and rabbit γG. Combining ratios were determined by quantitatively determining the amount of complex formation by analytical ultracentrifugation. Each monomeric subunit had only one effective antigen-binding site, and there were no inactive subunits. All of the 3.7 S Fabµ fragments, prepared by trypsin digestion of the subunits, were active and each had at least one effective binding site per fragment. These results indicate that there are 10 potential combining sites in the intact molecule although only five are available for antigen binding. Studies were performed also on the nature of the cross-reaction of this antibody with rabbit γG. These experiments showed that the amount of complex formed with rheumatoid factor subunits and their antigen was the same whether the antigen consisted of rabbit γG alone, human γG alone, or a mixture of the two. Since there is no evidence for antibody subunits which combine with only one species of γG, the binding sites appear to resemble those in other cross-reacting systems in which each combining site may be occupied by either antigen. In contrast to the excellent binding activity of γM antibody subunits for rabbit γG, Fabµ fragments showed little or no binding activity for rabbit γG.