Detection of IgG-Associated Determinants in Reduced and Alkylated Preparations of Human IgG3 by Monoclonal Antibodies

Abstract

Using classical typing antisera, previous experiments have failed to demonstrate IgG3 in partially reduced and alkylated preparations of human IgG intended for intravenous application (IGIV). To establish that IgG3 is actually present in such preparations and to investigate the effects of reduction and alkylation on IgG3-associated epitopes, an enzyme-linked immunosorbent assay (ELISA) was designed using monoclonal antibodies as solid phase reagents and protein A-purified IgG3 as antigen. Three different samples of reduced and alkylated antigen were used: 1) IgG3 isolated from a ready-for-infusion IGIV; 2) IgG3 which was purified from an intramuscular (Cohn Fraction II) IgG solution before being subjected to a mild reduction and alkylation procedure; and 3) completely reduced and alkylated IgG3. The reduction and alkylation procedure did not affect the solubility of IgG3, indicating that IGIV prepared in this manner should contain normal quantities of IgG3. In the ELISA, solid-phase monoclonals which were cross-reactive with multiple IgG subclasses clearly reacted with reduced and alkylated IgG3. Furthermore, there was no substantial difference between the quantities of modified and native antigen required for 50% maximal ELISA signal. In contrast, solid-phase monoclonals with IgG3-restricted specificity did not recognize reduced and alkylated material. These results indicate that IGIV prepared by reduction and alkylation has a normal IgG3 content and confirm that some IgG3-specific determinants are altered by the modification procedure.