The use of protein A-sepharose affinity chromatography for separation and detection of specific IgM antibody in acquired rubella infection: A comparison with absorption by staphylococci containing protein A and density gradient ultracentrifugation

Abstract

Protein A-Sepharose affinity chromatography has been investigated as a means of removing IgG from sera before testing for rubella-specific IgM. The method was compared with whole staphylococci containing protein A and with sucrose density gradient ultracentrifugation. Approximately 98% of IgG was removed by protein A-Sepharose absorption and 95% by whole staphylococcal absorption, but 50–60% of IgM was also removed. One hundred and two sera were tested — 49 had been shown to contain rubella-specific IgM by ultracentrifugation and 53 to be negative. Accepting 2-fold or greater reduction in antibody titre with 2-mercaptoethanol as the criterion for positivity after absorption, 4 of 49 (8%) sera were negative with protein A-Sepharose and 31% whith whole staphylococci. Of the 4 sera negative after protein A-Sepharose treatment three were obtained between 48 and 62 days after infection. It is essential to confirm positive results by demonstrating reduction of IgM antibody remaining after absorption by treatment with 2-mercaptoethanol. It is also essential that all post-absorption antibody be treated with 2-mercaptoethanol to eliminate false positives. Protein A-Sepharose affinity chromatography was more sensitive than absorption with whole staphylococci but less sensitive thn ultracentrifugation. The method has a place in screening convalescent sera collected less than 28 days after infection.