An immunoblotting procedure following agarose gel electrophoresis for subclass typing of IgG paraproteins in human sera

Abstract

A method for subclass typing of IgG paraproteins in human sera following agarose gel electrophoresis is presented. After electrophoretic separation, serum proteins were blotted by capillary diffusion onto nitrocellulose. Mouse anti-human IgG1-IgG4 monoclonal antibodies were exposed to bound IgG. Goat anti-mouse IgG alkaline phosphatase conjugate was employed as second antibody. Thirty six sera were examined, in which the presence of an IgG paraprotein had previously been proved by agarose gel electrophoresis and subsequent immunofixation with anti-IgG antiserum. The subclass frequency distribution was 27 IgG1, 6 IgG2, 2 IgG3 and 1 IgG4. By means of IgG subclass typing in 8 out of these 36 sera, a total of 12 additional monoclonal bands (1 to 2 bands per serum) were detected which were not seen after immunofixation electrophoresis with anti-IgG antiserum. Eleven of 12 additional bands belonged to an IgG subclass different from that of the bands already detected by immunofixation electrophoresis. Light chain typing was performed in 9 of 12 bands found additionally. Two of these 9 bands belonged to a light chain class different from that of the bands already detected by immunofixation electrophoresis. The method described can be employed to further elucidate the possible diagnostic and prognostic significance of the subclass type of an IgG paraprotein.