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Monoclonal IgG rheumatoid factors (RFs) are identified using a novel immunoblot assay which detects RFs that bind to SDS-denatured Fc on nitrocellulose. Recognition of these self-associating antibodies is by the use of F(ab′) 2 fragments of light chain-specific antisera. In this way, IgG RFs can be easily identified and the precise binding characteristics to different isotypes of IgG, or other antigens, further specified. The assay can also be used to detect other classes of RFs such as IgM RFs. Although less sensitive than the standard ELISA, the use of this immunoblot RF assay (IRFA) will identify IgG RFs and their target antigen with precision.

A method for determining the affinity and the concentration of mouse monoclonal antibodies against human IgG has been developed. The method comprises two steps. First, monoclonal antibodies are allowed to combine with radioiodinated human IgG. Secondly, mouse monoclonal IgG with and without complexed IgG is precipitated with rabbit IgG against mouse IgG. As the antigen is divalent complexes of varying composition are formed in this system leading to deviations from linearity in plots obtained in commonly employed analytical systems. The theoretical background of these systems has been studied by computer simulation, and a concentration effect on the formation of immune complexes was demonstrated. The affinities (in terms of the association constants) of 6 monoclonal antibodies were estimated and found to be in the range from 2 × 106 M−1 to 5 × 108 M−1.

Murine IgG subclasses show different affinities when they react with protein A. The temperature of application of the sample to protein A-Sepharose markedly influences the interaction of certain antibodies with this specific adsorbent. Elution of weakly bound mouse IgG could be carried out by mild temperature changes instead of lowering the pH, and this should be advantageous with certain immunoglobulins that are labile under acidic conditions.