Determination of the affinity of monoclonal 19 S IgM rheumatoid factor for IgG by modified immunoassay (ELISA)

Abstract

In rheumatoid arthritis (RA), the pathogenicity of IgM rheumatoid factor (RF), an autoantibody whose antigen is IgG, is still unclear although RF-IgG complexes appear to be important mediators of immune injury. The polyclonality of RF in RA makes it difficult to characterize certain qualitative properties such as specificity and affinity which may be very important in determining pathogenicity. Monoclonal IgM RF can be used to circumvent this problem. Monoclonal RF secreting cells can be produced via hybridizations with RA B lymphocytes fused with mouse or human myeloma cell lines. Another source of monoclonal RF is the sera of patients with Waldenström's macroglobulinemia (WM). One particular WM IgM RF (Kas) was chosen for our experiments to measure affinities and specificities to eight different monoclonal IgGs (three IgG1s, three IgG3s, one IgG2, and one IgG4). 19 S IgM RF, a pentavalent molecule, was mildly reduced with DTT to make 7 S univalent fragments (7 S IgM RF). 7 S IgM RF was incubated with each of the different IgGs at several different concentrations. These mixtures were allowed to come to equilibrium. An aliquot was then used to determine the amount of free 7 S IgM RF by ELISA. By plotting the reciprocal of the fraction of bound RF versus the reciprocal of the concentration of free antigen at equilibrium, different affinities were determined. The results of these determinations compare favorably with published IgM RF affinities determined by more traditional methods. This method can also be used with proteolytic digest fragments of IgG and short synthetic peptides of the IgG molecule to better locate the antigen binding site. The technique may also help us to determine whether there are select clones of RSC producing RF with different affinities that could complex to a particular type of IgG which, in vivo, could produce greater inflammatory tissue damage. Furthermore, this methodology should be useful in the study of other autoimmune diseases characterized by pathogenic autoantibodies of differing affinities.