A primary standard for the ELISA of thyroglobulin and microsomal autoantibody IgG subclass associated activity.

A primary standard for the assay of thyroid autoantibody subclass distribution was prepared by removing all but one IgG subclass from a standard serum by negative affinity chromatography. Affinity columns were prepared using murine monoclonal antibodies showing restricted specificity for human IgG (clone TM10-non-IgG1; HP6019-non-IgG2; VC9-nonIgG3 and 1a1-non-IgG4). Aliquots of patient serum were chromatographed and the eluted fractions assayed for IgG subclass concentration and thyroid autoantibody activity by ELISA. Recoveries of the desired IgG subclasses were: TM10 26.7%, HP6019 77.3%, VC9 53.0% and 1a1 46.0%. Contamination with unwanted subclasses was usually less than 1% but there was some "breakthrough" of 3 and 4 with HP6019. The thyroid autoantibody distribution, corrected for recovery and dilution, was thyroglobulin autoantibody (TgAb) IgG1 61.2%, IgG2 37.7%, IgG3 2.7%, IgG4 2.9% and thyroid microsomal autoantibody (MicAb) IgG1 73.7%, IgG2 19.8% IgG3 3.6% IgG4 3.8%. Several other serum samples were also analyzed by this technique and also by subclass ELISA calibrated with this standard serum. Regression analysis of the data obtained by the two assay methods gave correlation coefficients of r = 0.88 for TgAb and r = 0.82 for MicAb.