Isolation of thyroid stimulating immunoglobulins by affinity chromatography using protein A sepharose

Sera from a reference pool, 29 controls and 18 patients with Graves' disease were fractionated on sepharose CL-4B protein A. After a primary elution of the unbound fraction at normal pH, IgG subclasses 1, 2 and 4 were eluted using a discontinuous pH gradient. The eluate was monitored by UV-absorption at 280 nm and three peaks were observed within narrow pH ranges in all sera tested. From the reference pool (n = 10) peak A was eluted at pH 6.6 +/- 0.1, peak B at 4.2 +/- 0.03 and peak C at 3.8 +/- 0.01. This distribution was partially related to the IgG subclasses with IgG1 recovered in peak C, while IgG2 and IgG4 were distributed in both peak B and C. Thyroid adenylate cyclase stimulating antibodies (TSAb) and thyrotrophin binding inhibiting immunoglobulins (TBII) were measured in the pooled fractions under these peaks. In the reference pool total assay variations (SD) were 10-22 per cent. The 95 percentiles of the 29 control values were used as reference range. In the 18 patients TSAb were positive in 4 in fraction A, 9 in B and 12 in C. TBII were found in one in fraction A, none in B and 13 in C. No correlation between TSAb and TBII values were found before or after fractionation. In most cases TSAb activity was highest in fraction C, however, in 3 patients fraction A was the more active and in these patients TBII were only found in fraction C. It is concluded, that the present results indicate a heterogeneity of the thyroid stimulating immunoglobulins with separation of the TSAb and TBII activities in some patients.