Sensitive assay to detect thyroid stimulating antibody (TSAb) in the presence of thyroid stimulation blocking antibody (TSBAb) in serum

The detection of thyroid stimulating antibody (TSAb) activity in the presen ce of thyroid stimulation blocking antibody (TSBAb) in Graves' serum is dif ficult because TSBAb blocks TSAb activity, We recently demonstrated that po lyethylene glycol (PEG) augments TSAb activity in porcine thyroid cells (PT C) assay. This PEG-induced augmentation makes it possible to develop a sens itive assay to detect TSAb in the presence of TSBAb. We studied the effects of PEG on TSAb- and TSBAb-activities in PTC using 4 different preparations of the samples; (1) crude IgG using PEC 22.5% precipitated fraction (PF) f rom Graves' serum (0.2 ml), (2) crude IgG using PEG 12.5% PF, (3) serum (50 mul). and (4) serum (50 mul) in the presence of 5 % PEG (final). When the effects of PEG on TSAb activity using crude IgG were examined, PEG 22.5% PF showed significantly higher TSAb activity than PEC 12.5 % PF as reported p reviously, The augmentative effect of PEG on TSAb activity was also observe d by the addition of 5% PEG to serum. We also demonstrated that PEG augment ed TSAb-activities even in TSBAb-positive serum by two methods (crude IgG u sing PEG 22.5 % PF and the addition of 5 % PEG to serum). TSBAb activities were expressed by two calculation methods(A = [1 - (a - b) / (c - d) x 100] and B = [1 - (a - d) / (c - d) x 100], where a is cAMP produced in the pre sence of bTSH and patient's IgG, b is cAMP produced in the presence of pati ent's IgG, c is cAMP produced in the presence of bTSH and normal IgG, and d is cAMP produced in the presence of normal IgG). In the presence of TSAb, the values of A method were always higher than those of B method, since TSA b stimulated cAMP synthesis. We have developed two sensitive methods to det ect TSAb even in the presence of TSBAb in serum using PEG; 1) incubation of crude IgG using PEG 22.5 % PF from serum (0.2 ml), and 2) co-incubation of 5 % PEG with test serum (50 mul).