Polyethylene glycol method is superior to ammonium sulfate and protein-A sepharose-4B method in fractionating thyroid stimulating immunoglobulin in Graves' disease.

BACKGROUND

The purpose of this study is to compare the performance of three methods, i.e., ammonium sulfate, polyethylene glycol (PEG) and protein A sepharose 4B in fractionation of thyroid stimulating immunoglobulin.

METHODS

Twelve patients with Graves' disease and twelve age-, sex-matched normal controls were recruited. 0.9 ml of saturated ammonium sulfate was added slowly to 1.1 ml of sera of all subjects and was stirred until reaching 45% saturation. The precipitation was allowed to form at 4 degrees C overnight. The solution was centrifuged at 10,000 x g for 5 minutes. The precipitate was washed twice with 45% ammonium sulfate, dissolved in 0.55 ml of distilled water equal to half of the initial volume of the serum and dialyzed against 200 volumes of phosphate-buffered saline (PBS) for 48 hours. Another 0.5 ml of serum sample from the same patients was mixed with 1.5 ml of 20% polyethylene, followed by centrifugation at 2,800 g for 20 minutes. The pellet was dissolved in 0.6 ml of Hanks' medium without NaCl containing 1.5% bovine serum albumin, 20mM HEPES: 1 ml of sera was applied to 0.75 g of protein A sepharose-4B in PBS buffer and eluted with glycine, pH 2.3. By adding immunoglobulin (Ig) from the different methods to the FRTL-5 tissue culture, we determined the cAMP generation.

RESULTS

Patients with Graves' disease were found to have higher serum thyroxine and triiodothyronine concentrations (p < 0.001) and significantly higher free thyroxine concentration than normal subjects. Ig elicited stronger response in cyclic AMP generation when prepared with PEG method, as compared with the other two methods (p < 0.001). With sepharose-4B Ig preparation, 10 out 12 patients (83.33%) showed positive thyroid stimulating immunoglobulin (TSI) while with ammonium sulfate precipitation, nine out 12 patients (75%) showed positive TSI. On the other hand, all patients showed positive TSI with PEG precipitation. When the degree of thyrotoxicosis was classified in terms of thyroid hormone concentrations, there was no correlation between TSI titers and thyroid hormone concentration.

CONCLUSIONS

Polyethylene glycol is superior to both ammonium sulfate and protein A sepharose-4B in fractionating TSI in terms of sensitivity and convenience in operation.