Increased sensitivity of triiodothyronine antibodies for radioimmunoassay after removal of endogenous antigen by simple laboratory procedures

Abstract

Conventionally produced antibodies against triiodothyronine (T₃) are known to possess high amounts of endogenously produced T₃ associated with them. We felt that such antibodies would work better for T₃ radioimmunoassay (RIA) after prior removal of the antigen. With this in view, we attempted dissociation and subsequent removal of T₃ from antisera by two different methods, viz. dialysis and alcohol extraction. It was possible to remove T₃ to an extent of 77% by alcohol extraction and 60% by dialysis. Resultant antisera fail to demonstrate any increase in the titre. However, when standard curves were generated using these antisera, the assays became more sensitive and it was possible to detect T₃ in concentrations as low as 6.25 pg. The affinity constants of these antisera calculated from the respective Scatchard plots were found to have increased after both dialysis treatment was well as alcohol extraction. This was thought to be due to rendering some of the high affinity binding sites on the antibodies free of antigen after treatment. Serum T₃ levels were measured in 68 patients with various thyroid status using both treated as well as untreated antiserum. The difference between the average values of serum T₃ concentration estimated using various antisera before and after the treatment was not statistically significant. Our results suggested that a simple procedure like stripping of antigen from antibodies could be of help for acquiring high affinity and high sensitivity antibodies for this purpose.