Examination of Some Factors Affecting Sensitivity and Reproducibility in Radioimmunoassay of Thyrotropin

Abstract

Conditions for measuring human thyrotropin by radioimmunoassay have been investigated, to improve the sensitivity and reproducibility of the assay. The Chloramine-T method of iodination was used, the reaction time being 20 s. Doubling the iodination reaction volume from 55 to 95 mul did not affect the immunoreactivity. Purification of labeled hormone by use of anion-exchange resin followed by silica (Quso G-32) instead of Sephadex gel-filtration or anion exchange alone yielded a product that was the least (less than 4%) contaminated with Na125 I. Human serum albumin (2.5 g/liter) in phosphate-buffered saline, instead of bovine serum, should be used as diluent for unknowns; within-assay variance was 3% for the former, 62% for the latter. The assay worked equally well for both pregnant and nonpregnant patients, with use of 50 to 100 mul of serum per determination. A five-week-old labeled hormone yielded higher values than did two-week-old material. Use of sequential saturation techniques (tracer added on day 3) resulted in a greater than 50% drop in B/B-0 ratio in the standard curve between 0.39 and 50 microunits/ml. Somatotropin, choriomammotropin, and procine insulin did not cross react in this system. Human follitropin and lutropin did cross react, and this cross reaction could not be prevented with as much as 40 international units of human choriogonadotropin per tube. With a total reaction volume of 0.4 ml before addition of second antibody, 0.2 ml of 10-fold diluted second antibody yielded a standard curve with lower nonspecific binding and higher maximum precipitation than one constructed by using only 20 mul of second antibody. In 29 euthyroid patients the mean thyrotropin value was 5.7 microunits/ml (range 2.8-11); nine hypothyroid patients had a mean of 112 microunits/ml (range 38-267); and 13 hyperthyroid subjects showed suppressed thyrotropin with a mean of 3.1 microunits/ml (range 2.2-4.5). Following these suggestions, one can expect a more highly purified immunoreactive tracer and a more sensitive assay than is obtained with the procedure from the Pituitary Agency of the National Institutes of Health.