Thyrotropin-releasing hormone radioimmunoassay in human serum and its application in thyroidal pathology

Abstract

This paper presents the methodology of a specific radioimmunoassay of the Thyrotropin-Releasing Hormone (TRH) and the results obtained in patients with thyroid disorders.

Synthetic TRH was labeled by iodine 125 at room temperature using a technique with Chloramine-T. The labeled compound was purified on a column chromatograph, and it was found the immunoreactivity of labeled-TRH was dependent on the elution fraction. Anti-TRH antibodies were obtained in rabbits after coupling the molecule with albumin using a diazotization method. Good antibody titers were obtained within 45 days.

Blood samples were collected on a EDTA-8 hydroxyquinoline sulfate-Tween 20 mixture and kept at 4° C. TRH was extracted from the serum by methanol precipitation and 60° C evaporation. The assay methodology included incubation at 4° C for 48 h, and separation of free and bound fractions was achieved by immunoprecipitation with antirabbit goat antiglobulins. This methodology gave a very high specificity and the range was from 10 to 2,000 pg/ml with an Ekins sensitivity of 20 pg/ml.

In 62 normal patients, plasma TRH mean value and SD were found to be 137±53 pg/ml. The clinical results show a significant decrease of TRH levels in hyperthyroid patients (Graves' disease and multinodular toxic goiter), and and increase in the few hypothyroid subjects examined, arguing strongly for the negative feed-back hypothesis. However, TRH levels were found to be increased in cold nodules (although with euthyroid function) and in the normal range in Plummers' disease, stating the problem of its pathogeny.

Thus, we think that this TRH radioimmunoassay is very suitable and appropriate for routine use in human patients with thyroidal disorders. In the future it should be used as a dynamic test for the investigation of the kinetics of the hypothalamic TRH release.