Abstract
Radioimmunoassay for human TSH by means of double antibody technique was investigated.
Human pituitary thyrotrophic hormone standard was a gift from the National Institute for Medical Research, London, and both human TSH for a radioiodination and anti-human TSH rabbit serum were gifts from the National fnstitute of Health, Bethesda, Md., U. S. A.
Labeling of human TSH was achieved with 125I in accordance with Hunter and Greenwood's method. Isolation and purification of the labeled HTSH were performed by using Sephadex G-50 and G-75 chromatocolumn.
In a preliminary study, the optimal ratio between labeled hormone and antiHTSH serum in various concentrations was determined by DEAE paper chromato-electrophoresis, and the ratio for 1: 200,000 dilution of antiserum vs. 20,000 cpm of labeled hormone or 1: 300,000 dilution of antiserum vs. 15,000 cpm of labeled hormone were adopted for the radioimmunoassay.
As a rule, the immunoassay was performed in accordance with the method reported by Odell et al., however the following two points were modified. Firstly, purified bovine serum albumin (Armour) was added to a buffer solution (0.01 M phosphate, 0.15 M NaCl, 0.1 % Na-azide) instead of normal rabbit serum, and 0.1 ml of normal rabbit serum was added when anti-rabbit γ goat serum was added to complete the assay system, in much the same manner as radioimmunoassay methods for other hormones such as insulin and growth hormone. Secondly, 0.1 ml of various concentrations of standard TSH was added to each assay system instead of adding varied volumes from a constant HTSH solution.
1. Fundamental investigations. Sufficient counts percent in B/T was not obtained in normal rabbit serum added buffer system, and the method reported by Hales and Randle failed to give any good results. A high sensitive standard curve was obtained by addition of EDTA solution, and a higher sensitive standard curve was also obtained when labeled hormone was added 24 hours after addition of non-labeled HTSH as reported by Odell et al., as compared with simultaneous addition of both labeled and non-labeled HTSH.
No influence was seen on the sensitivity of the standard curve by an addition of TSH negative serum to the assay system.
A dilution test of hyper-TSH serum showed an exact relation.
2. Clinical investigation.
Hyperthyroidic sera showed the lowest TSH level, and relatively high levels were seen in Hashimoto's and chronic thyroiditis and the highest levels were seen in hypothyroidic sera. A negative correlation between TSH levels and T7 values was seen in various thyroid diseases.
Little or no change in TSH levels was seen in comparison between pre and post treated patients with hyperthyroidism, while a remarkable decrease of TSH levels was observed in hypothyroidic patients after administration of desiccated thyroid powder.
Acromegaly, diabetes insipidus and dwarfism were analysed, and the TSH level in cord serum showed higher values than in pregnant serum.
Influences of administrations of vasopressin, glucagon, adrenocortical steroid, and synthetic ACTH upon TSH and corticosteroid levels in serum were investigated. Elevations in corticosteroid levels were observed in synthetic ACTH and glucagon administration, and on the other hand, no influence has so far been seen in TSH levels in the serum.
