[Standardization of a double antibody radioimmunoassay technic for the determination of growth hormone in the rat plasma and pituitary].
Bioassay techniques are not sufficiently sensitive to measure plasma growth hormone nor are they entirely specific for the growth hormone molecule. All these requirements are fulfilled by the radioimmunoassay technique. Successful performance of the GH radioimmunoassay technique is dependent upon the good quality of the protein reagents (antibodies) used and the great care taken in handling both the reagents and the labelled hormone. The described method is based on displacement of the radioiodinated (125I) hormone bound to the first antibody (Ab1 = monkey serum anti-rat's GH) by the sample hormone. The hormone Ab1 complex is precipitated by the Ab2 (rabbit serum anti-monkey gammaglobulin). The radiation of the insoluble precipitate is then counted and the hormone quantitated through a standard curve. In the present paper we dealt with Ab2 obtention, hormone radiolabelling, and the purification of the radioiodinated hormone. The accomplished specific tests showed values of 70% for the hormonal radioiodination efficiency, with 88.7% of the processed hormone being recovered in the pure state. The highest precipitability value of the system was 84.6% whereas using optimal dilutions of the labelled-hormone (1:250) and Ab1 (1:4x10(4)) this value achieved 26.9%. These results are very much in agreement with those notified in the literature. Applying the present protocol to the analysis of plasma-and anterior-pituitary-gland-GH content from young-adult Wistar male rats kept on normal feeding schedule and rest conditions the values obtained were 82 +/- 28 ng/ml (plasma) and 63 +/- 15 ng/mg (pituitary), respectively.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D005455:Fluorescent Antibody Technique; D013006:Growth Hormone / Q000032:analysis* / Q000097:blood; D007457:Iodine Radioisotopes; D008297:Male; D010903:Pituitary Gland, Anterior / Q000032:analysis*; D011863:Radioimmunoassay / Q000592:standards*; D051381:Rats
D000906:Antibodies; D007457:Iodine Radioisotopes; D013006:Growth Hormone
Padronizacão da técnica de radioimunoensaio com duplo anticorpo para determinacão do hormonio de crescimento en plasma e hipófise de rato.