[en] The labelling reaction of human growth hormone (hGH) with ¹²⁵I and its chromatographic purification have been studied with emphasis on the reproducibility of the yields, quantitaTive recoveries and resulting activities. Through the accurate standardization of a monitoring technique, it is confirmed that there are no significant losses in radioactivity or protein during the labelling or purification process. By strict control of the reaction conditions a fairily good reproducibility is also obtained in the labelling of various hGH extracts with diferent ¹²⁵I shipments used after short or long storage. Finally, the specific activity (or absolute mass) of the radioiodinated protein is determined by this Analysis of the Reaction Mixture and compared to the widely used radioimmunological assay (Self-displacement). (M.A.C.)

[en] We describe here the standardization of a technique for the estimation of ¹²⁵I in the thyroid gland of laboratory workers involved with ¹²⁵I labelling techniques. It is based on a comparison with a standard curve obtained by placing in a thyroid neck phantom various calibrated standard sources of ¹²⁵I. Its sensitivity was calculated around 0,8 - 1,7 nCi. The precision of our measurements was avaliated in a range of 1.35 - 310.5 nCi and the coefficient of variation obtained varied from 2,2% to 26,3%. The accuracy of our ''in vivo'' measurements was checked by analyzing the influence of the shape and the size of the thyroid in the neck phantom. (author)

[en] We present here a technique for the purification of proteins carried out by a quantitative analytical method used in conjunction with a preparative gel electrophoresis. Both methods employ densitometric ultraviolet scanning of unstained protein bands, a procedure wich is particulary suitable for the purification and recovery of biologically active polypeptides. In short, the purified extracted protein, isolated in a segment cut out from a preparative gel, is recovered by a second (reversed) electrophoresis. We performed the extractions and recoveries of different amounts of two standard proteins (BSA and STI) and a polypeptide hormone (hGH). Our main interest, especially for the hormone is the complete protein recovery with retention of bio and immunoactivity and high purity. For the proteins tested, the mean recovery was of 93 ⁺- 5% obtaining a mean purity of 95 ⁺- 7%. We conclude that the proposed method should have interesting applications, particularly in the obtention of very pure hormones, as are needed for radioligand assays, for radiolabelling and specific antibody raising. We emphasize the simplicity and rapidity of the method (the entire preparative process: first electrophoresis, UV scanning and reversed electrophoresis can be performed in approximately six hours) and its efficiency in recovering pure proteins even on a milligram scale. We thank the support from the IAEA (4299/RB) and FINEP (43.86.0351.00) and CENE (Brazil). (author)

[en] Human growth hormone (hGH) extracted from pituitary glands is often heterogeneous, presenting, besides the native isohormone - B (IH-B) up to four different isohormones. To obtain more homogeneous preparations, essential for reproduceble radioligand assay results, the original method was modified, on a small scale, and by adding bacteriostatic and enzime inhibiting agents to all buffers (NaN₃, EDTA, Trasylol) in order to minimize isohormone formation. After 3 to 4 homogeneizations and extractions of the frozen glands (10-20), hGH is precipitated, by 50% ammonium sulphate immediatly purified by Sephadex G 100 molecular sieve chromatography and hGH fractions are lyophilized. The whole process is completed in one week. Using 20 hypophises, 1.3 mg/gland was obtained. Several labellings with 125-I were performed using this purified hGH stored for more than one year. Two labelling techniques were employed: the classical method which uses 50 μg of Chloramine T and the stoichiometric iodination, which uses limiting amounts of chloramine T. For this preparation only 1.5 μg Chloramine T were necessary to achieve the desired specific activity. (author)

No abstract available

[en] From a preparation of human growth hormone its integral variant (hGH-22K) was isolated by isoelectric focusing, having a pI of 5,20 and relative mobility (Rm) of 0,621 in the polyacrylamide gel electrophoresis. Several experiments for the characterization of the isolated variant were carried out. The immunological properties was tested by radioimmunoassay (RIE), in which the activity of the isolated variant and the activity of the total preparation were compared. The dose response-curves obtained by RIE were found to be considered parallels (p < 0,01). It was checked using the F test between the slope of the two curves. The parallelism shown the immunochemical identity of the two preparation and indicates that the separation process developed does not produce alterations in the immunological properties of the variant. (Author)

[en] Short communication

[en] Short communication

No abstract available

[en] A human thyrotropin (hTSH) extract has been prepared in the laboratory of the National Nuclear Energy Commission (IPEN-CNEN), Sao Paulo. The purified and ampouled reference preparation was first submitted to an eight-month preliminary intra-laboratory quality control. Its potency, determined via immunoradiometric assay was 58.8 ± 6.6 μIU/ampoule, while ampoule variability showed an intra-assay inter-ampoule (n = 6) coefficient of variation of 4.1%. After testing this preparation, a certain number of ampoules were distributed to each one of the 14 participating Latin American countries, to six more ARCAL VIII participating Brazilian laboratories and to the WHO Reference Laboratory (Hammersmith Hospital, London, United Kingdom). A total of 185 unknown samples were analysed by 14 different laboratories using the Latin American and NETRIA (North East Thames Region Immunoassay Unit, London, United Kingdom), hTSH reference preparations. A linear regression analysis carried out on these data presented a correlation coefficient r = 0.954 and a slope = 1.03, with a highly significant correlation (p < 0.001) between the two sets of results. The stability of the Latin American Reference Preparation was also confirmed through a 20 month intralaboratory study. The WHO Reference Laboratory found it sufficiently pure, the standard curve running parallel to a well-know commercial preparation which is calibrated against WHO-IRP-hTSH-80/558. 4 refs, 2 figs