Preparation and Purification of High Specific Activity Insulin-131iodine

Abstract

The preparation of high specific activity insulin-131I in a relatively pure state is essential for the immuno-assay of plasma insulin (Berson & Yalow, 1960). The micro\x=req-\ diffusion technique (Banerjee & Ekins, 1961) has provided a safe method for preparing insulin-131I with relatively little chemical damage but is limited in iodination efficiency to a theoretical maximum of 50%. Hunter & Greenwood (1962) have prepared human growth hormone-131I, using chloramine T as the oxidizing agent, with an iodination efficiency over 60%.One of the main difficulties in preparing high specific activity insulin-131I by such methods has been the associated chemical damage of the insulin due to direct contact of the hormone with the oxidizing agent. This would be overcome if a suitable purification technique could be found. A new method of purification of the insulin\x=req-\ 131I has been developed in which the damaged iodinated insulin is removed by buffer elution on a suitable grade dextran column. In this way, using chloramine oxi¬ dizing system and dextran column purification technique, it has been possible to prepare insulin-131I with a specific activity of 1 c/mg., the final product containing a damaged fraction of < 3 %.Freshly dissolved crystalline beef insulin (24-5 u./mg., Burroughs Wellcome Ltd) 2-5 µg. in 0-025 ml. borate buffer, pH 8-0,4-5 mc (0-05 ml.) carrier free, and thiosulphate free 131I in sodium hydroxide (Radiochemical Centre, Amersham) were mixed with 50 µg. chloramine (British Drug Houses Ltd) in 0-025 ml. borate buffer for 1 min. The reaction was terminated by adding 120/xg. sodium metabisulphite in 0-1 ml. buffer. The reaction was carried out in a 10 ml. stoppered glass tube in a glove box. The free 131I was removed by dialysing the iodinated-insulin mixture against distilled water for 2 hr. at 4°c. 1 ml. Fresh human serum was incubated with 0-5 % iodoacetamide for 30 min. at room temperature. The treated serum was stained with 0-01 % bromphenol blue indicator in barbitone buffer pH 8-5 (v:v/5:l) and was then dialysed against 11. barbitone buffer for 2 hr. to remove any excess dye and iodoacetamide. 0-1-0-2 ml. of the stained serum was added to 0-3-0-4 ml. of the iodinated insulin solution as the binding protein for the damaged iodo-compounds. The sample was passed through a 1 g. Sephadex G-75 medium grade (Pharmacia, Uppsala, Sweden)

SELECTIVE UPTAKE OF RADIOACTIVITY BY THE ADRENAL CORTEX OF DEXAMETHASONE-TREATED GUINEA-PIGS AFTER THE ADMINISTRATION OF 131i-Labelled Α1–24 ADRENOCORTICOTROPHIN

“…125I-Labelled porcine insulin. Crystalline porcine insulin (Lilly) was labelled with [125I]iodine (IMS3, Radiochemical Centre, Amersham) by the method of Banerjee & Gibson (1962). The labelled material was analysed by chromatoelectrophoresis (Berson & Yalow, 1968) and by trichloroacetic acid precipitation.…”

Insulin response to a rapid intravenous injection of dextrose in patients with anorexia nervosa and obesity

“…Tracer insulin was iodinated with 131I using the chloramine T method (Banerjee & Gibson, 1962;Hunter & Greenwood, 1962). The antibodies to pork insulin and to guinea-pig gamma globulin used in the assay were prepared in the Middlesex Hospital Medical School using the technique described by Robinson & Wright (1961).…”

Studies on Experimental Insulin Immunity: I. Dynamics of Insulin Immunity in the Guinea Pig

“…If not immediately used, the antisera obtained from these rabbits were frozen and stored at -18 0 C. The titer of the antisera was obtained by measuring the total protein content of the precipitate, 48 or by a radioisotopic technic, 49 using I-125 labeled guinea pig y, | S and a globulins. 50 For the insulin antibody assay radioactive insulin was diluted with a 1 per cent bovine serum albumin in borate buffer (^H 8.5, 0.13M) and with carrier crystalline beef insulin; the final concentration of insulin-I-125 w a s 3-8 uc. per milliliter, and insulin concentrations were adjusted to 1.0, 0.1 and 0.01 U. per milliliter with the carrier insulin.…”

“…To determine conditions necessary for the quantitative precipitation of the antibodies with R-AGPG or R-AGPS, I-125-labeled guinea pig 3 and y globulins were prepared according to the method of Banerjee and Gibson, 50 after the separation of normal guinea pig serum fractions by column electrophoresis. Various amounts of the R-AGPG were added to these globulin preparations.…”