A Method for Assay of Insulin Using Alloxan Diabetic Hypophysectomized Mouse

Measurement of Small Quantities of Insulin-Like Activity Using Rat Adipose Tissue. I. A Proposed Procedure*

“…The bioassay procedures used have been based on the hypoglycemic effect of insulin in animals whose sensitivity was increased by endocrine manipulation (1)(2)(3) or on the effects of insulin on isolated tissue, in particular the mammary gland of lactating rats (4), and the diaphragm of rats (5)(6)(7)(8) or mice (9). The largest body of information currently available concerns procedures based upon the effect of insulin on glucose uptake by rat diaphragm muscle (10).…”

“…Without chilling, and with a minimum of handling, each pad was then taken up at its base and further cut into three equal segments. 2 As it was cut, each segment was dropped into its appropriate preweighed flask which had been kept stoppered. The segments were so distributed as to lead to the "balanced segment design" described below.…”

“…1 For example, serum, glucose content having been brought to 250 mg per 100 ml by the addition of dry glucose. 2 The segment closest to the base (i.e., closest to the epididymis) is termed the "proximal" segment, followed by the "medial" and the "distal" segments. The proximal segment did not include the portion which had been handled with forceps.…”

Method of Extraction and Bio-assay of Insulin in Blood

“…The method to be described here is modified from one previously published (Anderson et al 14 ), in which the mouse was used. Male rats (SpragueDawley strain) six to seven weeks of age and weighing 130 to 150 gm.…”

“…The various in vitro bio-assays are based on the effect of insulin on glucose metabolism of excised tissue, such as the diaphragm of the rat 4 " 6 and mouse, 7 epididymal fat pad of the rat 8 " 10 and mammary tissue of the rat. 11 The basis of the in vivo bio-assay is a fall in blood sugar in rats 12 -13 and mice 14 rendered highly sensitive to insulin by hypophysectomy. On the other hand, the immunochemical assays for insulin are based on inhibition of hemolysis of insulin-sensitized red cells, 15 competitive inhibition of binding of labeled insulin on antibody as measured by electrophoresis, 16 and a per cent change in the binding of labeled insulin to insulin antibodies in the presence of varying quantities of unlabeled insulin.…”