AN IMMUNOCHEMICAL ASSAY OF TOTAL EXTRACTABLE INSULIN IN MAN *

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Abstract
The new method for the immunochemical assay of circulating human insulin described in this report is based on the per cent change in binding of insulin-ll31 to insulin antibodies in the presence of varying quantities of unlabeled insulin. The degree of binding is measured quantitatively after preferential precipitation of bound insulin with sodium sulfite. Preliminary extraction of serum with acid alcohol resulted in an 80% recovery of insulin-ll31 regardless of the presence of endogenous antibodies. The method is therefore applicable to serum from subjects who have previously had insulin therapy. Nonspecific binding caused by extraneous proteins in the extracts was effectively removed with 15% urea. Affinity of insulin to its antibodies was pH-sensitive, being negligible at acid pH and reaching a maximum at neutral or alkaline pH. Measurements of insulin extracted from human pancreas by the described chemical method and by mouse convulsion assay were in close agreement, indicating that effective cross reaction occurs between human insulin and antibodies made against beef insulin in the guinea pig. Circulating levels of human insulin were lower than the minimum sensitivity of the assay (20 [mu]U per ml of serum). Elevated insulin levels were detected in normal subjects after oral administration of glucose and in patients with active acromegaly. Studies with the immunochemical assay demonstrated a rapid disappearance from serum of injected crystalline bovine insulin, confirming the results of others who employed the clearance of an insulin-I131 tracer. Insulin-resistant diabetics still retain large amounts of circulating insulin 24 hrs. after intramuscular injection of the hormone, despite severe hyperglycemia, glycosuria and ketonuria.