Studies on the insulin and Anti-Insulin Antibodies in Sera of Insulin-Treated Patients:1) A Method for Determinination of Free and Total Insulin Levels in Serum by Use of Polyethylene Glycol Precipitation and Enzyme Immunoassay

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A method has been developed for the determination of free and antibody-bound insulin levels in the serum of patients with anti-insulin antibody.<BR>The assay procedure for the free insulin consists of antibody precipitation by polyethylene glycol (PEG, final 12.5%) and enzyme immunoassay (EIA) for immunoreactive insulin (IRI) in the supernatant. For the assay of total insulin (free insulin and antibody-bound insulin), serum was first acidified with HCI to dissociate bound insulin, and then PEG (final 9%) was mixed to precipitate antibody. After neutralization with NaOH, 10% BSA solution was added to compensate total protein concentration. Following centrifugation, IRI in the supernatant was measured using EIA kit.<BR>The standard solutions were prepared by dissolving lyophilized porcine insulin in human serum containing low level of IRI.<BR>The coefficients of variation of intra- and inter-assay of the free and total insulin were 5.9-19.2%, and the recovery tests were satisfactory (93.3-113.3%).
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