Abstract
A method has been developed for the determination of free and antibody-bound insulin levels in the serum of patients with anti-insulin antibody.
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.
The standard solutions were prepared by dissolving lyophilized porcine insulin in human serum containing low level of IRI.
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%).
