Sandwich elisa kit

Insulin levels in brain and plasma were determined using a commercially available sandwich ELISA kit (Alpco) designed for the quantitative measurement of human insulin and proinsulin in serum, plasma, and cell culture supernatants. Plasma samples diluted with “zero standard” and brain insulin extracts (as obtained above) were assayed per the manufacturer’s instructions. Briefly, insulin in standards, samples, and controls was captured using an anti-human insulin primary antibody coated on 8-well microplate strips (in a 96-well format). A secondary anti-human insulin antibody conjugated to biotin and HRP-conjugated streptavidin was also added along with the samples for the initial incubation. After an hour of incubation at room temperature, with shaking at 700–900 rpm on a microplate shaker, the HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added to the wells and color was allowed to develop for 30 min with further shaking. Color development was stabilized by the addition of sulfuric acid stop solution, and absorbance intensity was measured at 450 nm. The resulting stabilized color was proportional to the amount of insulin standards, and the curve was fit to a five parameter logistic fit. Insulin levels in the known diabetic control and in the samples were determined using the standard curve.