Sandwich elisa mouse insulin assay system

Plasma and total brain insulin content was measured using the sandwich ELISA mouse insulin assay system (Millipore) following the instructions provided by the manufacturer. Briefly, plasma or brain lysates were added to microtitre plate well pre-coated with anti-insulin antibody. After incubation and washing, a biotinylated anti-insulin antibody was added. This biotinylated antibody reacts against a distinctive epitope to that of the coated anti-insulin. The reaction was incubated for 15 minutes and the absorbance was read at 370 nm. The stop solution provided by the assay kit was added to the sample when the absorbance was read at 1.8. Immnoreactivity was immediately determined by measuring the absorbance at 450 nm and 590 nm.

The method used to measure tissue insulin level is the same as described in our recent study [19] (link). Total liver insulin content was measured using the sandwich ELISA mouse insulin assay system (Millipore) following the instructions provided by the manufacturer. Briefly, liver lysates were added to microtiter plate well pre-coated with anti-insulin antibody. After incubation and washing, a biotinylated anti-insulin antibody was added. This biotinylated antibody reacts against a distinctive epitope to that of the coated anti-insulin. The reaction was incubated for 15 min and the absorbance was read at 370 nm. The stop solution provided by the assay kit was added to the sample when the absorbance was read at 1.8. Immunoreactivity was immediately determined by measuring the absorbance at 450 nm and 590 nm.