Method Description

The glucagon enzyme-linked immunosorbent assay (ELISA) is a quantitative two-step sandwich type immunoassay. In the first step, calibrators, controls, and unknown samples are added to glucagon antibody-coated microtiter wells and incubated with biotinylated glucagon antibody. After the first incubation and washing, the wells are incubated with streptavidin horseradish peroxidase conjugate (SHRP). After the second incubation and washing step, the wells are incubated with substrate solution (tetramethylbenzidine: TMB). After TMB incubation, an acidic stopping solution is added. In principle, the antibody-biotin conjugate binds to the solid phase antibody-antigen complex, which in turn binds to the streptavidin-enzyme conjugate. The antibody-antigen-biotin conjugate-SHRP complex bound to the well is detected by enzyme-substrate reaction. The degree of enzymatic turnover of the substrate is determined by dual wavelength absorbance measurement at 450 nm as primary test filter and 630 nm as reference filter. The absorbance measured is directly proportional to the concentration of glucagon in the samples and calibrators.(Package insert: Glucagon ELISA IFU. Ansh Labs, Revision 09, 01/2023)