Immunoenzymometric assay for insulin involving column chromatography and insulin immobilized on Sepharose

This practical assay for measuring insulin involves use of a 4 X 8 mm chromatographic column. Serum samples are incubated at 30 degrees C for 1 h with beta-D-galactosidase-labeled antibody to insulin, then passed through the 0.1-mL column containing insulin immobilized on Sepharose 4B. After the column is washed to remove the bound label, the buffer in the column is replaced with a solution of o-nitrophenyl-beta-D-galactoside. The column is then incubated at 30 degrees C for 1 h, the enzyme reaction is stopped by washing the column with sodium carbonate solution, and the absorbance of the eluate is measured at 420 nm. Results obtained by this method were compared with those by a radioimmunoassay and a solid-phase enzyme immunoassay.