Recombinant aequorin with a reactive cysteine residue for conjugation with maleimide-activated antibody.

The mutated recombinant aequorin with a reactive cysteine residue (Cys-aequorin) was highly purified and then conjugated with a maleimide-activated antibody without significant loss of luminescence activity. The conjugate ratio of Cys-aequorin to heavy chain of immunoglobulin G (IgG) was estimated to be 1:1. To test the bioluminescent immunoassay with aequorin-labeled antibody, alpha-fetoprotein (AFP), a serological marker of liver cancer, was used as a model analyte. The measurable range of AFP was 0.02 to 200 ng/ml with the coefficient of variation between 2.1 and 4.5%.