Development of highly sensitive bioluminescent enzyme immunoassay with ultra-wide measurable range for thyroid-stimulating hormone using firefly luciferase

A bioluminescent enzyme immunoassay (BLEIA) of thyroid-stimulating hormone (TSH) using biotinylated firefly luciferase as a labelling enzyme for an antibody was developed. This immunoassay, of which method was one step sandwich method, had high sensitivity and very wide measurable range. The detection limit for TSH was found to be 0.0025µUml-1 and the measurable range for TSH was 0.0025-200µUml-1. The conjugate for this BLEIA was prepared by binding the antibody to the end of cross-linking reagent and streptavidin (SA)biotinylated luciferase complex to the opposite end of the cross-linking reagent, respectively. The stability of the conjugate was excellent. It showed 70% conjugate activity at the storage of a week at 37°C and 100% at the storage of 6 months at 4°C.