Heterogeneous bioluminescence binding assay for an octapeptide using recombinant aequorin

An assay was developed for an octapeptide by using recombinant aequorin as the label. Aequorin (AEQ) is a bioluminescent protein found in the jellyfish (Aequorea victoria). A plasmid was designed by fusing the gene of AEQ to an oligonucleotide sequence that codes for the octapeptide of interest. The plasmid was transformed into Escherichia coli, and the octapeptide–aequorin fusion protein was expressed and purified. The properties of the octapeptide–aequorin conjugate were studied, and it was observed that the fusion protein retained the bioluminescence characteristics of native aequorin. The octapeptide–AEQ fusion protein was employed in the development of a heterogeneous bioluminescence immunoassay for the octapeptide. The detection limit of the assay was 5×10−10M. This study also demonstrates that aequorin can be used as a sensitive label in bioluminescence immunoassays for small biomolecules even when the binding constant between the biomolecule and its corresponding antibody is relatively low.