Method for rapid conjugation of recombinant photoprotein aequorin with streptavidin and application as a universal detection reagent for binding assays Original Research Article

A binding assay utilizes the highly specific molecular recognition properties of a biological macromolecule, e.g. an antibody, receptor or DNA/RNA probe for the detection/quantification of a target analyte in a complex mixture. The recognition event is linked to a signal-producing system. Because biomolecules can be easily labeled with biotin, the biotin–streptavidin interaction has been established as a general system for linking molecular recognition with signal generation. To this end, we report a rapid and simple method for conjugation of the highly detectable recombinant photoprotein aequorin with streptavidin, thus generating a universal reagent for binding assays. The method is based on the use of aequorin fused to a hexahistidine tag at the amino terminus. Thiol groups were introduced to aequorin whereas streptavidin was derivatized with maleimide groups. The conjugate was purified in a single step by immobilized metal ion affinity chromatography, thus avoiding laborious chromatographic procedures. The performance of aequorin–streptavidin conjugate was tested in a DNA hybridization assay as a model. The limit of detection was 0.3 pmol l−1 and the analytical range extended up to 500 pmol l−1. The CV was about 8%. Besides the high detectability, the assay is rapid (completed in just 25 min) due to the flash-type (3 s) bioluminescent reaction of aequorin, which avoids substrate incubation steps that are common to binding assays employing enzyme labels.