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

摘要

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 50pmol 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.