Bioluminescence immunoassay for thyroxine employing genetically engineered mutant aequorins containing unique cysteine residues.

Genetically engineered one-to-one conjugates between an analyte and a protein label have been demonstrated to yield assays with better detection limits and performance characteristics than those prepared by conventional chemical conjugation methods. To date, the preparation of these conjugates has been limited to fusion techniques where a peptide analyte is fused in frame to the protein label. To further expand the range of analytes that can be detected by using genetic engineering techniques coupled with bioanalytical methods, we have employed site-directed mutagenesis to prepare one-to-one analyte-label conjugates that include nonpeptidic analytes such as drugs, vitamins, and hormones. Specifically, we have prepared mutants of the photoprotein aequorin containing single cysteine residues suitable for site-specific conjugation. Aequorin is a photoprotein that emits light at 469 nm and has been employed as a highly sensitive bioluminescent label in the development of binding assays for important biomolecules. We have performed polymerase chain reaction-based site-directed mutagenesis on apoaequorin to yield four mutant aequorins containing unique cysteine residues at positions 5, 53, 71, and 84 in the polypeptide chain for the purpose of site-specific conjugation to a model analyte. A maleimide-activated thyroxine was selected as the model analyte and site-specifically conjugated to the mutants through their unique cysteine residues. A heterogeneous assay for thyroxine was then developed by employing the genetically engineered aequorin mutants.