Naturally occurring luminescent proteins as labels in the development of binding assays for small biomolecules

The naturally luminescent proteins aequorin, obelin and the green fluorescent protein (GFP) have been employed in the development of highly sensitive immunoassays for small biomolecules. Two of these proteins (aequorin and GFP) were isolated from the jellyfish Aequorea victoria located in the Pacific Northwest. Obelin was isolated from the marine polyp Obelia longissima . The cDNA has been determined for each of these proteins allowing for their genetic manipulation. We have employed recombinant DNA techniques on each of the luminescent proteins to produce protein-labeled analyte conjugates that are homogeneous in nature (e.g., one-to-one conjugates). Homogeneous populations of labeled-conjugates offer several advantages over heterogeneous populations in the development of immunoassays including superior assay performance.; Fluorescence and bioluminescence binding assays were developed for a small peptide employing fusion proteins between the peptide and the two proteins, GFP and obelin. The two proteins (GFP and obelin) act as labels to provide sensitive detection for the octapeptide. To the best of our knowledge, this is the first time that GFP and obelin have been used as quantitative labels in fusion proteins for the determination of small peptide analyte.; Additionally, a fluorescence microplate competitive binding assay based on fusion proteins between two model peptides and a red-shifted green fluorescent protein (rsGFP), as well as a blue fluorescent protein (BFP) was developed. The GFP mutant proteins act as labels to provide sensitive dual detection in the same sample of the two selected small peptides in a competitive assay format.; In order to further expand the range of genetically engineered one-to-one labeled-analyte conjugates to include nonpeptidic analytes such as drugs, vitamins, hormones, etc., we have prepared mutants of the photoprotein aequorin. We have performed polymerase chain reaction (PCR)-based site-directed mutagenesis on apoaequorin to produce 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 employing the genetically engineered aequorin mutants.