Using epitope-aequorin conjugate recognition in immunoassays for complex proteins.
Recombinant techniques are used to fuse biologically important molecules or peptides to the N-terminus of the photoprotein aequorin such that the binding characteristics of the molecule and the bioluminescent activity of aequorin are retained. This work demonstrates that the peptide region of a bulky protein can be used to develop an assay for the protein. A heterogeneous competitive binding assay was first developed for HPC4 epitope, the binding region of protein C, using HPC4-apoaequorin conjugate. It was observed that the binding of HPC4 epitope to its monoclonal antibody and the bioluminescence properties of aequorin were retained in the fusion protein. The same strategy and the same fusion protein were used to develop the assay for protein C. This project could potentially be a model for large biomolecules utilizing only the binding region of the protein in the labeled analyte. Also, this assay can be used in clinical diagnostics for the quantitative detection of protein C.
Desai
U A
UA
Department of Chemistry and Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Wininger
J A
JA
Lewis
J C
JC
Ramanathan
S
S
Daunert
S
S
D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D007118:Immunoassay / Q000379:methods*; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000925:Anticoagulants; D000939:Epitopes; D010455:Peptides; D011486:Protein C; D011993:Recombinant Fusion Proteins; D000331:Aequorin