Ca 2+ -triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay

The recombinant Ca 2+ -triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems. Figure 3D structures of obelin D12C ( a ) and CBP ( b ) (N ends), displaying the introduced SH group. c Plots of bioluminescence of obelin versus the amount of obelin ( open triangles ) and RmLuc versus the amount of CBP ( open circles ). d Simultaneous bioluminescence immunoassay of luteinizing hormone (LH; obelin signals, filled triangles ) and follicle-stimulating hormone (FSH; RmLuc signals, filled circles ) in standard sera