Surface plasmon resonance: a study of the effect of biotinylation on the selection of antibodies for use in immunoassays.
Surface plasmon resonance is a valuable optical phenomenon for monitoring biomolecular interactions in real time. In this project anti-mouse-Fc was coupled to the carboxymethyl dextran coating on the surface of a CM5 sensor chip (BIAcore) using amine coupling. Monoclonal antibodies (MAbs) to Luteinizing Hormone (LH) were then captured on this surface in the correct orientation for binding. LH (500 IU/l) was injected over the surface and the subsequent binding and dissociation events were monitored. The resulting optical response curves allowed fast analysis of the binding interactions of eight selected MAbs. It was possible to develop a two-site immunometric assay for LH using a pair of these MAbs. The effect of biotinylating the MAbs, using various biotin:antibody coupling ratios, on their subsequent binding to both LH and avidin conjugated alkaline phosphatase was also investigated. This approach has allowed rapid evaluation of the effect of changes in both reagent and reaction conditions on immunoassay performance and appears to be a valuable adjunct to immunosensor and immunoassay development.
Pearson
J E
JE
Department of Medicine (Clinical Biochemistry), University of Manchester, Hope Hospital, Salford, UK.
Kane
J W
JW
Petraki-Kallioti
I
I
Gill
A
A
Vadgama
P
P
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D007124:Immunoenzyme Techniques / Q000379:methods; D008024:Ligands; D007986:Luteinizing Hormone / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance / Q000379:methods*
D000911:Antibodies, Monoclonal; D008024:Ligands; D001360:Avidin; D001710:Biotin; D007986:Luteinizing Hormone; D000469:Alkaline Phosphatase