Flow injection renewable surface immunoassay for real time monitoring of biospecific interactions.
An automated system for performing rapid immunoassay, kinetic measurements, and affinity ranking of biomolecular interactions using fluorescence-labeled ligands is described. Its distinctive feature is the automated renewal of solid phase for each measurement, which avoids the need for regeneration of the sensing surface. This system--flow injection renewable surface immunoassay (FIRSI)--is used for the first time here for determination of rate constants for an antibody/antigen interaction and for affinity ranking of several related antigens against one antibody. The performance of the system is compared with a commercial BIAcore system that uses surface plasmon resonance for monitoring biomolecular interactions. While the values of association and dissociation rate constants for human serum albumin (HSA) with monoclonal anti-HSA antibody obtained by these techniques were comparable, it is shown that the FIRSI techniques requires simpler instrumentation, handles a broader size range of analytes, and does not suffer from disturbances caused by changes in the refractive index.
Willumsen
B
B
Department of Chemistry, University of Washington, Seattle 98195, USA.
Christian
G D
GD
Ruzicka
J
J
D000818:Animals; D001322:Autoanalysis; D002417:Cattle; D017022:Flow Injection Analysis; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D008024:Ligands; D010455:Peptides / Q000032:analysis
D005456:Fluorescent Dyes; D008024:Ligands; D010455:Peptides