CONCENTRATION MEASUREMENT OF UNPURIFIED PROTEINS USING BIOSENSOR TECHNOLOGY UNDER CONDITIONS OF PARTIAL MASS-TRANSPORT LIMITATION

Using biosensor technology, it is possible to measure protein concentr ation when the binding of the protein to an appropriate ligand immobil ized on the sensor surface is totally limited by diffusion and mass tr ansport, a condition difficult to achieve in practice, In such a case, the observed binding rate does not reflect the intrinsic binding capa city of the molecular partners, but is simply proportional to the conc entration of the protein analyte that is introduced in a continuous ho w over the ligand, We describe here a more general biosensor method fo r measuring protein concentration which is applicable to conditions wh ere mass transport is not totally but only partially rate limiting, Th e proposed method, which is based on measurements at different how rat es, does not require a standard of known protein concentration and can be used with unpurified proteins, The method is applicable to ligand- analyte pairs with an association rate constant as low as 10(3) M-1 s( -1) and requires only knowledge of the molecular weight and diffusion coefficient of the analyte, The method was used successfully to measur e the concentration of monoclonal antibodies, monoclonal antibody frag ments (Fab) obtained by papain cleavage, and recombinant Fab fragments of widely different affinities in crude Escherichia coli extracts. (C ) 1997 Academic Press.