Assay procedure optimization of a rapid, reusable protein C immunosensor for physiological samples.

Abstract

A fiber-optic immunosensor for quantifying the protein C (PC) amount in a plasma sample is being developed to provide accurate, fast, cost-effective diagnosis of heterozygous PC deficiency (0.5-2.5 microg ml(-1)). As a progress report on the sensor development, this paper focuses on optimizations of vanous assay steps. These include: (1) the primary antibody concentration; (2) the effect of the primary antibody leaching on the sensitivity; (3) the fluorophore to secondary antibody ratio; and (4) the sample and secondary antibody incubation times. The optimal primary antibody concentration for the PC sensor was determined to be 65 microg ml(-1); its leaching was minor, stabilized within 3 days, and the sensor sensitivity change after 30 days of storage was minor; sample and secondary antibody incubation times were reduced from 10 to 5 and from 5 to 3 min, respectively, while maintaining a reasonable signal-to-noise level. The immunosensor was also tested with (5) human serum albumin and human plasma and (6) with and without convective flow. High viscosity of human plasma decreased signal intensity, but clear signal discrimination was possible in the concentration range of interest. (7) Application of convective flow increased the signal intensity by increasing PC mass transport rate to the sensor surface. The optimized PC immunosensor provides a smaller (100 microl sample chamber) and faster (10-15 min) tool for PC detection in human plasma.