Effectiveness of protein A for antibody immobilization for a fiber optic biosensor

Abstract

The fiber optic biosensor performs fluoroimmunoassays at the surface of multimode optical fibers. The effectiveness of protein A, an immunoglobulin binding protein, for antibody immobilization on the surface of these fiber probes has been investigated. No difference was observed in the binding of fluorescently-labeled goat-IgG by rabbit anti-goat IgG regardless of whether the capture antibody was bound to the probe surface via protein A or covalently attached. However, in a sandwich immunoassay for the F1 antigen of Yersinia pestis, probes with rabbit anti-plague IgG bound to the surface via protein A generated twice the signal as probes with the antibody covalently attached. Assay regeneration was also examined with protein A probes since antibody-antigen complexes have been successfully eluted from protein A under low pH conditions. Protein A probes coated with rabbit anti-goat IgG obtained nearly identical signal levels at 500 and 5000 ng/ml of Cy5.5 goat IgG five consecutive times following regeneration with glycine-HCl, 2% acetic acid, pH 2·5.