[10] Use of enzyme-linked immunosorbent assay technique for quantitation of serum retinol-binding protein

This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) technique for quantitation of serum retinol-binding protein (RBP). The assay detects RBP via a double antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP, alkaline phosphatase linked to the same antibody, and p-nitrophenyl phosphate substrate. Colorimetric analysis of the hydrolysis of the substrate by the enzyme attached to the antigen is used for RBP quantitation. The ELISA assay represents an improvement over the existing methodologies for RBP analysis. With respect to the radioimmunoassay techniques, ELISA assay eliminates the need for radioactive counting and disposal, for a second antibody, and for high-speed centrifugation; the time constraint imposed by the inherent radioactive decay is also eliminated. The radial immunodiffusion technique requires a minimum of 3-days incubation time and most of all requires sample concentration for expected low ranges with possible loss of antigen.