Quantitation of serum retinol-binding protein by an enzyme-linked immunosorbent assay

Abstract

An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. 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 (antigen), alkaline phosphatase linked to the same antibody, and p-nitrophenyl phosphate (substrate). Colorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coefficients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assay can be performed in less than 7 h and has a sensitivity in the nanogram range (3–48 ng/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50–398 μg/ml) and urine RBP levels (mean 14, range 1–80 μg/ml) than normal donors (mean serum 43, range 30–60 μg/ml; mean urine RBP 0.06, range 0.04 – 0.13 μg/ml). Liver disease patients had lower than normal serum RBP values (mean 22, range 10–43 μg/ml).