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Development of a rapid enzyme immunoassay for the detection of retinol-binding protein^1,2,3

¹ From the Program for Appropriate Technology in Health, Seattle (JH, JM, and MT); the Institute of Nutrition for Central America and Panama, Guatemala City (CM); Vista Diagnostics, Kirkland, WA (IB); the Johns Hopkins School of Medicine, Baltimore (AS); and Helen Keller Worldwide, Jakarta, Indonesia (AS).

Background: Retinol-binding protein (RBP) was chosen as a surrogate marker for retinol because of the close correspondence between retinol and RBP.

Objective: To meet the need for rapid, cost-effective determination of vitamin A status in populations, a quantitative enzyme immunoassay (EIA) for detection of RBP was developed.

Design: The resulting RBP EIA, a competitive assay, uses RBP adsorbed to microtest strip wells to compete with RBP in serum. The assay takes 40 min.

Results: With a reference panel of sera, test accuracy was found to be within 4% of expected values through the calibrated range of 0.48–1.92 µmol RBP/L (10–40 µg RBP/mL). Intraassay and interassay variability averaged 6.7% and 8.9%, respectively. Specificity testing showed no interference from other serum proteins, prealbumin, rheumatoid factor, bilirubin, estrogen, or C-reactive protein. The RBP EIA provided linear results between 0.43 and 1.80 µmol RBP/L (9 and 38 µg RBP/mL). Preliminary laboratory evaluations indicated that the RBP EIA correlates well with radial immunodiffusion for RBP and with HPLC for retinol, the current reference standard. A field evaluation in a population at risk for vitamin A deficiency (VAD) resulted in close correlation between RBP EIA measures and retinol measures by HPLC (R² = 0.82).

Conclusions: The RBP EIA is as reliable in estimating VAD as is HPLC retinol. After successful validations, the test should enable public health authorities to rapidly monitor VAD and track vitamin A status in populations.

Key Words: Retinol-binding protein • vitamin A deficiency • enzyme immunoassay • vitamin A status • serum retinol

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