Measuring Ciliary Neurotrophic Factor (CNTF) in Lacrimal Fluid Using Optimized Commercial ELISA

Abstract

The concentration of ciliary neurotrophic factor (CNTF) was measured in lacrimal fluid (LF) using Human CNTF Quantikine ELISA kit (“R&D Systems”, USA) on a ChemWell 2910 automatic analyzer (“Awareness Technology Inc.”, USA). We initially attempted to use commercial kits, designed for serum and plasma CNTF detection, to quantify lacrimal CNTF. The results, however, were rarely above the minimum detection level of the kits, most likely due to matrix complexity and low concentrations of CNTF in diluted LF (LF had to be diluted because of the small volume of collected samples). The optimal sensitivity and the lowest background for the best minimum quantifiable value were determined empirically. Phosphate buffer solution containing 1% bovine serum albumin was selected as an optimal diluent for CNTF measurements in small fluid samples. A standard curve was produced using the calibrating solutions 0-250 pg/ml. Acid treatment of LF samples before the analysis allowed to increase the detectable concentration of the CNTF two-fold. The 1:3 dilution was selected based on the available volume of collected LF and a reasonable variation coefficient. The described protocol allowed to develop a sandwich ELISA optimized for lacrimal CNTF.