Biomedical Chemistry: Research and Methods, 2018, 1(4), e00079

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

T. A. Druzhkova1*, A. A. Shpak2, K. I. Kozlova2, A. A. Yakovlev1,3, A. B. Guekht1, N. V. Gulyaeva1,3

1Moscow Research and Clinical Center for Neuropsychiatry, Moscow Healthcare Department,
43 Donskaya street, Moscow, 115419 Russia; *e-mail: druzhkova.tatiana@mail.ru
2The S. Fyodorov Eye Microsurgery Federal State Institution,
Moscow, 59a Beskudnikovsky bulvar, Moscow, 127486 Russia
3Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences,
5A Butlerova street, Moscow, 117485 Russia

Keywords: ciliary neurotrophic factor; lacrimal fluid; sandwich enzyme immunoassay technique

DOI:10.18097/BMCRM00079

The whole version of this paper is available in Russian.

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.
Figure 1. Calibration curves obtained by diluting the standard solution with different diluents.
1 - RD6-3, 2 - PBS +4% BSA, 3 - PBS +1% BSA, 4 - PBS +0,1% BSA, 5 - RD5P.
Figure 2. . Increase in detectable lacrimal CNTF concentration (pg/ml) after acid treatment, %.

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Table 1. Parameters of the enzyme immunoassay system using different diluents.

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Table 2. Lacrimal CNTF concentration (pg/ml) in samples with and without acid treatment at different dilutions.

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Table 3. Reproducibility of control measurements obtained on the 0 to 250 pg/ml calibration curve with PBS and 1% BSA diluent.

ACKNOWLEDGEMENTS

This study was supported by the Russian Foundation for Basic Research, grant № 18-015-00355.

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