DEVELOPMENT AND OPTIMIZATION OF AN ELISA TEST SYSTEM FOR THE QUANTITATIVE DETERMINATION OF ALPHA-FETOPROTEIN
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Authors
S. Kan
M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Dosmukhamedov str. 86, 050012, tel. +7(727) 2937092, Almaty, Kazakhstan
R. Tleulieva
M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Dosmukhamedov str. 86, 050012, tel. +7(727) 2937092, Almaty, Kazakhstan
N. Abdolla
M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Dosmukhamedov str. 86, 050012, tel. +7(727) 2937092, Almaty, Kazakhstan
N. Nurgalieva
M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Dosmukhamedov str. 86, 050012, tel. +7(727) 2937092, Almaty, Kazakhstan
Yu. Perfilyeva
M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Dosmukhamedov str. 86, 050012, tel. +7(727) 2937092, Almaty, Kazakhstan
Ye. Ostapchuk
M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Dosmukhamedov str. 86, 050012, tel. +7(727) 2937092, Almaty, Kazakhstan
Abstract
This study presents the development of a prototype enzyme-linked immunosorbent assay (ELISA) test system for the quantitative determination of human alpha-fetoprotein (AFP) using monoclonal antibodies (mAbs). Hybridoma cell lines 1E3 and 1C7 were recovered after long-term storage, followed by cultivation, recloning, and evaluation of antibody production stability. Both 1E3 and 1C7 lines were shown to maintain stable secretion of highly specific anti-AFP monoclonal antibodies.
Preparative quantities of antibodies were obtained both in vitro and in vivo. The concentration of mAbs exceeded 1 mg/mL, and their specific activity was confirmed by indirect ELISA. Based on mAbs 1C7 and 1E3, key parameters of a sandwich ELISA were optimized, including selection of the capture antibody, determination of the optimal immobilization concentration, and establishment of the working dilution of the horseradish peroxidase-conjugated detection antibody. mAb 1C7 was selected as the capture antibody, as it provided higher signal intensity and improved linearity of the calibration curve.
The developed test system demonstrated a specific correlation between optical density and AFP concentration within the studied range, confirming the functional suitability of the selected antibody pair for the development of a diagnostic kit. The obtained results provide a foundation for further optimization of assay conditions aimed at improving analytical sensitivity and reproducibility.
Keywords
alpha-fetoprotein, enzyme-linked immunosorbent assay, diagnostics, test system
Article Details
References
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