Fluorescence immunoassay of HDL and LDL using protein A LB film

초록

A fluorometric detection technique for HDL (High Density Lipoprotein) and LDL (Low Density Lipoprotein) was developed for application in a fiber-optic immunosensor using a protein A Langmuir-Blodgett (LB) film. For the fluorescence immunoassay, antibodies specific to HDL or LDL were immobilized on the protein A LB film, and a fluorescence amplification method was developed to overcome their weak fluorescence. The deposition of protein A using the LB technique was monitored using a surface pressure-area (pi -A) curve, and the antibody immobilization of the protein A LB film was experimentally verified. The immobilized antibody was used to separate only HDL and LDL from a sample then the fluorescence of the separated HDL or LDL was amplified. The amount of LDL or HDL was measured using the developed fiber optic fluorescence detection system. The optical properties resulting from the reaction of HDL or LDL with o-phtaldialdehyde, detection range, response time, and stability of the immunoassay were all investigated. The respective detection ranges for HDL and LDL were sufficient to diagnose the risk of coronary heart disease. The amplification step increased the sensitivity, while selective separation using the immobilized antibody led to linearity in the sensor signal. The regeneration of the antibody-immobilized substrate could produce a stable and reproducible immunosensor.

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