Development Of Enzyme-Linked Immunosorbent Assay For Ketamine With Phage Simulated Epitope

Ketamine, first man-made in 1962, was one of ramification of Phencyclidine. Itaimed to instead of the PCP, as a new anaesthetic to decrease the side-effect of PCP.However, ketamine abuse was quickly spreading from country to country since it wasfirst reported in San Francisco and Los Angeles in 1971. At the end of 1990s,ketamine abuse was found in Asia, such as Japan, Thailand, etc, and subsequently wasfound in China. In recent twenty years, the rate of ketamine abuse was higher andhigher year by year, especially in HongKong special administrative region. For theserious of ketamine abuse, it is necessary to build the sensitive and accuratedetermination method.In this paper, there are three parts as follows:(1) Bioscreening of the ketamine simulated epitope by Ph.D.-7^TM phage displaypeptide library. The Bioscreening was carried out with anti-ketamine monoclonalantibody as the target to isolate the ketamine simulated epitope as the antigensubstitute. After four times' biopanning, the random 10 phage clones were selectedand identified by ELISA method. 5 clones were found to compete with the ketamineand represented four sequences. The four amino acid sequences were theAsn-Leu-Pro-Ser-Thr-Thr-His, the Ala-Ala-Leu-Pro-Pro-Tyr-Arg, the Thr-Leu-Ser-Asn-Arg-Pro-Pro and the Lys-Ala-Pro-Trp -Asn-Phe-Thr.(2) Establishment of the ketamine competitive ELISA method. The optimal conditionsof the indirect competitive enzyme-linked immunosorbent assay were as follows: theconcentration of coating antigen is 0.5μg/mL, the dilution concentration of antibodyis 1: 8000 and the indirect competitive curve was obtained. The IC₅₀ and the limit ofdetection of this method are 17.55 ng/mL and 0.68 ng/mL, respectively.(3) Establishment of ketamine phage ELISA method. The optimal conditions of theindirect competitive enzyme-linked immunosorbent assay were as follows: thedilution concentration of antibody is 1:4000, the phage concentration of P1,P2,P3,P4were 1:10⁴,1:10⁴,1:10³ and 1:10³, respectively, and the optimal method was developedwith P2 clone and the indirect competitive curve was obtained. The IC₅₀ and the limitof detection of this method are 27.27 ng/mL and 1.55 ng/mL, respectively.