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目的:制备氯胺酮单克隆抗体(McAb),并对其特性进行分析。方法:采用丁二酸酐偶联法将氯胺酮(Ket)与BSA和0VA偶联合成人工完全抗原Ket-BSA和Ket-OVA,选择Ket-BSA免疫BALB/C小鼠,用Ket-OVA作包被抗原进行间接ELISA和竞争ELISA检测免疫小鼠血清效价,从而选择小鼠脾细胞为融合备用。应用杂交瘤技术制备氯胺酮单克隆抗体(Ket-McAb),并对其效价、亲和性和特异性等进行鉴定。结果:成功地合成了氯胺酮人工完全抗原,用该抗原免疫小鼠产生高效价抗Ket抗体(1:12800),并能被Ket单体抑制;建立了一株分泌稳定的单克隆抗体细胞株,细胞培养上清液的抗体效价为1:6400,腹水抗体效价为2×106;同种型为IgG2a,50%抑制浓度为80ng/ml,经鉴定能与Ket特异性结合与其它毒品类药物如冰毒、摇头丸,大麻、吗啡等无交叉反应。结论:抗氯胺酮McAb是一种特异的探针,对吸毒和戒毒病例的检测具有潜在的应用价值。
Objective: To prepare monoclonal antibody (McAb) of ketamine and analyze its characteristics. Methods: Ket-BSA and Ket-OVA were synthesized by conjugating ketamine (Ket) with BSA and 0VA by succinic anhydride method. BALB / C mice were immunized with Ket-BSA and coated with Ket-OVA Antigen indirect ELISA and competitive ELISA were used to detect the serum titer of the immunized mice so as to select the mouse spleen cells for fusion. Ketamine monoclonal antibody (Ket-McAb) was prepared by hybridoma technology, and its potency, affinity and specificity were identified. RESULTS: Ketamine artificial total antigen was successfully synthesized. The mouse anti-Ket antibody (1: 12800) was immunized with the anti-Ket antibody and inhibited by Ket monomer. A monoclonal cell line secreting stable monoclonal antibody was established. The antibody titer of the cell culture supernatant was 1: 6400, the ascites antibody titer was 2 × 106, the isotype was IgG2a, and the 50% inhibitory concentration was 80 ng / ml. It was identified to bind specifically to Ket with other drugs Drugs such as methamphetamine, ecstasy, marijuana, morphine and other cross-reactions. CONCLUSION: Anti-ketamine McAb is a specific probe that has potential value in the detection of drug addiction and drug addiction.