氯胺酮单克隆抗体的研制及其免疫学特性研究

Study on the preparation and immunological characteristics of a monoclonal antibody against Ketamine

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 and identify monoclonal antibody （McAb） specific for ketamine（Ket）. Meth- ods：Succine anhydride coupling reaction were used to compose the ketmine complete antigen Ket-BSA and Ket-OVA . Specific antibody level were measured with traditional indirect ELISA and compete ELISA in Ket-BSA immune BALB/C mice. Spleen cell were isolated from the immunized mice for cell fusion.The McAb specific for Ket were prepared via hybridoma technique. Indirect ELISA was used to determine the activity of the McAb. Agarose double immunodiffusion test was performed to confirm subclass. Results： The complete antigen of Ket was successfully prepared . The concentration of the purified antigen after elution and dialysis was 2.8mg/ml and 3.0mg/ml respectively. The antibody titre in serum from immunized mice was up to 1：12800 and could be inhibited by Ket monomer. We established a hybridome cell line with hybridoma technique, which could steadily secrete anti-Ket McAb. The antibody titre of cells cultural supernatant fluid of monoclon was up to 1： 6400 and ascites of mice was up to 1：2＊ 106 The immunoglobulin isotype of McAb was determined as IgG2a. Inhibition test showed that the inhibition rate was 50%,when the concentration of the antigen was 80ng/ml. The McAb showed very high specificity by ELISA with Ket and with other narcotic drug no cross reaction example amphetamine chloride,MDMA,morphine et al. Conclusion： The results above indicate that the McAb is of potential value in the test of drug abuse and rehabilitation.