CAJ | 학술논문

目的：制备地西泮(diazepam, DZP)单克隆抗体,并且对制备的抗体进行一系列性质鉴定。方法利用1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐[1-ethyl-3-(3-dimethyllaminopropyl) carbodi imide hydrochloride, EDC·HCl]法合成免疫原和包被原,用免疫原免疫Balb/C小鼠,当效价到1：16000以后取小鼠脾脏与SP2/0进行细胞融合。然后采用竞争结合双阳性两步筛选法,筛选出能分泌特异性抗体的杂交瘤细胞,并且利用有限稀释亚克隆方法得到单株细胞,采用体内诱生法制备腹水型单克隆抗体。利用辛酸-饱和硫酸铵法对腹水型抗体进行纯化,利用酶联免疫吸附法、表面等离子共振仪(surface plasmon resonance, SPR)等方法对纯化后的抗体进行性质鉴定。结果成功合成了地西泮免疫原和包被原,免疫Balb/C小鼠7次后效价达到1：16000,最终制备出单克隆抗体,抗体解离常数(dissociation constants, KDs)为4.0985×10-7 mol/L,且与大部分结构类似物没有明显的交叉反应。应用此抗体建立间接竞争ELISA法,抗体的IC50=10.8 ng/mL,检测范围为0.45~862.00 ng/mL。结论本研究制备出了地西泮单克隆抗体,为地西泮的免疫学检测提供了有力的支持。
목적：제비지서반(diazepam, DZP)단극륭항체,병차대제비적항체진행일계렬성질감정。방법이용1-을기-(3-이갑기안기병기)탄이아알염산염[1-ethyl-3-(3-dimethyllaminopropyl) carbodi imide hydrochloride, EDC·HCl]법합성면역원화포피원,용면역원면역Balb/C소서,당효개도1：16000이후취소서비장여SP2/0진행세포융합。연후채용경쟁결합쌍양성량보사선법,사선출능분비특이성항체적잡교류세포,병차이용유한희석아극륭방법득도단주세포,채용체내유생법제비복수형단극륭항체。이용신산-포화류산안법대복수형항체진행순화,이용매련면역흡부법、표면등리자공진의(surface plasmon resonance, SPR)등방법대순화후적항체진행성질감정。결과성공합성료지서반면역원화포피원,면역Balb/C소서7차후효개체도1：16000,최종제비출단극륭항체,항체해리상수(dissociation constants, KDs)위4.0985×10-7 mol/L,차여대부분결구유사물몰유명현적교차반응。응용차항체건립간접경쟁ELISA법,항체적IC50=10.8 ng/mL,검측범위위0.45~862.00 ng/mL。결론본연구제비출료지서반단극륭항체,위지서반적면역학검측제공료유력적지지。
Objectives To establish a rapid and effective method for the detection of diazepam (DZP), we prepared anti-DZP monoclonal antibody and identified the characteristics of the antibody. Methods The im-munogen and coating antigen were synthesized by using the method of EDC and the mice were immunized with the immunogen until the titer reach 1:16000. To obtain the hybridoma, splenocytes from immunized mice were fused with SP2/0 under the condition of PEG4000. Then, hybridoma clones were screened by indirect complete enzyme-linked immunosorbent assay (ELISA) and the positive cell lines were subclone three times using the method of limit dilution assay. In order to prepare ascites monoclonal antibody, the positive cell lines were injected into the Balb/C mice which have been injected into paraffin oil seven days ago. The ascites mo-noclonal antibody was purified by bitterness-ammonium sulfate precipitation and the characteristics were de-termined by a method based on ELISA. Results The immunogen and coating antigen were synthesized suc-cessfully and the titer reach 1:16000 after seven times immunization. The results showed that the limit of de-tection (LOD) was 0.45 ng/mL and IC50=10.8 ng/mL. The dissociation constants (KDs) of the monoclonal an-tibody by surface plasmon resonance (SPR) was 4.0985×10-7 mol/L and the cross-reaction was not obvious ex-cept temazepam (35.4%). Conclusion The anti-DZP monoclonal antibody was prepared successfully which was very meaningful for the rapid detection of DZP.