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本研究拟建立肌酸激酶同工酶MB(CK-MB)特异性单克隆抗体(m Ab)的研制方法,对抗CK-MB单抗进行评价分类及性质鉴定,并初步建立CK-MB定量检测试剂。以CK-MB抗原免疫BALB/c小鼠,利用常规单抗制备技术,使用间接和捕获ELISA差异筛选法筛选单抗。利用肌酸激酶同工酶(CK-MM/BB/MB)抗原对所制备单抗的抗原识别表位进行鉴定,另通过免疫印迹法及合成CK-MM、CK-BB差异性的线性表位肽鉴定对所制备的单抗进行评价分类。使用双抗体夹心ELISA方法筛选检测CK-MB抗原的配对m Ab,并初步建立CK-MB定量检测试剂。使用74例临床标本初步评价该试剂与罗氏试剂的检测一致性。最终,我们成功筛选到22株稳定分泌抗CK-MB抗体的杂交瘤细胞株,这些单抗可以分为线性、偏构象的CK-MB和CK-MM或者CK-BB交叉的单抗以及与CK-MB特异反应的偏构象型单抗,并使用偏构象型单抗研制出CK-MB定量检测试剂,该试剂与罗氏试剂相关系数r达到0.930 9。综上所述,本研究建立了研制CK-MB偏构象型特异性单抗的筛选方法,通过对所筛选的单抗进行分析鉴定并建立了CK-MB定量检测试剂,与罗氏试剂检测结果符合率高。
In this study, it is proposed to develop a method for the determination of creatine kinase MB (CK-MB) specific monoclonal antibody (m Ab), to evaluate the classification and characterization of CK-MB McAb, and to establish a quantitative CK- Reagents. BALB / c mice were immunized with CK-MB antigen and monoclonal antibodies were screened by indirect and capture ELISA differential screening using conventional monoclonal antibody preparation techniques. The antigenic epitopes of McAbs were identified by CK-MB / BB / MB antigenicity. The immunoprecipitation and the linear epitopes of CK-MM and CK-BB Peptide identification The prepared monoclonal antibodies were evaluated for classification. The double-antibody sandwich ELISA method was used to screen the matched m Ab for detection of CK-MB antigen, and a reagent for quantitative detection of CK-MB was initially established. 74 cases of clinical specimens preliminary evaluation of the reagent and Roche reagent detection consistency. Finally, we successfully screened 22 hybridoma cell lines that stably secrete anti-CK-MB antibodies. These mAbs can be divided into linear, partial conformational CK-MB and CK-MM or CK-BB cross-linked mAbs, -MB specific reaction of hemizygous MAb and the use of partial conformation mAb developed CK-MB quantitative detection reagent, reagent and Roche reagent correlation coefficient r reached 0.930 9. In summary, this study established a method for screening CK-MB partial conformer specific McAb, by screening the identified monoclonal antibody identified and established CK-MB quantitative detection reagent, and Roche reagent test results in line with High rate.