33株抗心肌肌钙蛋白T (cTnT) 的单克隆抗体制备、鉴定及应用

Development, identification and application of 33 monoclonal antibodies against cardiac troponin T

摘要:

旨在制备抗心肌肌钙蛋白T (cTnT) 的单克隆抗体 (mAb)，对单抗进行初步评价鉴定，并建立 (cTnT) 的化学发光定量检测试剂。首先利用外购的cTnT抗原免疫BALB/c小鼠，利用常规mAb制备技术和间接ELISA法筛选mAb，以表达和合成的cTnT片段对筛选到的 mAb进行表位鉴定。使用双抗体夹心ELISA方法筛选检测cTnT抗原的配对mAb，并建立cTnT全自动化学发光定量检测试剂。使用220例临床标本评价该试剂与罗氏试剂的检测一致性，另外使用238例临床样本和784例体检人群样本评价该试剂的临床应用。我们成功筛选到33株稳定分泌抗cTnT抗体的杂交瘤细胞株，并对单抗的表位进行初步鉴定。我们筛选到能检测10 pg/mL cTnT抗原的配对mAb E16H8和C8G11，并使用该配对研制出全自动化学发光定量试剂。该试剂与罗氏试剂相关系数r达到0.959 9，检测一致率95%，利用该试剂盒检测临床样本灵敏度为97.5%，特异性为99.15%，99%体检人群的cTnT浓度分布小于0.080 6 ng/mL，符合WHO对急性心肌梗死的定义标准。综上，初步建立了cTnT诊断优势表位单抗，并利用这些优势表位的单抗建立全自动管式化学发光定量检测试剂，与罗氏试剂检测结果符合率高。

Abstract:

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs’ epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche’s was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche’s.