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旨在制备抗心肌肌钙蛋白T(cTnT)的单克隆抗体(m Ab),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m Ab制备技术和间接ELISA法筛选m Ab,以表达和合成的cTnT片段对筛选到的m Ab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m Ab,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/m L cTnT抗原的配对m Ab 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/m L,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。
The purpose of this study was to prepare a monoclonal antibody (m Ab) against cardiac troponin T (cTnT). The mAb was initially evaluated and identified, and a chemiluminescence quantitative detection reagent (cTnT) was established. First, BALB / c mice were immunized with purchased cTnT antigen, m Ab was screened by conventional m Ab production and indirect ELISA, and expressed and synthesized cTnT fragments were used to identify m Ab epitopes. The double-antibody sandwich ELISA method was used to screen the paired m Ab for detection of cTnT antigen and the cTnT automated chemiluminescence quantitative detection reagent was established. 220 clinical samples were used to evaluate the consistency of this reagent with Roche reagent. In addition, 238 clinical samples and 784 samples from the physical examination population were used to evaluate the clinical application of this reagent. We have successfully screened 33 hybridoma cell lines stably secreting anti-cTnT antibodies and initially identified the epitopes of the monoclonal antibodies. We screened the paired m Ab E16H8 and C8G11 that can detect 10 pg / mL of cTnT antigen and developed an automated chemiluminescence quantitative reagent using this pair. The correlation coefficient r between this reagent and Roche reagent reached 0.959 9, and the detection coincidence rate was 95%. The sensitivity and specificity of this kit for detecting clinical samples were 97.5% and 99.15%, respectively. The distribution of cTnT in 99% of the subjects was less than 0.080 6 ng / m L, in line with WHO definition of acute myocardial infarction standard. In summary, cTnT was initially established for the diagnosis of dominant epitopes, and a fully automated tube-chemiluminescence quantitative detection reagent was developed using these epitope-specific monoclonal antibodies, with a high coincidence rate with Roche reagent detection results.