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目的:制备抗脑钠肽(BNP32)单克隆抗体(mAb),并利用双抗体夹心ELISA法建立BNP抗原检测技术,应用于临床心脏患者脑钠肽水平的检测。方法:以基因工程原核重组表达BNP抗原免疫BALB/c小鼠,利用常规杂交瘤技术制备mAb,mAb经纯化和HRP标记后,利用双抗体夹心ELISA法筛选检测BNP32蛋白的最佳配对mAb,以其建立BNP32抗原检测技术,并与临床BNP检测的标准实验做平行比较。结果:成功筛选到16株稳定分泌抗BNP32mAb的杂交瘤细胞株,16株mAb的亚型分别为IgG1、IgG2a和IgM,并从中筛选出最佳mAb配对组合,该组合对BNP32蛋白的检测灵敏度为20ng/L。建立的双抗体夹心BNP检测ELISA法与临床BNP检测的标准实验平行比较具有很好的一致性(kappa值=0.828),两者没有统计学意义(P>0.05)。结论:成功地建立了BNP32抗原的双抗体夹心ELISA法检测技术,并能够很好地运用于临床心衰患者BNP指标的检测。
Objective: To prepare anti-brain natriuretic peptide (mAb) monoclonal antibody (mAb) and use the double antibody sandwich ELISA to establish the detection technology of BNP antigen for detection of brain natriuretic peptide in clinical cardiac patients. Methods: BALB / c mice were immunized with prokaryotic recombinant prokaryotic expression of BNP antigen. MAb was prepared by conventional hybridoma technique. The mAb was purified and labeled with HRP. The best matched mAb of BNP32 protein was screened by double antibody sandwich ELISA The establishment of BNP32 antigen detection technology, and with clinical BNP standard test to do parallel comparison. RESULTS: Totally 16 hybridoma cell lines secreting anti-BNP32 mAb were successfully screened. The subtypes of 16 mAbs were IgG1, IgG2a and IgM, and the best mAb pairing combination was screened out. The sensitivity of this combination to BNP32 protein was 20ng / L. The established double antibody sandwich BNP detection ELISA and clinical BNP standard test parallel comparison has good consistency (kappa = 0.828), both of which have no statistical significance (P> 0.05). Conclusion: The double antibody sandwich ELISA assay of BNP32 antigen was successfully established and can be applied to the detection of BNP index in patients with clinical heart failure.