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目的获得人脑钠肽前体N端(NT-pro BNP)的高亲和力、高特异性的抗体,建立双抗体夹心ELISA法检测人血浆中NT-pro BNP。方法通过Discovery Studio4.0软件预测抗原表位,合成其表位肽偶联载体蛋白免疫Balb/c小鼠,采用杂交瘤细胞融合技术获得分泌抗体的阳性杂交瘤细胞株,制备腹水型单抗。通过SDS-PAGE、ELISA等方法对获得的抗体性质进行鉴定,建立了双抗体夹心ELISA检测方法,并对120例临床血液样本中NT-pro BNP进行快速检测。结果获得6株能稳定分泌抗人NT-Pro BNP抗体的杂交瘤细胞株,其中4株单抗的腹水型效价高于1×10~(-6)。共筛选出4对能双抗夹心ELISA配对的抗体,其中Ab1与Ab5具有较高的灵敏度和较大的线性范围,其亲和力分别为1.0×10~(10)L/mol与5.9×109L/mol,检测线性范围:300~9 600 pg/ml。样本检测结果显示,自制试剂盒的阳性检出率为97.5%(78/80),阴性检出率为100%(40/40)。结论筛选获得1对高亲和力、高特异性的配对抗体,建立了双抗体夹心ELISA的检测方法,亦为新的快速免疫学检测方法的建立奠定了基础。
OBJECTIVE: To obtain NT-pro BNP high-affinity and high-specificity antibodies and to establish a sandwich ELISA for the detection of NT-pro BNP in human plasma. Methods The antigen epitopes were predicted by Discovery Studio software, and Balb / c mice were immunized with the epitope-peptide-coupled carrier protein. Hybridoma cells secreting antibodies were obtained by hybridoma cell fusion technique to prepare ascites monoclonal antibody. The antibody properties were identified by SDS-PAGE, ELISA and other methods. A double-antibody sandwich ELISA assay was established and NT-pro BNP was rapidly detected in 120 clinical blood samples. Results Six hybridoma cell lines secreting anti-human NT-Pro BNP were obtained. The ascites titer of the four monoclonal antibodies was higher than 1 × 10 -6. A total of 4 pairs of antibodies were screened by double-antibody sandwich ELISA. Ab1 and Ab5 showed high sensitivity and large linear range with affinity of 1.0 × 10 ~ (10) L / mol and 5.9 × 109 L / mol , Detection linear range: 300 ~ 9 600 pg / ml. The sample test results showed that the positive detection rate of homemade kit was 97.5% (78/80), the negative detection rate was 100% (40/40). Conclusion A pair of high-affinity and high-specificity paired antibodies was screened to establish a double-antibody sandwich ELISA assay, which laid the foundation for the establishment of a new rapid immunological assay.