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本研究采用双抗体夹心ELISA法,以前期研制的抗人表皮生长因子受体2(HER2)单克隆抗体Hu A21与生物素标记Her A分别为包被抗体与检测抗体,培养CHO细胞进行转染HER2胞外区(HER2-ECD)基因的表达载体,选择抗性克隆进行培养,并分离、纯化抗原作为标准抗原,建立可定量检测人血清中HER2含量的技术,为肿瘤患者的治疗及预后评估提供参考。建立的ELISA法检测灵敏度为7.8 pg/ml,检测范围为0~500 pg/ml,其批内变异系数为0.2%~10.9%,批间变异系数为1.4%~12.4%,在人血清中的抗原回收率为86.84%~116.40%。使用本方法检测临床标本,结果显示HER2阳性的转移性乳腺癌患者血清HER2含量明显高于早期乳腺癌患者及健康人的平均水平(P<0.05)。因此本研究成功建立了双抗体夹心ELISA法检测人血清HER2技术,其检测结果特异、稳定、可靠,可定量检测肿瘤患者血清HER2水平。
In this study, a double antibody sandwich ELISA method was used. The monoclonal antibody Hu A21 against anti-human epidermal growth factor receptor 2 (HER2) and the biotin-labeled Her A were coated antibody and antibody. The CHO cells were cultured and transfected HER2-ECD gene expression vector, select the resistant clones for culture, and isolate and purify the antigen as the standard antigen to establish a technique for quantitative determination of HER2 in human serum for the treatment and prognosis of cancer patients for reference. The sensitivity of the established ELISA was 7.8 pg / ml, the detection range was 0 ~ 500 pg / ml, the intra-assay coefficient of variation was 0.2% -10.9%, the inter-assay coefficient of variation was 1.4% ~ 12.4% Antigen recovery rate of 86.84% ~ 116.40%. Using this method to detect clinical specimens, the results showed that HER2-positive patients with metastatic breast cancer serum HER2 levels were significantly higher than those of patients with early-stage breast cancer and healthy people (P <0.05). Therefore, we successfully established the double antibody sandwich ELISA for the detection of HER2 in human serum. The detection results are specific, stable and reliable, and can detect the serum HER2 level of tumor patients quantitatively.