高灵敏血清HER2蛋白检测方法的建立及临床应用

Establishment and clinical application of a highly sensitive method to detect HER2 protein in human serum

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, using double antibody sandwich ELISA technology, the anti-human epidermal growth fac- tor 2 （HER2） monoclonal antibody HuA21 andbiotin-labeled HerA antibody were used as the capture antibody and detection antibody, respectively. HER2 extracellular domain（HER2-ECD） was transfected into CHO cell line and the resistant clones were selected to produce the antigen protein, which was isolated and purified as standard anti- gens in order to establish a technique for quantitatively detecting HER2 in human serum, and could provide refer- ence for the treatment and prognosis evaluation of cancer patients. The sensitivity of the method was 7.8 pg/ml, and the detection limit was from 0 to 500 pg/ml. The intra-assay coefficient of variation was 0. 2% - 10.9%, and the coefficient of variation was 1.4% - 12.4%. The recovery rate of the antigen in human serum was 86. 84% to 116.40%. Our method was used to determine the selected clinical samples that the serum levels of HER2 in pa- tients with HER2-positive metastatic breast cancer was significantly higher than those in patients with early-stage breast cancer and healthy subjects （P 〈 0. 05 ）. So a double-anti-sandwich ELISA method of human serum HER2 detection is established, and the determination result is specific, stable and reliable and can be used to quantita- tively detect the senim HER2 levels for the cancer natients.