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目的建立乳腺癌患者血清HER-2/neu胞外域蛋白(ECD)水平检测方法,为乳腺癌治疗及预后判断提供参考。方法以识别HER-2/neu不同抗原表位的2种抗HER-2/neu单克隆抗体(McAb)为基础,建立可定量检测血清HER-2/neuECD水平的双抗夹心酶联免疫吸附试验法(ELISA)。结果本研究建立的HER-2/neuECD水平检测ELISA所用试剂[包括抗HER-2/neuMcAb5A12包被的酶标板、辣根过氧化物酶(HRP)标记的HER-2/neuMcAb1-2及HRP底物四甲基联苯胺(TMB)溶液]在1年内保持稳定;批内、批间变异系数分别为3·6%±4·2%、6·9%±3·3%;与HER-2ECD同源性很高的人表皮生长因子受体(EGFR/HER-1)ECD不发生交叉反应。自制ELISA试剂盒与BenderMedsystems试剂盒检测人血清HER-2ECD水平的相关性较好(r>0·75)。健康人群(20名)与乳腺癌患者(140例)血清HER-2ECD阳性率之间的差异无统计学意义(P>0·05),但与转移乳腺癌患者(10例)血清HER-2ECD阳性率之间差异有统计学意义(P<0·01)。结论本研究建立的HER-2/neuECD双抗夹心ELISA检测方法特异、稳定、可靠,可定量检测乳腺癌患者血清HER-2/neuECD水平。
Objective To establish a method for detecting serum levels of HER-2 / neu extracellular domain protein (ECD) in breast cancer patients and provide references for the treatment and prognosis of breast cancer. Methods Based on the two anti-HER-2 / neu monoclonal antibodies (McAbs) that recognize different epitopes of HER-2 / neu, a double-antibody sandwich enzyme-linked immunosorbent assay Method (ELISA). Results The HER-2 / neuECD levels established in this study were used to detect the reagents used in ELISA [including anti-HER-2 / neuMcAb5A12 coated ELISA plates, HRP-labeled HER-2 / neuMcAb1-2 and HRP The substrate tetramethylbenzidine (TMB) solution] remained stable within 1 year. The intra-assay and inter-assay CVs were 3.6% ± 4.2% and 6.9% ± 3.3% 2 ECD human epidermal growth factor receptor (EGFR / HER-1) ECD does not cross-react. The correlation between home-made ELISA kit and BenderMedsystems kit for the detection of human serum HER-2ECD level was good (r> 0.75). There were no significant differences in the positive rates of HER-2ECD between healthy subjects (20 patients) and breast cancer patients (140 patients) (P> 0.05). However, the serum HER-2ECD levels in patients with metastatic breast cancer (10 patients) There was a significant difference between the positive rates (P <0.01). Conclusion The HER-2 / neuECD double antibody sandwich ELISA assay established in this study is specific, stable and reliable and can detect HER-2 / neuECD levels in patients with breast cancer.