Objective: To establish a quantitative assay of serum Golgi protein 73 (GP73) using xMAP technology and evaluate its performance. Methods: Monoclonal antibodies against GP73 were prepared and purified, and antibody pair screening was performed by double-antibody sandwich enzyme-linked immunosorbent assay. The screened antibodies were used to construct a Luminex liquid chip detection system, and the analysis performance of the detection system was evaluated. The serum levels of GP73 were detected in 90 clinical samples from healthy controls and patients with chronic hepatitis B infection (CHB) and hepatocellular carcinoma (HCC). Results: Five anti-GP73 monoclonal antibodies were prepared and purified, and 5 antibody pairs were successfully screened. The Luminex liquid chip detection system of GP73 was successfully constructed using 8F10D1 and 10B9F11 antibody pairs. The analytical performance evaluation showed that the sensitivity of this system was 0.25 ng/ml and the dynamic range was 0.25-100 ng/ml. No cross reactivity was observed. The intra- and inter-assay variation for GP73 was <8% and <11%, respectively. The recovery was 83%-92%. The linear regression equation was y=1.141x+ 6.436 (r(2)=0.998 4, P<0.001). The GP73 concentrations in the serum samples of healthy control, CHB group, and HCC group were 42.8 (38.68, 55.90) ng/ml, 61.49 (43.59, 81) ng/ml, and 122.78 (49.36 liter, 264.55) ng/ml, respectively. The levels of GP73 in HCC group were significantly higher than those in CHB group and healthy controls (P<0.05). Moreover, the levels of GP73 in CHB group were significantly higher than those in healthy controls (P<0.05). Conclusions: A liquid chip detection system of GP73 was successfully constructed. It provides a powerful tool for the clinical application of GP73 in the future.
目的： 构建高尔基体糖蛋白73(GP73)的液态芯片检测体系并评价其检测性能。 方法： 制备并纯化5株抗GP73的单克隆抗体，采用酶联免疫吸附试验双抗夹心法进行抗体配对筛选。应用筛选到的抗体对构建Luminex液态芯片检测体系，并对该检测体系进行分析性能评估。应用该体系检测正常对照、慢性乙型肝炎和肝细胞癌血液样本各30例，比较3组间GP73浓度的差异。 结果： 制备并纯化了5株抗GP73单克隆抗体，配对筛选成功5对。应用8F10D1和10B9F11抗体对成功构建了GP73的Luminex液态芯片检测体系。该检测体系的灵敏度为0.25 ng/ml，线性检测范围为0.25～100 ng/ml，特异度较好，批内精密度<8%，批间精密度<9%，回收率为83%～92%，线性稀释效应的线性回归方程为y＝1.141x＋6.436(r(2)＝0.998 4，P<0.001)。正常对照组、慢性乙肝组和肝细胞癌组血液样本的GP73浓度分别为42.8(38.68，55.90)ng/ml、61.49(43.59，81)ng/ml和122.78(49.36l，264.55)ng/ml。肝细胞癌组的GP73浓度高于慢性乙肝组和正常对照组(均P<0.05)，慢性乙肝组的GP73浓度高于正常对照组(P<0.05)。 结论： 成功构建了GP73的液态芯片检测体系，为GP73检测在临床上的应用奠定了基础。.
Keywords: Antibody screening; Golgi protein 73; Hepatocellular carcinoma; Liquid chip.