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目的建立重组抗表皮生长因子受体(epithelial growth factor receptor,EGFR)单克隆抗体生物学活性检测方法。方法利用A-431细胞,对系列梯度稀释的重组抗EGFR单克隆抗体参考品和供试品进行检测,通过四参数方程进行拟合,计算二者的半数有效浓度(EC50)及供试品的相对比活性,并对该方法进行专属性、精密度(重复性、中间精密度)和准确度验证。结果重组抗EGFR单克隆抗体供试品及参考品在该方法中均存在明显的量效关系,四参数拟合的曲线呈典型的反S型曲线。人Ig G1亚型免疫球蛋白(阴性对照)与A-431细胞无交叉反应,不呈现与重组抗EGFR单克隆抗体供试品相似的剂量-反应曲线;同一名试验人员重复6次检测结果的RSD为5%,同一名试验人员3个工作日检测结果的RSD为4%,3名试验人员3次检测结果的RSD为10%,不同浓度的3个供试品溶液生物学活性理论值和检测值的相对误差分别为10%、8%和12%,均满足方法验证的可接受标准。结论成功建立了重组抗EGFR单克隆抗体生物学活性检测方法,该方法专属性较强,精密度良好,准确度较高,可作为重组抗EGFR单克隆抗体生物学活性的常规检测方法。
Objective To establish a method for the detection of the biological activity of recombinant monoclonal antibody against epithelial growth factor receptor (EGFR). Methods A-431 cells were used to test a series of gradient anti-EGFR monoclonal antibody reference materials and test samples. The four-parameter equations were used to calculate the half effective concentration (EC50) Relative activity, and the method for specificity, precision (repeatability, intermediate precision) and accuracy verification. Results The recombinant anti-EGFR mAb showed obvious dose-response relationship in this method. The four-parameter fitting curve showed a typical anti-S curve. Human Ig G1 subtype immunoglobulin (negative control) did not cross-react with A-431 cells and did not exhibit a dose-response curve similar to that of the recombinant anti-EGFR monoclonal antibody; the same panelist repeated 6 test results RSD was 5%, RSD of test result was 4% for 3 working days of the same test person, RSD of 3 test results of 3 test persons was 10%, the theoretical value of biological activity of the three test solutions for different concentrations The relative errors of the measured values were 10%, 8% and 12% respectively, which all met the acceptable standard of method verification. Conclusion The method for detecting the biological activity of recombinant monoclonal antibodies against EGFR has been successfully established. The method is characterized by high specificity, good precision and high accuracy, and can be used as a routine detection method for the biological activity of recombinant anti-EGFR monoclonal antibodies.