重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白受体结合活性检测方法的改进

Improvement of a method for determination of binding activity of recombinant human typeⅡtumor necrosis factor receptor-Fc fusion protein

目的对重组人Ⅱ型肿瘤坏死因子受体(Tumor necrosis factor receptor,TNFR)-抗体融合蛋白的受体结合活性检测方法进行改进。方法采用夹心ELISA法对系列稀释的重组人Ⅱ型TNFR-抗体融合蛋白供试品和参比品进行检测,通过四参数方程拟合,计算二者的半数有效浓度(EC50),分析其受体结合活性。对改进的方法进行精密性和准确性验证,并与原方法进行比较。结果重组人Ⅱ型TNFR-抗体融合蛋白供试品和参比品均存在量效关系,符合四参数方程y=(A-D)/[1+(X/C)B]+D;3批重组人Ⅱ型TNFR-抗体融合蛋白经3次测定,受体结合活性分别为(133±8)、(114±4)和(138±9)BU,符合其质量标准中受体结合活性65～135 BU的规定,变异系数分别为6.02%、3.51%和6.52%;1批供试品经3次重复测定,回收率为(107.67±7.50)%;3批供试品采用原方法和改进方法分别重复测定3次,受体结合活性差异均无统计学意义(P>0.05)。结论改进的方法精密性好,准确性高,可作为重组人Ⅱ型TNFR-抗体融合蛋白受体结合活性的常规检测方法。

Objective To improve a method for determination of binding activity of recombinant human typeⅡ tumor necrosis factor receptor（TNFR）-Fc fusion protein.Methods Serially diluted test samples and references were determined by sandwich ELISA,of which the median effective concentrations（EC50） were calculated by four-parameter equation,and the binding activities were analyzed.The improved method was verified for precision and accuracy,and the results were compared with those of original method.Results Dose-response mode was observed in either samples or references of recombinant human typeⅡ TNFR-Fc fusion protein determined by the improved method,and the determination results complied with the following four-parameter equation： y =（A-D） / [1 ＋（X / C）B] ＋ D.The binding activities of three batches of recombinant human typeⅡ TNFR-Fc fusion protein were determined for 3 times,and the results were（133 ± 8） BU,（114 ± 4） BU and（138 ± 9） BU,with coefficients of variation of 6.02%,3.51% and 6.52%,respectively,which met the requirements in quality standard（65 ～ 135 BU）.The recovery rate of one batch of test samples determined for 3 times was（107.67 ± 7.50）%.Three batches of test samples were determined for binding activity by original and the improved methods for 3 times respectively,and the results showed no significant difference（P 0.05）.Conclusion The improved method showed high precision and accuracy,which might be used as a routine method for determination of binding activity of recombinant human type Ⅱ TNFR-Fc fusion protein.