检测重组人血小板生成素含量的改良双抗体夹心ELISA方法的建立

Development of A Modified Double Antibody Sandwich ELISA for Determination of Recombinant Human Thrombopoietin Content

目的建立检测重组人血小板生成素(rhTPO)含量的改良双抗体夹心ELISA方法。方法用棋盘滴定法确定包被单抗、一抗和酶标二抗的最佳工作浓度;绘制标准曲线,确定线性范围、检测限和定量限;检测该方法的准确性(回收率)、精密性和特异性,并与Lowry法检测结果进行比较。结果包被单抗的最佳包被浓度为1.0μg/ml,一抗和酶标二抗的最佳使用浓度分别为1∶5000和1∶10000;检测的线性范围为(125～4000)pg/ml,检测限为75pg/ml,定量限为189pg/ml;不同浓度rhTPO的回收率在(97.0±2.1)%～(105.0±8.5)%之间;6批样品的试验内变异系数在3.2%～8.1%之间,试验间变异系数在5.7%～10.4%之间,特异性良好;与Lowry法检测结果具有良好的相关性。结论已建立检测rhTPO含量的改良双抗体夹心ELISA法,准确性、精密性、特异性良好,可用于rhTPO研究及生产过程中的定量分析。

Objective To develop a modified double antibody sandwich ELISA for determination of recombinant human thrombopoietin （rhTPO） content. Methods Select the optimal working concentrations of McAb for coating, first antibody and the enzyme-labeled second antibody by chessboard titration. Plot the standard curve to obtain the linear range, detection limit and quantification limit for determination of rhTPO. Evaluate the accuracy （recovery rate）, precision and specificity of the developed method and compare with those of Lowry method. Results The optimal working concentrations of McAb for coating, first antibody and the enzyme-labeled second antibody were 1.0 μg/ml, 1 ： 5 000 and 1 ： 10 000 respectively. The linear range, detection limit and quantification limit for determination of rhTPO were 125 ～ 4 000, 75 and 189 pg/ml respectively. The recovery rates of rhTPO at various concentrations ranged from （97.0 ± 2. 1 ）% to （105.0±8. 5 ）%, the variation coefficients of inter- and intra-assays of 6 batches of samples were （3. 2 ～ 8. 1 ）% and （5.7 ～ 10. 4）% respectively. The developed method showed good specificity, and its determination result showed good relationship to that by Lowry method. Conclusion A modified double antibody sandwich ELISA for determination of rhTPO content was developed, which showed good accuracy, precision and specificity and might be used for the quantitative analysis of rhTPO during research and production.