曲妥珠单克隆抗体生物学活性检测方法的建立及比较

目的建立4种检测曲妥珠单克隆抗体Trastuzumab生物学活性的方法,并进行比较。方法利用表面等离子共振技术(Surface plasmob resonance,SPR)建立Trastuzumab的BIAcore分析方法,同时建立抗体对人乳腺癌SKBR3细胞增殖抑制活性的检测方法、基于SKBR3细胞的抗体结合特性检测方法及抗体与ErbB2抗原结合活性的ELISA检测方法,验证各方法的重复性,并对不同检测方法进行比较。结果 BIAcore法的偶联最佳pH值为5.0,样品浓度为0.05～5.00 nmol/L,动力学常数(KD)为0.19 nmol/L;Trastuzumab体外抑制SKBR3细胞增殖的半数有效浓度(EC50)为0.145μg/ml(R2>0.98);基于SKBR3细胞的抗体结合特性的EC50值为0.124μg/ml(R2>0.98);Trastuzumab抗体与ErbB2抗原结合活性的EC50值为0.143μg/m(lR2>0.98);4种检测方法重复性较好,检测结果基本一致。结论 4种检测方法均能有效地反映Trastuzumab的生物学活性,为快速、简便地筛选曲妥珠单克隆抗体提供了多种检测方法。

Development and Comparison of Methods for Determination of Biological Ativity of Trastuzumab

Objective To develop and compare four methods for determination of biological activity of Trastuzumab.Methods BIAcore method for Trastuzumab was developed by surface plasmob resonance(SPR);a method for determination of activity of Trastuzumab in inhibiting the proliferation of human breast cancer SKBR3 cells was developed;a SKBR3 cells-based method for determination of binding activity of Trastuzumab as well as an ELISA method for determination of binding activity of Trastuzumab to ErbB2 antigen were developed.The four methods were verified for reproducibility and compared.Results The optimal pH value and sample concentration for BIAcore method were 5.0 and 0.05 ～ 5.00 nmol / L respectively,while the kinetic constant(KD) was 0.19 nmol / L.The median effective concentration(EC50) of Trastuzumab for inhibiting the proliferation of SKBR3 cells in vitro was 0.145 μg/ml(R2 > 0.98).The SKBR3 cells-based binding activity(EC50) of Trastuzumab was 0.124 μg/ml(R2 > 0.98).The binding activity(EC50) of Trastuzumab to ErbB2 antigen was 0.143 μg/ml(R2 > 0.98).All the four methods showed high reproducibility,by which the determination results were basically in agreement.Conclusion All the developed methods reflected the biological activities of Trastuzumab effectively,which provided several tools for rapid and simple screening of monoclonal antibodies.