目的: 建立抗CD79b单抗及抗CD79b单抗-vc-MMAE的表面等离子共振(surface plasmon resonance, SPR)法和酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)法评价其结合活性。方法: 采用SPR法,使用Protein A芯片,测定抗CD79b单抗及抗CD79b单抗-vc-MMAE与其靶点CD79b蛋白之间的动力学常数包括结合常数(Ka)和解离常数(Kd)、亲和力常数(KD)、半最大效应浓度(half maximal effective concentration, EC50)及相对效价;采用经典ELISA法测定抗CD79b单抗及抗CD79b单抗-vc-MMAE与CD79b的EC50及相对效价。结果: 经3次实验测定,抗CD79b单抗与CD79b的Ka为(2.61±0.14)×106 L·mol-1·s-1,Kd为(1.87±0.12)×10-2 s-1,KD为(7.17±0.30)×10-9 mol·L-1,RSD均＜10%;抗CD79b单抗-vc-MMAE与CD79b的Ka为(2.99±0.26)×106 L·mol-1·s-1,Kd为(1.46±0.04)×10-2 s-1,KD为(4.88±0.31)×10-9 mol·L-1,RSD均＜10%。经3次实验测定,SPR法测得抗CD79b单抗与CD79b的EC50为(78.68±6.35)ng·mL-1,相对效价为(84.62±6.63)%,RSD均＜10%;测得抗CD79b单抗-vc-MMAE与CD79b的EC50为(35.93±0.75) ng·mL-1,相对效价为(84.74±1.76)%,RSD均＜5%。经3次实验测定,ELISA法测得抗CD79b单抗与CD79b的EC50为(22.66±0.41) ng·mL-1,相对效价为(95.70±1.74)%,RSD均＜5%;测得抗CD79b单抗-vc-MMAE与CD79b的EC50为(69.19±1.71) ng·mL-1,相对效价为(97.64±2.39)%,RSD均＜5%,SPR和ELISA 2种方法结果基本一致。结论: 建立了抗CD79b单抗及抗CD79b单抗-vc-MMAE的SPR法,并对靶点结合的动力学、亲和力进行评价,与ELISA方法在EC50及相对效价方面进行了对比,为该类抗体偶联药物的研发提供参考。
Objective: To study the binding activity of anti-CD79b antibody and anti-CD79b antibody-vc- MMAE by establishing surface plasmon resonance(SPR) and enzyme linked immunosorbent assay(ELISA) measures. Methods: In this experiment, SPR and Protein A chip were used to determine the kinetic constants, including binding constant(Ka), dissociation constant (Kd), affinity constant(KD), half maximal effective concentration(EC50) and relative potency between anti-CD79b antibody and anti-CD79b antibody-vc-MMAE and their target CD79b protein. Classical ELISA was used to determine the EC50 and relative potency of anti-CD79b antibody and anti-CD79b antibody-vc-MMAE with CD79b. Results: After three experiments, the kinetic and affinity parameters between anti-CD79b antibody and CD79b include: Ka, Kd and KD, which were (2.61±0.14)×106 L·mol-1·s-1, (1.87±0.12)×10-2 s-1, (7.17±0.30)×10-9mol·L-1 respectively, RSDs were<10%. The kinetic and affinity parameters between anti-CD79b antibody-vc-MMAE and CD79b include: Ka, Kd and KD, which were(2.99±0.26)×106 L·mol-1·s-1, (1.46±0.04)×10-2 s-1, (4.88±0.31)×10-9 mol·L-1 respectively, RSDs were<10%. After three experiments, the EC50 and relative potency between anti-CD79b antibody and CD79b determined by SPR were (78.68±6.35) ng·mL-1, (84.62±6.63)% respectively, RSDs were<10%. The EC50 and relative potency between anti-CD79b antibody-vc-MMAE and CD79b determined by SPR were (35.93±0.75) ng·mL-1, (84.74±1.76)% respectively, RSDs were<5%. The EC50 and relative potency between anti-CD79b antibody and CD79b determined by ELISA were (22.66±0.41) ng·mL-1, (95.70±1.74)% respectively, RSDs were<5%. The EC50 and relative potency between anti-CD79b antibody-vc-MMAE and CD79b determined by ELISA were (69.19±1.71) ng·mL-1, (97.64±2.39)% respectively, RSDs were <5%. The results of SPR and ELISA were basically consistent. Conclusion: In this study, the kinetic and affinity parameters of anti-CD79b antibody and anti-CD79b antibody-vc-MMAE against target binding were determined by SPR.The EC50 and relative potency analyzed by SPR and ELISA are compared. The study provides a reference for the development of this kind of ADC drugs.
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