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目的利用Biacore T200生物分子相互作用分析系统研究阿达木单抗的生物学活性。方法将人源阿达木单抗偶联于CM5传感芯片,利用Biacore T200系统观察重组人肿瘤坏死因子α(TNF-α)和食蟹猴TNF-α与阿达木单抗结合的动力学常数,并采用不同种属的重组TNF-α验证种属特异性,进一步研究阿达木单抗抑制重组TNF-α的细胞毒作用,以确认阿达木单抗的生物活性。结果阿达木单抗与重组人TNF-α的结合常数(K_a)为(2.114±0.007)×105,解离常数(K_d)为(5.315±0.220)×10~(-5),亲和力常数(K_D)为(2.515±0.107)×10~(-10);与重组食蟹猴TNF-α的K_a为(2.574±0.068)×105,K_d为(1.554±0.124)×10~(-5),K_D为(5.320±1.271)×10~(-10)。阿达木单抗与50 nmol/L重组人TNF-α、重组食蟹猴TNF-α、重组小鼠TNF-α和重组大鼠TNF-α结合的响应值分别为84.3、66.4、1.3和-0.8 RU,呈现明显的种属差异。阿达木单抗可抑制重组人TNF-α和重组食蟹猴TNF-α对L929细胞的毒性作用,其半数有效浓度分别为194.0和685.5 pmol/L,但对重组小鼠或重组大鼠TNF-α导致的细胞毒作用无影响,细胞水平检测结果与Biacore T200系统检测结果一致。结论用Biacore T200生物分子相互作用分析系统研究抗原抗体结合简便快速准确,该方法适合用于重组人TNF-α单抗生物学活性分析与药效学评价。
Objective To study the biological activity of adalimumab by using Biacore T200 biomolecular interaction analysis system. Methods Human adalimumab was conjugated to CM5 sensor chip. The kinetic constants of binding of recombinant human TNF-α and adalimumab to cynomolgus monkey TNF-α were observed by Biacore T200 system. Species specificity was tested using different species of recombinant TNF-α, further study adalimumab inhibition of recombinant TNF-α cytotoxicity in order to confirm the biological activity of adalimumab. Results The binding constants (K_a) of adalimumab to recombinant human TNF-α were (2.114 ± 0.007) × 105, the dissociation constants (K_d) were (5.315 ± 0.220) × 10 -5 and the affinity constants ) Was (2.515 ± 0.107) × 10 ~ (-10), K_a was (2.574 ± 0.068) × 105 and K_d was (1.554 ± 0.124) × 10 ~ (-5) (5.320 ± 1.271) × 10 ~ (-10). The response of adalimumab to 50 nmol / L recombinant human TNF-α, recombinant cynomolgus monkey TNF-α, recombinant mouse TNF-α and recombinant rat TNF-α were 84.3, 66.4, 1.3 and -0.8 RU, showing obvious species differences. Adalimumab inhibited the toxic effects of recombinant human TNF-αand recombinant cynomolgus monkey TNF-α on L929 cells, the effective concentrations of which were 194.0 and 685.5 pmol / L, respectively, but the recombinant mouse or recombinant rat TNF- α cytotoxicity caused by no effect, cell-level test results and Biacore T200 system test results. Conclusion Biacore T200 biomolecular interaction analysis system is simple, rapid and accurate for antigen-antibody binding. This method is suitable for the biological activity analysis and pharmacodynamic evaluation of recombinant human TNF-α monoclonal antibody.