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重组人源化抗TNF-α单抗WLB303猴重复给药毒性试验 血清中和抗体确证方法的建立
重组人源化抗TNF-α单抗WLB303猴重复给药毒性试验 血清中和抗体确证方法的建立
陈红霞1,李 雪1,罗 涛1,李 乐1,于玉根1,蒙志超2,刘思思2

1 深圳万乐药业有限公司,深圳 518029;2 北京昭衍新药研究中心股份有限公司,北京 100176
Development of a method for detection of serum neutralizing  antibodies in cynomolgus monkeys undergoing repeated dose toxicity  study of humanized anti-TNF-α monoclonal antibody WLB303
(1 Shenzhen Main Luck Pharmaceuticals Inc., Shenzhen 518029, China;  2 JOINN Laboratories (Beijing), Beijing 100176, China)

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起始页:2386

摘要:[摘要] 目的:建立一种快速、灵敏、高效和易操作的方法,用于检测重组人源化抗TNF-α单抗WLB303猴重复给药毒性试验血清中产生的中和抗体。方法:对食蟹猴静脉输注WLB303,连续给药4周。采取给药期和恢复期不同时间的血清,随后采用桥式酶联免疫吸附法对血清中的抗药抗体进行定性筛选,对筛选为阳性的血清样本进行预处理,然后以WLB303生物学活性测定法为基础的中和反应法对中和抗体进行确证。结果:建立了WLB303中和抗体的确证方法,确定了检测的临界点。确证方法的检出率为100%,在血清中检测到具有中和活性的抗体,中和抗体的中和作用强度总体与给药时间、给药剂量呈正相关。结论:建立的检测方法能够直接体现出剂量差异性和个体差异性,也能反映中和抗体的滴度差别。该方法的特异性、灵敏度和有效性能够满足试验要求,可以用于单抗药物重复给药动物毒性试验血清中和抗体的检测。

关键词:[关键词] 桥式酶联免疫吸附法;中和抗体;生物学活性;重复给药毒性试验

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Abstract:[Abstract] Objective: To establish a rapid, sensitive, highly-efficient and easy-to-use method for detection of neutralizing antibodies against WLB303 in serum of animals undergoing repeated dose toxicity study. Methods:The cynomolgus ms were continuously infused with recombinant humanized anti-TNF-α monoclonal antibodies intravenously for 4 weeks. Serum samples were taken at different time points before and after administration and at different stages of recovery period. Then a bridging-ELISA method was used to qualitatively screen anti-drug antibody (ADA). A sample pre-treatment procedure was conducted to eliminate the non-specific interference in serum samples prior to verification of neutralizing activity. Neutralization activity was verified in a cell-based neutralization antibody assay. Results:A cell-based neutralizing antibody assay was established and the cut-off point was confirmed. The detection rate of the confirmed neutralizing antibodies was 100%. Specific neutralizing antibodies were detected in serum samples, and the neutralization activity of neutralizing antibodies had positive correlation with the time and dosage of administration. Conclusion: The established assay has proven to be capable of demonstrating dose-dependent effect difference and inter-individual difference. It also reflects difference in titers of neutralizing antibodies. The cell-based neutralizing antibody assay was found to have desired properties of specificity, sensitivity and validity, and can be used as a feasible method for detection of neutralizing antibodies in repeated dose toxicity study.

Key words:[Key words] bridging-ELISA method; neutralizing antibody; biological activity; repeated dose toxicity study

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