重组人白细胞介素-1受体拮抗剂免疫大鼠ELISA抗体阳性血清中和抗体活性检测方法的建立及验证  

Development and verification of a detection method for activity of neutralizing antibody in ELISA antibody positive serum of rats immunized with recombinant human interleukin⁃1 receptor antagonist

作　　者：彭炳淮 刘景会 刘玉林 孙中涛 孙基伟 梁冠桥 俞露 PENG Binghuai;LIU Jinghui;LIU Yulin;SUN Zhongtao;SUN Jiwei;LIANG Guanqiao;YU Lu(Changchun Institute of Biological Products Co.,Ltd.,Changchun 130062,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130062

出　　处：《中国生物制品学杂志》2024年第2期215-220,共6页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划(20210204092YY)。

摘　　要：目的 建立用于检测重组人白细胞介素-1受体拮抗剂(recombinant human interleukin-1 receptor antagonist,rhIL-1Ra)免疫大鼠ELISA抗体阳性血清中和抗体活性的方法,并进行验证。方法 分别用3、20、100 mg/kg的rhIL-1Ra注射液经皮下免疫SD大鼠,10只/组,雌雄各半,每天给药2次,间隔至少4 h,连续给药13周,于给药期及恢复期经大鼠颈静脉采血,分离血清,ELISA法检测血清抗体效价,Protein A层析柱纯化经ELISA筛选为rhIL-1Ra抗体阳性的血清样本。以D10G4·1细胞生物学活性试验为基础建立中和抗体活性检测方法,并验证方法的专属性、灵敏度及精密性。采用建立的方法检测ELISA筛选为rhIL-1Ra抗体阳性血清的中和抗体活性。结果 随着给药次数的增加,各剂量组大鼠血清抗体滴度逐渐增强,至恢复期时仍有抗体存在,且滴度仍较高。兔抗rhIL-1Ra单抗对rIL-1Ra有明显的中和作用,兔抗rIFN-2b单抗与rIL-1Ra无剂量效应关系;该方法的灵敏度为171.93μg/mL;精密性验证CV均≤20%。ELISA筛选为阳性抗体血清对rhIL-1Ra注射液均可产生中和效应,与ELISA法检测结果相符。结论 本研究建立了用于检测rhIL-1Ra免疫大鼠血清中和抗体活性的方法,具有良好的特异性、专属性及较高的灵敏度,可用于rhIL-1Ra重复给药动物血清中和抗体活性的检测。Objective To develop and verify a method for detecting the activity of neutralizing antibodies in ELISA antibody positive serum of rats immunized with recombinant human interleukin-1 receptor antagonist(rhIL-1Ra).Methods The SD ratsweresubcutaneously immunized with 3,20 and 100 mg/kg rhIL-1Ra injection respectively,10 rats in each group,half male and half female,twice a day at an interval of at least 4 h between each dose for 13 consecutive weeks.The blood samples were collected from the jugular vein of rats during the administration period and the recovery period.The serum samples were isolated and detected for the antibody titers by ELISA,and the samples positive for rhIL-1Ra antibody were purified by Protein A chromatographic column.Based on,D10G4·1 cells biological activity assay,a method for the detection of neutralizing antibody activity was developed and verified for the specificity,sensitivity and precision.The neutralizing antibody activity of rhIL-1Ra antibody positive serum determined by ELISA was detected by using the developed method.Results With the increase of doses,the serum antibody titers of rats in various dose groups gradually increased,and there were still antibodies in the recovery period,and the titer was still high.Rabbit anti-rhIL-1Ra monoclonal antibody showed obvious neutralizing effect on rIL-1Ra,while rabbit anti-rIFN-2b monoclonal antibody had no dose-effect relationship with rIL-1Ra.The sensitivity of the method was 171.93μg/mL;The CVs of precision verification were not more than 20%.The positive antibody sera detected by ELISA all had neutralizing effect on rhIL-1Ra injection,which was consistent with the results detected by ELISA.Conclusion The method developed in this study has good specificity and high sensitivity in the detection of serum neutralizing antibody activity in rats immunized with rhIL-1Ra,which can be used to detect the serum neutralizing antibody activity of animals with rhIL-1Ra repeated administration.

关 键 词：重组人白细胞介素-1受体拮抗剂 中和抗体 D10G4·1细胞生物学活性试验 

分 类 号：Q331[生物学—遗传学]