抗猴痘病毒A35R蛋白单克隆抗体的制备及双抗体夹心ELISA检测方法的建立  

Preparation of monoclonal antibody against monkeypox virus A35R protein and establishment of double antibody sandwich ELISA detection method

作　　者：欧阳蕾 吴佩璇 师江龙 潘勇兵 OUYANG Lei;WU Peixuan;SHI Jianglong;PAN Yongbing(Antibody Laboratory,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司抗体研究室,湖北武汉430207

出　　处：《微生物学免疫学进展》2024年第4期1-8,共8页Progress In Microbiology and Immunology

摘　　要：目的制备抗猴痘病毒(monkeypox virus,MPXV)A35R蛋白单克隆抗体并建立其双抗体夹心ELISA检测方法,同时对方法进行验证。方法原核表达MPXV A35R蛋白并免疫BALB/c小鼠,经杂交瘤技术制备抗A35R蛋白单克隆抗体,对单克隆抗体的特异性、结合活性进行鉴定;建立双抗体夹心ELISA检测方法,对该方法的线性范围、检测限、专属性、准确性、精密度进行验证。结果制备了7株抗MPXV A35R单克隆抗体。Western blot鉴定结果显示,7株单克隆抗体均与MPXV A35R蛋白及灭活MPXV发生特异性反应;7株单克隆抗体结合活性为11.35~27.73 ng/mL;抗体经配对筛选后,确定以7G5和2F5为最佳配对抗体,用于建立双抗体夹心ELISA方法。该方法对灭活MPXV抗原最低检测限为1.250×10~4 TCID50/mL,对MPXV A35R蛋白最低检测限为1.95 ng/mL,线性范围为3.91~125.00 ng/mL,线性回归方程为y=1.7320x-0.9208,R2=0.9843;该方法专属性较强且不与其他病毒及蛋白发生交叉反应;准确性验证结果显示,病毒抗原回收率为96.94%~110.04%;批间CV为0.26%~8.13%,批内CV为2.10%~5.48%。结论成功制备了抗MPXV A35R蛋白单克隆抗体,建立了双抗体夹心ELISA检测方法。验证结果显示,该方法具有较高的灵敏度、准确度、重复性及专属性,可用于以A35R蛋白为基础的猴痘亚单位疫苗的生产质量控制。Objective Preparation of monoclonal antibody against monkeypox virus(MPXV)A35R protein and a related double antibody sandwich ELISA assay was established and validated.Methods The monkeypox virus A35R protein was expressed using prokaryotic expression system and immunized BALB/c mice,the anti-A35R monoclonal antibody was prepared by hybridoma technique,the specificity and binding activity of the monoclonal antibody were charaterized;a double-antibody sandwich ELISA assay was established,and its linear range,detection limit,specificity,accuracy and precision were verified.Results Seven monoclonal antibodies against monkeypox virus A35R were prepared.Western blot analysis demonstrated that all seven monoclonal antibody strains exhibited specific reactivity with the MPXV A35R protein and inactivated MPXV.The monoclonal antibody binding activity of the seven strains were from 11.35 ng/mL to 27.73 ng/mL.After screening for antibody pairs,7G5 and 2F5 were identified as the optimal antibody pair for the double-antibody sandwich ELISA method.The minimum detection limits for inactivated MPXV antigen were 1.250×10~4 TCID_(50)/mL and 1.95 ng/mL for MPXV A35R protein,the linear range was 3.91-125.00 ng/mL,the linear regression equation was y=1.7320x-0.9208,R~2=0.9843;the method is highly specific and does not cross-react with other viruses and proteins;the accuracy validation results showed that the recovery of viral antigens was between 96.94%-110.04%;inter-batch CV ranged from 0.26%to 8.13%,and intra-batch CV ranged from 2.10%to 5.48%.Conclusion An anti-monkeypox virus A35R protein monoclonal antibody was successfully prepared,and a related double-antibody sandwich ELISA method was established and fully validated,the method can be employed for the purpose of quality control in the production of subunit monkeypox vaccines based on the A35R protein.

关 键 词：猴痘病毒 A35R蛋白 单克隆抗体 双抗体夹心ELISA 抗原检测 蛋白质亚单位疫苗 质量控制 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]