摘要
为制备非洲猪瘟病毒CD2v蛋白阻断ELISA候选单克隆抗体,并探究其免疫学特性,以真核表达的CD2v蛋白免疫BALB/c小鼠,经两次免疫后采集小鼠血清,以间接ELISA检测小鼠多抗血清效价。对血清效价最高的小鼠进行加强免疫后,取脾脏进行细胞融合。通过有限稀释法筛选能够稳定分泌CD2v单克隆抗体的杂交瘤细胞株。采用小鼠体内诱生腹水法和辛酸-硫酸铵法获得纯化的CD2v单克隆抗体,对其进行免疫学特性检测。间接ELISA试验显示,4号小鼠血清效价最高(1:72900),选取该小鼠脾脏进行细胞融合,经过筛选得到4株杂交瘤细胞,分别命名为21D10、21G8、36A3和38G8,亚型鉴定均为IgG_(1)/κ,经检测36A3 ELISA效价最高,可达1:2.187×10^(5)。免疫荧光试验显示,36A3抗体能与细胞内表达的CD2v蛋白发生特异性反应,而与携带His标签的p30蛋白无交叉反应;特异性鉴定结果显示,36A3抗体特异性良好,与猪伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪瘟病毒以及非洲猪瘟病毒p72蛋白均无反应;亲和力检测显示,36A3抗体亲和力较高,亲和常数(Ka)为9.68×10^(8)L/mol。初步建立的阻断ELISA试验显示,36A3能够有效区分阴阳性血清,N/P高达12.53,提示其为非常有效的阻断ELISA候选抗体。本试验结果为深入研究非洲猪瘟病毒CD2v蛋白功能及研发非洲猪瘟病毒阻断ELISA血清学检测试剂盒奠定了基础。
In order to prepare monoclonal antibodies against CD2v protein of African swine fever virus(ASFV)by blocking ELISA,and to identify its immunological characteristics,BALB/c mice were immunized with CD2v protein expressed in eukaryotic cells,sera were collected from the mice immunized for two times to detect their polyantiserum titer by indirect ELISA.After booster immunization in the mice with the highest serum titer,their spleens were taken out for cell fusion.Hybridoma cell strains that could regularly secrete CD2v monoclonal antibodies were selected through a limited dilution method.Purified CD2v monoclonal antibodies were obtained using mouse ascites inducers and caprylic acid-ammonium sulfate(CA-AS)to detect immunological characteristics.The indirect ELISA test results showed that No.4 mouse was with the highest titer(1:72900),its spleen was selected for cell fusion,and four strains of hybridoma cells were obtained,named as 21D10,21G8,36A3 and 38G8,respectively,all of which belonged to IgG_(1)/κ subtype,as it was tested that the titer of 36A3 was highest by ELISA,which could be up to 1:2.187×10^(5).Immunofluorescence assay results showed that 36A3 could specifically reacted with CD2v protein expressed in cells,but failed with p30 protein carrying His tag.Specificity test results showed that 36A3,with good specificity,failed to react with porcine pseudorabies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV)and p72 protein of African swine fever virus(ASFV).Affinity test results showed that 36A3 had a high affinity,with an affinity constant(Ka)of 9.68×10^(8)L/mol.The preliminary blocking ELISA test results showed that negative and positive sera could be distinguished by 36A3,with a N/P value of 12.53,indicating an effective candidate antibody against ASFV by blocking ELISA.In conclusion,a basis was laid for further researches on ASFV CD2v protein and for development of a blocking ELISA kit of ASFV.
作者
王彩霞
史喜菊
冯春燕
于浩洋
仇松寅
刘晓飞
陈冬杰
贾红
吴绍强
林祥梅
Wang Caixia;Shi Xiju;Feng Chunyan;Yu Haoyang;Qiu Songyin;Liu Xiaofei;Chen Dongjie;Jia Hong;Wu Shaoqiang;Lin Xiangmei(Animal Inspection and Quarantine Institute,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;Science and Technology Research Center of China Customs,Beijing 100026,China;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100091,China)
出处
《中国动物检疫》
CAS
2023年第7期95-102,共8页
China Animal Health Inspection
基金
中国检验检疫科学研究院基本科研业务费项目(2022JK21)。
作者简介
通信作者:林祥梅。E-mail:linxm@caiq.org.cn;通信作者:吴绍强。E-mail:sqwu@sina.com。
