摘要
为建立区分口蹄疫病毒(FMDV)疫苗免疫和自然感染动物的血清学鉴别检测方法,本研究采用杆状病毒表达的FMDV非结构蛋白3ABC免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,并以原核表达的FMDV非结构蛋白3B作为抗原筛选融合细胞的培养上清液,获得一株稳定分泌抗3B蛋白单克隆抗体(MAb)的杂交瘤细胞。经间接免疫荧光鉴定其为一株抗FMDV非结构蛋白3B的特异性MAb,为IgG1/κ亚类,间接ELISA检测杂交瘤细胞上清液和腹水的抗体效价分别为1∶12 800和1∶106。硫氰酸盐洗脱法测定其相对亲和力常数为1.5 mol/L。采用该MAb作为包被抗体并特异性的结合原核表达的3B重组蛋白用于检测血清中抗FMDV的非结构蛋白抗体,初步建立了以MAb为基础的ELISA方法。其MAb包被浓度为5μg/mL,3B重组蛋白浓度为1.1μg/mL,被检血清1∶100稀释,通过检测猪血清并与3B间接ELISA和prioCHECKRNSP ELISA试剂盒进行比较,表明该方法具有更强的特异性。
To establish a detection method of foot-and-mouth disease virus （FMDV） 3B antibody for differential diagnosis between infected and vaccinated animals, the SP2/0 cells were fused with spleen cells immunized with the purified FMDV nonstructural protein 3ABC expressed by baculovirus to prepare monoclonal antibody （MAb） against the nonstructural protein followed by screening MAb using 3B antigen expressed by E.coli. One hybridoma cell line that secreted MAb against the 3B was obtained. The antibody titers in cell medium and ascite were 1∶12 800 and 1∶106, respectively. The relative affinity index of the MAb was 1.5 mol/L determined by thiocyanate elution measurement. Furthermore, an ELISA was established based on the direct binding of antibodies to the 3B trapped by the MAb with 5 μg/mL of coating concentration and 1.1 μg/mL of trapped concentration. This ELISA was more sensitive than 3B-iELISA through detecting swine sera in the differential diagnosis.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2014年第6期448-452,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
中央级公益性科研院所基本科研业务费专项(0302013012)
关键词
口蹄疫病毒
3B重组蛋白
MAB
ELISA
foot-and-mouth disease vires
recombinant 3B protein
monoclonal antibody
ELISA
