摘要
目的建立能够分泌针对肠出血性大肠杆菌O157∶H7表面抗原的特异性单克隆抗体,以期用于该病原菌的检测。方法以敲除毒力基因的O157突变株F25全菌免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞Sp2/0融合,分别以全菌抗原和热酚水法纯化的O157 LPS抗原用间接ELISA筛选能够分泌特异性抗体的杂交瘤细胞株,纯化抗体并鉴定。结果建立了稳定分泌抗O157 LPS抗原单克隆抗体的杂交瘤细胞株3D6,分泌抗体属IgG3亚类,轻链为κ型。腹水抗体ELISA效价达1∶3.2×106。以辛酸/饱和硫酸铵法纯化后抗体效价为1∶1.6×106,亲和常数Ka大于6.14×10-11mol/L。用于协同凝集和间接荧光抗体试验具有良好的特异性。结论该单克隆抗体针对O157的LPS抗原,特异性强,亲和力高,可用于O157的检测或分离鉴定。
Spleen cells of BALB/c mice immunized with the antigen of Enterohemorrhagic Escherichia coli （EHEC） O157 ： H7 （F25） were fused with murine Sp2/0 myeloma cells. A hybridoma cell line specific to EHEC O157 LPS was established after screening and subcloning and was designated as 3D6. The isotype and the specificity of monoclonal antibodies （McAb） were determined and identified by ELISA. The results showed that the subtype of the McAb belonged to mouse IgG3. and the titer of the ascitic was 1 ： 3.2 ×10^6. The titer of the purified antibody was 1 ： 1.6 ×10^6. Affinity constant exceeded 6.14×10^-11 mol/L. This McAb could react with all 3 strains of EHEC O157 ： H7 and a Staphylococcus aureus strain ATCC29213 but not with other strains of bacterial, including 7 strains of E. coli and 11 strains of others bacterial genera. As demonstrated by indirect fluorescent antibody test, it had been proved that the McAb process high degree of specificity. It concludes that this McAb can be used as a useful reagent to detect the presence of EHEC O157 ： H7.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第10期971-973,977,共4页
Chinese Journal of Zoonoses
