摘要
用牛血清白蛋白(BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。
The monoclonal antibodies against BSA were generated by fusion spleen cells from immunized BALB/c mouse and SP2/O mouse myeloma cells, which can be used in determination of BSA contamination in biological preparations. The culture of hybridoma lines were first screened by sandwich-ELISA in detection of mouse IgG, then mouse ascites were prepared. Four hybridoma lines which secreted monoclonal antibodies against BSA were determined and developed, named as 2A5,3A3,3G6,4A8 and isotyping as IgG2a/κ,IgG1/κ,IgG1/κ,IgG1/κ respectively. These McAbs with titers over 10.5 to BSA by indirect ELISA, and purity of purified ascites attained more than 90%. Forthermore, the MCAbs showed a high specificity to BSA with a MW 68000 Da by Western blotting and had a low reactivity to serum albumin from human and other animals, which can recognize two different epitopes on BSA. Both of their relative affinity and sensitivity are in an order 3A3 〉 2A.5 〉 3G6 〉4A8. These hybridoma lines could be cultured for continuous three months and kept in a stable condition.
出处
《微生物学免疫学进展》
2007年第1期30-34,共5页
Progress In Microbiology and Immunology
