牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

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。

Preparation and characterization of monoclonal antibody(McAb) against BSA

The monoclonal antibodies against BSA were generated by fusion spleen cells from immunized BALB/c mouse and SP2/0 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>2A5>3G6>4A8.These hybridoma lines could be cultured for continuous three months and kept in a stable condition.