抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG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,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。

Preparation and characterization of hybridomas secreting monoclonal antibodies against bovine serum IgG

The monoclonal antibodies against bovine serum IgG were prepared by fusion of the bovine IgG immunized BALB/c mouse’s spleen cells with SP2/0 myeloma cells,the fusion cells were cultured by the culture medium containing goat sera,the supernatants were screened by an indirect-ELISA.Four hybridoma cell lines secreting anti-bovine IgG McAbs were established and named as 1G5、2A8、3F5、4C5.By analysis of Ig subclass,the 1G5,3F5,4C5 are classified as IgG1,and the 2A8 as IgG2a.McAbs titers of the ascites of four hybridoma lines were over 10-5.The four McAbs showed high specificity to bovine IgG and no cross-reactivity to sera globulins from human,horse,swine,goat,rabbit,guinea pig and other animals,except that 3F5 had a high reactivity to goat serum IgG.They can recognize two different epitopes of 160kD on the bovine IgG molecule.The orders of their relative affinity are 4C5>2A8>1G5>3F5 and the order of their relative sensitivity are 2A8>4C5>3F5>1G5.Each of these hybridomas with chromosome numbers of over 90 was proved to be capable of secreting McAb stably after consecutive culture,cryopreservation and resuscitation.The double antibody sandwich ELISA was developed by using these McAbs to detect the residual bovine IgG in preparation of biological products.