牛种布氏菌单克隆抗体的研究

RESEARCH OF MONOCLONAL ANTIBODY TO BR. ABORTUS

应用酚杀死的光滑型牛种布氏菌544A抗原免疫的BALB/C小鼠,融合前一周用超声波处理后高速离心制备的可溶性抗原加强免疫,腹腔内注射3次,以无菌手术取脾,制备成免疫脾细胞悬液,取10~8个免疫脾细胞与10~7个SP^2/0小鼠骨髓瘤细胞混合,在分子量4000的PEC 0.8ml(w/v)融合剂的作用下,两分钟后速加无血清的1640液25ml,离心沉淀再悬浮于20%FBSHAT1640培养基中,分装六块96孔细胞培养板,定期补加培养液。10天后开始测其孔上清,其检出阳性孔44孔(ELISA OD值>0.3),初步筛选出11株效价较高、特异性较好者,经反复检测,抗体分泌水平比较稳定,扩大培养,冻存液氮中。克隆后的细胞染色体计数平均为94条/细胞,无血清培养的细胞上清液在PAGE中呈现一条带,符合Mc.cell株仅分泌一种抗体的特性。

The BALB/c mice were immunized with smooth Br, abortus544A antigen killed by phenol. A week before hybridizing, the animals were booster immunized with soluble antigen prepared by hypercentrifugation after ultrasonication four times weekly ID. Taking their spleens under axenic operation.We preparated suspension of immune spleen cells, mixed 10~8 immune spleen cells with 10~7 SP2/0 mice myeloma cells treatmented with 0.8ml PEG, molecular weight 4,000, two minutes. Then, added 25ml 1640 medium without serum,suspended the mixed cells in 20%FBS HAT 1640 medium after centrifugalization again, then equally placed into six piece cell of culture plates with 96 wells. Supplemented culture liquid on time. Supernatant of the wells was tested after 10 days. 44 cell strains of better specificity were primary screened.They were detected repeatly,increased the cultures which had better specificity and stability for production of antibody to antigen,and were stored in liquid Nitrogen.The chromosomal count of the colony cells was94 each cell on an average. The cells supernatant of serumfree culture showed a belt in PAGE. This is in keeping with the character that monoclonal strain only secrete a antibody.