对猪卵透明带抗原杂交瘤制备的初步研究

抗透明带抗体对透明带溶解、精子穿透以及透明带脱落的抑制效应已被证实,本实验应用细胞融合技术制备猪卵透明带(PZ)单克隆抗体,探讨应用抗PZ 单克隆抗体对PZ 抗原结构及特性进行研究的途径。先后进行了六次细胞融合,五次融合获得成功。共接种1728孔,有克隆生长255孔,平均融合率15%。ELISA 检测246孔,初筛抗体阳性9孔,连续多次检测抗体持续阳性2孔。随后采用有限稀释,进行克隆化培养。卵细胞表面沉淀试验表明,阳性杂交瘤培养上清液与无卵丘卵子表面形成明显的沉淀层。分别采用分子量为1000和4000的PEG 作细胞融合,克隆生长孔各为34与29,阳性克隆孔数分别为2和3。对融合后无饲养细胞培养的观察说明,无饲养细胞生长克隆孔数是13,含饲养层生长克隆孔数为34,融合率各为4.5%与11.8%。

A PRELIMINARY RESEARCH ON THE PREPARATION OF HYBRIDOMA FROM PORCINE ZONA PELLUCIDA ANTIGEN

The purpose of this experiment is to manufacture the monoclonal antibody of porcinezona pellucida(PZ)by using cell fusion technique,and find out the way of how to study thestructure and properties of PZ by the monoclonal antibody.6 cell fusion experiments wereperformed,of which 5 were successful.In the experiment,1728 wells in total were inoculated.It was found that 255 wells have growing clones with 15% average fusion rate.Among the255 wells,246 were detected by Enzyme Linked Immunosorbent Assay.After preliminaryscreening,9 of them were found to be antibody positive.Among these 9 wells,2 appearedto be persistently antibody positive with constant and repeated detection.Then the mater-ials were treated with limited dilution and cultured in a way to obtain monoclone.Twoseperate experiments were performed simultaneously with the use of PEG of molecularweight 1,000 and 4,000 resptectively in order to fuse the cells.As a result,34 and 29 wellsgot growing clones with antibody positive in 2 and 3 weeks respectively.The fused cellswere then cultured.The one with non-feeding layer got 13 wells with clonal growth inthem and the one with feeding layer got 34 wells.The cell fusion rates were 4.5 and 11.8per cent respectively.