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以人胎盘提纯Ⅳ型胶原为抗原,免疫 BALB/c 小鼠,制备了 McAB,并对其特异性进行了鉴定,为临床广泛应用和研究奠定了基础.一、材料和方法实验动物由北医动物部提供,主要试剂为 Sigma 或国产分析纯试剂.方法:用本室提纯的 COL Ⅳ免疫动物,一周后尾部取血,经 ELISA 方法检测其血清抗体滴度.本实验共免疫四次,血清抗体滴度大于1/12 000.最后一次免疫两周后参照Zala,H 方法,取免疫好的 BALB/c 小鼠脾细胞与 SP2/0细胞在50%PEG 4 000溶液介导下进行细胞融合.ELISA 方法检测杂交瘤细胞上清液,筛选分泌抗 COL ⅣMcAB 的杂交瘤细胞株.结果共筛出46株阳性克隆,经有限稀释法将抗体滴度高的五个阳性株分别作了四次克隆化,其中三株亚克隆产物阳性率连续三次达100%,命名为3E6、3D4、4G8,三株细胞连续传代培养5个月分泌抗体能力不变,冻存6个月细胞复苏后仍能分泌抗体.
The human placenta purified type Ⅳ collagen as antigen, BALB / c mice were immunized, prepared McAB, and its specificity was identified for the clinical application and research has laid the foundation for a wide range of applications and research materials, Animal Department, the main reagent is Sigma or domestic analytical reagent.Methods: COL IV immunized animals in this room, a week after the tail blood, ELISA serum antibody titers.This experiment was immunized four times, the serum Antibody titer greater than 1/12 000. Two weeks after the last immunization with reference to Zala, H method, immunized BALB / c mouse spleen cells and SP2 / 0 cells in 50% PEG 4 000 solution-mediated cell fusion ELISA method was used to detect the hybridoma cell supernatant, screening anti-COL ⅣMcAB secretion of hybridoma cell lines.Results A total of 46 positive clones were screened by limiting dilution of the antibody titer of five positive strains were made four times The positive rates of the three subclones were 100% for three times in a row, named as 3E6, 3D4 and 4G8. The ability of three strains to continue to subculture for 5 months was the same with the ability of secreting antibody. After 6 months of cryopreservation, Secretion of antibodies.