抗人Cu/Zn SOD单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibody against human Cu/Zn SOD

目的制备抗人Cu/Zn SOD单克隆抗体并对其生物学特性进行鉴定。方法以人Cu/Zn SOD作为免疫原免疫Balb/c小鼠,取其脾细胞与SP2/0细胞融合,经多次筛选及克隆化,建立可稳定分泌抗人Cu/Zn SOD单克隆抗体的杂交瘤细胞株。用ELISA、斑点ELISA、Western Blot、免疫组化鉴定单克隆抗体的特性,并测定其效价、亚型及相对亲和力。结果筛选到一株可稳定分泌抗人Cu/Zn SOD单克隆抗体的杂交瘤细胞株4H10H4B12,亚型鉴定重链为IgG2a,轻链为kappa链。ELISA法测定腹水效价为1∶256,000,抗体相对亲和力为1.101×109mol/L。斑点ELISA、Western Blot显示抗体能特异识别免疫原、人肝细胞株HL-7702和人红细胞中的天然Cu/Zn SOD;不与鼠Cu/Zn SOD和人Mn SOD发生交叉反应。免疫组化进一步显示了人Cu/Zn SOD均匀分布于细胞的胞浆中。结论成功制备了抗人Cu/Zn SOD单克隆抗体,为进一步研究其与腹主动脉瘤的关系奠定了基础。

Objective To prepare and identify the monoclonal antibody （MAb） against human Cu/Zn SOD. Methods BALB/C mice were immunized with immunogen human Cu/Zn SOD. The spleen cells of the immunized mice were i- solated and fused with SP2/0 cells. After several rounds of detecting and cloning, a hybridoma cell line secreting anti-hu- man Cu/Zn SOD MAb was obtained. Its specificity was evaluated with indirect-ELISA, spot-ELISA, Western blot and immunohistochemistry. The titer, immunoglobulin subtype and affinity of the MAb were measured. Results One cell line of hybridoma named 4H10H4B12 was obtained, which was identified that the heavy and light chains of anti-human Cu/Zn SOD MAb were IgG2a and κ, respectively, with high affinity of 1. 101 × 10^9 L/mol and high titer of 1：256,000. The spot-ELISA, Western blot revealed that the MAb was specifically against the immunogen and native proteins expressed in HL-7702 cells, human hematids and didn＇t react with rat Cu/Zn SOD or human Mn SOD. The cell immuno- histochemistry proved that the MAb could recognize the human Cu/Zn SOD located in kytoplasm of HL-7702 cells. Conclusion The success in mouse anti-human Cu/Zn SOD MAb preparation provides the basis for further study of AAA.