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目的 :制备纤溶酶 α2 抗纤溶酶复合物 (PAP)的单克隆抗体(mAb)。方法 :以从血浆中纯化的PAP免疫BALB/c小鼠。按常规方法融合 ,以固相等分子浓度的纤溶酶原、α2 抗纤溶酶(α2 AP)及PAP为抗原 ,建立间接ELISA筛选杂交瘤细胞培养上清 ,并对杂交瘤细胞分泌的mAb的特异性和亲和力进行鉴定。结果 :共获得 2 4株可稳定分泌特异性mAb的杂交瘤细胞。其中 ,针对PAP分子中纤溶酶结构的mAb 16株 ,针对α2 AP结构的mAb 1株 ,针对新抗原 (PAP分子中新出现的不同于前体分子纤溶酶原及α2 AP的抗原决定簇 )结构的mAb 7株。这些腹水中抗PAPmAb的滴度为 2× 10 -4～ 1× 10 -8,其中 4株mAb的亲和常数为 5 .6 2× 10 -9～ 3.5 8× 10 -11mol/L之间。结论 :成功地制备针对PAP新抗原的具有高亲和力的mAb ,为建立不受其前体分子干扰的PAP特异性检测方法 ,研究纤溶系统的激活状态提供了工具。
AIM: To prepare a monoclonal antibody (mAb) for plasmin α2 antiplasmin complex (PAP). Methods: BALB / c mice were immunized with purified PAP from plasma. According to the conventional method, an indirect ELISA was used to screen the supernatant of the hybridoma cell culture with the plasminogen, α2 anti-plasmin (α2 AP) and PAP as the antigen of the same molecular weight. The mAbs secreted by the hybridoma cells The specificity and affinity were identified. Results: A total of 24 hybridomas were obtained which could stably secrete specific mAb. Among them, 16 mAbs directed against plasmin structure in PAP molecule and 1 mAb directed against α2 AP structure were targeted against a novel antigen (PAP antigen, which is different from the antigenic determinants of plasminogen and α2 AP ) Structure of mAb 7 strains. The titer of anti-PAPmAb in these ascites was 2 × 10 -4 ~ 1 × 10 -8, of which the affinity constant of 4 mAbs was between 5.2 × 10 -9 ~ 3.5 8 × 10 -11 mol / L. CONCLUSION: The successful preparation of mAb with high affinity against PAP neoantigen provides a tool for establishing PAP-specific detection method that is independent of the precursor molecule and investigating the activation state of fibrinolytic system.