纤溶酶-α2抗纤溶酶复合物单克隆抗体的制备及鉴定

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。

Preparation and characterization of monoclonal antibodies against human plasmin-α2-antiplasmin complexes

AIM: To prepare monoclonal antibodies (mAbs) against human plasmin-alpha(2)-antiplasmin complexes (PAP). METHODS: BALB/c mice were immunized with PAP purified from human fresh plasma. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by indirect ELISA with equimolar plasminogen, alpha(2)-antiplasmin and PAP as immobilized antigen, respectively. The specificity and affinity of mAbs in ascitic fluids were characterized by Western blot and ELISA, respectively. RESULTS: Among the 24 specific mAbs obtained, 7 were directly against neoantigens (new emerging epitopes that were different from PAP precursor plasminogen and alpha(2)-antiplasmin, which are formed during PAP generation), 16 against plasmin domain and 1 against modified alpha(2)-antiplasmin in PAP molecule. Titers of all the mAbs to PAP were from 2 x 10(-4) to 1 x 10(-8), and 4 of them could strongly recognize PAP or plasminogen (K(d) from 5.62 x 10(-9) to 3.58 x 10(-11) mol/L). CONCLUSION: The mAbs against PAP neoantigens with high affinity was acquired successfully, which provided a tool for determination of plasma levels of PAP without interferences from plasminogen and alpha(2)-antiplasmin and for research on fibrinolytic status in-vitro.