纤溶酶-α_2抗纤溶酶复合物单克隆抗体的制备及鉴定  

Preparation and characterization of monoclonal antibodies against human plasmin-α_2-antiplasmin complexes

作　　者：李建新[1] 王红[2] 杭勤[2] 郑佐娅[2] 罗伟[2] 李稻[2] 王鸿利[2] 

机构地区：[1]北京大学深圳医院检验科,广东深圳518036 [2]上海第二医科大学医学检验重点实验室,上海200023

出　　处：《细胞与分子免疫学杂志》2004年第3期372-375,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：上海市科技发展基金资助项目 (No .9941 1 90 0 3)

摘　　要：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 monoclonal antibodies (mAbs) against human plasmin α 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, α 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 α 2 antiplasmin, which are formed during PAP generation), 16 against plasmin domain and 1 against modified α 2 antiplasmin in PAP molecule. Titers of all the mAbs to PAP were from 2×10 -4 to 1×10 -8 , and 4 of them could strongly recognize PAP or plasminogen (K d from 5.62×10 -9 to 3.58×10 -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 α 2 antiplasmin and for research on fibrinolytic status in vitro .

关 键 词：PAP 单克隆抗体 新抗原 

分 类 号：R392.11[医药卫生—免疫学]