抗人PAI-1单克隆抗体制备、鉴定和应用

Preparation, Characterization and Application of Monoclonal Antibodies Against PAI-1

以重组PAI-（rPAI-1）为抗原,通过杂交瘤技术获得6株阳性杂交瘤细胞（AP1、AP2、AP3、AP4、AP5和AP6）,并用SPA亲和层析纯化了抗PAI-1单克隆抗体（McAb）。所有McAb均能识别rPAI和天然PAI-1,腹水滴度均为106以上。6种McAb对PAI-1亲和常数在3.45×107—1.05×109mol/L之间。AP2、AP3 McAb能完全抑制PAI-1活性,AP4、AP和AP6只能部分抑制PAI-1活性,AP1则不抑制PAI-1活性。6种McAb中只有AP1、AP4和AP5能识别PAI-1/t-PA复合物。利用AP1、AP3、和AP4 McAb制备免疫亲和柱一步纯化HepG2细胞分泌的PAI-1,纯度大于98％,回收率92％,纯化倍数51倍。利用抗PAI-1 McAb建立了夹心法ELISA,测定了人血浆PAI-1水平,正常人血浆PAI-1平均含量（±D）为24.7±7.75ng/ml。

Six hybridoma cell lines (API, AP2, AP3, AP4, AP5 and AP6) secreting monoclonal amtibodies (McAb) against PAI-1 were obtained by fusing the murine myeloma cell line SP2/0 with the spleen cells from BALB/c mouse immunize.d with re-combinant PAI-1 expressed in E. coli . These antibodies were purfied by SPA affinity chromatography. All McAbs recognized rPAI-1 and PAI-1 from the human hepatoma cell line HepG2, The titers of ascites were more than 106. The antibody-antigen affinity constants (Kaff) for anti-PAI-1 McAb measured by ELISA were between 3. 45×107-1. 05 × 1010mol/L. AP2 and APS McAbs were quite effective in quenching the activity of PAI-1. Partial quenching of PAI-1 activity was achieved with AP4, AP5 and AP6 McAbs , respectively. API McAb had no effect upon PAI-1 activity. Three of the six McAbs (AP1, AP4 and AP5 ) bound to the PAI-1/t-PA complex, while the others did not. The PAI-1 was purified 51 folds to homogeneity from serum free medium of HepG2 with the recovery rate of 92 % by one-step procedure using Sepharose 4B conjugated with anti-PAI-1 McAb (AP1, AP3 and AP4). A sandwich ELISA for the measurement of PAI-1 antigen in human plasma was developed based on anti-PAI-1 McAb against non-overlapping epitopes. The mean value of plasma PAI-1 for the healthy donors was 24. 7 ± 7. 75ng/ml measured by ELISA.