人D-二聚体分离纯化及鉴定

Isolation,purification and identification of human D-dimer

目的分离纯度较高和人D-二聚体(D-dimer),为制备抗人D-二聚体单克隆抗体提供可靠抗原。方法以凝血酶、凝血因子等处理人纤维蛋白原,得到交联纤维蛋白,经纤溶酶消化,得到交联纤维蛋白降解产物(FDP);对FDP作Sephacryl S-300凝胶过滤层析和聚焦层析,分离纯化得到D-dimer。采用免疫比浊法测定D-dimer含量、染料结合法测定纯化蛋白含量,以SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察D-dimer的纯化情况,WestonBlot法确认D-dimer的存在。结果制备得到的FDP SDS-PAGE图谱显示有多条蛋白条带,1条分子量为180～250kD蛋白条带在Western Blot图谱中显示可以被D-dimer单克隆抗体特异性地识别。FDP经Sephacry S-300凝胶层析和聚焦层析后得到纯度较高的D-dimer(n=3):总蛋白含量比为(77.82±7.98)%,蛋白回收率(25.27±6.35)%;SDS-PAGE图谱显示:经过两步层析得到的终产物为分子量180～250 kD的蛋白;Western Blot证实:经过Sephacryl S-300凝胶层析和聚焦层析处理,最终得到分子量为180～250 kD的终产物可被D-dimer单克隆抗体特异性识别。结论采用Sephacryl S-300凝胶层析和聚焦层析能够分离纯化获得较高纯度的D-dimer,可作为制备D-dimer单克隆抗体的抗原。

Objective To prepare isolated and purified D-dimer,which would be the antigen to prepare the mono- clonal antibody （McAb） in the future research. Methods Turned human fibrinogen into cross-linked fibrin via thrombin and Factor III. Then used plasmin to digest cross-linked fibrin into fibrin degradation products （FDP）. A 2 steps flow com- prising of Sephaeryl S -300 gel filtration chromatography and focusing chromatography were used to isolate and purifie D-di- mer from FDP. The eontent of D-dimer was determined by Turbidimetrie ImmunoAssay. Ultraviolet speetroseopy was used to determine the protein content of products. The purity of products was detected by sodium dodecyl sulphate-polyaerylamide gel electrophoresis （SDS-PAGE）. Western Blot was used to identify D-dimer. Results The FDP contained several kinds of protein,which were displayed in SDS-PAGE image. The western blot displayed that the monoclonal antibody against D-di- mer could specifically recognize the protein which molecular weight was between 180 kD and 250 kD in the FDP. After Sephacryl S -300 gel filtration chromatography and focusing chromatography,we got the final product, which percentage be- tween D-dimer and protein was （77. 82 ± 7. 98 ） %, and it was concentrated in （25.27 ± 6. 35 ） % yield from FDP. And the main composition of this product was the protein which molecular weight was between 180 kD and 250 kD showed in the SDS-PAGE image. The protein mentioned above which molecular weight was between 180 kD and 250 kD could be specific- ally recognized by the monoclonal antibody against D-dimer in Western Blot. Conclusion The two step-flows could well separate and purified D-dimer from FDP.