Preparation And Application Of Monoclonal Antibody Against Human D-dimer

Objective:(1) Isolated and purified D-dimer, a degradation product of cross-linked fibrin, and identified the common features of the D-dimer. (2) Prepared hybridoma cell lines which could secrete monoclonal antibody against human D-dimer. (3) Purified and preliminarily applied the monoclonal antibody which was against human D-dimer.Methods:(1)A two steps flow comprised Sephacryl S-300 gel filtration chromatography and focusing chromatography was used to isolate and purify the D-dimer from human fibrinogen degradation products (FDP). (2)BALB/c mice were immunized with the purified D-dimer. Used Hybridoma technique and ELISA to prepare and screen hybridoma cell lines, which secreted monoclonal antibody against human D-dimer. Hybridoma cells were injected to abdominal cavity of BALB/c mice for ascites. Monoclonal antibodies were purified from ascites with ammonium sulfate fractionation and DEAE-affi-gel-blue chromatography. The purity and subclasses were identified by sodiumdodecylsulfate-polyactylamide gel electrophoresis (SDS-PAGE) and Mouse Monoclonal Antibody Isotyping Kit.The characteristics of these antibodies were analyzed, including the cross reaction between monoclonal antibody and fragment D, fragment E, fibrinogen, thrombin and factor ⅩⅢ (F ⅩⅢ), and the effect of divalent mental ion (Ba2+、Zn2+、Mg2+、Ca2+、 Mn2+) on the combination between monoclonal antibody and D-dimer. (3) 03,05,07 D-dimer McAb were covalently coupled to Carboxyl Microspheres, and then analyzed the effect of this coupling. Used the Carboxyl Microspheress, which were coupled with the D-dimer McAb, to react with the purified D-dimer, analyzed the effect of the reaction.Results:(1) After two steps of chromatography,D-dimer was concentrated in 25.27±6.35% yield from FDP, and the percentage between D-dimer and protein was 77.82±7.98%,(n=3). (2)8 Cell lines of hybridoma secreting monoclonal antibodies specifically against D-dimer were established and named 01～08 respectively. They could steadily secrete antibodies after the cell lines had been continuously cultured for five months. The number of chromosomes range was 79 to 94. (3) 8 monoclonal antibodies were purified from ascites. The immunoglobulin subclass of all the monoclonal antibodies were all IgG2b. Zn2+could remarkably restrain the combination of D-dimer with all eight antibodies against D-dimer; Mn2+ could restrain the combination of D-dimer with 06,07 monoclonal antibodies against D-dimer, but the restraining effect was weaker than Zn2+. All those D-dimer McAb were against the same antigen-binding epitopes on the surface of D-dimer or the epitomes were very close. The reaction between fragment D, fragment E fibrinogen, thrombin, factor XIII (F XIII) with the monoclonal antibodies against D-dimer were negative. The gradation of comparatively affinity is 03>07≥05>04≥01≥02≥06>08. (4) Antibodies were efficiently coupled with Carboxyl Micospheres.But the coupling rates between those three antibodies were different,03 antibodies was 52.16%,05 was 29.80%,07was 77.41%.The Carboxyl Micospheres coupled with D-dimer McAb could react with D-dimer, after the reaction, the average absorbance at 280 nm of the supernate decreased by 59.54%.Conclusions:The D-dimer which could bring high response of BALB/c mice was separated and purified by the way included two chromatography steps effectively. Established 8 clones of hybrid cells,which secreted monoclonal antibodies against D-dimer, and had made a certain amount of antibodies. The D-dimer McAb could couple with Carboxyl Micspheres effectively, and could react with D-dimer, layed the root for developing D-dimer detecting kits.