ABSTRACT
To easily obtain plasmin-alpha(2)-antiplasmin complex (PAP) with high purity, the purification procedure was improved by authors. After urokinase activated fresh human platelet-deficient plasma had been applied to Lysine-Sepharose 4B affinity chromatography column, elutions were sequentially performed by several eluents with different ingredients. The results showed that 3.0 mg PAP could harvested from 100 ml fresh plasma by this method and the whole procedure could be finished within several hours. In conclusion, this procedure is simple, rapid, economical and high-yield.
Subject(s)
Fibrinolysin/isolation & purification , Sepharose/analogs & derivatives , alpha-2-Antiplasmin/isolation & purification , Blotting, Western , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Fibrinolysin/analysis , Humans , alpha-2-Antiplasmin/analysisABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Fibrinolysin/immunology , alpha-2-Antiplasmin/immunology , Animals , Antibody Affinity , Antibody Specificity , Enzyme-Linked Immunosorbent Assay , Female , Hybridomas/immunology , Hybridomas/metabolism , Immunization , Male , Mice , Mice, Inbred BALB CABSTRACT
AIM: To prepare monoclonal antibodies (mAb) against recombinant human nucleoside diphosphate kinase-A(NDPK-A) and characterize their properties. METHODS: BALB/c mice were immunized with rhNDPK-A, and mAb was prepared by hybridoma technique. Ig subclass and specificity was analysed by double immunodiffusion and western blot respectively. The titres of mAbs in ascitic fluid, relative affinity and epitopes recognized by mAbs were determined by indirect ELISA. RESULTS: 6 hybridoma cell lines secreting anti-rhNDPK-A mAbs were obtained. Their Ig subclass belonged to IgG1. The titers of 6 mAbs in ascitic fluid were 1x10(-4)-5x10(-6). Relative affinity of mAbs were 4.5x10(-9)-2.8x10(-10) mol/L. They recognized 3 different epitopes on rhNDPK-A molecule. CONCLUSION: 6 mAbs against rhNDPK-A have been prepared successfully which provide useful reagent for clinical diagnosis and further research.
