[Preparation and characterization of monoclonal antibodies against human plasmin-alpha 2-antiplasmin complexes].

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.