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Published on: June 2, 2023
[Preparation and characterization of monoclonal antibodies against human plasmin-alpha 2-antiplasmin complexes]
Jian-xin Li1, Hong Wang, Qin Hang
1Department of Clinical Lab., Shenzhen Hospital,Peking University, Shenzhen 518036, China. ljxms@citiz.net
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.
