Objective: To generate bispecific single-chain Fv (bis-scFv) antibody fragment SZ-2/SZ-21 against platelet glycoprotein (GP) Ib alpha and GP IIIa.
Methods: The SZ-2/SZ-21 bispecific single-chain antibody expression vector pET22-21-L-2scFv was constructed by gene recombination technique with a interdomain linker fragment, SZ-21 single-chain antibody (anti- GP IIIa) gene and SZ-2 single-chain antibody (anti- GP Ib alpha) gene, identified by sequencing, and introduced into Escherichia coli strain BL21 (DE3) Plys. The bispecific protein accumulated in bacteria as insoluble inclusion bodies was harvested, denatured, refolded, and affinity-purified in vitro. SZ-2/SZ-21 bispecific single-chain antibody was added into platelet-rich plasma (PRP) and the binding of SZ-2/SZ-21 bispecific single-chain antibody with platelets was examined by histochemistry and cytometry, ELISA, or Western blotting. Restocetin, ADP, or thrombin was added into PRP with SZ-2/SZ-21 bispecific single-chain antibody to observe the platelet aggregation.
Results: The expression vector pET22-21-L-2scFv was correctly constructed. Its recombinant protein was expressed mostly in the form of inclusion bodies, and the yield accounted for 14% of the total cell protein. The bispecific protein could bind to platelet. The platelet aggregation induced by restocetion, ADP, or thrombin in PRP with SZ-2/SZ-21 bispecific single-chain antibody was significantly reduced.
Conclusion: An SZ-2/SZ-21 bispecific single-chain antibody against two target antigens in platelet was developed and characterized with the ability to inhibit platelet aggregation induced by restocetin, ADP, and thrombin.