[Construction and characterization of bispecific single-chain antibody fragments SZ-2/SZ-21 against platelet glycoprotein Ib alpha and beta3].
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.


CONCLUSIONS
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.
Dai
Kesheng
K
Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
An
Guangyu
G
Ruan
Changgeng
C
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D018033:Antibodies, Bispecific; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D039661:Integrin beta3 / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D018033:Antibodies, Bispecific; D007128:Immunoglobulin Fragments; D039661:Integrin beta3; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D013917:Thrombin