[Expression of antibody Fab against platelet IIb/IIIa in E.coli and characterization of its biological activity].
OBJECTIVE
To develop a Fab antibody against platelet GPIIb/IIIa.


METHODS
A mouse anti-human GPIIb/IIIa monoclonal antibody (mAb) P140 was selected by the indirect immunofluorescence assay (IFA) and platelet aggregation inhibition test. The Fd and light chain genes were cloned from the hybridoma cells secreting the mAb P140. The genes of P140 Fab were inserted into plasmid p3MH to construct recombinant expression plasmid p3MH/P140kappa-Fd. After digestion with the restriction enzyme, the recombinant plasmid was transformed into E.coli XLI-Blue. The expressed product was purified by TALON metal affinity resin. Purified P140 Fab was characterized by SDS-PAGE, ELISA, Western blot and platelet aggregation inhibition test.


RESULTS
SDS-PAGE analysis showed that relative molecular mass (M(r)) of P140 Fab was 47x10(3). The results of ELISA, Western blot and platelet aggragation inhibition test indicated that P140 Fab could specifically bind to platelet and inhibit platelet aggragation in dose-dependent manner. The mean value of IC(50) was 16.85 mg/L.


CONCLUSIONS
A soluble anti-platelet GPIIb/IIIa antibody P140 Fab was prepared successfully, which lays the foundation for further clinical application.
Yang
Yong-chang
YC
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Lai
Chun-ning
CN
Wang
Yan
Y
Shen
Bei-fen
BF
Li
Yan
Y
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D010974:Platelet Aggregation / Q000187:drug effects*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007145:Immunoglobulin kappa-Chains; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins