[Characterization and functional studies of vWF A3 domain monoclonal antibodies that inhibit binding of vWF to collagen].
OBJECTIVE
To prepare anti-von Willebrand factor A3 (vWF-A3) domain monoclonal antibodies(mAbs) which block vWF-A3 binding to collagen, and characterize their biochemical properties and functions.


METHODS
BALB/c mice were immunized with purified recombinant vWF-A3 protein (rvWF-A3). Murine anti-human vWF-A3 mAbs were developed by standard hybridoma technology and identified with ELISA. The recognition of the mAbs with rvWF -A3 and reduced human vWF was identified by Western-blot. The effect of mAbs on binding of purified human vWF to human placenta or calf skin collagen III was studied with collagen binding inhibition test.


RESULTS
A group of 30 murine anti-human vWF-A3 mAbs was obtained, from which 2 clones were identified as inhibitory ones and designated as SZ-123 and SZ-125. SZ-123 and SZ-125 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that SZ-123 and SZ-125 could recognize a 27 x 10(3) band of rvWF-A3 and 2 reduced human vWF bands at 250 x 10(3) and 170 x 10(3). SZ-123 and SZ-125 not only inhibited the binding of purified human vWF (1.5 and 3.0 microg/ml) to human type III collagen and to calf skin collagen III in a dose dependent manner, but also inhibited the binding of plasma vWF from human, rhesus monkeys or Beagle dogs to the two collagens.


CONCLUSIONS
SZ-123 and SZ-125 are neutralizing mAbs against vWF-A3 domain and may have therapeutic potential as an antithrombotic agent.
Zhao
Yi-Ming
YM
Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Dong
Ning-Zheng
NZ
Shen
Fei
F
Xie
Li-Qian
LQ
Ruan
Chang-Geng
CG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D003094:Collagen / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D014841:von Willebrand Factor / Q000276:immunology*
D000911:Antibodies, Monoclonal; D014841:von Willebrand Factor; D003094:Collagen