Inhibition assay for the binding of biotinylated von Willebrand factor to platelet-bound microtiter wells in the presence of ristocetin or botrocetin.
We developed a solid-phase inhibition assay for the binding of biotinylated von Willebrand factor (vWF) to platelet glycoprotein (GP)Ib based on an enzyme-linked immunosorbent assay (ELISA) with platelet-bound microtiter wells. Washed platelets were immobilized onto the microplates via the anti-GPIIb/IIIa monoclonal antibody (mAb) VNR-5. In the presence of an antibiotic ristocetin (1 mg/ml, final) or the snake venom botrocetin (2 micrograms/ml, final), biotinylated vWF bound to the affixed platelets with half-maximal binding occurring at a vWF concentration of approximately 2 micrograms/ml. Several specific inhibitors of the binding of vWF to GPIb abolished the interaction of biotinylated vWF with GPIb in a dose-dependent fashion, with comparable protein concentrations to those seen in the liquid-phase inhibition assay using 125I-vWF. These inhibitors included mAb against vWF (NMC-4), mAb against GPIb (AP-1), and two vWF fragments containing vWF's putative GPIb-binding domain, namely a 39/34-kDa dispase-digested vWF fragment and a recombinant vWF fragment. Thus, this new assay is a convenient alternative to the conventional inhibition assay using 125I-vWF.
Miura
S
S
Department of Blood Transfusion, Nara Medical University, Kashihara City, Japan.
Nishida
S
S
Makita
K
K
Sakurai
Y
Y
Shimoyama
T
T
Sugimoto
M
M
Yoshioka
A
A
Ishii
K
K
Kito
M
M
Kobayashi
T
T
Fujimura
Y
Y
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D001710:Biotin / Q000737:chemistry; D003435:Crotalid Venoms / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006388:Hemagglutinins / Q000494:pharmacology*; D006801:Humans; D007457:Iodine Radioisotopes; D008832:Microchemistry; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000187:drug effects*; D011485:Protein Binding; D012310:Ristocetin / Q000494:pharmacology*; D017186:Titrimetry; D014841:von Willebrand Factor / Q000737:chemistry / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D006388:Hemagglutinins; D007457:Iodine Radioisotopes; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D012310:Ristocetin; D001710:Biotin; C030961:botrocetin