Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin

We have developed a rapid [H-3]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the c olchicine-binding site on tubulin. The premise of our assay is that compoun ds will compete with radiolabeled colchicine for the tubulin-binding domain . Biotin-labeled tubulin is incubated first with unlabeled compound and rad iolabeled ligand Streptavidin-labeled SPA beads are added, and the radiolab el associated with tubulin is directly counted with no separation steps. Un der our experimental conditions, the dissociation constant of binding (K-d) for colchicine to tubulin was determined to be 1.4 mu M, which was consist ent with previously reported values. Assay validation was performed by comp etitively inhibiting [H-3]colchicine binding to tubulin with known microtub ule inhibitors and comparing their inhibition constants (K-i). Our SPA bead method is a powerful tool since it overcomes the disadvantage of tradition al filtration techniques, as there are no separation steps. It is extremely easy to set lip, multiple samples can be assayed and supply and labor cost s are reduced because of the minimal volume and test reagents used.