A one-step enzyme-linked immunosorbent assay for a novel osteoblast differentiation-promoting compound, TAK-778 in serum.
TAK-778, ((2R,4S)-(-)-N-[(4-diethoxyphosphorylmethyl)phenyl]- 1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2- carboxamide), is a novel osteoblast differentiation-promoting compound, and its sustained-release formulation is expected to be clinically used for the enhancement of fracture healing. To date, TAK-778 levels in serum have been measured using conventional reverse-phase HPLC with inferior sensitivity and time-consuming procedures. We have produced polyclonal antibodies against TAK-778 by using one of its derivatives coupled with a carrier protein, and developed a one-step ELISA for the determination of TAK-778 in serum. The antibodies had minimal cross-reactivities to the biologically inactive metabolites including the oxidized-form (0.36%) and the cleavaged-form (0.00083%). The competitive ELISA was accomplished within three hours with a detection limit of 0.5 ng/ml serum, and the coefficients of variations for samples ranging from 1 ng/ml to 80 ng/ml were 1.1-4.4% in an intra-assay and 3.1-9.6% in an inter-assay. The present ELISA is so rapid, sensitive and selective for TAK-778 that it could be conveniently used in a clinical field for the determination of many serum specimens.
Kurokawa
T
T
DDS Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Hoshino
T
T
Oda
T
T
Saito
K
K
Ogawa
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001582:Benzothiepins / Q000032:analysis / Q000097:blood* / Q000276:immunology; D002454:Cell Differentiation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008297:Male; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D001582:Benzothiepins; C120450:TAK 778