A picogram BA-ELISA quantification assay for rLj-RGD3, a platelet fibrinogen receptor antagonist, in the rat plasma and its application to a pharmacokinetic study

Abstract

Abstract rLj-RGD3, a new member of RGD-motif toxin protein family derived from Lampetra japonica by means of DNA recombinant technique, has been demonstrated to be a platelet fibrinogen receptor antagonist. The present article aims to report an innovative validated highly sensitive and specific biotin-avidin enzyme linked immunosorbent assay (BA-ELISA) in order to provide a bio-analytical method for its pharmacokinetic (PK) study. The concentration of rLj-RGD3 in rat plasma was measured at a picogram level by the developed double sandwich BA-ELISA assay, which used two different mouse anti-rLj-RGD3 monoclonal antibodies as capture antibody and detection antibody, respectively, directed at different epitopes. The method was validated to be highly specific (no interference with the detection from blank plasma), precise (RSD <10%) and accurate (94%-104%). The absolute recovery was as high as 94%-105%. The calibration curve showed good linearity ranging from 50 to 1600 pg/mL. The LOQ was found to be as low as 50 pg/mL. The above validated assay was successfully employed for the assessment of PK disposition of rLj-RGD3 in rats. After i.v. and s.c. dosing 30 µg/kg the rLj-RGD3 plasma concentration declined biexponentially with time, which could be best fitted by the two-compartment model. In conclusion, the BA-ELISA method reported here was proved to completely meet requirements for PK study of rLj-RGD3 with effective pharmacological dose at µg/kg BW level.