Immunoassay for the determination of 7-hydroxycoumarin in serum using 'real-time' biosensor analysis

An inhibition immunoassay for the determination of total 7-hydroxycoumarin (7-OHC) in human serum samples using the BIACORE biosensor is described. 7-OHC-BSA was immobilised onto BIACORE sensor chips and 7-OHC was premixed with a polyclonal anti-7-OHC antibody and injected over this surface. Excess antibody bound to the immobilised conjugate, generating a binding response that was inversely proportional to the amount of 7-OHC in the sample. The sample buffer was formulated to minimise non-specific binding of serum components to the sensor surface. Regeneration of the sensor surface was optimised such that the conjugate-coated surface retained acceptable antibody-binding capacity over 64 binding-regeneration cycles. The recovery of free drug from samples spiked with 7-hydroxycoumarin-glucuronide (7-OHCG) approximated 100%, while the inter- and intra-day coefficients of variation for the assay were less than 8.76% for 5 replicates. The assay had a measuring range of 0.5 - 80 mu g/ml. This BIACORE-based immunoassay for 7-OHC could be performed using crude samples and both the reproducibility and sensitivity compared well to established methods of analysis.