Influence of the hapten-fluorophore bridge on binding parameters in a fluoroimmunoassay for carbamazepine

Raising antibodies to a hapten (drug or steroid) requires that it be coupled to a carrier protein through a bridge such that the hapten has maximum exposure with minimum changes in its configuration. In raising antisera in six sheep against carbamazepine (CBZ) coupled to bovine albumin, we found that some subpopulations of the antibodies recognized the bridge linking the drug to the carrier protein. To study the influence of the bridge on a fluoroimmunoassay for CBZ, we prepared four tracers by linking the carbamyl nitrogen of CBZ to fluorescein via four alkyl bridges of different lengths and structures. We calculated various binding parameters--including antibody affinity, binding capacity, and heterogeneity index--for each tracer and chose for the final fluoroimmunoassay the tracer that gave the best displacement with CBZ. We then optimized and validated the assay for direct measurement of CBZ in serum or plasma. The antibodies are coupled to magnetizable particles, which greatly facilitates separation and ensures removal of endogenous interferents.