The production of antibodies to coumarin and its major human metabolites

Abstract

Two strategies were applied to the production of antibodies to coumarin and its phase I metabolite, 7-hydroxycoumarin. One system was the production of Fab antibody fragments using the combinatorial phage display library, employing the phagemid vector, pComb3. Although a library of murine heavy and light chain genes was established following immunisations with coumarin and 7-hydroxycoumarin conjugates, preparation of the library in pComb3 was not possible due to associated problems with this vector. An alternative antibody library system was obtained. This was the naive semi-synthetic human scFv library of Nissim et al. (1994). The production of antibodies to both coumarin and 7-hydroxycoumarin was investigated. Various production and isolation techniques were explored. Best results were obtained by isolating correctly folded scFv protein from the periplasm or culture supernatants of E. coli. In the case of coumarin, three soluble antibody clones were isolated and characterised. These clones showed the characteristics of poor affinity for free coumarin, but did bind well to coumarin-protein conjugates. Isolated anti-7-hydroxycoumarin clones also showed the same characteristics of no binding to free drug. It was theorised that, due to the structural simplicity of these molecules, affinity selection and screening is biased against the detection of clones with high affinity for free drug. This conclusion was in broad agreement with other findings (Danilova, 1994). Recommendations are made as to how the selection and screening procedures can be improved to isolate antibodies to such molecules. Techniques for the in vitro production of the phase II metabolite 7-hydroxycoumaringlucuronide were developed. These were accompanied by the development of analytical methods (capillary electrophoresis and HPLC) for the glucuronide. Purification methodologies using 7-hydroxycoumarin present in these metabolic mixtures and in urine were also developed. The coupling of 7-hydroxycoumarin-glucuronide to proteins for the production of antibodies was also explored. A 7-hydroxycoumarin-glucuronide-bovine serum albumin conjugate was successfully synthesised for use in further antibody production strategies