Sequence variability of Clostridium botulinum serotypes C and D is particularly complex.Some serotype C and D strains have unique gene structures that encode mosaic isoforms ofbotulinum neurotoxin (BoNT) containing components of both BoNT type C1 (BoNT/C1) andBoNT type D (BoNT/D). Such sequence variability and the potential for cross neutralisationmust be taken into consideration when developing serotype C and D detection andidentification assays. Three fusion proteins containing either a fragment from thecarboxyl-terminal domain of the heavy chain (HC) of BoNT/C1 (strain 573), a fragment fromthe HC of BoNT/D (strain BVD/-3) or a fragment from the amino-terminal domain of theheavy chain (HN) of BoNT/C1 (strain 573) were expressed in Escherichia coli, andadministered as immunogens to mice. Monoclonal antibodies (mAbs) against therecombinant BoNT fragments were prepared by three fusions. MAbs recognising nativeBoNT/C1 and BoNT/D were detected by enzyme-linked immunosorbent assay (ELISA). Ninemonoclonal antibodies (mAbs) were produced, six of which recognised a BoNT fragmentthat is highly conserved across all serotype C and D producing strains. We conclude thatthese mAbs and this approach to mAb production may facilitate the development ofimmunological diagnostic techniques that are not constrained by the existence of mosaicisoforms for the detection and identification of serotypes C and D.