Ochratoxin A is a potent toxic fungal metabolite whose undesirable presence in food commodities constitutes a problem of public health, so it is strictly regulated and controlled. For the first time, two derivatives of ochratoxin A (OTAb and OTAd) functionalized through positions other than the native carboxyl group of the mycotoxin, have been synthesized in order to better mimic, during the immunization process, the steric and conformational properties of the target analyte. Additionally, two conventional haptens making use of that native carboxyl group for protein coupling (OTAe and OTAf) were also prepared as controls for the purpose of comparison. The immunological performance in rabbits of protein conjugates based on OTAb and OTAd overcome that of conjugates employing OTAe and OTAf as haptens. After immunization of mice with OTAb and OTAd conjugates, a collection of high-affinity monoclonal antibodies to ochratoxin A was generated. In particular, one of those antibodies, the so-called OTAb#311, is very likely the best antibody produced so far in terms of selectivity and affinity to ochratoxin A.

Ochratoxin A (OTA) is a carcinogenic mycotoxin produced by Aspergillus and Penicillium, which contaminates cereals like corn and wheat, in addition to beverages such as coffee, wine and beer. Chromatography is the preferred analytical method for OTA determination, but unfortunately this method is still elaborate, expensive and tedious. Alternative mycotoxin detection techniques involve assays and sensors based on molecularly imprinted polymers nanoparticles (nanoMIPs) as synthetic polymeric receptors mimicking antibodies. Herein we designed nanoMIPs for specific recognition of OTA using computational screening and docking of different acryl functional monomers. Subsequently, different nanoMIPs were prepared by solid phase synthesis using two OTA haptens, OTAe‐MIPs without carboxylic group and OTAb‐MIPs bearing the carboxylic group. NanoMIPs affinity towards OTA was analyzed by surface plasmon resonance, the results displayed dissociation constants for OTAb‐MIPs at 0.4 μM and for OTAe‐MIPs at 5.1 μM. Therefore, the results demonstrate that nanoMIPs recognition depends on the immobilization position of the OTA hapten as observed using antibodies.

Bioorthogonal reactions have revolutionized the way low-molecular-weight compounds are coupled to biomolecules. Organic chemistry, polymer science, and chemical biology are among the disciplines that have benefited the most from this breakthrough. Despite the reliability of the click chemistry concept for the efficient and chemoselective functionalization of biomacromolecules with haptens at preferred positions, the fact that azide–alkyne cycloaddition reactions originate new chemical moieties as part of the linker may have delayed their application in the immunodiagnostic field. Using the mycotoxin ochratoxin A as a model compound, we herein demonstrate for the first time that bioconjugates arising from the ligation between an azido-bearing hapten and an alkyne-modified carrier protein are able to elicit the generation of high-affinity monoclonal antibodies suitable for the development of rapid methods for the immunodetection of small organic molecules.

Fluxapyroxad high-affinity antibody generation and sensitive immunoassay development from a suitable hapten design.