Click chemistry-based bioconjugates for direct immunochemical analysis of the mycotoxin citrinin

BACKGROUND: Over the past few decades, immunoassays have been developed for all regulated mycotoxins. This scientific and technological achievement has significantly impacted the protection of human health and has remarkably contributed to the growth of the food immunodiagnostic industry. Among these mycotoxins, citrinin is of particular relevance, especially given the recent tightening of regulatory limits for its presence in food supplements within Europe. Commercial ELISA kits for mycotoxin detection typically employ monoclonal antibodies in conjunction with an enzyme tracer. Notably, however, no direct competitive immunoassays based on monoclonal antibodies for the analysis of citrinin have been published to date. RESULTS: To address this gap, a novel hapten featuring an azide-functionalized linker for click chemistry conjugation was designed and synthesized. Remarkably, this hapten preserves the carboxyl group of citrinin at the C-7 position, and molecular modelling studies confirmed that the resulting functionalized derivative is an outstanding structural mimic of the native mycotoxin. Using this strategy, a functional enzyme tracer suitable for immunoassay development and an immunizing conjugate capable of generating a robust immune response were prepared and characterised by MALDI-TOF analysis. Subsequently, a collection of mouse monoclonal antibodies with affinity values in the low nanomolar range (2.62 - 18.1 nM) were produced through a double-screening process involving antibody-coated microplates for hybridoma selection. An ELISA prototype was successfully developed to determine citrinin in a quality-control material with an assigned value of 90.8 μg/kg, as well as in eight commercial food samples fermented with M. purpurea, where citrinin concentrations ranged from 4.7 to 67.6 μg/kg. SIGNIFICANCE: This paper reports the first direct assay for citrinin based on monoclonal antibodies. This goal was made possible by integrating a pioneering click chemistry enzyme tracer with newly generated anti-citrinin monoclonal binders. Upon further optimisation and validation, the resulting immunoassays will enable the sensitive determination of citrinin in regulated commodities, including food supplements containing fermented red rice.