Josep Vicent Mercader

Antifungal proteins from fungal origin (AFPs) are small, secreted, cationic, cysteine‐rich proteins with potential as novel biofungicides. However, their application requires safe and cost‐effective production platforms. PeAfpA is a highly active AFP naturally produced in large quantities by the phytopathogenic fungus Penicillium expansum and biotechnologically produced by the Generally Recognized As Safe fungus Penicillium chrysogenum although at lower yields . Here, the production of PeAfpA using the P. chrysogenum strain PCMG11552 was optimised in 0.5 L and scaled up to 5 L in stirred‐tank bioreactors. A competitive enzyme‐linked immunosorbent assay (cELISA) was developed to evaluate the influence of process parameters on PeAfpA production, identifying medium pH and agitation rate as key factors, while dissolved oxygen proved critical during scale‐up. The PeAfpA yields obtained were tenfold higher than those achieved previously in flask cultures, demonstrating the feasibility of scaling AFP production from laboratory to pilot scale for diverse applications. Finally, the P. chrysogenum marker‐free PeAfpA‐producing strain obtained via recyclable CRISPR/Cas9 system guarantees regulatory compliance and biosafety, making it suitable for its use in any commercial environment.

Dihydroanatoxins are emerging contaminants produced by cyanobacteria that proliferate in continental surface waters. Recent studies suggest that the oral toxicity of dihydroanatoxin-a may surpass that of anatoxin‑a, a congener of the same family of homotropane alkaloids. Currently, climate change and anthropogenic contamination of continental water reservoirs contribute to more frequent and extensive cyanobacterial blooms, which release these hazardous secondary metabolites. This underscores the need for sensitive and reliable analytical methods capable of detecting dihydroanatoxins at trace levels. In this study, novel bioconjugates based on dihydroanatoxin-a haptens were synthesized and thoroughly characterized, enabling the generation of a panel of high-affinity monoclonal antibodies. Several antibodies exhibited IC50 values below 1 nM for both dihydroanatoxins, dihydroanatoxin-a and dihydrohomoanatoxin-a. Using these immunoreagents, we developed, for the first time, a highly sensitive monoclonal antibody-based direct competitive ELISA with a limit of detection of 0.016 ng/mL for dihydroanatoxin-a. The optimized immunoassay showed excellent precision and accuracy in a variety of fortified freshwater samples (lake, river, water well, and irrigation pond) across concentrations from 0.5 to 200 ng/mL, with recoveries between 79% and 112%, and coefficients of variation below 20%. Validation using naturally contaminated samples revealed good statistical agreement with a reference chromatographic method. Additionally, a lateral-flow immunochromatographic assay was developed for the on-site analysis of both dihydroanatoxins, providing a visual limit of detection of 3 ng/mL. Overall, the developed immunochemical methods offer rapid, reliable, and cost-effective approaches for monitoring dihydroanatoxins in environmental water samples. Keywords Emergent cyanotoxins; Homotropane alkaloids; Monoclonal antibodies; Rapid Methods; Freshwater Monitoring

Immunoassays have been developed for all regulated mycotoxins, including citrinin, whose presence in food supplements has recently undergone additional restrictions in Europe. Immunoassays for mycotoxins most commonly use monoclonal antibodies alongside an enzyme tracer, resulting in the so-called direct antibody-coated competitive ELISA format. Notably, no immunoassay of this kind has been published for citrinin to date. To address this, a novel hapten featuring an azide-functionalised linker for click chemistry conjugation has been designed. Following confirmation by molecular modelling that this derivative is an excellent structural mimic of the mycotoxin, a functional enzyme tracer and an immunising conjugate were prepared and characterised by MALDI-TOF. Subsequently, high-affinity monoclonal antibodies were produced through a double screening process for hybridoma selection. In combination with the click chemistry tracer, these novel antibodies show promise as immunoreagents for setting up innovative direct immunoassays for the sensitive determination of citrinin in food supplements containing fermented red rice

Flow cytometry-based immunoassays are valuable in biomedical research and clinical applications due to their high throughput and multianalyte capability, but their adoption in areas such as food safety and environmental monitoring is limited by long assay times and complex workflows. Rapid, simplified bead-based cytometric immunoassays are needed to make these methods viable for point-of-need applications, especially with the increasing accessibility of miniaturized cytometers. This work introduces superparamagnetic hybrid polystyrene-silica core-shell microparticles as promising alternatives to conventional polymer beads in competitive cytometric immunoassays. These beads, featuring high specificity, sensitivity, and excellent handling capabilities via magnetic separation, were evaluated with three different antibodies and binding methods, showing variations in signal intensity based on the antibody and its attachment method. The optimal performance was achieved through a secondary antibody binding approach, providing strong and consistent signals with minimal uncertainty. The optimized protocol made it possible to achieve a detection limit of 0.025 nM in a total assay time of only 15 min and was successfully used to detect ochratoxin A (OTA) in raw flour samples. This work highlights the potential of these beads as versatile tools for flow cytometry-based immunoassays, with significant implications for food safety, animal health, environmental monitoring, and clinical diagnostics.

Mycotoxins are globally pervasive contaminants that threaten food safety worldwide. Regulatory authorities have established maximum permissible levels for certain mycotoxins, and their presence is routinely monitored throughout the food chain to ensure the provision of healthy food and safe feed for humans and animals. While immunoanalytical methods are essential for mycotoxin screening, monoclonal antibodies for the detection of patulin are notably absent. Moreover, leading immunodiagnostic companies currently do not offer rapid tests for patulin in their product portfolios. This deficiency in mycotoxin testing is primarily due to the electrophilic reactivity of patulin. In this study, we exploit this reactivity to develop an innovative strategy that targets the stable adduct formed by the reaction of patulin with aryl-1,2-dithiolates, rather than analyzing the mycotoxin itself. Based on this previously unknown reaction, we present the first collection of monoclonal antibodies, enabling the long-sought goal of sensitive, simple, and user-friendly immunosensing of patulin.

Proquinazid is a new-generation fungicide authorized in the EU for combating powdery mildew infections in high-value crops. Due to the perishable nature of fruits, alternative analytical methods are necessary to protect consumer’s health from pesticide residues. Currently, immunoassays are a well-established approach for rapidly monitoring chemical contaminants. However, the production of high-quality immunoreagents, such as antibodies and bioconjugates, is essential. This study presents a newly designed hapten that maintains the characteristic moieties of proquinazid unmodified. The linear aliphatic substituents of this molecule were used to introduce the spacer arm. A three-step synthesis strategy was optimized to prepare a hapten that displays the entire 6-iodoquinazolin-4(3H)-one moiety with excellent yields. The N-hydroxysuccimidyl ester of the hapten was activated and purified to prepare a protein conjugate with high hapten density, which was used as an immunogen. Antibodies were raised and competitive enzyme-linked immunosorbent assays were developed. To enhance the assay’s sensitivity, two additional heterologous haptens were prepared by modifying the halogenated substituent at C-6. The optimized assays demonstrated low limits of detection in buffer, approximately 0.05 μg/L. When applied to the analysis of proquinazid in QuEChERS extracts of strawberry samples, the immunoassays produced precise and accurate results, particularly in the 10–1000 μg/kg range. Graphical Abstract