Immunodiagnostic systems for on-site screening are increasingly in demand but require expensive reagents such as chemically modified antibodies. To obtain biomolecules as ready-to-use reagents for diagnostics, new recombinant fluorescent immunoglobulins G were generated by fusion of the heavy chain of a monoclonal antibody, 5H3, to two different fluorescent proteins: Green Fluorescent Protein (5H3GFP) and Cyan-excitable Orange Fluorescent Protein (5H3CyOFP1). The engineered antibodies were expressed by transient agroinfiltration in Nicotiana benthamiana plants, used as bioreactor sustainable, cost-effective and easy to scale up, with a yield of 80 and 35 mg/kg of purified 5H3GFP and 5H3CyOFP1, respectively. Both recombinant proteins retained the ability to recognize the antigen of the original mAb (aflatoxin M1, a contaminant of dairy products), while simultaneously being able to emit fluorescence in the green or orange light. To demonstrate their practical application in the diagnostic field, fluorescent antibodies were exploited to set up two fast screening systems for aflatoxin M1 detection: a lateral flow immunoassay and an OLED-based device. For both diagnostic systems, antigen-specific fluorescence signals as well as a first competitive assay were obtained. Although these assays will need a further implementation, our results demonstrate a concrete possibility to develop novel and fast analytical systems based on fluorescent recombinant antibodies produced in plants. © 2025 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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