Genetically engineered integrated aflatoxin B1 and deoxynivalenol bispecific nanobody as surrogate antigens for constructed time-resolved immunoassay dual detection methods

There is a phenomenon of combined contamination of fungal toxins, of which aflatoxin B₁ (AFB₁) is the most toxic, and deoxynivalenol (DON) contamination is common. The use of antigens for double or multiple testing of mycotoxins is easy to cause environmental pollution, and surrogate antigens have become necessary. The small molecule and susceptibility to genetic modification of nanobodies can be used to develop alternative antigens for mycotoxins. In this study, using the nanobody gene sequences of the heavy chain recognition regions of anti-aflatoxin and deoxynivalenol monoclonal antibodies, recombinant plasmids were successfully constructed by one-step cloning, and low-temperature-induced bispecific nanobodies against AFB₁-DON were obtained, which can be used as alternative antigens to reduce the pollution of the environment from mycotoxin detection. Enzyme-linked immunosorbent assay validated the bispecific nanobody, and the semi-inhibitory concentration (IC₅₀) of the bispecific nanobody were 0.47 μg/L and 149 μg/L for AFB₁ and DON, respectively. Finally, a time-resolved fluorescent dual-detection test strip was constructed by this bispecific nanobody as a surrogate antigen for AFB₁ and DON, which was capable of detecting AFB₁ and DON at the same time, and the limits of detection (LOD) for the two toxins were 0.0254 μg/L and 21.4 μg/L, respectively. This method has satisfactory sensitivity and does not require antigen, which reduces the toxicity of using antigen.