Anti-idiotypic nanobody: A strategy for development of sensitive and green immunoassay for Fumonisin B1

• We evidenced that Ab2β Nbs can be used as a substitute for hapten-conjugates.
• We developed and optimized a green and rapid Nb-ELISA for the determination of FB1.
• The sensitivity of Nb-ELISA was improved 20-fold than that of the hapten-conjugate.
• Ab2β Nb showed lower affinity than the hapten-conjugates to mAb.
• Ab2β Nb was more fit for working as coating antigen than the hapten-conjugates.

Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2β Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2β Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95±0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27–5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1–BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2β Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2β Nb replacing the antigen–carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens.

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