Hotspot Mutagenesis Strategy Yielding a Highly Sensitive Nanobody against Parathion for Immunoassay Development

Nanobodies (Nbs) hold great potential as affinity reagents for the immunoassays of small-molecule contaminants. However, there is an urgent need for better approaches to discover higher-affinity Nbs and expand their application in food analysis. Herein, with the antiparathion nanobody (VHH9) as a model, a novel hotspot mutagenesis technique for Nb affinity maturation is reported, which targets specific motifs within Nb DNA sequences and bypasses the need for traditional 3D protein modeling to navigate candidate mutated residues. Specifically, seven residues Ser28, Tyr29, Ser32, Lys102, Phe103, Arg105, and Ala106 encoded by two AGY and two RGYW codons were selected and randomized to construct a saturation mutation library. Enhanced mutants G4, H6, and G2 were successfully isolated and then characterized by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), with half-maximal inhibition concentrations (IC₅₀) of 4.7, 3.5, and 2.9 ng/mL, respectively, 2.1-, 2.9-, and 3.5-fold lower than that of the original Nbs. Subsequently, a biotin-streptavidin-amplified ELISA (BA-ELISA) was developed for the detection of parathion based on biotinylated H6. The developed BA-ELISA showed an IC₅₀ value as low as 1.5 ng/mL, which was 2.3-fold improvement in sensitivity compared to H6-based ic-ELISA. The average recoveries in vegetable and fruit samples ranged from 87.0% to 113.0%. In summary, this work demonstrated that hotspot mutagenesis is effective for maximizing the efficiency to obtain Nbs with improved sensitivity, and the developed BA-ELISA is a robust tool for the routine screening of parathion.