Chicken‐Derived Single‐Chain Variable Fragments Targeting Monkeypox Virus A29L Protein

Monkeypox (mpox), a zoonotic disease, has rapidly spread globally, prompting the WHO to declare it a public health emergency. The long incubation period, early symptoms resembling respiratory infections, and diagnostic challenges hinder timely epidemic control and accurate clinical diagnosis. The monkeypox virus (MPXV) encodes the A29L protein, which binds to cellular heparan sulfate to facilitate infection and serves as a target for treatment and diagnostics. Thus, developing effective diagnostic tools and treatments is critically important. In this study, we expressed and purified Escherichia coli-derived A29L protein, which was used for chicken immunization to generate specific polyclonal IgY antibodies. The results demonstrated a successful elicitation of a humoral immune response. Subsequently, two single-chain variable fragments (scFv) antibody libraries were constructed using phage display technology, comprising 2.6 × 10⁸ and 3.8 × 10⁸ transformants. After bio-panning, phage-based ELISA indicated the enrichment of specific clones. Three scFv-expressing clones, including cA29LS1, cA29LS5, and cA29LS13, were selected from 13 randomly chosen clones and classified based on nucleotide sequence analysis. Their binding activities were evaluated through ELISA and Western blot, followed by purification for affinity determination via competitive ELISA. Among the selected clones, cA29LS5 demonstrated the highest binding affinity (1.3 × 10⁻⁶ M), followed by cA29LS1 (5.3 × 10⁻⁶ M). Additionally, both IgY and all three clones demonstrated binding activity to cell-derived and commercially purchased A29L proteins, as confirmed by Western blot and ELISA. Overall, these findings suggested that the IgY and scFv antibodies developed hold promise as potential diagnostic and therapeutic agents against MPXV infections.