Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against <em>Fusarium verticillioides</em> by Phage Display

Abstract

<em>Fusarium</em> <em>verticillioides</em> is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from <em>F. verticillioides</em> were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of <em>F. verticillioides</em>, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10⁻² μg/mL and contaminating mycelium at a quantity of 10⁻² mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.

Keywords

• chicken antibody
• enzyme-linked immunosorbent assays (ELISAs)
• &lt;em&gt;Fusarium&lt;/em&gt; &lt;em&gt;verticillioides&lt;/em&gt;
• single-chain variable fragment (scFv)
• phage display
• immunofluorescence labeling