Direct rescue and rapid confirmation of antigen-binding of dominant VH and VL of chickens.

The most frequent sequences of immunoglobulin heavy (VH) and light (VL) chain variable regions are likely associated with immunodominant B cell clones selected during humoral immunity, yet a simple method is needed to define their antigenic specificity. Here, we used an E. coli extract-based cell-free expression system (CFES) to evaluate single-chain variable fragments (scFvs) constructed from dominant VH and VL sequences of immunized chickens. CFES was validated with a reference mouse anti-hemagglutinin scFv, employing V5tagging instead of Histagging to reduce nonspecific signals. Dominant VH and VL sequences were identified by deep sequencing from spleens of chickens immunized with smooth or rough Salmonella enterica serovar Gallinarum biovar Gallinarum (VH 2.79%, VL 1.02%; VH 1.13%, VL 1.36%, respectively). Sequence-based scFvs were expressed in CFES and tested for antigenic specificity using a commercial Salmonella group D O-antigen ELISA. VH/VL orientation slightly influenced protein yield but not binding. Only rough strain-derived scFvs were positive, likely binding to inner- or outer-core lipopolysaccharide in the ELISA kit, whereas smooth strain-derived scFvs were negative, possibly due to disrupted disulfide bond formation in VH CDR3. These results demonstrate that CFES provides a simple, rapid, and reliable platform to characterize antigenic specificity of dominant VH and VL sequences.