Direct rescue and rapid confirmation of antigen-binding of dominant V _H and V _L of chickens

The most frequent sequences of immunoglobulin heavy (V_H) and light (V_L) 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 <i>E. coli</i> extract-based cell-free expression system (CFES) to evaluate single-chain variable fragments (scFvs) constructed from dominant V_H and V_L sequences of immunized chickens. CFES was validated with a reference mouse anti-hemagglutinin scFv, employing V5tagging instead of Histagging to reduce nonspecific signals. Dominant V_H and V_L sequences were identified by deep sequencing from spleens of chickens immunized with smooth or rough <i>Salmonella enterica</i> serovar Gallinarum biovar Gallinarum (V_H 2.79%, V_L 1.02%; V_H 1.13%, V_L 1.36%, respectively). Sequence-based scFvs were expressed in CFES and tested for antigenic specificity using a commercial <i>Salmonella</i> group D O-antigen ELISA. V_H/V_L 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 V_H CDR3. These results demonstrate that CFES provides a simple, rapid, and reliable platform to characterize antigenic specificity of dominant V_H and V_L sequences.