Identification and characterization of a high-affinity antigen-binding fragment against intestinal bacteria from non-immunized rabbit single B cells using Ecobody technology

Antibodies play essential roles in mediating interactions between the host and microbiota. Despite their importance, the specificity and functional properties of naturally occurring antibodies reactive to gut microbiota remain poorly characterized, particularly at the monoclonal level. Here, we employed Ecobody technology, a single-B-cell-based antibody screening platform, to identify intestinal bacteria-reactive antigen-binding fragments (Fabs) from a non-immunized rabbit. Using Bacteroides cellulosilyticus as a model antigen, we identified and expressed Fabs using cell-free protein synthesis (CFPS) and subsequently produced a selected Fab in Brevibacillus choshinensis for detailed functional characterization. The purified Fab exhibited broad reactivity toward various intestinal bacteria, including both gram-positive and gram-negative bacteria. Conversely, it showed no binding to Saccharomyces cerevisiae or HEK293T cells, indicating specificity toward bacterial-associated antigens. Notably, the selected Fab demonstrated high binding affinity toward B. cellulosilyticus (KD = 2.41 nM). These results provide monoclonal-level evidence that antibodies reactive to intestinal bacteria are present in the peripheral B-cell repertoire of non-immunized hosts. Furthermore, this study demonstrates that the integration of Ecobody technology with the B. choshinensis expression system provides a feasible strategy for facilitating further investigation into host antibody-microbiota interactions.