Generation and NGS profiling of naïve rabbit antibody libraries for efficient monoclonal antibody selection.

Rabbit monoclonal antibodies (mAbs) are highly valued for their exceptional affinity and specificity, yet naïve rabbit antibody libraries generated by phage display remain rarely described, limiting the in vitro exploitation of the unique rabbit immune repertoire. To address this gap, we constructed two naïve scFv phage display libraries-one incorporating κ and the other λ light chains-assembled from bone marrow, spleen, and peripheral blood of rabbits from two breeds. Together, they yielded a combined theoretical diversity of 7 × 10⁹ independent clones, with the λ library providing access to a rarely explored component of the rabbit repertoire. Next-generation sequencing (NGS) confirmed the high genetic diversity of the libraries and allowed exploration of antibody repertoire regions in rabbits that have been scarcely characterized. A diverse panel of scFvs specific to two major sesame allergens (11S globulins and 2S albumins) have been isolated from both libraries as a proof of concept of their functionality. Selected scFvs were successfully reformatted into full-length rabbit IgG retaining target specificity and expressed in CHO cells at high expression yields. Overall, the constructed libraries provide a robust and versatile resource for antibody discovery, enabling the development of high-quality rabbit mAbs for applications in diagnostics, food safety, and broader analytical applications.