Data are presented as mean values (SD) for each group of mice (n?=?5). important PCR ribotypes. The goal of this study was to generate monoclonal antibodies recognizing various epidemic ribotypes of To do so, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole HGF bacteria from current ribotypes with different cross-specificities. This first collection of anti-mAbs represent valuable tools for basic and clinical research. Supplementary Information The online version contains supplementary material Carboxypeptidase G2 (CPG2) Inhibitor available at 10.1186/s13099-023-00592-7. Keywords: is an anaerobic, gram-positive, and spore-forming bacterium that is the main agent responsible for antibiotic-associated diarrhea and pseudomembranous colitis in adults [1]. In the past decades, there was a drastic increase in the incidence of both healthcare-associated infection (CDI) and community-acquired CDI [2]. There is a large phylogenetic diversity of with more than 300 distinct PCR-ribotypes (RT) reported worldwide, including epidemic lineages associated with increased transmission and mortality [3C6]. The latest epidemiology data worldwide reported that 5 ribotypes i.e., RT001, RT002, RT014, RT027 and RT078, account for approximately 50% of the infections [7]. Whereas several advances such as fluorescent mutants and novel fingerprinting techniques have contributed to a better understanding of diversity and physiology [8C10], basic research still relies on one single strain 630 that belong to RT012. An increasing number of studies has been performed on the epidemic ribotype 027, which caused major outbreaks in the United States and Europe at the end of the 2010s [11, 12]. Other ribotypes remain largely unexplored even though some are associated with antibiotic resistance and increased severity [3], which can be partly explained by the lack of genetic and immunological tools to study these strains. surface is composed of adhesins e.g., the flagellar cap protein FliD, the flagellin FliC, the cell wall protein Cwp66, the surface layer protein SlpA, and the protease Cwp84 [13]. SlpA is expressed on the bacterial surface of all ribotypes and plays a crucial role in the pathogenesis and virulence of by mediating interactions with the host cells and the surrounding environment [14C17]. SlpA contains two biologically distinct entities, the high-molecular weight (HMW) and the low Carboxypeptidase G2 (CPG2) Inhibitor molecular weight (LMW) subunits that assemble on the bacterial surface into a paracrystalline lattice [18]. Sequence variations of SlpA have been reported Carboxypeptidase G2 (CPG2) Inhibitor for the LMW that correlate with the diversity of clinical isolates, whereas the HMW is less variable [19, 20]. SlpA is highly immunogenic, meaning it can trigger an immune response in the host [21]. Indeed, antibodies against SlpA have been detected in the sera of patients infected with ribotypes i.e., RT001, RT002, RT014, RT027 and RT078. Hybridomas were generated and their corresponding IgG mAbs bound both recombinant LMW in vitro and LMW naturally expressed on the bacterial surface. At least one mAb was identified against each of the five ribotypes used for immunization, with 6 mAbs being cross-reactive between LMW subunits of two different ribotypes. The reduced sequence identity of LMW between different ribotypes [25] allows for specific identification of bacterial ribotypes by this anti-LMW mAb collection that represents a novel toolkit for research. Results LMW SlpA subunits from 5 predominant ribotypes of i.e., RT001, RT002, RT014, RT078 and RT027 (Fig.?1a), were recombinantly produced from transformed as his-tagged soluble proteins and affinity-purified. As anti-LMW antibodies may potentially be of therapeutic interest for the treatment of CDIs, we used knock-in mice in which the endogenous genes encoding the heavy chain variable domain (VH) and the kappa light chain variable domain (V) were replaced by their human counterparts (Velocimmune?mice) [23, 24] with one modification, i.e., only one allele of the endogenous V locus was replaced by human V segments, and the second allele of the endogenous V locus was replaced by human V segments (Fig.?1b). As the Vk locus expresses 95% of the light chains in mice [26], placing human V segments at the Vk locus increases the variability of light chain expression. Thus, after hybridoma identification, cloning of these VH and VL into vectors containing human heavy and light chain constant domains, allows for direct developmenttoxin. Four days after the last boost, spleens were collected and hybridoma generated. d Sera titers at day 42 of immunized mice for recombinant LMW-RT001, LMW-RT002, LMW-RT014, LMW-RT078, LMW-RT027 measured by ELISA. OD.