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10.1042/BJ20121305. bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype. KEYWORDS: nanobodies, paratopes, antibiotic resistance, CMY-2 -lactamase, new antibiotic scaffold, class C beta-lactamase, structural biology INTRODUCTION Bacterial resistance to antibiotics is recognized as a major threat in human and veterinary medicine. Antimicrobial resistance accounts for 700,000 deaths per year in the world, including 30,000 deaths in Europe. This physique could exceed 10 million deaths in 2050 if no new treatments are designed (1). Among the different classes of antimicrobials, the -lactam antibiotics are extensively used because of their wide spectrum of action and their low toxicity for eukaryotic cells (2). They are able to specifically neutralize the enzymatic activity of penicillin binding protein (PBP) involved in biosynthesis of the bacterial cell wall ARRY-380 (Irbinitinib) (3, 4). Bacteria have developed different mechanisms in order to affect the biological activity of the -lactam antibiotics (5,C9). In Gram-negative bacteria, the most common mechanism is usually hydrolysis of the -lactam ring by hydrolases called -lactamases. The misuse and the intensive use of ARRY-380 (Irbinitinib) antibiotics lead to the selection of multidrug-resistant (MDR) bacteria, which are unaffected by the presence of antibiotics belonging to at least three different families (10). Therefore, it is essential to develop new rapid diagnostic assays in order to detect faster the presence of -lactamases that favor the rapid implementation of contamination control steps to circumvent nosocomial infections. In addition, it is essential to develop new inhibitor scaffolds able to block -lactamase activity by targeting binding sites that are not tolerant to mutations. To develop new inhibitors, one strategy is to select inhibitory antibodies that serve in the development of new -lactamase inhibitors by peptidomimetics (11). VHH, also Rabbit Polyclonal to NCBP1 referred as nanobody, ARRY-380 (Irbinitinib) is the single-domain fragment corresponding to the binding domain name of camelid heavy-chain antibodies (HCAbs). They constitute potential candidates for obtaining inhibitory antibodies against -lactamases. They are exclusively found in camelids (VHH) or in cartilaginous fish (V-NAR) (12). Despite their small size (15?kDa), they are able to interact with their antigen with a high affinity and specificity (13). In addition, VHHs present unique properties, including a generally easy recombinant production in ARRY-380 (Irbinitinib) genes in bovine isolates in Wallonia, Belgium, highlighted that this strains expressing this -lactamase were isolated from animals (18) and human sources (19). Phenotypic assessments cannot distinguish between different subclasses of AmpC -lactamases, for which molecular approaches such as PCR or DNA microarray are required (20). Nevertheless, those methods cannot identify the different subclasses of AmpC. The assays must be complemented by molecular approaches such as PCR or DNA microarray when available (21). Those methods are expensive, time-consuming, and generally used only for reference laboratories and research settings (22). Furthermore, expression of multiple -lactamases (23) or other resistance features such as the decrease of porin expression could result in more complex susceptibility patterns (24). Altogether, those observations clearly demonstrate the real necessity to develop new diagnostic approaches for the veterinarian and the clinician in order to detect AmpC easily and with a high specificity. On the other hand, treatments generally employed to treat infections with expressing AmpC consist of the use of carbapenems, cefepime, or recently approved -lactamC-lactamase inhibitor combinations (25). Newer -lactamase inhibitors as avibactam or vaborbactam have also a high potency against AmpC activity (26). However, it is usually expected to favor the appearances of resistance against those antibiotics and inhibitors, specifically against carbapenems (27). In this work, we developed nanobodies (VHHs) in order to set up a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 and to find inhibitors able to neutralize -lactamase activity. We first isolated eight VHHs belonging to three families that acknowledged CMY-2. The results highlighted a high specificity for their antigen but rapid dissociation rates of the VHH/CMY-2 complexes. We could stabilize the complex with the development of a homobivalent VHH. Competition assays exhibited also an overlapping epitope of the VHHs for CMY-2. With the help of an ELISA, we could detect the production of CMY-2-like enzymes in a collection of human and veterinary bacterial isolates. The.