We made 4 separate epitope maps using recombinant BoNT/Ci, Di, CDi or DCi (Table 5). for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with of 14C99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1C19 pM), six of which also bind BoNT/DC with lower affinity (93C508 pM). In addition to Hydrocortisone acetate application in diagnostic assessments, these VHHs could be used for the development of novel therapeutics for animals or humans. Keywords: botulinum neurotoxin group III, mosaic toxin, botulism, toxin A and tetanus neurotoxin (TeNT), are similarly composed of three functional domains [5,6]. BoNT/A, B, E, F and G are mainly associated with botulism in humans whereas BoNT/C, D, CD and DC are associated with animal botulism. BoNT/DC and CD are naturally existing mosaic toxins related to BoNT/C and D [7]. The BoNT/DC LC and HN domains are almost identical to BoNT/D, whereas its HC domain name (HC/DC) shares about 77% amino acid sequence identity with HC/C. The protein sequences of BoNT/CD are almost identical to Hydrocortisone acetate LC/C, HN/C and HC/D. strains can be classified into different groups based on various characteristics. The so-called group III strains produce BoNT/C and D and their mosaic variants [8,9]. The work presented here focuses on diagnostics for group III BoNTs. Due to their high toxicity, there is a need for highly sensitive diagnostics for BoNT detection and identification. For laboratory detection of BoNTs the mouse bioassay is still the gold standard, despite international efforts to develop alternative in vitro assessments to reduce animal testing. Although many alternative technologies have been developed, reliable detection of all (sub)serotypes in complex clinical and environmental matrices remains a challenge. One of the most promising methods is the mass spectrometry (MS)-based endopeptidase BoNT activity assay known as Endopep-MS [10]. Endopep-MS detects the enzymatic action of the LC on a peptide KLF1 substrate which mimics BoNTs Hydrocortisone acetate in vivo protein target. By using BoNT-specific peptide substrates and monitoring the cleavage position by examining the mass of the N-terminal (NT) and C-terminal (CT) cleavage products, the various BoNTs can be differentiated. An immunoaffinity step, with capture antibodies on magnetic beads, prior to incubation with the peptide substrate, is usually a crucial aspect for detecting and differentiating BoNTs in clinical specimens and culture supernatants [11,12,13]. The immunoaffinity not only increases the sensitivity of the test by concentrating BoNT, but it also allows a washing step to remove non-specific proteases that are often present in clinical samples. The sensitivity of Endopep-MS has been shown to equal or exceed that of the mouse bioassay [13,14,15,16,17,18,19], has shown good performance in an international proficiency test for detection of BoNT/A, B and E [20], and compares favorably to other in vitro methods for routine diagnostics of clinical specimens and food [21,22]. While most advances have been made to improve the Endopep-MS assay for BoNT types related to human botulism [12,14,23], detection and identification of group III BoNTs related to animal botulism has also progressed [13,14,16]. A major challenge is the interference of non-specific proteases that are present in many complex sample matrices, like gastrointestinal contents, liver, feed and environmental samples [24]. These proteases not only may degrade the capture antibodies around the magnetic beads but can degrade the peptides used as a substrate for the enzymatic cleavage by BoNT. A wash with 2 M NaCl in PBS (high salt wash) after immunocapture of BoNTs from complex matrices has been shown to reduce non-specific protease activity [13,16,25]. Endopep-MS was also improved by the optimization of peptide substrates [19,26]. For efficient immunocapture, especially for BoNT/A, B, E and F, monoclonal antibodies (mAbs) have been optimized by increasing affinity using various molecular evolution approaches. Large increases in affinity have been obtained, resulting in Hydrocortisone acetate up to 1000-fold lower affinity constants (toxin A [45] and BoNT/A [46]. To enable development and, ultimately, implement in-house routine diagnostics for BoNTs, novel antibodies were generated. In this work, 34 VHHs were isolated that bind group III BoNTs (30 VHHs), BoNT/A (3 VHHs) or BoNT/B (1 VHH). These 34 VHHs and 2 published VHHs [30] were yeast-produced and their antigenic specificity was characterized. VHH binding impartial antigenic sites were used for generating 52 VHH multimers by.