We thank Dr. for further development. Keywords:Phage display, Botulinum neurotoxin, monoclonal antibody, neutralizing antibody Botulinum neurotoxins (BoNTs) are produced by various strains of the anaerobic bacterium,Clostridium Botulinum,and are classified into seven serotypes based on their distinct antigenicity (designated as serotype AG).1They are causative agents of botulism, which is characterized by flaccid paralysis, and are the most potent toxins known to humans. CAY10505 Botulinum neurotoxins/A,/B, CAY10505 and/E account for nearly all recorded cases of human botulism, and almost all infant botulism in the United States results from either BoNT/A or BoNT/B.2These neurotoxins have a similar structure, consisting of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC) linked by a disulfide bond1. The HC is mainly involved in the cell-binding, internalization and translocation. More specifically, the 50 kDa C-terminal portion of the heavy chain (HC) is believed to preferentially target the BoNTs to the peripheral presynaptic termini at the neuromuscular junction.3Once engulfed inside a neuronal cell, the N-terminal half of the HC (HN) facilitates translocation of the LC into the cytosol. [4] and [5] The LC domain is a group of Zinc-dependent endoproteases6that specifically cleave SNARE proteins (SNAP-25, VAMP and syntaxin) that are essential for release of the neurotransmitter acetylcholine. Due to their extreme potency and high lethality, BoNTs are classified as one of the six highest-risk threat agents for bioterrorism by the US Centers for Disease Control and Prevention (CDC). Currently, human or equine antisera is considered the most effective immunotherapeutic for BoNT exposure,[7] and CAY10505 [8] however either supply is a limiting factor or severe side effects9(e.g., allergic reactions, serum sickness, anaphylaxis) are problematic. Advancements in monoclonal antibody (mAbs) generation/engineering has overcome these barriers by providing highly specific human antibodies with unlimited supply, reduced allergic effects, and improved pharmacological properties.10 Phage display is a powerful technique in which peptides or proteins can be expressed on the surface of bacteriophage and selected against a target antigen. In general, phage display method has been proven to be a fast, cost-effective alternative for mAb generation. Attractively, these selected mAbs can be easily manipulated to improve their affinity or converted into various antibody formats based on clinical utility. Additionally, if human antibodies are desired, a human antibody gene repertoire as the source for phage display libraries, i.e. antibodies with human origin can be directly isolated and applied to downstream clinical trials, bypassing tedious humanization procedures. Here, we report the use of a human nave scFv phage display library for the CAY10505 production of human neutralizing mAbs against BoNT/B. BoNT HCdomain contains regions thought to bind to presynaptic neuronal receptors, the first requisite step for intoxication, and results in protective immunity when used as an immunogen.[11] and [12] Hence, we used BoNT/B HCdomain as antigen to screen BoNT/B neutralizing antibodies. BoNT/B HCdomain (10881295) was prepared by PCR and subsequently inserted into the region between the NdeI and NotI sites on pET28b vector (Novagen). Rosetta2 (DE3) E.coli (Novagen) were transformed with the recombinant plasmid and amplified CCR3 in SB medium to an OD600 of approximately 0.60.8. The culture was induced for expression with 0.1 mM IPTG and was incubated at 25C overnight. The overnight culture was centrifuged at 5,000 g, 4C for 10 min; while cell pellets were resuspended in 40 mL PBS buffer supplemented with protease inhibitor (phenylmethylsulfonyl fluoride) and were lysed with a sonicator. The lysate was centrifuged at 35,000 g, 4C for 20 min and the BoNT/B HCdomain protein within the supernatant was purified with a Ni-NTA resin column by IMAC chromatography. The construction of a human nave scFv phage display library has been described previously.13To reduce the susceptibility of BoNT denaturation on a solid phase surface,14we developed a solution phase selection approach for panning. In brief, phage library panning was performed on Ni-NTA resin coated with purified N-terminal hexahistidine tag BoNT/B HCdomain and blocked with BlockerCasein in PBS (Pierce). Upon incubation with the scFv-phage library, the Ni-NTA resin was washed 5 times with PBS and bound phage were eluted with 1 mL elution buffer (300 mM imidazole in PBS). The eluted phage were used to infect log-phaseE.coliTG1. Titration of the phage libraries, phage rescue and preparation were performed as described previouly.15 Upon completion of the fourth round of panning, 96 single colonies were randomly picked for scFv-phage amplification and ELISA analysis. BoNT/B HCwas diluted to 5 g/mL in PBS and immobilized on Costar ELISA plates (Corning) at 4C overnight. After blocking for 1h at room temperature, the plates were incubated with diluted phage, which were at a concentration of 1 1:1.