doi: 10.1016/j.jmb.2007.05.022. bound at occluded sites on the particles. The flexible hinge region, which contained 10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble BL21 cells. Transformed cells were grown at 37C in LB medium for 2 h. Expression was induced with isopropyl–d-thiogalactopyranoside (IPTG) (0.75 mM) at an optical density at 600 nm (OD600) of 0.6 for 18 h at 22C. Cells were harvested by centrifugation and disrupted by sonication on ice. A His-tagged fusion-P domain protein was eluted from a Ni-nitrilotriacetic acid (NTA) column after a Mouse monoclonal to EGR1 series of washing steps. The fusion protein was digested with HRV-3C protease (Novagen) overnight at 4C, and then the P domain was separated on the Ni-NTA column and dialyzed in gel filtration buffer (GFB; 0.35 M NaCl and 25 mM Tris-HCl [pH 7.4]) overnight at 4C. The P domain was further purified by size exclusion chromatography with a Superdex-200 column and stored in GFB at 4C. Nanobody production. A single alpaca was injected subcutaneously on days 0, 7, 14, 21, 28, and 35 with 115 g GII.10 VLP protein per injection (VIB Nanobody Service Fluralaner Facility, Vrije University, Brussels, Belgium). The alpaca immunization was performed by the VIB Nanobody Service Facility with the approval of the Ethical Commission of Vrije Universiteit, Fluralaner Brussels, Belgium. A VHH library was constructed and screened for the presence of antigen-specific nanobodies. A VHH library of about 108 independent transformants was obtained. Three consecutive rounds of panning were performed on a solid-phase coating with GII.10 VLPs (20 g/well). In total, 143 individual colonies were randomly selected. Crude periplasmic extracts were analyzed for the presence of specific antigens using ELISA. Forty-seven colonies were positive, and nucleotide sequencing revealed these represented 35 different nanobodies that belonged to 17 distinct groups based on sequence alignments. In this study, we examined two nanobodies (termed Nano-25 and Nano-85) that represented two distinct groups. The nanobodies were cloned into a pHEN6C expression vector and grown in WK6 cells overnight at 28C. Expression was induced with 1 mM IPTG at an OD600 of 0.7 to 0.9. Nanobodies were extracted from periplasm and the supernatant collected. Nanobodies were eluted from a Ni-NTA column after a series of washing steps and purified by size-exclusion chromatography using a Superdex-200 column. Nanobodies were concentrated to 2 to 5 mg/ml and stored in GFB. Nanobody reactivities using ELISA. The nanobody reactivities against norovirus VLPs and P domains were determined using a direct ELISA as previously described, with slight modifications (18), i.e., His tag nanobodies were detected with a secondary horseradish peroxidase Fluralaner (HRP)-conjugated anti-His IgG. Microtiter plates (Maxisorp, Denmark) first were coated with 100 l (2 g/ml) of VLPs (GII.10, GII.12, GII.4, and GI.1) or 100 l (7 g/ml) of GII.10 P domain in PBS (pH 7.4). Wells were washed three times with PBS (pH 7.4) containing 0.1% Tween 20 (PBS-T) and then blocked with 300 l of PBS containing 5% skim milk (PBS-SM) for 1 h at room temperature. After washing, 100 l of serially diluted nanobodies in PBS (from 10 M) were added to each well. The wells were washed, and then 100 l of a 1:3,000 dilution of secondary HRP-conjugated anti-His IgG (Sigma) was added to wells for 1 h at 37C. After washing, 100 l of substrate of 7 nM; GII.4 P domain and Nano-85, of 3.5 nM). The interactions were largely enthalpy driven, which suggested the net formation of noncovalent.