Ni nta agarose beads

Sequences for the variable regions of antibody heavy and light chains were obtained from deposited antibody structures on the PDB and cloned into expression vectors to make full-length human IgG1 antibodies. Sequences for the heavy chain were modified with a C-terminal ybbR tag for enzymatic labeling (63 (link)) and, in the case of Fab fragments, a His6 tag for affinity purification using Ni-NTA Agarose Beads (Thermo Scientific Pierce). Full-length antibodies were purified using protein A agarose beads (Thermo Scientific Pierce). Antibodies were expressed in HEK-293T following transfection with heavy and light chains at >70% confluency. Cells were subsequently cultured for seven days in Opti-MEM with 1x Anti-Anti and with or without 2% FBS for Fab and IgG antibodies, respectively. Supernatants from the HEK-293T cells were collected for affinity purification. Full details on antibody purification and enzymatic labeling are described elsewhere (63 (link)).
Human convalescent sera was obtained through BEI Resources (NR-18964 and NR-18965 for Serum 001 and Serum 002, respectively) and used without further purification in virus counting assays and microneutralization assays. IC50 values for serum neutralization were measured as fold-dilutions from the initial undiluted stock.

Sequences for the variable regions of antibody heavy and light chains were obtained from deposited antibody structures on the PDB and cloned into expression vectors to make full-length human IgG1 antibodies. Sequences for the heavy chain were modified with a C-terminal ybbR tag for enzymatic labeling (56 (link)) and, in the case of Fab fragments, a His6 tag for affinity purification using Ni-NTA Agarose Beads (Thermo Scientific Pierce). Full-length antibodies were purified using protein A agarose beads (Thermo Scientific Pierce). Antibodies were expressed in HEK-293T following transfection with heavy and light chains at >70% confluency. Cells were subsequently cultured for 7 days in Opti-MEM with 1× Anti-Anti and with or without 2% FBS for Fab and IgG antibodies, respectively. Supernatants from the HEK-293T cells were collected for affinity purification. Full details on antibody purification and enzymatic labeling are described elsewhere (57 (link)).
Human convalescent sera were obtained through BEI Resources (NR-18964 and NR-18965 for Serum 001 and Serum 002, respectively) and used without further purification in virus counting assays and microneutralization assays. IC50 values for serum neutralization were measured as fold dilutions from the initial undiluted stock.

To construct the plasmids corresponding to GST-MoAA91 and GST-MoAA91^DTB, His-MoMSN2, and His-CEBiP, full-length cDNA of MoAA91 lacking the signal peptide was amplified and inserted into the vector pGEX4T-2, full-length cDNA of MoMSN2 was amplified and inserted into the vector pET-32A, and full-length cDNA of CEBiP lacking the signal peptide and the transmembrane domain was amplified and inserted into the vector pET-32A, respectively. These plasmids were then expressed in E. coli strain BL21, and bacterial cells were collected and treated with lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100). To confirm the expression of the GST or His fusion proteins, bacterial lysates were separated by the use of SDS-PAGE gel followed by Coomassie blue staining (see Fig. S7A to D in the supplemental material). In the assay used for purification of protein (His-MoMsn2/CEBiP and GST-MoAa91/MoAa91^DTB), bacterial lysate containing His-MoMsn2/CEBiP protein was incubated with 30 μl nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Invitrogen, Shanghai, China) and bacterial lysate containing GST-MoAa91/MoAa91^DTB protein was incubated with GST beads for 1 h at 4°C. The beads were then washed five times, and the elution was reduced using glutathione (Abmart, Shanghai, China).