Histrap excel column

cDNAs encoding the BCR heavy and light variable regions were synthesized by GeneScript and subcloned into pFUSE-CHIg-HG1 and pFUSE2ss-CHIg-hK vectors (InvivoGen), respectively. Full-length antibodies were expressed using ExpiCHO cells according to standard protocol. Cells were transfected with 0.6 μg/mL light chain plasmid and 0.4 μg/mL heavy chain plasmid. The medium was collected at Day 8 post-transfection. Antibodies from the medium were purified using Pierce Protein A/G Agarose (ThermoFisher, Cat# 20424) with the standard protocol. Filtered medium (0.22 μm) was loaded onto Protein A/G Agarose pre-equilibrated with binding buffer (25 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA). After washing with the same buffer for three times, the protein was eluted with 5 CV of 0.1 M Glycine pH 2.7 and the pH of eluent was immediately adjusted with 0.5 CV 1 M Tris pH 8.0. Protein was concentrated and further applied to Superdex 200 10/300 Increase (Cytiva) in Dulbecco’s PBS (Sigma) buffer. For SW186 Fab protein, the cDNA of heavy chain and light chain was subcloned into modified pCAG-His vector with an IL2 signal peptide at N-terminus. A stop codon was introduced to the C-terminus of light chain to make it a tag-free protein. The protein was then expressed the same way as full-length antibodies, and purified with HisTrap Excel column (Cytiva) using the AKTA system.

A soluble extracellular cadherin repeat 1 (sEC1, GenBank: NM_002587, residues 1 to 172) construct was synthesized and cloned into the pCDNA3.1(+) mammalian cell expression vector by GenScript with a C-terminal GSG linker and decahistidine tag. sEC1 was expressed in ExpiCHO cells and purified through a HisTrap Excel column (Cytiva) using anÄKTA pure protein purification system (Cytiva), and then labeled with Alexa Fluor 647 (Thermo Fisher). Expi293F cells transfected with a plasmid encoding full-length ANDV M segment, as described previously, were incubated with 10 μg/mL of each mAb for 1 hour at 4°C. A control mAb, DENV 2D22 directed to dengue virus envelope protein, was added as a negative control, and unlabeled sEC1 was added at 50 μg/mL as a positive control. Labeled sEC1 was added directly to the cell suspension and first mAb at a final concentration of 200 ng/mL and incubated for an additional hour at 4°C. Cells then were washed with flow cytometry buffer and stained with 0.5 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI). Alexa Fluor 647 and DAPI staining were measured with an iQue Screener Plus flow cytometer (Intellicyt) and quantified using the manufacturer’s ForeCyt software. Binding in the presence of antibody was divided by the maximal binding signal of labeled sEC1 alone to determine the % receptor blocking.

A synthetic gene encoding an anti-human CD3ε monoclonal antibody (clone UCHT1) in single-chain Fv format was synthesized (Integrated DNA Technologies). To construct expression vectors encoding scDbs, genes encoding the variable domains of heavy and light chains of the HapImmune™ antibodies and UCHT1 with a His-tag at the C-terminus were cloned into the mammalian expression vector pBCAG. Expi293F cells (Thermo Fisher) were transiently transfected with expression vectors using the ExpiFectamine 293 Transfection Kit (Thermo Fisher), according to the manufacturer’s protocol. Transfected cells were incubated at 37°C with 8% CO₂ for 7 days, and scDbs were purified from supernatants using a HisTrap excel column (Cytiva) followed by size exclusion chromatography using a Superdex 200 10/300 column (Cytiva). The purity of the scDb proteins was analyzed by SDS-PAGE.