To produce human monoclonal antibodies, the spleen cells from KM mice vaccinated with the S791 fragment were fused with the mouse myeloma cell collection SP2/O-Ag14, and hybridomas were screened for their ability to bind to recombinant S791 protein by conventional enzyme-linked immunosorbent assay as described elsewhere [23]

To produce human monoclonal antibodies, the spleen cells from KM mice vaccinated with the S791 fragment were fused with the mouse myeloma cell collection SP2/O-Ag14, and hybridomas were screened for their ability to bind to recombinant S791 protein by conventional enzyme-linked immunosorbent assay as described elsewhere [23]. from KM mice vaccinated with the S791 fragment were fused with the mouse myeloma cell collection SP2/O-Ag14, and hybridomas were screened for their ability to bind to recombinant S791 protein by standard enzyme-linked immunosorbent assay as explained elsewhere [23]. Mass production of 5H10 was performed in fed-batch culture of CHO cells, as described elsewhere [25]. Epitope mapping of mAbs was performed using a set of overlapping peptides (>70% purity) spanning the entire sequence of the S791 protein, each consisting of 15 amino acids overlapping the next peptide by 5 amino acids, as described elsewhere [24]. Neutralization of SARS-CoV In Vitro by 5H10 All experiments using SARS-CoV were performed in BSL3 level BC 11 hydrobromide facilities. The neutralizing activities of mAbs against SARS-CoV Frankfurt01 were determined by plaque reduction assay. Monolayers of Vero E6 cells were produced in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 5% fetal calf serum in 6-well plates at 37C with 5% carbon dioxide. Samples (100 mL) of 5H10 were incubated with an equal volume of SARS-CoV suspension (made up of 200 plaque-forming models of computer virus) in a water bath at 37C for 1 h. Samples of the incubated combination were used to infect Vero E6 monolayers. After adsorption for 1 h, the cultures were overlaid with 1% methylcellulose-containing media and incubated for 6C8 days at 37C. The cells were then fixed with 20% formalin, stained with 0.1% crystal violet in 0.1 M citric acid, Rabbit Polyclonal to K6PP and irradiated with UV light for 1 h. After drying, the numbers of computer virus plaques were counted. The neutralizing titer was defined as the reciprocal dilution of serum causing a 50% reduction in the plaque count, compared with the unfavorable BC 11 hydrobromide control. BC 11 hydrobromide In the neutralization assay against SARS-CoV PUMC01, cell monolayers were stained with 100 L of 0.15% neutral red (Sigma) in DMEM for 1 h at 37C. After washing, 100 L of acid alcohol (1% acetic acid in 50% ethanol) was added to each well. After incubation for 30 min at room heat, BC 11 hydrobromide the absorbance of neutral red-stained plates was go through at 450 nm. CellCCell Fusion Assay Mediated by Conversation Between SARS-S and Human ACE2 The full-length human gene in pcDNA3.1 (?) (kindly provided by Dr H. Choe, Harvard Medical School), the human codon-optimized gene of SARS-CoV in the VRC8304 vector (kindly provided by Dr G. J. Nabel, National Institutes of Health), and pGL4.35 encoding luciferase, pACT-MyoD, and pBIND-Id plasmids (Promega) were used in the fusion assay. The human ace2 gene and the spike gene of SARS-CoV were inserted into pIRES2 DdRed-Express2 and pIRES2-AcGFP1 (Clontech), respectively, and used in the syncytium formation assay. The spike gene and pBIND-Id plasmids were transfected into HEK293T cells with Lipofectamine LTX Reagent (Invitrogen) in one group. The human ace2 gene, pACT-MyoD, and pGL4.35 were transfected into the other HE293T cell group. Two days after transfection, HEK293T cells expressing S protein were detached with trypsin/EDTA answer (Sigma) or EDTA answer. HEK293T cells expressing ACE2 protein were detached with EDTA answer. The treatments with anti-SARS antibody were performed before and after detachment. These cells were mixed and cocultured for 1 day at 37C. The cells were harvested with lysis buffer and centrifuged. The cell lysates were then mixed with the substrate PicaGene (Toyo Ink), and luciferase activity was measured using a plate reader (Infinite F200; Tecan). In Western blot BC 11 hydrobromide analysis, HEK293T cells expressing S protein were treated in the presence or absence of 5H10. Cells were detached using trypsin/EDTA or EDTA answer. After antibody treatment and detachment, these cells were lysed with radioimmunoprecipitation assay answer (0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Nonidet P – 40, 150 mM sodium chloride, 50 mM Tris-HCl, pH 8.0). The cell lysates were separated using sodium dodecyl sulfateCpolyacrylamide gel electrophoresis and transferred onto Immun-Blot P polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were incubated with a mixture of in-house anti-S1-N, S1-C, S791, and S2 antibodies explained above and anti-rabbit IgG horseradish peroxidase (GE Healthcare). After washing, the membranes were incubated with SuperSignal West Substrate (Thermo Scientific) according to the manufacturers instructions. Syncytium Formation Assay and Inhibition by Anti-SARS Antibodies The gene in pIRES2-AcGFP1 and gene in pIRES2 DdRed-Express2 were transfected into HEK293T cells separately. Two days after transfection, HEK293T cells expressing S protein.