Recombinant Human Anti-SFTSV Antibody (MAb4-5) (CAT#: HPAB-0032-WJ)

• Immunogen
• Purified SFTSV virus

• Host Species
• Human

• Derivation
• Phage display library

• Type
• Human IgG1

• Specificity
• SFTSV Gnl domain of glycoprotein N

• Species Reactivity
• SFTSV

• Clone
• MAb4-5

• Applications
• Neut, ELISA, WB, SPR, IF, EM

• Related Disease
• SFTSV infection diseases

• Application Notes
• The SFTSV antibody has been reported in applications of Neutralization, Enzyme-linked Immunosorbent Assay, Western Blot, Surface Plasmon Resonance, Immunofluorescence, Electron Microscopy.
  For Neutralization: Fifty-microliters of twofold serial-diluted MAb 4–5 or control antibody (Ebola monoclonal antibody 13C6) was mixed with equal volume of 100 TCID50 SFTSV (SDYY007), or RVFV (BJ01) at 310 K for 1 h. The virus–antibody mixture was then transferred to 80% confluent Vero cells in a 96-well plate and incubated at 310 K for 1 h. The plate was washed with DMEM three times, and incubated at 310 K in a 5% CO2 incubator. Cytopathic effects were observed every 24 h for 6 d. The antibody was considered as having neutralizing capacity if 100% of viral cytopathic effects was inhibited.
  For SPR: For the SPR assays, a BIAcore 3000 spectrometer was used to measure kinetic constants at room temperature (298 K). All proteins were exchanged into a buffer of 20 mM Hepes, pH 7.4, 150 mM NaCl and 0.005% (vol/vol) Tween 20. MAb 4–5 proteins were immobilized on CM5 chips at approximately 1,000 response units and analyzed for real-time binding by flowing through gradient concentrations (ranging from 7.8 to 1,000 nM) of Gn head proteins. For the Octet RED96 biosensor method, samples of buffer were dispensed into polypropylene 96-well black flat-bottom plates at a volume of 200 μL per well, and all measurements were performed at 298 K in HBS-EP buffer with the plate shaking at the speed of 1,000 rpm. Anti-human IgG Fc (AMC)-coated biosensor tips were used to capture antibody MAb 4–5 from 10 ng/μL stock buffer. The buffer was 20 mM Hepes, pH 7.4, 150 mM NaCl and 0.005% (vol/vol) Tween 20. Kinetic measurements for antigen binding were performed by exposing biosensors to a series of analyte concentrations (15.6−250 nM for Gn wild-type, and 15.6–500 nM for Gn mutants) and background subtraction was used to correct for sensor drifting. All sensors were generated with 10 mM Glycine-HCl, (pH 1.7,).
  For Western Blot: To determine the binding target of the selected MAb 4-5 IgG1, purified SFTSV strain JS-2010-003 virions were lysed with sample buffer and proteins were fractionated by 10% SDS-PAGE. The separated proteins were electrotransferred to a nitrocellulose (NC) membrane and incubated consequently with MAb 4-5 IgG1 and horseradish peroxidase (HRP)-conjugated goat anti-human immunoglobulin. The blots were visualized by 3,3′diaminobenzidine (DAB) according to the manufacturer's instructions.
  For IF: Reactivity between human MAb 4-5 IgG1 and SFTSV-infected cells was assessed using indirect immunofluorescence assay (IFA). The infected and noninfected (SFTSV, JS-2010-003) Vero cells were grown on an 8-well Millicell EZ slide for 36 h at 37°C, and cells were fixed by treatment with acetone for 10 min at −20°C. Human MAb 4-5 IgG1or an irrelevant human IgG1 (described above) were incubated with the fixed cells for 30 min at 37°C. Bound antibodies were detected using fluorescein isothiocyanate (FITC)-conjugated anti-human antibodies (diluted by phosphate-buffered saline PBS containing 0.01% [wt/vol] Evens blue) and observed under an immunofluorescence microscope.
  For EM: SFTSV JS-2010-003 from the supernatant of infected Vero cells was adsorbed to copper grids coated with carbon and Pioloform. These were incubated with MAb 4-5 IgG1 by floating on droplets for 30 min at room temperature, and an irrelevant human IgG1 was used as a negative control. Bound monoclonal antibodies were detected by incubation on droplets of anti-human IgG gold 10-nm conjugates. The grids were negatively contrasted with 2% phosphotungstic acid and assessed with a Hitachi 7650A transmission electron microscope.
  For ELISA: In order to measure the binding activities of the MAb4-5 IgG1 antibodies, 96-well half-area microplates were coated with Gn-Fc fusion protein and incubated at 4°C overnight. Plates were blocked with 3% skim milk in PBS for 1 h at room temperature. The plates were then washed with PBS and received antibodies that were 10-fold serially diluted from 1 μM to 10 μM in blocking buffer. The plates were then incubated for 2 h at room temperature and washed three times with 0.05% Tween20 in PBS solution. Then, 50 μL of HRP-conjugated anti-human Ig kappa light chain antibody diluted in blocking buffer (1:5000) was added into each well. Then, plates were incubated for 1 h at room temperature. After washing, each well received 50 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The color reaction was stopped by adding 50 μL of 2 M sulfuric acid. The absorbance of each well was measured at 450 nm using a microplate spectrophotometer .

• Alternative Names
• thrombocytopenia syndrome virus; SFTSV

• HPAB-0032-WJ-F(E)
• Recombinant Human Anti-SFTSV Antibody Fab Fragment (MAb4-5)

• HPAB-0032-WJ-S(P)
• Recombinant Human Anti-SFTSV Antibody scFv Fragment (MAb4-5)

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