mouse monoclonal antibody against sftsv np  (Applygen Technologies)

Journal: eBioMedicine

Article Title: Discovery and characterization of potent broadly neutralizing antibodies from human survivors of severe fever with thrombocytopenia syndrome

doi: 10.1016/j.ebiom.2024.105481

Figure Lengend Snippet: Diverse lineages of SFTSV Gn-reactive mAbs isolated from donors who have recovered from SFTS . (A) Flow cytometry analysis of Gn-specific B cells isolated from four donors who have recovered from SFTS. Flow cytometry plots showed the gated on CD14/CD16/CD3/Zombie/IgM/IgD − CD19 + IgG + CD27 + Gn + B cells. Inset denotes donor ID (1–4) and time between the onset of illness and blood draw. (B) Frequency of class-switched B cells among Gn-reactive B cells. (C) Frequency of CD27 + B cells among Gn-reactive class-switched IgG + B cells. (D) The binding of 283 antibodies to SFTSV measured by ELISA. The OD 450 value was shown for each antibody tested at a concentration of 1 μg/mL. (E) Somatic hypermutation rate determined by the number of VH nucleotide substitutions away from the predicted VH germline. Comparison was performed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗∗, P < 0.001). (F) Clonal analysis of isolated antibody genes. Clones that encode the same germline genes, CDR3 lengths, and have >80% nucleotide sequence homology within HCDR3 are considered to be within the same lineage. The colored portion indicates expanded clonal lineage and unique clones are shown as a single gray segment. The numbers above the columns represent the total number of mAbs with the binding activity verified by ELISA. (G and H) The germline and rearrangement usage for heavy (G) and light (H) chains of the 146 high-confidence candidates.

Article Snippet: The staining process involved the use of a mouse monoclonal antibody against SFTSV NP as the primary antibody, followed by an anti-mouse HRP conjugated secondary antibodies (Applygen, Cat. C1308).

Techniques: Isolation, Flow Cytometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison, Clone Assay, Sequencing, Activity Assay

Journal: eBioMedicine

Article Title: Discovery and characterization of potent broadly neutralizing antibodies from human survivors of severe fever with thrombocytopenia syndrome

doi: 10.1016/j.ebiom.2024.105481

Figure Lengend Snippet: Broadly neutralizing activities of Gn-reactive mAbs against SFTSV in vitro . (A) Neutralizing activity of 146 mAbs against SFTSV at a concentration of 250 ng/mL, as measured by reduction in the number of plaques in Vero cells compared to control antibody using a focus reduction neutralization test (FRNT). Each circle represents a different mAb. (B and C) The neutralization activity (FRNT 50 ; B) and curves (C) of 18 selected mAbs against five SFTSV strains belonging to distinct viral clades. Two mAbs (JK-44 and JK-65) without negligible neutralization activity were also tested as controls. The FRNT 50 was calculated using the equation of log [inhibitor] versus response method. Data was shown as mean values from three biologically independent replicates. (D) Representative images of Vero cells infected with SFTSV (clade I; MOI = 0.1) treated with mAbs at indicated concentrations. Green (stained with SFTSV nucleocapsid protein [NP] antibody) indicates viral infected cells and blue represents cell nuclei. Experiment was performed in three biological replicates with similar results.

Article Snippet: The staining process involved the use of a mouse monoclonal antibody against SFTSV NP as the primary antibody, followed by an anti-mouse HRP conjugated secondary antibodies (Applygen, Cat. C1308).

Techniques: In Vitro, Activity Assay, Concentration Assay, Control, Neutralization, Infection, Staining

Journal: eBioMedicine

Article Title: Discovery and characterization of potent broadly neutralizing antibodies from human survivors of severe fever with thrombocytopenia syndrome

doi: 10.1016/j.ebiom.2024.105481

Figure Lengend Snippet: Antigen specificity and affinity of Gn-reactive mAbs . (A) Competition binding of Gn-specific mAbs. Eighteen selected mAbs and two mAbs were subjected to competition-binding assays. The mAbs are displayed in 6 groups (designated A [with A1, A2, or A3 subgroups], B, C, or D) based on their ability to compete for binding with each other. The values shown are the percentage of binding that occurred during competition compared to non-competed binding of the mAb using biolayer interferometry (BLI) platform with the In-Tandem method. The values are also indicated by the box fill color; pink colors toward red indicate higher competition, and lighter colors toward white indicate less competition, on a gradient scale. Data are shown as mean values from three biologically independent replicates. (B) Binding kinetics of the six candidate mAbs (KD < 1 nM) with the Gn protein of SFTSV measured by BLI assay. JK-59, JK-8, JK-12, and JK-3 belong to groups A1, A3, B and C, respectively; JK-2 and JK-70 belong to group A2.

Article Snippet: The staining process involved the use of a mouse monoclonal antibody against SFTSV NP as the primary antibody, followed by an anti-mouse HRP conjugated secondary antibodies (Applygen, Cat. C1308).

Techniques: Binding Assay

Journal: eBioMedicine

Article Title: Discovery and characterization of potent broadly neutralizing antibodies from human survivors of severe fever with thrombocytopenia syndrome

doi: 10.1016/j.ebiom.2024.105481

Figure Lengend Snippet: Interference of the binding of SFTSV to cells by mAbs . (A) Effects of mAbs on the binding of SFTSV to THP-1 cells that were measured by RT-qPCR. SFTSV virions (10 6 FFUs) were incubated with each mAb at indicated concentrations for 1 h at 37 °C and subsequently incubated with THP-1 cells (10 5 ) for 1 h at 4 °C. The bound virions (relative viral RNA levels) were measured via RT-qPCR (n = 3 biologically independent replicates). Comparison analysis was severally performed among each mAb with four indicated concentrations and the vehicle control. (B and C) Effects of mAbs on the binding of SFTSV to THP-1 cells that were measured by flow cytometry analysis. SFTSV virions (2 × 10 7 FFUs) were incubated with each mAb at indicated concentrations for 1 h at 37 °C and subsequently incubated with THP-1 cells (10 5 ) for 1 h at 4 °C. SFTSV infection rates of the cells were measured via flow cytometry with a mouse monoclonal antibody against SFTSV NP (n = 3 biologically independent replicates). Comparison analysis was severally performed among each mAb with three indicated concentrations and the vehicle control (B). Representative images of flow plot of SFTSV infection in THP-1 cells were shown in panel C. (D and E) Surface expression of CCR2 on THP-1 cells. SFTSV virions (10 7 FFUs) were incubated with vehicle or each mAb at 25 μg/mL for 1 h at 37 °C and subsequently incubated with THP-1 cells (10 5 ) for 1 h at 4 °C. The mean fluorescence intensity (MFI) of CCR2 on THP-1 cells were determined by flow cytometry (n = 3 biologically independent replicates). Representative images indicating the cell surface fluorescence shift of CCR2 are shown in panel E. Data were presented as mean ± SD (A, B, and D). One-way ANOVA followed by Tukey's multiple comparisons test was performed for comparison of variables among multiple groups (A, B, and D). ∗, P < 0.1; ∗∗, P < 0.01, ∗∗∗, P < 0.001; ns, no significant.

Article Snippet: The staining process involved the use of a mouse monoclonal antibody against SFTSV NP as the primary antibody, followed by an anti-mouse HRP conjugated secondary antibodies (Applygen, Cat. C1308).

Techniques: Binding Assay, Quantitative RT-PCR, Incubation, Comparison, Control, Flow Cytometry, Infection, Expressing, Fluorescence

Journal: eBioMedicine

Article Title: Discovery and characterization of potent broadly neutralizing antibodies from human survivors of severe fever with thrombocytopenia syndrome

doi: 10.1016/j.ebiom.2024.105481

Figure Lengend Snippet: Analysis of binding epitope of JK-2/JK-8/JK-12 on SFTSV Gn head . (A) The binding epitopes of JK-2/JK-8/JK-12/Mab4-5/Ab10 on SFTSV Gn head are depicted. JK-2/JK-8/JK-12/Mab4-5 is colored in cyan, bright orange, violet, and yellow, respectively. The binding sites of Ab10 on SFTSV Gn head are colored in wheat. Gn is colored in gray. (B) Docking of JK-8 onto the surface of SFTSV virion (5-fold of SFTSV virion, EMD-35178), with hexon and penton highlighted in color.

Article Snippet: The staining process involved the use of a mouse monoclonal antibody against SFTSV NP as the primary antibody, followed by an anti-mouse HRP conjugated secondary antibodies (Applygen, Cat. C1308).

Techniques: Binding Assay

Journal: eBioMedicine

Article Title: Discovery and characterization of potent broadly neutralizing antibodies from human survivors of severe fever with thrombocytopenia syndrome

doi: 10.1016/j.ebiom.2024.105481

Figure Lengend Snippet: Structural analysis of JK-8 and JK-12 interaction with the SFTSV Gn head . (A–B) The binding region of JK-8/JK-12 on the SFTSV Gn head. Left: The SFTSV Gn are shown by surface and colored in grey. HCDRs and LCDRs interacting to the Gn are displayed by cartoon and colored. Middle: The SFTSV Gn are shown by surface and the residues interacting with antibodies are colored. Right: The variable regions of heavy chains and light chains are exhibited by surface. The SFTSV Gn are displayed in cartoon with primary interacting residues in sticks and colored in grey. JK-8 heavy chain, bright orange; JK-8 light chain, slate; JK-12 heavy chain, violet; JK-12 light chain, splitpea. (C–D) Detailed interactions between HCDRs/LCDRs of JK-8/JK-12 and Gn. Key residues are labeled and shown in sticks with identical colors to panels A–B. The glycan molecules are shown in sticks and colored cyan. Yellow dash lines, hydrogen bond interactions; cyan dash lines, salt bridge interactions.

Article Snippet: The staining process involved the use of a mouse monoclonal antibody against SFTSV NP as the primary antibody, followed by an anti-mouse HRP conjugated secondary antibodies (Applygen, Cat. C1308).

Techniques: Binding Assay, Labeling

Journal: eBioMedicine

Article Title: Discovery and characterization of potent broadly neutralizing antibodies from human survivors of severe fever with thrombocytopenia syndrome

doi: 10.1016/j.ebiom.2024.105481

Figure Lengend Snippet: Protective efficacy of mAbs in a lethal SFTSV infection mouse model . (A and B) Survival probability (A) and relative body weight (B) in six-week-old male IFNAR1 −/− C57BL/6 mice (n = 8 per group) that were treated with the indicated mAbs (10 μg per mouse) or human IgG1 isotope control antibody and then challenged with SFTSV (20 FFUs per mouse) at 1 day post treatment. (C) Viral loads in serum from IFNAR1 −/− C57BL/6 mice (n = 8 for each group) tested by RT-qPCR at 3 and 5 days post infection (dpi). (D and E) Survival probability (D) and relative body weight (E) in six-week-old male IFNAR1 −/− C57BL/6 mice (n = 8 per group) that were challenged with SFTSV (20 FFUs per mouse) and then treated with the indicated mAbs (10 μg per mouse) or control antibody at 1, 2, or 3 dpi. (F) Viral loads in serum from IFNAR1 −/− C57BL/6 mice (n = 8 for each group) tested by RT-qPCR at 3 and 5 dpi. (G) Viral titers in tissue samples (spleen, lung, liver, and kidney) from IFNAR1 −/− C57BL/6 mice (n = 4 per group) tested by immunological focus assay at 5 dpi. (H and I) Representative images of immunohistochemistry (H) and histopathology (I) analysis of spleen sections collected from mAb- or control antibody-treated IFNAR1 −/− mice with SFTSV infection at 5 dpi. Sections were stained with a rabbit polyclonal antibody against SFTSV NP for immunohistochemistry analysis and with hematoxylin and eosin (H&E) for histopathology analysis. Experiment was performed in triplicates. The asterisk indicates white pulp atrophy in the spleen. Data were presented as mean ± SD (B, C, E, F, and G). One-way ANOVA followed by Tukey's multiple comparisons test was performed for comparison of variables among multiple groups (C, F, and G). The Kaplan–Meier method and log-rank test were used to analyze time-to-event data (A and D). ∗, P < 0.1; ∗∗, P < 0.01, ∗∗∗, P < 0.001; ns, no significant.

Article Snippet: The staining process involved the use of a mouse monoclonal antibody against SFTSV NP as the primary antibody, followed by an anti-mouse HRP conjugated secondary antibodies (Applygen, Cat. C1308).

Techniques: Infection, Control, Quantitative RT-PCR, Immunohistochemistry, Histopathology, Staining, Comparison