Journal: PLOS Neglected Tropical Diseases

Article Title: Development of novel monoclonal antibodies for detection of pan- Lassa virus

doi: 10.1371/journal.pntd.0014326

Figure Lengend Snippet: A) Cross-reactivity of mAbs to different LASV lineages NP was measured using cell lysate in indirect ELISA. OD 450 for each mAb is summarized as heat map. B) Representative of binding of anti-LASV NP mAb with cell lysate of HEK293T expressing LASV NPs (lineage I-VII) using WB, each lysate was diluted 1:100 times and used as an antigen for measurement of binding of mAb 28-2-3. C) Representative images of IFA showing binding of mAb 57-2 to the HEK293T cells expressing LASV NP, 24 hours post transfection cells were fixed, permeabilized and incubated with mAb 57-2 and detected by Alexa488 anti-mouse IgG antibody. Hoechst was used for nucleus staining. HEK293T cells transfected with pCAGGS empty vector were used as negative control (NC).

Article Snippet: The membrane was incubated with 1 μg/ml of mouse anti-LASV NP mAbs which were generated in house or commercial mAb from Zalgen (NP LASV MAb1474) was also used to detect LASV NP in the cell lysate.

Techniques: Indirect ELISA, Binding Assay, Expressing, Transfection, Incubation, Staining, Plasmid Preparation, Negative Control

Journal: PLOS Neglected Tropical Diseases

Article Title: Development of novel monoclonal antibodies for detection of pan- Lassa virus

doi: 10.1371/journal.pntd.0014326

Figure Lengend Snippet: The cross-competition profile of mAbs for binding to LASV NP was determined using Ab competitive ELISA. HEK293T cell lysate expressing LASV lineage III (Ojoko) NP was diluted to 1:100 in PBS and coated on the ELISA plate surface. Serially diluted unconjugated mAbs were used as primary antibody as represented in different colors in each graph and then 1µg/ml of HRP-conjugated mAbs for LASV NP were used as secondary antibody for each measurement. Anti-LASV GPC mAb was used as an IgG control.

Article Snippet: The membrane was incubated with 1 μg/ml of mouse anti-LASV NP mAbs which were generated in house or commercial mAb from Zalgen (NP LASV MAb1474) was also used to detect LASV NP in the cell lysate.

Techniques: Binding Assay, Competitive ELISA, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: PLOS Neglected Tropical Diseases

Article Title: Development of novel monoclonal antibodies for detection of pan- Lassa virus

doi: 10.1371/journal.pntd.0014326

Figure Lengend Snippet: A) 2x10 5 PFU/ml of LASV lineages I-IV were serially diluted and used as an antigen for sandwich ELISA with 1µg/ml of each capturing and detection mAbs. Binding of different pairs of capturing mAb/detection mAb with LASV NP are denoted in the graph as OD 450 . B) Sandwich ELISA for detection of LASV NP from lineages V-VII were measured using VLPs, lineage IV VLPs was used as positive control for this measurement. C) Measurement of NP mAbs binding with rVSV, EBOV, MARV, and LCMV at different concentration of authentic virus using different combinations of mAbs. Data represents means and standard deviations (SDs) of OD 450 from three biological triplicates. The threshold of sandwich ELISA was calculated as the mean + 3SDs of OD 450 value of negative control and represented as a black dotted horizontal line in the graphs.

Article Snippet: The membrane was incubated with 1 μg/ml of mouse anti-LASV NP mAbs which were generated in house or commercial mAb from Zalgen (NP LASV MAb1474) was also used to detect LASV NP in the cell lysate.

Techniques: Sandwich ELISA, Binding Assay, Positive Control, Concentration Assay, Virus, Negative Control

Journal: npj Viruses

Article Title: Site-1 protease mediated GPC processing is required for persistence of LCMV Clone 13

doi: 10.1038/s44298-026-00184-7

Figure Lengend Snippet: a – c Multi-step growth kinetics of rCl13, rCl13-RRRR, and rCl13-RRLL in WT ( a ), S1P-KO ( b ), and SP2-KO ( c ) HAP1 cells. Cells were seeded at 3 × 10 5 cells/well in a 24-well plate and 24 h later infected (MOI = 0.01) with the indicated virus. Cell culture supernatants (CCSs) were collected at indicated hours post infection (hpi), and infectious virus titers were determined by focus-forming assay (FFA). d Processing of GPC in WT, S1P-KO, and SP2-KO HAP1 cells infected (MOI = 1) with rCl13, rCl13-RRRR, or rCl13-RRLL. At 24 hpi, cell lysates were prepared and analyzed by western blotting using the mouse monoclonal G204 to GPC. e Multi-step growth kinetics of rCl13, rCl13-RRRR, and rCl13-RRLL in furin-deficient LoVo cells and LoVo-furin cells with reconstituted furin expression. Cells were seeded at 3 × 10 5 cells/well in a 24-well plate and 24 h later infected (MOI = 0.01) with the indicated virus. CCSs were collected at the indicated hpi, and infectious virus titers were determined by FFA. f , g Susceptibility of rCl13 and rCl13-RRRR to furin inhibitor BOS-318 ( f ) and S1P inhibitor PF-429242 ( g ). A549 cells were seeded in 96-well plates at 2 × 10 4 cells/well and 24 h later infected (MOI = 0.01) with the indicated virus and treated with serial dilutions (1:2, 6 replicates per dilution) of either BOS-318 (starting at 2 µM), or PF-429242 (starting at 40 µM). At 72 hpi, cell viability was determined by CellTiter 96 Aqueous One Solution (purple), then cells were fixed with 4% PFA. The level of infection was assessed by IF using the rat monoclonal antibody VL4 to NP (green), and cell viability was also alternatively determined by DAPI staining (blue). Raw values of CellTiter, NP, and DAPI staining were normalized to vehicle-treated and infected control cells present in each plate. Dose-response curves for BOS-318 and PF-429242 were generated using a nonlinear regression model with log(inhibitor) vs response – variable response (four parameters) equation in GraphPad Prism. The dashed line represents 50% of vehicle control. h Effect of PF-429242 on multi-step growth kinetics of rCl13 and rCl13-RRRR in A549 cells. A549 cells were seeded at 2.5 × 10 5 cells/well in a 24-well plate and 24 h later infected (MOI = 0.01) with the indicated virus. After 90 min adsorption, the virus inoculum was removed, and medium containing the indicated concentration of PF-429242 was added to the cells. At the indicated hpi, CCSs were collected and virus titers determined by FFA. Cells lacking S1P or treated with PF-429242 were grown in media supplemented with lipids (1 mM sodium mevalonate, 20 μM sodium oleate, 5 μg/ml cholesterol).

Article Snippet: Cells were fixed with 4% PFA at 72 hpi and stained with rat mAb VL4 against LCMV NP (Bio X Cell), then with anti-rat AF488-conjugated antibody.

Techniques: Infection, Virus, Cell Culture, Focus Forming Assay, Western Blot, Expressing, Staining, Control, Generated, Adsorption, Concentration Assay

Journal: npj Viruses

Article Title: Site-1 protease mediated GPC processing is required for persistence of LCMV Clone 13

doi: 10.1038/s44298-026-00184-7

Figure Lengend Snippet: a Eight-week-old B6 mice were infected with rCl13, rCl13-RRRR ( n = 5/group, 2 × 10 6 FFU/mouse, IV), or mock-infected ( n = 2, 200 µL PBS, IV). Spleens were harvested at 24 hpi and processed to generate single-cell suspensions. Specific cell types were identified by flow cytometry through surface marker staining: B cells: B220+, CD3−, CD4−, Siglec-H−; CD4 T cells: B220−, CD3+, CD4+; CD8 T cells: B220−, CD3+, CD8+; plasmacytoid dendritic cells (pDCs): B220+, CD3−, CD4+, Siglec-H+; natural killer (NK) cells: B220−, CD3−, NK1.1+; CD8+ dendritic cells (DCs): B220−, CD3−, NK1.1−, Ly6G−, CD11c high , MHC-II+, CD11b low , CD8+; CD11b+ cDCs: B220−, CD3−, NK1.1−, Ly6G−, CD11c high , MHC-II+, CD11b+, CD8−; macrophages: B220−, CD3−, NK1.1−, Ly6G−, CD11c low , CD11b−, F4/80+; Ly6C+ monocytes: B220−, CD3−, NK1.1−, Ly6G−, CD11c low , CD11b high , Ly6C high ; Ly6C- monocytes: B220−, CD3−, NK1.1−, Ly6G−, CD11c low , CD11b high , Ly6C low . Intracellular NP was stained with AF488-conjugated VL4 rat monoclonal antibody. b shows the representative contour plots of NP + gates from the macrophage populations. c , d Infection of B6 mice and processing of spleen samples were done as in ( a ). Specific cell types were identified by flow cytometry through surface marker staining: metallophilic marginal-zone macrophages (MMM): lin-(CD19−, CD3−, NK1.1−), Ly6G−, Ly6C−, F4/80+, CD11b high , CD169 high ; red pulp macrophages (RPM): lin-(CD19−, CD3−, NK1.1−), Ly6G−, Ly6C−, F4/80+, CD11b low , CD169−. Intracellular NP was stained with AF488-conjugated VL4 rat monoclonal antibody. Representative contour plots of NP + gates from the parental gates are shown. A statistical test (unpaired t -test) was done between rCl13 and rCl13-RRRR groups in GraphPad Prism. P values are shown or represented by asterisks (ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). e , f Eight-week-old B6 mice were infected as in ( a ). Spleens were collected at 24 hpi and split in half. One half was flash frozen in OCT and 8 µm tissue sections were stained with human anti-GPC antibody (green), rat anti-CD169 antibody (red) and DAPI (blue). Sections were prepared for four mice per group, and representative images from each group are shown. The other half of the spleen was homogenized and virus titers determined by FFA. A statistical test (unpaired t -test) was done between rCl13 and rCl13-RRRR groups in GraphPad Prism. P values are represented by asterisks (ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Article Snippet: Cells were fixed with 4% PFA at 72 hpi and stained with rat mAb VL4 against LCMV NP (Bio X Cell), then with anti-rat AF488-conjugated antibody.

Techniques: Infection, Single Cell, Flow Cytometry, Marker, Staining, Virus