maxisorp high-binding elisa plate  (Thermo Fisher)

Journal: Nature Communications

Article Title: Antibodies targeting the Crimean-Congo Hemorrhagic Fever Virus nucleoprotein protect via TRIM21

doi: 10.1038/s41467-024-53362-7

Figure Lengend Snippet: Sera stocks for adoptive transfer were confirmed to have CCHFV-specific antibodies via ( a ) whole virion IgG ELISA and isotype/subtype whole virion IgG ELISA. Naïve WT C57BL6/J mice were treated with sera from repNP or sham vaccinated mice on day −1 (1tx) or days 0 and +3 (2tx) relative to lethal challenge with 100 TCID 50 CCHFV strain UG3010. Mice ( N = 8) were ( b ) weighed daily and monitored for ( c ) survival until day 14 p.i. Dashed lines indicate limit of detection. Significance was calculated using one-way ANOVA; ns P > 0.05, **** P < 0.0001. Data shown as mean plus standard deviation.

Article Snippet: Briefly, whole CCHFV antigen was used to coat NUNC MaxiSorp high protein-binding capacity ELISA plates (ThermoFisher) overnight before blocking with milk, incubation with mouse sera, and detection with secondary goat anti-mouse IgG antibody (Southern Biotech) and ABTS (SeraCare).

Techniques: Adoptive Transfer Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature Communications

Article Title: Antibodies targeting the Crimean-Congo Hemorrhagic Fever Virus nucleoprotein protect via TRIM21

doi: 10.1038/s41467-024-53362-7

Figure Lengend Snippet: To investigate antibody-dependent intracellular neutralization (ADIN), L929 cells were electroporated with antibody and efficiency of electroporation was verified via FLOW cytometry measuring internalization of control anti-mouse AF488 antibody with and without electroporation (EP) andcells were gated by AF488- or AF488+ (Supplementary Fig. ). Next, L929 cells were electroporated with ( a ) mouse sham or NP-immune sera, as used in adoptive transfer studies, and infected with MA-CCHFV. Viral growth was monitored via TCID 50 72 h p.i., Study was performed with four technical replicates. Pre-immune and NP-immune sera from cynomolgus macaques vaccinated with our repNP vaccine was pooled and evaluated for CCHFV-specific antibody via ( b ) ELISA and then electroporated into L929 cells to assess ( c ) ADIN capacity in MA-CCHFV infected cells as above. Human sera was confirmed positive or negative for CCHFV specific antibodies via ELISA and assessed for ( d ) ADIN capacity as above. Data graphed to show inhibitory concentrations in mean plus standard deviation. Lower dashed line indicates limit of detection (LOD) of assay and upper dashed lines indicate the average TCID 50 (Log 10 ) of sham, pre-immune, or negative sera samples. Significance was calculated using one-way ANOVA; ns P > 0.05, **** P < 0.0001. Exact p -values: ( d ) * P = 0.0113; ** P = 0.0049; *** P = 0.0004.

Article Snippet: Briefly, whole CCHFV antigen was used to coat NUNC MaxiSorp high protein-binding capacity ELISA plates (ThermoFisher) overnight before blocking with milk, incubation with mouse sera, and detection with secondary goat anti-mouse IgG antibody (Southern Biotech) and ABTS (SeraCare).

Techniques: Neutralization, Electroporation, Flow Cytometry, Control, Adoptive Transfer Assay, Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cell Discovery

Article Title: Specialized Tfh cell subsets driving type-1 and type-2 humoral responses in lymphoid tissue

doi: 10.1038/s41421-024-00681-0

Figure Lengend Snippet: a C57BL/6 mice were immunized subcutaneously in the footpad with OVA either emulsified in IFA (IFA), admixed with CpG (CpG), or incorporated with CpG in nanoparticles (NP-CpG). Eleven days later, blood and draining lymph nodes (LNs) were collected for analysis. b ELISA quantification of anti-OVA IgG1 and IgG2a, and IgG1/IgG2a ratio in serum of the immunized mice. c , d Representative flow cytometry plots ( c ) and quantification of GC B cells (CD19 + CD95 + GL7 + ), Tfh cells (CD4 + Foxp3 − CD25 − CXCR5 + PD1 + ), and GC-Tfh cells (CD4 + Foxp3 - CD25 - CXCR5 hi PD1 hi ) ( d ) in the draining LNs. Data from one experiment ( n = 4), each dot representing one sample and bars representing mean values, analyzed by one-way ANOVA and Tukey’s multiple comparisons tests: ** P < 0.01, *** P < 0.001. e T cells from OVA-specific TCR-transgenic mice were adoptively transferred into congenic recipients, immunized on the following day with OVA associated with different adjuvants. f On day 11, OVA-specific activated Th and Tfh cells were isolated by FACS for RNA-seq according to the represented gating strategy. g Principal component analysis (PCA) from the RNA-seq datasets of Th and Tfh cells, isolated from the two strains, and three types of immunization. PC1 explained 15% of the variance, discriminating datasets from the two strains. h PC2 and PC3 have a similar impact on the variance, with PC2 segregating Tfh cells from activated non-follicular T cells, and PC3 separating the samples based on the type of adjuvant used (type-1 vs type-2).

Article Snippet: Briefly, high protein-binding ELISA plates (Nunc MaxiSorp, #44-2404-21) were coated overnight at 4 °C with OVA (Invivogen, #vac-pova) at 10 μg/mL in coating buffer (eBioscience, #00-0044-59).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Transgenic Assay, Isolation, RNA Sequencing, Adjuvant