biotinylated anti ifn γ detection antibody  (Mabtech Inc)

Journal: iScience

Article Title: Extensive evolution and T cell escape by SARS-CoV-2 in a 2.5-year persistent infection of an immunocompromised host

doi: 10.1016/j.isci.2026.114917

Figure Lengend Snippet: Magnitude and specificity of T cell memory responses generated by 899-day infection (A) Ex vivo IFNγ-ELISpot well images. DMSO control unstimulated wells and wells stimulated with overlapping peptides covering S1 and S2 region of Spike (duplicates shown), or single peptides S51 and S134. (B) Total magnitude of SARS-CoV-2-specific memory T cell response to structural proteins Spike, membrane (M), nucleoprotein (NP) and open reading frame 3a (ORF3a) and RTC proteins (NSP7, NSP12 polymerase, and NSP13 helicase) colored by protein targeted and measured after persistent infection (>900 days). (C and D) Total magnitude of SARS-CoV-2-specific T cell response (C) or T cell response to Structural and RTC proteins (D) in pre-pandemic samples (pre-August 2019), in exposed healthcare workers (HCW) who remained seronegative, including abortive infections, and in HCW with laboratory confirmed SARS-CoV-2 infections (samples 4 months post-exposure/infection in June to July 2020) for comparison to T cell response in persistently infected patient. (E) Ratio of the magnitude of the T cell response to RTC/structural T cells. Percentage of cohort with a response above 1 (stronger response to RTC than structural proteins) shown below. (F) Magnitude of T cell response to a pool of epitopes from Flu, EBV, and CMV. (B–E) Subset of data previously published in Swadling et al. (A–F) IFNγ-ELISpot. (C and D) Box and Whisker, Tukey. (C–F) Statistical analysis was performed using Kruskal-Wallis tests with Dunn’s correction. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p < 0.0001. (E and F) Bars, geomean.

Article Snippet: ELISpot plates were developed with human biotinylated IFNγ detection antibodies (7-B6-1, Mabtech; 1 μg/ml) for 3 h at room temperature, followed by incubation with goat anti-biotin alkaline phosphatase (Vector Laboratories; 1:1,000) for 2 h at room temperature, both diluted in PBS with 0.5% BSA by volume (Sigma-Aldrich), and finally with 50 μl per well of sterile filtered BCIP/NBT Phosphatase Substrate (Thermo Fisher Scientific) for 7 min at room temperature.

Techniques: Generated, Infection, Ex Vivo, Enzyme-linked Immunospot, Control, Membrane, Comparison, Whisker Assay

Journal: iScience

Article Title: Extensive evolution and T cell escape by SARS-CoV-2 in a 2.5-year persistent infection of an immunocompromised host

doi: 10.1016/j.isci.2026.114917

Figure Lengend Snippet: Ex vivo functionality and proliferative potential of T cell memory generated by a 899-day persistent infection (A) The IFNγ and/or TNF⍺ producing CD4 and CD8 T cell memory response to SARS-CoV-2 proteins after persistent infection. Percentage of CD4 or CD8 is shown. Spike divided into S1 and S2 peptide pools. DMSO, unstimulated control. FEC, response to Flu, EBV, and CMV epitope pool. (B) Summed CD4 or CD8 IFNγ+ response to spike (S1, S2) or the replication-transcription proteins (RTC; NSP7/12/13). (C) The proportion of SARS-CoV-2-specific CD4 or CD8 T cells to spike or RTC that co-produce several cytokines/effector molecules are shown as pie charts. Pie arcs show the proportion producing each single cytokine. (D) Proliferation and expansion of SARS-CoV-2-specific memory CD4 and CD8 T cells post-persistent infection when in vitro stimulated with Spike, Flu EBV and CMV epitopes, or PHA, as shown by CTV dilution and IFNγ production after 8-day stimulation. Percentage of CD4 or CD8 shown for each quadrant. PHA, phytohemagglutinin.

Article Snippet: ELISpot plates were developed with human biotinylated IFNγ detection antibodies (7-B6-1, Mabtech; 1 μg/ml) for 3 h at room temperature, followed by incubation with goat anti-biotin alkaline phosphatase (Vector Laboratories; 1:1,000) for 2 h at room temperature, both diluted in PBS with 0.5% BSA by volume (Sigma-Aldrich), and finally with 50 μl per well of sterile filtered BCIP/NBT Phosphatase Substrate (Thermo Fisher Scientific) for 7 min at room temperature.

Techniques: Ex Vivo, Generated, Infection, Control, In Vitro

Journal: iScience

Article Title: Extensive evolution and T cell escape by SARS-CoV-2 in a 2.5-year persistent infection of an immunocompromised host

doi: 10.1016/j.isci.2026.114917

Figure Lengend Snippet: Failure of host T cells to recognize emerging virus (A) Ex vivo magnitude of the T cell response to individual ancestral sequence peptides in which mutation arose over persistent infection by IFNγ-ELISpot. (B) Magnitude of the CD4 and CD8 T cell responses after 10-day in vitro peptide expansion with individual ancestral sequence peptides. Percentage of CD4 and CD8 producing IFNγ, TNF, or both are shown. (C) Magnitude of IFNγ+, IFNγ+TNF+, and CTV lo IFNγ+ CD4 and CD8 T cells after 8-day expansion with a pool of 12 peptides corresponding to ancestral sequence epitopes or variant sequence epitopes containing mutations in the virus isolated at day 899 of infection. (D) Magnitude of the IFNγ+TNF+ CD4 or CD8 T cell response after 10-day in vitro peptide stimulation with individual epitopes using ancestral sequence or variant sequence peptides for culture and re-stimulation on day 9. Summary data showing all T cell responses that were detectable (left) or shown for individual peptides (right) for CD4 then CD8 T cells. Duplicate and triplicate stimulations are shown for CD8 T cells for M14 and NSP2, respectively. (D) Bars, mean. Statistical analysis was performed using Kruskal-Wallis tests with Dunn’s correction. ∗ p ≤ 0.05; ∗∗∗ p ≤ 0.001. Peptide sequences shown in .

Article Snippet: ELISpot plates were developed with human biotinylated IFNγ detection antibodies (7-B6-1, Mabtech; 1 μg/ml) for 3 h at room temperature, followed by incubation with goat anti-biotin alkaline phosphatase (Vector Laboratories; 1:1,000) for 2 h at room temperature, both diluted in PBS with 0.5% BSA by volume (Sigma-Aldrich), and finally with 50 μl per well of sterile filtered BCIP/NBT Phosphatase Substrate (Thermo Fisher Scientific) for 7 min at room temperature.

Techniques: Virus, Ex Vivo, Sequencing, Mutagenesis, Infection, Enzyme-linked Immunospot, In Vitro, Variant Assay, Isolation