biotinylated affinity purified goat igg anti human interferon γ detection antibody  (R&D Systems)

Journal: Lab on a chip

Article Title: High-resolution low-cost LCD 3D printing for microfluidics and organ-on-a-chip devices.

doi: 10.1039/d3lc01125a

Figure Lengend Snippet: Fig. 5 LCD 3D printed microfluidic ELISA chip. (a) ELISA-on-a-chip designed for LCD 3D printing with structurally encoded sequential delivery of reagents to autonomously perform an assay and coupled with a built-in chip-to-assay connection. (b) ELISA workflow showing the sandwich immunoassay designed for the detection of IFN-γ by sequentially delivering assay reagents and wash buffer to a nitrocellulose membrane pre- spotted with anti-human IFN-γ capture antibody; (1) sample containing IFN-γ, (2) biotinylated anti-human IFN-γ detection antibody, (3) streptavidin-conjugated enzyme pHRP, and (4) enzyme substrate in the presence of hydrogen peroxide to generate the colorimetric readout. (c) Autonomous CC workflow for on-chip reagent aliquoting by metering the correct reagent volumes and drainage of the excess, followed by (d) MCR-based sequential delivery of assay reagents with wash steps in between. Arrows show the direction of flow. Scale bar = 5 mm. (e) Binding curve of the on-chip assay for the detection of IFN-γ with a limit of detection of 12 pg mL−1 (CV: 6.8%) across triplicate chips for each tested concentration point; line shows a 4-point logistic fit.

Article Snippet: Purified mouse monoclonal IgG antihuman interferon-γ capture antibody (Cat. #MAB2852, lot #FIO1022021, R&D Systems, Minneapolis, Minnesota, United States), biotinylated affinity purified goat IgG anti-human interferon-γ detection antibody (Cat. #BAF285, lot #ZX2721071, R&D Systems, Minneapolis, Minnesota, United States), recombinant human interferon-γ protein (Cat. #285- IF, lot #RAX2422031, R&D Systems, Minneapolis, Minnesota, United States), Pierce streptavidin poly-horseradish peroxidase (pHRP) (Cat. #21140, lot #XJ360080, Thermo Fisher Scientific, Waltham, Massachusetts, United States), SIGMAFAST 3,3′-diaminobenzidine tablets (Cat. #D4293, lot #SLCG5357, Sigma-Aldrich, Oakville, Ontario, Canada), bovine serum albumin (BSA) (Cat. #001-000-162, lot #162191, Jackson ImmunoResearch Labs, West Grove, Pennsylvania, United States), BSA-biotin (Cat. #A8549, Sigma-Aldrich, Oakville, Ontario, Canada), Tween 20 (Cat. #P7949, lot #SLBX0835, Sigma-Aldrich, Oakville, Ontario, Canada).

Techniques: Enzyme-linked Immunosorbent Assay, Membrane, Binding Assay, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Utilization of primary tumor samples for cancer neoantigen discovery

doi: 10.1136/jitc-2024-010993

Figure Lengend Snippet: Example of screening TIL for recognition of cancer neoantigens (patient 4371). ( A ) The number and distribution of all tumor-specific mutations detected in the primary formalin-fixed, paraffin-embedded sample and three lung metastases resected at the National Cancer Institute. ( B ) TIL cultures from 24 tumor fragments (F1-24) were co-cultured with autologous DCs that were either pulsed with ten 25-mer peptide pools (PPs) or transfected with corresponding TMGs representing 144 mutations detected in metastatic tumors, as well as 13 mutations detected exclusively in primary tumors. Names of PPs and TMGs containing the latter are underlined. A TMG encoding point mutations from another patient (Irrel. TMG) and DMSO were used as negative controls for TMG and PP testing, respectively. Results of IFN-γ ELISpot are depicted. ( C ) F13 TIL were co-cultured with DCs that were pulsed with individual peptides contained in PP9; names of genes harboring mutations exclusive to the primary tumors are underlined. Numerical suffixes represent different transcripts of the same genes or, in the case of indels, different overlapping peptides. PMA/ionomycin (PMA) was used as a non-specific positive control. Results of IFN-γ ELISpot and flow cytometric analysis of 4-1BB expression on the cell surface are depicted together. ( D ) 4-1BB upregulation was analyzed using flow cytometry following an overnight co-culture of F13 TIL with autologous DCs that were pulsed either with PP9 (red dots) or DMSO (black dots). Data was gated on live CD8 + T cells. Cells in the gated area were subjected to single-cell sorting and TCR sequencing. ( E ) T cells from an unrelated healthy donor were transduced with the PP9-reactive TCR and co-cultured with the patient’s DCs cells that were pulsed with decreasing concentrations of BIRC6 P1505T (black squares) and corresponding wildtype peptide (empty squares). The 9-mer BIRC6 P1505T peptide was identified from the 25-mer sequence as the minimal epitope in a separate experiment (not shown). IFN-γ production was measured the following day using IFN-γ ELISA. DCs, dendritic cells; DMSO, dimethylsulfoxide; ELISpot, enzyme-linked immunosorbent spot assay; IFN, interferon; PMA, phorbol 12-myristate 13-acetate; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; TMG, tandem minigene.

Article Snippet: The plates were then processed as detailed previously, using biotinylated anti-human IFN-γ detection antibody (clone 7-B6-1) and streptavidin-ALP (both from Mabtech, Cincinnati, Ohio, USA).

Techniques: Immunopeptidomics, Formalin-fixed Paraffin-Embedded, Cell Culture, Transfection, Enzyme-linked Immunospot, Positive Control, Expressing, Flow Cytometry, Co-Culture Assay, FACS, Sequencing, Transduction, Enzyme-linked Immunosorbent Assay, ELISpot Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Utilization of primary tumor samples for cancer neoantigen discovery

doi: 10.1136/jitc-2024-010993

Figure Lengend Snippet: Example of screening PBL-derived memory T cells for recognition of cancer neoantigens (patient 4400). ( A ) Experimental design. ( B ) The number and distribution of all tumor-specific mutations detected in the primary FFPE sample and two lung metastases resected at the National Cancer Institute. ( C ) Memory T cells from PBL were separately stimulated with two PPs (PP-PRIM1 and PP-PRIM2) and a TMG (TMG-PRIM) representing mutations exclusive to primary tumors, along with a control PP and a TMG containing mutations previously recognized by autologous TIL from six tested patients (PP-CTRL and TMG-CTRL). Expanded CD8 + and CD4 + cells were then tested for recognition of B cells that were pulsed or electroporated with corresponding PPs or TMGs. Results of IFN-γ ELISpot are depicted. Sterile water alone (Mock) and DMSO were used as negative controls for TMG and PP testing, respectively. PMA/ionomycin (PMA) was used as a positive control. ( D ) Reactive cells from ( C ) were co-cultured with B cells pulsed with individual 25-mer peptides from the three PPs. Results of IFN-γ ELISpot and flow cytometric analysis of surface 4-1BB expression are depicted. ( E ) 4-1BB upregulation was analyzed using flow cytometry following an overnight co-culture of CD4-PP-PRIM1 cells with autologous B cells that were pulsed either with DDX5 R355C or DDX5 WT peptide. Peptides used in this and the following assays were high-performance liquid chromatography-purified. Data was gated on live CD4 + T cells. Cells in the shaded areas were subjected to single-cell sorting and TCR sequencing. ( F ) Results of TCR sequencing performed on the populations from ( E ). A single TCR, named 4400 TCR1 (CDR3b sequence CASTDPPVNEQYF), was highly enriched in 4-1BB-positive versus negative cells. ( G ) T cells from an unrelated healthy donor were transduced with TCR1 and were co-cultured with the patient’s DCs pulsed with serial dilutions of either DDX5 WT (WT) or DDX5 R355C peptide (MUT). IFN-γ concentration was measured in co-culture supernatants by ELISA. Squares represent average reads from duplicate co-culture wells; error bars represent SD. The same procedure was repeated with another healthy donor, yielding consistent results (not shown). Panel ( A ) was created with www.BioRender.com . DCs, dendritic cells; DMSO, dimethylsulfoxide; ELISpot, enzyme-linked immunosorbent spot assay; FFPE, formalin-fixed, paraffin-embedded; IFN, interferon; PBL, peripheral blood; PMA, phorbol 12-myristate 13-acetate; PP, peptide pool; TCR, T-cell receptor; TIL, tumor infiltrating lymphocyte; TMG, tandem minigene; WT, wild type.

Article Snippet: The plates were then processed as detailed previously, using biotinylated anti-human IFN-γ detection antibody (clone 7-B6-1) and streptavidin-ALP (both from Mabtech, Cincinnati, Ohio, USA).

Techniques: Derivative Assay, Immunopeptidomics, Control, Enzyme-linked Immunospot, Sterility, Positive Control, Cell Culture, Expressing, Flow Cytometry, Co-Culture Assay, High Performance Liquid Chromatography, Purification, FACS, Sequencing, Transduction, Concentration Assay, Enzyme-linked Immunosorbent Assay, ELISpot Assay, Formalin-fixed Paraffin-Embedded

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations

doi: 10.1007/s00262-024-03729-y

Figure Lengend Snippet: Identification of HLA-A*11:01-restricted T cell epitopes of mutant PI3Kα. a Stabilization analysis of HLA-A on TAP1-deficient K562 cells expressing HLA-A*11:01, which b median fluorescent intensity (MFI) from three independent experiments. c Naïve CD8 + T cells were isolated from HLA-A*11:01 positive healthy donors (HDs) and stimulated with pE542K and pH1047L peptides. After 2 rounds of stimulation, the response of T cells was determined by IFN-γ ELISpot after pulsed with wild type and mutant peptides. d Tetramer staining showing the percentage of gated tetramer + CD8 + T cells. e Experimental procedure for the isolation of pH1047L-specific T cells from HDs. CD8 + T cells were repetitively stimulated with pH1047L-pulsed DCs. pH1047L-specific T cells were sorted by flow cytometric analysis, and selected TCR clones were sequenced. TCR-T cells were engineered and characterized by cytokine production and cytotoxicity. IFN-γ, interferon gamma; ELISpot, enzyme-linked immunospot. N-P64: SARS-CoV-2 N protein 361-369 (KTFPPTEPK). Differences were tested by student’s t -test ***: p < 0.001; ****: p < 0.0001; ns: denotes not significant

Article Snippet: Subsequently, the plates were incubated with anti-human biotinylated IFN-γ detection antibody (Clone: 7-B6-1, 1 mg/ ml, Mabtech), followed by incubation with Streptavidin-AP (1:1000, Mabtech) and BCIP/NBT-plus substrate (Mabtech).

Techniques: Mutagenesis, Expressing, Isolation, Enzyme-linked Immunospot, Staining, Clone Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations

doi: 10.1007/s00262-024-03729-y

Figure Lengend Snippet: Validation of pH1047L-specific TCR functionality in primary T cells. Primary CD8 + T cells were transfected on day 2 post-stimulation with TRAC/TRBC knockout (dKO) or not (no KO). Direct comparison of a mTCRβ expression and b tetramer binding rate between primary CD8 + T cells dKO with no KO. Flow cytometric analysis of c the expression and d the percentage of CD137 + pH1047L-specific TCR-T cells after co-cultured with HLA-A*11:01 + K562 cells pulsed with wild type or mutant peptide. DMSO was used as control. e Flow cytometric analysis of dose-dependency of CD137 + cell proportions, pulsed with titrated pH1047L peptide. ELISA of f IFN-γ and g IL-2 release from pH1047L-specific TCR-T cells. Differences were tested by student’s t-test. *: p < 0.05; **: p < 0.01; ****: p < 0.0001

Article Snippet: Subsequently, the plates were incubated with anti-human biotinylated IFN-γ detection antibody (Clone: 7-B6-1, 1 mg/ ml, Mabtech), followed by incubation with Streptavidin-AP (1:1000, Mabtech) and BCIP/NBT-plus substrate (Mabtech).

Techniques: Biomarker Discovery, Transfection, Knock-Out, Comparison, Expressing, Binding Assay, Cell Culture, Mutagenesis, Control, Peptide ELISA

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations

doi: 10.1007/s00262-024-03729-y

Figure Lengend Snippet: pH1047L-specific TCR-T cells recognize endogenously present PIK3CA H1047L . ( a , b ). Flow cytometric analysis of the percentage of CD137 + dKO-CD8 + T cells following overnight co-culture with K562, MDA-MB-231 and SKOV3 cells expressing PIK3CA H1047L at an effector-to-target (E:T) ratio of 1:1. ELISA of c IFN-γ and d IL-2 release from activated pH1047L-specific TCR-T cells. e The pH1047L-specific TCR1-T cells were co-cultured with HCC827 cancer cells endogenously expressing HLA-A*11:01 overnight. The percentage of CD137 + TCR-T cells was analyzed via flow cytometry. An MHC blocker (clone W6/32) was added to block the TCR–pMHC interaction. Differences were tested by student’s t -test. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001

Article Snippet: Subsequently, the plates were incubated with anti-human biotinylated IFN-γ detection antibody (Clone: 7-B6-1, 1 mg/ ml, Mabtech), followed by incubation with Streptavidin-AP (1:1000, Mabtech) and BCIP/NBT-plus substrate (Mabtech).

Techniques: Co-Culture Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Clonal structure and the specificity of vaccine-induced T cell response to SARS-CoV-2 Spike protein

doi: 10.3389/fimmu.2024.1369436

Figure Lengend Snippet: Study workflow and T cell response in vaccination and placebo groups measured by ELISPOT and flow cytometry. (A) Workflow for processing samples received from clinical trial participants. Blood samples collected from each individual at each time point were assessed for T cell responses by ELISPOT and intracellular cytokine staining followed by flow cytometry. Workflows for T cell expansion followed by IFNγ + sorting, sequencing of total PBMC fractions, and peptide-specific expansion followed by IFNγ ELISA (enzyme-linked immunosorbent assay) and further activation assay (AIM assay) cell sorting are shown in red, green, and blue, respectively. Fractions of expanded cultures that were also collected for TCR β-sequencing are indicated by the gray arrow. Details of T cell expansion, cell sorting, and sequencing are described in Materials and Methods. C Negative control well, cultivated without Spike protein; +S - T cell expansions with added Spike protein. (B) IFNγ T cell response to Spike protein, measured by ELISPOT in vaccinated participants (n = 50) and PL (n = 19). (C, D) Intracellular production of IFNγ by CD4 + (D) and CD8 + (C) T cells after stimulation with Spike-derived peptide pools, measured by flow cytometry in vaccinated participants (n = 50) and placebo (PL, n = 19). (E) Spearman сorrelation between levels of IgG and T cell response, measured by flow cytometry and ELISPOT on the 14th day for all vaccinated participants. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. In B-D Mann-Whitney U-test was used for testing statistical significance. The median is plotted on the graphs from independent experiments.

Article Snippet: After the restimulation, 3 × 10 6 cells of the restimulated expansion were then transferred to warm medium (37°C) for 45 minutes to reinitiate IFNγ secretion, washed with ice-cold PBS containing 2 mM EDTA and 0.5% BSA, and stained with surface markers in 100 mcl: CD3-AF700 (0.6 mcl; clone OKT3; Sony; 2186700); CD4-FITC (0.6 mcl; clone RPA-T4; Sony; 2102690); CD8-PerCP/Cy5.5 (1.25 mcl; clone RPA-T8; Sony; 2105160)) and IFNγ detection antibody-APC (10 mcl; Miltenyi Biotec; 130-090-762) for 10 minutes at 4°C.

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Staining, Sequencing, Enzyme-linked Immunosorbent Assay, Activation Assay, FACS, Negative Control, Derivative Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Clonal structure and the specificity of vaccine-induced T cell response to SARS-CoV-2 Spike protein

doi: 10.3389/fimmu.2024.1369436

Figure Lengend Snippet: Identification of vaccine-induced Spike-specific clonotypes. A, B Cumulative frequency of Vac-clonotypes (orange), Ubiquitous (turquoise), or Unique (grey) in different samples of a representative donor (p1800) (A) or in all vaccinated donors ( B , only Vac and Ubiquitous clonotypes are shown). Spike-expansion 6M - expanded Spike-specific culture from samples collected 6 months post-vaccination. (C) A representative enrichment plot for donor p1800, showing frequencies of CDR3β sequences in the culture well, stimulated by Spike protein versus untreated control culture. Red dots represent clonotypes that are enriched in the culture well. (D) The comparison of cumulative frequencies of individuals Vac-clonotypes in different fractions. (E) Venn diagram for representative donor illustrating the overlapping clonotypes in enriched fractions of the T cell after the Spike-specific expansion on day 14, with sequenced IFNγ + T cell clonotypes. (F) Number of clonotypes in different groups as illustrated on E across donors. Undef - Undefined clonotypes. (G) Abundance of the Spike-specific clonotypes found in the total repertoire on day 14 for all donors. In (B) Mann-Whitney U-test was used for testing statistical significance. In D , F , G , one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used. The median is shown on the graphs of independent experiments.

Article Snippet: After the restimulation, 3 × 10 6 cells of the restimulated expansion were then transferred to warm medium (37°C) for 45 minutes to reinitiate IFNγ secretion, washed with ice-cold PBS containing 2 mM EDTA and 0.5% BSA, and stained with surface markers in 100 mcl: CD3-AF700 (0.6 mcl; clone OKT3; Sony; 2186700); CD4-FITC (0.6 mcl; clone RPA-T4; Sony; 2102690); CD8-PerCP/Cy5.5 (1.25 mcl; clone RPA-T8; Sony; 2105160)) and IFNγ detection antibody-APC (10 mcl; Miltenyi Biotec; 130-090-762) for 10 minutes at 4°C.

Techniques: Control, Comparison, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Clonal structure and the specificity of vaccine-induced T cell response to SARS-CoV-2 Spike protein

doi: 10.3389/fimmu.2024.1369436

Figure Lengend Snippet: Identified epitope-specific clonotypes had the same dynamics as Spike-specific clonotypes. (A) For each epitope number of IFNγ- responders (pink) within the total number of screened donors (grey) is depicted. IFNγ production was measured with Elisa after restimulation of expansion derived from PBMC collected on day 14 with peptides. (B) Number of identified epitope-specific unique TCR clonotypes per donor per each epitope. (C) Dynamic of cumulative frequency of all epitope-specific clonotypes, found in the total repertoires in 4 time points. (D) Dynamics of a number of all epitope-specific clonotypes found in the total repertoires in 4 time points. (E) Cumulative frequency of all epitope-specific clonotypes, found in Spike-specific expansion on the day 14 and 6 months after vaccination. (F) The number of all epitope-specific clonotypes found in Spike-specific expansion on the 14th day and 6 months. The interquartile range is plotted on the graph. Mann-Whitney U-test (in B , E , F ) and significant Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test (in C and D ) were used to test statistical significance.

Article Snippet: After the restimulation, 3 × 10 6 cells of the restimulated expansion were then transferred to warm medium (37°C) for 45 minutes to reinitiate IFNγ secretion, washed with ice-cold PBS containing 2 mM EDTA and 0.5% BSA, and stained with surface markers in 100 mcl: CD3-AF700 (0.6 mcl; clone OKT3; Sony; 2186700); CD4-FITC (0.6 mcl; clone RPA-T4; Sony; 2102690); CD8-PerCP/Cy5.5 (1.25 mcl; clone RPA-T8; Sony; 2105160)) and IFNγ detection antibody-APC (10 mcl; Miltenyi Biotec; 130-090-762) for 10 minutes at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, MANN-WHITNEY, Comparison