elisa kit  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: Inflammation and the NKG2D system in H. pylori infection and gastric cancer. Stomach biopsies from healthy controls (Healthy), H. pylori gastritis cases (HpG) and stomach adenocarcinoma cases (Cancer) were immune-phenotyped. (A, B) H&E and IHC staining of leucocytes (CD45), CTLs (CD8) and NK cells (CD56, NKp46). (A) Representative images, scale bars: 100 µm. (B) Quantification of positive cells per mm 2 of tissue n=10-30 per group, the data do not follow a normal distribution, median ± interquartile range, Kruskal-Wallis test and Dunn’s test. (C) qPCR analysis of NKG2D , MICA and MICB , n=8-16 per group. The data passed Shapiro-Wilk normality test, mean ± SD, one-way ANOVA and Tukey’s test. *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001. ns, not significant.

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Infection, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: H. pylori -dependent modulation of NKG2D-L expression and soluble release via proteolytic shedding. MKN28 cells were challenged with C. acnes , butyrate, and H. pylori WT for 24 and 48 h (A) MICA and MICB mRNA levels were determined by qPCR. (B) Soluble MICA and MICB levels in cell culture supernatants were determined by ELISA. Experiments were performed three times. Mean ± SD, one-way ANOVA and Tukey’s test (ns, not significant, *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001). (C) MKN28 and AGS cells were challenged with H. pylori WT or 2 mM butyrate for 48 h Simultaneously, cells were treated with either 10 µmol/L batimastat dissolved in DMSO (= batimastat +) or with DMSO alone as solvent control (= batimastat –). Soluble MICA and MICB proteins in cell culture supernatants were determined by ELISA. Experiments were performed three times. Mean ± SD, t-test of batimastat – vs. batimastat +, for each treatment group (ns, not significant, *P <0.05; ***P <0.001; ****P <0.0001).

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Solvent, Control

Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: H. pylori virulence factor-dependent modulation of NKG2D-L expression and soluble release. MKN28 cells were challenged with H. pylori WT and isogenic mutants ΔcagA , ΔcagL and ΔvacA for 24 and 48 h (A) MICA and MICB mRNA levels were determined by qPCR. (B) Soluble MICA and MICB levels in cell culture supernatants were determined by ELISA. (C–E) AGS-MICA cells were challenged with H. pylori WT and isogenic mutants ΔcagA , ΔcagL and ΔvacA for 24 h (C) MICA mRNA levels were determined by qPCR. (D) MICA protein in cell lysates was determined by Western Blot. (E) Soluble MICA protein in cell culture supernatants was determined by ELISA. Experiments were performed three times. Mean ± SD, one-way ANOVA and Tukey’s test (ns, not significant, *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001).

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection

doi: 10.3389/fimmu.2024.1282680

Figure Lengend Snippet: Scheme highlighting the NKG2D system modulation by H. pylori . Healthy epithelia store MICA and MICB proteins intracellularly. Upon stress, MICA/B are expressed at the cell surface and bound by the immunoreceptor NKG2D, which activates immune attack by NKG2D-harboring effector cells. During H. pylori infection, virulence factors CagA and VacA modify the expression and proteolytic shedding of MICA/B. Soluble MICA/B proteins lead to immune evasion by binding NKG2D, which results in internalization and downregulation of NKG2D and a suppression of lymphocyte cytotoxicity. Hypofunctional NK and cytotoxic T cells in the stomach lamina propria could allow transformed cells to escape immune surveillance and facilitate tumor development. Scheme drawn with BioRender ( https://biorender.com ).

Article Snippet: ELISA was performed using the Human MICA Duoset ELISA kit (R&D) and the Human MICB Duoset ELISA kit (R&D), according to the manufacturers protocol.

Techniques: Infection, Expressing, Binding Assay, Transformation Assay

Journal: Med (New York, N.Y.)

Article Title: A chimeric antigen receptor uniquely recognizing MICA/B stress proteins provides an effective approach to target solid tumors.

doi: 10.1016/j.medj.2023.04.004

Figure Lengend Snippet: Figure 1. 3MICA/B CAR targets the conserved alpha-3 domains of MICA/B stress ligands and is resistant to binding competition by shed MICA/B A novel MICA/B-targeting synthetic receptor capable of high-affinity pan-allelic binding, surface MICA/B density augmentation, and resistance to inhibition via shed MICA and MICB peptides. (A) Domain-specific amino acid sequence similarity was scored across the 21 most prevalent MICA and MICB alleles using the BLOSUM method highlighting greater sequence conservation within the membrane-proximal a3 domain relative to the membrane-distal a1-2 domains. (B) Pan-allelic reactivity of 3MICA/B mAb, an a1-2 MICA/B-reactive mAb (6D4), and NKG2D:Fc fusion protein was assessed across an extended panel of 28 prevalent MICA alleles. (C) MM1-R cells, pre-treated with trypsin to cleave baseline surface-expressed MICA/B ligands, were incubated with 3MICA/B mAb, or relevant isotype, and surface MICA/B expression kinetics was measured using a commercial mAb (6D4) over a 96-h time course. In parallel, MM1.R cells were incubated

Article Snippet: The concentration of shed MICA/B was measured using a human MICA ELISA (Ab59569, Abcam) or human MICB DuoSet ELISA kit (DY1599, R&D Systems), following the manufacture’s recommendation.

Techniques: Binding Assay, Inhibition, Sequencing, Membrane, Incubation, Expressing