Anti-IFN gamma Antibody Picoband®

Figure . Western blot analysis of IFN gamma using anti-IFN gamma antibody (RP1002).

Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.

Lane: Recombinant Human IFN gamma Protein 0.5ng

After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-IFN gamma antigen affinity purified polyclonal antibody (Catalog # RP1002) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for IFN gamma at approximately 17KD. The expected band size for IFN gamma is at 17KD.

Percentages of NK (CD3 − CD56 + ) cells ( a – d , m ) in PBMC, NK cell group 2D receptor (NKG2D) + ( e – h , n ) and IFN-γ + ( i – l , o ) NK cells within total NK cells. HCs, healthy controls; IT, chronic HBV carriers; IA, CHB patients; IH, HBV-ACLF patients. Student-Newman-Keuls q test following one-way ANOVA were used for comparing percentages of NK cells, and Nemenyi test following Kruskal-Wallis H test were used for comparing percentages of NKG2D + and IFN-γ + NK cells between two groups. Compared with HCs group, ◆ ◆ ◆ P < 0.01; Compared with IT group, ★ ★ ★ P < 0.01, ★ ★ P < 0.05; Compared with IA group, ▲▲▲ P < 0.01.
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Intrahepatic expression of NKG2D mRNA ( a ) and IFN-γ mRNA ( b ). HCs, healthy controls (the relative expression were defined as 1.00); IT, chronic HBV carriers; IA, CHB patients; IH, HBV-ACLF. Nemenyi test following Kruskal-Wallis H test were used for comparing mRNA expressions of NKG2D and IFN-γ between two groups. Compared with HCs group, ◆ ◆ ◆ P < 0.01; Compared with IT group, ★ ★ ★ P < 0.01; Compared with IA group, ▲▲▲ P < 0.01.
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Representative graphs of intrahepatic IFN-γ + cells (A, 200×) and NKG2D + cells (B, 200×) expressions. ( a ) HCs, healthy controls, ( b ) IT, chronic HBV carriers, ( c ) IA, CHB patients, ( d ) IH, HBV-ACLF patients. ( e ) Collective analysis of results from all 4 groups. IFN-γ + cells were distributed mainly in the inflammatory sites and periportal areas that were infiltrated with lymphocytes. NKG2D + cells were mainly distributed in Disse’s space of hepatic lobule in HCs and chronic HBV carriers, and mainly in periportal areas in CHB and HBV-ACLF group. Nemenyi test following Kruskal-Wallis H test were used for comparing intrahepatic IFN-γ + and NKG2D + cells expressions between two groups. Compared with HCs group, ◆ ◆ ◆ P < 0.01; Compared with IT group, ★ ★ ★ P < 0.01; Compared with IA group, ▲▲▲ P < 0.01.
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Analysis of NKG2D and IFN-γ mRNA levels in co-cultured cells (NK + HepG2/HBV-HepG2) of Group A ( a , NK + HepG2) and Group B ( b , NK + HBV-HepG2). Nemenyi test following Kruskal-Wallis H test were used for comparing mRNA expressions of NKG2D and IFN-γ between two compared groups. Compared with Control group (NK + HepG2 or NK + HBV-HepG2), ◆ ◆ ◆ P < 0.01; Compared with Control + NKG2D mAb group, ★ ★ ★ P < 0.01; Compared with Control + IFN-α group, ▲▲▲ P < 0.01; Analysis of the levels of NKG2D and IFN-γ protein in different groups ( c ). The density of NKG2D and IFN-γ protein was the highest in group of NK + HBV-HepG2 + IFN-α, followed by group of NK + HBV-HepG2 + IFN-α + NKG2DmAb and group of NK + HBV-HepG2 + NKG2DmAb.
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Serum Levels of IFN-γ, TNF-α, perforin and granzyme B in different clinical stages of chronic HBV-infected patients. HCs, healthy controls; IT, chronic HBV carriers; IA, CHB patients; IH, HBV-ACLF. Nemenyi test following Kruskal-Wallis H test were used for comparing IFN-γ, TNF-α, perforin and granzyme B levels between two compared groups. Compared with HCs group, ◆ ◆ ◆ P < 0.01, ◆ ◆ P < 0.05; Compared with IT group, ★ ★ ★ P < 0.01, ★ ★ P < 0.05; Compared with IA group, ▲▲▲ P < 0.01.
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Levels of TNF-α ( a ) and IFN-γ ( b ) in the supernatants with or without co-cultured NK cells. Student-Newman-Keuls q test following one-way ANOVA were used for comparing IFN-γ, TNF-α, perforin and granzyme B levels between two compared groups. Compare with HepG2 cells group, ◆ ◆ ◆ P < 0.01; Compare with HBV-HepG2 cells group, ◇ ◇ ◇ P < 0.01; Compare with NK cells group, ★ ★ ★ P < 0.01. Levels of perforin ( c ) and granzyme B ( d ) in the supernatants in the presence IFN-α with or without NKG2D blocking. Compare with control group, ▲▲▲ P < 0.01; Compare with + IFN-α group, ■■■ P < 0.01.
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Serum levels of IP-10, IFN-γ, IL-4, and TGF-β1 as well as the IFN-γ/IL-4 ratio in chronic hepatitis B patients with or without fibrosis. The levels of serum IP-10, IFN-γ, IL-4, and TGF-β1 in CHB patients with or without liver fibrosis were determined by ELISA, and the IFN-γ/IL-4 ratio was calculated. ♦♦♦ Differs from controls (the F0 group), P < 0.05; ★ ★ ★ differs from mild or minimal fibrosis (the F1–2 group), P < 0.05; ▴ ▴ ▴ differs from moderate fibrosis (the F3–4 group), P < 0.05. ( a ) IP-10; ( b ) IFN-γ; ( c ) IL-4; ( d ) TGF-β1; ( e ) the IFN-γ/IL-4 ratio.
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Statistical analysis of the correlation between the serum IP-10 level or the IFN-γ/IL-4 ratio with liver fibrosis among chronic hepatitis B patients. Spearman’s correlation analysis of the association between ( a ) IP-10; ( b ) IFN-γ; ( c ) the IFN-γ/IL-4 ratio and TGF-β1. Spearman’s correlation analysis of the association between serum ( d ) IFN-γ; ( e ) IL-4; ( f ) the IFN-γ/IL-4 ratio and IP-10.
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ROC curve analysis for evaluating the sensitivity and specificity of the IP-10 level, IFN-γ/IL-4 ratio, or their combination to predict significant fibrosis among CHB patients. ( a ) ROC curve analysis for serum IP-10 (with the cut-of f value of 300 pg/mL), the serum IFN-γ/IL-4 ratio (with the cut off value of 1.8), and the combination of IP-10 and the IFN-γ/IL-4 ratio; ( b ) Specificity and sensitivity for IP-10, the IFN-γ/IL-4 ratio, and their combination to predict significant liver fibrosis among patients with CHB.
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Intrahepatic mRNA levels of IP-10, IFN-γ, and IL-4 in chronic hepatitis B patients with or without fibrosis. Real-time qRT-PCR was conducted to quantify the mRNA levels of intrahepatic IP-10, IFN-γ, and IL-4 in the CHB patients without or with fibrosis as described in the Materials and Methods section. The relative mRNA levels of intrahepatic IP-10, IFN-γ, and IL-4 were calculated by comparative Ct analysis after normalization for the quantity of GAPDH in the same samples and were represented as 2 - △ △ Ct values for controls (the F0 group), which were set equal 1. ♦♦♦ Differs from controls (the F0 group), P < 0.05; ★ ★ ★ differs from mild or minimal fibrosis (the F1–2 group), P < 0.05; ▴ ▴ ▴ differs from moderate fibrosis (the F3–4 group), P < 0.05. (a) IP-10; (b) IFN-γ; (c) IL-4.
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Intrahepatic protein expression of IP-10, IFN-γ, IL-4, TGF-β1, and α-SMA as well as the IFN-γ/IL-4 ratio in chronic hepatitis B patients with or without fibrosis. The protein expression of intrahepatic (a, b, c, and d) IP-10, (e, f, g, and h) IFN-γ, and (i, g, k, and l) IL-4. In addition, the protein levels of intrahepatic IP-10, IFN-γ, IL-4, TGF-β1, and α-SMA were quantified based on the value of integrated optical density (IOD) and represented as histograms, from which the IFN-γ/IL-4 ratio was calculated. ♦♦♦ Differs from controls (the F0 group), P < 0.05; ★ ★ ★ differs from mild or minimal fibrosis (the F1–2 group), P < 0.05; ▴ ▴ ▴ differs from moderate fibrosis (the F3–4 group), P < 0.05.
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IHC analysis of IFN gamma using anti-IFN gamma antibody (RP1002).
IFN gamma was detected in a paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 μg/ml rabbit anti-IFN gamma Antibody (RP1002) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.