Ifn γ elisa kit

HepG2 and 293 T cell lines were obtained from the Cell Bank of Chinese Academy of Sciences. Top10 Escherichia coli Top10 were stored in our laboratory, and lentiviral vector pLVX-IRES-ZsGreen1 (Cat #. 632,187) was from Clontech. High-fidelity PCR kit (11,304,029), DNA purification kit (K310001), plasmid extraction kit (K210002), FBS (12,676,029), RPMI1640 (C11875500BT), DEME medium (C11995500BT), lipofectamine2000 (11,668,019) were all purchased from Invitrogen. PEG-it (LV810A-1) was purchased from System Biosciences, restriction enzyme (DL5000) and DNA Marker (DL2000) were from TaKaRa, lymphocyte separation medium (LTS1077) was from Tianjin TBD science, CD3 monoclonal antibody (300,314) was from BioLegend, CD3 Microbeads (130–050-101) were from Miltenyi, DELFIA cytotoxicity kit (AD0116) was from PerkinElmer, and IFN-γ ELISA kit (ab46025) was from Abcam. Oligonucleotide primers and rhodamine-labeled C-terminal polypeptides of GPC3 were synthesized by Shanghai Shine Gene Molecular Biotechnology Co., Ltd.

For cytokine assays, serum samples were collected from three chickens at 2, 4, 6, and 8 weeks post 1st immunization. The amounts of IFN-γ and IL-2 in the collected serum samples were determined using the IFN-γ ELISA Kit (Abcam Co. Ltd., Shanghai, China) and IL-2 ELISA Kit (Santa Cruz Co. Ltd., Santa Cruz, CA, USA), respectively. All the operations were performed according to the procedures described for the cytokine ELISA kits.

Heart tissues were harvested 24 hours after reperfusion and homogenate was prepared on ice using a glass homogenizer. Caspase 3 activity was detected using a Caspase 3 Activity Assay Kit (C1116, Beyotime, China) based on manufacturer instructions. To detect TNF-α, IFN-γ, IL-4 and IL-10 levels, the Mouse TNF alpha ELISA kit (ab208348, abcam, UK), IFN-γ ELISA Kit (ab100689, abcam, UK), IL-4 ELISA Kit (ab100710, abcam, UK) and IL-10 ELISA Kit (ab255729, abcam, UK), were used, respectively.

To investigate the level of IFN-γ production in mice, the IFN-γ ELISA kit from Abcam, Cambridge, UK (ab100689 IFNγ Mouse ELISA Kit) was used following the manufacturer’s instructions. In short, 100 μL of each recombinant mouse IFN-γ standard and sera from immunized mice were added into wells and incubated overnight at 4 °C with gentle shaking. A total of 100 μL of biotinylated IFN-γ detection antibody was added to each well after four washes and incubated for 1 h at room temperature. In total, 100 μL of HRP-Streptavidin solution was then added and incubated for 45 min at room temperature, followed by 100 μL of TMB One-Step Substrate Reagent to each well and incubated for 30 min at room temperature in the dark with gentle shaking. After incubation, 50 μL of stop solution was added to each well, and the results were read at 450 nm. A standard curve was generated and used to calculate the concentration of the test sera. A GraphPad prism was used to plot the graph of the IFN-γ concentration, and one-way ANOVA was used to analyze the difference between wtTMV and TMV(-A, -D, -F, -H, -L, and -S21P2).