In order to verify whether the vaccine polypeptide can stimulate helper T cell immunity, the ELISA experiment was conducted to determine IFN-γ. In 96-well plates with RPMI1640 medium containing 10% FCS, we co-culture the synthesized vaccine polypeptide (5 μg/ml) with PBMCs (2 × 105) of CE and HC groups. After culturing at 37°C for 20h under 5% CO2, the ELISA plate was coated with the specific anti-Human IFN-γ capturing antibody in coating buffer overnight at 4-8°C. The plate was blocked with phosphate buffer saline (PBS) containing 5% nonfat milk for at 37°C for 3 h and washed by PBS containing 0.05% Tween 20. Then, the collected cell culture supernatant was added and the plate was incubated at 37°C for 1 h. After washing the ELISA plate with PBS containing 0.05% Tween 20, the specific anti-IFN-γ secondary antibody labelled with HRP was added and incubated at 37°C for 1 h. Last, after washing again and the TMB solution (sigma) was added for color development. The OD450nm value was measured using a Microplate reader and the levels of IFN-γ was reflected by standard curve. The Human IFN-γ ELISA Kit (70-EK180-48, MultiSciences, Hangzhou, China) was adopted for the ELISA experiment.
Human ifn γ elisa kit
Manufactured by MultiSciences Biotech
Sourced in China
The Human IFN-γ ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human interferon-gamma (IFN-γ) levels in biological samples such as cell culture supernatants, plasma, and serum. The kit utilizes a specific antibody coated on a 96-well plate to capture the target analyte, and a detection antibody for quantification.
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3 protocols using human ifn γ elisa kit
1
Vaccine Polypeptide T Cell Immunity Verification
2
Quantifying Cytokine Production by ELISA
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To measure the levels of TNF-α and IFN-γ produced by cells, the supernatants were collected and the concentrations of TNF-α and IFN-γ were measured by the Human TNF-α ELISA Kit (MultiSciences, Hangzhou, China) and Human IFN-γ ELISA Kit (MultiSciences) according to the manufacturer’s guidelines. Optical densities were determined using a microplate reader (Bio-Tek Elx 800; Bio-Tek Instruments) at 450 nm.
3
Blocking IFN-γ Induction by N-IgY
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The project of this in vitro experiment was approved by the ethics committee of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2020–355). Samples of anticoagulated peripheral blood were donated by healthy human donors (n = 7). Basic characteristics of the donors were listed in Tab. S1. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll reagent (Haoyang, China) under density gradient centrifugation according to the operating manual. The induction of interferon-γ (IFN-γ) was then measured to identify the blocking ability of N-IgY.
The isolated PBMCs were incubated in RPMI 1640 culture media (Corning, USA) supplemented with 10% of FBS (Corning, USA). The cells were seeded (5 × 105 cells/500 μL/well) in 24-well plate (Corning, USA) and cultured at 37 °C in a 5% humidified CO2 atmosphere.
Cells were treated with NP (20 μg/mL) alone (groupN ), or NP premixed with N-IgY (1 mg/mL) for 30 min (group N + IgY ) for 18 h. Those treated with PBS were set as control (group Con ). After centrifugation at 4000 rpm and 4 °C for 3 min, the separated supernatant was immediately frozen at −20 °C until detected. Human IFN-γ ELISA kit (MULTI SCIENCES, China) was used to detect the secretion of IFN-γ in the culture supernatant according to the operating manual.
The isolated PBMCs were incubated in RPMI 1640 culture media (Corning, USA) supplemented with 10% of FBS (Corning, USA). The cells were seeded (5 × 105 cells/500 μL/well) in 24-well plate (Corning, USA) and cultured at 37 °C in a 5% humidified CO2 atmosphere.
Cells were treated with NP (20 μg/mL) alone (group
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