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IFN-γ Enzyme-Linked Immunospot Assay Kit (ELISPOT)
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- Description
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Principle of ELISPOT Technology
Enzyme-linked immunospot assay: The term “enzyme-linked” carries the same meaning as in ELISA, referring to an immunoassay that relies on enzymatic reaction amplification. As with ELISA, the antibodies in ELISPOT are immobilized (coated) onto a solid support; however, whereas ELISA antibodies capture target proteins that are already present and uniformly distributed in the sample solution, ELISPOT antibodies capture proteins freshly secreted by cells in response to dynamic stimulation. In ELISA, the detection signal is manifested as a colored solution, whereas in ELISPOT it appears as colored spots deposited on the solid substrate. Both ELISPOT and ELISA are based on the sandwich immunoassay principle, but ELISPOT further incorporates cell culture techniques. While ELISA provides quantitative measurement of the concentration of a target protein in the sample solution, ELISPOT offers a dynamic assessment of the immune status of live cells within the sample.
ELISPOT (enzyme-linked immunospot assay) enables the quantitative measurement of cytokine secretion by individual cells. As one of the most sensitive molecular detection methods available today, ELISPOT exhibits a sensitivity 20 to 200 times greater than that of ELISA. Because the analyte of interest binds immediately to the capture antibody upon secretion, it is protected from degradation by binding proteins and proteases. These advantages—high sensitivity, high reliability, high-throughput capability, and preservation of immune function—make ELISPOT a practical tool for studying small populations of live cells. It finds broad applications in basic immunological research, vaccine development, and immunogenicity studies, among other fields. In particular, the IFN-γ ELISPOT assay has become the “gold standard” for evaluating cell-mediated immune responses induced by vaccines. By measuring IFN-γ levels, one can indirectly assess the activity of helper T cells, which is crucial for understanding immune responses.
Relying on its extensive proprietary cell bank and ELISPOT R&D technology service platform, Heyousheng has launched the IFN-γ ELISA Spot Assay Kit (Catalog No.: DCT01004-kit). This product utilizes a pre-coated ELISPOT assay kit, reducing the assay duration from 3 days to 2 days, significantly streamlining sterile-handling procedures, alleviating the workload of laboratory personnel, and minimizing the risk of contamination. As a result, researchers can perform complex ELISPOT assays with ease and high efficiency.
Product Overview of the IFN-γ ELISpot Assay Kit
Kit Components
Product Advantages
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End-to-end localization of the assay kit: the original research antibodies are sourced from in-house screening, and both the coating process and coating reagents have been fully developed and localized.
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Wide sample applicability: Suitable for the in vitro quantitative determination of interferon-γ levels in human serum, plasma, cell culture supernatants, or other relevant biological fluids.
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High specificity: Utilizes a sandwich ELISA format, making it suitable for the detection of complex samples;
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High sensitivity: detection sensitivity is consistently in the single-digit range;
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Convenient operation: the sample does not require any special purification treatment;
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Good reproducibility: Repeated experiments demonstrate low intra-assay and inter-assay coefficients of variation, indicating excellent stability.
Product Usage and Operation Procedure
Day 1: Seed cells, add stimulants or test samples, and perform culture procedures (with strict adherence to aseptic technique).
1. Activation of the pre-coated plate: Add 200 μL of serum-free medium or RPMI-1640 medium to each well, incubate at room temperature for 5–10 minutes, then carefully aspirate the liquid.
2. Add cell suspension: Add the cell suspension adjusted to the desired concentration to each experimental well.
3. Add the stimulant: add PHA diluted 1:1000 to the culture medium.
4. Add the samples to be tested: According to the experimental requirements, add the samples to be tested that have been diluted in culture medium. Use the same volume of culture medium for the blank/negative control.
5. Incubation: After all samples and stimulants have been added, securely seal the plate with its lid. Incubate in a 37°C, 5% CO2 incubator for 16–20 hours.
Day 2: Post-Culturing Assay Procedures
1. Lyse the cells: Discard the cells and culture medium from the wells. Add 200 μL/well of ice-cold deionized water, and incubate at 4°C for 5–10 minutes to induce hypotonic lysis.
2. Plate washing: Discard the liquid from the wells, then wash 5 times with 1× PBS at 200 μL per well. For the final wash, blot dry on absorbent paper.
3. Antibody incubation: Dilute the biotinylated antibody working solution in PBS containing 1.0% BSA, and add 100 μL per well to each experimental well. Incubate at 37°C for 1 hour.
4. Plate washing: Discard the liquid from the wells, then wash 5 times with 1× PBS at 200 μL per well. For the final wash, blot dry on absorbent paper.
5. Streptavidin-HRP Incubation: Add 100 μL per well of the diluted Streptavidin-HRP working solution to each assay well. Incubate at 37°C for 1 hour.
6. Plate washing: Discard the liquid from the wells, then wash 5 times with 1× PBS at 200 μL per well. For the final wash, blot dry on absorbent paper.
7. Color Development: Add 100 μL per well of the TMB chromogenic substrate working solution to each assay well. Incubate at room temperature in the dark for 5–15 minutes.
8. Stop the color development: Decant the liquid from the wells, remove the plate base, and wash the plate 3–5 times on both sides and on the base with deionized water to stop the color development. Place the plate in a cool, shaded area at room temperature and allow it to air-dry naturally before replacing the base.
9. Count spots on the ELISPOT plate, record various spot parameters, and perform statistical analysis.
IFN-γ ELISpot Assay Kit Verification Data Display
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Hycells’ ELISPOT assay results demonstrate significantly higher spot counts compared with imported competing products, reflecting superior assay precision and accuracy.
Reproducibility: PBMCs + PHA (1.0 μg/mL), incubated for 16 hours, with 11 replicates.
Imported competing product CV value
Hycells CV value
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The data in the table clearly show that Hycells’ CV value is significantly lower than that of its foreign competitors, indicating more stable product performance.
Competing ELISPOT
Kit plate reading diagram
Hycells ELISPOT
Kit plate reading diagram
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As clearly evident from the images, Hycells’ IFN-γ ELISpot assay kit exhibits weak non-specific binding and a cleaner background.
For more details on HeYouSheng Bio’s IFN-γ ELISPOT assay kits, please contact our sales representative. Scan the QR code below to apply for a free sample. Contact: Mr. Xie, 15201775322.
Applications are welcome.
Long-press the QR code to apply for a trial.
Heyou Sheng is about to launch several new ELISPOT assay kits, such as:
Human IL-2 and IL-6 ELISA Dot Blot Kit
Mouse IFN-γ, IL-2, and IL-6 ELISPOT Assay Kit!
Stay tuned. ！
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