ELISPOTアッセイ:IFN-γ分泌脾細胞の検出
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Q1: What is the ELISPOT assay and how does it detect immune responses?
ELISPOT (Enzyme-linked Immunospot) is a method that detects and quantifies immune cell activation by measuring secreted proteins like cytokines. The assay uses a microplate coated with capture antibodies that immediately immobilize proteins secreted by live cells, preventing dilution or degradation. This high-accuracy approach allows detection of single activated cells, even within small subpopulations, making it ideal for studying cellular-level immune responses.
Q2: How does the capture and detection process work in a cytokine-based ELISPOT assay?
A capture antibody specific to the target cytokine coats the microplate wells. When T-cells are stimulated and secrete cytokines, the capture antibody immediately immobilizes them. A biotinylated detection antibody then binds to the captured cytokine, followed by streptavidin-conjugated horseradish peroxidase. Adding the AEC substrate produces a dark precipitate marking each secretory cell as a visible spot.
Q3: Can ELISPOT be used to measure antibody-secreting B cells?
Yes, ELISPOT can quantify memory B-cells after infection or vaccination by analyzing their antibody production. In antibody-based ELISPOT, a specific antigen replaces the antibody in either the capture or detection step. This variation allows researchers to assess antigen-specific memory B-cell responses, which are important for rapid protective antibody responses following pathogen re-exposure.
Q4: What is the purpose of serial dilution in the ELISPOT protocol?
Serial dilution creates decreasing cell concentrations across the plate wells, allowing researchers to establish a dose-response relationship. By plating cells at multiple concentrations and observing how spot numbers decrease with lower cell density, scientists can determine the frequency of activated cells within the total population and validate assay linearity.
Q5: Why is blocking with cell culture medium an important step in ELISPOT?
After coating the plate with capture antibody, blocking with cell culture medium prevents non-specific binding of cells and proteins to the plate surface. This step ensures that only the target cytokine captured by the specific antibody contributes to spot formation, improving assay specificity and reducing background noise during the detection phase.
Q6: How are ELISPOT spots counted and what do they represent?
After the AEC substrate produces dark precipitate spots, an automated plate reader or microscope counts them. Each spot represents one activated secretory cell, and spot size correlates with the amount of protein secreted from that cell. Results are typically expressed as spot counts per number of cells plated, indicating the frequency of activated cells within the population.
Q7: What are the key advantages of ELISPOT compared to other immune detection methods?
ELISPOT offers simplicity with straightforward protocols requiring minimal technical expertise, exceptional sensitivity detecting single cells among 300,000 cells, and quantitative functionality data on immune responses. Unlike flow cytometry, ELISPOT requires very few cells and provides direct enumeration of protein-secreting cells, making it a gold standard for measuring antigen-specific T cell immunity in clinical trials.