A Window into the World of Immunity: Detailed Explanation of ELISpot Technolog
With the development of the biomedical field, a deeper understanding and precise measurement of immune system functions have become increasingly important. Enzyme-Linked ImmunoSpot (ELISpot) technology serves as an efficient tool, playing a critical role in this process. Through ELISpot, researchers can accurately quantify cytokines or other molecules secreted by individual immune cells (including but not limited to T lymphocytes, B lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, among others), providing new insights for the study of immune responses.
In this issue, we will lead you into the world of ELISpot. Let's uncover the secrets behind immune reactions together!
Table of Contents
1. What is ELISPOT?
2. How does ELISPOT Assay Work?
3. Advantages of ELISPOT Assay
4. Applications of ELISPOT Assay
5. ELISPOT vs ELISA
6. FluoroSpot: The Next-Generation ELISpot Assay
01 What is ELISPOT?
Enzyme Linked ImmunoSpot Assay (ELISpot) is an innovative cell detection technique based on the principles of ELISA. The history of ELISpot can be traced back to 1978 when Professor Don Mason at Oxford University developed a "spot on film" technique as an early attempt to detect antibody-secreting cells. Later, in 1983, ELISpot was independently advanced by scientists J.D. Sedgwick and C. Czerkinsky. They introduced the concept of enzyme-linked immunoassays and applied it to spot formation technology, making it more practical with enhanced sensitivity [1, 2].
02 How does ELISPOT Assay Work?
ELISpot can detect the ability of individual cells to secrete cytokines/antibodies, enabling dynamic monitoring and evaluation of the immune status of live cells in samples. As shown in Figure 1, cells are cultured in a PVDF plate coated with specific capture antibodies. The target proteins secreted by the cells are captured by these specific antibodies. After appropriate incubation, cells are removed and biotinylated antibodies are added. An enzymatic color reaction then forms precipitates known as "spots" [3, 4]. Each spot corresponds to one active cell secreting that cytokine, enabling a powerful and intuitive quantitative analysis of cellular immune response functions .
The specific steps of the ELISPOT test include:
1. Antibody Coating: A "capture antibody" with high affinity and specificity for the target cytokine is pre-coated at the bottom of wells on a microplate (covered with a PVDF membrane).
2. Cell Incubation and Stimulation: The cells to be tested (e.g., peripheral blood mononuclear cells, PBMCs) are seeded into the wells and stimulated with a specific antigen, peptide, or non-specific stimulants (such as PMA/Ionomycin). Immune cells that recognize the stimulus become activated and secrete the target cytokine.
3. Cytokine Capture: The cytokines secreted by the cells are immediately captured by the adjacent capture antibodies on the well membrane before they can diffuse into the culture medium.
4. Cell Removal and Detection: After a period of incubation, the cells are removed, and a biotin-labeled "detection antibody" is added. This antibody binds to the captured cytokines.
5. Signal Amplification and Color Development: A streptavidin-enzyme complex (such as SA-ALP) is then added to bind with biotin. Finally, a colorimetric substrate is introduced, and the enzyme catalyzes an insoluble colored precipitate at the antibody capture site, forming distinct "spots."
6. Spot Analysis: Each spot represents a cell that actively secreted the target cytokine at that location. An automated ELISpot reader counts the spots to determine the number of active cells per million cells (SFU, Spot Forming Units per million cells).
Fig.1 Diagram of the ELISpot Assay Principle
03 Advantages of ELISPOT Assay
ELISPOT technology is considered the "gold standard" for immune monitoring due to its unparalleled technical advantages:
l Ultra-High Sensitivity: It can detect extremely small numbers of antigen-specific reactive cells (as few as one in a million) from hundreds of thousands or even millions of cells, surpassing traditional methods like ELISA.
l Single-Cell Resolution: It directly reflects the frequency of cells secreting specific factors, providing true functional single-cell data rather than average concentrations of cell populations.
l Functional Activity Detection: It assesses the ability of cells to actually secrete functional proteins upon stimulation, directly revealing the effector functional status of the cells.
l Quantitative and Intuitive: Results are presented as spots, offering strong visualization and precise quantification through instrumental counting.
l Low Cell Requirement: This makes it ideal for clinical sample analysis, especially when dealing with samples that are limited in cell quantity (such as pediatric or biopsy samples).
04 Applications of ELISPOT Assay
The ultra-high sensitivity of ELISpot technology and its ability to dissect functional immune responses at the single-cell level make it an indispensable tool in many frontier areas of biomedical research and clinical applications.
Vaccine Development and Evaluation
ELISpot plays a critical role in vaccine development and evaluation, precisely measuring the intensity and breadth of T cell immune responses induced by vaccines and providing key data on vaccine efficacy.
Infectious Disease Research[5]
In infectious disease research (such as HIV, tuberculosis, COVID-19, hepatitis, etc.), ELISpot is a core tool for assessing the immune memory to specific pathogen antigens, effectively monitoring disease progression and treatment outcomes.
Cancer Immunotherapy[6]
In the rapidly advancing field of cancer immunotherapy, ELISpot is used to discover and evaluate tumor antigen-specific T cell responses. This guides the development and clinical efficacy monitoring of cancer vaccines and adoptive T cell therapies (such as TIL and TCR-T therapy).
Autoimmune and Allergic Diseases
Additionally, this technology plays an important role in studying the mechanisms of autoimmune and allergic diseases. By detecting reactive T cells to autoantigens or allergens, it provides a unique window into understanding disease mechanisms and developing new therapies.
From exploring basic immune mechanisms to preclinical research and clinical trial immune monitoring, ELISpot has become a solid bridge connecting fundamental immunology research and clinical translation.
Application Case Studies:
Fig.2 Incubate PBMCs (1×105 cells/well) for 20 hours with or without the stimulant Anti-CD3 mAb (500 ng/mL). The number of spots indicates the cells secreting human IFN gamma.
05 ELISPOT vs ELISA
In essence, both ELISpot and ELISA are based on the sandwich assay principle. However, ELISA provides a quantitative measurement of the target protein concentration in a sample solution, while ELISpot offers dynamic detection and evaluation of the immune status of live cells within the sample. The detailed comparison is as follows:
Table 1. Differences Between ELISpot and ELISA
|
Differences |
ELISpot |
ELISA |
|
Principle/Method |
Sandwich Assay Principle |
Direct Method/Indirect Method/Sandwich Assay/Competitive Assay |
|
Detection Target |
Real-time Secretion Frequency of Cytokine or Antibody-Producing Cells |
Pre-existing Target Protein Concentration in the Sample |
|
Sample Type |
PBMC, Splenocytes, Other Cells |
Cell Supernatant, Cell/Tissue Lysate, Serum/Plasma, Other Secretions such as Urine, Saliva, Milk, etc. |
|
Sample Plate |
The Bottom of the Well Plate Needs to be Coated with a PVDF Membrane |
Standard Microplate |
|
Sensitivity |
Higher, capable of precisely detecting the cytokine secretion from a single activated cell among hundreds of thousands or even millions of lymphocytes |
2 to 3 Orders of Magnitude Lower than ELISpot |
|
Detection Equipment |
ELISpot Reader |
Microplate Reader |
|
Enzymatic Reaction |
1. BCIP/NBT Staining Based on ALP 2. AEC or Precipitating TMB Staining Based on HRP |
1. TMB, OPD, or ABTS Color Development Based on HRP 2. pNPP Color Development Based on ALP |
|
Detection Results |
Semi-quantitative |
Qualitative, Quantitative, Semi-quantitative |
|
Detection Method |
Through a color development reaction, distinct spots appear at the locations where cytokines are secreted. Each spot represents one cell, allowing for the calculation of the frequency of cells secreting that cytokine. |
Through a color development reaction, the absorbance is measured and compared against a standard curve to calculate the total amount of soluble protein. |
Recommended Elabscience® ELISPOT Kits
Leveraging the unique advantages of ELISPOT technology, Elabscience® has developed a series of high-performance ELISpot kits designed to provide researchers worldwide with sensitive, stable, and ready-to-use solutions, aiding in more efficient unraveling of immune responses.
Core Advantages of Our Products:
² Compatible with Multiple Samples: Supports human/mouse PBMCs, splenocytes, TILs, and other cell samples.
² Multi-Species and Multiplex Coverage: Includes human and mouse species, targeting various key cytokines/factors such as IFN gamma, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, TNF-α, and others.
² Flexible Experiment Compatibility: Pre-coated and ready-to-use, with detachable strips for diverse needs.
² Superior Detection Performance: Intra- and inter-batch CV < 10%, with >99% data accuracy verified.
² Worry-Free Service: One-on-one service with a 24-hour response.
Quick Overview of Popular Products
• Human ELISPOT Kit
|
Cat. No. |
Product Name |
Size |
