Lymphocytes stimulated by antigen locally produce cytokines, which are captured by specific monoclonal antibodies (precoated) on PVDF membrane at the bottom of ELISPOT plate. After the cells are removed, the captured cytokines are bound to monoclonal antibodies labeled with biotin, and then to avidin labeled with alkaline phosphatase or horseradish peroxidase. Purple or reddish brown spots appeared on PVDF membrane after adding substrate, which indicated that cells produced cytokines. Spots are automatically counted and analyzed by ELISPOT reader.
German AID company has been focusing on the research and development of enzyme-linked immunospot analyzer for many years. Since 1990s, AID has provided a large number of enzyme-linked immunosorbent assay (ELISA) dot readers for scientific research, clinical and pharmaceutical laboratories all over the world. Its application in various fields can be seen everywhere and it has become a brand recognized by researchers all over the world.
Using AID enzyme-linked immunospot analyzer to automatically collect, count and analyze the spot images of each hole on ELISPOT plate, not only can the number of spots be automatically obtained and the secretory activity value of cytokines be given, but also negative and positive results can be directly obtained according to the preset interpretation rules, and the results can be output to JPG, EXCEL, TXT, PPT, etc. It can also be connected to the LMS system of the laboratory for data management and output, thus becoming a reliable ELISPOT analysis tool.
In addition, the traditional ELISPOT also has limitations, and it lacks the simultaneous detection of various cytokines produced by T cells. In order to overcome this problem, based on fluorescent labeling, a fluorescent immune spot analysis (fluorescent spot) was developed. This new fluorescent western blot analysis technique can be used to detect lymphocytes expressing various cytokines at the single cell level, such as T cells expressing IFN-γ (green fluorescent label) and IL-2 (red fluorescent label).
Germany's AIDSPOT Reader first realized the application of an enzyme-linked immunospot analyzer to simultaneously analyze the fluorescent spots of traditional ELISPOT and fluorescent immunospot: AIDSPOT Reader collected one image of green fluorescent spots (FITC-labeled IFN-γ) and one image of red fluorescent spots (Cy3-labeled IL-2) on each hole of ELISPOT, and fused the two images by fluorescence image superposition technology patented by FluoroAID, which ensured the accuracy of * * staining spots counting.
The AID enzyme-linked immunospot analyzer adopts the products of Swedish Mabtech, a world-famous ELISPOT reagent manufacturer, and can be used to detect the T/B cell immune response of cattle, sheep, horses, pigs and other species, as well as the veterinary field. Besides IFN-γ and interleukin series, there are TNF-α, perforin, granzyme B and MIP- 1β.
① people: apoE, GM-CSF, granzyme B, IFN-α (Subt.2, 10, 14, 17), IFN-α(pan-specific), IFN-γ, IL-/kloc-0. IL- 12/-23(p40), IL- 12(p70), 13, 17A, 2 1, 23, 3 1, MIP.
② Monkey: IFN-γ, IL-2, IL-17;
③ Mice: GM-CSF, IFN-α, IL-4, 5, 6, IL- 12/-23(p40), IL- 13, IL-2 1, MIP- 1β, etc.
④ Others: Lyme disease spot, EBV-Spot, CMV-Spot, Trans Spot kit;
⑤ For more T cell /B cell ELISPOT and fluorescent spot kits, please consult.
High sensitivity: 200,000-300,000 1 positive cells detection rate;
Intuitive and reliable: single cell level detection, a positive cell has a spot, the number of spots represents the number of cytokines secreted by the cell, and the size of spots represents the level of cytokines secreted by the cell;
Qualcomm: One ELISPOT can be used for 96 tests, and researchers can detect hundreds of samples every day. Only a few cells, such as 45 ml of blood, are enough to test the reactions of 600 different antigens/peptides.
Functional detection of living cells: the detected cytokines do not exist, but are secreted by external stimuli during the detection process;
Clinical trials are applicable: cells that have been cryopreserved and revived do not lose their immune function, which is very important for clinical trials. Samples before and after treatment can be detected in parallel, and the results can be repeated; If the experiment involves multiple clinical institutions, blood samples can be frozen and transported to the core laboratory for testing.
ELISPOT is not only used as a scientific research tool, but also used for clinical diagnosis. Recently, the first diagnostic platform (T-Spot. TB) approved for listing by SFDA according to ELISPOT method. This test method for detecting tuberculosis infection is to detect specific T cell immune response by measuring IFN-γ secretion stimulated by tuberculosis specific antigen. It can replace the traditional skin test, and greatly improve the sensitivity and specificity, and is not affected by BCG vaccination. ELISPOT can also be used for anti-infective immunity research and diagnosis of other infectious diseases, such as hepatitis B, hepatitis C, EB virus, cytomegalovirus and Lyme disease.
ELISPOT method is an internationally recognized standard tool for detecting and evaluating the cellular immunity level of vaccines. Internationally, IFN-γ ELISPOT method is widely used to monitor immune response in HIV vaccine research, and ELISPOT method is used to evaluate the function of T cells in a large number of experiments to monitor new HIV vaccine candidates in the international AIDS vaccine action plan. ELISPOT method defines the effect of vaccine by measuring its ability to induce strong T cell response. ELISPOT method can be used to screen and determine the specific T cell epitopes of antigens in vaccine development and design. This information can also be used to evaluate appropriate immunization strategies and immunization routes.
China Institute for the Control of Pharmaceutical and Biological Products pointed out in the Technical Guidelines for Pre-clinical Research of Preventive DNA Vaccines and the Principles of Technical Indicators for the Prevention of Live Vaccine Preparations with Viruses that ELISPOT method is an effective method to detect and evaluate the cellular immune titer of vaccines. ELISPOT technology, as a more sensitive method to detect the specific secretion of T/B cells, is particularly important to detect a small number of antigen-specific memory T cells, and has been widely used in the development and evaluation of preventive vaccines and therapeutic vaccines, such as AIDS vaccines, hepatitis B vaccines, influenza vaccines and various tumor vaccines.
According to the different cytokines detected, ELISPOT can be used to distinguish different activated T cell subsets. For example, Th 1 cells mainly secrete IFN-γ, IL-2 and TNF-α, while other cytokines such as IL-4, IL-5 and IL- 13 are generally secreted by Th2 cells. A hot spot in allergic research is the determination of Th2 cytokines by ELISPOT, because Th2 reaction is the main reaction of allergic reaction, but it usually occurs at a low frequency and requires a highly sensitive method to successfully detect it.
Monitoring T cell immune response in immunotherapy for specific allergic reactions; In the application of cell-mediated in vitro detection and diagnosis of contact allergy, ELISPOT method has attracted more and more attention of allergy workers. Because factors such as IL-4 (the main cytokine of TH2 type reaction) are always difficult to be detected by other methods (such as flow cytometry and ELISA), it is particularly important to study allergen-specific immune response by using highly sensitive ELISPOT method.
ELISPOT technology is also widely used in the diagnosis and etiological research of autoimmune diseases, such as the etiological analysis of diseases such as type I diabetes, multiple sclerosis, myasthenia gravis and systemic lupus erythematosus, disease prediction, early diagnosis, guiding treatment and prognosis analysis.
Although ELISPOT is mainly used for T and B cell analysis, it is also applicable to any experiment that wants to study protein secretion at the single cell level. This method is especially suitable for those experiments where the number of effector cells is critical or of biological significance, such as the quantitative analysis of monocytes secreting IL-6 and IL- 1β after LPS stimulation of PBMC; In addition, it is especially suitable for studying secretory cells, such as plasma cell-like dendritic cells, which are particularly rare in blood, but IFN-α secreted by them after activation can be easily detected by ELISPOT.
take for example
Drug screening: screening of compounds and immune response of drugs;
Tumor immunity research: it has been used to analyze and evaluate the vaccine test of inducing tumor-specific T cells in tumor patients. The detection of specific cellular immune response level before and after treatment can effectively evaluate the therapeutic effect and provide important reference information for treatment and medication.
Transplantation immunology research: the prediction of transplant rejection.
Since its launch, more than 65,438+0,500 laboratories around the world have used AID's enzyme-linked immunospot analyzer. The hardware and software of AID ELISPOT reader fully meet the EU ce certification, FDA 21CFR Part11specifications and GMP/GLP standards.
Speckle image acquisition and counting
Fully automatic reading board, which automatically collects the image of each hole; Automatically calculate the number of spots and spot related information, such as size, area, optical density, etc. Built-in different factor parameter settings, directly call; CountAID function is helpful to optimize reading parameters and has intelligent parameter memory function.
result analysis
Directly judge the results of yin-yang test; The diagnostic module AIDiagnostic is suitable for interpreting the results of ELISPOT experiment for clinical diagnosis, and outputting speckle images and result reports of each patient respectively; The distribution of spots in each well was statistically analyzed, and the immune response and cytokine secretion were compared quantitatively.
Data output and quality control
Single-hole image, arbitrary-hole image and whole-board image can be output; A variety of save formats, Excel, Txt, PPT, etc. Output data can be customized to include point number, SFU, optical density intensity, activity, point distribution map, etc. Comprises a quality control module, which automatically records the operation history and conforms to GLP specifications.
Fluorescent two-color ELISPOT analysis -Fluorospot, using patented fluorescent image superposition technology, ensures accurate identification of polychromatic factor * * * secretion spots and avoids misjudgment caused by uneven secretion of different cytokines in enzyme-labeled two-color detection. 4-8-bit filter wheel, fully automatic switching, at least FITC and Cy3 fluorescent filters are standard, other wavelengths can be added.
Fully automatic translation
ISpot images the fluorescent spots of various colors in each well on the plate separately, such as detecting the secretion of IFN-γ labeled by FITC and IL-2 labeled by Cy3. The software superimposes different fluorescent images by itself to accurately identify the secretion of * * * (IFN-γ&: IL-2) cells. The whole process is fully automatic without manual intervention.
Stable performance, good compatibility and expansibility.
High-energy excitation ensures weak fluorescence signal detection, narrow-band filter ensures specific signal detection, and multi-color expansion only needs to be equipped with filters.
technical progress
1996, the world's first commercially available enzyme-linked spot analyzer, invented the patent of "ELISPOT method for detecting blood cell activity"; The only patent PCT/EP 2008/009112 ‐ fluoroacid is a fluorescent multicolor ELISPOT detection system based on fluorescent filter.
Academic publishing authority
From the launch of fluorescent products in 2007 to now, 80% of SCI documents on the application of fluorescent ELISPOT have been published by AID, which is recognized as a good choice for fluorescence detection.
High international recognition
Developed in close cooperation with Oxford Immunology Laboratory of Oxford University, it is a special tool for T-Spot diagnosis of tuberculosis. International AIDS Vaccine Action Organization (IAVI) designated brand; World-famous brand of ELISPOT kit development laboratory (such as Swedish Mabtech).
Please contact us 1352 1452266 to apply for the trial of the AID enzyme-linked immunospot analyzer.
—— German first-class aid agent in China, Zeping, Beijing.