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In the field of immunology, disease and vaccine research are not limited to humoral immune responses (B cell immunity), and cell-mediated immune response (CMI) is also of concern, while T cells are in CMI. Play a key role. In the study of immune response mechanism, enzyme-linked immunosorbent assay (ELISA) was used to detect free cytokines (CK) or antibodies in body fluids. However, due to the different half-mourning period of free circulating antibodies or CK, it is continuously in body fluids. It is metabolized or binds to target organs, but does not accurately reflect the levels of antibodies and CK in the body. In the 1980s, foreign researchers established a solid-phase enzyme-linked immunospot technique (ELISPOT) for the detection of specific antibody-secreting cells and CK-secreting cells in vitro based on the basic principles of ELISA. As a novel immunoenzyme technology, the enzyme linked immunospot assay (ELISPOT) is a cellular immunology that detects secretory antibody cells (ASC) or secreted cytokine (CK) cells from a single cell level. Detection Technology. Because of its high specificity and sensitivity, the method is easy to operate, and the cost is relatively low compared with flow cytometry. It has been widely used in the secretion of CK cell assay or ASC assay to explore the pathogenesis of autoimmune diseases. Significance.
The ELISPOT method is derived from ELISA and breaks through the traditional ELISA method, which is an extension and new development of quantitative ELISA technology. Both are cytokines or other soluble proteins produced by the cells, the biggest difference being:
(1) The ELISA measures the absorbance on a microplate reader by color reaction, and compares with the standard curve to obtain the total amount of soluble protein.
(2) ELISPOT also displays clear and discernible spots at the corresponding positions where the cells secrete this soluble protein through the color reaction. The spots can be counted directly under the microscope or the spots are counted by the ELISPOT analysis system. One spot represents One cell is used to calculate the frequency of cells secreting the protein. (Some studies not only measure the amount of cytokine production, but also the frequency of cells that secrete this cytokine)
Because it is a single-cell level test, ELISPOT is more sensitive than ELISA and limiting dilution methods, and can detect a cell secreting this protein from 200,000-300,000 cells. The capture antibody is a high affinity, high specificity, low endotoxin monoclonal antibody, and does not affect the secretion of cytokines by activated cells when the investigator activates the cells with a stimulant.
The following sections will introduce the development, principles, specific experimental operations, technical advantages and applications.
Development of ELISPOT technology
(1) The past of ELISPOT technology
ELISPOT is an old technology that has been successfully developed more than 20 years ago. Czerkinsky et al. pioneered the use of this technique in 1983 to successfully detect the frequency of B cells secreting antibodies due to stimulation. Since then, immunization scholars around the world have started a long-term ELISPOT Cytokine technology competition. Each team hopes to take the lead in promoting this technology to study T cell immunity. The wonderful thing is to provide an environment close to in vivo experiments. The clonal size group of Precursor T, the initiator of the immune response in the body, is quantified by detecting various cytokines secreted by T cells, and predicts the downstream immune response of the immune system in the body.
Although ELISPOT Cytokine technology is simple to operate, it has not been successfully applied to ex vivo T cell function studies as expected. The first major problem in making ELISPOT technology from B cell immunity to T cell immunity is the increase in sensitivity because T cell cytokines produce very little compared to B cell immunoglobulin. The early ELISPOT analysis conditions were not very satisfactory. The technical problems of the quality of the paired antibodies and the material of the color-based film made it difficult for most research teams to obtain clear spots. The result was insufficient sensitivity and data analysis. Low sex. This problem was not until the introduction of the PVDF film by the Paul Lehmann team at Case Western Reserve University in Ohio, USA (Forsthuber et al., Science, 1996), which solved the lack of low sensitivity of the technique due to spot coloring problems. Compared with the original standard membrane surface, the PVDF membrane provides 1,000 times larger area to adsorb individual antibodies, so that various cytokines secreted by T cells can be captured near the cells, allowing the spots to be concentrated, clear, and contrasting. Greatly improve the sensitivity of ELISPOT Cytokine analysis. Figure 1 shows the results of a parallel experiment using a PVDF-BASED modified film (Panel A) and a conventional standard film (Panel B). The same human PBMC sample, through the exact same experiment, Panel A presents a clearer small color point.
The second technical problem is that the basic assumptions of ELISPOT Cytokine's experimental analysis lack scientific evidence, and the technology has not received much attention at the time. No researchers have tried to clarify some basic but important issues, such as:
Is the spot obtained from the experiment produced by antigen-specific T cells, or is the secondary effect of the bystander cells stimulated by Cytokine?
What is the physiological significance of small or large spots; how to determine the cutoff value?
Can you believe that spots are caused by a single cell?
What is the exact frequency range that ELISPOT Cytokine analysis technology can measure?
If cytokine, which has antagonistic effects, is produced at the same time, will they interfere with each other? And to what extent?
(2) The current ELISPOT technology
Since 1996, with the improvement of antibody preparation, PVDF membrane rafts and other technologies, ELISPOT analysis technology has gradually reached the expectations of researchers. It has been recognized as the most sensitive antigen-specific T cell in vitro detection technology. By examining the fingerprint of cytokine secreted by freshly isolated PBMC cells, ELISPOT analysis can suggest two important parameters of the T cell immune system: the size of the clone population of antigen-specific T cells; and the signaling pathway Th1/Th2 of immune effector cells.
After years of research by dozens of research teams, there have been scientific verifications on several issues of the ELISPOT analysis technology itself. It is now generally accepted that ELISPOT technology allows researchers to identify antigen-specific T cells at the single cell level, primarily for immune responses via CD4 or CD8 cells. Under appropriate experimental design, the quantitative analysis of ELISPOT Cytokine spots showed that the spots were generated by a single cell, while the spot morphology and Time Response analysis showed that the size of the spot directly reflected the productivity of the cell population. Because of this, ELISPOT is an immunological labeling technology that allows researchers to observe the actual process of cell secretion of Cytokine under almost natural physiological conditions, and to obtain pharmacological information on how immunomodulatory drugs affect T cell secretion. The rate of various types of cytokine. Drug-inhibited T-cell immunity can generally be achieved by two distinct processes; the Precursor T cell population that secretes Cytokine becomes smaller, or the cytokine capacity per Precursor T cell is reduced, while ELISPOT technology provides dual information.
Several features of ELISPOT analysis technology have made it the leader in antigen-specific T cell immunology. This technology is the only one that allows for an accurate analysis of 1:1000,000 in a millionth of a lifetime. Such a powerful analytical force makes flow cytometry also look back, with conventional intracellular cytokine or Dimer/tetramer staining at home and abroad. The sensitivity of the technology is generally limited to 1:10,000. Such high sensitivity is necessary for T cell immunology studies because antigen-specific T cells typically occur in the circulatory system around the body at a frequency that is typically less than one. In addition, since ELISPOT Cytokine technology has been proven to be suitable for human lymphocytes recovered after freezing, PMBC has a comparable potency to freshly isolated samples after thawing, without loss of function. This is very important for clinical trials. It enables researchers to analyze blood samples of patients before and after immunotherapy in parallel. If the trial involves multiple clinical trials across the country, the patient's blood samples can be stored in ice and concentrated in the “central laboratoryâ€. test. Similarly, a blood sample or standard reference can be divided into small portions and sent to different inspection centers for testing, for interactive comparison, and at the same time, the standardization and standardization of the ELISPOT Cytokine technical process is beneficial.
(3) ELISPOT Future Outlook
ELISPOT analysis technology is playing its full potential in Europe and the United States, allowing immunologists to directly understand the T cell-related physiology or pathology in vivo, just like the electrocardiogram in the hands of a cardiac surgeon. The living body directly enters an organ system, where it inspects and excavates and gains new insights.
It can be seen from the literature of various countries that ELISPOT technology has reached the requirements of standardization and standardization, and is widely used in many fields, including monitoring the response of cancer patients undergoing immunotherapy (Lewis, 2000 and Janetzki, 2000), and infectious diseases (Moss). , 2000, Rahman, 2000, Rowland-Jones, 1998, Herr, 1997 and Lechner, 2000), Tumor (Nagorsen, 2000), or a specific form of immune response in autoimmune patients (Pelfrey, 2000). ELISPOT technology is also used as an important end point in several human trials for vaccine titer evaluation. In January 2003, the World Health Organization will use the ELISPOT technology to monitor HIV vaccine research immune response technology in cooperation with the Beijing Sex Center, which will be the first to use ELISPOT technology for HIV vaccine research and clinical trials in Asian countries. Related research. We look forward to the future application of this technology in the field of immunology in China.
principle
ELISPOT is very simple in terms of its principle. In essence, the principle of ELISA is the same, and it is not difficult to understand. The cells are stimulated to locally produce cytokines, which are captured by specific monoclonal antibodies. After the cells are decomposed, the captured cytokines bind to the biotinylated secondary antibody and then bind to the alkaline phosphatase-labeled avidin. After incubation of the BCIP/NBT substrate, a "purple" spot on the PVDF wells indicates that the cells produced cytokines, and the spots were analyzed by the ELISPOT enzyme-linked speckle analysis system. The traditional ELISA and ELISPOT are based on the principle of enzyme immunoassay, and the high catalytic frequency of the enzyme is used to amplify the reaction effect, thereby achieving a highly sensitive detection effect.
The full name of ELISPOT is Enzyme-linked Immunospot Assay, and its technical principle is similar to ELISA. The experimental design was to coat a PVDF membrane at the bottom of a 96-well culture plate to adsorb a monoclonal antibody that was specifically selected and non-toxic (excluding sodium azide, endotoxin). After appropriate separation of the venous blood PBMC cells, they are distributed to the microplate, and then subjected to appropriate antigen stimulation, and the microplate is placed in an incubator for a period of time. In general, memory T cells begin to secrete cytokines after several hours of antigen stimulation, at which point local cytokines secreted (next to the secretory cells) are captured by specific antibodies on the PVDF membrane. After the cells in the microplate are removed and washed, the captured cytokine can be further labeled with a Biotin-labeled secondary antibody, followed by StreptAvidin, which binds to the enzyme, and is added to the enzyme. The color makes it color, and the reactive cells leave stain spots.
The reaction steps can be roughly divided into:
(1) coating a 96-well plate with a monoclonal antibody specific for the cytokine, and blocking the remaining blank sites;
(2) adding a cell suspension, stimulating and activating the cells with a specific antigen to produce a cytokine, and the cytokine will diffuse nearby to specifically bind to the previously coated antibody;
(3) Wash off the cells;
(4) Adding the enzyme-labeled secondary antibody specifically binds to the cytokine, and by the color reaction of the substrate, a spot appears at the position of a cell, and finally the sample can be calculated by using a microscope or a specific reader. The number of activated cells in the cell.
The above is a procedure for detecting cells secreting cytokines. If it is a cell that detects a specific antibody, it is only necessary to change the coating-specific antibody into a coating-specific antigen.
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ELISPOT technology experiment steps
In principle, ELISPOT is very simple, but there are many conditions to be explored during the operation, so here is a brief introduction to the ELISPOT operation steps.
(1) Appropriate amount of capture antibody was pre-coated on 96-well ELISPOT plates, taped and incubated overnight at 4 °C. The plates were washed 6 times with PBS, blocked with medium containing 10% fetal bovine serum (FCS), and incubated for at least 1 h at room temperature. FCS can block the FCS receptor of the coated antibody to reduce non-specific reactions. There is a special 96-well plate bottom medium that is a PVDF membrane. The porous structure of the membrane allows a single cell to sit in it and then secrete cytokines or specific antibodies, thus making the final detection of single cell sensitivity possible. At the same time, the opacity of the PVDF membrane is also the difference between ELISPOT and ELISA. In theory, it is possible to coat two different antibodies at the same time. Two different antibodies can display different colors using different color development systems, but in this case, the incompatibility between different antibodies must be considered, and each type must be guaranteed. The amount of antibody coating should be sufficient. If you are using a pre-coated board for the kit, this step can be omitted.
(2) Negative control and cell samples, such as peripheral blood mononuclear cells (PBMC), purified CD8+ T cells or PBMCs that remove a specific cell population, using medium containing 10% FCS Dilute to 5 × 104 ~ 2 × 105 / hole If conditions can reach a higher number of cells, it is recommended to try 3 × 105 ~ 5 × 105 / hole, especially in secretory cells), dispensed in the ELISPOT plate hole Then add specific stimuli, such as peptide or gene expression products, extracted antigens, etc., and only the medium is added to the negative control wells. It is best to use serum-free medium in the experiment, so as to avoid differences due to different serum batches. The resulting error, while also avoiding certain components of the serum can cause non-specific reactions), the positive control wells are added with phytohemagglutinin (PHA, 2μg/mL) or positive polypeptide, cultured at 37 ° C, volume ratio of 5% CO2 24 to 48h. The cells to be subjected to the ELISPOT assay must be prepared in the form of a suspension. Detection using ELISPOT is very sensitive, and secretory cells with frequencies as low as 10-12 can be detected. This method can also be used for cells that adhere to the wall. Although the cells are not easily washed away after adhering to the wall, they do not interfere with the color development of the last spot. As long as the cells can normally secrete products that specifically bind to the antibody, these products will gradually spread around the cells and eventually show spots.
(3) Wash 6 times with PBS containing 0.01% Tween-20 to discard the cells, add biotinylated anti-cytokine detection antibody, and incubate for 3 hours. After a further 6 washes, AP or HRP-labeled streptavidin was added and incubated for 3 h at room temperature.
(4) Wash again with PBS-Tween-20 for 5 times, add the substrate to develop color at room temperature, and when purple (product of AKP and substrate) or red (product of HRP and substrate) appears, use stereoscopic anatomy. The number of spots was calculated by a microscope or computer-aided imaging analysis system, and the results were recorded in speckle forming units (sfu/L million added to cells). Each spot represents a cell that specifically secretes CK. If the antigen is pre-coated, it can be used for ASC assay, in which case each spot represents a cell that secretes a specific antibody.
After completing the above steps, it was only half of the experiment. After you continue to explore the conditions of the reaction, you get a plate full of spots, how to analyze these spots. If the use of microscope artificial technology is undoubtedly an experiment with a personal factor in Taishen, the experimental results will certainly be questioned by the reviewers. Subsequently, after using a digital camera to take pictures, using photoshop for processing and counting, this method also has many personal factors. A technology must be popularized and accepted by researchers. It is necessary to minimize human error. Therefore, in recent years, many companies have begun to develop computer-aided systems that use artificially set thresholds to manually count spots. In this way, the non-specific reaction spots in the negative control are removed by the computer and counted by the computer, and the confidence of the experimental results is greatly improved. The primary criterion for judging is the size of the spot, but researchers who have done computer imaging of electrophoresis, isotope, etc. have encountered a problem: in many cases, the experiment produces some light spots, which are absolutely background by the naked eye. Therefore, it is necessary to consider the factors of the speckle when designing the software. The computer can analyze the kinetic characteristics of cell secretion by using the characteristics of the depth of the spot, whether the center is the origin and the surrounding lightening. The maturity of this technology is believed to make the application range of ELISPOT wider.
(5) ELISPOT Spot Correction Like most Cell-based analysis techniques, ELISPOT Cytokine technology is also a time consuming and labor intensive task. In fact, the visual interpretation of the characteristics of a small number of cell populations such as the activation of T cell hormone secretion has been regarded as a major technical challenge in immunology. The interpretation of the ELISPOT results often requires a dedicated and experienced technician; sometimes even with a full-time technician, the use of a conventional microscope to calculate the number of spots is inevitably subjective and biased.
The essence of scientific evidence lies in how to make the experimental results as objective, accurate and reproducible as possible. The traditional manual calculation method obviously cannot meet this requirement. Therefore, several research teams have developed Plate Scanner and automatic analysis software, among which the ImmunoSpot Analyzer automatic analyzer researched and developed by Professor Paul Lehmann is the most representative. In the process of analyzing the ELISPOT experimental data, the instrument first captures the color image on the film in the micro hole with a high-resolution lens and then stores it into a TIF file; these images can be further manually or automatically calculated for the number of spots, if using ImmuneSpot analysis Software, you can first set the conditions, and then analyze the size of the spots to estimate the amount of hormone secretion. It is expected that an automatic image scanning analysis instrument such as the ImmunoSpot Analyzer will overcome the shortcomings of the traditional microscope interpretation method, such as manpower and human objectivity, completely eliminate the bottleneck problem of the ELISPOT analysis technology, and further promote the novel application of the technology.
The ImmunoSpot@ Image Scanning Analyzer can provide different data formats, including unprocessed and processed film surface images, the number of spots in each well, the average number of spots in each well, and the size of spots in each well. The histogram of the histogram.
The emergence of the ELISPOT analyzer solved the following problems
Which spots are produced by antigen-specific T cells (this spot has further analytical value) and which are produced by unrelated cells (this spot has no T cell research value)
The meaning of the size of the spot.
How to define the maximum and minimum size of the spot when counting and analyzing different cytokines.
How to analyze from a single cell basis.
How accurate is the experiment? If a cell produces similar cytokines, to what extent it interferes with the results of the analysis.
Repeat the experiment several times to make the results more meaningful.
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ELISPOT technology advantages and applications
The history of ELISPOT dates back to the hemo-lytic plaque forming cell assay (HPF) created by Jerne et al in 1963, which can be used to detect and count individual antibody-forming cells. With the maturity of monoclonal antibody technology, in 1983, Czerkinsky et al. used this method to detect the number of cells secreting specific antibodies in splenocytes, and found that the method is highly sensitive and simple. Later, they used this method to quantify the number of cells secreting IFN2γ in peripheral blood stimulated by mitogens. Subsequently, the ELISPOT method detecting secretion of other cytokines at the single cell level (e.g., IL-2, IL-4, IL-5, IL-10, TNFα, and IL-6) number of means are also set up. Nordstrom et al. used biotin-avidin biomagnification to increase the sensitivity of this method. Herr used computer-assisted video image analysis (CVIA) to automatically analyze the size and number of TNF2α enzyme-linked immunospots. The number of antigen-specific CD8+ T cells in peripheral blood mononuclear cells (PBMC) after contact with target cells loaded with antigenic peptides was calculated, which not only improved the resolution of background staining but also made large-scale studies possible. Vaquerano et al. combined a digital camera with a computer to develop a similar speckle counting system, which is considered to be more objective, reproducible and time-saving when the number of spots per hole is greater than 100.
With the continuous development of ELISPOT technology, its advantages are gradually revealed. The advantages of ELISPOT compared with other traditional technologies are listed below.
ELISPOT analysis visualizes single cell secreted products. These analyses are extremely sensitive because they capture directly around the secretory cells and are either surface diluted or captured by receptors on adjacent cells, or before degradation. In vitro frequency measurement up to 1:1 million cell precision. This resolution has far exceeded the accuracy measured by tetramer staining and ELISA using intracellular cytokines. This high sensitivity is critical because antigen-specific T cells are typically low frequencies in vitro. High yield T cell analysis became feasible. A trained team of related laboratories can test the response of dozens of antigens in hundreds of samples. Only a small number of cells are needed and can be used for comparison by other cytological methods. For example, 45 ml of blood is sufficient to test the response of 600 different antigen/peptide species. Immune factor indicators for CD4 and CD8 cells can be defined. This is because only a few cells are needed and the analysis can be performed in high yield mode. ELISPOT analysis is an ideal tool for screening peptide libraries and mapping immune factors. Used to determine the functional affinity of antigen-specific T cells. This is most likely to be affected by peptide drugs up to 50%. It can be used to study the secretory process of cells that have not been treated with drugs. If a net reduction in production is observed, it can be determined whether this difference is due to a decrease in secretory cells or a decrease in secretion production per cell. Lymphocytes survived after ELISPOT analysis. This allows for additional value after analysis for further analysis, cloning or cryopreservation. It is possible that human lymphocytes after cryopreservation can be used for detection without losing their function. Samples before and after treatment can be tested side by side and the results can be repeated.
(1) and ELISA
The ELISA measures the absorbance on a microplate reader by color reaction, and compares with the standard curve to obtain the total amount of soluble protein. ELISPOT also displays clear and discernible spots at the corresponding positions where the cells secrete this soluble protein by color reaction. The spots can be counted directly under the microscope or by computer-aided analysis system. One spot represents 1 Cells, thereby calculating the frequency of cells secreting the protein. (Some studies not only measure the amount of cytokine production, but also the frequency of cells that secrete this cytokine.) Because it is a single-cell level test, ELISPOT is more sensitive than ELISA, and can detect 1 secretion from 200,000-300,000 cells. The cells of this protein. The capture antibody has a high affinity, high specificity, low endotoxin monoclonal antibody, and does not affect the secretion of cytokines by activated cells when the investigator activates the cells with a stimulant.
And limiting dilution analysis (LDA)
LDA is able to quantify specific CTL cells and has been considered the “gold standard†for CTL testing for many years. It enables detailed understanding of immune kinetics and memory cytotoxic T lymphocyte (mCTL) subpopulations. Cell cycle. However, this method requires a high strength antigen stimulation, which will speed up the effect of CTL (eCTL) apoptosis, and can not be quantified or measured value eCTL low cell numbers. Secondly, the method is cumbersome, and the culture time can be as long as 2 to 3 weeks, and it is easy to cause a loss of T cell number.
(2) and tetramer method (Tetramer)
In recent years, the MHC2 class I molecular antigen peptide tetramer method has appeared. The advantage of this method is that it is rapid, direct, sensitive and specific. Even when the mCTL level in the body is less than 1%, the MHC2I antigen peptide tetramer method can directly measure the accurate value, which is higher than the LDA sensitivity. 10 times. However, this technology also has many application difficulties: in addition to the technical difficulties of synthesizing and stabilizing MHC class I molecules, the main disadvantage is the choice of epitopes. Since each reaction can only analyze a single epitope, and the various epitopes bound by MHC class I molecules can form a CTL response, but it changes with the background and time of the gene, so the correct epitope is selected. Particularly important. Screening for dominant epitopes can be done with Elispot, but scanning the entire peptide library is not an easy task. Of course, the use of bioinformatics has greatly helped the screening of epitopes. At the same time, some studies have shown that not all positive cells identified by Tetramer have exact functions. Tetramer-positive cells are 10 times more likely to secrete INF2γ cells by ELISPOT analysis, many of which are inert cells that do not function. Represents a memory CTL precursor cell. Tetramer analysis only increases the sensitivity of the assay, and it is important to determine its function.
(3) the Cr51 release assay
This method is a more classical method. Although it is very effective in detecting the antigen diversity recognized by CTL, and can accurately clarify the best epitope recognized by CTL, it is at most a semi-quantitative level, and the spontaneous development of Cr51-labeled cells. The release rate is high, and it is not suitable for long-term culture; the concentration of cells required for labeling is high, which brings a lot of inconvenience to the test; in addition, the Cr51 release method measures the characteristics of a cell population rather than a single cell.
(4) intracellular staining CK
Compared with the ELISPOT method, intra-cellular CK staining (ICS) is mainly applied to the staining of intracellular cumulative cytokines and the FACS method of multicolor parameters, which is feasible for detecting antigen-specific T lymphocytes in circulating lymphocytes. method, the ELISPOT method was to detect all secreted CK eCTL.
application
At present, ELISPOT has been used to detect the characteristics and functions of drugs, chemical agents and other CK complexes, thus providing a basis for the regulation of immune function in vivo. In recent years, ELISPOT technology has been increasingly used in tumor vaccine evaluation tests, immunological monitoring and immunology research.
(1) Application in anti-tumor immunity and related fields
In many studies, this technique has been used to quantify the number of antigen-specific cytotoxic T cells (CTLs) in peripheral blood lymphocytes of animals. Recently, ELISPOT technology has been used to analyze and evaluate peptide-specific T lymphocytes from PBMCs of patients with infectious diseases and vaccine trials to induce tumor-specific T cells in cancer patients.
ELISPOT detects T cell responses with high sensitivity, specificity, and reproducibility. It is simple and rapid to operate, requires a small amount of specimens, and reflects the function of antigen-specific cells while also reflecting its function, and is related to clinical outcomes. The ELISPOT plate can be prepared in advance, and the pipetting, washing and reading plates can be automated, so large-volume inspections can be done quickly and efficiently. After comparison with other methods, ELISPOT is likely to be the first choice for quantitative analysis of tumor or virus-specific T cell responses.
(2) Application in anti-infective immunity
CTL plays a major role in the immune response against viral infection, and the balance of Th1/Th2 in the CD4+ helper T cell subset plays an equally important role in combating viral infection. In HIV research, the recognition of different antigens by MHC class 1 molecules by CTL and the generation of an immune response also play an important role in controlling HIV-1 infection. Using the ELISPOT method to detect CK such as IFN2γ, we can further understand the specific CTL response and related immune information for HIV.
In addition, the ELISPOT method was also used to evaluate the immunogenicity of a mucosal CD4+ T cell immune response caused by HIV-1 infection and a DNA vaccine in which HIVgp 120g is co-expressed with cholera toxin. In other studies, ELISPOT was also used to detect cellular immune responses produced by rectal immunization with low-dose hepatitis A vaccine. In acute self-limiting hepatitis, a Th1-based response promotes cell-mediated immunity and plays an important role in controlling viral infections and delaying the development of chronic diseases.
This method also plays an important role in many antibacterial immunity studies. For example, the ELISPOT assay confirmed that the T cell response induced by recombinant BCG (rBCG.MalE) expressing E. coli MalE protein is CD4+ T cell-dependent, and there is a Th1/Th2 balance transition in rBCG.MalE-induced specific CD4+ T cell response. And gradually form a Th1/Th2 mixed response.
(3) Clinical aspects
Currently, the field of clinical testing has been the detection of tuberculosis (TB). The most recent research hotspot is the prediction of transplantation. Receptor-to-donor-specific HLA antibodies have long been a major obstacle to organ transplantation because their presence not only mediates hyperacute rejection but also causes acute rejection. Thus, the presence of HLA antibodies limits the need for donor organs in allogeneic antigen-sensitive patients, thereby increasing the time to wait for transplantation. Early clinical complications such as acute rejection and allogeneic immunity before transplantation in the heart, liver and kidney allografts urgently require an appropriate test method to understand receptor-specific donor body fluids before organ transplantation. The functional status of immunity.
In the recipients of the transplant, some of them have had multiple blood transfusions, or have had previous allogeneic graft failures or have had positive allogeneic antibodies in the history of multiple pregnancy. When they receive allogeneic transplantation, the existing test methods can only show that they are allogeneic. The immune function is normal. However, there are memory B lymphocytes in their body, and once they are stimulated by the corresponding HLA antigen, they will evoke an immune response, which not only affects the transplant results, but also the transplant-related complications that endanger the patient's life.
ELISPOT is a potent and effective method for detecting single antibody secreting cells with high sensitivity. Therefore, it is the best means to stimulate the proliferation of B lymphocytes, differentiate into plasma cells, and establish a specific ELISPOT method to detect HLA antibody-producing cells. If ELISPOT is to further promote its application, especially in clinical applications, its standardization will be a very important issue. The US FDA and NIH have begun to establish the standardization process of ELISPOT. It is believed that with the maturity of ELISPOT technology and the establishment of standardized procedures, its application will be more extensive.
ELISPOT experimental program
Kit for BD
Preparation of experimental materials
Not required in the kit but necessary
material:
• Disinfected pipette (Gilson). Single road 20ul, 100ul, 200ul, 1ml, 8/12 road 200ul
• Sterile disposable polystyrene pipette (1ml, 5ml, 10ml) (Axygen).
• Disposable latex gloves.
• Sterile disposable centrifuge tubes (15ml, 50ml) (Corning cat #430052 and 430290).
• absorbent paper (toilet paper)
• Sterile tip head, 200ul, 1ml,
Reagents:
1. PBS sterile 300ml/10 plate, bacteria 4000ml/10 plate Do not use commercial PBS when preparing coated antibodies
2. ddH2O (sterile) For 10 ELISPOT plates, 200 ml
3. Bacterial Wash Buffer (PBST): bacteria 3000ml/10 plate
Tween-20 imports
4. Bacterial Wash Buffer (PBS): There are bacteria 1000ml/10 plate
5. Culture medium: serum-free medium (P/S, 2 mM glutamine, and Hepes buffer).
6. PMA + ionomycin.
Phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., product nr. P8139 is recommended).
PMA
Storage solution: PMA is dissolved in DMSO at a concentration of 1 mg/ml and stored at -20 °C.
Working solution: stock solution 1:4000 diluted in serum-free medium
At this time it is 250ng/ml
Calcium ionophore (ionomycin; Sigma Chemical Co, product nr. I0634 is recommended).
Ionomycin
Stock solution: Ionomycin is dissolved in DMSO at a concentration of 1 mg/ml and stored at -20 °C.
Working solution: stock solution 1:200 diluted in serum-free medium
At this time, it is 5μg/ml
50ml mixed stimulant = 12.5ul of PMA working solution, 250ul of Ionomycin working solution, serum-free medium
7. 10% heat inactivated fetal bovine serum (Hyclone cat# ALH14384)
8. Antigen
equipment:
• Plate washer: automatic or manual (washing, manual dispenser, etc.).
• Dedicated CO2-cell incubator (37 ° C).
4°C refrigerator
Aseptic aseptic console
Preparation kit reagents
1. Coating antibody
50 μl was added to 10 ml of PBS. (Sufficiently coated with a 96-well plate, 100 μl/well).
For 10 boards, 500ul is added to 100ml PBS
2. Biotinylated detection antibody
Add 50 μl to 10 ml Dilution buffer. (After mixing, add ELISPOT plate, 100 μl/well).
For 10 boards, 500ul is added to 100ml Dilution buffer (PBS + 10% FBS)
3. STREP-HRP
Add 100 μl to 10 ml Dilution buffer. (After mixing, add ELISPOT plate, 100 μl/well).
For 10 boards, 1ml is added to 100ml Dilution buffer
4 color activation system (Activation system)
For one ELISPOT plate, 200ul AEC Chromogen + 10 ml AEC Substrate, mix and run out in 10 minutes. (100 μl/well).
ELSPOT experimental steps
Day 1
Wrapped ELISPOT board 16:30
1. Add 100 μl of coated antibody to each well in ELISPOT plate at 4 °C overnight.
Day 2
ELISPOT board closure 17:00
2. Remove the coated ELISPOT plate and discard the coated antibody solution from each well.
3. Wash once with serum-free medium.
4. Add 200 μl of complete serum-free medium to each well in the ELISPOT plate. Room temperature 2h. (8 lanes of guns)
5.
PBMC added to ELISPOT plate 20:00
6. Remove the ELISPOT plate for 2 hours and aspirate the blocking solution in each well. (Do not wash the board!)
7. In the ELISPOT plate, first add 30μl serum-free medium (12-channel gun) and 20μl peptide library (8-channel gun), then add 50μl cells (8-row gun) (the total number of viable cells per well is 2×105, each The total number of viable cells required is 10 × 106 and the volume is 2.5 ml. The final concentration of each peptide is 5 μg / ml, the working concentration of living cells is 4 × 106 / ml, and the final concentration of living cells is 2 × 106 / ml. ).
50 μl of cells and 30 μl of serum-free medium and 20 μl of PMA + Inomysin (working fluid concentrations of 250 ng/ml and 5 ug/ml, respectively, final concentrations of 50 ng/ml and 1 ug/ml, respectively) were added to each well of the positive wells.
8. Cover the ELISPOT plate with low evaporation lid and incubate in a 37 ° C CO2 incubator for 30 h.
Note: Do not shake the ELISPOT board.
Day 4
二抗和GABAæ ‡è®°ï¼Œæ˜¾è‰²å应 8：30
9. 培养30håŽï¼Œå–出ELISPOTæ¿ï¼Œç”©åŽ»ç»†èƒžåŽã€‚
10. 用去离å水洗涤2次。æ¯æ¬¡æµ¸æ³¡5分钟。
11. 用0.05ï¼…PBST洗涤3次。æ¯æ¬¡æµ¸æ³¡2分钟。
12. æ¯å”åŠ å…¥100μl ç”Ÿç‰©ç´ åŒ–çš„detector Ab(8é“排枪)。室温2å°æ—¶ã€‚
13. å–出ELISPOTæ¿ï¼Œå€’去detector Ab溶液，用PBST洗涤3次。æ¯æ¬¡æµ¸æ³¡2分钟
æ¯å”åŠ å…¥100μl Strep-HRP溶液(8é“排枪)。室温1å°æ—¶
14. å–出ELISPOTæ¿ï¼Œå€’去Strep-HRP溶液。
15. PBST洗涤4次。æ¯æ¬¡æµ¸æ³¡2分钟。
16. 用PBS洗涤2次。æ¯æ¬¡æµ¸æ³¡2分钟。
17. 洗涤完毕åŽï¼Œåœ¨å¸æ°´çº¸ä¸Šè½»è½»æ‹å¹²ELISPOTæ¿ã€‚
æ¯å—æ¿å200ul AEC Chromogen + 10 ml AEC Substrate，混匀，15分钟内用完。
18. æ¯å”åŠ å…¥100 µl é…好的substrate(8é“排枪)。在暗处ã€å®¤æ¸©ä¸‹åµè‚²ï¼ˆ5～60分钟）。
出现斑点åŽï¼Œç”¨è‡ªæ¥æ°´å†²æ´—，ä¸æ¢å应。室温下干燥，检测。
Quantitative detection of immunocytokines, Elispot enzyme-linked speckle analysis
ELISPOT technology principle
Env1
Pol1
Pol1
Vif
Vif
Env1
Env1
Pol1
Pol1
Vif
Vif
Env2
Env2
Pol2
Pol2
Yin pair
阴对
Env2
Env2
Pol2
Pol2
阴对
阴对
æ ·æœ¬1
Env3
Env3
Pol3
Pol3
阴对
阴对
æ ·æœ¬2
Env3
Env3
Pol3
Pol3
阴对
阴对
Env4
Env4
Pol4
Pol4
阴对
阴对
Env4
Env4
Pol4
Pol4
阴对
阴对
Env5
Env5
Pol5
Pol5
阴对
阴对
Env5
Env5
Pol5
Pol5
阴对
阴对
Gag1
Gag1
Nef
Nef
阴对
阴对
Gag1
Gag1
Nef
Nef
阴对
阴对
Gag2
Gag2
Tat
REV
Tat
REV
æ ·æœ¬é˜³å¯¹
æ ·æœ¬é˜³å¯¹
Gag2
Gag2
Tat
REV
Tat
REV
æ ·æœ¬é˜³å¯¹
æ ·æœ¬é˜³å¯¹
Gag3
Gag3
Vpu
Vpr
Vpu
Vpr
æ ·æœ¬é˜³å¯¹
æ ·æœ¬é˜³å¯¹
Gag3
Gag3
Vpu
Vpr
Vpu
Vpr
æ ·æœ¬é˜³å¯¹
æ ·æœ¬é˜³å¯¹