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Basic principles and experimental methods and precautions
1. Basic principles and experimental methods
2. ELISPOT experiment considerations
(1) Collection of test samples and samples for ELISPOT test
(2) Negative control, positive control and stimulator of test well in ELISPOT test
(3) The influence of other factors in the test on the experimental results
(4) Judgment, analysis and positive judgment criteria of ELISPOT test results
Enzyme-linked immunospot assay (ELISPOT) assay
The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for detecting cells secreting a particular protein in a single cell suspension and for quantifying the frequency of cells producing that particular protein. In 1983, Crekinsky et al. successfully detected the cell frequency of secretory specific antibodies using ELISPOT technology. After continuous development, this technology has been widely used to detect cells that produce and secrete various other effector molecules (such as cytokines). This method is simple to operate, but it provides an environment very similar to in vivo experiments, monitoring various types of cytokines secreted by T cells, and predicting further immune responses in the immune system in vivo. In the laboratory of the HIV Vaccine Trial Network (HVTN) and Vaccine Research Center (VRC) of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, the ELISPOT method has been standardized and validated and has been used to test HIV vaccine immunogens. A major method of sexual endpoints.
ELISPOT detects the number of activated T lymphocytes that release cytokines. In the ELSPOT method, a 96-well plate of a nitrocellulose membrane is pre-coated with an antibody against cytokines, and T cells and antigens or peptides are incubated together in a well, and stimulated T cells produce cytokines and secrete. The cytokine accumulates in the cell where it binds to the pre-coated antibody in the well, and then the enzyme-labeled secondary antibody is added. Finally, the substrate is added, and an insoluble color-colored spot is formed. Spots represent a cell that produces cytokines. This method is 200 times more sensitive than ELISA, is non-radioactive, can be quantitatively analyzed, and has the same accuracy as LDA (Miyahira et al_, 1995).
The basic principles of the ELISPOT method are the same, but there are still some changes, including the type of effector cells, either peripheral blood T lymphocytes or T cells cultured in vitro. The antigen stimulator may be a peptide or a protein, and the antigen presenting cell may be a peripheral blood mononuclear cell, a dendritic cell or a tumor cell line, and the detection index may be various cytokines or granzyme B. Figure 10.7 illustrates the principles and flow of ELIPOT.
The ELISPOT assay mimics the in vivo environment in vitro and detects the status of activated cytokines secreted by immune cells, so the subject is a functional cell. For humans, monkeys and other primates, mononuclear lymphocytes that are freshly isolated from peripheral blood are generally used, and spleen cells are often used in mice. Cell separation, preservation, resuscitation, culture, transportation, and detection of the environment and specific operations may affect the vitality and function of the cells. If the viability and function of the cells are not ensured, the cytokines secreted by the immune cells cannot be obtained. Real test results. At present, the isolation method of peripheral blood mononuclear cells is usually after adding the calcium-free PBS isotonic buffer to dilute the peripheral blood, and the cells are separated by using the lymphocyte separation solution, and the whole operation must be completed under aseptic conditions to the lymphocytes. The diluted whole blood should be slowly added to the separation solution, and should be gently handled during the whole operation to avoid artificially mixing the diluted whole blood with the separation solution. Once the peripheral blood is collected, it should be isolated immediately. According to reports in the literature, when stored at room temperature for more than 12 hours, and then separating peripheral blood mononuclear cells, the viability of the cells will decrease (Kierstead eta1., 2007).
The isolated cells should be subjected to the ELISPOT test as soon as possible. Otherwise, they should be stored in liquid nitrogen by the corresponding cryopreservation method. Studies have shown that 180 days of peripheral blood mononuclear cells are preserved in liquid nitrogen, and the activity of the cells remains after resuscitation. Above 80%, and the results of the ELISPOT assay are consistent with freshly isolated cells (Smith eta1., 2001). At present, many clinical trials of vaccines need to concentrate samples from different regions in central laboratories. Therefore, the cryopreservation of cells lays a foundation for this. The cryopreservation of cells by liquid nitrogen ensures the completion of clinical trials of vaccines. The fluctuation of temperature during cell transportation is a key factor affecting cell viability. To ensure the consistency of transport and preservation of cell temperature, liquid nitrogen should be used for transportation.
Negative controls and positive controls are usually required for the immunoassay. The same is true for the ELISPOT test. The negative control is the test cells plus the medium, the positive control is the test cells plus the positive stimulator, and a complete medium control should be established. For primates such as monkeys, the positive stimulator may be selected from staphylo-coccal enterotoxin B (SEB), phytohemagglutinin (PHA), CEF peptide library or a mixture of PMA and ionomycin (or A23187). CEF is a mixture of T cell MHC class I molecular epitope peptides containing 32 cytomegalovirus (CMV), Epstein-barr virus (EBV) and influenza virus (inflLlenzavirus) sources. A specific response is similar to the stimulatory effect of administering a specific peptide or antigen in vitro, and different humans respond differently to CEF (Mwau and McMichael, 2002). Concanavalin A (ConA) was used as a positive stimulator in mouse spleen cells. If there is no response to a positive stimulator, the cells are damaged during cryopreservation or resuscitation, and the test cells are not eligible for the ELISPOT test. Generally, cells that respond to the above stimuli can be collected in large quantities for isolation and cryopreservation. The cells are resuscitated in each experiment for parallel experiments as a positive control, and the positive control can also be used as an indicator for ELISPOT quality control. The stimulator of the test cell is a specific antigen or peptide library designed according to the requirements of the test, and the stimulation concentration depends on the purity of the antigen and the peptide library. Generally, the working concentration of the peptide library is 2 ug/ml, and the work of the protein The concentration is 10ug/ml, and the best conditions can be obtained through preliminary tests in actual work.
At present, the ELISPOT detection reagent has a reaction plate which has been coated with a cytokine-capture antibody, and a reagent which needs to be coated with an antibody in advance. The concentration of different coated antibodies affects the spot of the final reaction. If the concentration of the coated antibody is too low, the reaction spot It is too large and has a dispersion phenomenon. As the concentration of the antibody increases, the reaction spots will gradually become clear and dense. Therefore, it is necessary to first explore the concentration of the coated antibody before each test or test according to the recommendations of the manual. The number of cells in the test well affects the results of the ELISPOT test. Generally, the number of cells per well is between 200,000 and 400,000, which can form the maximum density of monolayer cells (Smith et al., 2001). According to different test stimuli, the concentration of test cells can be adjusted accordingly. Under the action of non-specific stimuli (SEB, PHA, PMA, ionomycin or ConA), the number of cells per well is generally 0.5 to 50,000. Under the action of the specific stimulant CEF peptide library, the corresponding antigen or peptide library, the number of cells per well is generally 50,000 to 400,000 (Sylvia et al., 2006).
Most cells can be directly subjected to the ELISPOT test without prior incubation, but pre-incubation of cells and stimuli can effectively remove the effects of macrophages and granulocytes on the experimental results, and for IL-10 or granzymes, etc. It is difficult to obtain good test results by direct method, and it is necessary to obtain better test results by pre-incubation. It has been reported in the literature that frozen cells can be more effectively removed from apoptotic cells if they are incubated overnight (Marta et al., 2007).
The cells are added to the ELISPOT reaction plate for incubation with the stimulator for a period of time, generally above 16 h, and up to 24 h or longer. In addition, when adding cells to the reaction well, it should be added vertically slowly to avoid cell aggregation at the edge of the plate. It is recommended to add the reaction stimulator before each experiment and then add the cells. For the reaction wells with the PVDF membrane at the bottom, the bottom of the plate holes should be avoided during the loading to avoid damage to the PVDF membrane. After the cells are added to the reaction plate, the reaction plate should be avoided as much as possible to prevent the tailing of the reaction spots. Although the ELISPOT assay has been relatively simple compared to other cellular immunoassay methods, its procedure is almost similar to the ELISA reaction, but it still needs to be handled carefully during the process of washing the plate. Currently there are manual washing and machine washing, in manual During the process of washing the plate, each time the plate is washed, the reaction plate should be lightly buckled to remove excess water in the hole of the plate, and the violent gusset may cause the reaction spots to fall off. Machine washing can increase the efficiency of the test, and the safety of the experimenter can be ensured when the infection test is carried out, but the aseptic operation is not guaranteed, so the washing step before the cell incubation requires manual washing. In addition, the type of medium can affect the background of the entire reaction, and if serum-free medium is selected, the background of the wells may be lowered.
During the operation of ELISPOT, the test medium, coating antibody concentration, detection antibody concentration, incubation time, washing operation and color development time may affect the results of each test. The operator can establish the corresponding ELISPOT reaction conditions according to the laboratory conditions, so as to obtain better test results. It is worth noting that different laboratories need to explore the same experimental conditions for the same cytokine. The same laboratory should also explore the experimental conditions of each cytokine when testing different cytokines.
The disadvantages of the ELISPOT method are that errors caused by manual operation counts, errors caused by changes in the size and shape of the points, and errors caused by subjective operations of the experimental operator can affect the experimental results. Commercial computer imaging analysis systems have been available, which greatly increase the accuracy and speed of counting immune spots. Among them, the US CTL company has a high product share. By analyzing the shape, size, and density of the spots, it is possible to exclude non-specific spots and determine the amount of cytokines produced by a single T cell.
ELISPOT, the test has been widely used in clinical trials of HIV vaccines to evaluate the cellular immune effects induced by HIV vaccines. The criteria for positive ELISPOT test results are currently classified into three categories: empirical criteria, Permutation-based resampling criteria, and the Distribution-free resampling criteria (Zoe et al., 2006).
The specific criteria for the empirical criteria are: in the 200,000 cells per well, the average number of reaction-cell spot-forming cells (SFCs) stimulated by the corresponding antigen or peptide is at least 11 and is the average number of SFCs in the negative control well. 4 times, if the above two conditions are met, the test group is a positive reaction. The empirical standard is the criterion for positive judgment by the tester based on the experimental experience of ELISPOT. The application is relatively simple, but there are some unavoidable shortcomings. For example, the accuracy of the empirical standard is poor. The results obtained according to the empirical standard are often specific and sensitive. Both are relatively poor. The conditions of ELISPOT, the method of operation of the test are diverse, each laboratory is not identical, and the empirical criteria are often based on a specific test condition. Strictly speaking, laboratories with the same test conditions can judge according to the standard. If the laboratories with different test conditions refer to the empirical criteria to judge the cellular immune effect of the vaccine, some laboratory judgment results may be inaccurate. Information exists.
The Permutation-based resampling standard applies statistical methods based on empirical criteria and relatively corrects some of the shortcomings of empirical standards. The specific specification of the standard is: the P value is obtained according to T=(YE-YC)/Sp, thereby judging the result. YE represents the average number of reaction well spotted cells (SFC) stimulated by the corresponding antigen or peptide, YE is at least 10 SFC/200 000 PBMC, Yc represents the average number of SFCs in the negative control well, SP represents the standard deviation, and the standard is applied The degree of detection is increased to a certain extent.
The Distribution-free resampling standard is an updated positive criterion that combines statistical methods and is simpler to calculate than the Permutation-based resampling standard. This method avoids the interaction of different peptides in the stimulating peptide library to some extent. Better applied to clinical trials of vaccines