Factors affecting the establishment of ELISPOT standardization

In order to achieve the standardization of ELISPOT, the color development is preferably carried out in an incubator, the temperature is controlled at 25 ° C ± 5 ° C, and color development is preferably 25 minutes. In addition, color development at 37 ° C is also a good method, but pay attention to the corresponding shortening of the color time to 12-18 minutes, and color inspection in the diligent. In order to improve the repeatability of ELISPOT test results and increase the credibility of the experiment, a standardized set of experimental practices must be established. Standardized experimental operation specifications are not only some experimental operations, but also a system engineering, which needs to be upgraded to the laboratory management and institutional framework. This includes the establishment of laboratory systems, training of experimental personnel, establishment and standardization of experimental procedures, selection and standardization of experimental equipment, reagents, consumables, and so on. As far as the experimental procedure is concerned, the standardized specification starts from the collection phase of the experimental sample and continues until the end of the entire experiment, that is, the completion of the data analysis processing. There are many factors that affect the ELISPOT spot frequency, let's analyze them one by one. 1. Cell type The cells we use for ELISPOT testing usually have blood-derived PBMC and spleen-derived lymphocytes. Some experimenters need bone marrow cells. Different sources have different cell composition, so these differences should be considered and cannot be directly compared. What factors lead to different cell sources, and the ELISPOT results are different? One factor is the distribution of positive cells in the body. We know that cellular immunity often occurs at local immune response sites. For example, when mucosal immunity occurs, various immune cells are enriched in mucosal organs; when an autoimmune reaction occurs, sensitized cells are often concentrated in the affected organs. Therefore, the proportion of positive cells in lymphocytes of local organs may be different from the ratio of positive cells in the blood or spleen to lymphocytes. There may also be differences between blood and spleen. Another factor is the amount of APC. There are also differences in the content of APC cells mixed in lymphocytes from different tissue and organ sources, and even different methods of isolation. In extreme cases (such as the cells to be tested are purified by magnetic beads), the APC content is insufficient, and APC (manually cultured DC cells or "feeder cells") needs to be added. Therefore, standardization must consider cell type and cell composition. 2. Cell state Cell state is the touchstone that reflects the technical level and experience of the experimenter. Many experimenters asked me why some people's ELISPOT results are very good, while others have done very badly. Regardless of the objective factors, the subjective factors are mainly reflected in the treatment of the detected cells. Some people have the basis for long-term cell maintenance and cytology experiments, handling cells to be handy, obtaining good cell status and high vitality, and the results of ELISPOT testing are naturally good. On the contrary, some cells have not been taken to do ELISPOT test, have been tossed to die or dying, can you make good experimental results? ! Cellular state involves aspects of cell origin (including tissue sampling), methods of cell separation, and whether cells require cryopreservation and recovery. See Section 4 for information on maintaining good cell status; Section 6 for cell separation methods; and Section 7 for cell cryopreservation. These links must be standardized, the state of the cells is guaranteed, and the repeatability and credibility of the experimental results are guaranteed. 3. Cell Count Cell counts seem simple, and many experimenters ignore it. But its impact on the credibility and repeatability of ELISPOT results cannot be overemphasized. Because ELISPOT detects the cell frequency, the number of molecular spots, and the denominator is the number of cells. If we go through a lot of hardships, we make the molecules (that is, the spots) very beautiful and make them very accurate, but the denominator is neglected. Can the score (positive cell frequency) be accurate? We at Dacco have a deep feeling for this. Our company had an ELISPOT research project in 2005-06 to study whether the same frozen cells and stimuli can produce the same results in different laboratories. We have made great efforts to study the cryopreservation and resuscitation of cells, study the long-distance transportation of cryopreserved cells in China, and also put a lot of effort into training the personnel skills and qualifications of the laboratories participating in the experiment. The final experimental results were impeccable from the spot and background, but the data from the two laboratories was significantly different from the data from the other eight laboratories. Finally, after detailed investigation, the reason actually appeared on the blood count board! The quality of the blood cell counting plates in these two laboratories is poor, and the error is large, resulting in a final cell count error of ±40%! The most common equipment for cell counting is the blood cell counting board. Although it does not look like a high, fine, sharp device, it also represents the level of glass finishing in a country. China can develop sophisticated high-power solid-state lasers, and also have high-quality blood cell counting boards. It is a pity that the ability does not necessarily have a desire. At present, there are large errors in the testing of various blood cells in China. It is recommended to buy an imported blood cell counting board, which is really easy to use. If you must use domestic, then please use the same blood count board. Cell dilution must also be carefully followed and carefully mixed before sampling. When doing important experiments, it is best to let two people independently dilute and count, the results should be controlled within 10%. 4. The irritant, kit and serum test should be repetitive, and the stimulating effect of the stimulant must be repeatable and reproducible. After a batch of irritants is used up, when a batch of stimuli is replaced, a confirmatory test is to be performed. In addition, some polypeptides, protein stimuli may be inactivated and degraded during storage, and repeated freezing and thawing may cause problems. This requires pre-planning a stimulant preservation plan, and a confirmatory experiment should be done after three or six months. See section 4 for related content. The ELISPOT kits currently on the market are from internationally renowned companies, and the quality of the antibodies used in the kits of different companies is guaranteed. However, people often overlook other components than antibodies, such as blocking solutions, antibody dilutions, and so on. These also aid reagents can cause significant differences. At the time of pursuing the repeatability and credibility of experimental results, attention must be paid. Serum and endotoxin are of course an important factor. In order to achieve ELISPOT standardization and improve the credibility and repeatability of the experiment, the fundamental method is to use serum-free ELISPOT technology. See Sections 3 and 5 for related content. 5, the experimental operation process and speckle color development The third section of this manual is the ELISPOT standardized operation process, as long as the implementation, there should be no problem. But there is one point that needs to be taken seriously, that is, standardization is also fixed. Often an experimental procedure has a lot of flexibility, so you can do that. If it is true, these practices have its own reasons. Theory and experiment can't prove who is better than anyone. This is why they can be kept at the same time. However, once the laboratory's method of operation is fixed, do not change it at will, even if it is an "equivalent" operation, do not change it at will. Standardization is also fixed. As long as there is no error in the standard operating plan, it must be strictly enforced. The reason why spot coloration is listed separately from the experimental process is because of its importance. At present, ELISPOT has three sets of color development systems, one is the HRP/AEC color development system on the PVDF film, showing red spots; one is the AP/BCIP·NBT color development system on the PVDF film, showing blue spots; The set is a GABA/silver dye system on a transparent plate with black spots. It should be said that there is no significant difference between the sensitivity and background control between these color rendering systems, but once a color rendering system is selected, it is best to stick to it for a long time to meet the needs of standardization. In addition to the choice of color development system, the color development problem should also pay attention to the color temperature and color development time. Color development is the only step in all ELISPOT operations that is done at "room temperature." However, please note that the "room temperature" in biology and chemicalization is strictly 25 °C ± 5 °C, rather than the so-called "indoor temperature". Since entering the autumn and winter, we have received a large number of customer calls, saying that the ELISPOT spot is light in color and it is suspected that the antibody is inactivated. Interestingly, most of these customers are concentrated in the Yangtze River Basin. After careful verification, their problems are almost always due to the lack of heating in the winter room, and the lack of color due to low room temperature. Special Note: Once the color development has been carried out for more than 10 minutes, it does not make sense to increase the temperature because the enzyme has been largely inactivated. In order to achieve the standardization of ELISPOT, the color development is preferably carried out in an incubator, the temperature is controlled at 25 ° C ± 5 ° C, and color development is preferably 25 minutes. In addition, color development at 37 ° C is also a good method, but pay attention to the corresponding shortening of the color time to 12-18 minutes, and color inspection in the diligent. 6. Spot count and data processing What kind of point is judged as ELISPOT effective spot, what kind of point is not judged or the count is invalid, which is the "scale" of the spot count. In order to achieve standardization, it is necessary to unify the "scale" and not to "double standards." The premise of the unified scale is to use machine automatic counting. For example, on the BioReader 4000, we have more than a dozen independent parameters for describing and determining spots. These parameters can be saved separately as "method files". As long as the parameters in the "Method File" are the same, the "scale" of the spot count is the same, and the standardized spot count is guaranteed. Data processing, including data synthesis, negative positive judgment, mathematical statistics and so on. Regardless of how it is handled, the same treatment should be taken, which is no doubt. In summary, the ELISPOT standardization idea is: in every step of the ELISPOT experiment, practical and effective steps and methods are adopted and fixed, so as to finally ensure the repeatability and credibility of the experimental results.

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