Introduction to ELISPOT technology principles and experimental methods

ELISPOT technology principle

With the wide application of enzyme-linked immunoassay technology in the fields of medicine and biology, new breakthroughs have been made in the detection of various cytokines and antibodies in vitro. 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 bound to target organs and 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 detecting specific antibody-secreting cells and CK-secreting cells in vitro based on the basic principles of ELISA technology. Because of its high specificity and sensitivity, it is currently widely used at home and abroad, and is of great significance for exploring the pathogenesis of autoimmune diseases.

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 by a color reaction, and compares with the standard curve to obtain the total amount of soluble protein.
2, ELISPOT through the color reaction, in the cell to secrete the corresponding position of the soluble protein on the clear discernible spots, you can manually count the spots under the microscope or through the ELISPOT analysis system to count the spots, 1 spot represents 1 The 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)
3. Because it is a single-cell level test, ELISPOT is more sensitive than ELISA and limiting dilution methods, and can detect a cell secreting the protein from 200,000-300,000 cells.
4. The capture antibody is a high affinity, high specificity, low endotoxin monoclonal antibody produced by BD, R&D, Mabtach, and does not affect the secretion of cytokines by activated cells when the researchers activate the cells with a stimulant.

ELISPOT detection principle: ELISPOT full name is Enzyme-linked Immunospot Assay, 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 is such that the reactive cells leave stains of about 10-20 mm in size.

ELISPOT technology applications: prediction of rejection in transplantation, vaccine development, Th1/Th2 analysis, autoimmune disease research, oncology research, allergic disease research, infectious disease research, antigenic determinant mapping, compound and drug immunological reactions Screening and more.

ELISPOT experimental method

The PVDF Eli-spot method for detecting human IFNγ is now introduced, and is for reference only.
Kit contents: PVDF96 well plate, stored at room temperature; 0.1ml capture antibody, stored at 4°C; 0.1ml biotin-labeled detection antibody, stored at 4°C; 15ul avidin alkaline phosphatase, stored at 4°C; 0.25g bovine serum Albumin, stored at 4 ° C; 0.25 g skim milk, stored at 4 ° C; 11 ml substrate buffer, stored at 4 ° C; 11 ml concentrated PBS (10X), stored at room temperature; 11 ml concentrated washing solution (200x), stored at room temperature

Self-contained materials/reagents: Cell culture medium, CO2 incubator, 70% alcohol direct and indirect Eli-spot method

Cells can be directly stimulated in wells that have been coated with antibodies (direct method), or cells can be stimulated first in a 24-well plate or flask and then placed in the coated wells (indirect method). Which method is used is based on: 1) detecting the type of cells; 2) the amount of cells desired. If only a small amount of cytokine is required to produce cells, use the direct method; if the cytokine produces more cells, it is best to use the indirect method. The other steps are the same.

Stimulation method: It is recommended to stimulate PBMC with PMA and quinolone to produce IFNγ.

PBMC were diluted in culture (eg RPMI 1640 plus 2 mM glutamate and 10% heat-inactivated calf serum) containing 1 ng/ml PMA and 500 ng/ml 娄nomycin (Sigma, Saint Louis, MO) ). Add 2.104 to 5.104 cells to the antibody-coated PVDF wells and incubate for 10-15 hours in an incubator. Other stimulant incubation times may vary, based on the amount of cytokine-producing cells, and are optimally selected for different situations.

Preparation of reagent: 1. 10 ml of phosphate buffered saline (PBS, 10X) diluted with 90 ml of distilled water; 2. 0.22 g of skim milk was dissolved in 11 ml of diluted PBS, the final concentration was 2%; 3. 0.22 g of BSA dissolved in 22 ml of diluted PBS The final concentration is 1%; 4. 10ml concentrated washing solution (200x) diluted with 1990ml distilled water; 5. 10ul avidin alkaline phosphatase diluted with 10ml PBS-1%, BSA 6. 7ml alcohol diluted with 3ml distilled water, and finally The concentration is 70%.

Eli-spot operation process:
1. Incubate the PVDF well plate with 100 ul of 70% alcohol and incubate for 10 minutes at room temperature.
2. Pour off the alcohol and wash 3 times with 100 ul PBS.
3. Add 100 ul of capture antibody to 10 ml PBS, mix, add 100 ul per well, cover with a plate, and overnight at 4 °C.
4. Pour off the liquid and wash once with 100 ul PBS.
5. Add 100 ul of 2% skim milk PBS to each well (see reagent preparation), cover with a plate, and incubate for 2 hours at room temperature.
6. Tap on the side of the sink and absorbent paper to pour off the liquid.
7. Wash once with 100 ul PBS.
8. Add 100 ul of cell suspension per well (containing the appropriate amount of cells and the appropriate concentration of stimulant). The cells can be pre-excited in vitro (indirect Eli-spot). Cover with a standard 96-well plate plastic plate and incubate for a certain period of time (15-20 hours) in a 37 ° C CO2 incubator. Do not shake or move the orifice during this time.
9. Tap on the side of the sink and absorbent paper to pour off the liquid.
10. Add 100 ul of wash buffer to each well and incubate for 10 minutes at 4 °C.
11. Wash the wells 3 times with 100 ul of wash buffer.
12. Dilute 100 ul of detection antibody in 10 ml PBS-1% BSA, which is the amount of one plate. Add 100 ul of this liquid to each well, cover the plate, and incubate for 1 hour and 30 minutes at 37 °C.
13. Pour off the liquid and wash 3 times with 100 ul of wash buffer.
14. Dilute 10 ul of avidin alkaline phosphatase per plate with 10 ml PBS-1% BSA. 100 ul of this liquid was added to each well, and the plate was capped and incubated at 37 ° C for 1 hour.
15. Pour off the liquid and wash 3 times with 100 ul of wash buffer. Pat on absorbent paper to blot out the remaining wash solution.
16. Add 100 ul of BCIP/NBT to each well.
17. The reaction is carried out for 5-15 minutes at room temperature. After full color development, pour this solution into the appropriate tray.
18. Wash both sides of the membrane thoroughly with distilled water. Tap on absorbent paper to dry the film. When storing, invert the plate to prevent residual liquid from flowing back onto the film. Once the film is dry, read the number of dots. Placed at 4 ° C for one night, the point will be more obvious.

This plate is stored in the dark at room temperature.

When using, it should be noted that BCIP/NBT buffer is a potential to cancerous substance. Gloves should be taken during the test and treated properly after use. On the MAIPAN4510 plate, the reagent penetrates into the membrane due to capillary action during incubation, and the liquid is difficult to wash away, thus causing an increase in background. To avoid this, it is recommended to remove the bottom of the plate at step 12, invert the membrane, rinse with a stream of distilled water, and wash with washing buffer in the normal procedure. In step 14, since the bottom of the plate has been removed, it is recommended to place the MAIP4510 plate on an empty 96-well plate, with the subsequent steps unchanged.

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