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ELISA is an abbreviation for Enzyme-Linked Immunosorbent Assay. It is an immunoenzyme technology developed after immunofluorescence and radioimmunoassay. Since its inception in the early 1970s, this technology has developed rapidly and is now widely used in many fields of biology and medical science.

(a) Principle

ELISA is a highly sensitive test technique based on immunological reactions that combines the specific reaction of antigens and antibodies with the efficient catalytic action of enzymes on substrates. Since the reaction of the antigen and the antibody is carried out in the well of a solid phase carrier, a polystyrene microtiter plate, after each reagent is added, the excess free reactant can be removed by washing to ensure the specificity of the test result. And stability. In practical applications, there are many specific method steps through different designs. Namely: an indirect method for detecting antibodies (panel a), a double antibody sandwich method for detecting antigens (panel b), an antigen competition method for detecting small molecule antigens or haptens, and the like. More commonly used are ELISA double antibody sandwich method and ELISA indirect method.

(2) Operation steps

Method 1 Double antibody sandwich method for detecting unknown antigens:

1. Coat: The antibody was diluted to a protein content of 1 to 10 μg/ml with 0.05 M PH9. 牰 carbonate coating buffer. 0.1 ml was added to the reaction well of each polystyrene plate at 4 ° C overnight. The next day, the solution in the well was discarded and washed 3 times with washing buffer for 3 minutes each time. (referred to as washing, the same below).

2. Loading: Add 0.1 ml of the sample to be tested to the above-mentioned coated reaction well, and incubate at 37 ° C for 1 hour. Then wash. (Do blank holes, negative control wells and positive control wells at the same time).

3. Add enzyme-labeled antibody: Add 0.1 ml of freshly diluted enzyme-labeled antibody (diluted titration) to each well. Incubate at 37 ° C for 0.5 to 1 hour and wash.

4. Add substrate solution to develop color: Add 0.1 ml of temporarily prepared TMB substrate solution to each reaction well at 37 ° C for 10 to 30 minutes.

5. Stop the reaction: Add 0.05 ml of 2M sulfuric acid to each reaction well.

6. Judgment of results: The results can be observed directly on the white background with the naked eye: the darker the color in the reaction well, the stronger the positive degree, the negative reaction is colorless or very light, according to the depth of the color, with "+", The "-" sign indicates. The OD value can also be measured: on the ELISA detector, at 450 nm (if the color is developed by ABTS, 410 nm), the OD value of each well is measured after zero adjustment of the blank control well, if it is greater than 2.1 times the OD value of the specified negative control. Is positive.

Method 2 Indirect method for detecting unknown antibodies:
Dilute the known antigen to 1 to 10 μg/ml with a coating buffer.
Add 0.1 ml to each well and overnight at 4 °C. Wash 3 times the next day.
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Add 0.1ml of the sample to be tested (unknown antibody) to the above-mentioned coated reaction well
Incubate at 37 ° C for 1 hour and wash. (while doing blank, negative and positive hole comparison)
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Add 0.1 ml of freshly diluted enzyme-labeled secondary antibody (anti-antibody) to the reaction well.
Incubate at 37 ° C for 30-60 minutes, wash, and wash with DDW for the last time.
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The remaining steps are the same as the "double antibody sandwich method" 4, 5, and 6.

(3) Reagent equipment

Reagent

(1) Coating buffer (pH 9.6 0.05M carbonate buffer):
NaHCO3 1.59g
NaHCO3 2.93g
Add distilled water to 1000ml

(2) Wash buffer (pH 7.4 PBS): 0.15M
KH2PO4 0.2g
Na2HPO4•12H2O 2.9 g
NaCl 8.0 g
KCl 0.2 g
Tween-20 0.05% 0.5ml
Add distilled water to 1000ml

(3) Diluent:
Bovine serum albumin (BSA) 0.1 g
Add washing buffer to 100ml
Or use serum such as sheep serum, rabbit serum and washing liquid to prepare 5 to 10%.

(4) Stop solution (2M H2SO4):
178.3 ml of distilled water, 21.7 ml of concentrated sulfuric acid (98%) was added dropwise.

(5) Substrate buffer (pH 5.0 phosphoric acid citric acid):
0.2M Na2HPO4 (28.4 g / L) 25.7ml
0.1M citric acid (19.2 g / L) 24.3ml
Add 50 ml of distilled water.

(6) TMB (tetramethylbenzidine) use solution:
TMB (10mg/5ml absolute ethanol) 0.5ml
Substrate Buffer (pH 5.5) 10ml
0.75% H2O2 32μl

(7) ABTS use liquid:
ABTS 0.5mg
Substrate Buffer (pH 5.5) 1ml
3% H2O2 2μl

(8) Antigen, antibody and enzyme-labeled antibodies.

(9) Normal human serum and positive control serum.

2. Equipment:

(1) Polystyrene plastic plate (referred to as microplate) 40-well or 96-well, ELISA detector, 50μl and 100μl sampler, plastic dripper, small towel, wash bottle.

(2) Small beakers, glass rods, test tubes, straws, and measuring cylinders.

(3) 4 ° C refrigerator, 37 ° C incubator.

(four) matters needing attention

1. In the formal test, the test conditions should be controlled by the positive control and the negative control respectively, and the samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is higher, indicating a non-specific reaction, which can be blocked by sheep serum, rabbit serum or BSA.

2. In the ELISA, it is important to select the various experimental conditions, including:

(1) Selection of solid phase support: Many substances can be used as solid phase carriers such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. The form may be a flat plate, a test tube, a bead or the like. Currently used is a 40-well polystyrene recessed plate. Regardless of the carrier, screening can be carried out before use: the reaction is carried out under the same experimental conditions by coating with an equal amount of antigen, and whether the coloration reaction is uniform or not, and whether the adsorption performance is good.

(2) Selection of coated antibody (or antigen): When the antibody (or antigen) is adsorbed on the surface of the solid phase carrier, the purity is required to be good, and the pH is generally required to be between 9.0 and 9.6. The adsorption temperature, time and the amount of protein also have a certain influence, and generally use 4 ° C for 18 to 24 hours. The optimum concentration of protein coating is titrated: after coating with different protein concentrations (0.1, 1.0, and 10 μg/ml, etc.), the OD value of the positive specimen is observed when the other test conditions are the same. The concentration with the highest OD value and the least amount of protein is selected. It is usually 1 to 10 μg/ml for most proteins.

(3) Selection of working concentration of enzyme-labeled antibody: First, titration of preliminary titer is carried out by direct ELISA (see enzyme-labeled antibody fraction). Then, other conditions are fixed or the "square matrix method" (the coating, the reference sample of the sample to be tested, and the enzyme-labeled antibody are respectively different dilutions) are accurately titrated in the formal experimental system.

(4) Enzyme substrate and hydrogen donor selection: The choice of hydrogen donor is cheap, safe, and has obvious color reaction, but it is colorless. Some hydrogen donors (such as OPD) have potential carcinogenic effects and should be protected. Those who are qualified should use hydrogen donors that are not carcinogenic and sensitive. For example, TMB and ABTS are currently satisfactory hydrogen donors. After the substrate has been applied for a while, a strong acid or a strong base should be added to terminate the reaction. Usually the substrate action time is preferably 10-30 minutes. The substrate used must be freshly prepared, especially H2O2 before use.

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