The experimental principle and operation steps of ELISA

1. Experimental principle

ELISA is a highly sensitive test technique based on immunological reaction, which combines the specific reaction of antigen and antibody with the efficient catalysis of enzyme to substrate. Since the reaction of antigen and antibody is carried out in a solid phase carrier — polystyrene microtitration plate, after each addition of a reagent to incubate, excess free reactants can be removed by washing, so as to ensure the specificity and stability of the test results.In practical application, through different designs, specific methods and steps can have a variety of.The indirect method used to detect antibodies, the double antibody sandwich method used to detect antigens, and the antigen competition method used to detect small molecule antigens or haptens, etc.More commonly used are double antibody sandwich ELISA method and indirect ELISA method.

2. Experimental materials

1, Reagent

(1) encapsulation buffer (PH9.6 0.05M carbonate buffer):

Naco3:1.59g, NaHCO3:2.93g, add distilled water to 1000ml

(2) Washing buffer (PH7.4 PBS) : 0.15mkH2PO4 0.2g Na2HPO4· 12H2O2.9g NaCl 8.0g KCl 0.2g Tween-20 0.05% 0.5mL distilled water to 1000mL

(3) Diluant: Bovine serum albumin (BSA) 0.1g add washing buffer to 100ml or mix sheep serum, rabbit serum and other serum with washing solution to 5-10% for use.

(4) Termination solution (2M H2SO4) : 178.3ml distilled water, adding 21.7ml concentrated sulfuric acid (98%) drop by drop.

(5) Substrate buffer (PH5.0 phosphate jujube citric acid) : 0.2m Na2HPO4(28.4 g /L) 25.7ml 0.1m citric acid (19.2 g /L) 24.3mL plus distilled water 50mL.

Solution: TMB(10mg/ 5mL absolute ethanol) 0.5mL Substrate buffer (PH5.5) 10mL 0.75% H2O232μ L

(7)ABTS solution: ABTS0.5mg substrate buffer (PH5.5) 1ML 3% H2O22μ L

(8) Antigen, antibody and enzyme labeled antibody.

(9) Normal serum and positive control serum.

2. The equipment:

(1) Polystyrene plastic plate (referred to as ELISA plate)40 or 96 well, ELISA tester, 50μ L and 100μ L sampler, plastic dripper, small towel, washing bottle.

(2) Small beaker, glass rod, test tube, straw and measuring cylinder, etc.

(3)4℃ refrigerator, 37℃ incubator.

3. Experimental steps

1. Double antibody sandwich method for detecting unknown antigens:

2. Coating: Use 0.05M PH9. Carbonate coating buffer to dilute the antibody to a protein content of 1-10μg/ml. Add 0.1ml to the reaction well of each polystyrene plate, overnight at 4°C. The next day, discard the solution in the well and wash with washing buffer 3 times, 3 minutes each time. (Referred to as washing, the same below).

3. Adding samples: add 0.1ml of a certain diluted sample to be tested into the above-mentioned coated reaction well, and incubate at 37°C for 1 hour. Then wash. (Make blank holes, negative control holes and positive control holes at the same time).

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

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

6. To terminate the reaction: add 0.05ml of 2M sulfuric acid to each reaction well.

7. Judgment of the result: You can directly observe the result with the naked eye on a white background: the darker the color in the reaction hole, the stronger the positive degree, and the negative reaction is colorless or extremely light. The “-” sign means. O·D value can also be measured: on an ELISA tester, at 450nm (if ABTS is used for color development, 410nm), use the blank control hole to zero and then measure the O·D value of each hole, if it is greater than the specified negative control OD 2.1 times the value is considered positive.

8. Indirect method for detecting unknown antibodies:

Dilute the known antigen to 1-10μg/ml with coating buffer, add 0.1ml to each well, overnight at 4°C. Wash 3 times the next day. Add 0.1ml of diluted sample (unknown antibody) to the above-mentioned coated reaction well, incubate at 37°C for 1 hour, and wash. (Control the blank, negative and positive wells at the same time) Add 0.1ml of freshly diluted enzyme-labeled secondary antibody (anti-antibody) to the reaction well, incubate at 37°C for 30-60 minutes, wash, and then wash with DDW. The rest of the steps are the same as 4, 5, and 6 of “Double antibody sandwich method”.

4.  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 high, indicating a non-specific reaction, which can be blocked with goat serum, rabbit serum or BSA.

2. 　In ELISA, the selection of various experimental conditions is very important, including:

(1) “The choice of solid-phase carrier: Many substances can be used as solid-phase carriers, such as polyvinyl chloride, polystyrene, polyacrylamide, and cellulose. The form can be a concave plate, test tube, beads, etc. At present, a 40-hole polystyrene concave plate is commonly used. No matter what kind of carrier, it can be screened before use: Coating with the same amount of antigen, reacting under the same experimental conditions, observing whether the color reaction is uniform, and judging whether its adsorption performance is good.

(2) The choice of coating antibody (or antigen): when the antibody (or antigen) is adsorbed on the surface of the solid phase carrier, the purity is required, and the pH is generally required to be between 9.0 and 9.6 during adsorption. Adsorption temperature, time and protein content also have a certain influence, generally 4℃ for 18-24 hours. The appropriate concentration of protein coating needs to be titrated: that is, after coating with different protein concentrations (0.1, 1.0 and 10μg/ml, etc.), under the same other test conditions, observe the OD value of the positive sample. Choose a concentration with a large OD value and a small amount of protein. For most proteins, it is usually 1-10μg/ml.

(3) “Selection of the working concentration of enzyme-labeled antibody: First, use the direct ELISA method to perform preliminary titer titration (see the section of enzyme-labeled antibody). Then fix other conditions or adopt the “square matrix method” (different dilutions of the coating material, the reference product of the sample to be tested, and the enzyme-labeled antibody) in the formal experimental system to accurately titrate its working concentration.

(4) The choice of enzyme substrate and hydrogen donor: The choice of hydrogen donor is cheap, safe, and has obvious color reaction, and it is colorless. Some hydrogen donors (such as OPD, etc.) have potential carcinogenic effects and should be protected. Those who have the conditions should use non-carcinogenic and highly sensitive hydrogen donors. For example, TMB and ABTS are currently more satisfactory hydrogen donors. After the substrate has acted for a period of time, a strong acid or a strong base should be added to stop the reaction. Usually the substrate action time is 10-30 minutes. The substrate liquid must be freshly prepared, especially H2O2 is added before use.

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