1、 Experimental principle

ELISA is a highly sensitive experimental technique that combines the specific reactions of antigens and antibodies with the efficient catalytic activity of enzymes on substrates, based on immunological reactions. Due to the reaction of antigens and antibodies taking place in the pores of a solid-phase carrier - polystyrene microtiter plate, after incubation with each reagent added, excess free reactants can be removed by washing to ensure the specificity and stability of the test results. In practical applications, there can be multiple specific method steps through different designs. Namely, indirect methods for detecting antibodies, double antibody sandwich methods for detecting antigens, and antigen competition methods for detecting small molecule antigens or haptens, etc. The commonly used methods are ELISA double antibody sandwich method and ELISA indirect method.

2、 Experimental materials

1. Reagent

(1) Coating buffer (pH 9.6 0.05M carbonate buffer):

NaCO3: 1.59g, NaHCO3: 2.93g, add distilled water to 1000ml

(2) Wash buffer (PH7.4 PBS): 0.15M KH2PO4 0.2g Na2HPO4 · 12H2O 2.9g NaCl; 8.0g KCl 0.2g Tween-20 0.05%; Add 0.5ml distilled water to 1000ml

(3) Dilution solution: Add 0.1 grams of bovine serum albumin (BSA) to 100 milliliters of washing buffer, or use sheep serum, rabbit serum, etc; Mix serum and detergent to make 5-10% for use.

(4) Termination solution (2M H2SO4): Distilled water 178.3ml, add concentrated sulfuric acid (98%) 21.7ml dropwise.

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

(6) TMB (Tetramethylbenzidine) Solution: TMB (10mg/5ml anhydrous ethanol) 0.5ml substrate buffer (pH 5.5) 10ml 0.75% H2O2 32 μ l

(7) ABTS solution: ABTS 0.5mg substrate buffer (pH 5.5) 1ml 3% H2O2 2 μ l

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

(9) Normal human serum and positive control serum.

2. Equipment:

(1) Polystyrene plastic plate (referred to as enzyme-linked immunosorbent assay plate) with 40 or 96 wells, ELISA detector, 50 μ l and 100 μ l sampler, plastic dropper, small towel, washing bottle。

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

(3) 4 ℃ refrigerator, 37 ℃ incubator.

3、 Experimental steps

（一）Double antibody sandwich method for detecting unknown antigens:

1. Coating: Dilute the antibody with 0.05M PH9. carbonate coating buffer to a protein content of 1-10 μ g/ml. Add 0.1ml to each reaction well of polystyrene board and overnight at 4 ℃. The next day, discard the solution in the well and wash it three times with washing buffer for 3 minutes each time. (referred to as washing, the same below).

2. Sample addition: Add 0.1ml of the sample to be tested with a certain dilution to the coated reaction well mentioned above, and incubate at 37 ℃ for 1 hour. Then wash it. (Simultaneously perform blank wells, negative control wells, and positive control wells).

3. Add enzyme-linked immunosorbent assay (ELISA) antibody: Add 0.1ml of freshly diluted ELISA antibody (diluted by titration) to each reaction well. Incubate at 37 ℃ for 0.5-1 hour and wash.

4. Add substrate solution for color development: Add 0.1ml of temporarily prepared TMB substrate solution to each reaction well and incubate at 37 ℃ for 10-30 minutes.

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

6. Result judgment: It can be directly observed with the naked eye on a white background. The darker the color inside the reaction well, the stronger the positivity. Negative reactions are colorless or extremely light, and are represented by "+" and "-" according to the depth of the color. The O · D value can also be measured: on an ELISA detector, at 450nm (410nm if ABTS color is used), zero the blank control well and measure the O · D value of each well. If it is greater than 2.1 times the specified negative control OD value, it is considered positive.

（two）Indirect method for detecting unknown antibodies:

Dilute the known antigen with coating buffer to 1-10 μ g/ml, add 0.1ml to each well, and overnight at 4 ℃. Wash three times the next day. Add 0.1ml of the test sample (unknown antibody) with a certain dilution to the coated reaction well mentioned above, incubate at 37 ℃ for 1 hour, and wash. (Simultaneously perform blank, negative, and positive well controls) Add 0.1ml of freshly diluted enzyme-linked secondary antibody (anti antibody) to the reaction well, incubate at 37 ℃ for 30-60 minutes, wash, and then wash again with DDW. The remaining steps are the same as steps 4, 5, and 6 of the "double antibody sandwich method".

4、 Precautions

1.During the formal experiment, positive and negative controls should be used to control the experimental conditions, and two copies of the test sample should be prepared to ensure the accuracy of the experimental results. Sometimes a high background indicates non-specific reactions, which can be blocked with sheep serum, rabbit serum, or BSA.

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

(1) Selection of Solid Phase Carriers: Many substances can be used as solid phase carriers, such as polyvinyl chloride, polystyrene, polyacrylamide, and cellulose. Its form can be concave hole flat plate, test tube, bead, etc. The currently commonly used is 40 hole polystyrene concave hole plate. Regardless of the type of carrier, it can be screened before use: coat it with an equal amount of antigen, conduct the reaction under the same experimental conditions, observe whether the color reaction is homogeneous, and determine whether its adsorption performance is good based on this.

(2) Selection of coated antibodies (or antigens): When adsorbing antibodies (or antigens) on the surface of a solid carrier, good purity is required, and the pH during adsorption is generally required to be between 9.0 and 9.6. The adsorption temperature, time, and protein content also have a certain impact, generally using 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 10 μ g/ml, etc.), observe the OD value of positive specimens under the same experimental conditions. Choose a concentration with a high OD value and low protein content. For most proteins, it is usually 1-10 μ g/ml.

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

(4) The selection of substrates and hydrogen donors for enzymes: The requirements for selecting hydrogen donors are low cost, safety, clear color reaction, and colorless nature. Some hydrogen donors (such as OPD) have potential carcinogenic effects and should be protected. Conditional individuals should use non carcinogenic and highly sensitive hydrogen donors, such as TMB and ABTS, which are currently satisfactory hydrogen donors. After a period of substrate action, strong acid or strong base should be added to terminate the reaction. Normally, the substrate action time should be 10-30 minutes. The substrate solution must be freshly prepared, especially H2O2 added before use.

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