Indirect ELISA Protocol


ELISA is a highly sensitive test technique based on immunological reactions that combine 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-polystyrene microtiter plate, after each reagent is added, the excess free reactant can be removed by washing to ensure the specificity and stability of the test result. In ELISA, indirect ELISA is useful for antibody screening, epitope mapping, and protein quantification. The secondary antibody serves to enhance the signal of the primary antibody, which makes it more sensitive than direct ELISA. However, it also produces a higher background signal and potentially decreases the overall signal.

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Indirect ELISA Protocol

Figure 1. Indirect ELISA for the colorimetric detection of antigen-specific antibodies.


  • Coating buffer (pH 9.6 0.05M carbonate buffer): Na2CO3 1.59g, NaHCO3 2.93g, add distilled water to 1000ml.
  • Wash buffer (pH 7.4 0.15M PBS): KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.05% 0.5ml, add distilled water to 1000ml.
  • Diluent: Bovine serum albumin (BSA) 0.1g, add washing buffer to 100ml or use serum of sheep serum, rabbit serum and other washing liquid to prepare 5-10%.
  • Stop solution (2M H2SO4): 178.3 ml of distilled water was added dropwise with concentrated hydrochloric acid (98%) 21.7 ml.
  • Substrate buffer (pH 5.0 phosphorylated citric acids): 0.2M Na2HPO4 (28.4g/L) 25.7ml, 0.1M citric acid (19.2g/L) 24.3ml, add 50 ml of distilled water.
  • TMB (tetramethylbenzidine) solution: TMB (10mg/5ml absolute ethanol) 0.5ml, Substrate Buffer (pH 5.5) 10ml, 0.75% H2O2 32μl.
  • ABTS liquid: ABTS 0.5mg, Substrate Buffer (pH 5.5) 1ml, 3% H2O2 2μl.
  • Antigen, antibody and enzyme-labeled antibodies.
  • Normal human serum and a positive control serum.


  • Polystyrene plastic plate (referred to as microplate) 40 holes or 96 holes.
  • ELISA detector.
  • Incubators.


1. Dilute the known antigen to 1 to 10 μg/ml with a coating buffer, add 0.1 ml per well, and overnight at 4 °C. Wash 3 times the next day.

2. Add a certain dilution of the sample to be tested (unknown antibody) 0.1 ml in the above-mentioned coated reaction well, incubate at 37 ° C for 1 hour, and wash. (while doing blank, negative and positive hole comparison)

3. Add 0.1 ml of freshly diluted enzyme-labeled secondary antibody (anti-antibody) to the well, incubate at 37 ° C for 30-60 minutes, wash, and wash with DDW for the last time.

4. Adding substrate liquid color: 0.1 ml of a temporarily prepared TMB substrate solution was added to each reaction well at 37 ° C for 10 to 30 minutes.

5. Termination reaction: 0.05 ml of 2 M sulfuric acids was added to each reaction well.

6. Determination of results: Measured OD value: on the ELISA detector, at 450 nm (if ABTS color development, 410 nm), the blank control well is adjusted to zero and then the OD value of each well is measured. If the value is 2.1 times greater than the specified negative control, which is positive.


1. In the formal test, the test conditions should be controlled by the positive control and negative control respectively, and the sample on the detecting 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.

Indirect ELISA Protocol

Figure 2. Antibody–antigen checkerboard titration format.

(3) Selection of working concentration of enzyme-labeled antibody

First, titration of preliminary titer is carried out by direct ELISA. 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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