ELISA Assays: Indirect, Sandwich, and Competitive

1. Indirect ELISA

An indirect ELISA is one where the primary antigen-specific antibody is recognized by a secondary conjugated antibody. The following protocol is an example of an indirect ELISA method, where the serum samples of of influenza A virus (IAV)-infected mice are tested for the presence of IAV-specific IgG antibody. One strength of this example is that different secondary antibodies can be used that recognize all antibody isotypes or specific isotypes (e.g., IgG).

Coating antigen to the microplate

1. Coat the wells of a 96-well ELISA plate with purified antigen by pipetting 50 µL of purified antigen (2 mg/mL of purified A/PR/8 Influenza A virus in 0.05M Tris-HCl buffer (pH 9.5)) into each well of the plate.
2. Cover the plate with an adhesive cover and incubate it overnight at 4°C to allow the antigen to bind to the plate.
3. Upon incubation completion, remove the coating solution by flicking the plate over a sink.

Blocking

4. Block the remaining protein-binding sites in the coated wells by adding 200 µL blocking buffer, 5% donkey serum in 1X PBS is used here, per well. Alternative blocking reagents include 5% non-fat dry milk or BSA in PBS or normal serum from an animal in which the secondary antibody was generated.
5. Incubate for at least 2 hours at room temperature or overnight at 4°C.
6. Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.

Incubation with the primary antibody

7. Prepare a serial dilution of the serum sample, which contains the primary antibody, to obtain a dilution range of 1 to 204,800, using 1X PBS. To do this, first dilute the serum 1:12.5 and then perform a 4X dilution (dilution range - 1:12.5 to 1:204,800).
8. Add 100 µL of the serially-diluted serum samples to the wells.
9. Cover plate with adhesive cover and incubate at room temperature for 1-2 h.
10. Following the incubation, flick the plate over a sink and wash plate with PBS containing 1% Tween-20.

Incubation with the secondary antibody

11. Add 100 µL of an enzyme-conjugated secondary antibody, horseradish peroxidase, HRP-conjugated donkey anti-mouse secondary in this experiment, to each well.
12. Incubate the plate for 1 hour at room temperature.
13. Following the incubation, flick the plate over a sink and then wash plate with PBS containing 1% Tween-20.

Detection

14. Add 100 µL of the indicator substrate (3,3',5,5'-tetramethylbenzidine (TMB)) at a concentration of 1 mg/mL to each well.
15. Incubate the plate with the substrate for 5-10 min at room temperature.
16. After 10 min, stop the enzymatic reaction by adding 100 µL 2N Sulfuric acid (H₂SO₄).
    Within 30 min of adding the stop solution, read the plate using a microplate reader at 405 nm to determine the absorbance of the wells.

2. Sandwich ELISA

In this ELISA version, the experimental sample is "sandwiched" between an unconjugated capture antibody and a conjugated detection antibody, both of which are specific to the same protein but at different epitopes. In the following sandwich ELISA example, concentration of human TNFα was determined in unknown sample using a standard curve generated from 2.5X serial dilution of a known standard, recombinant human TNFα (stating at concentration of 75 pg/mL).

Coating capture antibody to the microplate

1. Coat the wells of a 96-well ELISA plate with purified capture antibody by adding 100 µL of capture antibody (1-10 µg/mL range) to each well of the plate.
2. Cover plate with an adhesive plate cover and incubate it overnight at 4°C.
3. After incubation, remove the coating solution from plate by flicking the plate over a sink.

Blocking

4. Block the remaining protein-binding sites in the antibody coated wells by adding 200 µL blocking solution, 5% nonfat dry milk containing PBS, to the wells.
5. Incubate for at least 2 h at room temperature or overnight at 4°C.
6. Following the incubation, remove the blocking buffer by flicking the plate and then wash plate with PBS containing 1% Tween-20.

Add antigen containing test samples

7. Add 100 µL of the test sample to the wells. Seal the plate with an adhesive cover.
8. Incubate for 1-2 h at room temperature or overnight at 4°C.
9. After incubation, remove the samples by flicking the plate over the sink and then wash the wells with 200 µL 1X PBS containing 1% Tween-20.

Add enzyme-conjugated detection antibody

10. Add 100 µL of enzyme-conjugated detection antibody to the wells at a preoptimized concentration.
11. Seal the plate with an adhesive cover and incubate at room temperature for 2 h.
12. Remove the unbound detection antibody by flicking the plate over a sink and wash the wells with 200 µL 1X PBS containing 1% Tween-20.

Detection

13. Add 100 µL of the indicator substrate at a concentration of 1 mg/mL. Any bound enzyme-conjugated detection antibody will convert the substrate to a detectable signal.
14. Incubate the plate for 5-10 min at room temperature.
15. After 5-10 min, stop the enzymatic reaction by adding 100 µL 2N H₂SO₄ to the wells. Within 30 min of adding the stop solution, read the plate using a microplate reader to determine the absorbance of the wells.

3. Competitive ELISA

The steps of a competitive ELISA are different from those used in indirect and sandwich ELISA, with the main difference being the competitive binding step between the sample antigen and the "add-in" antigen. The sample antigen is incubated with the unlabeled primary antibody. These antibody-antigen complexes are then added to the ELISA plate, which has been pre-coated with the same antigen. After an incubation period, any unbound antibody is washed away. There is an inverse correlation between the amount of free antibody available to bind the antigen in the well and the amount of antigen in the original sample. For example, a sample with abundant antigen would have more antigen-primary antibody complexes, leaving little unbound antibody to bind to the ELISA plate. An enzyme-conjugated secondary antibody specific to the primary antibody is then added to the wells, followed by the substrate.

Coating antigen to the microplate

1. Coat the wells of a 96-well ELISA plate with 100 μL of purified antigen at a concentration of 1-10 μg/mL.
2. Cover plate with an adhesive plate cover and incubate the plate overnight at 4°C.
3. Following incubation, remove the unbound antigen solution from the wells by flicking the plate over a sink.

Blocking

4. Block the remaining protein-binding sites in the coated wells by adding 200 μL of blocking buffer to each well, which can be either 5% non-fat dry milk or BSA in PBS.
5. Incubate the plate for at least 2 h at room temperature or overnight at 4°C.

Incubation sample (antigen) with the primary antibody

6. While blocking the wells, prepare the antigen-antibody mixture by mixing 150 μL sample antigen and 150 μL of primary antibody for each well in the assay.
7. Incubate this mixture for 1 h at 37°C.

Add antigen-antibody mixture to the well

8. Now, remove the blocking buffer from the wells by flicking the plate over a sink.
9. Then, wash the wells with 1X PBS containing Tween-20.
10. Add 100 μL of the sample antigen-primary antibody mixture.
11. Incubate the plate at 37°C for 1 h.
12. Remove the sample mixture by flicking the plate over a sink.
13. Then, wash the wells with 1X PBS containing 1% Tween-20 to remove any unbound antibody.

Add the secondary antibody

14. Add 100 μL of an enzyme conjugated secondary antibody, which in this case is AP-conjugated antibody, to each well.
15. Incubate the plate for 1 h at 37°C.
16. Following incubation, wash the plate with 1X PBS containing 1% Tween-20.

Detection

17. Add 100 μL of the substrate solution to each well.
18. Wait for 5-10 min.
19. After 10 min, stop the enzymatic reaction by adding 100 μL 2N sulfuric acid to the wells. Then, measure the absorbance in a microplate reader within 30 min of adding the stop solution

In the following example of an indirect ELISA, the presence of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice was determined. C57Bl/6 mice were infected with influenza A virus (A/PR/8; 10⁵ PFU in 100 µL PBS i.p.) and serum was collected 28 days later. To quantitate the amount of IAV-specific IgG in the serum, 96-well ELISA plates were coated with purified A/PR/8 Influenza A virus (50 µL/well of 2 mg/ml PBS virus) overnight at 4°C. Coated plates were blocked for 1 hour at room temperature with 5% normal donkey serum in PBS, followed by incubation with diluted serum samples from IAV-challenged mice overnight at 4°C. The serum was initially diluted 1:12.5, followed by 1:4 dilutions (dilution range - 1:12.5 to 1:204,800). After washing, plates were incubated with an alkaline phosphatase (AP)-conjugated donkey anti-mouse IgG for 1 h. The plates were washed, and then p-Nitrophenyl Phosphate (PNPP; 1 mg/mL, 100 µL/well) was added. The colorless PNPP solution turns to a yellow color when AP is present. After 5-10 min, the enzymatic reaction was stopped by adding 100 µL/well 2N H₂SO₄. The plate was read on a microplate reader at 405 nm. The results obtained are shown in Table 1 and Figure 1.

Table 1: Indirect ELISA assay data. Serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-infected mice containing IAV-specific IgG, optical density (OD) (405 nm) values and mean OD₄₀₅ values.

Figure 1: Indirect ELISA assay scatter plot of mean OD₄₀₅ values(+ S. D.) and serum dilutions (from 1:12.5 to 1:204,800), of influenza A virus (IAV)-specific IgG in the serum of IAV-infected mice. The OD₄₀₅ values can be inversely correlated to the serum dilutions.

In the following example of a sandwich ELISA, a 1:2.5 dilution of recombinant human TNFα standards (starting at a concentration of 75 pg/mL) was added to the indicated wells of a 96-well flat-bottom plate. These standards led to a corresponding 2.5-fold change in the absorbance readings.

Table 2: TNFα Sandwich ELISA standard curve data. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL), OD (450 nm) values, mean OD₄₅₀ values, back concentration calculations and their averages.

Figure 2: Standard Curve for TNFα sandwich ELISA. A 1:2.5 dilution of recombinant human TNFα standards (75 to 0.3 pg/mL) was analyzed using sandwich ELISA.The OD₄₅₀ values can be directly correlated to the standard dilution concentrations. The amount of TNFα protein in the test sample was determined using the standard curve, which corresponds to a concentration of 38.72 pg/mL.

Once the standard curve was generated, the amount of TNFα protein in the test sample was determined. In this sandwich ELISA example, the test samples gave OD₄₅₀ readings of 0.636 and 0.681, which give an average of 0.6585. When plotting this OD450 reading on the above chart, this corresponds to a TNFα concentration of 38.72 pg/ml.

As demonstrated, a range of immunoassays (with slight variation in protocols) fall within the ELISA technique family. Determining which version of ELISA to use depends on a number of factors, including what antigen is being detected, the monoclonal antibody available for a particular antigen, and the desired sensitivity of the assay (5). Some strengths and weaknesses of the different ELISAs described herein are:

Table 3: Summary. A summary of the strengths and weaknesses of the different ELISA techniques.

While a simple and useful technique, there are also some drawbacks to any ELISA. One is the uncertainty of the amount of the protein of interest in the test samples. If the amount is too high or too low, the absorbance values obtained by the microplate reader may fall above or below the limits of the standard curve, respectively. This will make it difficult to accurately determine the amount of protein present in the test samples. If the values are too high, the test sample can be diluted prior to adding to the wells of the plate. The final values would then need to be adjusted according to the dilution factor. As mentioned, homemade kits often require careful optimization of the antibody concentrations used to yield a high signal-to-noise ratio.