Experimental Protocols for Direct, Indirect, & Sandwich ELISAs

Enzyme-linked immunosorbent assays (ELISAs) use the catalytic properties of enzymes to detect and quantify immunologic reactions in research applications and clinical analyses. In ELISA a specific reaction component is nonspecifically adsorbed or covalently bound to, typically, a 96-well plate. Depending on the ELISA, testing includes a primary and/or secondary detection antibody (coating), an analyte and antigen, addition of buffer to remove nonspecific binding (blocking), wash steps, and substrate or chromogen detection.

The substrates used in ELISA will generate a color that can be assessed once the reaction is complete; common substrates include horseradish peroxidase (HRP) and alkaline phosphatase (AP) which will turn the reaction plate blue and yellow, respectively. Upon analysis, the fluorescent absorbance of these colors is directly proportional to the concentration of the detection antibody within each sample. Meaning, the deeper the color the more protein present.

Construction of a Standard Curve

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To precisely quantitate the amount of antigen present in each well, an experimental standard curve must be created during each experiment. Typically, an ELISA measures protein concentrations in the range of 0.01-0.1 ng, however this depends on the antibody-antigen interaction. Therefore the classic standard curve ranges from 0-1000 or 0-2000 pg/ml. The protocol is as follows:

1. Label twelve 1.5 mL microcentrifuge tubes 1-12.
2. Add the appropriate amount of PBS or buffer to each microcentrifuge tube, listed in the table below.
3. Reconstitute the standard with an 1mL of PBS or buffer. The concentration will be 1 µg/mL.
4. Add 10 µL of standard to tube 1. Gently Mix by pipetting.
5. Add 200 µL of tube 1 to tube 2. Gently Mix by pipetting.
6. Add 500 µL of tube 2 to tube 3. Gently Mix by pipetting.
7. Moving forward, continue the process for tubes 4-11 per the table below. Reference the table below for the amount to transfer from one tube to the next.
8. Do not add anything to tube 12 as this will be used as the blank control.

Step	Procedure	Samples
1	Label tubes	1	2	3	4	5	6	7	8	9	10	11	12
2	Add PBS (µL)	990	800	500	500	500	500	500	500	500	500	500	500
4-8	Transfer amount from previous tube into current tube (µL)	10 Std	200 Tube 1	500 Tube 2	500 Tube 3	500 Tube 4	500 Tube 5	500 Tube 6	500 Tube 7	500 Tube 8	500 Tube 9	500 Tube 10	N/A
N/A	Concentration (pg/mL)	1e5	200	100	500	250	125	62.5	31.2	15.6	7.8	3.9	0

Tool:

Serial Dilution Calculator

Direct ELISA Protocol

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In direct ELISA, plates are coated with a purified antigen and only one antibody is used. In direct ELISA, the antigen binds directly with the enzyme conjugated primary antibody. The complex formed onto the plate is: antigen + primary antibody. If using already coated plates, skip to step 4.

1. Antigen preparation
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the antigen in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL)
2. Coat the plate with antigen
   
   1. Coat each well of a microtiter plate with 100 µl of the diluted antigen.
   2. Seal the plate and incubate overnight at 4°C, or for at least 30 minutes at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS, Wait 5 minutes between each wash.
      1. After each wash, invert and flick the back of the plate over the sink.
      2. After each wash, pat the plate onto a paper towel to remove remaining drops.
      3. Do not allow the plate to completely dry.
3. Blocking
   1. Pipet 200 µl blocking buffer or BSA to each well.
   2. Cover the plate with an adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4°C.
4. Prepare the standard solutions, as shown in Construction of a Standard Curve.
5. Add 100 µL of each standard into appropriate wells of the plate.
6. Prepare the primary antibodies
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the primary antibody in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL).
7. Preparing the plate with primary antibody
   1. Add 100 µL of the diluted primary antibody to each well.
   2. Seal the plate and incubate for 2 hours at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS, following the same steps in section 2.
8. Prepare the substrate solution immediately before use according to the manufacturer's protocol
9. Add 90 µL substrate solution into each well including the standard.
10. Seal the plate and incubate at room temperature in the dark.
    1. Covering the plate with aluminum foil is recommended.
    2. Incubation times depend on the manufacturer's protocol, generally 15 - 30 minutes.
11. If necessary, add 100 µL of stop solution to each well.
12. Immediately read the optical density (OD) of each well using a plate reader.
    1. Prepare a standard curve using the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale)
    2. Direct ELISA will yield a normal curve, meaning higher values of antigen in the samples yield a higher amount of color change.

Indirect ELISA Protocol

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In indirect ELISA, plates are coated with a purified antigen and a primary and secondary antibody are used. In indirect ELISA, the antigen binds to an unconjugated primary antibody, which then binds to an enzyme conjugated secondary antibody. The complex formed onto the plate is: antigen + primary antibody + secondary antibody. If using already coated plates, skip to step 4.

1. Antigen preparation
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the antigen in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL)
2. Coat the plate with antigen
   1. Coat each well of a microtiter plate with 100 µl of the diluted antigen.
   2. Seal the plate and incubate overnight at 4°C, or for at least 30 minutes at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS, Wait 5 minutes between each wash.
      1. After each wash, invert and flick the back of the plate over the sink.
      2. After each wash, pat the plate onto a paper towel to remove remaining drops.
      3. Do not allow the plate to completely dry.
3. Blocking
   1. Pipet 200 µl blocking buffer or BSA to each well.
   2. Cover the plate with an adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4°C.
4. Prepare the standard solutions, as shown in Construction of a Standard Curve.
5. Add 100 µL of each standard into appropriate wells of the plate.
6. Prepare the primary antibody
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the antibody in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL)
7. Preparing the plate with primary antibody
   1. Add 100 µL of the diluted primary antibody to each well.
   2. Seal the plate and incubate for 2 hours at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS, following the same steps in section 2.
8. Prepare the secondary antibody
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the secondary antibody in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL).
9. Preparing the plate with secondary antibody
   1. Add 100 µL of the diluted secondary antibody to each well.
   2. Seal the plate and incubate for 2 hours at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS, following the same steps in section 2.
10. Prepare the substrate solution immediately before use according to the manufacturer's protocol.
11. Add 90 µL substrate solution into each well, including the standard.
12. Seal the plate and incubate at room temperature in the dark.
    1. Covering the plate with aluminum foil is recommended.
    2. Incubation times depend on the manufacturer's protocol, generally 15 - 30 minutes.
13. If necessary, add 100 µL of stop solution to each well.
14. Immediately read the optical density (OD) of each well using a plate reader.
    1. Prepare a standard curve using the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale)
    2. Competitive ELISA will yield an inverse curve, meaning higher values of antigen in the samples yield a smaller amount of color change.

Sandwich ELISA Protocol

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In sandwich ELISA, plates are coated with a capture antibody (not the antigen, the primary, or secondary antibody). Sandwich ELISA can be direct if the primary antibody is enzyme conjugated, or indirect if the primary antibody is unconjugated requiring the use of a secondary antibody that is enzyme conjugated.

In direct sandwich ELISA the complex formed onto the plate is: capture antibody + antigen + primary antibody complex. In indirect sandwich ELISA the complex formed onto the plate is: capture antibody + antigen + primary antibody + secondary antibody. If using already coated plates, skip to step 4.

1. Capture antibody preparation
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the capture in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL)
2. Coat the plate with the capture antibody
   1. Coat each well of a microtiter plate with 100 µl of the diluted capture antibody.
   2. Seal the plate and incubate overnight at 4°C, or for at least 30 minutes at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS. Wait 5 minutes in between each wash.
      1. After each wash, invert and flick the back of the plate over the sink.
      2. After each wash, pat the plate onto a paper towel to remove remaining drops.
      3. Do not allow the plate to completely dry.
3. Blocking
   1. Pipet 200 µl blocking buffer or BSA to each well.
   2. Cover the plate with an adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4°C.
4. Prepare the standard solutions, as shown in Construction of a Standard Curve.
5. Add 100 µL of each standard into appropriate wells of the plate
6. Prepare the antigen
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the antigen in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL)
7. Preparing the plate with the antigen
   1. Add 100 µL of the diluted antigen to each well.
   2. Seal the plate and incubate for 2 hours at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS, following the same steps in section 2.
8. Prepare the primary antibody
   1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
   2. Dilute the primary antibody in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL)
9. Preparing the plate with primary antibody
   1. Add 100 µL of the diluted primary antibody to each well.
   2. Seal the plate and incubate for 2 hours at room temperature.
   3. Invert the plate, carefully but deliberately, over the sink.
   4. Wash plate 3 times with 200 µL PBS, following the same steps in section 2.
10. Prepare the secondary antibody
    1. Calculate the total volume needed for the assay by multiplying 100 µL per well. Add 200 - 300 µL extra to account for possible pipetting errors.
    2. Dilute the secondary antibody in PBS or buffer appropriately, depending on the protocol (e.g., for a final concentration of 10 µg/mL)
11. Preparing the plate with secondary antibody
    1. Add 100 µL of the diluted secondary antibody to each well.
    2. Seal the plate and incubate for 2 hours at room temperature.
    3. Invert the plate, carefully but deliberately, over the sink.
    4. Wash plate 3 times with 200 µL PBS, following the same steps in section 2.
12. Prepare the substrate solution immediately before use according to the manufacturer's protocol.
13. Add 90 µL substrate solution into each well, including the standard.
14. Seal the plate and incubate at room temperature in the dark.
    1. Covering the plate with aluminum foil is recommended.
    2. Incubation times depend on the manufacturer's protocol, generally, 15 - 30 minutes.
15. If necessary, add 100 µL of stop solution to each well.
16. Immediately read the optical density (OD) of each well using a plate reader.
    1. Prepare a standard curve using the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale)
    2. Direct sandwich ELISA will yield a normal curve, meaning higher values of antigen in the samples yield a higher amount of color change.
    3. Competitive sandwich ELISA will yield an inverse curve, meaning higher values of antigen in the samples yield a smaller amount of color change.

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References

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