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  • Antibody Validation Using the Indirect ELISA Method

Antibody Validation Using the Indirect ELISA Method

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Introduction

This protocol describes how to run an indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) against a purified antigen to validate an antibody. This protocol will describe how to create a standard curve using known concentrations of the antigen and how to probe with an antibody against the target to show a dose response.

Sharing speeds science. We believe that sharing the full details of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should produce similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this information is solely for transparency intended to support reproducibility in science.

General Considerations

The ELISA can be completed in a single day or broken up over several days by incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes when there are different incubation options available.

Caution
Use the Stop Solution in a well ventilated area and avoid breathing in the vapors.

Workflow Timeline

Day 1:
Antigen Coating
Day 2:
Blocking
Day 3:
Primary antibody incubation
Day 4:
Secondary antibody incubation and plate read

Tips and Troubleshooting

The optimal concentration of primary antibody to use will vary and needs to be empirically determined. If possible, run an initial test with serially diluted primary antibody and determine the best concentration for your assay.

Washing steps can be done by hand but if available, use an automated ELISA plate washer.

Equipment

  • Spectrophotometer compatible with 96-well plates
  • 1–10 µL single channel pipette
  • 2–20 µL single channel pipette
  • 20–200 µL single channel pipette
  • 200–1000 µL single channel pipette
  • 20–200 µL multichannel pipette
  • Multichannel pipette reagent reservoirs
  • Pipette controller
  • Microcentrifuge
  • Microplate shaker
  • ELISA plate washer (optional)
  • Scale
  • Microsoft Excel or similar software

Reagents

  • Purified antigen
  • Purified recombinant antibody
  • Isotype control antibody
  • HRP-conjugated isotype-specific secondary antibody
  • ELISA 96-well microplate, Corning 9018
  • Bovine serum albumin (BSA), VWR 10273-264
  • 50 mL conical tubes, VWR 89039-656
  • 96-well polyester (clear) microplate seal, Thermo Scientific 5701
  • Microcentrifuge tubes, Neptune 3745.X
  • Pipette tips, 1000 µL, VWR 76322-154
  • Pipette tips, 10 µL, VWR 76322-132
  • Pipette tips, 200 µL, VWR 76322-150
  • Pipette tips, 20 µL, VWR 76322-134
  • Pipettes, 10 mL, VWR 89130-898
  • 1 L polystyrene bottle, Corning 430518
  • PBS, 1X pH 7.4, VWR 45000-446
  • TMB (3,3',5,5'-tetramethyl-[1,1'-biphenyl]-4,4'-diamine) substrate kit, Pierce 34021
  • Tween-20, VWR 0777-1L
  • Foil

Before Starting

Warm reagents to room temperature.

Procedure

Section 1: Prepare the Antigen Standard and coat the plate

  1. Dilute purified antigen to 20 ng/µL in a microfuge tube and vortex.
    2. Pro-Tip
    You may need to try a few different concentrations of antigen to determine the ideal concentration.
  2. Prepare serial dilutions of the purified antigen as follows:
    • 2 ng/µL: Add 100 µL of 20 ng/µL stock into 900 µL PBS in a microfuge tube and vortex.
    • 1 ng/µL: Add 450 µL of 2 ng/µL stock into 450 uL PBS in a microfuge tube and vortex.
    • 0.5 ng/µL: Add 450 µL of 1 ng/µL stock into 450 µL PBS in a microfuge tube and vortex.
    • 0.25 ng/µL: Add 450 µL of 0.5 ng/µL stock into 450 µL PBS in a microfuge tube and vortex.
    • 0.125 ng/µL: Add 450 µL of 0.25 ng/µL stock into 450 µL PBS in a microfuge tube and vortex.
    • 0 ng/µL: 450 µL PBS only.
  3. Pipette 50 µL into the first three wells of each row, using a different standard solution for each row of a 96-well ELISA microplate.
  4. Cover the plate with a 96-well plate seal and incubate at 37 °C for 30 min, or overnight at 4 °C.

Section 2: Block the plate

  1. Prepare the wash buffer (0.05% Tween-20 in PBS) as follows:
    • Pipette 0.5 mL of Tween-20 to into 999.5 mL 1X PBS
  2. Cap the bottle and invert several times to mix.
  3. Carefully remove the plate seal from the 96-well plate and set aside.
  4. Aspirate the wells and use a multichannel pipette to add 200 µL wash buffer to each well. Washing can be done by hand but if available, use an automated ELISA plate washer.
  5. Seal the plate.
  6. Place the plate on a microplate shaker set at 400 rpm and shake for 1 min.
  7. Repeat steps 8–10 twice for a total of three washes.
  8. After the final wash, aspirate the wash buffer from the wells.
  9. Prepare the blocking buffer (1% BSA in PBS) as follows:
    • Add 250 mg of BSA to 25 mL of PBS.
  10. Cap the tube and invert several times to mix.
  11. Using a multichannel pipette, add 200 µL of blocking buffer to each well.
  12. Cover the plate with a plate seal and incubate on a microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C.

Section 3: Primary antibody incubation

  1. Carefully remove the plate seal from the 96-well plate and set aside.
  2. Aspirate the wells and use a multichannel pipette to add 200 µL wash buffer to each well. Washing can be done by hand but if available, use an automated ELISA plate washer.
  3. Seal the plate
  4. Place the plate on a microplate shaker set at 400 rpm and shake for 1 min.
  5. Repeat steps 18–20 twice for a total of three washes.
  6. After the final wash, aspirate the wash buffer from the wells.
  7. Prepare antibody dilution buffer (0.1% BSA in PBS) as follows:
    • Dilute 50 mg BSA into 50 mL PBS.
  8. Cap the tube and invert several times to mix.
  9. Dilute the primary antibody and an isotype control antibody to the desired concentrations in the antibody dilution buffer.
    10. Pro-Tip
    The ideal antibody concentration will vary and needs to be empirically determined. We suggest starting with three concentrations between 1–10 µg/mL.
  10. Cap and invert the tubes several times to mix.
  11. Using a multichannel pipette, add 100 µL of primary antibody solution to the appropriate wells.
  12. Cover the plate with a plate seal and incubate on a microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C.

Section 4: Secondary antibody incubation

  1. Carefully remove the plate seal from the 96-well plate and set aside.
  2. Aspirate the wells and use a multichannel pipette to add 200 µL wash buffer to each well. Washing can be done by hand but if available, use an automated ELISA plate washer.
  3. Seal the plate
  4. Place the plate on a microplate shaker set at 400 rpm and shake for 1 min.
  5. Repeat steps 30–32 twice for a total of three washes.
  6. After the final wash, aspirate the wash buffer from the wells.
  7. Dilute the HRP-conjugated isotype-specific secondary antibody to the desired concentrations in the antibody dilution buffer.
  8. Cap and invert the tube several times to mix.
  9. Using a multichannel pipette, add 100 µL of secondary antibody to each well.
  10. Cover the plate with a plate seal and incubate on a microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C.

Section 5: TMB reaction

  1. Carefully remove the plate seal from the 96-well plate and set aside.
  2. Aspirate the wells and use a multichannel pipette to add 200 µL wash buffer to each well. Washing can be done by hand but if available, use an automated ELISA plate washer.
  3. Seal the plate
  4. Place the plate on a microplate shaker set at 400 rpm and shake for 1 min.
  5. Repeat steps 40–42 twice for a total of three washes.
  6. After the final wash, aspirate the wash buffer from the wells.
  7. Immediately before use, mix 5 mL TMB Solution and 5 mL Peroxide Solution from the TMB substrate kit.
  8. Cap the tube and invert several times to mix.
  9. Using a multichannel pipette, add 100 µL of the TMB/Peroxide mix to each well.
  10. Cover the plate with a plate seal and wrap in foil.
  11. Incubate plate at room temperature on a microplate shaker set at 400 rpm for 15–30 min.
      12. Pro-Tip
      Check the plate periodically to determine when the desired color change has occurred. The color will change from yellow to blue and generally takes between 15 to 30 min. High concentrations of HRP yield a greenish solution. Stop the reaction before any wells display a green product. A precipitated product indicates the presence of too much HRP and the need to optimize experimental conditions.
  12. When the desired color change has occurred, gently remove the plate seal.
  13. Using a multichannel pipette, add 100 µL of TMB Stop Solution directly to the wells to stop the reaction.
  14. Measure the absorbance of each well at 450 nm on the spectrophotometer. Use the spectrophotometer to generate a standard curve. If you don’t see any color change or dose response, your antibody concentration may have been too low. In this case, use higher antibody concentrations. If the reaction is saturated and you don't see any dose response, your antibody concentration may have been too high. In this case, use lower antibody concentrations. The ideal range of the standard curve will vary between targets and needs to be empirically determined. The dilution series created in section 1, step 2 is a suggested starting point. If your unknown sample’s absorbance falls above the range of the standard curve you will need to either include higher concentrations in the standard curve or dilute the unknown sample (don’t forget to factor in that dilution when calculating the original sample concentration!).

Sample Data

Alt text: A graph of Micrograms (mg) of Protein Loaded versus Absorbance at 450 nm. The negative control protein shows a flat line with absorbance of less than 0.1 nm measured even with up to 2 mg protein loaded. The absorbance measured for human recombinant purified Desmin increased sharply between 0 and 0.5 mg of protein loaded (0 to 1.75 nm) and then increased more slowly between 0.5 and 2 mg of protein loaded (to about 2.5 nm).
Figure 1: The plate was coated with serial dilutions of human recombinant purified Desmin (blue) or a negative control protein, Apolipoprotein L3 (red) overnight, blocked, and incubated overnight with Anti-Desmin [D7]. Plates were incubated with HRP-linked anti-Human IgG secondary antibody for 2 hours, reacted with TMB substrate and the reaction stopped after 15 minutes. Absorbance was read at 450 nm and plotted against the micrograms of antigen loaded.

Last reviewed on: October 8, 2024