Introduction

Enzyme-linked immunosorbent assays (ELISAs) are plate-based assays used to detect and quantify a specific protein in complex mixture. In a sandwich ELISA, highly specific antibodies immobilize the target protein (antigen) to the plate and indirectly detects the presence of the target protein. This type of capture assay is called a sandwich assay because the analyte is bound between two primary antibodies, each detecting a different epitope of the antigen—the capture antibody and the detection antibody.

Find protocols below for a standard sandwich ELISA using a 96-well plate for the detection techniques—colorimetric (chromogenic), chemiluminescent, and fluorescent detection.

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Overview of ELISA method

  1. Attachment of capture antibody specific to target protein to a microplate
  2. Addition of standards and samples containing unknown amount of the target protein which binds to the capture antibody
  3. Washing to remove unbound substances
  4. Addition of a detection antibody that binds to the immobilized target protein
  5. Washing away excess detection antibody and addition of horseradish peroxidase (HRP) conjugate
  6. Addition of HRP substrate for indirect detection of bound protein
Colorimetric sandwich ELISA protocol

This protocol represents an example colorimetric sandwich ELISA using direct detection with an HRP-conjugated antibody or indirect detection with a biotinylated antibody and streptavidin-HRP.

Materials


Additional materials required


Procedure

  1. Prepare Coating Solution by diluting the Capture antibody in Coating buffer. Refer to Antibody Dilution Recommendations table below for dilution recommendations or refer to the manufacturer’s instructions.
  1. Coat plates with 100 µL per well of Coating Solution. Cover plates and incubate for 1 hour at room temperature or overnight (12–18 hours) at 2–8°C.
  1. Aspirate contents and wash once with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid.
  1. Block the plate with 300 µL per well of Blocking buffer for 1 hour at room temperature.
  1. Aspirate blocking buffer then invert, and tap the plate on absorbent paper to remove excess liquid.
  1. Prepare standards and sample dilutions in Blocking buffer.
  1. Pipette 100 µL of standards (in duplicate) and samples into designated wells. Incubate for 1–2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times.
  1. Prepare detection antibody solution by diluting the Detection antibody in Blocking buffer. For recommended antibody dilution, refer to the manufacturer's instructions.
  1. Add 100 µL of the detection antibody solution into each well. Incubate for 2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times. If the Detection antibody is HRP conjugated, proceed to step 15.
  1. If Detection antibody is biotinylated: Prepare a working solution of Streptavidin-HRP in Blocking buffer at 1:5,000 dilution. For example, to make enough for one plate, add 2 µL of Streptavidin-HRP to 9.998 mL of Blocking buffer.
  1. If Detection antibody is biotinylated: Add 100 µL of working streptavidin-HRP solution into each well. Incubate for 1 hour at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times.
  1. Add 100 µL of TMB substrate solution to each well. Incubate plate for 30 minutes at room temperature or when the desired color intensity is reached.
  1. Add 100 µL of Stop solution to each well.
  1. Measure absorbance at 450 nm within 30 minutes of adding the Stop solution.
  1. Calculate results using a log-log or 4-parameter curve fit. See ELISA Data Analysis.

Using an alkaline phosphatase system

If using alkaline phosphatase (AP) instead of HRP for the enzyme conjugate, an AP-specific substrate must be used. Substitute TMB substrate solution in step 15 with p-nitrophenyl phosphate (PNPP) (1-Step PNPP Substrate Solution). Incubate at room temperature for 15–30 minutes. Substitute the stop solution with 50 µL of 2 N NaOH to stop the reaction. Measure absorbance of each well at 405 nm.

Performing direct antigen immobilization

When immobilizing the antigen-containing sample directly to the plate, there is no need for a capture antibody. Different concentrations of the sample should be prepared in coating buffer and identical volumes added directly to the plate. The rest of the protocol should be performed as previously described using a detection antibody and enzyme conjugate plus substrate.

Using an enzyme-labeled secondary antibody

When using a non-biotinylated detection antibody followed by an enzyme-labeled secondary antibody, there will be slightly less amplification of enzyme signal compared to using a biotinylated detection antibody with streptavidin-HRP. Therefore, it may be necessary to use a slightly higher concentration of secondary antibody-enzyme conjugate than one would normally use for a streptavidin-enzyme conjugate.

Chemiluminescent sandwich ELISA protocol

This protocol represents an example of a chemiluminescent sandwich ELISA using direct detection with an HRP-conjugated antibody or indirect detection with a biotinylated antibody and streptavidin-HRP. A luminol-based substrate is used for detection.

Materials

Additional materials required

Protocol tips

Chemiluminescence detection is recommended when detecting and quantifying low abundant proteins or when sample and primary (capture and detection) antibodies are limited.

Protocol tips

White or black plates can be used for chemiluminescent detection. White plates typically display higher signal than black plates, and black plates should be used when background signal is an issue.

Procedure

  1. Prepare Coating Solution by diluting the Capture antibody in Coating buffer. Refer to Antibody Dilution Recommendations table below for dilution recommendations or refer to the manufacturer’s instructions.
  1. Coat plates with 100 µL per well of Coating Solution. Cover plates and incubate for 1 hour at room temperature or overnight (12–18 hours) at 2–8°C.
  1. Aspirate contents and wash once with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid.
  1. Block the plate with 300 µL per well of Blocking buffer for 1 hour at room temperature.
  1. Aspirate blocking buffer then invert, and tap the plate on absorbent paper to remove excess liquid.
  1. Prepare standards and sample dilutions in Blocking buffer.
  1. Pipette 100 µL of standards (in duplicate) and samples into designated wells. Incubate for 1–2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times.
  1. Prepare detection antibody solution by diluting the Detection antibody in Blocking buffer. For recommended antibody dilution, refer to the manufacturer's instructions.
  1. Add 100 µL of the detection antibody solution into each well. Incubate for 2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times. If the Detection antibody is HRP conjugated, proceed to step 15.
  1. If Detection antibody is biotinylated: Prepare a working solution of Streptavidin-HRP in Blocking buffer at 1:5,000—1:20,000 dilution. For example, to make enough for one plate, add 2 µL of streptavidin-HRP to 9.998 mL of Blocking buffer. The optimal dilution should be determined empirically.
  1. If Detection antibody is biotinylated: Add 100 µL of working streptavidin-HRP solution into each well. Incubate for 1 hour at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times.
  1. Prepare a working solution of Chemiluminescent substrate by mixing equal parts of Luminol and Stable Peroxide Solution.
  1. Add 100 µL of working Chemiluminescent substrate solution into each well. Incubate for 1 minute at room temperature.
  1. Measure relative light units (~425 nm) using a luminometer between 1–5 minutes after adding the substrate. Longer periods between adding the substrate and measuring the plate may result in decreased signal intensity.

Utilizing an alkaline phosphatase (AP) system

When substituting horseradish peroxidase (HRP) with alkaline phosphatase (AP) for the enzyme conjugate, it is necessary to use an AP-specific substrate. Replace the TMB substrate solution in step 15 with p-nitrophenyl phosphate (PNPP) (1-Step PNPP Substrate Solution). Incubate the reaction mixture at room temperature for 15 to 30 minutes. To terminate the reaction, add 50 µL of 2 N NaOH as the stop solution. Subsequently, measure the absorbance of each well at 405 nm.

Conducting direct antigen immobilization

When directly immobilizing the antigen-containing sample onto the plate, a capture antibody is not required. Prepare varying concentrations of the sample in coating buffer and add equal volumes directly to the plate. Follow the remainder of the protocol as previously outlined, utilizing a detection antibody along with an enzyme conjugate and a substrate.

Utilizing an enzyme-labeled secondary antibody

When employing a non-biotinylated detection antibody followed by an enzyme-labeled secondary antibody, the amplification of the enzyme signal will be somewhat reduced compared to using a biotinylated detection antibody with streptavidin-HRP. Consequently, it may be required to use a slightly higher concentration of the secondary antibody-enzyme conjugate than typically used for a streptavidin-enzyme conjugate.

Fluorescent sandwich ELISA protocol

This protocol represents an example of a fluorescent sandwich ELISA using direct detection with an HRP-conjugated antibody or indirect detection with a biotinylated antibody and streptavidin-HRP. A fluorogenic peroxidase substrate is used for detection.

Materials

Additional materials required

Procedure

  1. Prepare Coating solution by diluting the Capture antibody in Coating buffer to a concentration of 5–10 μg/mL.
  1. Coat plates with 50–100 µL per well of coating solution. Cover plates, and incubate 1 hour at room temperature or overnight (12–18 hours) at 2–8°C.
  1. Aspirate contents and wash wells once with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block the plate with 300 µL per well of Blocking buffer for 1 hour at room temperature.
  1. Aspirate blocking buffer, invert, and tap the plate on absorbent paper to remove excess liquid.
  1. Prepare standards and sample dilutions in Blocking buffer.
  1. Pipette 100 µL of standards (in duplicate) and samples into designated wells. Incubate for 1–2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times.
  1. Prepare detection antibody solution by diluting Detection antibody to a concentration of 0.05–0.1 μg/mL in Wash Buffer.
  1. Add 100 µL of the detection antibody solution into each well. Incubate for 1–2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times. If the Detection antibody is HRP conjugated, proceed to step 15.
  1. If Detection antibody is biotinylated: Prepare a working solution of Streptavidin-HRP in Wash buffer by diluting to a concentration of 0.05–0.1 μg/mL.
  1. If Detection antibody is biotinylated: Add 50–100 µL of working streptavidin-HRP solution into each well. Incubate for 1 hour at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate contents and wash the wells with >300 µL of Wash buffer per well. Following wash, invert and tap the plate on absorbent paper to remove excess liquid. Repeat the wash step five times.
  1. Prepare working solution of QuantaBlu substrate by mixing nine parts of QuantaBlu Substrate Solution with one part of QuantaBlu Peroxide Solution.
  1. Add 100 µL of working QuantaBlu solution into each well. Incubate for 1.5–90 minutes at room temperature or 37°C.
  1. Add 100 μL of QuantaBlu Stop Solution; the enzymatic activity is immediately stopped (an incubation is not required).
  1. Avoid bubbles. Bubbles in assay solutions cause light scattering and erroneous signals. Briefly centrifuge the microplate, or pop large bubbles with a pipette tip.
  1. Measure relative fluorescent units (RFU). The excitation and emission maxima for QuantaBlu substrate is 325 nm and 420 nm. Wavelengths between 315 nm and 340 nm for excitation and 370 nm and 470 nm for emission can also be used for detection.

Using a fluorophore-conjugated detection antibody

With a fluorophore-conjugated detection antibody, no enzyme conjugate or substrate is required. The plate fluorescence units can be measured directly after step 11. The working concentration of labeled antibody or protein is typically 2–4 μg/mL.

Executing direct antigen immobilization

For direct immobilization of the antigen-containing sample onto the plate, a capture antibody is not needed. Prepare the sample in varying concentrations using the coating buffer and dispense equal volumes directly onto the plate. Continue with the rest of the protocol as previously detailed, applying a detection antibody along with an enzyme conjugate and substrate.

Applying an enzyme-labeled secondary antibody

When utilizing a non-biotinylated detection antibody followed by an enzyme-labeled secondary antibody, the enzyme signal amplification will be lower compared to the use of a biotinylated detection antibody with streptavidin-HRP. Therefore, you might need to increase the concentration of the secondary antibody-enzyme conjugate slightly more than what is usually required for a streptavidin-enzyme conjugate.

Antibody dilutions

Antibody dilution recommendations

The following tables provide recommended ranges for different ELISA components. Concentrations are guidelines only; for best results, optimize each component individually.

Recommended starting concentration ranges for coating and detection antibodies for ELISA optimization. The use of non-purified antibodies will work but may result in higher background. It is generally recommended to use affinity-purified antibodies for optimal signal-to-noise ratio.

SourceCoating antibodyDetection antibody
Polyclonal serum5–15 μg/mL1–10 μg/mL
Crude ascites5–15 μg/mL1–10 μg/mL
Affinity-purified polyclonal antibody1–12 μg/mL0.5–5 μg/mL
Affinity-purified monoclonal antibody1–12 μg/mL0.5–5 μg/mL

Recommended detection antibody concentration for ELISA in different systems. Check the user guide for the substrate as it may recommend a more defined concentration range for the enzyme conjugate.

EnzymeSystemConcentration
HRPColorimetric20–200 ng/mL
Chemifluorescent25–50 ng/mL
Chemiluminescent10–100 ng/mL
APColorimetric100–200 ng/mL
Chemiluminescent40–200 ng/mL

Employing an enzyme-labeled secondary antibody

When a non-biotinylated detection antibody is used after an enzyme-labeled secondary antibody, the resulting enzyme signal amplification is slightly reduced compared to the use of a biotinylated detection antibody with streptavidin-HRP. As a result, it may be necessary to use a slightly higher concentration of the secondary antibody-enzyme conjugate than typically used for a streptavidin-enzyme conjugate.

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Ordering information for related products

Colorimetric sandwich ELISA products
Chemiluminescent sandwich ELISA products
Fluorescent sandwich ELISA products

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For a complete set of ELISA reagents, Invitrogen ELISA Buffer Kit includes 2 Coating Buffers (pH 7.4 and pH 9.4), 5X Assay Buffer (blocking reagent and diluent), 25X Wash Buffer, Stabilized TMB, and Stop Solution. Pair this kit with Matched Antibody Pair kits that contain matched antibody pairs, standards and Streptavidin-HRP conjugate.

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Stylesheet for Classic Wide Template adjustments

For Research Use Only. Not for use in diagnostic procedures.