Introduction to sandwich ELISA protocols

Enzyme-linked immunosorbent assays (ELISAs) are plate-based assays for detecting and quantifying a specific protein in a complex mixture. The detection and quantification of target-specific protein in a sandwich ELISA is accomplished by using highly specific antibodies that immobilizes 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 being measured is bound between two primary antibodies, each detecting a different epitope of the antigen—the capture antibody and the detection antibody. There are many different types of components, such as substrates, plates, and other reagents, that the choices can sometimes be overwhelming. We’ve made general suggestions in the protocols below, and you can also visit our ELISA builder online selection guide that asks a few simple questions about your specific ELISA and then recommends all the components you’ll need, from plates to stop solution.

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

Overall procedure

  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 HRP conjugate
  6. Addition of HRP substrate for indirect detection of bound protein 
     
Colorimetric protocol

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

Protocol tips

For a complete set of ELISA Buffers, Invitrogen Antibody Pair Buffer Kit, Cat. No. CNB0011, includes: Coating Buffer (pH 7.4 and pH 9.4), Assay Buffer (Blocking Buffer), Wash Buffer, Stabilized TMB, and Stop Solution.

Protocol tips

To help ensure reliable results with accuracy and precision, high-quality washing is essential. Wash plates securely with automatic plate washers such as the Thermo Scientific Wellwash Microplate Washer, an easy-to-use, reliable microplate-strip washer for routine ELISA applications.

Procedure

  1. Prepare Coating Solution by diluting the Capture antibody in Coating buffer. Refer to Antibody Dilution Recommendations table 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 one hour at room temperature or overnight (12–18 hours) at 2–8°C.
  1. Aspirate contents and wash one time with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block plate with 300 µL per well with Blocking buffer for one hour at room temperature.
  1. Aspirate blocking buffer then invert, and tap 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 one to two hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Prepare detection antibody solution by diluting the Detection antibody in Blocking buffer. For recommended antibody dilution, refer to the manufacturer's instruction.
  1. Add 100 µL of the detection antibody solution into each well. Incubate for two hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wells wash five times with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid. If the Detection antibody is HRP conjugated, proceed to step 15.
  1. If Detection antibody is biotinylated: Make working solution of Streptavidin-HRP with Blocking buffer by diluting 1:5,000. For example, to make enough for 1 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 one hour at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  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 Stop solution.
  1. Calculate results using a log-log or 4-parameter curve fit.

Using an alkaline phosphatase system

If alkaline phosphatase (AP) is to be used 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) (Thermo Scientific 1-Step PNPP Substrate Solution, Cat. No. 37621). Incubate at room temperature for 15–30 minutes. Substitute the stop solution with 50 µL 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 protocol

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

To help ensure reliable results with accuracy and precision, high-quality washing is essential. Wash plates securely with automatic plate washers such as the Thermo Scientific Wellwash Microplate Washer, an easy-to-use, reliable microplate-strip washer for routine ELISA applications.

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 for dilution recommendations or manufacturer’s instructions.
  1. Coat plates with 100 µL per well of coating solution. Cover plates, and incubate one hour at room temperature or overnight (12–18 hours) at 2–8°C.
  1. Aspirate contents and wash wells one time with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block plate with 300 µL per well with Blocking buffer for one hour at room temperature.
  1. Aspirate blocking buffer, invert, and tap 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 one to two hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap plate on absorbent paper to remove excess liquid.
  1. Prepare detection antibody solution by diluting the Detection antibody in Blocking buffer. For recommended antibody dilution, refer to manufacturer's instruction.
  1. Add 100 µL of the detection antibody solution into each well. Incubate for two hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid. If the Detection antibody is HRP conjugated, proceed to step 15.
  1. If Detection antibody is biotinylated: Make a working solution of Streptavidin-HRP with Blocking buffer by diluting 1:5,000- 1:20,000. 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 one hour at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap plate on absorbent paper to remove excess liquid.
  1. Make working solution of Chemiluminescent substrate solution by mixing equal parts of Luminol and Stable Peroxide Solution.
  1. Add 100 µL of working Chemiluminescent substrate solution into each well. Incubate for one minute at room temperature.
  1. Use a luminometer to measure relative light units (~425 nm) from one to five minutes after adding the substrate. Longer periods between adding the substrate and measuring the plate may result in decreased signal intensity.

Using an alkaline phosphatase system

If alkaline phosphatase (AP) is to be used 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) (Thermo Scientific 1-Step PNPP Substrate Solution, Cat. No. 37621). Incubate at room temperature for 15–30 minutes. Substitute the stop solution with 50 µL 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.

Fluorescent protocol

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

  1. Prepare Coating solution by diluting the Capture antibody in Coating buffer to 5–10 μg/mL.
  1. Coat plates with 50-100 µL per well of coating solution. Cover plates, and incubate one hour at room temperature or overnight (12–18 hours) at 2–8°C.
  1. Aspirate contents and wash wells one time with >300 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block plate with 300 µL per well with Blocking buffer for one hour at room temperature.
  1. Aspirate blocking buffer, invert, and tap 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 one to two hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap plate on absorbent paper to remove excess liquid.
  1. Prepare detection antibody solution by diluting Detection antibody 0.05–0.1 μg/mL in Wash Buffer.
  1. Add 100 µL of the detection antibody solution into each well. Incubate for one to two hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap plate on absorbent paper to remove excess liquid. If the Detection antibody is HRP conjugated, proceed to step 15.
  1. If Detection antibody is biotinylated: Make a working solution of Streptavidin-HRP in Wash Buffer by diluting to 0.05–0.1 μg/mL. The optimal dilution should be determined empirically.
  1. If Detection antibody is biotinylated: Add 50–100 µL of working streptavidin-HRP solution into each well. Incubate for one hour at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate contents and wash wells five times with >300 µL of Wash buffer per well. Following wash, invert and tap plate on absorbent paper to remove excess liquid.
  1. Make working solution of QuantaBlu substrate by mixing nine parts of QuantaBlu Substrate Solution and one part of QuantaBlu Peroxide Solution.
  1. Add 100 µL of working QuantaBlu solution into each well. Incubate for 1.5 minutes to 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 and 340 nm for excitation and 370 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.

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.

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

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.

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