Introduction

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.

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Find protocols below for a standard sandwich ELISA using a 96-well plate for the detection techniques–colorimetric (chromogenic) and chemiluminescent 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 Sandwich ELISA Protocol

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.

Procedure

  1. Prepare Coating Solution by diluting the Capture antibody in Coating buffer. Refer to manufacturer for dilution recommendations.
  1. Coat plates with 100 µL per well of Coating Solution. Cover plates, and incubate overnight (12–18 hours) at 2–8 °C.
  1. Aspirate wells and wash 1 time with >200 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block plate with 200 µL per well with Blocking buffer for 1 hour at room temperature.
  1. Aspirate, 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 1 hour at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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 manufacturer's instruction.
  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 and wash 5 times with >200 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. 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. Add 100 µL of working streptavidin-HRP solution into each well. Incubate for 30 minutes at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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.
  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.

Protocol tips

To 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.

Chemiluminescent Sandwich ELISA Protocol

Materials

Additional materials required

Procedure

  1. Prepare Coating solution by diluting the Capture antibody in Coating buffer. Refer to manufacturer for dilution recommendations.
  1. Coat plates with 100 µL per well of coating solution. Cover plates, and incubate overnight (12–18 hours) at 2–8 °C.
  1. Aspirate wells and wash 1 time with >200 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block plate with 200 µL per well with Blocking buffer for 1 hour at room temperature.
  1. Aspirate, 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 1 hour at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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 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 2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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- 1:20,000. For example, to make enough for 1 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 30 minutes at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate and wash 5 times with >200 µL of Wash buffer per well. Following wash, invert and tap 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 1 minute at room temperature.
  1. Use a luminometer to measure relative light units (~425 nm) from 1 to 5 minutes after adding the substrate. Longer periods between adding the substrate and measuring the plate may result in decreased signal intensity.

Protocol tips

To 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, black plates should be used when background signal is an issue.
Colorimetric Detection

Colorimetric Sandwich ELISA Protocol

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.

Procedure

  1. Prepare Coating Solution by diluting the Capture antibody in Coating buffer. Refer to manufacturer for dilution recommendations.
  1. Coat plates with 100 µL per well of Coating Solution. Cover plates, and incubate overnight (12–18 hours) at 2–8 °C.
  1. Aspirate wells and wash 1 time with >200 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block plate with 200 µL per well with Blocking buffer for 1 hour at room temperature.
  1. Aspirate, 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 1 hour at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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 manufacturer's instruction.
  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 and wash 5 times with >200 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. 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. Add 100 µL of working streptavidin-HRP solution into each well. Incubate for 30 minutes at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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.
  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.

Protocol tips

To 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.
Chemiluminescent Detection

Chemiluminescent Sandwich ELISA Protocol

Materials

Additional materials required

Procedure

  1. Prepare Coating solution by diluting the Capture antibody in Coating buffer. Refer to manufacturer for dilution recommendations.
  1. Coat plates with 100 µL per well of coating solution. Cover plates, and incubate overnight (12–18 hours) at 2–8 °C.
  1. Aspirate wells and wash 1 time with >200 µL of Wash buffer per well. Following wash, invert and tap on absorbent paper to remove excess liquid.
  1. Block plate with 200 µL per well with Blocking buffer for 1 hour at room temperature.
  1. Aspirate, 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 1 hour at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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 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 2 hours at room temperature with gentle continual shaking (~500 rpm).
  1. Aspirate and wash 5 times with >200 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- 1:20,000. For example, to make enough for 1 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 30 minutes at room temperature with gentle continual shaking (~500 rpm).
  1. If Detection antibody is biotinylated: Aspirate and wash 5 times with >200 µL of Wash buffer per well. Following wash, invert and tap 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 1 minute at room temperature.
  1. Use a luminometer to measure relative light units (~425 nm) from 1 to 5 minutes after adding the substrate. Longer periods between adding the substrate and measuring the plate may result in decreased signal intensity.

Protocol tips

To 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, black plates should be used when background signal is an issue.

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