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
- Attachment of capture antibody specific to target protein to a microplate
- Addition of standards and samples containing unknown amount of the target protein which binds to the capture antibody
- Washing to remove unbound substances
- Addition of a detection antibody that binds to the immobilized target protein
- Washing away excess detection antibody and addition of HRP conjugate
- Addition of HRP substrate for indirect detection of bound protein
Colorimetric Sandwich ELISA Protocol
Materials
- Clear 96-well plate (e.g. Eight-well strip MaxiSorp microplates, Cat. No. 468667)
- Coating buffer (10 mM phosphate buffer, pH 7.4 or 50 mM carbonate buffer, pH 9.4, Cat. No. 28382)
- Blocking buffer (Assay Buffer) (e.g. ELISA Assay Buffer, Cat. No. DS98200)
- Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
- Plate sealers (e.g. Sealing Tape for 96-Well Plates, Cat. No. 15036)
- Reagent reservoirs (e.g. ELISA Reagent Reservoirs, Cat. No. 15075)
- Capture (coating) antibody
- Detection antibody
- Streptavidin-HRP (e.g. HRP-Conjugated Streptavidin, Cat. No. N100)
- TMB substrate solution (e.g. TMB Stabilized Chromagen, Cat. No. SB01)
- Stop solution (1.8 N H2SO4, e.g. Cat. No. SS03)
Additional materials required
- Absorbance-based microplate reader (e.g. Multiskan FC microplate reader)
- Distilled or deionized water
- Samples (see ELISA Sample preparation protocols)
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
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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
- Opaque 96-well plate (e.g. White MaxiSorp microplates, Cat. No. 437591)
- Coating buffer (10 mM phosphate buffer, pH 7.4 or 50 mM carbonate buffer, pH 9.4, Cat. No. 28382)
- Blocking buffer (e.g. StartingBlock T20 PBS Blocking Buffer, Cat. No. 37539 or StartingBlock T20 TBS Blocking buffer, Cat. No. 37543)
- Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
- Chemiluminescent substrate (e.g. SuperSignal ELISA Pico Chemiluminescent Substrate, Cat. No. 37070)
- Capture (coating) antibody
- Detection antibody
- Streptavidin-HRP (Detection antibody is biotinylated) (e.g. HRP-Conjugated Streptavidin, Cat. No. N100)
- Reagent reservoirs (e.g. ELISA Reagent Reservoirs, Cat. No. 15075)
- Plate sealers (e.g. Sealing Tape for 96-Well Plates, Cat. No. 15036)
Additional materials required
- Luminometer-based microplate reader (e.g. Luminoskan microplate reader)
- Distilled or deionized water
- Samples (see ELISA Sample preparation protocols)
Procedure
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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 Sandwich ELISA Protocol
Materials
- Clear 96-well plate (e.g. Eight-well strip MaxiSorp microplates, Cat. No. 468667)
- Coating buffer (10 mM phosphate buffer, pH 7.4 or 50 mM carbonate buffer, pH 9.4, Cat. No. 28382)
- Blocking buffer (Assay Buffer) (e.g. ELISA Assay Buffer, Cat. No. DS98200)
- Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
- Plate sealers (e.g. Sealing Tape for 96-Well Plates, Cat. No. 15036)
- Reagent reservoirs (e.g. ELISA Reagent Reservoirs, Cat. No. 15075)
- Capture (coating) antibody
- Detection antibody
- Streptavidin-HRP (e.g. HRP-Conjugated Streptavidin, Cat. No. N100)
- TMB substrate solution (e.g. TMB Stabilized Chromagen, Cat. No. SB01)
- Stop solution (1.8 N H2SO4, e.g. Cat. No. SS03)
Additional materials required
- Absorbance-based microplate reader (e.g. Multiskan FC microplate reader)
- Distilled or deionized water
- Samples (see ELISA Sample preparation protocols)
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
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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
- Opaque 96-well plate (e.g. White MaxiSorp microplates, Cat. No. 437591)
- Coating buffer (10 mM phosphate buffer, pH 7.4 or 50 mM carbonate buffer, pH 9.4, Cat. No. 28382)
- Blocking buffer (e.g. StartingBlock T20 PBS Blocking Buffer, Cat. No. 37539 or StartingBlock T20 TBS Blocking buffer, Cat. No. 37543)
- Wash buffer (Tris-buffered or phosphate-buffered saline with 0.05% Tween 20, Cat. No. 28360 or 28352)
- Chemiluminescent substrate (e.g. SuperSignal ELISA Pico Chemiluminescent Substrate, Cat. No. 37070)
- Capture (coating) antibody
- Detection antibody
- Streptavidin-HRP (Detection antibody is biotinylated) (e.g. HRP-Conjugated Streptavidin, Cat. No. N100)
- Reagent reservoirs (e.g. ELISA Reagent Reservoirs, Cat. No. 15075)
- Plate sealers (e.g. Sealing Tape for 96-Well Plates, Cat. No. 15036)
Additional materials required
- Luminometer-based microplate reader (e.g. Luminoskan microplate reader)
- Distilled or deionized water
- Samples (see ELISA Sample preparation protocols)
Procedure
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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.