Sandwich ELISA Protocol (R&D Systems DuoSet Kit)

Youssef Farhat, Written 3/5/12, Last Updated 5/6/14

Download the PDF or Microsoft Word versions of this protocol

Introduction

Enzyme-linked immunosorbent assays (ELISA) are a very common family of techniques used to measure the presence of proteins in tissues or cell culture samples.  Their sensitivity and specificity depends on the types of antibodies used and the concentration of each reagent.

A number of commercial ELISA kits are available that provide the reagents necessary to detect a protein of interest with great sensitivity and specificity.  The following is a protocol that was developed for the TGF-β1 ELISA kit manufactured by R&D Systems (#DY240), and may serve as a model for other ELISA kits.

It’s important to note the following:

• ELISAs are performed with 96-well plates that have a high affinity for proteins (for example, Costar, #3590).  Do not use 96-well plates designed for tissue culture.
• Capture antibodies are diluted in DPBS, while the other reagents are diluted in “Reagent Diluent” which usually contains some Bovine Serum Albumin (BSA) to prevent non-specific protein interactions.
• The reagents used to make Block Buffer and Reagent Diluent in this protocol are recommended by the manufacturer of this particular kit.  Other ELISA kits may use different reagents to make these solutions.
• The volume of each reagent may need to be adjusted for other ELISA kits based on the manufacturer’s recommendations of final antibody concentrations, etc.

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Materials

-        Human TGF-β1 DuoSet® ELISA Development System (R&D Systems, #DY240).  Store at 4°C.  This kit includes the following:

• Two vials of protein standard
• One vial of capture antibody
• One vial of detection antibody
• One vial of Streptavidin-HRP

-        ELISA plate (Costar, #3590)

-        1X DPBS (Can be made from our 20X DPBS Stock Solution)

-        Tween 20 (Bio-Rad, #170-6531)

-        Sodium Azide (NaN₃)

-        BSA (Gibco, #15260)

-       Substrate Reagent Pack (R&D Systems, #DY999).  Store at 4°C.  This pack contains:

• 8 vials Color Reagent A
• 8 vials Color Reagent B

-        Stop Solution (2 N H₂SO₄ [R&D Systems, #DY994])

-       Reagent Diluent Concentrate 1 (R&D Systems, #DY997)

Procedure

Performing an ELISA typically takes several days.  Briefly, the process consists of these steps: 1) Prepare Solutions, 2) Aliquot Reagents, 3) Prepare Plate, 4) Perform Assay

1) Solution Preparation (Estimated time: 30-60 min)

5% Tween 20 Solution – Not used directly in the assay, but convenient for making other solutions.  Prepare by combining 25 mL of Tween 20 with 475 mL of DPBS

Wash Buffer (0.05% Tween 20 in DPBS, pH 7.2-7.4) – Combine 10 mL of 5% Tween 20 Solution with 990 mL of DBPS

Block Buffer (5% Tween 20 in DPBS with 0.05% NaN₃) – Combine 100 mg of NaN₃ with 200 mL of 5% Tween 20 Solution

Reagent Diluent (1.4% Reagent Diluent Concentrate 1, 0.05% Tween 20 in DPBS, pH 7.2-7.4) – Combine 700 μL of Reagent Diluent Concentrate 1 with 50 mL of Wash Buffer.  Use within one day.

2) Aliquot Reagents (Estimated time: 30 min)

Note: the first time you use a new DuoSet ELISA Kit, you must aliquot the reagents and store them appropriately.  The following is one of many ways to aliquot and store the reagents of R&D Systems’ TGF-β1 ELISA kit and only serves as an example. 

Capture Antibody – Comes as 360 µg of powder.  Reconstitute in 1 mL of DPBS (concentration becomes 360 µg/mL).  Aliquot 60 µL into separate tubes and store at -80°C.

Detection Antibody – Comes as 54 µg of powder.  Reconstitute in 1 mL of Reagent Diluent (concentration becomes 54 µg/mL).  Aliquot 60 µL into separate tubes and freeze at -80°C.

Standard – Comes as two vials containing 60 ng of TGF-β1 in each.  Reconstitute in 500 µL of Reagent Diluent (concentration becomes 120 ng/mL).  Aliquot 16.7 µL into 1.5 mL Eppendorf Tubes (i.e. 2 ng TGF-β1 per tube) and store at -80°C.

3) Prepare Plate (Estimated time: 3 hours over 5 days or 10 hours over two days)

The following procedure is for a single ELISA plate, but it can be repeated or scaled for multiple plates.

1. Thaw a Capture Antibody aliquot
2. Take 55.6 µL* of Capture Antibody and place it in a clean reagent reservoir
3. Add 10 mL of DPBS to the reservoir directly over the drop of antibody (final working concentration of 2 µg/mL)
4. Using a multichannel pipettor, add 100 µL of capture antibody to each well of an ELISA plate
5. Seal the plate and refrigerate it overnight at 4°C

The next day…

1. Evacuate the ELISA plate and blot with a clean paper towel
2. Wash with 200 µL of Wash Buffer three times
3. Add 200 µL of Block Buffer to each well of the plate and incubate it for 2 hours at room temperature or refrigerate it overnight at 4°C

Two hours later or the next day…

4) Perform Assay

Using Reagent Diluent, dilute the samples so that they are at a concentration which is within the range of the standard curve.  To prepare the standard curve, add 983 µL of Reagent Diluent to the 16.7 µL standard aliquot (final concentration of standard becomes 2 ng/mL of TGF-β1).  Then, perform serial dilutions (1 part standard to 1 part water) starting from pure standard (2 ng/mL) down to the seventh well (31 pg/mL).  Let the eighth well should contain pure reagent diluent (blank control).

1. Evacuate the ELISA plate and blot with a clean paper towel
2. Wash with 400 µL of Wash Buffer five times to ensure thorough removal of the Block Buffer
3. Add 100 µL of samples and standards
4. Seal the plate and incubate it for 2 hours at room temperature or refrigerate it overnight at 4°C

Two hours later or the next day…

1. Thaw a Detection Antibody aliquot
2. Take 55.6 µL of the aliquot and place it in a clean reagent reservoir
3. Add 10 mL of Reagent Diluent to the reservoir directly over the drop of antibody (final working concentration of 2 µg/mL)
4. Evacuate the ELISA plate and blot with a clean paper towel
5. Wash with 200 µL of Wash Buffer three times
6. Add 100 µL of Detection Antibody to each well of the ELISA plate
7. Seal the plate and incubate it for 2 hours at room temperature or refrigerate it overnight at 4°C

Two hours later or the next day…

1. Dilute the Streptavidin-HRP 200 times by placing 50 µL in a clean reagent reservoir and adding 10 mL of Reagent Diluent
2. Evacuate the ELISA plate and blot with a clean paper towel
3. Wash with 200 µL of Wash Buffer three times
4. Add 100 µL of Streptavidin-HRP to each well of the ELISA plate
5. Protect the plate from light and incubate it for 20 min at room temperature

20 minutes later…

1. Prepare the Substrate Solution by combining 5 mL of Color Reagent A with 5 mL of Color Reagent B into a clean reagent reservoir and protect it from light
2. Evacuate the ELISA plate and blot with a clean paper towel
3. Wash with 200 µL of Wash Buffer three times
4. Add 100 µL of Substrate Solution to each well of the ELISA plate
5. Protect the plate from light and incubate it for 20 min at room temperature
6. Meanwhile, set up the plate-reader to shake the plate at medium setting for 15 seconds and then measure absorbance at 450 nm.
7. Bring the ELISA plate and Stop Solution near the plate reader for immediate assay when the 20 min has passed

20 minutes later or when the lowest nonzero concentration on the standard curve begins to turn blue…

1. Add 50 µL of Stop Solution to each well of the ELISA plate
2. Gently tap the plate ensure mixing and read absorbance at 450 nm in the plate reader

Citing this Protocol

Farhat, Y. “ELISA Protocol (R&D Systems DuoSet Kit).” The Protocol Place. March 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com/>