General Sandwich ELISA Protocol

Sandwich ELISA (enzyme-linked immunosorbent assay) involves attachment of a capture antibody to a microplate. Then, samples containing unknown amount of the target protein or analyte of interest are added and bind to the capture antibody. After washing steps to rid the microplate of unbound substances, an HRP conjugate is added for detection.

The ELISA method is a benchmark for quantitation of antigens. ELISAs are adaptable to high-throughput screening because results are rapid, consistent and relatively easy to analyze. The best results have been obtained with the sandwich format, utilizing highly purified, pre-matched capture and detection antibodies. The resulting signal provides data which is very sensitive and highly specific. Ready-to-use ELISA kits are commercially available for hundreds of commonly investigated proteins and other biological molecules. See Find ELISA Kits by Target for more information about Thermo Scientific Pierce ELISA Kits.

ELISA protocols (Figure 1) begin with a capture antibody, specific for a protein of interest, coated onto the wells of microplates. Samples, including a standard containing protein of interest, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the protein antigen binds to the capture antibody. After washing, a detection antibody is added to the wells, and this antibody binds to the immobilized protein captured during the first incubation. After removal of excess detection antibody, an HRP conjugate (secondary antibody or streptavidin) is added and binds to the detection antibody. After a third incubation and washing to remove the excess HRP conjugate, a substrate solution is added and is converted by the enzyme to a detectable form (color signal). The intensity of this colored product is directly proportional to the concentration of antigen present in the original specimen.

Note: The sandwich ELISA protocol provided here is representative of most ready-to-use ELISA kits for measurement of cytokines, chemokines, growth factors, and other extracellular targets. Depending on the protein of interest, antibodies, buffers, or substrates being used, this general protocol may need to be optimized. For more information see Overview of ELISA and ELISA Development and Optimization.

Typical ELISA Kit Components

• Antibody-coated 96-well microplate
• Detection antibody (usually biotinylated)
• Standard
• HRP conjugate (antibody or streptavidin)
• Diluent buffers
• Wash buffer
• Chromogenic substrate (usually TMB)
• Stop solution
• Plate covers

 Additional Materials Required

• Absorbance-based microplate reader
• Distilled or deionized water
• Squirt wash bottle or an automated 96-well plate washer
• Sample (See ELISA Sample Preparation Protocols)

Run time: 4 hours – 30 minutes hands-on time
Note: A standard curve must be run with each assay for quantitation

1. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.
2. Add 50-100 µL of prepared standard and sample to wells. Cover plate and incubate at room temperature for 2 hours.
3. Thoroughly aspirate or decant solution from wells and discard the liquid.
4. Wash wells 4 times using a squirt wash bottle or an automated 96-well plate washer.
5. Add 100 µL of diluted detection antibody to wells. Cover plate and incubate at room temperature for 1 hour.
6. Thoroughly aspirate or decant solution from wells and discard the liquid.
7. Wash wells 4 times.
8. Add 100 µL of diluted HRP conjugate to each well. Cover plate and incubate at room temperature for 30 minutes.
9. Thoroughly aspirate or decant solution from wells and discard the liquid.
10. Wash wells 4 times.
11. Add 100 µL of chromogenic substrate to each well.
12. Develop plate at room temperature in the dark for 30 minutes.
13. Add 100 µL of stop solution to each well. The solution in the wells should change from blue to yellow.
14. The plate must be evaluated within 30 minutes of stopping the reaction. Read the absorbance of each well at 450 nm and 550 nm. Subtract 550 nm values from 450 nm values to correct for optical imperfections in the microplate.
15. Use curve-fitting statistical software to plot a four-parameter logistic curve fit to the standards and then calculate results for the test samples.
    

Note: If using a Thermo Scientific Pierce Antibody Pair Kit, you will need to coat the capture antibody onto a 96-well microplate yourself. For this process you will need a 96-well microplate, capture antibody, and blocking buffer (usually BSA or milk diluted in PBS). You will follow these steps before starting the above protocol:

1. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.
2. Add 100 µL of diluted capture antibody to each well. Cover plate and incubate at 4^°C overnight.
3. Bring plate to room temperature. Thoroughly aspirate or decant solution from wells and discard the liquid.
4. Wash wells 4 times.
5. Add 200 µL of blocking buffer to each well. Cover plate and incubate at room temperature for 1 hour.
6. Thoroughly aspirate or decant solution from wells and discard.

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