ELISA Protocol-Fast and Easy


Our ELISA protocol allows you to run an ELISA assay. Enzyme-Linked Immunosorbent Assays (ELISA’s) are used to measure an unknown concentration of antigen or antibody. There are several types of ELISA’s: Indirect / Sandwich / Competitive

  • INDIRECT ELISA– the easiest ELISA is the “Indirect ELISA”. In this assay, the protein of interest is usually bound to the ELISA plate, followed by a primary antibody that is directed the protein of interest. Lastly, a labeled secondary antibody is added to the plate. The labeled secondary antibody is directed to the primary antibody. See Figure 1. IR TIP- In some case the term “direct” can be used interchangeably with “indirect”.
Indirect ELISA

Figure 1- Indirect ELISA









  • SANDWICH ELISA– this type of ELISA utilizes two primary antibodies that are “sandwiched” between the protein of interest.  See Figure 2.     IR TIP- In some case the term “Sandwich ELISA” can be used interchangeably with “Capture ELISA”.

Figure 2- Sandwich ELISA









ELISA Protocol

ELISA’s are a relatively quick and accurate testing method. When detecting a protein from a crude mixture (serum/CSF/Cell culture sups) a sandwich ELISA is most desirable. In a true sandwich ELISA, a purified primary antibody is coated to the bottom of a well in a 96-well plate. The target antigen is then added and binds to the antibody coated to the ELISA plate. An additional primary antibody is added on top of the antigen to create the “sandwich”. A labeled secondary antibody is added which binds the top primary antibody. Unbound products are removed in between each step with a wash. Finally, a colorimetric substrate is added to react with the label on the secondary antibody. For quantitative results, a purified antigen standard can be used as a comparison to determine the absolute amount of protein in the unknown sample.

Below is an example of a Sandwich ELISA protocol. There are many variables in this assay and caution should be used to have controls in place to have optimal results. For an ELISA 96 Well Loading Template, click here.


  • 96-well plate
  • Sample to be tested
  • Calibrated standards of the target antigen
  • Primary Antibody 1 (Capture Antibody that is bound to the ELISA plate)
  • Primary Antibody 2 (Detector Antibody that completes the “sandwich”)
  • Labeled secondary antibody that will bind to Primary Antibody 2.
  • Coating Buffer (50 mM Sodium Carbonate, pH 9.5)
  • Wash Buffer (10mM Tris, 1M Sodium Chloride, pH 7.2, 0.05% (v/v) Kathon)
  • Blocking Buffer (10 mM Tris, pH 7.2, 10% (w/v) D-Gluconic Acid, 5% (w/v) Bovine serum albumin) or (PBS/Tween-20)
  • Antibody diluent (PBS)
  • Colorimetric substrate
  • Stop solution
  • ELISA plate reader

Antigen Coating

  1. Prepare a 1 µg/ml dilution of the Primary Antibody 1 (Capture Antibody) using the Coating Buffer as the diluent. IR Tip- The coating antibody is referred as the “Capture Antibody”. 
  2. Add 100 µl of the Capture Antibody to as many wells of the plate as needed. If using a standard curve, be sure to coat plates for those too. Remember to run your samples in triplicate to ensure the most accurate results.
  3. Add 100 µl of Coating Buffer (without antigen) to a set of wells for the standard curve as a negative control.
  4. Incubate the plate for 1hr at 37º C, 3 hours at 18-25º C, or overnight at 4°C.
  5. After coating, flood each well of the plate with Wash Buffer and flick out the liquid. Wash the plate 3 times and pat dry on a paper towel.


  1. Add 375 µl of Blocking Buffer to each well.
  2. Incubate the plates for 1hr at 37º C, 3 hours at 18-25º C, or overnight at 4°C.
  3. Flick out the blocking buffer and pat the plate dry. Blocked plates can be stored covered at 4°C for up to two weeks.

Adding the Sample and Standard Controls

  1. To prepare a standard curve, dilute the control antigen to 1 µg/ml in Phosphate Buffered Saline (PBS). Prepare serial dilutions ranging from 1 µg/ml to 0.0039 µg/ml. Include a blank of PBS as a negative control. Add a total of 100 µl of each dilution into the coated wells. Each dilution (including the blank) should be added in triplicate.
  2. Dilute the antibody sample to appropriate dilutions in PBS. Add 100 µl of each dilution of the test sample to coated wells. Include a blank as a negative control.
  3. Incubate the plate for 1hr at 37º C, 3 hours at 18-25º C, or overnight at 4°C
  4. Wash the plate 3 times with wash buffer and pat dry.

Labeled Secondary Antibody

  1. Dilute the labeled secondary antibody (i.e., Goat anti-Mouse IgG HRP) to 0.5 µg/ml in PBS. IR Tip- If the stock antibody is at 1.0 mg/ml prepare a 1:2000 dilution to make a 0.5 µg/ml dilution. Add 100 µl of the diluted antibody to each well.
  2. Incubate the plate for 60 minutes at room temperature.
  3. Wash the plate 5 times with wash buffer and pat dry. IR Tip- Ensure you properly wash the plate with 5 washes. This will help prevent high background.

Antibody Detection and Plate Reading

  1. Immediately before use, prepare the colorimetric reagent (i.e. TMB Extreme for HRP conjugates)  and add 100 µl to each well.  Incubate until color develops, and stop the reaction with the appropriate solution. For example, if an HRP conjugate is used as the labeled secondary antibody, prepare a 1:1 mixture of TMB Solution. Add 100 µl of the mixture to each well and incubate for approximately 15 minutes until the wells turn blue. Stop the reaction by adding 100 µl of 2N Sulfuric Acid or TMB Stop Solution to each well.
  2. Read the plate at an appropriate wavelength. For HRP, read the plate at 450 nm.
  3. Plot the absorbance of the standard antibody dilutions against their concentrations and draw a line of best fit.
  4. Compare the absorbance of the test sample to the standard curve to determine the concentration. Multiply the values by the appropriate dilution factor.

Troubleshooting Your ELISA Protocol Assay:

  1. Negative control gives positive results- Check for contamination of the substrate solution, the labeled secondary antibody, or the controls.
  2. No color in the positive controls or samples- Check the expiration dates, concentrations, and storage conditions of the reagents.
  3. Very little color in the positive controls or samples- Check the dilution of the labeled secondary antibody and the concentration of the substrate.
  4. Color in the test samples but not in the positive controls – Check the expiration dates and storage conditions of the positive controls.
  5. Color is seen but absorbance is lower than expected – Check the wavelength setting.

ImmunoReagents, Inc. is a leading global manufacturer of quality antibodies and reagents used in pharmaceutical research, life science research and in vitro diagnostics. Product offerings include a wide range of immunochemistry reagents such as purified immunoglobulins, primary antibodies, and secondary antibodies covering a broad spectrum of immunoglobulins from various species. ImmunoReagents also provides custom manufacturing to meet specific customer requirements while adhering to cGMP guidelines and ISO quality systems requirements. The company is located in Raleigh, North Carolina.