ELISA (enzyme-linked immunosorbent assay) is a powerful method for detecting and quantifying specific proteins. ELISA typically requires that the antigen of interest be captured or immobilized on a solid surface and then be complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme’s activity via incubation with a substrate to produce a measurable product. ELISA development involves choosing a format, gathering the needed components, and constructing a working protocol. ELISA optimization involves systematically adjusting and testing the many components and variables to help ensure results are robust and accurate. The following image provides a generalized workflow for performing a sandwich ELISA.
ELISA Development and Optimization

Figure 1. Generalized scheme of a typical sandwich ELISA protocol.

ELISA development

There are many things to consider when developing an ELISA. The first is what ELISA format to use—direct, indirect, or sandwich. Formats differ in how the target antigen is captured and detected. Direct and indirect ELISA both immobilize the antigen on the assay plate and then use either a labeled primary antibody (direct) or primary antibody and labeled secondary antibody (indirect) to detect the antigen. Sandwich ELISA is considered the most robust format because the antigen is “sandwiched” between two primary antibodies (capture and detection) and then detected using a labeled secondary antibody (or labeled streptavidin, if the detecting antibody is biotinylated). 

The following videos describe detection methods in sandwich ELISA, as well as an entirely different ELISA format called competitive ELISA.

Different ELISA formats provide different levels of specificity, sensitivity, simplicity, and speed (i.e., number of steps). They also require different numbers and varieties of components. For example, sandwich ELISA requires a mutually compatible pair of specific antibodies, called antibody-matched pairs. If a researcher is developing an ELISA from scratch, it is often because the target of interest is “new” to research, in which case a matched pair of antibodies may not be available. Custom antibody production may be required.

For a more detailed description of ELISA formats and the various components of ELISA (e.g., microplates, wash buffers, blocking buffers, sample diluents, enzyme conjugates, and substrates), see “Overview of ELISA” or one of our technical guides or handbooks. For more information on custom antibody production, please see the Custom Antibodies technology page on our website.

Generally, unless new ELISA development is necessary for specialized research needs, one should check the availability of commercial ELISA kits for the target of interest. We offer more than 1,000 ready-to-use ELISA kits that have already been developed and optimized to specifically detect targets. The kits include antibody-precoated plate(s) and other components to perform the assay. Each kit undergoes validation and quality testing, so laborious optimization steps (described below) are not needed.  The representative data that follows was produced using a TNF alpha colorimetric ELISA kit to detect mouse TNF alpha mouse recombinant protein.

Detection of recombinant mouse TNF alpha protein by ELISA

Figure 2. Detection of recombinant mouse TNF alpha protein by ELISA. Sandwich ELISA analysis of mouse TNF-alpha was performed using a Mouse TNF alpha Colorimetric ELISA kit by loading 50 ml per well of Mouse TNF-alpha Recombinant Protein in dodecuplicate at 2450, 350, 50, and 0 pg/ml across a 3 µg/mL rat anti-mouse TNF-alpha pre-coated plate and incubating for 2 hours at room temperature along with 50 ml per well of rat anti-mouse TNF-alpha biotinylated antibody. The plate was washed and incubated with 100 ml per well of Streptavidin-HRP in all test wells at 1:30,000 for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB Substrate for 30 minutes at room temperature in the dark. The plate was then stopped with 0.16M sulfuric acid. Absorbances were read on a spectrophotometer at 450-550 nm.

Biomarker quantitation assay guide

This 72-page guide provides detailed information about different tools for protein and RNA quantitation. Download this valuable technical resource that covers technologies useful for cancer and inflammation research, immunology, neurology and more. Learn more about how antibody pairs, ELISA kits, and multiplex kits for the Invitrogen Luminex platform may help advance your research.

Download the Biomarker quantitation assay guide

Biomarker quantitation assay guide cover
ELISA optimization

Once a working ELISA has been developed, optimization procedures can be done to improve its performance. Below, steps for optimization of each component of the assay—from the capture antibody to the enzyme conjugate and choice of substrate—are provided. These steps assume the use of a sandwich ELISA format.

The most important part of optimization is testing different concentrations (i.e., dilutions) of antibodies, samples, and buffers. Although each component is described separately, in many instances it is possible to optimize two components simultaneously by performing a checkerboard titration as shown in the figure below.

Example of a checkerboard titration experiment to optimize two ELISA parameters at once

Figure 3. Example of a checkerboard titration experiment to optimize two ELISA parameters at once. This example shows primary antibody vs. detection antibody with all other reagents constant. After the capture and detection antibodies have been optimized, the enzyme conjugate can then be subjected to titration.

Optimizing the concentration of capture antibody
  1. Prepare different concentrations of the capture antibody in coating buffer (see ranges described in Table 1 below).
  2. Apply an equal volume of each concentration to the plate and proceed with the ELISA protocol.
  3. Check for strong signal vs. low background.

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Optimizing the blocking buffer
  1. Prepare different blocking solutions. If the blocking solution is not pre-formulated (i.e., it is a single protein such as BSA), try different concentrations of the protein.
  2. Apply an equal volume of each to the plate and proceed with the ELISA protocol.
  3. Check for strong signal vs. low background.
Optimizing the sample concentration
  1. Try to match the standard diluent as closely as possible to the matrix of the sample. If the matrix itself cannot be exactly duplicated, then test different standard diluent solutions.
  2. Apply an equal volume of each to the plate and proceed with the ELISA protocol.
  3. Check for good dynamic range for the standard curve, and linearity of dilution for the sample. 
  4. If the standard curve has poor dynamic range, then it may be necessary to choose a different diluent. If the sample has poor linearity of dilution when diluted, there may be an imbalance between the sample matrix and the standard diluent. In such cases spike-and-recovery or linearity-of-dilution experiments should be performed.

Select products 

Optimizing the concentration of detection antibody
  1. Prepare different concentrations of the detection antibody in standard diluent (see ranges described in Table 1 below).
  2. Apply an equal volume of each concentration to the plate and proceed with the ELISA protocol.
  3. Check for strong signal vs. low background.
Optimizing the concentration of enzyme conjugate
  1. Prepare different concentrations of the enzyme conjugate in standard diluent according to the range described in Table 2. Ensure the concentration is in accordance with the range described for the substrate.
  2. Apply an equal volume of each concentration to the plate and proceed with the ELISA protocol.
  3. Check for strong signal vs. low background.
Optimizing signal detection
  1. Select substrate(s) based on the likely amount of antigen in the sample and ability to detect it with appropriate instruments.
  2. Apply the working solution to the plate and proceed with the ELISA protocol.
  3. If the antigen can clearly be detected over a dynamic range, then the substrate is appropriate. If the antigen is below the threshold for detection, then select a more sensitive substrate.
Optimizing the standard diluent
  1. Prepare different concentrations of the enzyme conjugate in standard diluent according to the range described in Table 2. Ensure the concentration is in accordance with the range described for the substrate.
  2. Apply an equal volume of each concentration to the plate and proceed with the ELISA protocol.

  3. Check for strong signal vs. low background.

The tables that follow provide detailed information that is useful for determining the best experimental conditions to optimize ELISA results. Parameters to consider include antibody concentrations, sample source, and enzyme system of choice

Table 1. Recommended concentration ranges for coating and detection antibodies for ELISA optimization. The use of unpurified antibodies will work but may result in higher background. The use of affinity-purified antibodies is generally recommended, for optimal signal-to-noise ratio. The concentrations are guidelines only; for best results, optimize each component individually.

SourceCoating AntibodyDetection Antibody
Polyclonal serum5–15 µg/mL1–10 µg/mL
Crude ascites5–15 µg/mL1–10 µg/mL
Affinity-purified polyclonal1–12 µg/mL0.5–5 µg/mL
Affinity-purified monoclonal1–12 µg/mL0.5–5 µg/mL

Table 2. Recommended enzyme conjugate concentrations for ELISA in different systems. For a more defined concentration range for the enzyme conjugate, review the manufacturer’s instructions.\

EnzymeSystemConcentration
HRPColorimetric system20–200 ng/mL
 Chemifluorescent system25–50 ng/mL
 Chemiluminescent system10–100 ng/mL
APColorimetric system100–200 ng/mL
 Chemiluminescent system40–200 ng/mL

Recommended reading

  1. John R. Crowther, Methods in Molecular Biology, the ELISA Guidebook. Second Edition. Humana Press, a part of Springer Science + Business Media, LLC 2009.
  2. Butler J.E. The Behavior of Antigens and Antibodies Immobilized on a Solid Phase. In: M.H.V. Van Regenmortel, ed. Structure of Antigens. Boca Raton, FL: CRC Press, 1992: 209-259. Vol.1, 209; CRC Press, Inc.
  3. Lequin, Rudolf M. "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)." Clinical chemistry 51.12 (2005): 2415-2418. 

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