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.


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.

Video — ELISA: Direct vs. Indirect Methods

Video — Competitive ELISA: Single-Epitope Detection

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.

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. 

Assay Development Technical Handbook

The revised Assay Development Technical Handbook is an essential resource for any laboratory using enzyme-linked immunosorbent assay (ELISA) and related plate-based assay methods. The handbook describes the essential techniques and tools for designing and optimizing ELISA Assays. Featured products include coated microplates, standards, blockers, buffers, probe-labeling reagents, secondary antibodies and detection substrates.

Contents include: Introduction to ELISA, Selecting an ELISA Plate, Thermo Scientific Pierce Microplates, Thermo Scientific Pierce Coated Microplates, Blocking and Washing, Blocking and Washing Reagents, Detection Probes, Antibody Labeling, Choosing a Substrate, Bulk and Custom Offerings, and Recommended Reading.


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 Figure 1. 

Example of a checkerboard titration

Figure 1. 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.
  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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  1. Prepare different blocking solutions. If the blocking solution is not preformulated (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.
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  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.
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  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.
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  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.
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  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.
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  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.
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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.
Source Coating Antibody Detection Antibody
Polyclonal serum 5–15 µg/mL 1–10 µg/mL
Crude ascites 5–15 µg/mL 1–10 µg/mL
Affinity-purified polyclonal 1–12 µg/mL 0.5–5 µg/mL
Affinity-purified monoclonal 1–12 µg/mL 0.5–5 µg/mL

Table 2. Recommended enzyme conjugate concentrations for ELISA in different systems. Check the instructions for the substrate, as they may recommend a more defined concentration range for the enzyme conjugate.

Enzyme System Concentration
HRP Colorimetric system 20–200 ng/mL
  Chemifluorescent system 25–50 ng/mL
  Chemiluminescent system 10–100 ng/mL
AP Colorimetric system 100–200 ng/mL
  Chemiluminescent system 40–200 ng/mL