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Figure 1. Generalized scheme of a typical sandwich ELISA protocol.
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.
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.
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.
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.
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.
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.
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. For a more defined concentration range for the enzyme conjugate, review the manufacturer’s instructions.\
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 |
For Research Use Only. Not for use in diagnostic procedures.