ELISA stands for enzyme-linked immunosorbent assay, also often referred to as enzyme immunoassay (EIA). ELISA is a type of immunological assay used to detect and quantify specific biomolecules, such as proteins, antigens, or antibodies, in biological samples. An ELISA, like other types of immunoassays, relies on antibodies to detect a target antigen using highly specific antibody-antigen interactions. In an ELISA assay, the antigen must be immobilized to a solid surface. This is done either directly or via the use of a capture antibody itself immobilized on the surface. The antigen is then complexed to a detection antibody conjugated with a molecule amenable for detection such as an enzyme or a fluorophore. This immunosorbent assay format allows for sensitive, specific, and reproducible detection, making ELISA a preferred method for identifying disease biomarkers, monitoring immune responses, and screening for various conditions.

An ELISA is typically performed in a multi-well plate (96- or 384-wells), which provides a solid surface to immobilize the antigen. The immobilization of analytes facilitates the separation of the antigen from the rest of the components in the sample, making ELISA one of the easiest assays to perform on multiple samples simultaneously. The resulting measurable signal, such as a color change or fluorescence, allows signal detection, which is essential for accurately interpreting ELISA results.

Figure 1. The basic setup of an ELISA assay. A capture antibody on a multi-well plate will immobilize the antigen of interest. This antigen will be recognized and bound by a detection antibody conjugated to biotin and streptavidin-HRP.

ELISA equipment and materials

Performing an ELISA test requires a set of specialized equipment and carefully selected materials to ensure accurate and reliable results. The process begins with ELISA plates, which are typically 96- or 384-well microplates designed to provide a solid surface for the assay. Each well is coated with a capture antibody that binds specifically to the target antigen present in the sample. After the sample is added, a detection antibody—often labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase—is introduced to bind the captured antigen. The addition of an enzyme substrate triggers a chemical reaction, producing a measurable signal, usually a color change, that correlates with the amount of target antigen.

Other essential materials include blocking buffer to prevent non-specific binding, wash buffer to remove unbound antibodies and reagents, and a standard curve to enable quantitative analysis of results. Accurate pipetting, proper use of a microplate washer, and reading the results with a plate reader are all critical steps in the ELISA workflow. The choice and quality of detection antibody, capture antibody, and enzyme substrate can significantly impact the sensitivity and specificity of the assay. Proper storage and handling of all reagents and equipment are vital to maintain the integrity of the ELISA test and ensure reproducible outcomes.

ELISA advantages and disadvantages

Advantages

• High sensitivity and specificity: it is common for ELISAs to detect antigens at the picogram level in a very specific manner due to the use of antibodies.
• High throughput: commercial ELISA kits are normally available in a 96-well plate format. However, the assay can be easily adapted to 384-well plates.
• Easy to perform: protocols are easy to follow and involve little hands-on time.
• Quantitative: it can determine the concentration of antigen in a sample.
• Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine, and saliva among others.

Disadvantages

• Temporary readouts: detection is based on enzyme/substrate reactions and therefore readout must be obtained in a short time span.
• Limited antigen information: information limited to the amount or presence of the antigen in the sample.

These are the general ELISA advantages and disadvantages. There are other advantages and disadvantages depending on the type of ELISA used as explained in the next section.

Types of ELISA

ELISAs come in different formats, each one with its own advantages and disadvantages. The diagram below illustrates the four main different types of ELISA. The ELISA method encompasses these formats–direct, indirect, sandwich, and competitive ELISA–each offering unique applications for detecting and quantifying proteins and antigens.

Figure 2. The different types of ELISA (direct, indirect, sandwich, and competitive).

Direct ELISA

The antigen is immobilized on the surface of the multi-well plate and detected with an antibody specific to the antigen that is directly conjugated to HRP or another detection molecule. In direct ELISA, this detection antibody is often referred to as a labeled primary antibody, which enables direct identification of the target antigen.

Indirect ELISA

Similar to direct ELISA assays, the antigen is immobilized on the surface of the multi-well plate. However, a two-step process is required for detection whereby a primary antibody specific for the antigen binds to the target, and a labeled secondary antibody against the host species of the primary antibody binds to the primary antibody for detection. In indirect ELISA, primary antibodies provide high specificity for the target antigen, while secondary antibodies, often enzyme-conjugated, bind specifically to the primary antibodies to amplify the detection signal and enhance assay sensitivity.

This method can also be used to detect specific antibodies in a serum sample by substituting the serum for the primary antibody.

Explore indirect ELISA kits and reagents

Sandwich ELISA

Sandwich ELISA (or sandwich immunoassay), the most commonly used ELISA format, requires two antibodies specific to different epitopes of the antigen. These two antibodies are commonly referred to as matched antibody pairs. One of the antibodies is used to coat the surface of the multi-well plate where it serves as a capture antibody to facilitate the immobilization of the antigen. The other antibody is conjugated and facilitates the detection of the antigen. In this format, the detection antibody binds to a second epitope on the antigen after the capture antibody has bound the antigen, allowing for highly specific and sensitive detection. Monoclonal antibodies are often used in sandwich ELISA assays due to their monospecificity, stability, and engineered capabilities, which provide improved assay specificity and sensitivity compared to polyclonal antibodies.

Explore sandwich ELISA kits and reagents

Competitive ELISA

Also known as inhibition ELISA or competitive immunoassay, this assay measures an antigen’s concentration by detecting signal interference. Each of the previous formats can be adapted to the competitive format. The sample antigen competes with a reference antigen for binding to a specific amount of labeled antibody. The reference antigen is pre-coated on a multi-well plate. The sample is added to the plate wells with a labeled antibody, which is typically an enzyme-conjugated detecting antibody. This assay orientation is referred to as ”antigen down.” Depending on the amount of antigen in the sample, more or fewer free antibodies will be available to bind the reference antigen. This means that the more of the antigen there is in the sample, the less reference antigen will be detected and the weaker the signal will be. Some competitive ELISA kits use labeled antigens instead of labeled antibodies, an assay orientation referred to as “antibody down”. In this orientation, the labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody, which is immobilized on the plate. The lower the amount of antigen in the sample, the stronger the signal due to more labeled antigen in the well.

Explore competitive ELISA kits and reagents

SimpleStep ELISA® Kits

Our SimpleStep ELISA® kits provides improved speed and performance while retaining the familiar process and standard data output of a traditional ELISA kit. SimpleStep ELISA® kits reduce the number of wash steps by enabling the sandwich complex formation in one step rather than sequentially. Total time required is less than two hours.

Browse all SimpleStep ELISA® kits

Competitive SimpleStep ELISA® Kits

Our Competitive SimpleStep ELISA® Kits provide improved speed and performance while retaining the familiar process and standard data outputs of a traditional competitive ELISA® kit. Competitive SimpleStep ELISA® kits reduce the number of wash steps by enabling the antibody analyte complex formation in one step rather than sequentially.

The total time required is less than two hours.

Browse all competitive SimpleStep ELISA® kits

Which type of ELISA should I use?