ELISA Assays: Indirect, Sandwich, and Competitive

Posted by Jack on November 24, 2022
Table of Contents


    ELISA is an abbreviation for enzyme-linked immunosorbent assay. It's a test that uses antibodies to detect specific antigens in a sample. The ELISA assay is often used to determine the concentration of molecules such as viruses or drugs in biological fluids. ELISA assays are divided into three main types: indirect, sandwich, and competitive. Each type has its own advantages, so it's important to choose the most applicable format based on your needs.

    The different ELISA assay types each have their own advantages, so it's important to choose the most applicable format.

    Indirect ELISA Assay

    The most commonly used type of assay is the indirect enzyme-linked immunosorbent assay (ELISA), which can be performed with a commercial kit or by following a protocol. In this method, the antigen is immobilized onto an antibody-coated microtiter plate and then incubated with diluted patient sample or an enriched immune complex produced from an initial reaction. An enzyme conjugate solution containing either horseradish peroxidase (HRP) or alkaline phosphatase (AP) is added to detect bound antibodies in each well. This mixture is incubated for at least 2 hours before measuring absorbance at selected wavelengths. The optical density units are proportional to the amount of specific antibody present in each sample, allowing you to compare results between samples without having to perform additional reactions from each sample separately.

    Indirect ELISA Assay

    • The indirect ELISA assay is used to detect the presence of an antigen in a sample. In this assay, an antibody is first attached to a solid surface such as glass or plastic. A known amount of the antigen is then added to the solid phase antibody and incubated for a period of time. The next step involves adding a secondary antibody that has been labeled with an enzyme (for example, alkaline phosphatase). This enzyme catalyzes a reaction that causes it to emit light at one wavelength when excited by another wavelength of light. After washing away any unbound material, the amount of signal produced by each bead can be quantified using a plate reader instrument equipped with dual-wavelength capability and appropriate filters.

    Sandwich ELISA Assay

    The sandwich ELISA assay is a more sensitive assay than the direct ELISA. It also has better specificity than the direct ELISA. However, this assay is more expensive and time consuming than the direct ELISA.

    The sandwich ELISA assay uses two antibodies that recognize two different epitopes of your target antigen in order to detect your target protein (Figure 1). The first antibody is coated onto a well of an microtiter plate which binds to your antigen when you add it to that well. The second antibody recognizes an epitope on your antigen directly bonded or indirectly bonded through labeled proteins (biotin-Streptavidin binding) onto one end of small beads that have been coated with anti-streptavidin monoclonal antibodies. These beads are mixed with unbound conjugate, washed excess away and incubated under gentle shaking conditions for a period of time that allows for specific binding between conjugate and biotin-conjugated secondary antibodies while washing away any non-specifically bound material from both solid phase and solution phase components. After washing away excess unbound conjugate, individual wells can be read optically or by scintillation counting techniques to determine amount bound per well

    Competitive ELISA Assay

    A competitive ELISA assay is a variation of the sandwich ELISA that can be used to detect a specific antibody in a mixture of antibodies.

    In this assay, an unlabelled primary antibody (Ab1) binds to the antigen on a microplate. A specific secondary antibody (Ab2) is then added to the plate and allowed to bind with the unbound primary Ab1. Finally, the amount of Ab2 bound will be inversely proportional to the concentration of antigen present in the sample; hence "competitive".

    nzyme-Linked Immunosorbent Assay - What is it?

    An ELISA is an enzyme-linked immunosorbent assay, which is a test used to detect and measure the presence of an antigen in a sample.

    The ELISA test involves the use of antibodies (i.e., proteins produced by your body that help fight infections) to bind with antigens (i.e., proteins or other molecules that cause an immune system reaction). The bound antibody is then detected using an enzyme-linked antibody (which reacts with peroxidase, a chemical found in many organisms), producing a signal that can be measured quantitatively using various methods such as spectrophotometry or fluorimetry.

    History of ELISA

    ELISA (enzyme-linked immunosorbent assay) is a technique for detecting and measuring the presence of an antigen or antibody in a sample. The ELISA test uses antibodies to bind to the antigens they recognize, forming a complex that can be detected by colorimetric reactions such as a colored dye or fluorescence tag. By using different combinations of antibodies, scientists are able to detect the presence of many different types of molecules, including proteins, lipids, carbohydrates and even nucleic acids like DNA and RNA.

    Indirect ELISA

    For example, an indirect ELISA can be used to detect a specific antigen or antibody in a sample.

    This type of immunoassay is performed by first producing an antigen-antibody complex (as in the direct ELISA). The excess unbound antibody is washed away and then the bound complexes are detected using one or more labels. This method is indirect because you're using labeled secondary antibodies to detect the presence of your target protein instead of directly labeling your target protein itself with a fluorescent dye, which is what happens in direct ELISAs.


    In sandwich ELISA, the capture antibody is immobilized on a solid support, such as a microplate. The target antigen is added to the plate and binds to the capture antibody through its specific binding site (antigen-antibody interaction). Next, a detection antibody specific for that particular target antigen is added to the mixture. Finally, an enzyme-labeled conjugate that reacts with both detection and detection antibodies washes over each well on the plate in order to detect any remaining free protein bound at this time by adding substrate reagent containing chromogenic enzymes that catalyze an enzymatic reaction resulting in color development proportional to amount of bound protein present.

    Competitive ELISA

    A competitive ELISA is a variation of the standard indirect ELISA which can be run in either a sandwich or direct format and uses different antibodies directed against an antigen. The purpose behind this assay is to detect as little of the competitor as possible, while maximizing your own signal. To put it simply: you will want to get rid of all non-specific binding sites before they contaminate your final results.

    The steps involved in running a competitive ELISA are similar to those used for other types of assays except that there are additional steps taken to remove unbound competitors and maximize your own signal.

    ELISA Alternative Assay Techniques

    ELISA alternative assay techniques include:

    • Western blot (WB) - also called an immunoblot or blotting, this is a technique used in molecular biology to transfer proteins from gels onto membranes, which are then probed with antibodies or protein probes. The result is similar to an ELISA but uses gel electrophoresis instead of chromatography.
    • Radioimmunoassay (RIA) - RIA is essentially the same as an ELISA test but uses radioactive material rather than colorimetric or fluorescent materials.

    More to come.

    • You've probably heard of ELISA, and you know that it's an important test in the field of immunology. But did you know that there are a few different types?
    • The following sections will give you some background on ELISA assays: Indirect, Sandwich, and Competitive.
    • We'll also go over some ways to optimize the performance of your ELISA assay by changing certain parameters like incubation time or washing conditions.


    While ELISA assays have many advantages, they are not always the best choice for every application. It's important to understand the differences between indirect, sandwich, and competitive ELISA assays and choose the most appropriate method for your needs.

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