ELISA Assay Principles, Protocols & Kits

Posted by Jack on November 24, 2022
Table of Contents


    An ELISA is a very useful method for detecting the presence of specific molecules in your sample. It can be used to detect proteins, antigens or antibodies in serum or plasma samples. The ELISA assay principle is based on the use of specific monoclonal antibodies (mAbs) which bind to their target antigen (Ag). The antibodies are attached to the surface of microtiter plates and binding between the Ab-Ag complex and the specific antibody occurs through this attachment. This can be detected by adding a substrate that is converted into an insoluble colored product when it reacts with the product formed by reaction between Ag-Ab complex present on the plate surface and labelled secondary antibody (Ab-HRP).

    What is an ELISA?

    ELISA stands for Enzyme-Linked ImmunoSorbent Assay. ELISAs are a type of immunoassay that uses an antibody to detect and quantify an antigen (for example, a protein). ELISAs use antibodies bound to a plate, while the sample containing the antigen is added. The antigen will react with its specific antibody and bind to it, forming a bond between them. The bound complexes are detected by an enzyme conjugated with another substance in solution or on paper strips.

    How to do an ELISA.

    ELISA stands for enzyme-linked immunosorbent assay. It's a widely used technique in biochemistry and molecular biology laboratories because it lets you detect and quantify small amounts of antigen (substance that stimulates antibodies) in a sample, such as blood or tissue culture cells.

    ELISAs are typically performed in plate format, with one plate per well. In an ELISA experiment, the antigen is fixed to the solid phase (usually via a coating antibody), then exposed to your sample either by adding it directly or by washing over the surface. Your specific antibody is added to each well afterwards—if your antibody binds its target antigen, it will stay attached; otherwise, it will wash off into solution where it can be detected directly or through an additional reaction step involving HRP/enzymes.

    Antigen Coating and Blocking

    As you know, ELISA assays involve the use of an antigen and a secondary antibody. This can be done in two ways:

    • The antigen can be coated onto the wells of the microtiter plate and then incubated with a solution containing free antibodies
    • The antigens can be captured by adding a specific antibody solution to coat each well

    Primary antibody incubation.

    The primary antibody incubation step is the first step of an ELISA assay. It is important for two reasons:

    • It allows the primary antibody to bind to its target antigen (the analyte of interest).
    • It allows for secondary antibody detection by adding a conjugated secondary antibody as part of the ELISA kit protocol.

    Secondary antibody incubation.

    Your secondary antibody will be added to the plate at this point.

    This step is important because it allows for detection of your primary antibody.

    The secondary antibody is conjugated (attached) to an enzyme, which can be any number of colors or dyes.

    When you add your conjugated enzyme-labeled antibody to the plate, it binds to the primary antibodies that are already present in the wells. Then you add substrate solution and watch as color develops on your plate!

    Detection and quantification of binding.

    Once the bound antibody is revealed by a chromogen, the amount of binding can be quantified using an ELISA plate reader. Typically, detection is done by adding a solution called a substrate (e.g., 3,3',5,5'-tetramethylbenzidine) that produces a colored product when heated or otherwise activated.

    Quantitative measurements are made by comparing the amount of binding to a standard curve to determine the quantity of analyte present in your sample. The more specific your antibody is for its target antigen/epitope, the less background binding you will see and therefore less signal generated at these wavelengths.

    Washing the plate prior to adding the substrate.

    Washing the plate prior to adding the substrate.

    Washing is essential to prevent false positives. We always wash the wells before adding the substrate because:

    • Unbound antibody will be washed away (false negative)
    • Unbound antigen will be washed away (false positive)
    • Unbound conjugate will be washed away (false positive)

    Substrate addition - enzyme conjugate or chromogen.

    The substrate is added to the plate. The substrate is usually a chromogen or enzyme conjugate, which is an enzyme that has been linked to a dye. When the enzyme binds to its specific antigen in the sample, it causes a reaction between the dye and the substrate that changes its color.

    Stop reagent addition.

    Stop reagent addition. Stop reagent is a solution that stops the reaction and allows the absorbance to be read at the end of the assay. It contains a dye that absorbs light at a specific wavelength and is therefore used to determine when you should stop adding steps in ELISA.

    Readout at a specific wavelength (410 nm or 450nm).

    The difference between 410nm and 450nm is not as significant as you might think. Both are used for ELISA assay results, but depending on the type of results you want to get, one may be more appropriate than another. The difference in wavelength has no effect on sensitivity or specificity. However, it does affect the accuracy of your results because both wavelengths give different absorbance values for each color channel (blue vs green).

    The advantages of using 450nm instead of 410nm are:

    • You can measure turbidity directly from your sample buffer without adding any additional reagents or dilutions
    • It's easier for beginners to set up their assay since there is only one color channel (green)

    ELISA assays are particularly effective when you need to detect a limited number of molecules in your samples.

    ELISA is a great way to detect a specific material in a sample. However, it is not an absolute measure of that material's presence. ELISA works by detecting the binding of an antibody (a protein produced by B cells) to an antigen (a substance that stimulates an immune response). The latter can be any molecule or particle with which you want to detect; for example, you could use it to detect antibodies against certain types of cancer cells or viruses.

    ELISA has many applications outside of diagnostics as well. For example, scientists often use this technique to determine if certain compounds are present in samples from plants and animals without having access to those organisms themselves—something that might prove difficult if they're located thousands of miles away!

    ELISA can also be used when dealing with small amounts of material because the reaction occurs at such high levels that even tiny amounts will show up clearly on your results!


    We hope this article has been informative and helpful for you. ELISA is a powerful tool for detecting a wide variety of materials in your samples, including antibodies, antigens and other molecules that bind to antibodies. With these principles, you can run an ELISA experiment on any organism or cell type that you want!

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