ELISA Assay Solutions & Protocol

Posted by Jack on November 24, 2022
Table of Contents


    ELISA assay solutions are a critical part of the development and analysis of an ELISA. The components used in ELISA assay solutions are typically proprietary, which means that they are not available for purchase separately. However, the principles behind these solutions can be applied to other assays and applications. The following sections will outline the different components of an ELISA solution and explain why each is important for measuring antigen-antibody interactions in an immunoassay.

    Principles of ELISA Assay Development

    ELISA is a quantitative assay that detects the presence of an antigen in a sample. Antigens are molecules known to stimulate the production of antibodies, while antibodies are proteins produced by B-lymphocytes that bind to antigens and help initiate immune responses. By measuring this process, ELISA allows researchers to detect the presence of an antigen in a sample and determine whether it's present at high enough levels to trigger an antibody response.

    The majority of ELISA assays involve a sandwich configuration: A capture reagent binds specifically to its target molecule (antigen or antibody), then another reagent is added which binds only if there is also interaction between these two components. The detection reagent can be used as either an enzyme or as a colored dye; whichever one you choose will indicate whether binding has occurred between your target molecule and capture reagents—and thus whether you're detecting any given substance being sought out by your assay method

    ELISA Plate Coating

    The purpose of the coating step is to attach your antigen (the protein you are testing for) onto the plates. It's a simple process that involves incubating the plate with your antigen and washing off any unbound proteins.

    To start, you'll need to wash off any dust particles from your plates, add some coating buffer (usually PBS or TBS), then add an aliquot of antigen solution. Then you'll incubate overnight at room temperature, wash off any unbound proteins, add more coating buffer and incubate again overnight. You can repeat these steps until you've reached sufficient coverage (usually two times over).

    During incubation periods it's important to keep track of which wells have been coated because this will save time later on when doing your ELISA assay!

    ELISA Antibody Detection Solutions

    ELISA Antibody Detection Solutions:

    • Clear, colorless solutions that do not contain any preservatives.
    • No proteins or lipids. If you use a serum sample, it must be heat-treated to deactivate serum proteins and lipids before adding it to your assay buffer.
    • No detergents, which can interfere with detection of antibodies.

    ELISA Plate Washing

    Wash the plates with wash buffer. Wash the coated plates three times, each time for 5 minutes with 200 mL of wash buffer. Rinse the plates twice with distilled water and again with distilled water. Dry the coated plates by placing them upside down on a paper towel and blotting away excess moisture until all liquid has been removed (this may take several minutes). Repeat this process until all wash buffers are removed from the plate surface (about 10 times).

    Substrate Development and ELISA Color Development

    • Substrate development: In this step, the sample is incubated in a solution containing the ingredients to react with the antigen. The substrate used should be one that produces a visible color change when it reacts with bound antigen.
    • Color development: This involves adding a buffer solution containing an enzyme called peroxidase (POD). The POD cleaves hydrogen peroxide into water and oxygen gas as part of an oxidation-reduction reaction, producing an insoluble dye that forms visible crystals on top of the solid phase of your ELISA plate.

    ELISA Plate Reading and Data Analysis

    ELISA data analysis can be performed using different methods. The ELISA assay development team will determine which method is most appropriate for their application, but all methods have the same overall goal: to quantify the concentration of analyte in a sample.

    While the exact tools used to perform ELISA data analysis vary between applications, they are all complex processes that require significant time and effort to complete. Regardless of which software you choose, it is essential that you are comfortable with its capabilities before beginning your analysis project. Experts recommend that you take some time to familiarize yourself with how an ELISAsoftware performs calculations and displays results—this way, once a project begins in earnest, there won't be any surprises when running into problems along the way!

    Different components of the ELISA assay protocol are very important.

    Let's talk about the various components of an ELISA assay protocol:

    • ELISA plate coating
    • ELISA antibody detection solutions
    • ELISA plate washing
    • Substrate development and color development (background correction)
    • Plate reading and data analysis

    Reagent preparation

    • Prepare all reagents in a laminar flow hood using sterile, single-use disposable pipetting tips to avoid contamination and ensure consistency in your assay.
    • Use a clean, dry, disinfected surface for all handling of tubes and containers, then cover with a contact isolation film (e.g., Saran Wrap) or another type of barrier that is effective at preventing airborne particulates from contacting the solution inside the tube or container (we recommend “Saran Laminating Film”; see Resources). In general, you should never need to open more than one tube at once if you are working in a laminar flow hood—and even then it is best not to open more than two adjacent tubes at once!
    • Always wear proper PPE while working with infectious materials such as blood samples: gloves, goggles/face shield/face mask/lab coat (depending on what level of protection you need), shoe covers made out of Tyvek paper (not cotton), etc..

    ELISA Plate Setup

    • Prepare the plates by adding a known amount of standard or calibrator to each well.
    • Prepare the reagents according to manufacturer's protocol. In this example we are using an anti-human IgG monoclonal antibody (MAb) for detection and biotinylated goat anti-mouse IgG for secondary detection.
    • Prepare samples by diluting them in sample buffer (1:4) if necessary for your assay, as per manufacturer’s protocol or as needed for your particular application (for example, if you are using an ELISA kit from Pierce).
    • Add samples to wells in duplicate or triplicate depending on the number of colorimetric reaction measurements required by your specific application and how many replicates you wish to perform on each plate configuration (see below). Use enough volume so that there is at least 30 percent remaining volume after adding all samples; this will help ensure good results when performing either endpoint titration assays or sandwich assays where one antibody binds noncovalently with another molecule like fluorophore-labeled antibodies in immunoassay protocols

    Sample Preparation

    When you are ready to prepare your samples, you should use aseptic technique. This means that you will work in a clean space and use gloves and safety glasses to protect yourself against cross-contamination. You also should make sure that the equipment is clean before starting each step of the assay procedure. We recommend using a microtiter plate reader for this step because it allows you to easily measure absorbance at different wavelengths.

    Gloves can be used during all steps except when pipetting solutions into wells on plates or when removing blanks from their plates prior to reading absorbance values; however, we strongly recommend wearing them throughout all steps so that you do not accidentally contaminate your samples with DNA from previous experiments or other contaminants found outside in nature (e.g., in water).

    Sample addition, incubation and wash

    The sample is added to the plate in a volume of 25 µl per well. The sample should be added to each well no more than 1 hour prior to the end of incubation. Remove any air bubbles from the sample wells by gently tapping on the top of your plate with a pipet tip or finger.


    ELISA assay is a very sensitive and specific method to detect the target molecules in samples. ELISA is widely used in the medical, pharmaceutical and research industries. The protocol of an ELISA assay consists of several steps that need to be performed in order to ensure accurate results. These include plate coating, antibody detection solutions, plate washing protocols etcetera

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