Types of ELISA Assays
An Introduction to the Different Types of ELISA Assay Tests
Enzyme-linked immunosorbent assay (ELISA) is a method used to identify and quantify substances such as proteins, peptides, antibodies, hormones, and other analytes. ELISA Kits have become one of the most used assay techniques for research laboratories, clinical diagnostics companies and more. ELISA kits are used in other formats such as testing individual samples and automated screening. ELISA is used as a common diagnostic tool for medicine, biotechnology, plant pathology, and quality assurance in many industries.
Surmodics IVD provides assay developers a one-stop-shop for Enzyme-linked immunosorbent assays. This can be seen in our full line of dried protein stabilizers and blockers, sample or assay diluents, in-solution protein stabilizers, ELISA substrates, stop solutions and accessory reagents, and supply of DIARECT™ antigens and antibodies.
Please review some of the key technical advantages of our Components of an ELISA below.
Surmodics IVD's StabilCoat™, StabilBlock™ and StabilGuard™ Immunoassay Stabilizers are formulated to minimize non-specific binding interactions with the surface and stabilize the dried capture protein over time. These immunoassay reagents offer assay developers the gold standard for stability and blocking efficacy. They provide improved assay performance in a one-step process for streamlined manufacturing.
Surmodics IVD's StabilZyme™ Stabilizers offer assay developers the gold standard for stability of protein conjugates at working strength concentration for ELISA/EIA, ELISpot, RIA and immunoblot/Western Blot applications.
The signal generation step of immunoassay systems is critical to obtaining an accurate measurement of the target protein. Surmodics IVD’s portfolio of colorimetric substrates and chemiluminescent substrates offers the stability, low background and sensitivity needed to meet the demands of assay manufacturers.
Gold Standard BioFX™ stop solutions for colorimetric substrates are available as dry blends or in liquid formulations including our Nova-Stop solution that provides minimal drift and a safer alternative to assay developers as it is non-corrosive to both skin and eyes.
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There are three main types of Enzyme-linked immunosorbent assay (ELISA), broken down into tests based on how the antibodies and analytes are bonded, Direct ELISA, Indirect ELISA, and Sandwich ELISA. In the following section, these types of ELISA’s will be explored further along with a Competitive ELISA.
The Direct ELISA process is when an antigen or other sample of interest is immobilized directly onto the plate. It is then detected by a conjugated antibody that binds to the target protein. A substrate is then added, which produces a signal proportional to the amount of analyte concentration within the sample. These tests are less specific than the common sandwich ELISA. This is because only one antibody is being used in a direct ELISA.
The Indirect ELISA requires a two-step process for detection using both a primary antibody and labeled secondary antibody. The process is similar when looking at the additional immunoassay reagents required for signal generation, including but not limited to blocking buffers, Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP) Conjugate Stabilizers, and TMB Substrates.
Direct vs. Indirect ELISA’s
The main difference between a Direct and Indirect ELISA can be found in the use of a secondary antibody for detection within an Indirect ELISA. Moreover, an Indirect ELISA uses a primary antibody in combination with a secondary antibody which is conjugated with the detection enzyme. Whereas a Direct ELISA only uses a single primary antibody.
Sandwich ELISA assays are the most commonly used type of ELISA. This process uses two specific antibodies, which then sandwich the antigen or antibody of interest. These are commonly referred to as matched antibody pairs. The capture antibody is then coated on a microplate, and if present target protein binds to the captured antibody. A conjugated antibody is added and binds to detect the target protein. An ELISA substrate is added, producing a signal proportional to the analyte in the sample. These ELISA are highly specific assays, but often need additional research. This is due to risk for false positive results.
Competitive ELISA’s are typically used for small molecules. This ELISA is used when the target protein is too small to sandwich with two antibodies. A capture antibody is coated on a microplate. Then a conjugated antigen is used to compete with the sample antigen to bind with the capture antibody on the plate. The more antigen that is present in a sample, the less conjugated antigen will bind to the captured antibody. A substrate is then added, so the signal produced is inversely proportional to the amount of protein in the sample.
How to overcome non-specific binding in an assay
Non-specific binding is going to be an issue in many assay formats, especially those utilizing complex biological samples. Surmodics Protein Stabilizers, Blockers and Diluents help to cut back non-specific binding while maintaining and even increasing signal-to-noise ratios.
Addressing lot-to-lot inconsistency in an assay:
Lot-to-Lot inconsistencies can be a very common issue for assay developers that can directly lead to false positives or false negatives. Surmodics™ dedication to quality is demonstrated by the ISO 9001:2015 and the ISO 13485:2016 certifications and helps ensure lot-to-lot consistency of our high performance reagents.
Increasing the sensitivity and specificity of an assay:
Sensitivity and specificity are critical measurements when determining the accuracy and reliability of an assay. Surmodics IVD’s Protein Stabilizers, Blockers and Diluents help to increase the sensitivity and specificity of the assay and in turn increase the diagnostic accuracy of the assay.
Increasing the stability of an assay:
Choosing the most effective substrate for an assay:
Detection limit, dynamic range, and reproducibility are cornerstones in the development of a successful immunoassay application. Surmodics IVD’s portfolio of BioFX™ colorimetric and chemiluminescent substrates offers the stability, low background and sensitivity needed to meet the demands of assay manufacturers.
How many ELISA Assays are There?
ELISA’s have been in the market as a trusted resource for diagnostics for several decades now. There are likely over thousands of FDA approved ELISA kits in the market. ELISA technology is continuing to evolve and advance every day. With the help of Surmodics™ IVD’s immunoassay reagents, we can help assay developers achieve just that. At the end of the day, our goal is your goal, accurate and reliable results every time for every patient.
What is the most Common type of ELISA?
Sandwich ELISA assays are the most commonly used type of Enzyme-linked immunosorbent assay (ELISA). Surmodics™ IVD acts as a one-stop-shop for sandwich ELISA’s. We offer solutions at each step of the ELISA all of which are designed to increase the sensitivity, stability, and specificity of ELISA kits. It’s important to note, Surmodics™ IVD’s immunoassay reagents can be used in a variety of protein-based immunoassays, including lateral flow/POC applications, Western Blot, and more.
To learn more and/or request free samples for evaluation, contact our team today!