Enzyme-Linked Immunosorbent Assay (ELISA)




What is an ELISA?

ELISA, or enzyme-linked immunosorbent assay, is a method used to quantitatively detect an antigen within a sample. An antigen is a toxin or other foreign substance, for example a flu virus or environmental contaminant, that causes the vertebrate immune system to mount a defensive response. The range of potential antigens is vast, so ELISAs are used in many areas of research and testing to detect and quantify antigens in a wide variety of sample types. Cell lysates, blood samples, food items, and more can be analyzed for specific substances of interest using ELISAs.

Steps to run a sandwich ELISA assay

Most ELISAs are run in microplates, with the bottom of the microplate wells serving as the solid surface to which antibodies and other reagents bind. A microplate washer is used to wash away non-specific material in the wells, and a microplate reader detects the color change produced when target antigen is present. An ELISA’s plate reader software is used to plot standard curves and calculate results.

Here are the steps used for a typical sandwich ELISA assay:

ELISA analysis and techniques

ELISA is a commonly used analytical technique performed in many research and biotech labs. Below is a collection of application notes, research and technology related to significant ELISA assays and applications. We’ve started with a brief introduction of the four common types of ELISAs including direct, indirect, competitive, and sandwich.


  • Direct ELISA

    The antigen is bound to the bottom of the microplate well, and then it is bound by an antibody that is specific to the antigen and also conjugated to an enzyme or other molecule that enables detection.

    Indirect ELISA

    The antigen is bound to the bottom of the microplate well, then an antibody specific to the antigen is added. A secondary antibody, conjugated to an enzyme or other detection molecule, is then bound to the first antibody.

  • Competitive ELISA

    In this type of ELISA, a reference antigen is bound to the bottom of microplate wells. Sample plus antibody are added to the wells, and if there is antigen present in the sample, it competes with reference antigen for binding to the antibody. Unbound material is washed away. The more antigen was in the sample, the less antibody ends up bound to the bottom of the wells by the reference antigen, and the lower the signal.

    Sandwich ELISA

    For this type of ELISA, two antibodies specific to two different epitopes on the target antigen are used. The capture antibody is bound to the bottom of the microplate well and binds one epitope of the antigen. The detection antibody binds to the antigen at a different epitope and is conjugated to an enzyme that enables detection. (If the detection antibody is unconjugated, then a secondary enzyme-conjugated detection antibody is required).

  • Faster ELISA Results

    Faster ELISA results with CatchPoint SimpleStep ELISA kits

    A typical ELISA protocol is time-consuming, with multiple incubation and wash steps. CatchPoint SimpleStep ELISA kits give users a 90-minute ELISA workflow, a nearly two-thirds reduction in time compared to conventional ELISAs.

    Read Application Note  

    Quantitate Interleukin-8 ELISA

    Quantitate interleukin-8 concentrations in just 90 minutes

    Learn how to run the Human IL-8 SimpleStep ELISA on the SpectraMax® ABS Plus Microplate Reader using a convenient preconfigured protocol.

    Read Application Note  

  • Melamine Detection with ELISA Kit

    High-throughput melamine detection with Abraxis Melamine ELISA Kit and Molecular Devices Absorbance readers

    The Abraxis Melamine enzyme-linked immuno-sorbent assay (ELISA) is an immunoassay for quantitative screening of melamine; based on the recognition of melamine by antibodies.

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    ELISA – Cytokine Interleukin-22

    Protein quantitation with the EMax Plus Microplate Reader

    A performance comparison is made between the EMax Plus Microplate Reader and the EMax Endpoint Reader using a sandwich ELISA to quantitate levels of the cytokine Interleukin-22 (mouse/rat IL-22).

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  • Chemiluminescent VEGF ELISA

    Chemiluminescent VEGF ELISA Using the SpectraMax L Microplate Luminometer

    Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides that have been implicated in mammalian vascular development and in disease processes involving abnormal blood vessel growth.

    Read Application Note  

General ELISA protocol

A typical ELISA protocol has multiple reagent addition, incubation and wash steps. The ELISA workflow shows how a typical sandwich ELISA is run. Each step breaks down the ELISA process and highlights the instrumentation and tools needed to conduct the ELISA assay including a microplate washer, absorbance reader and plate reader software.

 

ELISA Assay Protocol Workflow
 

CAPTURE ANTIBODY BINDS TO WELLS

First, the capture antibody is bound to the bottom of the microplate well.

ELISA Plate

Most ELISAs are run in 96- or 384- well microplates, a 96-well plate being the most common. The bottom of the microplate wells serve as the solid surface to which antibodies and other reagents attach. Microplates are typically included in an ELISA kit.

01

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ADD SAMPLE

Sample is added to the well, and antigen within the sample binds to the capture antibody.

02

WASH MICROPLATE

Unbound material is washed away, leaving only the antigen of interest and minimizing the potential for high background signal.

ELISA Plate Washer

A microplate washer is used to wash away non-specific material in the wells.

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03

ADD DETECTION ANTIBODY

Enzyme-conjugated detection antibody binds to a second site on the antigen of interest, providing the means to detect the antigen.

04

WASH MICROPLATE

Unbound antibodies are washed away, leaving only those specific for the target of interest and again minimizing the potential for background signal.

AquaMax Microplate Washer

Aspiration and dispensing of 96- and 384-wells occur simultaneously in all wells leading to high-precision assays and faster microplate processing without mechanical plate indexing or quadrant processing.

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05

ADD SUBSTRATE

Substrate is converted by the enzyme on the detection antibody, producing a color change, with intensity proportional to the amount of antigen present.

Depending on the enzyme and substrate used, the readout can also be fluorescent or luminescent.

06

READ PLATE

The microplate reader detects the colored reaction product and outputs optical density (OD) values that indicate how much light is absorbed by the contents of each well.

ELISA Plate Reader

A microplate reader detects the color change produced when target antigen is present. It does so by measuring how much of the light passed through the wells of the microplate is absorbed by the material within the wells. The more antigen is present, the higher the absorbance value.

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07

ELISA Plate Reader Software

Software is used to plot standard curves and calculate results from the absorbance values provided by the microplate reader.

A standard curve is run so that the amount of antigen in each sample can be accurately calculated. A preconfigured protocol, helps save time by calculating results automatically.

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CALCULATE RESULTS

The amount of antigen in each sample is calculated, and dierent samples—for example, cells subjected to dierent treatment conditions—can be compared.

08

 

Resources for ELISA

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