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Enzyme-Linked Immunosorbent Assay (ELISA)

What is ELISA?

ELISA (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.

There are four major types of ELISAs: direct, indirect, competitive and sandwich. Each type is described below with a diagram illustrating how the analytes and antibodies are bonded and used.

  • Direct ELISA

    Direct ELISA

    In a 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

    Indirect ELISA

    In an 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 a competitive 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

    Sandwich ELISA

    For the sandwich 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).

Steps to run a sandwich ELISA assay

Most sandwich ELISAs are run in microplates, with the bottom of the plate 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 an absorbance ELISA microplate reader detects the color change produced when target antigen is present. And, a plate reader software is used to plot standard curves and calculate results.

The illustration below shows a workflow for a typical sandwich ELISA assay:

elisa-graph-wrkflow

 

Step 1: Capture antibody binds to ELISA plate wells

Step 2: Add sample to well – antigen within the sample binds to the capture antibody.

Step 3: Wash microplate – Unbound material is washed away, leaving only the antigen of interest

Step 4: Add detection antibody – Enzyme-conjugated detection antibody binds to a second site on the antigen of interest

Step 5: Wash microplate – Unbound antibodies are washed away, leaving only those specific for the target of interest

Step 6: Add substrate – Substrate is converted by the enzyme on the detection antibody, producing a color change

Step 7: Read plate – The microplate reader detects the colored reaction product and outputs optical density (OD) values

Step 8: Calculate results – The amount of antigen in each sample is calculated and analyzed

View ELISA Protocol

 

ELISA assays and applications

 

Enzyme-linked immunosorbent assay 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.

 

Workflow of an ELISA protocol

The workflow of a typical sandwich ELISA protocol has multiple reagent addition, incubation and wash steps. Here we’ve highlighted each step and the instrumentation and tools needed to conduct the ELISA assay including a microplate washer, absorbance ELISA plate reader and software.

 

Workflow: ELISA Assay Protocol
 
Antibody Binds to Well

CAPTURE ANTIBODY BINDS TO WELLS

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

Microplate wells

ELISA Plate

Most ELISAs are run in 96- or 384- well microplates, a 96-well plate being the most common and sometimes referred to as an ELISA plate. 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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spectra
spectra mobile
add sample
add sample mobile

ADD SAMPLE

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

02
wash microplate
wash microplate mobile

WASH MICROPLATE

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

ELISA Plate Reader and Washer

ELISA Plate Reader And Washer

Complete solution for your ELISA workflow using the EMax plus Microplate Reader with SoftMax Pro data and acquisition software and MultiWash+ Microplate Washer.

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03
add detection antibody
add detection antibody mobile

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 microplates
wash microplates mobile

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

ELISA Plate Washer

The 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
add substrate mobile

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 microplate
read microplate mobile

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 Principle

ELISA Plate Reader

An ELISA plate reader, like the SpectraMax ABS Plus Absorbance ELISA 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

ELISA Plate Reader Software

An ELISA plate reader software, like our SoftMax Pro data analysis 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 result
calculate result mobile

CALCULATE RESULTS

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

08

 

Resources for ELISA

Videos and Demos

SpectraMax ABS and ABS Plus Absorbance Microplate Readers

SpectraMax ABS and ABS Plus Absorbance Microplate Readers

SoftMax Pro 7 Software

SoftMax Pro 7 Software

AquaMax Microplate Washer

AquaMax Microplate Washer

ELISA Workflow using the EMax plus Microplate Reader

ELISA Workflow Using the EMax plus Microplate Reader and MultiWash+ Microplate Washer

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