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.
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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
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.
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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
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:
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
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.
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Chemiluminescent VEGF ELISA
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: Chemiluminescent VEGF ELISA Using the SpectraMax L Microplate Luminometer
Chemiluminescent VEGF ELISA
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: Chemiluminescent VEGF ELISA Using the SpectraMax L Microplate Luminometer
Determine Aflatoxin in Cannabis
Here, we demonstrate how the AgraQuant Total Aflatoxin 4/40 ELISA kit can be used together with the SpectraMax® ABS Plus Microplate Reader and SpectraMax® iD5 Multi-Mode Microplate Reader to test for levels of aflatoxins B1, B2, G1, and G2 in one of the most common cannabis products, the flower.
Read Application Note: Determine total aflatoxin in cannabis using an ELISA method
Determine Aflatoxin in Cannabis
Here, we demonstrate how the AgraQuant Total Aflatoxin 4/40 ELISA kit can be used together with the SpectraMax® ABS Plus Microplate Reader and SpectraMax® iD5 Multi-Mode Microplate Reader to test for levels of aflatoxins B1, B2, G1, and G2 in one of the most common cannabis products, the flower.
Read Application Note: Determine total aflatoxin in cannabis using an ELISA method
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Faster Results with 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: Faster ELISA results with CatchPoint SimpleStep ELISA kits
Faster Results with 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: Faster ELISA results with CatchPoint SimpleStep ELISA kits
Gluten Quantification in Beer
Gliadin levels in six commercially available beers were tested to determine gluten levels with the RIDASCREEN Gliadin competitive ELISA.
Read Application Note: Quantifying gluten in beer using an ASBC-approved ELISA method
Gluten Quantification in Beer
Gliadin levels in six commercially available beers were tested to determine gluten levels with the RIDASCREEN Gliadin competitive ELISA.
Read Application Note: Quantifying gluten in beer using an ASBC-approved ELISA method
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Melamine Detection with ELISA Kit
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.
Read Application Note: High-throughput melamine detection with Abraxis Melamine ELISA Kit and Molecular Devices Absorbance readers
Melamine Detection with ELISA Kit
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.
Read Application Note: High-throughput melamine detection with Abraxis Melamine ELISA Kit and Molecular Devices Absorbance readers
Protein Quantitation
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).
Read Application Note: Protein quantitation with the EMax Plus Microplate Reader
Protein Quantitation
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).
Read Application Note: Protein quantitation with the EMax Plus Microplate Reader
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Quantitate Interleukin-8
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: Quantitate interleukin-8 concentrations on the SpectraMax ABS Plus Microplate Reader with the SimpleStep ELISA kit
Quantitate Interleukin-8
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: Quantitate interleukin-8 concentrations on the SpectraMax ABS Plus Microplate Reader with the SimpleStep ELISA kit
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.
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 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.
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ADD SAMPLE
Sample is added to the well, and antigen within the sample binds to the capture antibody.
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
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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ADD DETECTION ANTIBODY
Enzyme-conjugated detection antibody binds to a second site on the antigen of interest, providing the means to detect the antigen.
WASH MICROPLATE
Unbound antibodies are washed away, leaving only those specific for the target of interest and again minimizing the potential for background signal.
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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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.
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
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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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 RESULTS
The amount of antigen in each sample is calculated, and different samples—for example, cells subjected to different treatment conditions—can be compared.
Resources for ELISA
Application Note
Quantifying gluten in beer using an ASBC-approved ELISA method
Quantifying gluten in beer using an ASBC-approved ELISA method
With the recent rise in the prevalence of celiac disease, monitoring gluten levels in food and beverage has become increasingly important as more people strive to avoid gluten.
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Application Note
Faster ELISA results with CatchPoint SimpleStep ELISA kits
Faster ELISA results with CatchPoint SimpleStep ELISA kits
CatchPoint SimpleStep ELISA kits give users a 90-minute ELISA workflow, a nearly two-thirds reduction in time compared to conventional ELISAs.
-
Application Note
Quantitate interleukin-8 concentrations on the SpectraMax ABS Plus Microplate Reader with the SimpleStep ELISA kit
Quantitate interleukin-8 concentrations on the SpectraMax ABS Plus Microplate Reader with the SimpleStep ELISA kit
Learn how to run the Human IL-8 SimpleStep ELISA on the SpectraMax® ABS Plus Microplate Reader using a convenient preconfigured protocol.
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Application Note
Chemiluminescent VEGF ELISA Using the SpectraMax L Microplate Luminometer
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…
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Application Note
Protein quantitation with the EMax Plus Microplate Reader
Protein quantitation with the EMax Plus Microplate Reader
Endpoint readers are prolific in the laboratory since absorbance has become the detection of choice for many applications. Examples include ELISAs for quantitation of cytokines and protein…
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Application Note
High-throughput melamine detection with Abraxis Melamine ELISA Kit and Molecular Devices Absorbance readers
High-throughput melamine detection with Abraxis Melamine ELISA Kit and Molecular Devices Absorbance readers
The organic base melamine is used to make a number of products, including plastics, flame retardants, pigments, and fertilizers. The practice of adding melamine to animal feed and foods for…
Videos and Demos
SpectraMax ABS and ABS Plus Absorbance Microplate Readers
SoftMax Pro 7 Software
AquaMax Microplate Washer
ELISA Workflow Using the EMax plus Microplate Reader and MultiWash+ Microplate Washer