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.
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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.
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.
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 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.
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).
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).
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Faster ELISA Results
Résultats ELISA plus rapides avec les trousses de test ELISA CatchPoint SimpleStep
A typical ELISA protocol is time-consuming, with multiple incubation and wash steps. Les trousses CatchPoint SimpleStep ELISA permettent d’exécuter le flux de travail ELISA en 90 minutes, soit le tiers à peine du temps nécessaire pour l’exécution d’un test ELISA conventionnel.
Faster ELISA Results
Résultats ELISA plus rapides avec les trousses de test ELISA CatchPoint SimpleStep
A typical ELISA protocol is time-consuming, with multiple incubation and wash steps. Les trousses CatchPoint SimpleStep ELISA permettent d’exécuter le flux de travail ELISA en 90 minutes, soit le tiers à peine du temps nécessaire pour l’exécution d’un test ELISA conventionnel.
Quantitate Interleukin-8 ELISA
Quantitate interleukin-8 concentrations in just 90 minutes
Apprenez à exécuter le test Human IL-8 SimpleStep ELISA sur le lecteur de microplaques SpectraMax® ABS Plus grâce à un protocole préconfiguré pratique.
Quantitate Interleukin-8 ELISA
Quantitate interleukin-8 concentrations in just 90 minutes
Apprenez à exécuter le test Human IL-8 SimpleStep ELISA sur le lecteur de microplaques SpectraMax® ABS Plus grâce à un protocole préconfiguré pratique.
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Melamine Detection with ELISA Kit
Détection haut débit de mélamine avec le kit Melamine ELISA d'Abraxis et les lecteurs d'absorbance Molecular Devices
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.
Melamine Detection with ELISA Kit
Détection haut débit de mélamine avec le kit Melamine ELISA d'Abraxis et les lecteurs d'absorbance Molecular Devices
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.
ELISA – Cytokine Interleukin-22
Quantification des protéines avec le lecteur de microplaques EMax Plus
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).
ELISA – Cytokine Interleukin-22
Quantification des protéines avec le lecteur de microplaques EMax Plus
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
VEGF ELISA chimiluminescent avec le luminomètre de microplaques SpectraMax L
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.
Chemiluminescent VEGF ELISA
VEGF ELISA chimiluminescent avec le luminomètre de microplaques SpectraMax L
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.
ELISA for Gluten Quantitation
Quantifying gluten in beer using an ASBC-approved ELISA method
Gliadin levels in six commercially available beers were tested to determine gluten levels with the RIDASCREEN Gliadin competitive ELISA.
ELISA for Gluten Quantitation
Quantifying gluten in beer using an ASBC-approved ELISA method
Gliadin levels in six commercially available beers were tested to determine gluten levels with the RIDASCREEN Gliadin competitive ELISA.
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.
CAPTURE ANTIBODY BINDS TO WELLS
First, the capture antibody is bound to the bottom of the microplate well.
plaque elisa
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.
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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.
laveur de plaques elisa
A microplate washer is used to wash away non-specific material in the wells.
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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.
Laveur de microplaques AquaMax
L’aspiration et la distribution des 96 et 384 puits se font simultanément dans tous les puits, ce qui permet d’avoir des tests de grande précision et un traitement plus rapide des microplaques sans indexage mécanique des plaques ni traitement en quadrant.
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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.
lecteur de plaques elisa
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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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 different samples—for example, cells subjected to different treatment conditions—can be compared.
Resources for ELISA
Note d'application
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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Note d'application
Résultats ELISA plus rapides avec les trousses de test ELISA CatchPoint SimpleStep
Faster ELISA results with CatchPoint SimpleStep ELISA kits
Les trousses CatchPoint SimpleStep ELISA permettent d’exécuter le flux de travail ELISA en 90 minutes, soit le tiers à peine du temps nécessaire pour l’exécution d’un test ELISA conventionnel.
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Note d'application
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
Apprenez à exécuter le test Human IL-8 SimpleStep ELISA sur le lecteur de microplaques SpectraMax® ABS Plus grâce à un protocole préconfiguré pratique.
-
Note d'application
VEGF ELISA chimiluminescent avec le luminomètre de microplaques SpectraMax L
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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Note d'application
Quantification des protéines avec le lecteur de microplaques EMax Plus
Protein quantitation with the EMax Plus Microplate Reader
Les lecteurs de points finaux abondent en laboratoire, car l’absorbance est devenue la détection de choix pour de nombreuses applications. Des exemples incluent les tests ELISA pour la quantification des cytokines et…
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Note d'application
Détection haut débit de mélamine avec le kit Melamine ELISA d'Abraxis et les lecteurs d'absorbance Molecular Devices
High-throughput melamine detection with Abraxis Melamine ELISA Kit and Molecular Devices Absorbance readers
La base organique mélamine est utilisée pour fabriquer un certain nombre de produits, notamment les plastiques, les ignifugeants, les pigments et les engrais. The practice of adding melamine to animal feed and foods for…
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