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[Home](https://www.sigmaaldrich.com/US/en)[Applications](https://www.sigmaaldrich.com/US/en/applications)[Protein Biology](https://www.sigmaaldrich.com/US/en/applications/protein-biology)(ELISA) Enzyme-Linked Immunosorbent Assay
# (ELISA) Enzyme-Linked Immunosorbent Assay
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Typically performed in multi-well microtiter plates, ELISAs are a molecular biology assay commonly used for the detection and quantification of diverse molecules, including peptides, proteins, and antibodies. Such assays can detect molecules of interest at the pg/mL level and are critical for both basic research and disease research application needs.
## ELISA Assays: Antibody-Antigen Interactions
The fundamental molecular components of an ELISA typically include the use of antibody conjugated to an enzyme, an immobilized molecule(s) of interest, and a detection substrate. A critical aspect that determines the success and quality of data obtained from an ELISA is dependent on the affinity and specificity of antibody-antigen interactions. Antigen-antibody interactions are influenced by numerous factors, including pH, temperature, and ionic strength.
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Overview
Related Articles & Protocols
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## ELISA Detection Formats
ELISAs that use direct detection methods require an immobilized antigen that is bound directly to the surface of an assay plate or indirectly by a capture antibody, followed by an antigen-specific primary antibody conjugated to an enzyme, and the detection substrate.
The more commonly used indirect detection format incorporates both an unconjugated primary antibody, followed by a conjugated secondary antibody that is specific to the detection of the primary antibody. Indirect detection benefits from increased immunoreactivity with the target antigen as the conjugated enzyme element is only present on the secondary antibody.
In addition to direct and indirect detection methods, capture or “sandwich” assays use an additional antigen-capturing antibody that is first attached to the microplate surface, followed by the use of both a primary and an enzyme-conjugated secondary antibody, similar to the indirect method previously described.
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## Related Articles
- [Conferma® ELISAs: Pursuing a “Pharmagrade” Immunoassay](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/conferma-sandwich-elisas)
Learn about the Conferma® ELISA development and manufacturing methods that provide strong sample detection and long-term assay and lot consistency, giving you confidence in your research.
- [Antibody Generation | Monoclonal vs Polyclonal Antibodies](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/generation-of-antibodies)
Learn differences in monoclonal vs polyclonal antibodies including how antibodies are generated, clone numbers, and antibody formats.
- [ELISA Troubleshooting Guide](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/elisa-troubleshooting-guide)
Troubleshoot and optimize ELISAs using this guide that includes solutions to some of the most common sources of problems for assay development.
- [ELISpot Assay: Functional Cellular Immunology](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/elispot-assays-state-of-the-art-tools-for-functional-analysis-of-cellular-immunology)
ELISpot assay provides qualitative and quantitative information on immune responses, visualizing multiple secretory products from single responding cells.
- [Complete Solutions for IVD Chemiluminescent Immunoassay (CLIA/CLEIA) Development](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/ivd-manufacturing/clia-chemiluminescent-immunoassay-development)
Accelerate your time to market with fit-for-use products which offer the quality, consistency, and documentation necessary for every step of your IVD development and manufacturing needs.
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## Related Protocols
- [ELISA Protocols](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/elisa/elisa-protocols)
This article offers 4 popular ELISA protocols: Sandwich ELISA protocol, Phosphorylation Assay Procedure, EIA Assay Procedure, & Cell-based Assay Procedure.
- [ELISA Procedures](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/elisa/elisa-procedures)
Detailed ELISA protocols cover indirect and capture ELISA techniques, recommending products for sandwich and indirect ELISA experiments.
- [Cell Proliferation ELISA, BrdU (colorimetric) Protocol & Troubleshooting](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/elisa/cell-proliferation-elisa-brdu-colorimetric)
Cell Proliferation ELISA, BrdU (colorimetric) Protocol & Troubleshooting
- [Conjugating Alkaline Phosphatase to Antibodies and Other Proteins](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-labeling-and-modification/conjugation)
Protocol for the one-step, glutaraldehyde coupling method to conjugation alkaline phosphatase to antibodies and other proteins.
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__Featured Articles__
[An Introduction to Antibodies: Antibody-Antigen Interaction](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/antibody-antigen-interaction)
The strength of interaction between antibody and antigen at single antigenic sites can be described by the affinity of the antibody for the antigen.
[An Introduction to Antibodies: Antigens, Epitopes and Antibodies](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/antigens-epitopes-antibodies)
Antibodies combine with specific antigens to generate an exclusive antibody-antigen complex. Learn about the nature of this bond and its use as a molecular tag for research.
[Antibody Generation | Monoclonal vs Polyclonal Antibodies](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/generation-of-antibodies)
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[ELISpot Assay: Functional Cellular Immunology](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/elisa/elispot-assays-state-of-the-art-tools-for-functional-analysis-of-cellular-immunology)
ELISpot assay provides qualitative and quantitative information on immune responses, visualizing multiple secretory products from single responding cells.
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