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A rapid method to improve protein detection by indirect ELISA
Robert Hnasko1, Alice Lin, Jeffery A McGarvey
1800 Buchanan St., USDA-ARS, Western Regional Research Center, Albany, CA 94710, USA. Robert.Hnasko@ars.usda.gov
Biochemical and Biophysical Research Communications
|June 18, 2011
Summary
A new method improves indirect ELISA (iELISA) by using chemical denaturation to enhance antibody binding to immobilized antigens. This boosts both qualitative and quantitative protein detection, expanding iELISA applications in research.
Area of Science:
- Biochemistry
- Immunology
- Assay Development
Background:
- Enzyme-linked immunosorbent assay (ELISA) is a key quantitative immunoassay for antigen detection.
- While capture ELISA (cELISA) offers high sensitivity, limited antibody pairs restrict its use in antigen discovery.
- Indirect ELISA (iELISA) uses a single antibody but often suffers from poor performance due to antibody epitope accessibility issues.
Purpose of the Study:
- To develop a method to improve antibody utility in microplate-based indirect ELISA (iELISA).
- To overcome limitations in quantitative antigen detection caused by antibody epitope binding impediments.
- To enhance both qualitative and quantitative protein detection in iELISA.
Main Methods:
- Investigated the effect of chemical denaturation on antibody binding to immobilized antigens in microplates.
- Developed a rapid method to assess antibody suitability for iELISA.
- Optimized assay conditions for chemical denaturation and subsequent iELISA.
Main Results:
- Chemical denaturation significantly improved antibody epitope accessibility for immobilized antigens.
- The method enhanced both qualitative and quantitative protein detection in iELISA.
- Demonstrated improved performance of antibodies that previously failed in microplate-based iELISA.
Conclusions:
- Chemical denaturation is an effective strategy to overcome antibody binding limitations in iELISA.
- This approach expands the utility of iELISA for quantitative protein expression analysis during discovery.
- The developed method provides a valuable tool for assessing and improving antibody performance in iELISA.
Related Concept Videos
01:33Enzyme-Linked Immunosorbent Assay
In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.
01:15Western Blotting
Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.