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A modified enzyme-linked immunosorbent assay adapted for immunodetection of low amounts of water-insoluble proteins
- Source :
- Journal of Immunological Methods. 326:83-92
- Publication Year :
- 2007
- Publisher :
- Elsevier BV, 2007.
-
Abstract
- A mixture of thiourea, urea and CHAPS (TUC) is an excellent solvent compatible with isoelectrofocusing (IEF) separation of water-insoluble protein extracts, and their subsequent two-dimensional gel electrophoresis is an important step in proteomic studies. The main aim of this work was to quantify extremely low amounts of water-insoluble proteins contained, for instance, in samples collected in bio-aerosol samplers. High CHAPS concentrations solubilize many proteins. However, enzyme-linked immunosorbent assay (ELISA), which is the most popular immunodetection method of quantifying antigens, is unfortunately not compatible with these high CHAPS concentrations and with the low protein concentrations of TUC extracts. The most common mixture used to solubilize these proteins contains 2 mol l − 1 thiourea, 7 mol l − 1 urea and 5% w/v CHAPS. This paper shows that these components inhibit the adsorption and/or recognition of proteins on microtitration plates, preventing antigen quantification under classic ELISA conditions. We have tried several solvents (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins stick to the ELISA plates, and ethanol was shown to be the most appropriate. In this study, we have defined a new ELISA protocol allowing rapid and sensitive detection of low concentrations (60–500 ng ml − 1 ) of water-insoluble proteins extracted with high concentrations of TUC.
- Subjects :
- Low protein
Detergents
Immunology
Enzyme-Linked Immunosorbent Assay
Immunoenzyme Techniques
chemistry.chemical_compound
Chaps
Animals
Chemical Precipitation
Urea
Immunology and Allergy
Trichloroacetic acid
Dactylis
Gel electrophoresis
chemistry.chemical_classification
Chromatography
Ethanol
Plant Extracts
Thiourea
Proteins
Water
Cholic Acids
Solvent
Kinetics
Enzyme
Solubility
chemistry
Biochemistry
Pollen
Rabbits
Subjects
Details
- ISSN :
- 00221759
- Volume :
- 326
- Database :
- OpenAIRE
- Journal :
- Journal of Immunological Methods
- Accession number :
- edsair.doi.dedup.....c113ec235846f7c28c947acdd619bab8