Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis

Immunoassays are the most commonly used quantitative techniques to determine the gliadin content of food aimed at coeliac patients. Though the minimal amount of gliadins inducing the typical histopathological changes at the intestinal mucosa in coeliacs is still a matter of debate, current research...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Doña, Vanina Valeria, Fossati, Carlos Alberto, Chirdo, Fernando Gabriel
Formato:	Articulo
Lenguaje:	Inglés
Publicado:	2008
Materias:	Ciencias Exactas Gliadin analysis ELISA Coeliac disease Antigen denaturation
Acceso en línea:	http://sedici.unlp.edu.ar/handle/10915/131277
Aporte de:	SEDICI (UNLP) de Universidad Nacional de La Plata

id	I19-R120-10915-131277
record_format	dspace
institution	Universidad Nacional de La Plata
institution_str	I-19
repository_str	R-120
collection	SEDICI (UNLP)
language	Inglés
topic	Ciencias Exactas Gliadin analysis ELISA Coeliac disease Antigen denaturation
spellingShingle	Ciencias Exactas Gliadin analysis ELISA Coeliac disease Antigen denaturation Doña, Vanina Valeria Fossati, Carlos Alberto Chirdo, Fernando Gabriel Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis
topic_facet	Ciencias Exactas Gliadin analysis ELISA Coeliac disease Antigen denaturation
description	Immunoassays are the most commonly used quantitative techniques to determine the gliadin content of food aimed at coeliac patients. Though the minimal amount of gliadins inducing the typical histopathological changes at the intestinal mucosa in coeliacs is still a matter of debate, current research is focussed on the development of methods having higher sensitivities. One of the main drawbacks in gliadin analysis is the low efficiency of the conventional extraction procedure using 60% ethanol. The use of reducing (2-mercaptoethanol) and denaturing (guanidinium chloride) agents has been recommended to improve the extraction efficiency. Owing to the well-known effects of these agents on native conformation of proteins, and their widely reported interference on the antigen/antibody interaction in other systems, we assessed whether gliadin detection by immunoassays is affected by the presence of those agents. Using two ELISA formats with a panel of polyclonal and monoclonal antibodies, we found that recognition by specific antibodies of partially or totally denatured gliadins is severely impaired. The magnitude of the interference depends on the antibodies used and the ELISA format. The impact of such interference was analysed for each step of the immunoassays. 2-mercaptoethanol had a stronger effect than guanidinium chloride, and the antigen became almost undetectable for some assays when both reagents were used in combination. Remarkably, since quantitative results are obtained by comparison with a calibration curve using a native antigen, there is no equivalence between the antigen/antibody interaction occurring in the sample and that in the standard gliadin, leading to underestimation of the actual gliadin content. Therefore, we suggest that not only the effects of reducing and denaturing agents on the antigen during the extraction procedure, but also the effects of residual amounts of these agents on the antigen/antibody interaction should be considered when a quantitative immunoassay is performed.
format	Articulo Articulo
author	Doña, Vanina Valeria Fossati, Carlos Alberto Chirdo, Fernando Gabriel
author_facet	Doña, Vanina Valeria Fossati, Carlos Alberto Chirdo, Fernando Gabriel
author_sort	Doña, Vanina Valeria
title	Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis
title_short	Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis
title_full	Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis
title_fullStr	Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis
title_full_unstemmed	Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis
title_sort	interference of denaturing and reducing agents on the antigen/antibody interaction : impact on the performance of quantitative immunoassays in gliadin analysis
publishDate	2008
url	http://sedici.unlp.edu.ar/handle/10915/131277
work_keys_str_mv	AT donavaninavaleria interferenceofdenaturingandreducingagentsontheantigenantibodyinteractionimpactontheperformanceofquantitativeimmunoassaysingliadinanalysis AT fossaticarlosalberto interferenceofdenaturingandreducingagentsontheantigenantibodyinteractionimpactontheperformanceofquantitativeimmunoassaysingliadinanalysis AT chirdofernandogabriel interferenceofdenaturingandreducingagentsontheantigenantibodyinteractionimpactontheperformanceofquantitativeimmunoassaysingliadinanalysis
bdutipo_str	Repositorios
_version_	1764820453582438400

Interference of denaturing and reducing agents on the antigen/antibody interaction : Impact on the performance of quantitative immunoassays in gliadin analysis

Ejemplares similares