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

摘要

Immunoassays are the most commonly used quantitative techniques to determine the gliadin contents of foods aimed at coeliac patients. Though the min. amount of gliadins capable of 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 with 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, this study sought to determine whether gliadin detection by immunoassay is affected by the presence of those agents. Using 2 ELISA formats with a panel of polyclonal and monoclonal antibodies, recognition by specific antibodies of partially or totally denatured gliadins was found to be severely impaired. The magnitude of the interference depended 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 were obtained by comparison with a calibration curve using a native antigen, there was 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, it is suggested 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.