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

DOÑA, VANINA; FOSSATI, ALBERTO; CHIRDO, FERNANDO

EUROPEAN FOOD RESEARCH AND TECHNOLOGY

Springer

Lugar: Berlin; Año: 2008 vol. 226 p. 591 - 602

1438-2377

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-GB;} p.MsoBodyTextIndent2, li.MsoBodyTextIndent2, div.MsoBodyTextIndent2 {margin-top:0cm; margin-right:0cm; margin-bottom:6.0pt; margin-left:14.15pt; line-height:200%; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-GB;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Immunoassays are the most commonly used quantitative techniques to determine the gliadin content of food aimed for 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 detectability. 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 in the presence of those agents. By 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 the underestimation of the actual gliadin content. Therefore, we suggest to consider not only the effects of the 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 when a quantitative immunoassay is performed.