H J Ellis

This study investigated the presence of mRNA coding for interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), and interleukins 2 (IL2) and 6 (IL6), in the mucosa of four coeliac patients in remission who had been challenged with either gliadin or synthetic gliadin oligopeptides. Jejunal biopsy specimens from these patients, taken before and at two, four, and six hours after challenge, were hybridised with specific 35S-labelled DNA oligonucleotide probes. The lamina propria of all the patients contained significantly increased numbers of cytokine mRNA expressing cells four hours after challenge with gliadin or an oligopeptide corresponding to amino acids 31-49 of A-gliadin (peptide A). No significant changes were seen with the peptides corresponding to aminoacids 202-220 (peptide B) or 3-21 (peptide C) of A-gliadin, with the exception of one patient who showed a significant increase in the number of TNF alpha mRNA expressing cells four hours after challenge with peptide B. In vivo studies in coeliac disease have shown that significant histological changes occur in the mucosa of treated coeliac patients four hours after challenge with either gliadin or peptide A. These findings suggest that the histological changes seen previously in the mucosa of coeliac patients after wheat peptide challenge may be caused by increased expression of cytokines within the mucosa.

Celiac disease is a gluten-sensitive enteropathy that affects as much as 1% of the population. Patients with celiac disease should maintain a lifelong gluten-free diet, in order to avoid serious complications and consequences. It is essential to have methods of analysis to reliably control the contents of gluten-free foods, and there is a definitive need for an assay that is easy to use, and can be used on site, to facilitate the rapid testing of incoming raw materials or monitoring for gluten contamination, by industries generating gluten-free foods. Here, we report on the development of an electrochemical immunosensor exploiting an antibody raised against the putative immunodominant celiac disease epitope, for the measurement of gliadin content and potential celiac toxicity of a foodstuff. To develop the gliadin immunosensor, we explored the use of two surface chemistries, based on the use of dithiols, 22-(3,5-bis((6-mercaptohexyl)oxy)phenyl)-3,6,9,12,15,18,21-heptaoxadocosanoic acid (1) and 1,2-dithiolane-3-pentanoic acid (thioctic acid) (2), for anchoring of the capture antibody. The different surface chemistries were evaluated in terms of time required for formation of self-assembled monolayers, stability, susceptibility to nonspecific binding, reproducibility, and sensitivity. The thioctic acid self-assembled monolayer took more than 100 h to attain a stable surface and rapidly destabilized following functionalization with capture antibody, while the heptaoxadocosanoic acid surface rapidlyformed (less than 3 h) and was stable for at least 5 days, stored at room temperature, following antibody immobilization. Both surface chemistries gave rise to highly sensitive immunosensors, with detection limits of 5.5 and 11.6 ng/mL being obtained for 1 and 2, respectively, with nonspecific binding of just 2.7% of the specific signal attained. The immunosensors were extremely reproducible, with RSD of 5.2 and 6.75% obtained for 1 and 2 (n = 5, 30 ng/mL), respectively. Finally, the immunosensor was applied to the analysis of commercial gluten-free and gluten-containing raw and processed foodstuffs, and excellent correlation achieved when its performance compared to that of an ELISA.