Antibody responses to deamidated gliadin peptide show high specificity and parallel antibodies to tissue transglutaminase in developing coeliac disease

Abstract

Coeliac disease (CD) is an enteropathy induced in genetically susceptible individuals by gluten components, gliadin, hordein and secalin, polypeptides present in cereals such as wheat, barley and rye, respectively. Although the disease starts as intolerance to gliadins, antibodies to tissue transglutaminase (tTG) in the gut epithelium are characteristic of the disease. Whereas serum autoantibodies against tTG (tTGA) are highly specific for CD, antibodies to gliadin are less informative as they can also be detected in other enteropathies, and even in healthy individuals. However, it was shown recently that antibodies to certain gliadin peptides occur with high specificity in CD patient sera. We developed a solid phase lanthanide-based immunofluorometric assay for simultaneous detection of serum IgA and IgG antibodies to a synthetic peptide derived from gamma gliadin of wheat comprising amino acids 86-103. Three glutamine residues of this native 18-mer peptide were replaced by glutamic acids and the peptide was biotinylated. Sera from 87 individuals who had undergone duodenal biopsy and were diagnosed with CD and from 81 healthy individuals were analysed for the presence of both IgA and IgG anti-gliadin peptide antibodies. The performance of the peptide AGA assay was excellent, showing a specificity and sensitivity of 90% and 92% for IgA, and 98% and 75% for IgG, respectively. The corresponding values for conventional anti-gliadin antibody (AGA) enzyme-linked immunosorbent assay (ELISA) tests were 72% specificity and 87% sensitivity for IgA, and 64% specificity and 78% sensitivity for IgG. In a prospective study, almost all the tTGA-positive sera drawn from children who later developed CD were also positive for gliadin peptide antibodies.

Fig. 1

The principle of the anti-gliadin peptide dual-label immunofluorometric assay (IFMA) assay. The C-terminally biotinylated gamma gliadin peptide (residues 86–103) is attached to a streptavidin-coated microtitre plate. After incubation with serum sample and subsequent washes the plate is developed using secondary Europium-labelled anti-human IgA and Samarium-labelled anti-human IgG antibodies. The fluorescence is measured in a time-resolved fluorometer (Wallac).

Fig. 2

Linearity of the anti-gliadin peptide antibody response of the assay for IgA (Fig. 2a) and IgG (Fig. 2b). A strongly IgA- and IgG-positive patient serum was serially diluted in assay buffer and measured with IFMA assay as described in Subjects and methods.

Fig. 3

Anti-gliadin peptide IgA (red symbols) and IgG (green symbols) antibodies immunofluorometric assay (IFMA) and anti-tTG antibodies (tTGA) enzyme-linked immunosorbent assay (ELISA) (blue symbols) were analysed in follow-up sera from children at genetic risk for type 1 diabetes (Finnish Diabetes Prediction and Prevention study). Coeliac disease (CD) antibody responses were determined in individuals who eventually developed CD (a), and in healthy individuals transiently positive in tTGA IgA ELISA (b). The arrows indicate the time-point of small intestine biopsy.