Affinity maturation of immunoglobulin A anti-tissue transglutaminase autoantibodies during development of coeliac disease

Abstract

Coeliac disease (CD) is an immune-mediated enteropathy triggered by ingestion of wheat gluten and related cereals in genetically predisposed individuals. Circulating immunoglobulin A (IgA) class autoantibodies against tissue transglutaminase (IgA-TGA) are highly specific and sensitive serological markers for CD, which is ultimately confirmed by duodenal biopsy. Although CD is considered a life-long disorder, transient or fluctuating IgA-TGA seropositivity has been observed in asymptomatic individuals on a gluten-containing diet. We set out to explore possible differences in the maturation of IgA-TGA avidity between individuals progressing to CD and subjects remaining healthy despite occasional expression of autoantibodies. We developed a time-resolved fluorometric IgA-TGA assay based on human recombinant tissue transglutaminase (tTG), and further modified the method to also measure urea-dependent avidity of the autoantibodies. We measured the autoantibody titres and avidities of sequential serum samples from 10 children developing CD and 10 children presenting transient or fluctuating autoantibodies. Both the initial titres at seroconversion and peak values of transient/fluctuating IgA-TGA were significantly lower than corresponding autoantibody titres in samples drawn from individuals with progressing CD (P = 0.004 and P = 0.0002, respectively). However, there were no statistically significant differences in the initial or peak avidity index values between the two groups of children. The avidity index values increased during the follow-up period (P = 0.013 for both groups) with no significant difference in the rate of avidity maturation between children with transient/fluctuating IgA-TGA and children developing CD. According to our results, high autoantibody titres have a higher predictive value than avidity maturation of TGA-IgA in screening for CD.

Fig. 1

(a,b) Correlation between the fluorometric assay and enzyme-linked immunosorbent assay (ELISA) (Celikey™). (a) The immunoglobulin A (IgA)–tissue transglutaminase (TGA) values of all the 259 follow-up samples analysed by the fluorometric assay were plotted against the corresponding ELISA results, R = 0·93. The cut-off level of the fluorometric assay is marked with a solid line, while the equivocal zone of the ELISA is indicated by dotted lines. (b) Two children showing fluctuating IgA–TGA in the ELISA (○) presented continuous autoantibodies from seroconversion onwards as measured in the fluorometric assay (•), cut-off levels marked as in (a).

Fig. 2

Competitive inhibition of binding with unlabelled tissue transglutaminase (tTG) of immunoglobulin A (IgA) tGT autoantibodies. Increasing amounts of unlabelled tTG were added to one IgA-tissue transglutaminase (TGA)-negative (○) and five positive serum samples in the second incubation of the fluorometric assay, in which IgA–TGA is captured by bio-tTG in SA-wells.

Fig. 3

Identification of variations in the avidity index level of immunoglobulin A (IgA) tissue transglutaminase (tTG) autoantibodies in independent serum samples using urea. IgA–anti-tissue transglutaminase (TGA) from 10 serum samples from different individuals were bound to bio-tTG in SA-wells and then challenged with 5 M urea before detection. Avidity indices were calculated as the ratio remaining fluorescence/maximal fluorescence, in per cent. The corresponding IgA–TGA titres in AU for the different samples were 43·5 (no. 3402), 194 (no. 4383), 92·8 (no. 4473), 8832 (no. 4591), 589 (no. 5436), 62·0 (no. 5565), 6035 (no. 5658), 287 (no. 5764), 20·3 (no. 5776) and 69·8 (no. 5790).

Fig. 4

(a,b) Monitoring of immunoglobulin A (IgA) tissue transglutaminase (tTG) autoantibody levels and avidities in children genetically predisposed to coeliac disease (C). Serum samples from 10 children diagnosed with CD (a) were analysed for autoantibody level (•) and avidity (○) during a period extending from seroconversion to 3·5–8·1 months after duodenal biopsy (x), diagnosis of CD and starting gluten-free diet (arrow). In two children (no. 13723 and no. 15805), gluten-free diet was started after the second biopsy. Similarly, autoantibody titres and avidities of follow-up serum samples from 10 children with transient or fluctuating IgA–anti-tissue transglutaminase (TGA) values (b) were measured during the periods of seropositivity. For children with fluctuating autoantibodies, negative titres are also shown. The dotted lines indicate the cut-off value of the fluorometric IgA–TGA assay.

Fig. 4

(a,b) Monitoring of immunoglobulin A (IgA) tissue transglutaminase (tTG) autoantibody levels and avidities in children genetically predisposed to coeliac disease (C). Serum samples from 10 children diagnosed with CD (a) were analysed for autoantibody level (•) and avidity (○) during a period extending from seroconversion to 3·5–8·1 months after duodenal biopsy (x), diagnosis of CD and starting gluten-free diet (arrow). In two children (no. 13723 and no. 15805), gluten-free diet was started after the second biopsy. Similarly, autoantibody titres and avidities of follow-up serum samples from 10 children with transient or fluctuating IgA–anti-tissue transglutaminase (TGA) values (b) were measured during the periods of seropositivity. For children with fluctuating autoantibodies, negative titres are also shown. The dotted lines indicate the cut-off value of the fluorometric IgA–TGA assay.

Fig. 5

(a,b) Tendency of changes in serum immunoglobulin A (IgA)–anti-tissue transglutaminase (TGA) concentration and avidity during seropostivity. For children with confirmed coeliac disease (C) (a), the autoantibody levels and avidity indices were measured at seroconversion and at diagnosis before introducing a gluten-free diet. Children with transient or fluctuating autoantibodies (b) were analysed at the first and last time-point of seropositivity. Children with fluctuating IgA–TGA positivity are indicated with dashed lines. The children marked with symbols (•) and (○) had fluctuating autoantibodies as determined by enzyme-linked immunosorbent assay (ELISA), but in the fluorometric assay they presented IgA–TGA positivity throughout the follow-up.