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B. of exposure to gluten. In conclusion, in coeliac disease there is a clear difference in avidity of the antibody responses directed against the auto- and alloantigens, indicating different regulation or site of initiation of these responses. Keywords: antibodies, avidity, coeliac disease, gliadin, transglutaminase Introduction In coeliac disease Rabbit Polyclonal to TACC1 (CD), an PD0325901 adaptive immune response is initiated directed against wheat gliadin and related gluten components from barley, rye and possibly oats as part of the diet 1. Gluten-specific T cells recognize (deamidated) gluten peptides in 98% of the cases presented by human leucocyte antigen (HLA)-DQ2 or DQ8 2,3. Interestingly, in these patients, several types of antibodies with different specificities that can function as diagnostic markers for the disease are found. These antibodies comprise antibodies directed against gliadin, deamidated gliadin, transglutaminase 2 (TG2) and endomysium. Interestingly, the most specific antibody for diagnosis of CD is directed against the autoantigen TG2 4. This enzyme deamidates proteins, and it has been found that in CD the immune response is directed mainly against deamidated gliadin, requiring the enzymatic function of TG2 for disease development. TG2 is present in the endomysium, the sheath of connective tissue surrounding muscle fibres in the small intestine. Immunofluorescent detection of these antibodies results in a very typical staining pattern, but nowadays antibodies directed against TG2 (TGA) are also determined with the use of recombinant TG2 in enzyme-linked immunosorbent assay (ELISA)-based techniques, and they play an important role in the diagnosis of CD. The pathogenic role of these antibodies is usually questionable and has so far not been shown, although presence in lesions in the intestine has been observed 5. In addition, it is also debatable whether or not TG2-specific T cells exist and drive the TG2-specific B cell response. It has been PD0325901 shown that TG2-specific B cells can receive help from gliadin-specific T cells because the B cell receptor recognizes the complex between TG2 and gluten peptides in a hapten-carrier configuration 4,6. These B cells may have escaped unfavorable selection on the basis of autoreactivity or, alternatively, they recognize a sterical (neo)epitope on TG2 or the gliadinCTG2 complex, leading to anti-TG2 antibody production 7. In the scenario where TG2-specific T cells are not involved, one could speculate that help from gliadin-specific T cells may not be as efficient, and consequently the affinity maturation of the antibody may therefore be less efficient 8,9; this may lead to an antibody pool with lower avidity. Information around the avidity of antibodies may provide insight into the development or even pathogenicity of the immune response 10,11. In this paper we compared the avidity of immunoglobulin (Ig)A antibodies directed against the autoantigen TG2 with that of IgA antibodies directed against the alloantigen gliadin in CD and against lysate (home-made) in NaHCO3 buffer (GA) or in TrisHCl buffer (TGA and antibody); 100 l of phosphate-buffered saline (PBS)/Tween/1% bovine serum albumin (BSA) was added to each well to block free binding places. Sera were diluted in PBS/Tween/1% BSA and incubated for 1 h to remove BSA reactivity and thereby prevent anti-BSA antibodies from binding to coated proteins. Without washing, 100 l of diluted serum, control sera (unfavorable, high and low concentrations), standard dilution or buffer (blank) was added to the wells. Sera were diluted in two-step dilutions starting at 1/25. All given dilutions are end-dilutions in the plate. After washing, bound IgA or IgG was detected with 100 l of 1/5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-IgA or IgG (Dako, Glostrup, Denmark) and developed with ortho phenylenediamine for a fixed amount of time. Absorbance at optical density (OD)450 was determined. The blank OD was subtracted from all measured values to calculate antibody concentration. The serum dilutions used to calculate the antibody concentration in aU/ml were chosen to be in the linear part of the reference line (Fig. ?(Fig.1).1). Cut-offs were the same as those used for diagnostic purposes and were determined by comparison with PD0325901 disease controls; these were 6 U/ml for TGA, 4 U/ml for GA and 29 U/ml for anti-antibody. Open in a separate window Fig. 1 Titration of serum and sodium thiocyanate (NaSCN). (a) Serum of one patient was chosen as an example and titrated in the three different enzyme-linked immunosorbent assays (ELISAs): for immunoglobulin (Ig)A transglutaminase-2 (TG2) antibodies (TGA), for IgA?antibodies and for IgA gliadin antibodies (GA). The dilution, resulting in 50% binding, was chosen to use in the avidity ELISA (indicated by the arrow). The.