It is well known that gliadin is directly or indirectly through immune mediated reactions, toxic to small bowel mucosa of relatively small population of genetically predisposed individuals who under this toxic action develop celiac disease (CD). These patients need to eat food without gluten, i.e., they need to be on gluten free diet (GFD). Therefore very reliable tests are needed to determine is the content of gliadin really below the accepted value (20 mg/kg). As gliadin isolated from various species used as the antigen may have different immunogenicity 1 that fact could be a problem in the immunological tests used for determination of gliadin content in food; i.e., results may greatly depend on the origin and type of gliadin that was used for calibration. In the aim to overcome this analytical problem, "prolamin working group" developed a PWG gliadin which represents protein fraction soluble in 60% ethanol from the mixture of twenty-eight wheat cultivars grown in Great Britain, France and Germany 123456. This gliadin is recommended for use as the standard antigen in immunological techniques for determination of gluten content in food.

At the same time, it was very important to standardize anti-gliadin antibodies that should be used in immunological tests for determination of gliadin content. In the recent time few monoclonal or polyclonal anti-gliadin antibodies were developed. Commercial kits often used polyclonal antibodies developed against wheat gliadin, or a monoclonal antibody made against ω-gliadins from the Australian wheat cultivar 'Timgalen'. There were also other antibodies developed in different laboratories such as monoclonal antibody PN3, raised against a synthetic peptide equivalent to the amino acids 31–49 of α-gliadin, i.e. the sequence of toxic peptide of α-gliadin, which has been shown to cause mucosal damage to the small bowel of celiac patients 78. The other polyclonal rabbit anti-gliadin antibody made for the same proposes was prepared against α-gliadin, isolated from the 'Kanzler' and other German wheat cultivars. At the present time the greatest attention has monoclonal antibody R5, developed against a secalin extract, which recognizes the potential celiac-toxic epitope QQPFP, which occurs repeatedly in α-, γ- and ω-gliadins, hordeins and secalins, of wheat, barley and rye to a similar degree. Applied in a sandwich ELISA, this antibody showed identical calibration curves for PWG- and Sigma-gliadin 6. This antibody is now recommended by Codex Alimentarius 910 as an antibody which has to be used in sandwich ELISA method for determination of gluten content in gluten free food. Today there are commercial kits available for determination of gluten content, that include R5 antibody as anti-gliadin antibody and PWG gliadin for calibration (Ridascreen, R-Biopharm, Germany).

The most toxic α-gliadin, mainly from the 6D genome of wheat, is the major contributor in both activating the adaptive immune response and the innate immune response. Tetraploid wheat (T. durum) do not contain the D genome. This wheat is hence already devoid of the known immunogenic sequences of α-gliadins 11. Molberg et al. 12 reported that several wheat lacking the D genome also lack the immunodominant 33-mer, but, they did not extend their conclusion to all durum wheat.

Therefore the first aim of this work was to determine the percentage of immunogenic gliadin isolated from ten bread wheat cultivars and three durum wheat cultivars grown commercially in Serbia and neighbouring countries.

Testing sera for IgA or IgG immunoreactivity to gliadin is usually one of the first steps in the complex process of diagnosing gluten intolerance, because it is well known that antibodies to native gliadin sequences are present in patients with celiac disease. This, second part of the study, was based on results of previous publication reporting that sera of some of multiple myeloma (MM) patients showed the presence of elevated levels of anti-gliadin IgA- without the enhanced levels of anti-gliadin IgG immunoglobulins, determined on commercial ELISA test. It was designed to assess is it possible to reveal is there any hidden, especially anti-gliadin IgG immunoreactivity, in serum of mentioned group of patients. For this purpose we tested MM patients' sera as well as celiac disease (CD) patients' sera for the antigliadin immunoreactivity using commercial ELISA test (Binding Site, Birmingham U.K), as well as home made ELISA tests using commercially available gliadin (alcohol soluble protein fraction from crude gliadin products of Sigma, and the native gliadin isolated from wheat species used from bread making in corresponding geographic region as the antigen.

Gliadin in ethanolic extract of wheat cultivars was assessed on the basis of its immunoreactivity with monoclonal antibody R5, using PWG gliadin as the standard.

Some differences in the content of gliadin regarding various wheat cultivars and their total protein contents could be seen on Table 1. Investigated non-bread wheat cultivars: B, Z and D are with the approximately three time lower content of immunogenic gliadin epitopes then cultivars S, R and P which are used for bread production.

Fifteen patients with histopathologically confirmed celiac disease were included in investigation: all of them were not on gluten-free diet (GFD).

Results obtained analyzing fifteen CD patients' sera before GFD, using commercial BS test, presented on Figure 1., showed the enhanced levels of anti-gliadin IgA and IgG immunity in seven and nine patients respectively. Data presented, showed that IgA immunoractyivity of CD patient's sera with gliadin isolated from tested wheat was in 3 patients more than three times higher than that obtained on BS commercial test, revealing that 10/15 CD patients had the enhanced serum antigliadin IgA antibodies.

It could be also seen that IgG immunoractivity of patient's sera with gliadin isolated from tested wheat and crude Sigma gliadin was in six patients more than three times higher than that obtained on BS commercial test, revealing that almost all analysed CD patients' sera had the enhanced levels serum antigliadin IgG antibodies.

The modest increase in IgA antigliadin antibodies were found by BS test in three MM patients' sera, as it could be seen on Figure 2. More then four times higher IgA immunoreactivity with gliadin isolated from local wheat was found in one patient with IgA(λ) myeloma. While the enhanced IgG immunity to gliadin in BS test was observed in one from sixteen myeloma patients, more then five times higher reactivity then that on commercial BS test was found in additional four MM patients' sera on tested wheat gliadin. It must be noted that two out from these four patients' sera did not show the enhancement in the IgG reaction with Sigma gliadin.

Immunity to food antigens (gliadin, cow's milk proteins, CMP) is in the centre of the attention of modern medicine 13 focused on the prevention of diseases, prevention which is based on the use of only appropriate restriction diet. Detection of the enhanced levels of the immune reactions in host, to antigen(s) present in food is from this point of view of great importance because some of premalignant conditions like monoclonal gammopathy of undetermined significance (MGUS), disappeared after the appropriate restriction diets: GFD diet 14 and CMP free diet 15. Thus detecting the enhanced immunity to some of proteins present in food in people with some of immunological health disturbances must be followed by more detailed examination of immunogenic antigens in order to prevent some more serious diseases like NHL, or MM.

It is well known that immunomediated toxicity of gliadin is present in relatively small population of genetically predisposed individuals, with class II human leukocyte antigens HLA-DQ2 or -DQ8 which are found in nearly all celiac patients. Non-HLA genes clearly may play a role in celiac disease as well. The immunologic response to gluten includes antibody reactivity to gluten proteins and the autoantigen TG2, CD4+ T cell reactivity to gluten, increased number of intraepithelial CD8+ T cells, NK cells, and elevated levels of a number of cytokines and chemokines 16

In recent papers an increase in the incidence of certain cancers among celiac disease patients, including non-Hodgkin lymphoma, enteropathy-associated T-cell lymphoma, small intestinal adenocarcinoma, and esophageal and oropharyngeal squamous carcinoma was reported, together with the data that strict adherence to gluten-free diet has been found to be effective at reducing the risk of some malignancies 17. The presence of anti-gliadin and of anti-tTG imunoreactivity was found in patients with multiple myeloma too 13, as well as the anti- CMP (results not shown).

Results obtained in this work using monoclonal antibody R5, clearly show that three of investigated wheat cultivars: B, Zi and D are with the approximately three time lower immunogenic content of gliadin then cultivars S, R and P. They are in accordance with the already published data that tetraploid wheat T. durum do not contain the D genome, are hence already devoid of the known immunogenic sequences of α-gliadins 11. They are also along with similar data that there is various content of immunogenic gliadin in some of wheat cultivars grown in EU countries 6.

In both patients groups IgA immunoreactivity to gliadin from different cultivars was almost homogenous and in correlation with results from commercial test (exept for one patient with IgA(λ) myeloma). But, results for IgG immunoreactivity were more frequently inhomogeneous, and especially for few MM patients they were increased and did not correlate with results obtained using Binding Site test.

Regarding durum wheat immunogenicity our results showed lower levels of humoral immunoreactivity to durum wheat gliadins than to bread wheat gliadin in CD patients and open an interesting area for further research that can elucidate whether observed differences in immunogenicity can be confirmed using peptic-tryptic gliadin digest or deaminated gliadin, as the antigen 18. But it must be noticed that in three myeloma patients the anti-gliadin immunity was preferentially pronounced on gliadin from one tested durum wheat Zi, in relation to Sigma gliadin.

This set up the question is there a need to develop new standard antigen, the representative mixture of gliadin isolated from wheat species used for bread and pasta production in corresponding geographic region for ELISA diagnostic tests.

Results obtained showed that there is different content of immunogenic gliadin epitopes in various species of wheat. They also point for new effort to elucidate whether more reliable data on the existence of immunoreactivity to wheat proteins in some people would be provided by testing their sera for the immunoreaction with gliadin from ethanolic extract of local wheat cultivars used for bread making in the appropriate geographic region.

ZJ designed the study, interpreted the data and wrote the first draft and last version of the manuscript. AKR isolated gliadin from wheat, performed determination of gliadin and total protein content in various wheat, made and perform ELISA tests, presented and interpreted data and wrote the manuscript too. DD grown the wheat specimens and revised critically the manuscript and added important points to the discussion. RK, NjJ, BBN, DP and SJ enrolled the patients with celiac disease and multiple myeloma respectively, revised critically the manuscript and added important points to the discussion. JR and AN have done immunophenotyping of "M" components in myeloma patients sera, IS, IB and MDj helped with ELISA serum testing and added important points to the discussion, too. All authors approved the final draft of the manuscript.