Definitions

• the invention relates to a method for detecting anti-transglutaminase antibodies in saliva for the diagnosis or therapeutic monitoring of celiac disease. Technological background of the invention
• Celiac disease is an autoimmune enteropathy induced by gluten in genetically predisposed subjects.
• Gluten is the protein fraction of the endosperm of wheat, rye, barley and oats.
• Wheat flour, made from the endosperm contains various proteins including gliadin, a protein from the prolamin family, which is the food antigen responsible for the disease.
• the active phase of the disease is associated with the production of anti-gliadin antibodies, and autoantibodies, anti-endomysium antibodies. These autoantibodies are directed against the connective tissue that surrounds the smooth muscle fibers.
• the antigen recognized by anti-endomysium antibodies is an enzyme, tissue transglutaminase, recently discovered by the Dieterich team. This enzyme seems to be involved in the pathogenesis of the disease: transglutaminase catalyzes the covalent bond of proteins between a lysine residue and a glutamine residue.
• transglutaminase to gliadin or to certain gliadin peptides induces the formation of neo-antigens recognized by the immune system and thus the production of autoantibodies against transglutaminase.
• celiac disease In its classic form, celiac disease is characterized by total or subtotal villous atrophy predominant in the proximal small intestine and secondary to the ingestion of gluten.
• Symptoms of the disease in adults are very variable. The usual signs are, as in children, diarrhea and worrying weight loss. More often than in the latter, the disease is mono-symptomatic (iron deficiency anemia, osteoporosis, ...) or atypical (manifested by muscle cramps, aphthous stomatitis, menstrual irregularities or even repeated miscarriages, a depressed state ).
• celiac disease In addition to the classic forms of the disease, which represent only a minority of cases, there are many patients who, in the absence of major clinical signs, exhibit significant villous atrophy of the jejunal mucosa. These pauci-symptomatic forms of celiac disease manifest themselves most often clinically in the form of anemia linked to an iron and / or folic acid deficiency or in the form of osteoporosis or osteomalacia. Some people are at high risk of celiac disease: family members of affected individuals, patients with herpetiform dermatitis, insulin-dependent diabetes, chronic autoimmune thyroiditis or selective IgA deficiency. These are mostly silent forms of celiac disease that predominate in these groups.
• celiac disease Early detection of crude and even subclinical cases of celiac disease is therefore difficult and, however, important in order to avoid on the one hand the complications linked to the nutritional deficit secondary to malabsorption and on the other hand, the development of lymphomas or digestive carcinomas which could appear in 15% of untreated patients. Celiac disease is observed especially in Caucasians and more frequently in women. In general, it is revealed in early childhood. The prevalence of celiac disease in Europe, between 1/1000 and 1/2000 inhabitants, could in fact be much higher if one took into account the pauci-symptomatic and silent forms of the disease, which would reduce the prevalence of the disease between 1/200 to 1/400.
• Anti-endomysium IgA represent the most reliable marker for the detection of celiac disease.
• the sensitivity of this test is more than 90% and its specificity is close to 100%.
• These antibodies are sought by an indirect immunofluorescence technique on sections of primate esophagus or on sections of human umbilical cords.
• the presence in the serum of patients with celiac disease of anti-gliadin antibodies has been known for a long time, the detection of these is done by immunoassays, but the lack of specificity of this test makes it a little diagnostic tool reliable if used alone.
• the search for anti-gliadin IgG is a sensitive but not very specific test, while that of IgA is more specific but less sensitive, among other things because of the high frequency of IgA deficiency in celiac patients.
• transglutaminase either from guinea pig liver or recombinant human, the latter making it possible to obtain a specificity comparable to that of anti-endomysium.
• the only element of formal diagnosis is based on the histology of the intestinal mucosa.
• the intestinal involvement is located in the proximal small intestine and characteristically associates subtotal or total villous atrophy, crypt hypertrophy, massive lymphocytic infiltration of the epithelium and lymphoplasmocytic infiltrate of the chorion, all of which are responsible. malabsorption syndrome.
• the basis of treatment for celiac disease patients is the strict gluten-free diet, which is particularly difficult to follow properly.
• the characteristic antibodies of celiac disease gradually disappear from the serum, thus making it possible to assess the degree of adherence to the diet.
• this again requires a blood test.
• the purpose of the present invention is to provide a non-invasive solution for the diagnosis and monitoring of celiac disease, by an economic and efficient diagnostic test, capable of remedying, at least partially the drawbacks of the solutions known in the art prior.
• the subject of the present invention is a method for detecting and / or assaying anti-transglutaminase antibodies in a saliva sample capable of containing said antibodies, characterized in that the saliva is collected in a saliva collector avoiding the degradation of the antibodies, that the saliva sample is subjected to a pretreatment, and then that said antibodies are detected in the saliva pretreated by an immune reaction with transglutaminase under conditions suitable for the formation of immunocomplex with said antibodies.
• the present invention comprises an effective non-radioactive immunoenzymatic technique for the detection of anti transglutaminase IgA in saliva including a pretreatment of saliva with a solution of mucolytic compound, preferably N-acetyl-cysteine, after collecting these appropriately.
• a pretreatment of saliva with a solution of mucolytic compound, preferably N-acetyl-cysteine after collecting these appropriately.
• the samples are collected in a particular way avoiding the degradation of the antibodies.
• the method comprises the collection of saliva in a sampling device and then the pretreatment of the sampled saliva which comprises bringing the saliva into contact with a mucolytic compound.
• the compound can be selected from the group consisting of N-acetyl-cysteine, nacystelyn, dithiothreitol, gelsolin, thioredoxin and EDTA.
• This involves treating saliva with a mucolytic compound, preferably N-acetylcysteine in order to degrade the chemical bonds between the IgA molecules and the mucins present in the saliva.
• This treatment makes it possible to release the IgA from the mucins, thus avoiding joint non-specific sticking of the mucins on the microtitration plates and increasing the detectability of the anti transglutaminase IgAs.
• This pretreatment makes it possible to reduce the non-specific fixation of samples not containing anti-transglutaminase antibodies and to increase the signal of the samples containing anti-transglutaminase antibodies, thus making it possible to greatly improve the discrimination between positive and negative saliva for presence of anti transglutaminase IgA.
• a prior step of the process according to the invention consists not only in collecting the saliva in special tubes provided for this purpose (sampling device), but also in subjecting the saliva to be pretreated before the search for antibodies by an immunoassay technique. enzyme.
• the saliva sampling device comprises: a sample collector which selectively receives a saliva sample; and an indicator activated by a preselected amount of the received saliva sample to indicate that the amount of the collected biological fluid sample is adequate.
• the method is characterized in that it comprises the collection of saliva in sampling devices as described in documents WO95 / 02996, US Patent No. 5,260,031, such as Omni -SAL ®, in WO97 / 24979, WO96 / 04850, WO95 / 27205 such as OraSure ®, in US Patent No. 4,774,962 as Salivette ®.
• the sampling device is selected from the group comprising: Omni-SAL ® (Saliva
• FIG. 1 represents a diagram illustrating the principle of anti-transglutaminase ELISAs according to a particular embodiment of the invention.
• FIG. 2 represents a graph comparing different concentrations of N-acetylcysteine according to a particular embodiment of the invention.
• FIG. 3 represents a graph comparing saliva untreated and treated with N-acetylcysteine according to a particular embodiment of the invention.
• FIG. 4 represents a graph comparing untreated saliva treated with N-acetylcysteine according to a particular embodiment of the invention with TG from guinea pig liver.
• Figures 5 and 6 show graphs comparing saliva untreated and treated with N-acetyl-cysteine according to particular embodiments of the invention with a pre-existing kit for assaying anti-TG antibodies in serum (Celikey, Pharmacia Diagnostics).
• Figures 7 and 8 show graphs comparing the level of anti-TG IgA in saliva not treated and treated with N-acetyl-cysteine in healthy control subjects ( Figure 7) and in untreated celiac patients (Figure 8), according to particular embodiments of the invention.
• FIG. 9 represents a graph comparing the level of anti-TG IgA in saliva not treated and treated with N-acetyl-cysteine in healthy control subjects and in untreated celiac patients, according to a particular embodiment of the invention.
• FIG. 10 represents a graph comparing the level of anti-TG IgA in saliva treated with N-acetyl-cysteine at the level of serum anti-TG IgA, according to a particular embodiment of the invention.
• FIG. 11 represents a graph comparing the level of anti-TG IgA in saliva treated with N-acetyl-cysteine in healthy control subjects, in pathological control subjects and in celiac patients not treated according to a particular embodiment of the invention.
• FIG. 12 represents a graph comparing the level of anti-TG IgA in saliva treated with N-acetyl-cysteine in healthy control subjects, in untreated celiac patients and in celiac patients on a diet, according to one embodiment particular of the invention. Detailed description of the invention.
• the method of the invention uses human saliva as a non-invasive source, instead of invasive serum or blood, to detect and / or assay anti-transglutaminase antibodies.
• IgA are the predominant immunoglobulins in secretions where they play a key role in the defense of the body against external aggressions. Most of the IgA in saliva is present in a dimeric form: secretory IgA or slgA, consisting of two monomeric IgA joined by a small molecule called "chain J" and a secretory glycoprotein called Secretory Component. IgA maintained as a dimer by the J chain is secreted by subepithelial plasma cells; it binds to the secretory component and then crosses the epithelial barrier. The secretory component facilitates the transport of IgA in the secretions and protects it against proteolysis.
• the present invention makes it possible to assay the anti-transglutaminase IgA antico ⁇ s by the ELIS A technique on salivary samples.
• the present invention relates to a method for detecting anti-transglutaminase antico ⁇ s in a saliva sample capable of containing said antico ⁇ s, characterized in that the saliva sample is subjected to a pretreatment.
• the method comprises on the one hand the collection of saliva in a sampling device such as Omni-SAL ® salivettes and on the other hand the pretreatment which comprises bringing the saliva into contact with a compound mucolytic such as N-acetyl cysteine.
• the transglutaminase used according to the invention can be of human, animal, synthetic or recombinant origin.
• Detection can be carried out in an immunoassay, for example, by direct or indirect coupling of a reaction partner having an easily detectable labeling substance.
• the detection is carried out in an ELIS A test.
• the immunocomplexes formed can be brought into contact with a conjugate consisting of a labeled antico ⁇ s directed against said antico ⁇ s, under conditions suitable for the formation of labeled immunocomplexes, then the labeled immunocomplexes are detected and quantified.
• the conjugate can be an anti-immunoglobulin antico ⁇ s labeled with alkaline phosphatase or peroxidase, then the formation of labeled immuno-complexes is detected and quantified by colorimetry or fluorimetry.
• the present method allows the detection of gluten-induced diseases such as celiac disease.
• the present process also makes it possible to follow a gluten-free diet during which the antico ⁇ s should normally disappear.
• the present invention therefore also relates to a method of diagnosis or therapeutic monitoring of celiac disease, characterized in that antico ⁇ s anti-transglutaminase is detected in a saliva sample capable of containing said antico ⁇ s, characterized in that one subjects the saliva sample to a pretreatment, then in that said antico ⁇ s is detected in the saliva pretreated by an immune reaction with transglutaminase under conditions suitable for the formation of immunocomplexes with said antico ⁇ s.
• the method is characterized in that it comprises the collection of saliva in sampling devices as described in documents WO95 / 02996, US Patent No. 5,260,031, such as Omni-SAL ® salivets then pretreatment which includes bringing saliva into contact with N-acetyl-cysteine.
• the present invention also relates to a diagnostic kit for the diagnosis or therapeutic monitoring of celiac disease.
• the present invention also relates to a method screening and / or detection and / or monitoring of celiac disease in an individual, said method comprising the detection of antico ⁇ s anti-transglutaminase in a saliva sample from the individual.
• the present invention relates to a method which, without the need to use a blood sample, is able to facilitate the diagnosis of celiac disease and monitor the progress of the disease.
• the present invention is suitable both for screening and for monitoring the development in celiac patients on a gluten-free diet. It also makes it possible to diagnose all diseases accompanied by an immune reaction against transglutaminase or corresponding antigenic structures or the like.
• the tests are carried out on saliva samples, taken using the Omni-SAL "salivettes.
• the salivette collecting pad is placed under the tongue until the indicator turns blue.
• the soaked tampon is then replaced in a tube containing a conservation liquid buffered to neutral pH and the saliva is extracted from it using a piston-type filter.
• the saliva is aliquoted and frozen at -80 ° C.
• the principle of anti-transglutaminase ELISA is schematically illustrated in Figure 1.
• the transglutaminase antigen either purified from guinea-pig liver (homemade technique), or recombinant human (Celikey TM PHARMACIA Diagnostics), has been adsorbed to well of a microtiter plate.
• the anti-transglutaminase antibodies which may be present in the samples are fixed to the transglutaminase on the solid phase.
• the apparatuses used for the implementation of the experiments include: ELISA plate reader, PC with processing software for the ELISA reader (KC 4 V3.1), Refrigerator 2-8 ° C, Freezer -25 ° C, 37 ° C oven, Vortex, Balance, pH meter.
• the small equipment used includes: ELISA plate (Nalge Nunc International) Nunc IMMUNO Plate Brand Products Maxisorb surface, Micropipettes from 0 to 10, from 10 to 40, from 50 to 200, from 200 to 1000 ⁇ l, Multichannel micropipettes (8 channels) from 50 to 200 ⁇ l, Aspirates and vacuum pipettes for pipettes from 2 to 10 ml, Disposable pipets from 2 to 10 ml, Plastic tubes with bottom conical from 5 to 10 ml, Microtubes.
• reagents used during these experiments include: Tris HCl, NaCl, CaCl 2; HCl 5N, EDTA (Kestranal A), Tween 20, Sodium citrate dihydrate, distilled H 2 O, purified guinea-pig liver transglutaminase (Sigma T5398), Standard IgA anti-transglutaminase at 10,000 AU dilution Vz in ethylene glycol ⁇ 5,000 AU, Ethylene glycol 99% (14,675.28 Janssen Chimica), Conjugate anti-human IgA-peroxidase (anti-IgA HRP) of rabbit (Dako P216), OPD (Sigma P6912), H 2 O 2 30%.
• the following buffers were used:
• Coating buffer pH 7.5 CaCl 2 (0.7g), brought to 1 liter with distilled H 2 O and adjusted to pH 7.5 with 5N HCl.
• Tris pH 7.4 dilution buffer Tris HCl (6.06g), NaCl (8.8g), EDTA (Kestranal A) (2.9g), brought to 1 liter with distilled H 2 O, supplemented with 1 ml of Tween 20, and adjusted to pH 7.4 with 5N HCl.
• OPD substrate Citrate buffer pH 4.2: C 6 H 5 Na 3 O 7 .2H 2 O (29.4g), brought to 1 liter with distilled H 2 O and adjusted to pH 4.2 with 5N HCl.
• the blank consists of the dilution buffer. 100 ⁇ l of each point on the curve (1/100, 1/400, 1/800, 1/1600, 1/6400), samples (undiluted saliva) and blank were distributed by well, then incubated for 2 h at room temperature, then washed 3 times with the dilution buffer.
• the conjugate was prepared at a 1/1000 dilution of the conjugate in the dilution buffer. 100 ⁇ l of this solution were distributed per well, then incubated for 1 hour at room temperature, then washed 3 times with the dilution buffer.
• the substrate was prepared at the rate of 20 ml of a solution of OPD at 1 mg / ml in the citrate buffer and in which 40 ⁇ l of H 2 O were added. 200 ⁇ l of this solution were distributed per well. The coloring is left to develop for 30 minutes at room temperature in the dark.
• the apparatuses used for the implementation of these experiments include: ELISA plate reader, PC with processing software for the ELISA reader (KC 4 V3.1), Refrigerator 2-8 ° C, Vortex.
• the small equipment used includes: Microtiter well covered with recombinant human transglutaminase, Micropipettes from 0 to 10, from 10 to 40, from 50 to 200, from 200 to 1000 ⁇ l, Multichannel micropipettes (8 channels) from 50 to 200 ⁇ l, Suction and vacuum pipettes for pipettes from 2 to 10 ml, Disposable pipettes from 2 to 10 ml, Plastic tubes with conical bottom from 5 to 10 ml, Microtubes.
• Reagents used during the experiments include: 6 anti-transglutaminase calibrators at concentrations of 0-3-7-16-40-100 U / ml in PBS containing BSA, 0.095% sodium azide ( w / v), detergent and human serum, 1 ml each, ready to use.
• the positive control consisted of a buffer containing BSA, 0.095% sodium azide (w / v), detergent and human serum, 1 ml each, ready to use.
• the negative control consisted of a buffer containing BSA, 0.095% sodium azide (w / v), detergent and human serum, 1 ml, ready to use.
• the concentrated wash buffer consisted of 20x concentrated PBS containing 0.095% sodium azide (w / v) and detergent, 50 ml.
• the HRP IgA conjugate consisted of anti-human HRP IgA conjugate HRP, 15 ml, ready to use.
• TMB substrate ready to use 15 ml.
• the stop solution consisted of H 2 SO 4 0.5 M, 10 ml, ready to use.
• the first tests were carried out using, strictly speaking, the ELISA protocol described below.
• NAC N-Acetyl-Cysteine
• the optimal concentration of NAC is 13.23 g / L of dilution buffer, in fact, it is at this concentration that the ODs of the controls are most bass. Comparison of treated and untreated saliva
• the sensitivity and specificity of the test with TG for guinea pig liver are therefore 30% and 100% respectively without treatment with NAC and 60% and 100% with treatment.
• Saliva pretreatment with NAC increased the signal of celiac patients since, out of 7 patients, 6 saw their signal increase with NAC (see Figure 5).
• the threshold value (1.6 U / ml) was calculated from a preliminary test carried out on a larger series of samples. This threshold value was also used for NAC-treated saliva.
• an ELISA has been developed for the detection of anti-transglutaminase (TG) IgA on saliva samples.
• Trials carried out using the anti-TG IgA research protocol in serum have shown little discrimination between the results of control individuals and celiac patients.
• One of the objectives therefore fried to reduce the signal of controls.
• the advantage of saturation with BSA or Casein has been evaluated and the uselessness of a saturation step has been demonstrated.
• Different concentrations (lml / L and 50 ml / L) of Tween 20 were compared using as dilution buffer either Tris or PBS. The decomplementation proved to be unsuitable, indeed, it causes a drop in the signal of positive controls.
• NAC N-Acetyl-Cysteine
• the value of saliva treatment with NAC has also been confirmed by using Celikey, an anti-transglutaminase ELISA using recombinant human TG as an antigen.
• the present invention allows screening for celiac disease using saliva samples.
• FIGS. 7 and 8 represent graphs comparing the level of anti-TG IgA in saliva not treated and treated with N-acetyl-cysteine in healthy control subjects (FIG. 7) and in untreated celiac patients (Figure 8).
• FIG. 9 represents a graph comparing the level of anti-TG IgA in saliva untreated and treated with N-acetyl-cysteine in healthy control subjects and in untreated celiac patients, the cut-off values being represented by the thin lines on the graph.

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