ITVR20120176A1 - METHOD AND KIT FOR DIAGNOSIS AND SENSITIVITY MONITORING AND INTOLERANCES IN FOOD - Google Patents

Description

METHOD AND KIT FOR DIAGNOSIS AND MONITORING OF SENSITIVITY AND INTOLERANCES TO FOOD.

The present invention relates to a method and a kit for the diagnosis and monitoring of sensitivities and intolerances to foods, more particularly to gluten.

State of the art

Most gastroenterologists agree on the possibility that there may be personal sensitivities to certain foods, probably related to innate immunity phenomena. These sensitivities are different from intolerances and adverse reactions to foods, which are evidenced by tests accepted by official science (immunological, biochemical and histological). To date, their existence cannot be ascertained with certainty, nor is it possible to diagnose them with conventional methods.

For these sensitivities there are therefore only unconventional methods, not accepted by the scientific community, which have very poor reproducibility, such as: the Voll EAV test or the Cytotest <TM>.

Of all these presumed sensitivities, the only one to have been ascertained is that of gluten, which has recently been defined as gluten sensitivity or â € œgluten sensitivityâ € (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med. 2012; 7: 10-13). Patients with this pathology are defined as gluten sensitive or â € œgluten sensitiveâ € or GS.

Sensitivity to gluten has been defined as a condition of intolerance to gliadin and glutenin present in cereals. For a long time there has been discussion about the existence or otherwise of this pathology, in which there is a loss of tolerance towards these proteins (or their digested fragments) with symptoms only partially similar to celiac disease, which however can be explained also at the extraintestinal level.

Sensitivity to gluten, for a long time considered a psychosomatic reaction, has only recently become a detectable pathology, following â € œdouble blindâ € studies that have shown its existence (see â € œGluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo-controlled trialâ € by Biesiekierski JR et al., Am J Gastroenterol. 2011; 106: 508-14).

By `` double-blind '' studies or analyzes, as is known, we mean investigations carried out by administering to patients substances, of which neither the treating physicians nor the patients know the composition, although, of course, the substances are identified by a code or abbreviation, which allows, at the end of the treatment, to ascertain the substances administered, while for studies or analyzes in `` blind '', we mean investigations carried out by administering substances to patients, of which only the treating doctors know the composition.

Recently, the scientific community has accepted gluten sensitivity as a pathological condition distinct from celiac disease on the basis of histopathological and serological investigations (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med. 2012; 7: 10-13).

The pathogenesis of gluten sensitivity is still unknown today (see â € œNew understanding of gluten sensitivityâ € by Volta U, D and Giorgio R. - Nat Rev Gastroenterol Hepatol. 2012; 9: 295-9). In a study conducted on celiacs, GS patients and healthy controls, it was observed that the intestinal permeability of celiac patients appears significantly increased compared to GS, in which instead it is normal (see â € œDivergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivityâ € by Sapone A, Fasano A. et al. BMC Med. 2011; 9: 23). In the same study it was shown that GS subjects show an increase in the expression of some adhesion molecules expressed by intestinal cells, such as claudin-1, ZO-1 and claudin-4, and that the expression of the innate immunity referred to as Toll-like receptor 2 is altered in the intestinal mucosa of GS patients.

Finally, the expression of the marker named FOXP3 on regulatory T lymphocytes was found to be decreased in GS patients. Until now, no other identifying marker has been proposed for the diagnosis of gluten sensitivity.

The symptomatology of gluten sensitivity is sometimes similar to that of celiac disease and gluten allergy and is resolved with the interruption of gluten intake. Symptoms mainly reported by patients with gluten sensitivity are: abdominal pain (68%), eczema and / or rash (40%), headache (35%), clouded mind (34%), fatigue (33%), diarrhea (33%), depression (22%), anemia (20%), feeling of numbness in the legs, arms or fingers (20%); and joint pain (11%) (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med. 2012; 7: 10-13).

The current diagnosis of gluten sensitivity is carried out exclusively using criteria for the exclusion of other pathologies, namely:

- the exclusion of allergic mechanisms typical of gluten allergies (negative immuno-allergic tests to wheat);

- the exclusion of the autoimmune mechanisms typical of celiac disease (anti - EMA and / or anti tTG negative); And

- the exclusion of damage to the duodenal mucosa typical of celiac disease (normal duodenal histopathology).

Furthermore, patients defined as GS must show a regression of symptoms from the moment they were introduced to a gluten-free or gluten-free diet and their reappearance when they are provided (blinded) with a load of gluten.

As for the other pathologies connected to the intake of gluten, a diet free of these proteins, which provides for the elimination from the diet of cereals containing gluten and all their derivatives, is currently the best solution therapeutic. However, for gluten sensitivity, the gluten free diet is effective even if less severe than that prescribed for celiacs, who must also avoid any cross-contamination or cross-contamination from foods containing gluten. It is believed that, after a period of 90-120 days of a gluten-free diet, a gradual reintroduction of gluten can be attempted, thus reactivating a kind of tolerance that allows avoiding gluten-free diets that last a lifetime (see â € œSpectrum of gluten-related disorders: consensus on new nomenclature and classificationâ € by Sapone A. et al. BMC Med.

2012; 7: 10-13).

The international application WO2011163258 teaches a method for diagnosing and monitoring food diseases and sensitivities, which is based on the use of synthetic peptides of known sequence and the response of the lymphocytes extracted from the patient to these synthetic antigens. The response is evaluated as lymphocyte proliferation or lymphocyte migration and also includes the measurement of the secretion of cytokines included in the group: IL-1b, IL-4, IL-6, IL-8, IL-10, IL-13, MCP-1 , G-CSF, IFN-g and TNFa.

However, a more objective diagnostic tool than symptoms has not been suggested so far, which indicates whether the reintroduction of gluten is tolerated well or not by the individual patient.

The main purpose of the present invention is to provide a method for an objective diagnosis and monitoring of food sensitivities and intolerances, in particular sensitivity to gluten.

Another object of the present invention is to provide a method as indicated above which is both highly reliable and easy to implement.

Another object of the present invention is to provide a kit for carrying out the method for diagnosing and monitoring food sensitivities and intolerances, in particular sensitivity to gluten, according to the present invention.

According to a first aspect of the present invention, an extracorporeal diagnosis method of the food sensitivity or intolerance of a patient is provided, comprising the following sequential steps:

- add at least one food to a sample of patient-derived peripheral blood or patient-derived peripheral blood mononuclear cells (PBMC);

- leave the sample thus added to stand for a predetermined time interval; And

- measure the quantity of at least one chemokine produced in the sample after the rest interval.

Advantageously, the method is for the diagnosis of gluten sensitivity and the food contains gluten.

Even more advantageously, at least one chemokine is chosen from the group consisting of CXCL10 (IP10), CXCL9 (MIG) and CXCL11 (I-TAC).

Preferably, the food is a total protein extract from at least one food.

According to another aspect of the present invention, a kit is provided for carrying out a method according to the present invention, comprising the following steps:

- at least one multi-well plate (multiwell) with a PVDF bottom, some of these wells having total proteins extracted from gluten-free flours pre-absorbed on the bottom, while other wells have total proteins extracted from gluten-free flours pre-adsorbed on the bottom;

- at least one first pack;

- at least a second pack of RPMI 1640 terremo containing 10% FBS and at least one antibiotic; And

- a plurality of test tubes.

Further aspects and advantages of the present invention will become more apparent from the following detailed description of specific examples of embodiment of a diagnostic method, the description being made with reference to the accompanying drawings, in which:

- Figure 1 is a diagram illustrating the results of tests conducted on peripheral blood samples from healthy patients and GS patients, evaluating CXCL10 as a cemoquine;

- Figure 2 shows test results conducted on a non-GS patient treated with sachets containing flours with gluten or placebo;

- Figure 3 shows the results obtained for a border-line patient treated with sachets containing flours with gluten or placebo;

- Figure 4 shows the results obtained in a GS patient treated with sachets containing flours with gluten or placebo;

Figure 5 schematically shows the method or operating sequence by means of a kit according to the present invention; And

Figure 6 illustrates the loading scheme of a multiwell plate supplied in a kit according to the present invention.

A method according to the present invention is an extracorporeal method which is based on the evaluation of the immunological responses of peripheral blood mononuclear cells (PBMCs) exposed in vitro to contact with protein extracts, preferably total protein extracts, of foods of which it is supposed to exist. sensitivity or intolerance. The same extracts are put in direct contact with the PBMCs by solubilizing the total proteins extracted in the culture medium in which the PBMCs (proteins in suspension) are kept or by adhering the total proteins to a substrate, for example a substrate of PVDF (polyvinyldenfluoride). o nitrocellulose or plastic materials capable of retaining proteins, which are found at the bottom of the wells in which the PBMCs are subsequently kept.

The PBMCs are then incubated for 4 - 24 hours in the presence of the protein extracts. The incubation time depends on the sensitivity of the chemokine assay method available. In fact, the PBMCs of GS patients, in contact with the protein extracts, produce chemokines that accumulate in the PBMC culture medium. After incubation with these extracts, the immunological responses of the PBMCs are evaluated by measuring the secreted chemokines by the PBMCs exposed to the total protein extracts of the foods under analysis.

Example 1 below refers to an incubation time of 16 hours, that is an intermediate value between the two limit values, time after which the quantity of chemokines that accumulate in the culture medium can be measured with Elisa systems o Luminex <TM>, which have a sensitivity of the order of 10 pg / ml. Longer times can be used for dosages with less sensitive methods, shorter times can be used combined with more sensitive dosage methods.

This method has been tested and developed for gluten sensitivity, using protein extracts of gluten-containing flours, but it can be easily adopted for any food (such as flours, meats, fruit, vegetables, milk and their derivatives), from which can be obtained total protein extracts.

With a high degree of probability, also because there are no contraindications whatsoever, the method described above is also applicable to other types of sensitivity (for example: sensitivity to tomato, soya, etc.) or food intolerances.

Currently, it has not so far been possible to test the above method for other types of sensitivities, because diagnostic criteria for sensitivities other than gluten sensitivity have not yet been defined.

The method according to the present invention is aimed at identifying some markers of sensitivity or intolerance to the protein component of foods (typically sensitivity to gluten or celiac disease), as well as to develop a test in the form of a kit, for the diagnosis and the therapy of these sensitivities.

The method according to the present invention is based on the purification of the mononuclear cells (PBMC) obtained from a peripheral blood sample from the patient. The PBMC cells thus obtained are put in direct contact, in vitro, with total protein extracts of foods, such as flours with a high gluten content (such as Manitoba flour, Claudio flour) and total extracts of gluten-free foods (such as flour of rice and corn flour). The variations in the differential secretion of chemokines, which are produced by PBMC cells as a consequence of their direct contact with food proteins containing gluten, are then measured. The chemokines, whose secretion can be altered following the contact of PBMC cells with food proteins such as gluten, are the following:

- CXCL10 (IP10);

- CXCL9 (MIG); And

- CXCL11 (I-TAC).

Among these, as regards the sensitivity to gluten, the chemokine CXCL10 has proved to be very sensitive, while as regards the CXCL9 and CXCL11, they have a high homology with the CXCL10 and bind the exact same cell receptor. It is therefore very probable and likely that their secretion is also altered in the PBMCs of patients sensitive to a particular food (such as wheat) exposed to the proteins extracted from that food (such as gluten).

The blood sampling, preferably peripheral venous blood for carrying out the extracorporeal method according to the present invention was carried out by extracting, for example, 5 ml of peripheral venous blood from each patient who undergoes the test, in tubes containing an anticoagulant, such as EDTA (ethylenediaminicotetraacetic acid).

For carrying out the method according to the present invention, arterial or other blood could also be used, provided it is not contaminated (for example fecal blood).

Total proteins are extracted from commercial flours according to the Osborne fractionation method, a method well known to an expert in the art, modified according to van den Broeck et al. for the extraction of gliadins and glutenins (see â € œA modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheatâ € By van den Broeck et al. - Journal of Chromatography B, 2009; 877: 975â € "982). The starting flours with a high gluten content are Manitoba flour (soft wheat) and Claudio wheat flour (durum wheat). The gluten-free starting flours are rice and corn flours certified as gluten-free foods. The protein extracts thus obtained are stored in the form of pellets dried in the freezer at -20 ° C.

Peripheral blood mononuclear cells (PBMCs) are obtained by a normal density gradient extraction, for example in Ficoll gradient of the collected blood. Commercial kits such as SepMate <TM> from Voden Spa, or Ficoll-Paqueâ „¢ gradients can be used to obtain PBMCs from peripheral blood, but other suitable procedures are not excluded. As is known, Ficoll is a highly water-soluble, high molecular weight, synthetic, branched copolymer synthesized starting from sucrose and epichlorohydrin and used to prepare density gradients for cell separation.

Example 1

PBMC cells from each individual sample were cultured using commercial RPMI 1640 medium (Sigma, USA) with the addition of 10% of decomplemented fetal bovine serum (a reagent well known to those skilled in the art and commonly used for cell cultures) and antibiotics (such as penicillin 100 U / ml and streptomycin 100 mg / ml).

PBMCs were distributed in 5 identical wells in 24-well plates, each well containing 300,000 cells and 1.5 mL of complete medium (RPMI 1640 fetal serum antibiotics).

Nothing (white) was added to the first well, while the remaining 4 wells were added 40 micrograms of total protein, respectively, of Rice, Corn, Manitoba and Claudio, dissolved in 500 microliters of complete medium.

The 24-well plates were placed for 16 hours in a thermostated CO2 incubator (Heraeus-b-5060-ek) in a controlled atmosphere (37 ° C, 6% CO2) under gentle agitation on a rocking plate.

At the end of the incubation, the contents of the wells were passed into 2 ml eppendorf tubes. The eppendorf tubes were then centrifuged at 500 g / min for 10 minutes at 4 ° C. At the end of the centrifugation, the supernatant or conditioned medium (1 ml) was withdrawn and transferred to a new eppendorf tube. The pellet, ie the semisolid mass of cells that thicken at the bottom of the tubes after centrifugation, containing the PBMCs was discarded.

The embodiment example described above was developed using 24-well plates and the protein extracts were added in suspension. It is also possible to use 96-well microplates with a PVDF (polyvinylidene fluoride) bottom, where the stimuli are previously adsorbed and adhered to the bottom of the well. The test performed in this mode has been shown to give identical results to the one described above.

The quantification of inflammatory cytokines in conditioned media can be performed with well-known technologies, such as ELISA technology, Luminex <TM> technology, radioimmunological methods (RIA) or other suitable quantitative methods with similar sensitivity. The inventors of the present invention used the Luminex <TM> technology with reagents supplied by BioRad.

The blank, containing PBMC in the absence of protein extracts provides the basal value of chemokine secretion by PBMCs, in the absence of protein extracts containing gluten.

The wells in which Rice and Corn protein extracts are added constituted the negative controls (gluten-free stimuli). The wells with Manitoba and Claudio constituted the production of chemokines in the presence of gluten. The reference value adopted is given by the formula below:

PG = P (manitoba) -P (rice)

PG = chemokine production caused by gluten

P (manitoba) = chemokine production in the presence of proteins extracted from manitoba

P (rice) = production of chemokine in the presence of proteins extracted from rice.

For the chemokine CXCL10 with reference to the sensitivity to gluten, based on the values obtained from the samples that were analyzed, it was established, on the basis of the analysis of the results that:

PG <50 pg / ml = patient negative for gluten sensitive (healthy)

PG> 100 = patient positive for gluten sensitive (suffering from GS)

50 â ‰ ¤ PG â ‰ ¤100 = border line patient (at the limit)

The values obtained for Mais and Claudio were used by internal controls of the experiment and must be the first similar to the value of the rice, the second similar to the value of the manitoba.

For a sample of 19 healthy individuals and 22 patients already diagnosed as GS on the basis of exclusion criteria, the method according to the present invention was carried out. By assaying the chemokine CXCL10 (see Figure 1), the results show that, while PBMCs obtained from the blood of healthy controls and exposed to gluten-containing protein extracts have secretion values lower than 100 pg / ml and in most cases lower than 50 pg / mL, PBMCs of GS patients exposed to contact with gluten-containing protein extracts showed much higher secretion values, with peaks often exceeding 250 pg / mL. Furthermore, the data obtained from the assays of CXCL10, applying a T-test well known to those skilled in the art between the two groups, proved to be statistically very significant with P values (probability of error) lower than 0.001, as reported in the table below. 1.

With reference to Figure 1, the indications “healthy patient” and “GS patient” were established on the basis of the negative or positive outcome of other traditional diagnostic criteria. In particular, if patients complained of complaints following the intake of foods including, among other things, gluten, but were healthy compared to other diseases or disorders other than gluten sensitivity, then these were referred to as GS patients and vice versa. A diagnostic test was conducted on the same subjects according to the present invention, following which the CXCL10 chemokine values indicated in Figure 1 were detected and therefore it was estimated that they were:

- healthy, i.e. not sensitive to gluten, patients 1, 3, 4 and 6 to 19 also previously considered healthy, as well as patients 4 to 7, 16, 18, 19 and 22 previously considered sensitive to gluten,

- border line, patient 2 previously considered healthy, as well as patient 14 previously considered sensitive to gluten, e

- sensitive to gluten, patient 5 previously considered healthy, as well as patients 1 to 3, 8 to 13, 15, 17, 20 and 21 previously considered sensitive to gluten.

An experiment based on the assumption of gluten or placebo in double blind was subsequently carried out on the same patients, which fully confirmed the results obtained with the method according to the present invention and largely denied the conclusions reached on the basis of the simple diagnosis of sensitivity to gluten by excluding other diseases / disorders.

Table 1

Statistical analysis (Unpaired T-test) applied to the obtained values of CXCL10, in the â € œhealthyâ € group compared with the group of patients confirmed as GS

The two-tailed P value obtained is less than 0.001.

GS Patient Health Checks Group

Average 18.30 pg / ml 269.64 pg / ml

SD 28.12 140.15

SEM 6.45 37.46

N 19 14

To further validate the test object of the present invention, a blinded study was carried out, where patients classified as GS and healthy controls were treated with sachets containing either gluten or placebo flours (gluten-free flours), to be added to foods meals. The study was carried out in collaboration with the Gastroenterology Unit (SVD), Prof. Alberto Lanzini and Dr. Chiara RICCI - A. O. Spedali Civili of Brescia, University of Brescia.

The results of the analyzed samples, once the blind was opened, showed that the test based on CXCL10 is able to accurately discriminate between healthy controls and GS patients and shows strongly positive values in correspondence with gluten intake and appearance of symptoms declared by patients. They also showed that the responses to the different flours used are affected by the gluten content present in them.

Figure 2 shows the results of tests conducted on a patient not suffering from GS, who declared moderate symptoms while taking placebo and absent symptoms while taking gluten. More specifically, the same Figure shows the analysis of the differential secretion of CXCL10 (flour with gluten-rice, in pg / ml) by the PBMCs of a possible GS patient, who was later classified as not GS . This patient was stimulated with protein extracts of flours with a high gluten content (Manitoba and Claudio), low gluten content (Kamut and Senatore Cappelli) or gluten-free (corn). Rice flour (gluten free) was used as a control value. At time T1 the subject had been on a gluten-free diet for at least 90 days, at time T2 he took placebo (declaring moderate symptoms), at time T1a he was again on a gluten-free diet and at time T1b he assumed a daily gluten load equal to 2 dishes of pasta of 100 g each (declaring the disappearance of symptoms).

Figure 3 shows the results obtained for a border-line patient, who declared moderate symptoms while taking gluten. More specifically, this Figure shows the analysis of the differential secretion of CXCL10 (manitoba-rice, in pg / ml) by PBMCs of border line GS patients stimulated with protein extracts of high gluten content flours (Manitoba and Claudio), low gluten content (Kamut and Senatore Cappelli) or gluten-free (corn). Again, gluten free rice flour was used as a control value. At time T1 the subject had been on a gluten-free diet for at least 90 days, at time T2 he took placebo (without symptoms), at time T1a he was again on a gluten-free diet and at time T1b he assumed a daily gluten load equal to 2 pasta dishes of 100 g each (mild symptoms).

Figure 4 shows the results obtained for a patient suffering from GS, who declared a strong symptomatology in correspondence with gluten intake. Also for this patient the differential secretion of CXCL10 (manitoba-rice, in pg / ml) by the PBMC of the patient stimulated with protein extracts of flours with a high gluten content (Manitoba and Claudio), low gluten content was analyzed (Kamut and Senatore Cappelli) or gluten-free (corn), while gluten-free rice flour was used as a control value. As it can be observed, at time T1 the subject had been on a gluten-free diet for at least 90 days, at time T2 he took placebo (without symptoms), at time T1a he was again on a gluten-free diet and at time T1b he assumed a daily gluten load equal to to 2 pasta dishes of 100 g each (strong symptoms). In this last patient, it is well known that grains with a high gluten content (Manitoba) give more positive responses, while grains with a lower gluten content (Senatore Cappelli) give less positive responses.

Corn does not provide any answers, confirming that the answers obtained are gluten-dependent.

Compared to the method taught by WO2011163258, the method object of the present invention differs both for the starting population, for the stimulus that is used and, above all, for what is measured as a response. The chemokines that are determined according to the present invention are in fact produced essentially by monocytes and granulocytes (see: Molecular mechanisms underlying the synergistic induction of CXCL10 by LPS and IFN-gamma in human neutrophils. Di Tamassia N, Calzetti F, Ear T, Cloutier A, Gasperini S, Bazzoni F, McDonald PP, Cassatella MA, Eur J Immunol. 2007 Sep; 37 (9): 2627-34) while according to WO2011163258 the dosed cytokines are obtained from lymphocytes. Furthermore, in the present invention total protein extracts from foods are used, while according to WO2011163258 synthetic peptides with known sequences are used.

A kit according to the present invention preferably comprises:

- a 96-well multiwell plate with PVDF bottom and total proteins extracted from gluten-free and gluten-free flour pre-adsorbed on the bottom (as shown in Figure 5);

- a first package of polymer solution for PMBC extraction in density gradient, for example a 100 ml bottle of Ficoll®;

- a second package, for example a bottle of RPMI 1640 medium containing 10% FBS and antibiotics; And

- 200 0.5 ml eppendorf type tubes.

For the realization of a diagnosis method according to the present invention with the kit indicated above, the procedure is as follows.

Phase 1: Extraction of PBMCs

5 ml of whole blood are diluted with 5 ml of PBS, as shown in the first image on the left in Figure 5.

The blood diluted in PBS is stratified on 5 ml of Ficoll® (second image from the left in Figure 5). It is centrifuged for 30 minutes at 900 g / min with the centrifuge set in "no brake" mode.

The PBMC ring is removed as shown in the third image from the left in Figure 5 and transferred to a new tube containing 10 ml of PBS to wash the PBMCs, shaking. It is centrifuged at 500 g / min for 15 minutes without brake.

Repeat the previous step discarding the supernatant and washing the pellet again in 10 ml of PBS. Spin at 500 g / min for 15 minutes with spin set in "no brake" mode.

The pellet is resuspended in 1 ml of complete RPMI1640 medium (provided in the kit).

Phase 2: Planting of PBMCs

The cells are counted with a blood cell counting chamber. RPMI 1640 is added until the concentration of 300,000 cells per ml is reached for each patient. While stirring the cell suspension obtained in the previous phase, 200µl of PBMC suspension is sown for each patient in each of the 6 wells relating to the patient, according to the scheme shown in Figure 6.

Figure 6 shows the loading diagram of a 96-well multiwell plate supplied in the kit. The wells marked with M have adsorbed on the bottom proteins extracted from flour with gluten (Manitoba), those marked with R have adsorbed on the bottom proteins extracted from gluten-free rice flour, while those marked with B have no proteins adsorbed on the bottom of the well . The kit allows to load PBMCs from 16 patients (indicated with numbers 1-16).

Step 3: Incubation and assay of CXCL10

After sowing, it is incubated for 16 hours in a humidified incubator with 5% CO2.

the contents of the wells are taken and transferred into 0.5 ml eppendorf tubes. Be centrifuged at 100 g / min for 10 minutes to eliminate PBMCs. The supernatant is collected and stored at -80 ° C until the moment of the assay (maximum 120 days) or the assay is carried out immediately.

The content in CXCL10 of the individual supernatants collected is measured using a highly sensitive method (Elisa, Luminex, RIA).

Phase 4: Analysis of the results

Stimulation of CXCL10 by gluten-containing protein extracts:

Patient 1 = (mean M1 values) - (mean R1 values)

The values are meant

positive, if the result exceeds 100 pg / ml;

border line for values between 50 and 100 pg / ml,

negative, if the result is less than 50 pg / mL.

The B values serve as controls and should be less than 50 pg / mL. Higher values of B indicate that the production of CXCL10 in that patient is high even in the absence of protein extracts and therefore make the test result unreliable for that patient.

The invention described above is susceptible of numerous modifications and variations within the scope of protection defined by the claims.

Claims (18)

CLAIMS 1. Method of out-of-body diagnosis of a patient's food sensitivity or intolerance, comprising the following steps in sequence: - add at least one food to a sample of patient-derived peripheral blood or patient-derived peripheral blood mononuclear cells (PBMC); - leave the sample thus added to stand for a predetermined time interval; And - measure the quantity of at least one chemokine produced in the sample after the rest interval. 2. Method according to claim 1, characterized in that it is a method for diagnosing sensitivity to gluten and in that the food contains gluten. 3. Method according to claim 1 or 2, characterized in that said at least one chemokine is selected from the group consisting of CXCL10 (IP10), CXCL9 (MIG) and CXCL11 (I-TAC). 4. Method according to any one of claims 1 to 3, characterized in that said food is a total protein extract from at least one food. 5. Method according to claim 4, characterized in that said at least one food comprises flour. 6. Method according to claim 5, characterized in that said flour comprises Manitoba flour or Claudio flour. 7. Method according to any one of the preceding claims, characterized in that said peripheral blood sample is a venous blood extract based on monocytes and granulocytes. 8. Method according to any one of the preceding claims, characterized in that said peripheral blood mononuclear cells are obtained with a density gradient extraction. 9. Method according to any one of the preceding claims, characterized in that said sample is incubated during the rest interval and the quantity of at least one chemokine produced in the sample is measured once incubated. 10. Method according to claim 9, characterized in that it comprises: the predisposition of said peripheral blood mononuclear cells (PBMC) in a culture medium; the addition to the culture medium of fetal decomplemented bovine serum, of at least one antibiotic and of total protein extracted from food; the incubation of said culture medium in a thermostated incubator at a controlled temperature; centrifugation of the incubation product; And the quantification of inflammatory cytokines in the supernatant which separated following centrifugation. 11. Method according to claim 10, characterized in that said at least one antibiotic is selected from the group formed by penicillin and streptomycin. Method according to claim 10 or 11, characterized in that approximately 300,000 PBMC cells are used for approximately 1.5 ml of culture medium. Method according to any one of the preceding claims, characterized in that a gluten-free protein extract is added to a portion of said at least one peripheral blood sample taken from a patient or peripheral blood mononuclear cells (PBMC) to obtain a negative control of the reference test results. 14. Method as claimed in claim 13, characterized in that said gluten-free protein extract is an extract of rice or corn flour. 15. Kit for carrying out the method according to any one of claims 1 to 14, characterized in that it comprises: - at least one multi-well plate (multiwell) with a PVDF bottom, some of these wells having total proteins extracted from gluten-free flours pre-absorbed on the bottom, while other wells have total proteins extracted from gluten-free flours pre-adsorbed on the bottom; - at least one first package of polymer solution for PMBC extraction in density gradient; - at least a second pack of RPMI 1640 medium containing 10% FBS and at least one antibiotic; And - a plurality of test tubes. 16. Kit according to claim 15, characterized in that said at least one first package is a bottle or bottle of 100 ml. Kit according to claim 15 or 16, characterized in that said polymer of said at least one first package is a branched synthetic copolymer of relatively high molecular weight, water-soluble and synthesized starting from sucrose and epichlorohydrin. 18. Kit according to any one of claims 15, 16 or 17, characterized in that each test tube is of the eppendorf type.