ES2548653A2 - Method and kit for the diagnosis of celiaquia (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Method and kit for the diagnosis of celiac disease. The present invention relates to methods and kits for the diagnosis of celiac disease by the combination by a single kit of the detection of serological and genetic markers. The present invention shows that the presence of antibodies against neoepitopes arising from the binding of the transglutaminase type 2 complex-deamidated gliadin peptides and the absence of the genetic markers hla-dq2 or -dq8, may indicate the presence of the disease . (Machine-translation by Google Translate, not legally binding)

Description

5 The present invention relates to methods and kits for diagnosis and / or monitoring of celiac disease. Therefore the present invention is encompassed within the technical field of medicine, specifically in the technical field of diagnosis clinically and more specifically in the diagnosis of patients suffering from celiac disease.

10 STATE OF THE TECHNIQUE

Celiac disease (CD) affects approximately 1% of the world's population, although there are differences depending on the populations. In Spain it is estimated that its frequency ranges between 1/71 in the child population and 1/357 in the adult population.

15 In addition, the frequency of the disease is higher in relatives of celiac patients and individuals with certain pathologies, such as type I diabetes mellitus, autoimmune thyroiditis, IgA deficiency, Down syndrome, Turner syndrome or Williams syndrome, which constitute the risk groups

20 Celiac disease is a chronic disease, therefore the lack of diagnosis implies the repeated visit of these patients to hospital consultations, mainly to Pediatrics and Digestive consultations, but they can also go to various specialists simultaneously since clinical symptoms are often unspecific, especially in adult individuals. If we consider that this

25 disease affects a large number of children, the health cost they can cause throughout their lives is very high. It must be taken into account that celiac disease has a treatment that is effective in the vast majority of cases and consists in eliminating gluten from the diet, so once diagnosed, they no longer imply a health cost, except for the controls of follow up they can

30 need.

In 2012, the European Society of Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) proposed new guidelines for the diagnosis of celiac disease in children and adolescents (Husby S, et al. J Pediatr Gastroenterol 35 Nutr 2012; 54: 136-60 ). The definition of the disease was also reviewed and CD became a systemic disorder, due to its great variability in

clinical manifestations, many of them extra-intestinal. This clinical variability implies the need for highly specific and sensitive diagnostic tools.

5 Currently, the diagnosis of celiac disease involves in the vast majority of cases the performance of three types of tests. Two of them (serological and genetic tests) are aimed at selecting the group of individuals who may suffer from the disease and therefore require the third test (duodenal biopsy), which is the one that offers the definitive diagnosis. However, given the

10 aggressiveness of said test, the serological and genetic tests are used as previous screening tests, so that with negative results in these tests the diagnosis of celiac disease can be ruled out without performing the intestinal biopsy. Diagnostic tests are carried out by different techniques and require different devices and / or commercial tests. Although currently

15 There are diagnostic algorithms based on the results of these tests, it is considered that only a small part of the patients are correctly diagnosed. The diagnosis of the disease has an immediate impact on the patient since after the start of treatment (gluten-free diet) the symptoms cease and the patient recovers a normal life in the vast majority of cases.

20 In this sense, the presence of specific antibodies against tissue transglutaminase type 2 (TG2), against endomysium (EMA) and against deamidated gliadin peptides (PDG), detected by specific kits for it, together with the presence of HLA (human leukocyte antigen) -DQ2 or -DQ8 haplotypes, detected

25 by means of specific probes and primers detected independently of the serological markers, they define the group of patients suffering from CD, when it coexists with a variable combination of clinical manifestations and gluten-dependent enteropathy. In usual clinical practice, there is a high availability of serological tests for the diagnosis of CD. However, the

The sensitivity and specificity thereof is based mainly on the specific antibodies comprising said kits, as well as on the commercial kit itself used for said diagnosis. Thus, it is known that the anti-TG2 and anti-EMA antibodies, both directed against the TG2 enzyme, are the most specific for the diagnosis of CD. The specificity of anti-EMA is superior, but since its determination

35 is carried out by indirect immunofluorescence, which is a laborious and subjective technique of interpreting by non-expert personnel, in the usual clinical practice,

It is recommended to test first the presence of anti-TG2 antibodies and then confirm the positive results for the presence of anti-EMA antibodies. In addition, the determination of anti-PDG antibodies is also used. The correlation of anti-TG2 and EMA antibodies is very high, but may decrease when

5 commercial kits are used that detect neoepitopes that emerge after the cross-linking reaction between TG2 and PDG. Commercial kits capable of detecting neoepitopes include the Aeskulisa® tTg-A kit (Aesku.Diagnostics, Wendelsheim, Germany), the Aeskulisa® CeliCheck kit (Aesku.Diagnostics, Wendelshiem, Germany) and the Quanta Lite® tTG- kit DGP screen (Inova Diagnostics, Inc, CA, USA).

10 On the other hand, it is currently considered that almost all celiac patients have alleles that encode the HLA-DQ2 or -DQ8 molecules. In addition, depending on the number of alleles and what precise alleles are present in the subjects, the risk of suffering from the disease varies. In this sense, the absence of

15 alleles encoding HLA-DQ2 or -DQ8 exclude or make the presence of CD very little possible. The DQ2 molecule is encoded by the joint presence of the DQA1 * 05 and DQB1 * 02 alleles and the DQ8 molecule by the joint presence of the DQA1 * 03 and DQB1 * 03: 02 alleles. In addition, it is believed that the DQB1 * 02 allele present in isolation also confers risk, although low. The absence of these alleles, in the

20 combinations described, practically rule out the disease.

The use of commercial kits for the detection of serological markers that include the detection of neoepitopes that arise after TG2-PDG binding, shows that there are cases of individuals who are positive for these markers and 25 negative for anti-EMA (no they present antibodies against EMA), so following the current diagnostic criteria it would be considered that they do not have celiac disease and that their ailment would be due to a different pathology. However, the inventors show that there are cases of subjects suffering from CD within this group of patients (TG2 + and EMA-), but who are diagnosed, a priori, 30 as subjects who do not suffer from the disease, that is, many They are misdiagnosed, since in fact, they do suffer from the disease. In addition, the inventors also show that some patients who have antibodies detected by the commercial kit Aeskulisa® tTg-A, which detects the formation of neoepitopes, but lack anti-EMA antibodies, show about 35 particular genetic characteristics that are not adapted to those commonly present in patients suffering from celiac disease, that is, they do not have markers

genetic, HLA-DQ2 or HLA-DQ8, since 33% of these patients lack complete HLA-DQ2 / DQ8 molecules, that is, they have only one or none of the alleles necessary for the formation of said molecules.

5 Therefore, taking into account all the aforementioned, there is a group of celiac patients who, today, using diagnostic guides and most commercial methods or kits available on the market, have very difficult to receive a correct diagnosis since they do not present the commonly established criteria for the diagnosis of the disease through serological tests

10 (anti-TG2 and anti-EMA) and genetic (HLA-DQ2 or -DQ8). Especially, it should be taken into account that the absence of antibodies against EMA is considered quite indicative of the absence of celiac disease. In addition, the main alleles of genetic risk that code for HLA-DQ2 or HLA-DQ8, do not appear at the same frequency as the scientifically recognized as characteristic for patients who

15 suffer from celiac disease, which contributes to make the diagnosis of these patients more difficult and makes the development of precise tools, with a high specificity and sensitivity, that allow the correct and rapid diagnosis of the disease more necessary. In this sense, there is an important need in the state of the art for the development of precise and reliable tools for the diagnosis of

20 celiac disease, specifically for the diagnosis of those patients who have rare serological and genetic characteristics that escape current diagnostic criteria, so that on the one hand these individuals are correctly diagnosed and on the other hand that the time elapsed is reduced since an individual begins to suffer the symptoms of the disease (debut)

25 until diagnosed correctly.

DESCRIPTION OF THE INVENTION

In order to overcome the problems existing in the state of the art for the

30 correct diagnosis of patients suffering from celiac disease, the present invention describes a method of obtaining useful data, as well as an in vitro diagnostic method, of patients suffering from celiac disease and that includes those patients who do not have the typical serological and genetic characteristics which are described in the clinical guidelines for the diagnosis of said disease and that therefore escape from

35 the current diagnostic criteria.

The term "celiac disease" or "celiac disease" in the present invention refers to a pathology mediated by the immune system characterized by chronic inflammation of the small intestine caused by exposure to gluten. Celiac disease involves a permanent intolerance to gluten. Gluten refers to 5 proteins present in cereals such as wheat (Triticum spp), barley (Hordeum vulgare), rye (Secale cereale) and possibly oats (Avena spp). In the subject suffering from celiac disease or celiac disease, after ingestion of gluten, the enzyme tissue transglutaminase or type 2 transglutaminase (TG2) modifies these proteins and triggers a response mediated by the immune system that leads to

10 histological intestinal lesion characterized in the great majority of cases by atrophy of the villi that cover the intestine and interference in the absorption of nutrients.

The development of celiac disease is determined both by factors

15 environmental (food) as genetic. Thus, celiac disease also implies a genetic predisposition since most celiacs have the human leukocyte antigen (HLA) of type DQ2 (HLA-DQ2) or DQ8 (HLA-DQ8) (Tjon JM. Et al. 2010 Immunogenetics 62 : 641-651).

Thus, in a first aspect, the present invention relates to a method of obtaining useful data, from here we will call it the first method of the invention, for the diagnosis of celiac disease in an isolated biological sample of a subject comprising the joint detection and / or quantification of serological and genetic markers, where serological markers are selected from: antibodies against

25 to transglutaminase type 2 (TG2), antibodies against deamidated peptides of gliadin (PDG) and antibodies against neoepitopes that arise from the binding and / or cross-linking of the transglutaminase complex type 2-deamidated peptides of gliadin, and where the genetic marker is HLA-DQ.

For the purposes of the present invention, human transglutaminase 2 (TG2) is defined as it is collected in the NCBI database with accession number AAT79353.1 (according to the August 4, 2004 version of said database) , encoded by the transglutaminase 2 gene as collected in the NCBI database with access number ID 7052 (reviewed on April 8, 2014 in that database). TG2 is the

35 responsible for the selective deamidation of gluten, which in turn causes the

generation of a series of gluten peptides that bind to HLA-DQ2 or DQ8 molecules with high affinity.

For the purposes of the present invention, PDGs are defined as gluten fragments that have suffered deamidation by human TG2 once ingested in the diet.

For the purposes of the present invention, the term "neoepitope" refers to those new epitopes that arise after covalent binding between gliadin peptides and TG2 as a result of a transamidation reaction catalyzed by it.

10 TG2. The term gliadin deamidated anti-peptide antibodies refers to those antibodies that are formed against gluten fragments that have undergone deamidation by the TG2 enzyme.

For the purposes of the present invention the term "HLA" or "human leukocyte antigen" or

"Human leukocyte antigen" refers to a segment of approximately 4 Mb located in the short arm of human chromosome 6 (6p21). The HLA region comprises a large number of genes, among which those that are classified in class I HLA and in class II, which present antigens to different subtypes of cells. Class II molecules, among which are HLA-DR and HLA-DQ, are

20 heterodimeric glycoproteins consisting of an alpha chain and a beta chain. MHC class II molecules are expressed on the cell surface of dendritic cells, macrophages, B cells and other cell types involved in the presentation of antigens to T cells that express CD4 cell surface glycoprotein.

For the purposes of the present invention the term "HLA-DQ" refers to heterodimeric glycoprotein that is expressed in certain cell types and that consists of an alpha chain encoded by the HLA-DQA1 gene and a beta chain encoded by the gene HLADQB1. The HLA-DQA1 protein is defined as it is collected in the database.

30 NCBI with access number AAH08585 (according to the October 3, 2003 version of said database) and with access number AAH73977 (according to the July 27, 2004 version of said database); encoded by the HLA-DQA1 gene, as collected in the NCBI database with access number ID 3117 (reviewed on April 8, 2014 in that database). The HLA-DQB1 protein is defined as it is

35 collected in the NCBI database with access number AAB60325 (according to the May 25, 1994 version of said database) and with access number

P01920.2 (according to the March 19, 2014 version of said database); encoded by the HLA-DQB1 gene, as collected in the NCBI database with access number ID 3119 (reviewed on April 8, 2014 in that database).

5 For the purposes of the present invention, genetic risk is understood as the risk that has a subject of celiac disease depending on the specific alleles DQA1 and / or DQB1 that you present, as well as if you submit one or two copies thereof.

For the purposes of the present invention the term "HLA-DQ2" refers to glycoprotein.

10 heterodimeric that is expressed in certain cell types of individuals presenting DQA1 * 05 and DQB1 * 02 alleles. In the present invention the DQA1 * 05 allele encompasses the DQA1 * 05: 01 and DQA1 * 05: 05 alleles; and the DQB1 * 02 allele encompasses the DQB1 * 02: 01 and DQB1 * 02: 02 alleles. Thus, as understood in the present invention, a subject has the HLA-DQ2 marker when expressing at least one of the alleles.

15 DQA1 * 05: 01 or DQA1 * 05: 05 together with at least one of the alleles DQB1 * 02: 01 or DQB1 * 02: 02.

For the purposes of the present invention, the term "HLA-DQ8" refers to heterodimeric glycoprotein that is expressed in certain cell types of individuals presenting

20 alleles DQA1 * 03 and DQB1 * 03: 02. In the present invention, the DQA1 * 03 allele encompasses the DQA1 * 03: 01 and DQA1 * 03: 02 alleles. Thus, as understood in the present invention, a subject presents the HLA-DQ8 marker when expressing at least one of the DQA1 * 03: 01 or DQA1 * 03: 02 alleles together with the DQB1 * 03: 02 allele.

25 The term "allele" refers to one of the two or more alternative forms of a gene, differing in the genetic sequence and found in the same place on a chromosome.

Thus, in a preferred embodiment, the first method of the invention is characterized in that the genetic marker HLA-DQ is selected from: HLA-DQ2 or HLA-DQ8.

Thus, in another preferred embodiment, the first method of the invention is characterized in that the HLA-DQ2 marker is encoded by the joint presence of the DQA1 * 05 and DQB1 * 02 alleles. In a still more preferred embodiment, the DQA1 * 05 allele is

35 is selected from variants DQA1 * 05: 01 and DQA1 * 05: 05 and the DQB1 * 02 allele is selected from variants DQB1 * 02: 01 and DQB1 * 02: 02.

In another preferred embodiment, the first method of the invention is characterized in that the genetic marker HLA-DQ2 is encoded by at least one of the DQA1 * 05: 01 or DQA1 * 05: 05 alleles together with at least one of the DQB1 alleles * 02: 01 or DQB1 * 02: 02.

5 In another preferred embodiment, the first method of the invention is characterized in that the genetic marker HLA-DQ8 is encoded by the joint presence of the alleles DQA1 * 03 and DQB1 * 03: 02. In an even more preferred embodiment, the DQA1 * 03 allele is Select between: DQA1 * 03: 01 and DQA1 * 03: 02.

In another preferred embodiment, the first method of the invention is characterized in that the genetic marker HLA-DQ8 is encoded by at least one of the DQA1 * 03: 01 or DQA1 * 03: 02 alleles together with the presence of the DQB1 * allele 03:02

In another preferred embodiment, the first method of the invention is characterized in that the detection of antibodies against neoepitopes arising from the binding and / or cross-linking of the transglutaminase type 2-deamidated gliadin peptides complex and the absence of alleles encoding for genetic markers HLADQ2 or HLA-DQ8 classifies the subject in a specific genetic risk group of

20 suffer from celiac disease.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a high genetic risk of suffering from the disease when antibodies against neoepitopes are detected that

25 arise from the binding and / or cross-linking of the transglutaminase complex type 2-deamidated peptides of gliadin and one or two DQA1 * 05 alleles and two DQB1 * 02 alleles.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a moderate genetic risk of

30 suffer from the disease when antibodies against neoepitopes arising from the binding and / or cross-linking of the transglutaminase type 2-deamidated gliadin peptides complex and a DQA1 * 05 allele and a DQB1 * 02 allele are detected.

In a more preferred embodiment of the first method of the invention, it is

35 characterized by the fact that a subject is considered to have a low genetic risk of suffering from the disease when antibodies to neoepitopes that are detected are detected.

arise from the binding and / or cross-linking of the transglutaminase complex type 2-deamidated peptides of gliadin and any combination of alleles in DQA1 and DQB1, different from the combinations described for the high and moderate genetic risk groups.

5 In a still more preferred embodiment of the group of patients classified as low genetic risk, these are characterized in that said subjects have a higher genetic risk of suffering from celiac disease if they have the DQA1 * 05 allele in the absence of the DQB1 * 02 allele than if they have any Another possible combination of alleles within the category of low genetic risk.

In another preferred embodiment of the first method of the invention, this is characterized in that the detection of antibodies against transglutaminase type 2 and the presence of at least one of the genetic markers HLA-DQ2 or HLA-DQ8 classifies the subject into a group of determined genetic risk of suffering from celiac disease.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a very high genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQA1 * 05 alleles are detected and two alleles DQB1 * 02.

In a more preferred embodiment of the first method of the invention, this is characterized in that a subject is considered to be at high genetic risk of suffering from the disease when antibodies against type 2 transglutaminase and a DQA1 * 05 allele and a DQB1 allele are detected. * 02.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a moderate genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQA1 * 03 and one alleles are detected. or two DQB1 * 03: 02 alleles.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a low genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQB1 * 02 alleles and no DQA1 * 05 allele.

In another preferred embodiment of the first method of the invention, this is characterized in that the detection of antibodies against deamidated gliadin peptides and the presence of at least one of the genetic markers HLA-DQ2 or HLA-DQ8 classifies the subject into a group of determined genetic risk of suffering from celiac disease.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a very high genetic risk of suffering from the disease when antibodies against deamidated peptides of gliadin and one or two DQA1 * 05 alleles are detected. and two DQB1 * 02 alleles.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to be at high genetic risk of suffering from the disease when antibodies against deamidated gliadin peptides and a DQA1 * 05 allele and an allele are detected. DQB1 * 02.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a moderate genetic risk of suffering from the disease when antibodies against deamidated gliadin peptides and one or two DQA1 * 03 alleles are detected and one or two alleles DQB1 * 03: 02.

In a more preferred embodiment of the first method of the invention, it is characterized in that a subject is considered to have a low genetic risk of suffering from the disease when antibodies to deamidated gliadin peptides and one or two DQB1 * 02 alleles are detected and no DQA1 * 05 allele.

In a preferred embodiment of the first method of the invention, it is characterized in that it further comprises the detection and / or quantification of at least one intestinal atrophy marker, preferably the detection of the REG1A marker (regenerating islet-derived 1 alpha).

30 Said REG1A protein is defined as collected in the NCBI database with accession number AAH05350 (according to the June 9, 2008 version of said database), and which is encoded by the REG1A gene as and as collected in the NCBI database with access number ID 5967 (revised on April 8,

35 2014 in said database).

In another preferred embodiment of the first method of the invention, this is characterized in that the antibodies detected have isotype A (IgA) or G (IgG).

The term "antibody", as used in the present invention, refers to a

5 glycoprotein that exhibits a specific binding activity for a particular protein, which is called "antigen." In the context of the present invention, the antibodies are anti-TG2, anti-PDG and anti-TG2-PDG antibodies, that is, they specifically recognize and bind TG2, PDG and neoepitopes that may arise from binding. TG2-PDG, respectively, said proteins, or fragments being

10 of them, the antigens.

Thus, in the context of the present invention any antibody capable of detecting the presence of anti-TG2, anti-PDG and anti-neoepitope antibodies that may arise from the TG2-PDG binding is used. Known antibodies for the detection of anti

TG2, anti-PDG and anti-neoepitopes that may arise from the binding and / or cross-linking between TG2-PDG, are for example, without limitation, any antibody designed by a research group or a commercial antibody that can be purchased in commercial houses: R&D System, Cell Signaling Technology, Orignene, GenScript, Abcam, Cloud Clone Corporation, LS Bio, among others.

In another preferred embodiment of the first method of the invention, this is characterized in that the detection of the serological markers is performed by an immunoassay and the detection of the genetic markers by the PCR technique, preferably, by the PCR-SSOP technique (Reaction in Polymerase Chain-probes

25 sequence specific oligonucleotides) or PCR-SSO (Polymerase chain reaction-sequence specific oligonucleotides).

The term "immunoassay", as used herein, includes any technique based on the formation or use of immunocomplexes, that is, complexes resulting from the binding of antibodies and antigens, as a reference for quantification of an analyte (substance under analysis ) determined, which may be the antibody or an antigen, using for measurement a molecule as a marker that produces a detectable signal in response to a specific binding. Said term includes both competitive and non-competitive immunoassays, as well as immunoassays.

35 heterogeneous and homogeneous.

Illustrative, non-limiting examples of markers include radioactive elements (e.g., sulfur, iodine, etc.); enzymes (e.g., peroxidase, glycosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, β-galactosidase, β-glucosidase, β-glucuronidase, etc.); fluorescent compounds or dyes (e.g., fluorescein, rhodamine, etc.), phosphorescent or chemiluminescent (e.g., dioxetanes, acridiniums, phenanthridines, ruthenium, luminol, etc.); latex or magnetic particles; colloidal particles of gold, silver, or selenium; metal chelates; coenzymes; etc. The selection of a particular marker is not critical, as long as it is capable of producing a signal by itself or in conjunction with one or more additional substances. Thus, the complex 10 formed can be detected or visualized by any appropriate technique, depending on the marker chosen, known to those skilled in the art, using the appropriate devices, for example, by techniques based on radioactive, colorimetric, fluorimetric methods, (chemo ) luminescent, etc., all known to those skilled in the art. By way of illustration, when the marker is

In an enzyme, the detection of the complex (antigen-antibody) / label can be carried out by contacting said complex with an appropriate substrate and, optionally, with the appropriate activators and / or enzymatic amplifying agents.

20 Illustrative, non-limiting examples of immunoassays suitable for the implementation of the methods of the present invention include Western blot, ELISA (enzyme-linked immunosorbent assay), DAS-ELISA ("Double Antibody Sandwich-ELISA"), competitive EIA (competitive enzyme immunoassay), DELFIA (lanthanide augmented dissociation fluoroimmunoassay), FPIA (immunoassay

25 of fluorescent polarization), CMIA (chemiluminescent magnetic immunoassay), RIA (heterogeneous and competitive radioimmunoassay), IRMA (heterogeneous and non-competitive radioimmunoassay), MEIA (microparticle immunoassay), luminoimmunoassays, hisochemical-immunochemical-immunochemical techniques use of spheres, nanoparticles, biomarker biochips, biosensors

30 (e.g., immunobiosensors) or microarrays, lab-on-a-chip that include specific antibodies, tests based on colloidal precipitation in formats such as dipsticks, etc.

As previously mentioned, the detection of genetic markers is performed by the polymerase chain reaction (PCR) technique, preferably by PCR-SSOP techniques (Chain Reaction of the

Sequence specific oligonucleotide polymerase probes) or PCR-SSO (Sequence specific polymerase oligonucleotide chain reaction).

In a still more preferred embodiment of the first method of the invention, the

5 joint determination of serological and genetic markers is carried out using Luminex technology (Tepnel Lifecodes Corp, Stamford, CT USA), using specific antibodies for the detection of anti-TG2, anti-PDG and anti-TG2-PDG antibodies and using specific probes or oligonucleotides for the detection of DQA1 * 05: 01, DQA1 * 05: 05, DQB1 * 02: 01 and DQB1 * 02: 02 alleles for

10 characterize the presence of the HLA-DQ2 marker and specific probes or oligonucleotides for the detection of DQA1 * 03: 01, DQA1 * 03: 02 and DQB1 * 03: 02 alleles to characterize the presence of the HLA-DQ8 marker.

The Luminex system is based on the combination of three technologies: the use of

15 microspheres labeled with fluorochromes, which act as identifiers and as a solid surface to develop the assay; an instrument based on flow cytometry and integrating different components (lasers, optics, advanced fluids ...); and software designed for data acquisition and analysis. This system allows different determinations to be carried out in a single well by means of the

The use of microspheres identified by the different proportion of more than one fluorochrome, preferably two fluorochromes and more preferably, more than two fluorochromes, which will allow them to be identified in isolation after being excited by the laser of the analysis equipment. In said technique, the corresponding molecules that capture the analytes of interest are attached to the surface of the spheres, in this

In the case of the antigens that detect the antibodies under study, the oligonucleotide probes that detect the alleles present in DQA1 and DQB1, or the antibodies directed against other molecules of interest, preferably against markers of intestinal atrophy. By using probes or secondary antibodies labeled with a molecule capable of emitting fluorescence, such as without

30 limit, phycoerythrin, we can detect by means of a second laser, if the sample contains the analyte sought (qualitative analysis), and even in the case of the detection of antibodies or protein antigens what quantity (quantitative analysis).

Luminex technology allows to analyze a multitude of proteins or nucleotide sequences to be detected in each well of a 96 or 384 well plate, using a very small sample volume. In the present invention, the

Luminex technology will be applied by anchoring the antigens that detect the presence of anti-TG2, anti-PDG or both antibodies to the surface of microspheres, as well as by anchoring the oligonucleotide probes that detect the alleles in DQA1 and DQB1 , to the surface of the microspheres. The anchoring of antibodies against specific proteins to the surface of the microspheres can also be considered. Such specific proteins may be, for example, but not limited to, markers of intestinal atrophy, such as the REG1A protein. The microspheres will be marked with different fluorochromes, so that a ratio of the fluorochromes is established in each of them, the combination of 10 fluorochromes in different proportions gives them a different color depending on the amount they possess of each fluorochrome. This procedure allows the creation of a large number of different populations of fluorospheres, and therefore, the presence of a large number of different probes for hybridization, one for each type of microsphere. The emission spectrum of each microsphere is unique, which allows the simultaneous identification of all of them and therefore, of the reaction or test that is being carried out on the surface of each of them. Subsequently, the microspheres are forced to pass through a flow of flow. Each of them is classified according to the ratio of its internal fluorescent label. In a particular embodiment, the TG2, PDG or both proteins or peptides are used as support antigens, in a solid phase, anchored to the surface of the microspheres. In another particular embodiment, the complementary probes that detect the alleles in DQA1 and DQB1 are anchored to the surface of the microspheres. In another particular embodiment, specific antibodies that recognize different proteins of interest, for example, markers, can be anchored to the surface of the microspheres

25 intestinal atrophy.

In the present invention, the term "fluorochrome" refers to a component of a molecule that makes it fluorescent. It is a functional group of the molecule that will absorb energy of a specific wavelength and will emit again in another

30 set of different wavelength. The amount of energy emitted and its wavelength depend on both the fluorochrome itself and its chemical environment. Some examples of fluorochromes that can be used in the present invention, but not limited to, are rhodamine, fluorescein or dansyl.

Thus, the immunoassay used for the implementation of the method of the present invention, preferably using the Luminex technique, can be used to

determine the quantity and thus quantify, the concentration or level of antiTG2, anti-PDG and anti-TG2-PDG antibodies, in a sample since the amount of antibody present in the test sample is proportional to the signal generated. In turn, by means of this technique, the presence of the alleles that characterize the

5 genetic markers HLA-DQ2 or HLA-DQ8.

The term "biological sample", as used in the present invention, refers to a sample, preferably, a biological fluid, more preferably, a sample of saliva, blood, peripheral blood and / or serum, isolated from a subject.

In another preferred embodiment of the first method of the invention, this is characterized in that the isolated biological sample is selected from blood, preferably peripheral blood, or serum.

15 The term "peripheral blood" is related to the volume of circulating blood distant from the heart, that is, the blood that circulates through the organism of a subject. The blood sample can be obtained by conventional methods known to the person skilled in the art. The term "serum", as used in the present invention, refers to the resulting blood component after blood clotting and

20 removal of the resulting clot. Methods of obtaining blood samples from a subject are widely collected in the state of the art, as well as methods of obtaining serum from blood samples.

In another preferred embodiment of the first method of the invention, this is characterized by the fact that the subject is human.

The term "subject", as used in the invention, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, for example, humans, primates. nonhumans, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a human being male or female of any age or race.

Another aspect described in the present invention relates to an in vitro diagnostic method, hereinafter second method of the invention, of celiac disease, in a

35 isolated biological sample of a subject, comprising:

a) the joint detection and / or quantification of serological and genetic markers where serological markers are selected from: antibodies against transglutaminase type 2, antibodies against deamidated gliadin peptides and antibodies against neoepitopes arising from

5 the binding of the transglutaminase type 2-deamidated peptide complex of gliadin, and the genetic marker is HLA-DQ in a biological sample isolated from a subject; Y

b) the association of the detection of serological and genetic markers 10 obtained in step a) with a determined genetic risk of suffering from the disease.

The term "in vitro" as used in the present invention, refers to the method of the invention being performed outside the subject's body.

The term "diagnosis", as used in the first method of the invention, comprises determining whether a subject currently has the disease. As the person skilled in the art will understand, such evaluation may not be correct for 100% of the subjects to be diagnosed, although

20 is preferably correct. However, the term requires that a statistically significant part of the subjects can be identified as suffering from the disease. If a part is statistically significant, it can be determined easily by the person skilled in the art using several well-known statistical evaluation tools, for example, the determination of confidence intervals,

25 the determination of p values, the Student t test, the Mann-Whitney test, etc. Details are provided in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80 %, at least 90%, at least 95%.

In a preferred embodiment of the second method of the invention, this is characterized in that the genetic marker HLA-DQ is selected from: HLA-DQ2 or HLA-DQ8.

In another preferred embodiment of the second method of the invention, this is characterized in that the genetic marker HLA-DQ2 is encoded by the joint presence of the alleles: DQA1 * 05 and DQB1 * 02.

In another preferred embodiment of the second method of the invention it is characterized in that the DQA1 * 05 allele is selected from DQA1 * 05: 01 and DQA1 * 05: 05 and the DQB1 * 02 allele is selected from DQB1 * 02: 01 and DQB1 * 02: 02.

In another preferred embodiment of the second method of the invention it is characterized why the genetic marker HLA-DQ2 is encoded by at least one of the alleles DQA1 * 05: 01 or DQA1 * 05: 05 together with at least one of the DQB1 * 02: 01 alleles or DQB1 * 02: 02.

In another preferred embodiment of the second method of the invention it is characterized in that the genetic marker HLA-DQ8 is encoded by the joint presence of the DQA1 * 03 and DQB1 * 03: 02 alleles. In another preferred embodiment, the DQA1 * 03 allele is selected from: DQA1 * 03: 01 and DQA1 * 03: 02.

In another preferred embodiment of the second method of the invention it is characterized in that the genetic marker HLA-DQ8 is encoded by at least one of the DQA1 * 03: 01 or DQA1 * 03: 02 alleles together with the presence of the DQB1 * allele 03:02

Thus, in a first stage of the second method of the invention [step a)], it is determined in a biological sample isolated from a subject, the detection and / or joint quantification of: i) serological markers: anti-TG2 antibodies, antibodies anti-PDG, anti-neoepitope antibodies that arise from the binding between TG2-PDG; and ii) the HLA-DQ genetic marker.

In a second stage of the second method of the invention [step b)], the specific genetic risk that a subject presents to suffer from the disease is determined, depending on the results obtained in step a) of the method. Thus, depending on the antibodies and alleles and / or oligonucleotides detected in the biological sample

After analyzing the subject, the genetic risk of whether or not the subject suffers from the disease is determined.

In a preferred embodiment of the second method of the invention, this is characterized in that the genetic marker HLA-DQ is selected from: HLA-DQ2 or HLA-DQ8.

In a preferred embodiment, the second method of the invention is characterized in that the detection of antibodies against neoepitopes that arise from the binding of the transglutaminase type 2-deamidated gliadin peptides complex and the absence of the complete genetic markers HLA-DQ2 or HLA -DQ8 classifies the subject in a group

5 determined genetic risk of suffering from celiac disease.

In another preferred embodiment of the second method of the invention, it is determined that a subject has a high genetic risk of suffering from the disease when it presents antibodies against neoepitopes that arise from the union of the complex.

10 transglutaminase type 2-deamidated peptides of gliadin, one or two DQA1 * 05 alleles and two DQB1 * 02 alleles.

In another preferred embodiment of the second method of the invention, it is determined that a subject has a moderate genetic risk of suffering from the disease when it presents

15 antibodies against neoepitopes that arise from the binding of the transglutaminase type 2-deamidated gliadin peptides complex, a DQA1 * 05 allele and a DQB1 * 02 allele.

In another preferred embodiment of the second method of the invention, it is determined that a

20 subject presents a low genetic risk of suffering from the disease when he / she presents antibodies against neoepitopes that arise from the union of the transglutaminase complex type 2-deamidated peptides of gliadin and any combination of alleles in DQA1 and DQB1 different from the combinations described for the high group and moderate genetic risk.

25 In a still more preferred embodiment, when the subject classified in the low genetic risk group presents the DQA1 * 05 allele in the absence of the DQB1 * 02 allele, it has a higher genetic risk of suffering from celiac disease than if it presents any other possible combination of alleles within the low genetic risk category.

In another preferred embodiment of the second method of the invention, this is characterized in that the detection of antibodies against transglutaminase type 2 and the presence of alleles that determine the presence of the genetic markers HLA-DQ2 or HLA-DQ8 classify the subject into a group of genetic risk determined to suffer from celiac disease. TO

35 these patients can be attributed a genetic risk depending on the HLA alleles present in the DQA1 and DQB1 loci. Said genetic risk coincides with the

known to the scientific community and differs from what the inventors have observed in patients who only have anti-TG2-PDG antibodies.

In a more preferred embodiment of the second method of the invention, it is

5 characterized in that a subject is considered to have a very high genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQA1 * 05 and two DQB1 * 02 alleles are detected.

In a more preferred embodiment of the second method of the invention, it is

10 characterized in that a subject is considered to be at high genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and a DQA1 * 05 allele and a DQB1 * 02 allele are detected.

In a more preferred embodiment of the second method of the invention, it is

15 characterized in that a subject is considered to have a moderate genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQA1 * 03 and one or two DQB1 * 03: 02 alleles are detected.

In a more preferred embodiment of the second method of the invention, it is

20 characterized in that a subject is considered to have a low genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQB1 * 02 alleles and no DQA1 * 05 allele are detected.

In another preferred embodiment of the second method of the invention, this is characterized

25 because the detection of antibodies against deamidated gliadin peptides and the presence of alleles that determine the presence of the genetic markers HLADQ2 or HLA-DQ8 classifies the subject in a group of genetic risk determined to suffer from celiac disease. These patients can be attributed a genetic risk based on the HLA alleles present in the DQA1 and DQB1 loci. Said risk

30 genetic coincides with that known by the scientific community and differs from that which the inventors have observed in patients who only have anti-TG2-PDG antibodies.

Thus, in another more preferred embodiment of the second method of the invention, it is characterized in that a subject is considered to have a very high genetic risk of

35 suffer from the disease when antibodies against deamidated peptides of gliadin and one or two DQA1 * 05 alleles and two DQB1 * 02 alleles are detected.

In a more preferred embodiment of the second method of the invention, it is characterized in that a subject is considered to be at high genetic risk of suffering from the disease when antibodies against deamidated gliadin peptides and a DQA1 * 05 allele and a DQB1 allele are detected. * 02.

5 In a more preferred embodiment of the second method of the invention, it is characterized in that a subject is considered to have a moderate genetic risk of suffer from the disease when antibodies against deamidated peptides are detected of gliadin and one or two DQA1 * 03 alleles and one or two DQB1 * 03: 02 alleles.

In a more preferred embodiment of the second method of the invention, it is characterized in that a subject is considered to have a low genetic risk of suffering from the disease when antibodies against deamidated peptides of gliadin and one or two DQB1 * 02 alleles are detected and no DQA1 * 05 allele.

In another preferred embodiment, in the absence of all the serological markers mentioned above, it is considered very unlikely that a subject may suffer from the disease.

In a preferred embodiment of the second method of the invention, it is characterized in that it further comprises the detection and / or quantification of at least one intestinal atrophy marker, preferably the detection of the REG1A marker (regenerating islet-derived 1 alpha).

In another preferred embodiment of the second method of the invention, this is characterized in that the antibodies detected have isotype A (IgA) or G (IgG).

In another preferred embodiment of the second method of the invention, this is characterized in that the detection of the serological markers is carried out by means of an immunoassay and the detection of the genetic markers is carried out by PCR, preferably, PCR-SSOP or PCR-SSO.

In another preferred embodiment of the second method of the invention, this is characterized in that the detection of serological and genetic markers is determined by Luminex technology, as explained above.

In another preferred embodiment of the second method of the invention, this is characterized in that the isolated biological sample is selected from blood, preferably peripheral blood, or serum.

In another preferred embodiment of the second method of the invention, this is characterized Because the subject is human.

The in vitro method described in the present invention is therefore a routine, simple, fast and considerably less invasive analytical method than the study.

10 of biopsies, for the previous screening and diagnosis of subjects suffering from celiac disease, being able to diagnose specific groups of patients that escape the criteria described in the guidelines for the diagnosis of the disease.

Another aspect described in the present invention relates to a kit comprising antibodies, probes or specific oligonucleotides capable of simultaneously detecting and / or quantifying serological and genetic markers, where the serological markers are selected from: antibodies against transglutaminase type 2, antibodies against deamidated gliadin peptides and antibodies against

20 neoepitopes that arise from the union of the transglutaminase complex type 2-deamidated peptides of gliadin, and the genetic marker HLA-DQ.

In a preferred embodiment, the kit of the invention is characterized in that the genetic marker HLA-DQ is selected from: HLA-DQ2 or HLA-DQ8.

In another preferred embodiment, the kit of the invention is characterized in that the genetic marker HLA-DQ2 is detected by specific probes or oligonucleotides that detect the alleles: DQA1 * 05 and DQB1 * 02.

In another preferred embodiment, the kit of the invention is characterized in that the specific probes or oligonucleotides detect the DQA1 * 05: 01, DQA1 * 05: 05, DQB1 * 02: 01 and DQB1 * 02: 02 alleles.

In another preferred embodiment, the kit of the invention is characterized in that the marker

Genetic HLA-DQ8 is detected by specific probes or oligonucleotides that detect the alleles: DQA1 * 03 and DQB1 * 03: 02.

In another preferred embodiment, the kit of the invention is characterized in that the specific probes or oligonucleotides detect the alleles: DQA1 * 03: 01, DQA1 * 03: 02 and DQB1 * 03: 02.

In another preferred embodiment, the kit of the invention is characterized in that the Detected antibodies have isotype A (IgA) or G (IgG). These methods of Antibody detection involves routine, simple, rapid and analytical methods considerably less invasive than the biopsy study for the diagnosis of Celiac disease The antibodies used in the present invention have been

10 previously described.

In another preferred embodiment, the kit of the invention is characterized in that serological and genetic markers are detected in a biological sample isolated from a subject.

In another preferred embodiment, the kit of the invention is characterized in that the biological sample is selected from blood, preferably peripheral blood, or serum.

In another preferred embodiment, the kit of the invention is characterized in that the subject is human.

The kit of the invention may further comprise, without any limitation, specific primary antibodies of the proteins of the invention, conjugated or unconjugated, peptides, buffers, conjugated secondary antibodies, streptavidin

25 conjugate, proteins or standard peptides, agents to prevent contamination, marker compounds, such as, but not limited to, fluorochromes, etc.

On the other hand, the kit of the invention can include all the supports and containers necessary for its implementation and optimization. The kit of the invention can

30 also contain other proteins or peptides that serve as positive and negative controls. Preferably, these kits further comprise the instructions for carrying out the methods described in the present invention.

Optionally, the antigens and probes of the invention are labeled or

35 immobilized in the kits of the invention. Preferably, these are marked with a tag selected from the list comprising: a radioisotope, a marker

fluorescent or luminescent, an antibody, an antibody fragment, a tag of affinity, an enzyme or a substrate of an enzyme. More preferably, the antigens and probes are immobilized in the kits of the invention. The term "immobilized", as used in the present invention, refers to the 5 antigens or oligonucleotide probes can be attached to a support without losing your activity. Preferably, the support may be the surface of a matrix (by for example, a nylon matrix), a microtiter plate (for example, 96 wells) or similar plastic support, or beads (spheres, for example, polystyrene microspheres, agarose spheres or small microspheres

10 superparamagnetic composed of biodegradable matrices).

Another aspect described in the present invention relates to the use of the kit described previously to carry out the methods of the invention, specifically for the in vitro diagnosis of celiac disease in a subject, said subject being preferably a

15 human being.

Another aspect described in the present invention relates to a method of diagnosing celiac disease in a subject by the joint detection of serological and genetic markers mentioned in the present invention, preferably,

20 by using the in vitro method and kit described in the present invention.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and characteristics of the

The invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

30 FIG. 1. Application of the two algorithms, for symptomatic and asymptomatic patients, developed by ESPGHAN to diagnose celiac patients. A) Asymptomatic patients with genetic risk of celiac disease (PAcRG). After testing IgA against TG2. In these individuals it is determined first

35 place the presence of HLA-DQ2 / DQ8. Two individuals with these characteristics are studied. Both individuals have HLA-DQ2 and / or HLA-DQ8, so

subsequently determines the presence of anti-TG2 antibodies and total IgA. With normal levels of IgA, none of the individuals have anti-TG2 antibodies, so they are diagnosed as "No EC", that is, they do not suffer from celiac disease.

5 B) Children / adolescents with a clinic suggesting celiac disease (CSEC). After testing IgA against TG2. In these individuals the presence of anti-TG2 antibodies and total IgA are determined. 13 individuals with these characteristics are studied. With normal levels of IgA, none of the 13 individuals have anti-TG2 antibodies, so they are diagnosed as "No EC"; that is to say they do not suffer

10 celiac disease

C) Asymptomatic patients with genetic risk of celiac disease (PAcRG). After testing IgA against TG2, PDG or neoepitopes that emerge from the TG2-PDG junction. In these individuals, the presence of HLA15 DQ2 / DQ8 is determined first. Two individuals with these characteristics are studied. Both individuals have HLA-DQ2 and / or HLA-DQ8, so the presence of anti-TG2, PDG or neoepitope antibodies and total IgA are determined. The two individuals have anti-TG2, PDG or neoepitope antibodies with a level lower than 3 times the upper limit of normal, so the presence of antibodies against

20 EMA The two individuals are EMA negative, so they are diagnosed as "No EC"; that is, they do not suffer from celiac disease.

D) Children / adolescents with a clinic suggesting celiac disease (CSEC). After testing IgA against TG2, PDG or neoepitopes that emerge from TG225 PDG. In these individuals the presence of anti-TG2, -PDG or neoepitope antibodies and total IgA are determined. 13 individuals with these characteristics are studied, all of them with anti-TG2, -PDG or -neoepitopes antibodies, so they are referred to the specialist. Nine of these individuals have values of these antibodies greater than 10 times the upper limit of normal, so the presence of antibodies against EMA and HLA-DQ2 / DQ8 is determined. Four of these individuals are EMA negative and HLA-DQ2 / DQ8 negative, so they are diagnosed as "No EC." Five of these individuals are EMA negative but have HLADQ2 and / or HLA-DQ8, so an intestinal biopsy is performed, the analysis of which reflects a Marsh 2 or 3 and therefore the five individuals are diagnosed with "CD"; that is to say

35 suffer from celiac disease.

E) Children / adolescents with a clinic suggesting celiac disease (CSEC). After testing IgA against TG2, PDG or neoepitopes that emerge from the TG2-PDG junction. In these individuals the presence of anti-TG2, PDG or -neoepitope antibodies and total IgA are determined. 13 individuals with these characteristics are studied,

5 all of them with anti-TG2, -PDG or -neoepitopes antibodies so they are referred to the specialist. Four of these individuals have TG2 values lower than 10 times the upper limit of normal, so an intestinal biopsy is performed, the analysis of which reflects a Marsh 2 or 3 and therefore the four individuals are diagnosed with "CD"; that is, they suffer from celiac disease.

10 N indicates the initial number of individuals examined. +: positive; -: negative; LS: upper limit of normal. The numbers in the squares indicate the number of individuals with the final decision indicated in the adjacent chart.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the product of the invention. The following examples serve to illustrate the invention and should not be considered as limiting the scope thereof.

Example 1. Study population.

The results of all serological tests performed for the diagnosis of CD in the Clinical Immunology Service of the 25th San Carlos Clinic Hospital (Madrid, Spain) for patients of the aforementioned hospital since January 2005, when the Aeskulisa kit began to be used, were retrospectively reviewed. ® tTg-A, which detects IgA against TG2, PDG and / or neoepitopes that arise after TG2-PDG binding, until June 2012. All individuals who presented positive antibodies with the aforementioned kit but lacked anti antibodies were selected -endomisio (EMA). The selection of such

30 group of individuals was based on the fact that according to existing diagnostic guidelines, the vast majority of these individuals would be considered not to have the disease, since celiac disease in individuals who are negative EMA is considered a very rare event. The objective was therefore to know if there were any cases of CD within the previously defined study group.

Taking these criteria into account, all individuals positive for anti-TG2-PDG antibodies, but negative for antibodies against EMA at the time of diagnosis were included in the study: 374 patients, 105 of them pediatric. All available data related to the histology of duodenal biopsies, genotyping HLA (human leukocyte antigens) for DRB1, DQA1 and DQB1 (including the presence of DQA1 * 05: 01, DQA1 * 05: 05 alleles, were recorded. DQB1 * 02: 01, DQB1 * 02: 02, DQA1 * 03: 01, DQA1 * 03: 02 and DQB1 * 03: 02) and the reason for consultation for the evaluation of CD (symptoms and clinical signs, conditions associated with EC), as well as the results of specific serological tests for

10 EC performed additionally.

In order to establish comparisons, a number of positive EMA patients with similar age of debut and sex were selected.

15 CD was diagnosed according to the ESPGHAN criteria (Husby S, et al. J Pediatr Gastroenterol Nutr 2012; 54: 136-60; Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition. Arch Dis Child 1990; 65: 909-11). For the diagnosis of adult subjects, other published clinical guidelines were also considered (AGA. Gastroenterology 2006; 131: 1977-80; Bai JC, et al. J Clin

20 Gastroenterol 2013; 47: 121-6). All patients diagnosed with CD also had such confirmation of the disease by duodenal biopsy with the exception of 18 adult subjects who refused endoscopy or their results were inconclusive and analytical and clinical remission after gluten-free diet was used to confirm the presence of EC.

Example 2. Serological tests, genotyping HLA and histological analysis.

Antibodies anti-TG2-PDG and IgG anti-PDG in serum were analyzed using commercial kits: Aeskulisa® tTg-A (Grifols, Wendelsheim, Germany), which uses 30 as recombinant human TG2 antigen and gliadin-specific peptides and which allows to recognize neoepitopes (cut-off point recommended by the manufacturer> 20 U / ml) and the Euroimmun kit (Euroimmun, Lübeck, Germany), which uses deamidated forms of gliadin peptides as an antigen (cut-off point recommended by the manufacturer> 25 U / ml), respectively. IgA anti-EMA antibodies were determined

35 by the immunofluorescence technique with a 1: 5 dilution in commercial tissue sections of monkey esophagus as an antigenic substrate.

For the HLA genotyping of the patients, DNA was extracted from fresh peripheral blood leukocytes by the salting out procedure (Miller, S.A. et al. Nucleic Acids Research. 1988; 16 (3): 1215). HLA genotyping was performed by PCRSSOP (Polymerase Chain Reaction-specific oligonucleotide probes

5 sequence) for DRB1, DQA1 and DQB1, or using PCR-SSO (Sequence-specific polymerase chain reaction-oligonucleotides) by Luminex technology (Tepnel Lifecodes Corp, Stamford, CT USA) for DRB1 and DQB1 (DQA1 it was extrapolated through the combination of DRB1 and DQB1). Some individuals were genotyped by both methods obtaining similar results.

10 Biopsies were performed by medical specialists and processed and examined by pathologists. Histopathological changes were classified according to the modified criteria of Marsh-Oberhuber (Oberhuber G, et al. Eur J Gastroenterol Hepatol 1999; 11: 1185-94).

15 Baseline characteristics between positive anti-TG2-PDG and negative EMA individuals, diagnosed or not with CD, were compared using the Mann-Whitney U test or the chi-square test, depending on whether they were continuous or categorical variables.

Example 3. Results.

Initially, two groups of IgA anti-TG2-PDG positive and EMA negative individuals were considered: 105 children or adolescents and 269 adults. After a review of

25 data collected, three adults were excluded because they died before a diagnosis was reached. Therefore, our sample was 371 individuals, 266 adults and 105 children or adolescents. Table 1 summarizes the main demographic characteristics and clinical and diagnostic characteristics related to CD of the individuals included in the study.

30 In said Table 1, individuals of any age are observed, showing a wide range of levels of anti-TG2-PDG and having or not presenting anti-PDG antibodies. Its clinical presentation is the one generally observed in children or adults who are being studied for possible CD.

Table 1. Description of the anti-TG2-PDG positive negative EMA classified individuals according to the time of study.

Pediatric

Adults

Number of individuals

105 266

Women (%)

37 (35.2) 158 (59.4)

Age serological tests (years) Mean ± ES Range

5.6 ± 0.4 1 -16 50.0 ± 1.1 17 -86

IgA Levels TG2-PDG Media ± ES Range

87.0 ± 9.3 20.1 -300.0 80.8 ± 4.8 20.0 -300.0

IgG PDGa

Positive b

21.7% 6.7%

Mean ± ES

58.0 ± 13.8 72.9 ± 14.4

Rank

25.0 -200.0 25.2 -188.0

Clinical presentation

Classic

Diarrhea

26.8 35.3

Abdominal pain

23.2 41.5

Short stature / stunted growth

28.0 -

Weightloss

18.3 8.0

Not classic

Iron deficiency anemia

8.5 25.0

Dermatitis herpetiformis

4.9 2.2

Neurological symptoms

1.2 3.6

Hypertransaminasemia

2.4 19.2

Deficit of folic acid, iron or Vit. B12

1.2 6.3

Osteoporosis

0 2.2

Others

7.3 7.6

Asymptomatic

First degree relatives

2.4 0

Down's Syndrome

7.3 0

Autoimmune thyroid disease

1.2 0

Diabetes type 1

0 0

Clinical data were available for 82 children / adolescents and 224 adults aIgG PDG was measured in 60 children / adolescents and 178 adults, since these antibodies began to be evaluated in August 2008, when they replaced anti-gliadin IgA, b37.5 % (6/16) of children under 2 years showed IgG PDG.

Table 2 shows the main characteristics of diagnosed and undiagnosed individuals of CD classified in pediatric or adults according to the time of EC screening. The percentage of cases of CD in the sample is 14% in children / adolescents and 15% in adults. In 8 children, the final diagnosis has not yet been established and monitoring is necessary, which means that the percentage of celiacs in children / adolescents could reach 22%. No gender differences are observed in children or adults diagnosed or not with CD. A similar average and age range is observed at the time of antibody detection for EC and non-EC individuals, both in children and adults.

When analyzing IgA levels against TG2-PDG, children with CD have a significantly higher mean than children not diagnosed with CD (188.6 in CD vs. 70.1 in non-EC, p1 tail = 0.002). No significant differences were observed in adults (78.3 in EC vs. 82.1 in non-EC, p1 tail = 0.39). There is a higher percentage of PDG positive individuals in the group with CD vs. the undiagnosed group of CD, although the difference is not significant. The mean levels of PDG IgG are significantly higher in children with CD than in those not diagnosed with CD (188.6 vs. 70.1 respectively, p1 tail = 0.002) and the same occurs in the adult group (138.0 vs 51.2, respectively, p1 tail = 0.018) Because PDG antibodies appear to be especially useful in the diagnosis of children under 2 years of age, this group was considered individually, but only comprised 3 children where only one was seropositive for PDG and showed elevated levels of the antibody (200).

A classic clinical presentation was also observed in all children diagnosed with CD except in three: two showed iron deficiency anemia and one was asymptomatic but was studied because it had Down syndrome. In adults, the most common clinical manifestations were iron deficiency anemia and abdominal pain, but other classic and non-classic symptoms were also found (Table 2).

For cases of CD, Table 3 shows the detailed HLA constitution of

according to the different categories of genetic risk. In order to establish

comparisons, we also show HLA data available from our patients with

EC (with serological tests performed before using Aeskulisa® tTg-A (n = 485)),

5 This group is identified in Table 3 as EC. Due to our low number of

patients with EMA-negative EC (15 pediatric patients and 21 adult patients),

we randomly choose a similar number of EMA-positive ECs of the same sex and age, to

determine if they are a representative group of the total EC. Finally, the

HLA of a group of individuals without diseases mediated by the immune system, 10 and therefore used as a control.

As shown in Table 3, 53% of pediatric celiac patients and 52%

of the adults presented the HLA-DQ2 molecule. These values remain

higher than those observed in the controls, but differ from 94-100% present in 15 groups of celiacs used to establish comparisons. When we look at

non-DQ2 individuals, the majority group of patients with IgA versus TG2-PDG

(which includes detection of neoepitopes) but negative EMA presented DQA1 * 05,

which appears in 41% of these non-DQ2 patients, but only in 0-4% of

non-DQ2 patients in the other groups of CD; and DQ8 was present in 61% of the 485 non-DQ2 celiacs, but only in 14% or 40% of our non-DQ2 sample,

depending on whether they are considered children / adolescents or adults.

Surprisingly, 8% of the children and adults studied do not have any allele that encodes DQ2 or DQ8 (Table 4). The absence of these alleles was observed only in one patient of the 485 patients available for comparison and the serological results showed that this patient was only seropositive for antibodies against gliadin.

5 The two groups of seropositive EMA patients show an HLA constitution similar to that observed in the 485 celiacs.

Two of the three patients lacking DQB1 * 02 and DQA1 * 05 show DQB1 * 05. However, this HLA allele is also very common in our controls. DQB1 * 06 10 is present in all non-DQ2 / DQ8 coeliacs, but it is also very common in controls.

Table 4. HLA constitution of non-DQ2 / DQ8 patients with celiac disease.

Debut

DRB1_1 DRB1_2 DQA1_1 DQA1_2 DQB1_1 DQB1_2

Pediatric

6 2 01:03 01:02 06:03 06:02

Pediatric

one 6 01:01 01:03 05:01 06:03

Adult

2 2 01:02 01 * 06:02 05:01

Finally, we wanted to determine the most probable diagnosis of children and adolescents with CD in our sample following the recent diagnostic algorithms proposed by ESPHGAN, after using most of the kits

20 for TG2 or the kit that recognizes TG2, PDG and neoepitopes that emerge after the TG2-PDG Aeskulisa® tTg-A junction, which is used in the present invention. This was carried out in the pediatric group, since the clinical guidelines mentioned are focused on children and adolescents. Figure 1 shows the application of two recently published algorithms:

25 1) for children and adolescents with unexplained symptoms and signs, which are suggestive of CD (13 of our 15 pediatric celiacs); 2) for children and adolescents without suggestive symptoms of CD belonging to a group of high genetic risk (2 of our 15 pediatric celiacs).

30 By using most commercial kits for TG2 (Figures 1 and 1B), which is the most widespread in routine clinical practice, the entire patient sample is

I would have considered TG2 negative. It should be noted that, given the high correlation between the presence of anti-TG2 and anti-EMA antibodies, it was assumed that all patients who were negative EMA would also be negative for anti-TG2. With the use of these kits, most likely

5 would have considered a cause other than CD in the 15 patients, both in the two asymptomatic patients belonging to risk groups (Figure 1A) and in the 13 patients with clinical symptoms suggestive of CD (Figure 1B).

When using a kit that detects neoepitopes that arise after TG2-PDG binding

10 (Figures 1C, 1D and 1E), the two asymptomatic celiacs would not have been diagnosed with CD, although more serological tests would have been recommended (Figure 1C); 9 of the 13 individuals with symptoms would have been diagnosed with CD and 4 would not have received a diagnosis of CD due to the seronegativity of the EMA and its HLA genetics (Figures 1D and 1E). If we add to this the genetic characteristics that

15 these patients present with antibodies against TG2-PDG neoepitopes but not against TG2, which the inventor team has observed, could be considered a diagnosis of CD in all patients.

A rough estimate of the weight that the group of patients with CD characterized

20 by the inventors (anti-TG2-PDG positive EMA-negative) represents in the total of patients with CD (for which all patients with positive EMA determined in the same period of time included in the group have been considered as patients with CD study), indicates that this group represents 13% of the total CD diagnosed. It should be noted that since most of these patients

25 are "out" of diagnostic criteria, we consider this value to be an underestimate of the real percentage.

Claims (75)

one.

Method of obtaining useful data for the diagnosis of celiac disease in an isolated biological sample of a subject that includes the simultaneous detection and / or quantification of serological and genetic markers, where the serological markers are: antibodies against transglutaminase type 2, antibodies against deamidated gliadin peptides and antibodies against neoepitopes that arise from the binding and / or cross-linking of the transglutaminase type 2 complex-deamidated gliadin peptides, and where the genetic marker is HLA-DQ.

2.

Method of obtaining useful data according to claim 1 characterized in that the genetic marker HLA-DQ is selected from: HLA-DQ2 or HLA-DQ8.

3.

Method of obtaining useful data according to claim 2 characterized in that the genetic marker HLA-DQ2 is encoded by the joint presence of the alleles: DQA1 * 05 and DQB1 * 02.

Four.

Method of obtaining useful data according to claim 3 characterized in that the DQA1 * 05 allele is selected from DQA1 * 05: 01 and DQA1 * 05: 05 and the DQB1 * 02 allele is selected from DQB1 * 02: 01 and DQB1 * 02 : 02.

5.

Method of obtaining useful data according to claims 2 to 4 characterized in that the genetic marker HLA-DQ2 is encoded by at least one of the DQA1 * 05: 01 or DQA1 * 05: 05 alleles together with at least one of the DQB1 alleles * 02: 01 or DQB1 * 02: 02.

6.

Method of obtaining useful data according to claim 2 characterized in that the genetic marker HLA-DQ8 is encoded by the joint presence of the DQA1 * 03 and DQB1 * 03: 02 alleles.

7.

Method of obtaining useful data according to claim 6, characterized in that the DQA1 * 03 allele is selected from: DQA1 * 03: 01 and DQA1 * 03: 02.

8.

Method of obtaining useful data according to claims 6 to 7 characterized in that the genetic marker HLA-DQ8 is encoded by at least one of the DQA1 * 03: 01 or DQA1 * 03: 02 alleles together with the presence of the DQB1 * 03 allele : 02.

9.

Method of obtaining useful data according to any of claims 2 to 8, characterized in that the detection of antibodies against neoepitopes arising from the binding of the transglutaminase type 2-deamidated gliadin peptides complex and the absence of alleles encoding the markers Genetic HLA-DQ2 or HLA-DQ8 classifies the subject in a group of genetic risk determined to suffer from celiac disease.

10.

Method of obtaining useful data according to claim 9 characterized in that the subject is classified as of high genetic risk when antibodies against neoepitopes that arise from the binding of the transglutaminase complex type 2-deamidated peptides of gliadin and one or two DQA1 alleles are detected * 05 and two DQB1 alleles * 02.

eleven.

Method of obtaining useful data according to claim 9 characterized in that the subject is classified as of moderate genetic risk when antibodies against neoepitopes that arise from the binding of the transglutaminase complex type 2-deamidated gliadin peptides and a DQA1 * 05 allele are detected. and an allele DQB1 * 02.

12.

Method of obtaining useful data according to claim 9, characterized in that the subject is classified as having a low genetic risk when antibodies against neoepitopes that arise from the binding of the transglutaminase type 2-deamidated gliadin peptides complex and any combination of alleles are detected. DQA1 and DQB1, different from the combinations described in claims 9 and 10.

13.

Method of obtaining useful data according to claim 12 characterized in that the subject classified in the low genetic risk group has a higher genetic risk of suffering from celiac disease if it presents the DQA1 * 05 allele in the absence of the DQB1 * 02 allele than if it presents any other Possible combination of alleles within the category of low genetic risk.

14.

Method of obtaining useful data according to any of claims 2 to 8 wherein the detection of antibodies against transglutaminase type 2 and the presence of at least one of the genetic markers HLA-DQ2 or HLA-DQ8 classifies the subject in a risk group Genetic determined to suffer from celiac disease.

fifteen.

Method of obtaining useful data according to claim 14, characterized in that the subject is classified as having a very high genetic risk of suffering from the disease when antibodies to transglutaminase type 2 and one are detected.

or two DQA1 * 05 alleles and two DQB1 * 02 alleles.

16.

Method of obtaining useful data according to claim 14, characterized in that the subject is classified as having a high genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and a DQA1 * 05 allele and a DQB1 * 02 allele are detected.

17.

Method of obtaining useful data according to claim 14 characterized in that the subject is classified as having a moderate genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one are detected

or two DQA1 * 03 alleles and one or two DQB1 * 03: 02 alleles.

18.

Method of obtaining useful data according to claim 14 characterized in that the subject is classified as having a low genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQB1 * 02 alleles and no DQA1 * 05 allele are detected.

19.

Method of obtaining useful data according to any of claims 2 to 8 wherein the detection of antibodies against deamidated gliadin peptides and the presence of at least one of the genetic markers HLA-DQ2 or HLA-DQ8 classifies the subject into a group of genetic risk determined to suffer from celiac disease.

twenty.

Method of obtaining useful data according to claim 19, characterized in that the subject is classified as having a very high genetic risk of suffering from the disease when antibodies are detected against deamidated peptides of gliadin and one or two DQA1 * 05 and two DQB1 * alleles 02.

twenty-one.

Method of obtaining useful data according to claim 19, characterized in that the subject is classified as having a high genetic risk of suffering from the disease when antibodies against deamidated gliadin peptides and a DQA1 * 05 allele and a DQB1 * 02 allele are detected.

22

Method of obtaining useful data according to claim 19 characterized in that the subject is classified as having a moderate genetic risk of suffering from the disease when antibodies are detected against deamidated peptides of gliadin and one or two DQA1 * 03 and one or two DQB1 alleles * 03: 02.

2. 3.

Method of obtaining useful data according to claim 19, characterized in that the subject is classified as having a low genetic risk of suffering from the disease when antibodies against deamidated peptides of gliadin and one or two DQB1 * 02 alleles and no DQA1 * 05 allele are detected. .

24.

Method of obtaining useful data according to any of claims 1 to 23, characterized in that it further comprises the detection and / or quantification of at least one marker of intestinal atrophy.

25.

Method of obtaining useful data according to claim 24 characterized in that the intestinal atrophy marker is the REG1A protein.

26.

Method of obtaining useful data according to any of claims 1 to 25 characterized in that the antibodies detected have isotype A (IgA) or G (IgG).

27.

Method of obtaining useful data according to any of claims 1 to 26, characterized in that the detection of the serological markers is carried out by means of an immunoassay and the detection of the genetic markers by the PCR technique.

28.

Method of obtaining useful data according to claim 27 wherein the detection of the serological and genetic markers is determined by Luminex technology.

29.

Method of obtaining useful data according to any of claims 1 to 28 characterized in that the isolated biological sample is selected from saliva, blood or serum.

30

Method of obtaining useful data according to claim 29 characterized in that the blood is peripheral blood.

31.

Method of obtaining useful data according to any of claims 1 to 30 characterized in that the subject is human.

32

In vitro method for the diagnosis of celiac disease in a subject comprising:

a) the simultaneous detection and / or quantification of serological and genetic markers where the serological markers are: antibodies against transglutaminase type 2, antibodies against deamidated peptides of gliadin and antibodies against neoepitopes that arise from the union of the transglutaminase complex type 2- deamidated gliadin peptides, and the genetic marker is HLA-DQ, in a biological sample isolated from a subject; Y b) the association of the detection of serological and genetic markers obtained in step a) with a determined genetic risk of suffering from the disease. 33. In vitro method according to claim 32 characterized in that the genetic marker HLA-DQ is selected from: HLA-DQ2 or HLA-DQ8. 34. In vitro method according to claim 33 characterized in that the genetic marker HLA-DQ2 is encoded by the joint presence of the alleles: DQA1 * 05 and DQB1 * 02.

35

In vitro method according to claim 34 characterized in that the DQA1 * 05 allele is selected from DQA1 * 05: 01 and DQA1 * 05: 05 and the DQB1 * 02 allele is selected from DQB1 * 02: 01 and DQB1 * 02: 02.

36.

In vitro method according to any of claims 33 to 35 characterized in that the genetic marker HLA-DQ2 is encoded by at least one of the DQA1 * 05: 01 or DQA1 * 05: 05 alleles together with at least one of the DQB1 * alleles 02:01 or DQB1 * 02: 02.

37.

In vitro method according to claim 33 characterized in that the genetic marker HLA-DQ8 is encoded by the joint presence of DQA1 * 03 and DQB1 * 03: 02 alleles.

38.

In vitro method according to claim 37 characterized in that the DQA1 * 03 allele is selected from: DQA1 * 03: 01 and DQA1 * 03: 02.

39.

In vitro method according to any of claims 37 to 38 characterized in that the genetic marker HLA-DQ8 is encoded by at least one of the DQA1 * 03: 01 or DQA1 * 03: 02 alleles together with the presence of the DQB1 * 03 allele: 02.

40

In vitro method according to any of claims 33 to 39, characterized in that the detection of antibodies against neoepitopes arising from the binding of the transglutaminase type 2-deamidated gliadin peptides complex and the absence of the HLA-DQ2 or HLA-DQ8 genetic markers classifies the subject in a group of genetic risk determined to suffer from celiac disease.

41.

In vitro method according to claim 40 characterized in that the subject is classified as of high genetic risk when antibodies against neoepitopes that arise from the binding of the transglutaminase complex type 2-deamidated peptides of gliadin and one or two DQA1 * 05 alleles are detected. two alleles DQB1 * 02.

42

In vitro method according to claim 40 characterized in that the subject is classified as of moderate genetic risk when antibodies against neoepitopes that arise from the binding of the transglutaminase complex type 2 deamidated gliadin peptides and a DQA1 * 05 allele and a DQB1 * allele are detected. 02.

43

In vitro method according to claim 40, characterized in that the subject is classified as of low genetic risk when antibodies against neoepitopes that arise from the binding of the transglutaminase type 2-deamidated gliadin peptides complex and any combination of alleles in DQA1 and DQB1 are detected. different from the combinations described in claims 29 and 30.

44.

In vitro method according to claim 43, characterized in that the subject classified in the low genetic risk group has a higher genetic risk of suffering from celiac disease if it has the DQA1 * 05 allele in the absence of the DQB1 * 02 allele than if it has any other possible combination of alleles within the low genetic risk category.

Four. Five.

In vitro method according to any of claims 33 to 39, wherein the detection of antibodies against transglutaminase type 2 and the presence of at least one of

The genetic markers HLA-DQ2 or HLA-DQ8 classifies the subject in a group of genetic risk determined to suffer from celiac disease.

46.

In vitro method according to claim 45 characterized in that the subject is classified as a very high genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQA1 * 05 alleles and two DQB1 * 02 alleles are detected.

47

In vitro method according to claim 45 characterized in that the subject is classified as having a high genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and a DQA1 * 05 allele and a DQB1 * 02 allele are detected.

48.

In vitro method according to claim 45 characterized in that the subject is classified as having a moderate genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQA1 * 03 and one or two DQB1 * 03: 02 alleles are detected .

49.

In vitro method according to claim 45 characterized in that the subject is classified as having a low genetic risk of suffering from the disease when antibodies against transglutaminase type 2 and one or two DQB1 * 02 alleles and no DQA1 * 05 allele are detected.

fifty.

In vitro method according to any one of claims 33 to 39, wherein the detection of antibodies against deamidated gliadin peptides and the presence of at least one of the genetic markers HLA-DQ2 or HLA-DQ8 classifies the subject into a specific genetic risk group. of suffering celiac disease.

51.

In vitro method according to claim 50, characterized in that the subject is classified as having a very high genetic risk of suffering from the disease when antibodies are detected against deamidated peptides of gliadin and one or two DQA1 * 05 and two DQB1 * 02 alleles.

52

In vitro method according to claim 50 characterized in that the subject is classified as having a high genetic risk of suffering from the disease when

detect antibodies against deamidated peptides of gliadin and a DQA1 * 05 allele and a DQB1 * 02 allele.

53.

In vitro method according to claim 50 characterized in that the subject is classified as having a moderate genetic risk of suffering from the disease when antibodies are detected against deamidated peptides of gliadin and one or two DQA1 * 03 and one or two DQB1 * 03 alleles: 02.

54

In vitro method according to claim 50, characterized in that the subject is classified as having a low genetic risk of suffering from the disease when antibodies against deamidated peptides of gliadin and one or two DQB1 * 02 alleles and no DQA1 * 05 allele are detected.

55.

In vitro method according to any of claims 32 to 54 characterized in that it further comprises the detection and / or quantification of at least one marker of intestinal atrophy.

56.

Method of obtaining useful data according to claim 55 characterized in that the marker of intestinal atrophy is the REG1A protein.

57.

In vitro method according to any of claims 32 to 56 characterized in that the antibodies detected have isotype A (IgA) or G (IgG).

58.

In vitro method according to any of claims 32 to 56 characterized in that the detection of the serological markers is carried out by means of an immunoassay and the detection of the genetic markers is carried out by PCR.

59.

In vitro method according to claim 58 wherein the detection of serological and genetic markers is determined by Luminex technology.

60

In vitro method according to any of claims 32 to 59 characterized in that the isolated biological sample is selected from saliva, blood or serum.

61.

In vitro method according to claim 60 characterized in that the blood is peripheral blood.

62

In vitro method according to any of claims 32 to 61 characterized in that the subject is human.

63.

Kit comprising antibodies and / or specific probes capable of detecting and / or quantifying serological and genetic markers, where the serological markers are: antibodies against transglutaminase type 2, antibodies against deamidated gliadin peptides and antibodies against neoepitopes that arise from the binding of the transglutaminase type 2-deamidated peptide complex of gliadin, and the genetic marker is HLA-DQ.

64.

Kit according to claim 63 characterized in that the genetic marker HLA-DQ is selected from: HLA-DQ2 or HLA-DQ8.

65

Kit according to claim 64 characterized in that the genetic marker HLA-DQ2 is detected by specific probes that detect the alleles: DQA1 * 05 and DQB1 * 02.

66.

Kit according to claim 65 characterized in that the specific probes detect any of the alleles DQA1 * 05: 01, DQA1 * 05: 05, DQB1 * 02: 01 and DQB1 * 02: 02.

67.

Kit according to claim 64 characterized in that the genetic marker HLA-DQ8 is detected by specific probes that detect the alleles: DQA1 * 03 and DQB1 * 03: 02.

68.

Kit according to claim 67 characterized in that the specific probes detect any of the alleles: DQA1 * 03: 01, DQA1 * 03: 02 and DQB1 * 03: 02.

69.

Kit according to any of claims 63 to 68 characterized in that it further comprises antibodies capable of detecting intestinal atrophy markers.

70.

Kit according to claim 69 characterized in that the intestinal atrophy marker is the REG1A protein.

71.

Kit according to any of claims 63 to 70 characterized in that the antibodies detected have isotype A (IgA) or G (IgG).

72.

Kit according to any of claims 63 to 71 characterized in that the serological and genetic markers are detected in an isolated biological sample of a subject.

73. Kit according to claim 72 characterized in that the biological sample is selected from saliva, blood or serum. 74. Kit according to claim 73 characterized in that the blood is peripheral blood. 10 75. Kit according to any of claims 63 to 74 characterized in that the subject is human. 76. Use of the kit according to any of claims 63 to 75 for diagnosis in celiac disease in a subject. fifteen 77. Use of the kit according to claim 76 wherein the subject is human. TO Fig. 1A B Fig. 1B Fig. 1C D Fig. 1D AND Fig. 1E