Patent Application
20170370911
The present invention relates to a method for the diagnosis of a disease comprising contacting a donor tissue section with a liquid capable of extracting an antibody from said donor tissue section and contacting said liquid with an acceptor material comprising an antigen, followed by detection of a complex comprising the antibody and the antigen, and a diagnostically useful carrier comprising a donor tissue section and an acceptor material comprising an antigen.
In most human beings, there exists, by default, a tolerance of all body components, i.e. they do not trigger the production of antibodies targeting their own tissue antigens. By contrast, disorders, referred to as autoimmune diseases, occur in which there is an apparent immunologic reaction of the host to his own tissues, often resulting in tissue injury.
The importance of an early and reliable diagnosis of autoimmune diseases cannot be overemphasized. Many autoimmune disorders cannot be cured, but therapies are available that may be used to significantly ameliorate their symptoms, often enabling patients to lead a normal life. The earlier the diagnosis, the better the chances to exploit the spectrum of therapies, such as administration of immunosuppressive drugs or a strict diet devoid of immunogenic antigens, to the full benefit of the patient.
The diagnosis of an autoimmune disease is frequently based on the output of a method for the detection of autoantibodies in a liquid sample comprising antibodies. Most frequently, blood serum routinely obtained from patients is used. In other cases, other liquids are used such as cerebrospinal fluid (CSF), in particular for the detection of autoantibodies associated with neurological symptoms.
Coeliac disease (CD) is a special example of an autoimmune disease. It is triggered by gluten which activates an immune reaction against the CD autoantigen, i.e. tissue transglutaminase (TTG), in genetically predisposed subjects. Following the finding that the presence of antibodies to gliadin and/or TTG is associated with CD it could be established that CD is not a rare, but a frequent condition with an expected prevalence higher than 1% in the worldwide population. A spectrum of diseases related to gluten sensitivity exists, including Non Coeliac Gluten sensitivity (NCGS) characterized by intestinal symptoms related to the ingestion of gluten-containing food, and in the case of a number of patients it remains difficult to thoroughly distinguish between them.
A pathologist with significant experience is required to examine tissue sections made from a bioptate as a basis for a reliable diagnosis. In many cases, such examination is inconclusive as characteristic features cannot be observed. However, the pathologist may not have at his disposal a serum sample from the patient to complement the histological analysis, or the concentration of antibodies in serum samples comprising peripheral blood may be insufficient.
In such a case, chances are significant that the patient will be misdiagnosed. False negative results may cause the patient to be denied adequate treatment, resulting in an increased risk of secondary disorders, among them cancers such as intestinal lymphoma, and a poor life quality.
Therefore, a problem underlying the present invention is to provide a method for diagnosing an autoimmune disease that may be used to corroborate an inconclusive histological examination of diseased tissue. Another problem is to provide an improved test having an increased sensitivity and reducing the likelihood of false negative results.
Not in the least a problem underlying the present invention is to provide an assay that may be run in the absence of serum samples and allows a pathologist to corroborate a diagnosis based on an inconclusive histological examination.
The problem underlying the present invention is solved by the subject-matter of the attached independent and dependent claims.
In a first aspect, the problem underlying the present invention is solved by a method for the diagnosis of a disease comprising contacting a donor tissue section with a liquid capable of extracting an antibody from said donor tissue section and contacting said liquid with an acceptor material comprising an antigen, optionally two or more antigens, followed by detection of a complex comprising the antibody and the antigen.
In a preferred embodiment, the donor tissue section and the acceptor material comprising an antigen are co-incubated in the liquid.
In a second aspect, the problem is solved by a diagnostically useful carrier comprising a donor tissue section and an acceptor material comprising an antigen, wherein the carrier is configured such that the donor tissue section and the acceptor material comprising an antigen may be co-incubated in a liquid capable of extracting an antibody from said donor tissue section and allowing the transport of the antibody to the acceptor material.
In a preferred embodiment, the donor tissue section and the acceptor material comprising an antigen are coated on the surface of the carrier and are preferably spatially separate.
In a preferred embodiment, the donor tissue section and the acceptor material comprising an antigen are on spatially separate biochips.
In a preferred embodiment, the carrier is configured such that a liquid capable of extracting an antibody from said donor tissue section may be placed on the surface of the carrier such that an antibody may be extracted from the donor tissue section and diffuse, via the liquid, to the acceptor material comprising an antigen.
In a preferred embodiment, the carrier comprises a first part having a surface coated with the donor tissue section and a second part having a surface coated with the acceptor material, wherein the first and the second part are separate and the carrier is configured such that the first and the second part may be contacted, preferably assembled such that the surface of either the first or the second part faces downwards on the surface of the other one of the first and the second part such that a drop of liquid may be placed between the surfaces of the first part and the second part to allow diffusion of any antibodies from the donor tissue section to the acceptor material.
In a preferred embodiment, the donor tissue section has been obtained from a patient to be diagnosed.
In a preferred embodiment, the acceptor material comprising an antigen is a tissue sample comprising the antigen, preferably in the form of a native polypeptide, a cell producing said antigen, or an isolated polypeptide.
In a preferred embodiment, the donor tissue section is a frozen tissue section.
In a preferred embodiment, the disease is a gastroenteropathy, preferably an inflammatory or autoimmune gastrointestinal disease, more preferably coeliac disease, the donor tissue section is a gastrointestinal, preferably duodenal tissue, and the antigen is tissue transglutaminase or deamidated gliadin or a variant thereof, preferably an oligomer of deamidated gliadin or a variant thereof.
In a preferred embodiment, the disease is pemphigus and/or pemphigoid, preferably bullous pemphigoid, the donor tissue section is diseased skin tissue, and the acceptor material comprises one or more antigens from the group comprising Dsg1, Dsg3, NC16A, BP180, BP 230, LAMA3 (SEQ ID NO 14), Laminin332, which is preferably a composition comprising polypeptides comprising LAMA3 (SEQ ID NO14), LAMBS (SEQ ID NO 15), and LAMC2 (SEQ ID NO 16) and, beta4 integrin (SEQ ID NO 17) and collagen type VII and a variant thereof, optionally in the form of a tissue, preferably selected from the group comprising primate esophagus, human salt split skin and rat urinary bladder.
In a preferred embodiment, the disease is Goodpasture syndrome or SLE, the donor tissue section is diseased kidney tissue, and the acceptor material comprising the antigen is or is derived from a material selected from the group comprising antiglomerular basement membrane, dsDNS, human epithelial cells (HEp-2), pLA2R and THSD7A and a variant thereof.
In a preferred embodiment, the disease is Crohn's disease, the donor tissue section is diseased intestinal tissue, and the acceptor material comprising the antigen is selected from the group comprising healthy pancreas tissue, CUZD1 and GP2 and a variant thereof, the latter two preferably in the form of a cell expressing CUZD1 and/or GP2 and a variant thereof.
In a preferred embodiment, the disease is Grave's and/or Hashimoto's disease, the donor tissue section is derived from thyroid gland, and the acceptor material comprising the antigen is or is derived from healthy thyroid gland tissue.
In a preferred embodiment, the disease is myositis, the donor tissue section is muscle or skin, preferably muscle tissue, and the acceptor material comprising the antigen is MUP-44 or a variant thereof.
The present invention is based on the inventors' surprising finding that autoantibodies may be detected in tissue sections. For example, this holds true for antibodies associated with CD.
Without wishing to be bound to any theory, the inventors hypothesize that autoantibodies accumulate in diseased tissues and organs and that their transfer to the patient's blood is incomplete and insufficient for a reliable analysis based on serology.
According to the present invention, a donor tissue section is provided. This is a piece of tissue obtained from a patient suffering from or suspected of suffering from a disease to be diagnosed. In a preferred embodiment, the donor tissue section is frozen, preferably by immersion in liquid nitrogen or melting isopentane. The tissue section may preferably have a thickness of 1 to 100, preferably 4 to 50 μm. The donor tissue section has preferably not been treated with any reagents or exposed to conditions substantially altering the antibodies present in the donor tissue section.
According to the present invention, the donor tissue section is contacted with a liquid capable of extracting an antibody from said donor tissue section. This is any liquid that, upon exposure to the donor tissue section, may take up antibodies present in the donor tissue section, conserves their chemical composition, structure or binding properties such as the ability to bind specifically to an antigen, and allows their passage to the acceptor material comprising an antigen. Preferably, the liquid is an aqueous liquid comprising a physiological buffer, more preferably at pH 5 to 9, more preferably 6 to 8, most preferably 6.2 to 7.8. In a most preferred embodiment, the liquid is PBS pH 7.4.
In a preferred embodiment, the term “binding specifically”, as used herein, means that the binding is stronger than a binding reaction characterized by a dissociation constant of 1×10−5 M, more preferably 1×10−7 M, more preferably 1×10−8 M, more preferably 1×10−9 M, more preferably 1×10−10 M, more preferably 1×10−11 M, more preferably 1×10−12 M, as determined by surface plasmon resonance using Biacore equipment at 25° C. in PBS buffer at pH 7.4.
It is preferred that the liquid capable of extracting the antibody comprises a detergent that helps dissociating any antibodies from tissue whilst conserving their chemical composition, structure or binding properties, in particular the ability to specifically bind to an antigen, and is preferably Tween. In a preferred embodiment, the liquid comprises 0.1 to 10%, preferably 0.5 to 5%, more preferably 1 to 4%, most preferably 1.5 to 2.5% of a detergent.
According to the present invention, the donor tissue section is contacted with a liquid capable of extracting an antibody from said donor tissue section, and said liquid is contacted with an acceptor material comprising an antigen. The acceptor material comprises the antigen in a state and conformation that allows for the specific binding of an antibody. The acceptor material may be selected from the group comprising tissue containing an antigen, preferably in the form of a native polypeptide, more preferably an endogenous, native polypeptide, a cell producing an antigen, preferably a recombinant cell overexpressing an antigen, and an isolated polypeptide. The cell may be intact or a lysed cell.
In a preferred embodiment, the donor tissue section and the acceptor material comprising an antigen are co-incubated in the liquid, i.e. both the donor tissue section and the acceptor material are contacted with the liquid at the same time as the antibody is extracted from the donor tissue section and transported to the acceptor material. This co-incubation step is carried out for at least 10, 20, 30, 60 minutes, 2, 3, 4, 6, 8, 10 or 12 hours.
Alternatively, the donor tissue section and the liquid may be contacted first, followed by separation of the donor tissue section and the liquid and contacting of the liquid and the acceptor material comprising an antigen. The liquid may be stored, frozen and/or concentrated prior to contacting it with the acceptor material comprising an antigen. Both contacting steps may be carried out for at least 10, 20, 30, 60 minutes, 2, 3, 4, 6, 8, 10 or 12 hours.
Subsequently, the acceptor material is separated from the liquid capable of extracting an antibody and/or the donor tissue section, for example by separating the first or second part of the carrier or by removing the liquid, for example by way of aspiration. The acceptor material may be washed at least once using a washing buffer. Fresh liquid capable of extracting the antibody may be used as washing buffer, preferably comprising a smaller concentration of detergent such as 10% of the concentration used in the incubation step or no detergent at all.
Subsequently, any complex comprising the antibody and the antigen may be detected. In a preferred embodiment, the complex is detected using a method selected from the group comprising immunodiffusion techniques, immunoelectrophoretic techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays such as those from the group comprising radiolabeled immunoassays, enzyme immunoassays such as ELISA, chemiluminscence immunoassays, and immunofluorescence techniques. The person skilled in the art is familiar with these methods.
The invention relates to a diagnostically useful carrier, which is preferably a solid, artificial carrier for contacting a donor tissue section and/or, preferably and an acceptor material comprising an antigen, which donor tissue section and/or acceptor material is coated on the surface of said carrier, with a liquid capable of extracting an antibody from the donor tissue section. Preferably, the donor tissue section and the acceptor material are surrounded by a hydrophobic surface to fix the position of the liquid capable of extracting an antibody. In a preferred embodiment, the solid carrier comprises two or more separate parts, one comprising the donor tissue section and one comprising the acceptor material, each separate part preferably selected from the group comprising a bead, a microtiter plate, a glass surface, a biochip and a membrane, most preferably a biochip.
In a preferred embodiment, the term “biochip”, as used herein, refers to a planar, thin slide having a thickness of 0.01 to 1 mm, preferably 0.02 to 0.5 mm, more preferably 0.05 to 0.4 mm. They are preferably made of glass or plastic. They are coated with a biological agent such as a donor tissue section or an acceptor material comprising an antigen.
In a preferred embodiment, the diagnostically useful carrier may be coated with donor tissue section and coated with the acceptor material comprising an antigen. The donor tissue section and the acceptor material, both preferably on a biochip, may be sized and placed in close proximity such that a drop of liquid capable of extracting the antibody, preferably having a volume of 10 to 80, preferably 20 to 60 μl, may be placed in contact with both the donor tissue section and the acceptor material at the same time for the incubation step. Coated donor tissue section and coated acceptor material comprising an antigen on the diagnostically useful carrier are preferably spatially separated, in particular such that one may be added or removed from the diagnostically useful carrier independent of the other.
In a preferred embodiment, the contacting step may be carried out as follows: a drop of liquid is placed on a surface and the diagnostically carrier is contacted with said drop such that the donor tissue section and the acceptor material, both on the same side of the diagnostically useful carrier, face downward when touching the drop sitting on the carrier. Owing to their hydrophilic natures, the surface, the donor tissue section and the acceptor material adhere to and fix the liquid drop for the entirety of the coincubation. The extraction and passage of antibodies through the liquid from the donor tissue section to the acceptor material may be supported by gently rocking the diagnostically useful carrier together with the surface. Devices for carrying out the incubation are described in US2010/0124750.
In another preferred embodiment, the carrier comprises a first part having a surface coated with the donor tissue section and a second part having a surface coated with the acceptor material comprising an antigen, wherein the first and the second part are separate and the carrier is configured such that the first and the second part may be contacted, preferably assembled such that the surface of either the first or the second part faces downwards on the surface of the other one of the first and the second part such that a drop of liquid may be placed between the surfaces of the first part and the second part to allow diffusion of any antibodies from the donor tissue section to the acceptor material.
In another preferred embodiment, the first part of the carrier is a bead coated with the acceptor material comprising an antigen, and this is contacted with the donor tissue section. The latter is preferably coated on the surface of a second part of the carrier, but may also be non-immobilized, for example floating in solution rather than being coated.
The acceptor material may comprise or consist of a polypeptide antigen, for example in a tissue, cell or in purified form represented by exact sequences referred to in this application explicitly, for example by function, name, sequence or accession number, or implicitly, but also including variants of such polypeptides.
In a preferred embodiment, the term “variant”, as used herein, may refer to at least one fragment of the full length sequence referred to, more specifically one or more amino acid which are, relative to the full-length sequence, truncated at one or both termini by one or more amino acids. Such a fragment comprises or encodes for a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or 200 successive amino acids of the original sequence or a variant thereof. The total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids. Variants may include full-length sequences or fragments that are at least 40, 50, 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to the reference amino acid sequence referred to or the corresponding fragment of said reference amino acid sequences.
Any variants thereof may, in addition, comprise chemical modifications, for example isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, methylation, hydroxylation and the like.
The variant of the polypeptide, preferably comprising an antigen, has biological activity. In a preferred embodiment, such biological activity is the ability to bind specifically to the antibodies, preferably autoantibodies of interest found in patients suffering from the respective disease. For example, the biological activity of a variant of an antigen such as deamidated gliadin may be the ability to bind specifically to an antibody from a patient to deamidated gliadin, the presence of which suggests that the patient suffers from a disease, such as a patient suffering from CD in the case of a deamidated gliadin variant.
The polypeptide or a variant thereof may be provided in any form and at any degree of purification, from liquid samples, tissues or cells comprising said polypeptide in an endogenous form, more preferably cells overexpressing the polypeptide, crude or enriched lysates of such cells, to purified and/or isolated polypeptide which is optionally essentially pure. In a preferred embodiment, the polypeptide is a native polypeptide, wherein the term “native polypeptide”, as used herein, refers to a folded polypeptide, more preferably to a folded polypeptide purified from tissues or cells, more preferably from mammalian cells or tissues, optionally from non-recombinant tissues or cells. In another preferred embodiment, the polypeptide is a recombinant protein, wherein the term “recombinant”, as used herein, refers to a polypeptide produced using genetic engineering approaches at any stage of the production process. In a preferred embodiment, a polypeptide is pure if at least 60, 70, 80, 90, 95 or 99 percent of the polypeptide in the respective sample consists of said polypeptide as judged by SDS PAGE followed by Coomassie blue staining and visual inspection. A recombinant polypeptide may comprise two or more copies of an antigen, preferably fused to one another.
If the antigen is provided in the form of tissue, it is preferred that the tissue is mammalian tissue, for example human, rat, primate, donkey, mouse, goat, horse, sheep, pig or cow.
According to the present invention, a method for the diagnosis of a disease is provided. In a preferred embodiment, the term “diagnosis”, as used herein, refers to any kind of procedure aiming to obtain information instrumental in the assessment of whether a patient suffers or is likely or more likely than the average or a comparative subject, the latter preferably having similar symptoms, to suffer from a certain disease or disorder in the past, at the time of the diagnosis or in the future, to find out how the disease is progressing or is likely to progress in the future or to evaluate the responsiveness of a patient with regard to a certain treatment, for example the administration of immunosuppressive drugs. In other words, the term “diagnosis” comprises not only diagnosing, but also prognosticating and/or monitoring the course of a disease or disorder.
In many cases the mere detection, in other words determining whether or not detectable levels of the antibody are present in the sample, is instrumental for the diagnosis as it indicates an increased likelihood that the patient suffers from a disease. In a preferred embodiment, the relative concentration of the antibody in the serum, compared to the level that may be found in the average healthy subject, may be determined. While in many cases it may be sufficient to determine whether or not autoantibodies are present or detectable in the sample, the method carried out to obtain information instrumental for the diagnosis may involve determining whether the concentration is at least 0.1, preferably 0.2, 0.5, 1, 2, 5, 10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000 times higher than the concentration found in the average healthy subject.
The person skilled in the art will appreciate that a clinician does usually not conclude whether or not the patient suffers or is likely to suffer from a disease, condition or disorders solely on the basis of a single diagnostic parameter, but also other aspects, for example the presence of other autoantibodies, markers, blood parameters, clinical assessment of the patient's symptoms or the results of medical imaging or other non-invasive methods such as polysomnography, to arrive at a conclusive diagnosis. (See Baenkler H. W. (2012), General aspects of autoimmune diagnostics, in Renz, H., Autoimmune diagnostics, 2012, de Gruyter, page 3.) The value of a diagnostic agent or method may also reside the possibility to rule out one disease, thus allowing for the indirect diagnosis of another.
The term “diagnosis” may also refer to a method or agent used to distinguish between two or more conditions associated with similar or identical symptoms. The term “diagnosis” may also refer to a method or agent used to choose the most promising treatment regime for a patient. In other words, the method or agent may relate to selecting a treatment regimen for a subject.
In a preferred embodiment, the antibody to be detected is an autoantibody. In another preferred embodiment, the antibody to be detected is an IgG or IgA class antibody, preferably an IgA class antibody.
Any data demonstrating the presence or absence of the complex comprising the antibody and the inventive polypeptide may be correlated with reference data. For example, detection of said complex indicates that the patient who provided the sample analyzed has suffered, is suffering or is likely to suffer in the future from a disease. If a patient has been previously diagnosed and the method for obtaining diagnostically relevant information is run again, the amount of complex detected in both runs may be correlated to find out about the progression of the disease and/or the success of a treatment. For example, if the amount of complex is found to increase, this suggests that the disorder is progressing, likely to manifest in the future and/or that any treatment attempted is unsuccessful.
The inventive teachings provide a kit, preferably for diagnosing a disease, comprising the diagnostically useful carrier according to the present invention. The diagnostically useful carrier may initially comprise the acceptor material comprising an antigen only, and is configured such that the end customer may add the donor tissue section prior to carrying out the inventive method. In addition, said kit may comprise instructions detailing how to use the kit and a means for contacting the inventive polypeptide with a bodily fluid sample from a subject, preferably a human subject. Furthermore, the kit may comprise a positive control, for example a batch of antibody or recombinant antibody known to bind to the inventive polypeptide and a negative control, for example a protein having no detectable affinity to the inventive polypeptide such as bovine serum albumin. Finally, such a kit may comprise a standard solution of the antibody or antigen for preparing a calibration curve.
In a preferred embodiment, the kit comprises a means for detecting an antibody binding to the antigen in the acceptor material, preferably by detecting a complex comprising the inventive polypeptide and an antibody binding to the inventive polypeptide. Such means is preferably an agent that binds to said complex and modifies the complex or carries a label such that makes the complex detectable. For example, said means may be a labeled antibody binding to said polypeptide, at a binding site other than the binding site recognized by the primary antibody or to a constant region of the primary antibody. Alternatively, said means may be a secondary antibody binding to the constant region of the antibody, preferably a secondary antibody specific for human antibodies.
In a preferred embodiment, the disease to be diagnosed is a gastroenteropathy, preferably an inflammatory or autoimmune gastrointestinal disease, more preferably coeliac disease, the donor tissue section is a gastrointestinal, preferably duodenal tissue, and the antigen is selected from the group comprising tissue transglutaminase (Uniprot data base: P21980, referring to the version online at the priority data, as all data base codes cited throughout this document) or a variant thereof or a gliadin variant selected from the group comprising SEQ ID NO 1, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5 and variants thereof, preferably more than one copy thereof fused, for example SEQ ID NO 2. The method or carrier may be used to diagnose NCGS, preferably to distinguish between gastroenteropathies related to gluten sensitivity, more preferably CD and NCGS. The antibody to be detected is preferably an IgA class antibody to TTG or deamidated gliadin, more preferably to deamidated gliadin.
In another preferred embodiment, the disease to be diagnosed is pemphigus and/or pemphigoid, preferably bullous pemphigoid, the donor tissue section is diseased skin tissue, and the antigen is selected from the group comprising Dsg1 (Uni prot data base code Q02413), Dsg3 (Uni prot data base code P32926), NC16A (SEQ ID NO 6), optionally in the form of a fusion comprising two or more copies of such as SEQ ID NO 7, BP180 (Uni prot data base code Q9UMD9), BP 230 (SEQ ID NO 8) and collagen type VII (Uni prot data base code Q02388) or a variant thereof.
In another preferred embodiment, the disease to be diagnosed is Goodpasture syndrome or SLE, the donor tissue section is diseased kidney tissue, and the acceptor material comprising the antigen is selected from the group comprising antiglomerular basement membrane, dsDNS, human epithelial cells (HEp-2), pLA2R (SEQ ID NO 9) and THSD7A (SEQ ID NO 10) or a variant thereof.
In another preferred embodiment, the disease to be diagnosed is Crohn's disease, the donor tissue section is diseased intestinal tissue, and the antigen is selected from the group comprising CUZD1 (SEQ ID NO 11) and GP2 (SEQ ID NO 12) or a variant thereof.
In another preferred embodiment, the disease to be diagnosed is Grave's and/or Hashimoto's disease, the donor tissue section is derived from thyroid gland, and the antigen is or is derived from healthy thyroid gland tissue.
In another preferred embodiment, the disease to be diagnosed is myositis, the donor tissue section is muscle or skin, preferably muscle tissue, and the antigen is or is derived from MUP-44 (SEQ ID NO 13) or a variant thereof.
The present invention is further illustrated by the non-limiting additional figures and the following non-limiting examples from which further features, embodiments, aspects and advantages of the present invention may be taken.
A biochip coated with a cryosection (4 μm) of the bioptate (frozen in liquid nitrogen) as tissue donor section was placed beside a biochip coated with dots of purified recombinant antigen GAF-3X (EUROPLUS) (SEQ ID NO 2) as acceptor material comprising an antigen within one reaction field on a microscopic slide. This was repeated with all bioptates obtained from other patients.
The microscopic slide comprising both biochips were incubated with a drop of PBST buffer (PBS with 2% (v/v) Tween-20, pH 7.4) placed in contact with both the bioptate and the antigen at 4° C. overnight. This way, antibodies eluted from the tissue could diffuse in the buffer and antibodies binding to deamidated gliadin could bind to the adjacent GAF-3X antigen dots and be detected. The next day, the biochips were washed using PBST (PBS with 0.2% (v/v) Tween-20, pH 7.4) for 5 minutes. Subsequently, the biochips were incubated with FITC-conjugated anti-human IgA antibodies (Euroimmun AG) for 30 minutes and again washed for 5 minutes. Finally, the slides were analyzed using a fluorescence microscope (EUROStar).
The analyzed cohort comprised 37 patients with coeliac disease diagnosed on the basis of histological examination of the bioptates by an experienced clinician and 35 healthy control persons. Duodenal biopsies from the Bulbus duodeni (36 CD patients, 34 control individuals) were obtained. Serum samples were available for all individuals. Samples were blinded for incubation and microscopic analyses and decoded for final evaluation of the data.
Serum samples were additionally tested using the commercial Anti-GAF-3X-IIFT (IgA) and Anti-GAF-3X ELISA (IgA) according to manufacturer's instructions (EUROIMMUN AG, Germany, products FV 3011-#A and EV 3011-9601A, respectively).
Using the immunofluorescence test, 28 samples were IgA anti-GAF-3X antibody positive among 37 tested CD patients, yielding a sensitivity of 76%. Of 35 control samples none was positive in the Anti-GAF-3X-IIFT (IgA). Therefore, the IIFT (IgA) reached a specificity of 100%.
Using Bulbus doudeni biopsies in the method according to the present invention, 32 out of 36 CD patients (samples of whom were available) were tested positive for IgA anti-GAF-3X antibodies. Among these 32 patients, five were tested negative in both the ELISA and the IIFT. 34 bulbus biopsies of control individuals were further tested of which only one exhibited anti-GAF-3X antibodies of class IgA.
In summary, the Co-Incubation test with Bulbus duodeni biopsies was 89% sensitive for IgA anti-GAF-3X, thereby reaching a specificity of 97%.
The results show that antibodies binding to deamidated gliadin are present within the small intestinal mucosa and may be detected using the inventive method in tissue section from patients serum samples of whom are negative. As a result, the number of false negative results may be reduced.
Bullous pemphigoid (BP) and pemphigus vulgaris (PV) are associated with circulating autoantibodies against BP180, BP230 and desmoglein (Dsg1, Dsg3).
Antibodies against BP 180 and/or BP230 give a serological indication of bullous pemphigoid. It may also be the rarely found lichen planus pemphigoides or the similarly unfrequent mucous membrane (only BP180) or cicatricial pemphigoid (only BP180), which predominantly occurs in elderly people. In pregnant women, pemphigoid gestationis should be taken into account.
Antibodies against Desmoglein 1 indicate the disease Pemphigus folicaceus, while antibodies against Desmoglein 3 (sometimes additionally anti Desmoglein 1) appear in Pemphigus vulgaris.
Direct immunofluorescence (DIF) on biopsies shows staining of desmosomes in pemphigus diseases and of the epidermal basement membrane in pemphigoid diseases. For this purpose, tissue sections from biopsy material of patients are made, incubated with a fluorescent dye-labeled anti-human monoclonal antibody and then evaluated using a fluorescence microscope.
Serological differentiation then has to be carried out with monospecific tests, e.g. by indirect immunofluorescence (IIF) with recombinant HEK cells (expressing specific antigens), and antigen preparations (EUROPLUS®). This step is important to determine the diagnosis, since several target antigens are suitable for the fluorescence of the basal membrane and the desmosomes respectively. BIOCHIP™-Mosaics, consisting of several small glass chips coated with tissue, cell substrates or preparations of small antigen dots (EUROPLUS®) in one reaction field are consecutively incubated with patient's sera and fluorescently labelled antisera. Afterwards they are microscopically evaluated according to the manufacturer's instructions.
The aim of this study was the monospecific determination of antibodies eluted from tissue by co-incubation of biopsies and cell preparations or antigen dots of recombinant proteins in IIF, contrary to conventional IIFT, which is performed with serum samples. This allows the search test (usually DIF) and confirmation test (usually IIFT) to be performed in one step.
Frozen sections from a biopsy of one PV and two BP patients, respectively, were combined to BIOCHIP™-Mosaics on one slide with HEK293 cells transfected with Desmoglein 1 (EUROIMMUN, substrate FD 1495-50; DSG1), Desmoglein 3 (EUROIMMUN substrate FD 1496-50; DSG3), BP230 (EUROIMMUN substrate FD 1502-56; BP230gC (SEQ ID NO 18)) or anti-BP180-NC16A-4X (EUROIMMUN substrate FD 1502-52; BP180-NC16A-4X) (
The individual mosaics were incubated with 30 μl PBS-Tween20 (2%) for 15 hours at 4° C. Bound antibodies were visualised by a FITC-labeled anti-human IgG antibody (EUROIMMUN, product no. AF 102, FITC-labelled anti-human-IgG (goat)). The incubation is carried out for 30 min. at room temperature (20-25° C.). The slides were washed with PBS-Tween20 (0.2%) for 5 min. after both incubation steps. Then they were covered and evaluated under a fluorescence microscope (Axio Scope A1, Zeiss, Jena, Germany, article no. 490035-9100-000).
Co-incubation of the PV biopsy showed parallel reactivity to desmosomes and Dsg3 (see
| Number | Date | Country | Kind |
|---|---|---|---|
| EP 16001395.9 | Jun 2016 | EP | regional |
