EP2235540A2 - New method for diagnosing sjogren's syndrome - Google Patents
New method for diagnosing sjogren's syndromeInfo
- Publication number
- EP2235540A2 EP2235540A2 EP08845893A EP08845893A EP2235540A2 EP 2235540 A2 EP2235540 A2 EP 2235540A2 EP 08845893 A EP08845893 A EP 08845893A EP 08845893 A EP08845893 A EP 08845893A EP 2235540 A2 EP2235540 A2 EP 2235540A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- hnrnp
- antibodies
- syndrome
- biological sample
- sjogren
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6875—Nucleoproteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
Definitions
- the present invention relates to a method of diagnosing Sjogren's syndrome. More particularly the present invention relates to methods and compositions for detecting anti-heterogeneous nuclear ribonucleoprotein-H1 (hnRNP-H1) autoantibodies for diagnosing Sjogren's syndrome.
- hnRNP-H1 anti-heterogeneous nuclear ribonucleoprotein-H1
- Sjogren's syndrome is an autoimmune exocrinopathy, characterized by dryness of the mouth and eyes resulting from a chronic loss of secretory function of the salivary and lacrimal glands.
- Extraglandular systemic manifestations are common and include abnormalities of skin, arthralgia, myalgia, thyroiditis, and pulmonary, renal, gastrointestinal, hematological, cardiac, and neurological abnormalities.
- the etiology of the disease remains unclear. The prevalence of the disease is estimated to be 0.5% and there is a female preponderance.
- Pathological findings involve focal lymphocytic infiltration of affected tissues.
- Anti-SSA antibodies are found in 45-90% of the patients with Sjogren's syndrome.
- Anti-SSB antibodies are found slightly less commonly.
- Anti-SSA antibodies are not specific for Sjogren syndrome as they are found in other systemic diseases such as systemic lupus erythematosus, subacute cutaneous lupus erythematosus and neonatal lupus. Sjogren's syndrome can occur in two forms: primary Sjogren's syndrome (not associated with other autoimmune diseases) and secondary Sjogren's syndrome (associated with other autoimmune diseases).
- the present invention shows hnRNP-H1 as a new target of autoantibodies in patients with Sjogren's syndrome. More in particular, the present invention identifies anti-hnRNP-H1 antibodies as a valuable diagnostic marker in Sjogren's syndrome.
- the present invention provides for a method for diagnosing
- the present invention provides methods and compositions for detecting anti-hnRNP-H1 autoantibodies for diagnosing Sjogren's syndrome.
- the method of this invention is applicable to a large group of patients, including patients having Sjogren's syndrome, patients being suspected as having Sjogren's syndrome or at risk of developing Sjogren's syndrome, or patients suffering from other autoimmune diseases.
- One aspect of the present invention relates to a method of diagnosing an autoimmune disease, more particularly Sjogren's syndrome, characterized in that the presence of autoantibodies against hnRNP- H1 is determined in an isolated biological sample derived from a subject.
- the present invention further relates to a method for detecting hnRNP-H1 antibodies in an isolated biological sample, the method comprising the steps of (a) obtaining an isolated biological sample from a patient; and (b) analyzing said isolated biological sample for the presence of antibodies against hnRNP-M.
- said method is characterized in that hnRNP-H1 proteins, peptides, variants or fragments thereof are used for determining the presence of the hnRNP-H1- antibodies.
- said method is characterized in that a hnRNP- H1-antigen, more specific an immobilized hnRNP-H1-antigen is used for determining the presence of the hnRNP-M -antibodies.
- said analysis or determination of the presence of autoantibodies against hnRNP-H1 is performed in an immunoassay.
- said immunoassay is selected from the group consisting of ELISA, FEIA, western blot, dot blot, bead-based assay, antigen array and Radio lmmuno Assay.
- the methods of the invention are used for predicting responsiveness to a medicament.
- Another aspect of the present invention relates to the use of hnRNP-H1 proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from a subject.
- the present invention further relates to the use of hnRNP-H1 proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from a patient for diagnosing an autoimmune disease, more particularly Sjogren's syndrome.
- said hnRNP-H1 proteins, peptides, variants or fragments thereof are used for carrying out the methods of the invention.
- the isolated biological sample is a fluid selected from the group consisting of blood, serum, plasma, saliva, tears, mucus and ascites fluid; and preferentially said biological sample is serum.
- said isolated biological sample is derived from blood, plasma, serum, saliva, tears, lymph, urine, cerebrospinal fluid, any biopsy material or tissue sample, including bone marrow, lymph nodes, nervous tissue, skin, hair, fetal material including amniocentesis material, uterine tissue, faeces or semen.
- said isolated biological sample is an antibody-containing isolated biological sample.
- test kit for detecting the presence of antibodies against hnRNP-H1 in an isolated biological sample.
- the test kit additionally comprises a solid phase onto which said antigen is or can be bound.
- the test kit additionally comprises a labeling group which is bound to said antigen or can be bound thereto.
- the test kit additionally comprises at least one other antibody class-specific test reagent.
- the test kit may additionally comprise other conventional reagents such as buffers, substrates and wetting solutions.
- the present ivention further relates to the use of said test kit for carrying out any of the methods of the invention.
- Another aspect of the present invention relates to a method of treating an autoimmune disease, more particularly Sjogren's syndrome, comprising administering to a patient an inhibitor that specifically binds to the hnRNP-H1-autoantibodies of said patient, in an amount effective to treat the autoimmune disease, more particularly Sjogren's syndrome.
- said inhibitor is selected from the group consisting of hnRNP-H1 proteins, peptides, variants, fragments, or epitopes thereof and an antibody.
- the present invention further relates to the use of a compound which specifically binds to hnRNP-H1 autoantibodies for the production of a medicine.
- said medicine is used for the treatment of an auto-immunedisease, more particularly Sjogren's syndrome.
- the subject or the patient is a human being.
- Said human being can be a patient having Sjogren's syndrome, a patient being suspected as having Sjogren's syndrome or at risk of developing Sjogren's syndrome, or a patient suffering from other autoimmune diseases.
- FIG. 1 Sequence alignments of hnRNP-H1 from mouse, rat and humans.
- the three sequences Q8VHV7_rat (SEQ ID NO:1), HNRH1_HUMAN (SEQ ID NO:2) and HNRH1_MOUSE (SEQ ID NO:3) show 99 % homology with exclusion of the missing 77 amino acids from hnRNP-H1Q8VHV7 from rat (SEQ ID NO:1), whereas sequence Q499R8_RAT (SEQ ID NO:4) has 72 % homology with HNRH1_HUMAN (SEQ ID NO:2) and HNRH1_MOUSE (SEQ ID NO:3) and 30 % with Q8VHV7_RAT (SEQ ID NO:1). Amino acids in bold were identified by MALDI-TOF ⁇ TOF mass spectrometry.
- Figure 2 Reactivity to hnRNP-H1 on Western blotting analysis.
- Rat hnRNP-H1 was subjected to SDS-PAGE and transferred onto a PVDF membrane.
- the membrane was incubated with purified antibody or with serum, as described in example 1.
- Lanes 1 , 2, and 3 show Western blotting analyses after incubation of the membranes with purified anti-hnRNP- H1 from rabbit, serum from a diseased person, and serum from a control person, respectively.
- Lane 4 shows the gel after SDS-PAGE and staining with Coomassie Brilliant Blue. The most left and most right lane shows the molecular weight marker.
- Figure 3 Indirect immunofluorescence pattern of anti-hnRNP-H1 antibodies. Effect of pre-incubation with recombinant hnRNP-H1.
- Panel A HEp-2 cells were incubated with anti-hnRNP-H1 antibodies from rabbit (0.25 ⁇ g/ml).
- Panels B-E HEp-2 cells were incubated with a serum sample that reacted with hnRNP-H1 (on Western blotting) after pre-incubation with phosphate buffered saline (Panel B) or with increasing concentrations of recombinant rat hnRNP-H1 (24 ⁇ g/mL, 63 ⁇ g/mL, or 126 ⁇ g/mL) (PanelC, D, and E, respectively).
- the figures show the indirect immunofluorescence analysis (magnification 400 X).
- Figure 4 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-H1 from rat.
- SSp primary Sjogren's syndrome
- SLE systemic lupus erythematosus
- MCTD mixed connective tissue disease
- SSc scleroderma
- DM dermatomyositis
- PM polymyositis
- RA rheumatoid arthritis
- * statistically significantly different from all other groups (Mann Whitney). For each group, the median 'peak x area' value is indicated.
- Figure 5 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing ELISA using recombinant hnRNP-H1 from rat.
- SSp primary Sjogren's syndrome
- SLE systemic lupus erythematosus
- MCTD mixed connective tissue disease
- SSc scleroderma
- DM dermatomyositis
- PM polymyositis
- RA rheumatoid arthritis
- * statistically significantly different from all other groups (Mann Whitney). For each group, the median absorbance value is given.
- the invention provides a method of detecting anti-hnRNP-H1 antibodies in a patient suffering from or susceptible to an autoimmune disease such as Sjogren's syndrome.
- the method of detecting anti-hnRNP-H1 antibodies of the invention includes the step of analyzing a biological sample for the presence of an antibody that specifically binds a hnRNP-H1.
- a biological sample that would normally be expected to contain immunoglobulins might be used.
- the sample would take the form of a bodily fluid, such as blood (and fractions thereof, such as serum or plasma), saliva, tears, mucus, and the like. Because immunoassays are known to generally work well with blood or blood fractions, these are preferred.
- Biological samples can be collected from a subject by any suitable method.
- a blood sample can be collected using conventional phlebotomy procedures; a saliva sample can be collected by spitting or merely by placing a stick in the mouth and is the preferred patient sample. Blood samples can be used for verification of detection of autoantibodies.
- a subject from which a biological sample can be obtained for analysis according to the invention is an animal such as a mammal, e.g. a dog, cat, horse, cow, pig, sheep, goat, primate, rat, or mouse.
- a preferred subject is a human being, particularly a patient suspected of having or at risk for developing an autoimmune disorder such as Sjogren's syndrome (e.g. an individual suffering from dry eye and/or dry mouth), or a patient with a connective tissue disease (e.g. an individual diagnosed with SLE, rheumatoid arthritis, or scleroderma).
- an agent to which an anti-hnRNP-H1 antibody specifically binds is used.
- agents might include a native (i.e. naturally occurring) hnRNP-H1 or fragments, mutants or variants thereof; or a non-hnRNP-H1 test reagent such as an anti-idiotypic antibody.
- native hnRNP-H1s have been characterized (e.g. amino acid sequenced), including those from human, mouse and rat.
- those hnRNP-H1s from the same species as the biological sample are preferred for particular variations of the method of the invention. Nonetheless, due to cross-reactivity, non-species matched assays may also be used.
- rat hnRNP-H1 can be used to detect human anti-hnRNP-H1 antibodies.
- test reagent that specifically binds to these proteins, a number of different methods might be used.
- the sample, or a purified portion thereof is contacted with the agent under conditions that allow agent- antibody binding.
- the presence of the formed agent-antibody complex is then detected as an indication that the sample contains an anti-hnRNP-H1 antibody.
- hnRNP-H1 is immobilized on a substrate, a human serum sample is placed on the substrate under conditions that would allow binding of anti-hnRNP-H1 antibodies to the immobilized hnRNP-H1.
- detectably labeled secondary antibody e.g. an anti-human immunoglobulin antibody if the biological sample was derived from a human subject
- detectable label e.g. an enzyme, fluorophore, or radioisotope
- antibodies contained within a biological sample are immobilized on a substrate, a detectably labeled hnRNP-H1 is then placed on the substrate under conditions that would allow binding of the immobilized anti-hnRNP-H1 antibodies to the hnRNP-H1.
- the presence of detectable label remaining on the substrate after washing indicates that the sample contained anti-hnRNP-H1 antibodies.
- the method of the invention can be carried out as qualitative or quantitative determination.
- hnRNP-H1 antibody concentrations which are above the so-called cutoff value are classified as positive.
- the cutoff value can be determined by calibrating the test system with positive and negative control samples. Alternatively, it is also possible to carry out a quantitative determination.
- test format is not in general critical. However, a test format can be preferred in which an immune complex comprising a hnRNP-H1 -antigen and the hnRNP-H1 autoantibody to be detected is bound to a solid phase.
- solid phases which can be employed are reaction vessels, microtiter plates, beads, biochips etc.
- the antigen can be immobilized on the solid phase by adsorptive interactions, covalent bonding or mediated by a high- affinity binding pair (streptavidin/biotin, hapten/anti-hapten antibody).
- the immobilized test reagent can be employed in a form which is already bound to a solid phase or else be immobilized only during the test.
- the method can be carried out as liquid test (e.g. in a reaction vessel) or else as dry test (e.g. on a test strip).
- the labeled test reagent may itself have a detectable or signal-emitting group (direct labeling) or be capable of binding to a detectable group (indirect labeling).
- the labeling group can be selected as desired from all labeling groups known from the prior art for immunological detection methods, for example from enzymes, metal particles or latex particles, and luminescent or fluorescent groups. It is particularly preferred for the labeling group to be selected from enzymes, e.g. peroxidase, ⁇ - galactosidase or alkaline phosphatase, and for the method to be carried out in the ELISA format.
- Purified or recombinant hnRNP-H1-antigen is spotted on a nitrocellulose or polyvinylidene difluoride (PVDF) membrane.
- the membrane strips are firstly blocked with blocking reagent. After a washing step to remove unbound proteins, they are incubated with diluted serum of the patient, a positive and a negative control serum. They are further washed with washing buffer and treated with alkaline phosphatase labeled protein A conjugate to bind the captured antibodies.
- the purified or recombinant hnRNP-H1-antigen is separated on a one dimensional sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) prior to blotting on a membrane.
- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
- the membrane strip is blocked in blocking buffer (BSA or skim milk powder).
- BSA blocking buffer
- the membrane is subsequently treated with diluted serum of the patient, anti-human IgG conjugated with horseradish peroxidase (HRP) or a fluorescent dye and a substrate to visualize the bound antibodies. Intermittent washing steps will remove the unbound proteins.
- This Western blot assay can be used in combination with other proteins of different molecular weight loaded in the same lane on SDS-PAGE (cfr. line assay). This technique is labor intensive but permits to screen the sera simultaneously for the presence of different auto-antibodies. It's a qualitative method.
- An Enzyme Linked Immunosorbent Assay provides a quantitative in vitro assay for the detection of antibodies.
- Polystyrene microplate strips are coated with purified, biochemically characterized pure or recombinant hnRNP-H1 -antigen as solid phase. If the sample is positive, specific antibodies in the diluted serum sample attach to the antigens coupled to the solid phase.
- the attached antibodies are detected with peroxidase-labeled anti-human antibodies.
- the bound antibodies are visualized using a chromogen/substrate solution which is capable of promoting a color reaction. The intensity of the color produced is proportional to the concentration of antibodies in the serum sample and is measured using a spectrophotometer with filter settings of a suitable wavelength.
- a Fluoro Enzyme lmmuno Assay provides a quantitative in vitro assay for the detection of antibodies.
- a carier e.g. polystyrene microplate strips
- a carier e.g. polystyrene microplate strips
- purified, biochemically characterized pure or recombinant antigen e.g. hnRNP-H1-antigen
- the attached antibodies are detected with an enzyme-labeled anti-human antibodies.
- the bound antibodies are visualized using a substrate solution which is capable of promoting a color reaction.
- the intensity of the color/fluorescence produced is proportional to the concentration of antibodies in the serum sample and is measured using a spectrophotometer with filter settings of a suitable wavelength.
- the fluorescence of the serum sample is than compared to a standard curve.
- Radio lmmuno Assays are immuno assays in which radioactive isotopes are used as labels for detection. These assays use gamma and beta counters for detection and are highly sensitive. The greatest disadvantage of radio immuno assays is that the amount of use and the kind of radioisotopes are limited by regulations and radioactive wastes are released in large amounts. Therefore, the RIA is becoming less popular as an immunoassay.
- Bead-based assays Human serum is treated with different sets of color-coded polystyrene beads, each bearing a different antigen (including hnRNP-H1). Individual beads bearing interacting antibodies are detected by flow cytometry. The beads are aligned into single files as they enter a stream of sheath fluid and then enter a flow cell. Once the beads are in single file within the flow cell, each bead is individually interrogated for bead color (analyte) and assay signal strength (fluorescence intensity). This is a fast, reproducible and reliable but expensive technique. Only a limited number of antigens can be screened.
- Antigen array Several antigens, including hnRNP-H1, are printed on nitrocellulose or superaldehyde coated slides. Printed microarray chips are then washed in wash buffer and probed with diluted patient's serum. Afterwards, fluorescently labeled anti-human IgG conjugate is added and the slides are washed again. The fluorescent signal is further quantified by scanning and image analysis software. These antigen arrays allow to simultaneously screen for the presence of an unlimited amount of autoantibodies.
- Autoimmune antibodies or inhibitor(s) of the present invention include polypeptides comprising the epitope of the antibody or biologically active fragment thereof, or polypeptide that is functional in conferring protection in the individual suffering from autoimmune disease, or functionally conserved fragments or amino acid variants thereof. Identification of the epitope is a matter of routine experimentation. Most typically, one would conduct systematic substitutional mutagenesis of the compound molecule while observing for reductions or elimination of cytoprotective or neutralizing activity. In any case, it will be appreciated that due to the size of many of the antibodies, most substitutions will have little effect on binding activity. The great majority of variants will possess at least some cytoprotective or neutralizing activity, particularly if the substitution is conservative.
- Conservative amino acid substitutions are substitutions from the same class, defined as acidic (Asp, GIu) 1 hydroxy- like (Cys, Ser, The), amides (Asn, GIn), basic (His, Lys, Arg), aliphatic-like (Met, He, Leu, VaI, GIy, Ala, Pro), and aromatic (Phe, Tyr, Trp).
- Homologous antibody or polypeptide sequences generally will be greater than about 30 percent homologous on an identical amino acid basis, ignoring for the purposes of determining homology any insertions or deletions from the selected molecule in relation to its native sequence.
- autoimmune inhibitors for administration to the patient with autoimmune disease and/or for removal, neutralization or inhibition of the autoimmunogen(s) by extracorporeal immunosorption in accordance with the present invention also include glycosylation variants as well as unglycosylated forms of the agents, fusions of the agents with heterologous polypeptides, and biologically active fragments of the agents, again so long as the variants possess the requisite neutralizing or cytoprotective activity.
- treatments involving administration of an autoimmune inhibitor to a patient may be performed alone or in combination.
- Administered autoimmune inhibitor of the invention binds to, neutralizes and/or inhibits the molecule(s) associated with or causing the autoimmune response in the patient. More specifically, administration of the autoimmune inhibitor to a patient results in suppression of pathological humoral and adaptive immunity in the patient. In other words, in accordance with the method of the present invention, treatment of a patient with the autoimmune inhibitor causes the humoral and adaptive immune response of the patient to be inhibited or neutralized over that which was, or would have been, present in the absence of treatment.
- a patient is in need of treatment with an autoimmune inhibitor, when the patient is suffering from an autoimmune disease or when the patient has produced autoantibodies.
- the autoimmune inhibitor antibody(ies) is also effective when immobilized on a solid support.
- solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art(Weir et al., "Handbook of experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chap. 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y.(1974)).
- autoantibody gene products may be generated which include proteins that represent functionally equivalent gene products.
- an equivalent hnRNP-H1 antibody gene product may contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues, but that result in a "silent" change, in that the change produces a functionally equivalent auto-hnRNP-H1 antibody gene product.
- Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
- nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;
- polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine;
- positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- deletion or non-conservative alterations can be engineered to produce altered anti-hnRNP-H1 antibody gene products.
- Such alterations can, for example, alter one or more of the biological functions of the autoantibody gene product. Further, such alterations can be selected so as to generate autoantibody gene products that are better suited for expression, scale up, etc. in the host cells chosen.
- cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. This applies to any autoimmune hnRNP-H1 molecule and allelic variants thereof, that are identified in an individual.
- the autoantibody gene products, peptide fragments thereof and fusion proteins thereof, of the invention can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors comprising auto-hnRNP-H1 antibody gene product coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook, et a/., 1989, and Ausubel, et a/., 1989.
- RNA capable of encoding auto-hnRNP-H1 antibody gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford.
- host-expression vector systems may be utilized to express the autoantibodies, such as anti-hnRNP-H1 gene products.
- Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the anti- hnRNP-H1 gene product of the invention in situ.
- the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner, et a/., 1987, Methods in Enzymol. 153, 516-544).
- a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of protein products may be important for the function of the protein.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
- Such mammalian host cells include but are not limited to CHO, JURKAT, Hep2, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138.
- a variety of methods can be employed for the diagnostic and prognostic evaluation of Sjogren's syndrome and for the identification of subjects having a predisposition to such autoimmune disorders. Such methods may, for example, detect the presence of hnRNP-H1 gene mutations, or the detection of either over-, under-, or no expression of hnRNP-H1 protein, or mutants.
- Mutations at a number of different genetic loci may lead to phenotypes related to autoimmune disorder, structural and synaptic abnormalities.
- the treatment of patients suffering from such disorders will be designed to target the particular genetic loci comprising the mutation mediating the disorder.
- Genetic polymorphisms have been linked to differences in drug effectiveness.
- identification of alterations in hnRNP-H1 molecules, such as, for example, gene or protein can be utilized to optimize therapeutic drug treatments.
- autoimmune related molecule such as, for example, hnRNP-H1, expression levels, mutations, polymorphisms
- a microassay of for example, hnRNP-H1 nucleic acid sequences immobilized to a substrate or "gene chip" for detection of hnRNP- H1 molecules see, e.g. Cronin, ef a/., 1996, Human Mutation 7:244-255. Preferred methods are detailed in the examples which follow.
- hnRNP-H1 or any hnRNP-H1 -related molecule gene expression can also be assayed as described in detail in the examples which follow. Additionally, it is possible to perform hnRNP-H1 gene expression assays "in situ", i.e. directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. For such in situ procedures (see, for example, Nuovo, G. J., 1992, “PCR In Situ Hybridization: Protocols And Applications", Raven Press, N.Y.) standard northern analysis can be performed to determine the level of mRNA expression of the hnRNP-H1 gene.
- Antibodies directed against hnRNP-H1 gene products may be used in vitro to determine, for example, the level of hnRNP-H1 antibody gene expression achieved in cells genetically engineered to produce such a gene product.
- an assessment is done, preferably, using cell lysates or extracts. Such analysis will allow for a determination of the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.
- the tissue or cell type to be analyzed will generally include those that are known, or suspected, to express the hnRNP-H1 gene.
- the protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
- the isolated cells can be derived from cell culture or from a patient.
- the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the hnRNP-H1 gene.
- Preferred diagnostic methods for the detection of autoimmune molecules may involve, for example, immunoassays wherein the hnRNP-H1 gene products or conserved variants or peptide fragments are detected by their interaction with an anti-hnRNP-H1 gene product-specific antibody.
- immunoassays wherein the hnRNP-H1 gene products or conserved variants or peptide fragments are detected by their interaction with an anti-hnRNP-H1 gene product-specific antibody.
- This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
- autoimmune disease refers to a disease that result from an aberrant immune response of an organism against its own cells and tissues due to a failure of the organism to recognise its own constituent parts (down to the sub-molecular level) as "self.
- autoimmune disease is intended to further include autoimmune conditions, syndromes and the like.
- antigen refers to a structure of a macromolecule, typically protein (with or without polysaccharides) or made of proteic composition comprising one or more hapten(s) and/or comprising at least one epitope.
- an “autoantigen” as used herein refers to a human or animal protein present in the body, which elicits an immune response within the same human or animal body, which may in turn lead to a chain of events, including the synthesis of other autoantigens or autoantibodies.
- An “autoantibody” is an antibody produced by an autoimmune patient to one or more of his own constituents which are perceived to be antigenic. For example, in SLE autoantibodies are produced to DNA, while in many other types of AD autoantibodies are produced to target cells.
- autoimmune diseases including, e.g., rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, systemic lupus erythematosus and others, are in need of treatment in accordance with the present invention.
- Treatment of patients suffering from these diseases by administration of autoimmune inhibitor and/or removal of compound(s) by extracorporeal immunosorption in accordance with the present invention will alleviate the clinical manifestations of the disease and/or minimize or prevent further deterioration or worsening of the patient's condition.
- epitope refers to one or several portions (which may define a conformational epitope) of an antigenic protein which is/are specifically recognised and bound by an antibody or a portion thereof (Fab 1 , Fab2', etc.) or a receptor presented at the cell surface of a B or T cell lymphocyte, and which is able, by said binding, to induce an immune response.
- An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids. Methods for determining the amino acids which make up an epitope include x-ray crystallography, 2 -dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by H. Mario Geysen et al. 1984. Proc. Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506. It is well known to a person skilled in the art that antibodies which recognize and bind an epitope can be monoclonal or polyclonal.
- the phrase "specifically (or selectively) binds" to an antibody when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
- the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
- Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
- polyclonal antibodies raised to marker "X" from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with marker "X” and not with other proteins, except for polymorphic variants and alleles of marker "X". This selection may be achieved by subtracting out antibodies that cross-react with marker "X" molecules from other species.
- a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
- solid- phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
- a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
- Immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g. a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
- Diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
- the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
- the "specificity” of a diagnostic assay is 1 (or 100 when indicated as percentage) minus the (percentage) false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
- nucleic acid delivery vector may be provided as naked nucleic acids or in a delivery vehicle associated with one or more molecules for facilitating entry of a nucleic acid into a cell.
- Suitable delivery vehicles include, but are not limited to: liposomal formulations, polypeptides; polysaccharides; lipopolysaccharides, viral formulations (e.g. including viruses, viral particles, artificial viral envelopes and the like), cell delivery vehicles, and the like.
- hnRNP-H1 as a new target of auto-antibodies in patients with primary and secondary Sjogren's syndrome.
- the specificity of anti-hnRNP-H1 antibodies 91.7% - 94.4%) tended to be higher than the specificity of anti-SSA antibodies (78.6% - 89%).
- the low specificity of anti-SSA antibodies is mainly related to the high prevalence of anti-SSA antibodies in patients with SLE.
- anti-hnRNP-H1 antibodies were not very much prevalent in patients with SLE.
- Anti-hnRNP-H1 antibodies are an interesting diagnostic marker in anti-SSA and/or anti-SSB seronegative patients with primary Sjogren's syndrome. We found that 5/11 (45%) anti-SSA/anti-SSB seronegative patients had anti-hnRNP-H1 antibodies.
- the negative predictive value is higher for anti-SSA antibodies than for anti-hnRNP-H1 antibodies.
- the analysis of the 188 consecutive (random) samples revealed relevant information on the specificity of anti-hnRNP-H1 antibodies and showed that these antibodies can be found, to a lesser extent in a number of other autoimmune diseases.
- One explanation is that in some of these patients, the detection of hnRNP-H1 antibodies is indicative for the development of secondary Sjogren's syndrome since we have shown (in example 2) that hnRNP-H1 auto-antibodies are an early marker for Sjogren's syndrome.
- anti-SSA antibodies are found, to a bigger extent compared with hnRNP-H1 antibodies, in a wide range of medical conditions with a possible autoimmune pathogenesis.
- Anti-hnRNP-H1 antibodies were not only found in samples with a speckled ANA pattern, but also in samples with a homogeneous staining (nuclear dots) pattern.
- Five of the 18 patients with anti-hnRNP- H1 antibodies received Infliximab, a TNF- ⁇ blocking drug, and displayed a homogeneous staining on ANA indirect immunofluorescence analysis. It is known that Infliximab can induce the formation of auto-antibodies, especially anti-dsDNA antibodies. We assumed that in such cases the typical speckled pattern caused by anti-hnRNP-H1 antibodies was masked by other antibodies.
- hnRNP-H1 we described hnRNP-H1 as a new target of autoantibodies in patients with Sjogren's syndrome. Therefore, we identified anti-hnRNP-H1 antibodies as a valuable diagnostic marker in Sjogren's syndrome.
- Recombinant his-tagged hnRNP-H1 protein from rat was prepared as described (Fijak M et al., J Pathol. 2005;207: 127-38).
- Recombinant GST-tagged hnRNP-H1 protein from human was prepared from cDNA according to Devogelaere et al. (Biochem. J. 2007;407:303-311). o
- PVDF Polyvinylidene difluoride
- Hybond-P Hybond-P
- IPG strips pH-range 3-10, ImmobilineTM DryStrip gels
- Rabbit thymus, goat anti-human IgG, and HPLC grade water were obtained from Sigma-Aldrich.
- Horseradish peroxidase conjugated rabbit anti- goat IgG was from DakoCytomation.
- C18 clean-up (ZipTip) was from Millipore Corporation.
- Purified rabbit anti-hnRNP-H1 antibodies were from Acris Antibodies.
- a second group of samples consisted of 188 consecutive serum samples submitted to the clinical laboratory for ANA testing (titer ⁇ 160).
- Antinuclear antibody testing identification of antibodies to extractable nuclear antigens, counterimmunoelectrophoresis
- ANA were determined by indirect immunofluorescence using HEp-2000 ® cells (Immunoconcepts) with an Axioplan 2 fluorescence microscope (Carl Zeiss Microimaging) and Cytovision 3.6 software (Applied Imaging Corporation). The presence of antibodies to ENAs was assayed by ENA dot blot (BioMedical Diagnostics), UniCaplOO and/or UniCap250 system (Phadia Diagnostics), ANA3 test (anti-SSA 52/anti-SSA 60 status, Euroimmun), or counterimmunoelectrophoresis with rabbit thymus as antigenic source.
- PVDF polyvinylidene difluoride
- Blotting signals were scanned using a Typhoon 9400 scanner set on green laser light (532 nm), filter 526 SP, PMT 640V at normal sensitivity and quantified by ImageQuant TL software (both from GE Healthcare).
- a positive and negative control serum sample were included in each run. The coefficient of variation of the positive control was 23.8% over 13 runs.
- Serum from the patient and from a control individual were used for Western blotting after 2D gel separation of rabbit thymus extract. Proteins to which there was reactivity were excised and identified by MALDI-TOF/TOF analysis, and for one of the proteins identified here, a significant score was obtained for the protein hnRNP-H1 from mouse (Mus musculus) mining the SwissProt database (Hn ⁇ pM, 035737) or Ratsgi from rat (Rattus norvegicus) screening the NCBI database ⁇ Hnrnphi, Q8VHV7) (see Fig.1).
- hnRNP-H1 heterogeneous nuclear ribonucleoprotein
- hnRNP-H1 heterogeneous nuclear ribonucleoprotein
- HnRNP-HI from rat has 99% similarity with human hnRNP-H1 (P31943
- anti-hnRNP-H1 antibodies give a speckled fluorescence pattern on indirect immunofluorescence analysis by two complementary approaches. First, incubating HEp-2 cells with a commercial rabbit anti-hnRNP-H1 antibody resulted in a fine speckled antinuclear antibody pattern (Fig. 3A).
- Anti-hnRNP-H1 antibodies are associated with Sjogren's Syndrome
- serum samples from 246 patients with well-defined autoimmune connective tissue disorders for reactivity to hnRNP-H1 in Western blot analysis. The results are shown in Fig. 4.
- a receiver operating characteristics analysis was performed for anti-hnRNP-H1 antibodies as a marker of primary Sjogren's syndrome.
- the sensitivity of anti-hnRNP-H1 antibodies for primary Sjogren's syndrome was 48.4% when a cutoff ('peak x area' 1.90) was used that corresponded to a specificity of 94.8% in diseased controls.
- a cutoff ('peak x area' 0.71) was used that maximized the sum of sensitivity and specificity, then the sensitivity of anti- hnRNP-H1 antibodies for primary Sjogren's syndrome was 74.2% and the specificity 81.7% (Youden index 0.559). In all subsequent analyses, the cutoff based on a specificity of 95% was used.
- Anti- hnRNP-H1 antibodies were found in 48% (45/93) of patients with primary Sjogren's syndrome, in 2.4% (1/41) of patients with SLE, in 22% (2/9) of patients with MCTD, in 7.7% (3/39) of patients with scleroderma, in 0% (0/15) of patients with dermatomyositis, in 0% (0/7) of patients with polymyositis, in 4.8% (2/42) of patients with RA, and in 5% (2/41) of healthy individuals.
- the sensitivity and specificity of anti-SSA antibodies for primary Sjogren's syndrome in the same patient cohort was 88.2% and 76.3%, respectively.
- Anti-SSA antibodies were found in 33% of MCTD, in 7.7% of patients with scleroderma, and in 63.4% of patients with SLE.
- the sensitivity of anti-hnRNP-H1 antibodies for Sjogren's syndrome was 43.7% and the specificity 94.4%.
- the area under the curve for anti-SSA antibodies was 0.82.
- the sensitivity and specificity of anti-SSA antibodies in the same patient group was 85.3% and 78.6%, respectively.
- the specificity of anti-hnRNP-H1 antibodies for Sjogren's disease was higher than the specificity of anti-SSA antibodies. This is mainly related to the high prevalence of anti-SSA antibodies in patients with SLE.
- the likelihood ratios were 7.81, 0.60, 3.98, and 0.19 for, respectively, hnRNP-H1 positive, hnRNP-H1 negative, SSA positive, and SSA negative.
- Eleven out of 93 patients with Sjogren's syndrome were seronegative for anti-SSA (both anti-SSA 52 and anti-SSA 60 kDa) and anti-SSB antibodies.
- Anti-hnRNP-H1 antibodies were found in 5 of these 11 seronegative patients (45%) (Table 1).
- FIG. 1 Another method for the evaluation of anti-hnRNP-H1 antibodies in a study cohort of 246 serum samples of patients with autoimmune connective tissue diseases and 41 healthy subjects is an ELISA using recombinant His-tagged rat hnRNP-H1. Using this method, the prevalence of anti-hnRNP-H1 antibodies was 22.5% for patients with primary Sjogren's disease.
- Figure 5 shows the reactivity of patient's serum samples to hnRNP-H1 by performing an ELISA using recombinant His-tagged hnRNP- H1 from rat.
- ELISA detected anti-hnRNPH1 antibodies in 2.4% (1/41) of patients with SLE 1 in 22% (2/9) of patients with MCTD, in 7.7% (3/39) of patients with scleroderma, in 13.3% (2/15) of patients with dermatomyositis and in 2.5% (1/40) of healthy individuals.
- No anti-hnRNP-H1 antibodies were detected in patients with RA (0/42) or polymyositis (0/7).
- a statistically significant difference between the prevalence of anti-hnRNP-H1 antibodies in patients with primary Sjogren's syndrome, healthy controls and diseased controls was found (p 0.0063, Fisher Exact Test).
- the sensitivity of anti-hnRNP-H1 antibodies for Sjogren's syndrome was 19.4% and the specificity 94.4% (cutoff 110, based on 95% specificity for diseased controls).
- Three of the 18 patients had secondary Sjogren's syndrome (one in combination with SLE and 2 in combination with RA [treated with anti-TNF- ⁇ ]).
- Primary Sjogren's syndrome was assumed in this patient, but he didn't fulfill the EU-US classification criteria.
- the sensitivity and specificity of anti-hnRNP-H1 for Sjogren's syndrome was 42.9% and 91.7%, respectively.
- the sensitivity and specificity were, respectively, 71.4% and 89.0%.
- the likelihood ratios were 5.16, 0.62, 6.46, and 0.32 for, respectively, hnRNP-H1 positive, hnRNP-H1 negative, SSA positive, and SSA negative.
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Abstract
The present invention relates to a method of diagnosing Sjögren's syndrome. More particularly the present invention relates to methods and compositions for detecting anti-heterogeneous nuclear ribonucleoprotein-H1 (hnRNP-H1 ) autoantibodies for diagnosing Sjogren's syndrome.
Description
NEW METHOD FOR DIAGNOSING SJOGREN'S SYNDROME
FIELD OF THE INVENTION
The present invention relates to a method of diagnosing Sjogren's syndrome. More particularly the present invention relates to methods and compositions for detecting anti-heterogeneous nuclear ribonucleoprotein-H1 (hnRNP-H1) autoantibodies for diagnosing Sjogren's syndrome.
BACKGROUND
Sjogren's syndrome is an autoimmune exocrinopathy, characterized by dryness of the mouth and eyes resulting from a chronic loss of secretory function of the salivary and lacrimal glands. Extraglandular systemic manifestations are common and include abnormalities of skin, arthralgia, myalgia, thyroiditis, and pulmonary, renal, gastrointestinal, hematological, cardiac, and neurological abnormalities. There is an increased propensity to develop B cell lymphoma. The etiology of the disease remains unclear. The prevalence of the disease is estimated to be 0.5% and there is a female preponderance. Pathological findings involve focal lymphocytic infiltration of affected tissues. Laboratory findings comprise hypergammaglobulinemia, rheumatoid factor and auto-antibodies against salivary ductal cells and SSA and SSB. Depending on the technique used, anti-SSA antibodies are found in 45-90% of the patients with Sjogren's syndrome. Anti-SSB antibodies are found slightly less commonly. Anti-SSA antibodies are not specific for Sjogren syndrome as they are found in other systemic diseases such as systemic lupus erythematosus, subacute cutaneous lupus erythematosus and neonatal lupus. Sjogren's syndrome can occur in two forms: primary Sjogren's syndrome (not associated with other autoimmune diseases) and secondary Sjogren's syndrome (associated with other autoimmune diseases).
Because of the non-specific symptoms (dry eyes and mouth), establishment of the diagnosis of Sjogren's syndrome may be difficult. There is an unmet need for additional accurate and valid diagnostic markers. A number of new autoantigens have recently been suggested: α-fodrin, muscarinic M3 acetylcholine receptor, and SS-56, which is structurally related to the 52 kDa SSA antigen. These markers, however, have not shown adequate specificity and sensitivity to serve as valuable clinical markers, or are not available for routine diagnostics (Ko vacs L et al., Rheumatology (Oxford). 2005;44:1021-5). The fact that there are patients with autoimmune diseases, particularly with sicca symptoms, who have antinuclear antibodies in high titer but in whom no antibodies to extractable nuclear antigens (e.g. SSA or SSB) can be identified is indicative for the need to identify novel and preferentially early diagnostic markers.
SUMMARY OF THE INVENTION
The present invention shows hnRNP-H1 as a new target of autoantibodies in patients with Sjogren's syndrome. More in particular, the present invention identifies anti-hnRNP-H1 antibodies as a valuable diagnostic marker in Sjogren's syndrome. The present invention provides for a method for diagnosing
Sjogren's syndrome. More particularly the present invention provides methods and compositions for detecting anti-hnRNP-H1 autoantibodies for diagnosing Sjogren's syndrome. The method of this invention is applicable to a large group of patients, including patients having Sjogren's syndrome, patients being suspected as having Sjogren's syndrome or at risk of developing Sjogren's syndrome, or patients suffering from other autoimmune diseases.
One aspect of the present invention relates to a method of diagnosing an autoimmune disease, more particularly Sjogren's syndrome, characterized in that the presence of autoantibodies against hnRNP- H1 is determined in an isolated biological sample derived from a subject.
The present invention further relates to a method for detecting hnRNP-H1 antibodies in an isolated biological sample, the method comprising the steps of (a) obtaining an isolated biological sample from a patient; and (b) analyzing said isolated biological sample for the presence of antibodies against hnRNP-M. In particular embodiments of the invention, said method is characterized in that hnRNP-H1 proteins, peptides, variants or fragments thereof are used for determining the presence of the hnRNP-H1- antibodies. In particular embodiments of the invention, said method is characterized in that a hnRNP- H1-antigen, more specific an immobilized hnRNP-H1-antigen is used for determining the presence of the hnRNP-M -antibodies. In particular embodiments of the invention, said analysis or determination of the presence of autoantibodies against hnRNP-H1 is performed in an immunoassay. In particular embodiments of the invention, said immunoassay is selected from the group consisting of ELISA, FEIA, western blot, dot blot, bead-based assay, antigen array and Radio lmmuno Assay. In a particular embodiment of the invention, the methods of the invention are used for predicting responsiveness to a medicament.
Another aspect of the present invention relates to the use of hnRNP-H1 proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from a subject. In particular embodiments of the invention, the present invention further relates to the use of hnRNP-H1 proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from a patient for diagnosing an autoimmune disease, more particularly Sjogren's syndrome. In a particular embodiment of the invention, said hnRNP-H1 proteins, peptides, variants or fragments thereof are used for carrying out the methods of the invention.
In particular embodiments of the invention, the isolated biological sample is a fluid selected from the group consisting of blood, serum, plasma, saliva, tears, mucus and ascites fluid; and preferentially said biological sample is serum. In another embodiment of the invention, said isolated biological sample is derived from blood, plasma, serum, saliva, tears, lymph, urine, cerebrospinal fluid, any biopsy material or tissue sample, including bone marrow, lymph nodes, nervous tissue, skin, hair, fetal material including amniocentesis material, uterine tissue, faeces or semen. In yet another embodiment of the invention, said isolated biological sample is an antibody-containing isolated biological sample.
Another aspect of the present invention relates to a test kit for detecting the presence of antibodies against hnRNP-H1 in an isolated biological sample. In a particular embodiment, the test kit additionally comprises a solid phase onto which said antigen is or can be bound. In a more particular embodiment of the foregoing, the test kit additionally comprises a labeling group which is bound to said antigen or can be bound thereto. In another embodiment of the foregoing, the test kit additionally comprises at least one other antibody class-specific test reagent. The test kit may additionally comprise other conventional reagents such as buffers, substrates and wetting solutions. The present ivention further relates to the use of said test kit for carrying out any of the methods of the invention.
Another aspect of the present invention relates to a method of treating an autoimmune disease, more particularly Sjogren's syndrome, comprising administering to a patient an inhibitor that specifically binds to the hnRNP-H1-autoantibodies of said patient, in an amount effective to treat the autoimmune disease, more particularly Sjogren's syndrome. In a particular embodiment, said inhibitor is selected from the group consisting of hnRNP-H1 proteins, peptides, variants, fragments, or epitopes thereof and an antibody.
The present invention further relates to the use of a compound which specifically binds to hnRNP-H1 autoantibodies for the production of a medicine. In a particular embodiment said medicine is used for the treatment of an auto-immunedisease, more particularly Sjogren's syndrome.
In particular embodiments of the foregoing the subject or the patient is a human being. Said human being can be a patient having Sjogren's syndrome, a patient being suspected as having Sjogren's syndrome or at risk of developing Sjogren's syndrome, or a patient suffering from other autoimmune diseases.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 Sequence alignments of hnRNP-H1 from mouse, rat and humans. The three sequences Q8VHV7_rat (SEQ ID NO:1), HNRH1_HUMAN (SEQ ID NO:2) and HNRH1_MOUSE (SEQ ID NO:3) show 99 % homology with exclusion of the missing 77 amino acids from hnRNP-H1Q8VHV7 from rat (SEQ ID NO:1), whereas sequence Q499R8_RAT (SEQ ID NO:4) has 72 % homology with HNRH1_HUMAN (SEQ ID NO:2) and HNRH1_MOUSE (SEQ ID NO:3) and 30 % with Q8VHV7_RAT (SEQ ID NO:1). Amino acids in bold were identified by MALDI-TOFΛTOF mass spectrometry.
Figure 2 Reactivity to hnRNP-H1 on Western blotting analysis.
Rat hnRNP-H1 was subjected to SDS-PAGE and transferred onto a PVDF membrane. The membrane was incubated with purified antibody or with serum, as described in example 1. Lanes 1 , 2, and 3 show Western blotting analyses after incubation of the membranes with purified anti-hnRNP- H1 from rabbit, serum from a diseased person, and serum from a control person, respectively. Lane 4 shows the gel after SDS-PAGE and staining with Coomassie Brilliant Blue. The most left and most right lane shows the molecular weight marker.
Figure 3 Indirect immunofluorescence pattern of anti-hnRNP-H1 antibodies. Effect of pre-incubation with recombinant hnRNP-H1.
Panel A: HEp-2 cells were incubated with anti-hnRNP-H1 antibodies from rabbit (0.25 μg/ml). Panels B-E: HEp-2 cells were incubated with a serum sample that reacted with hnRNP-H1 (on Western blotting) after pre-incubation with phosphate buffered saline (Panel B) or with increasing concentrations of recombinant rat hnRNP-H1 (24 μg/mL, 63 μg/mL, or 126 μg/mL) (PanelC, D, and E, respectively). The figures show the indirect immunofluorescence analysis (magnification 400 X).
Figure 4 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing a Western blotting analysis using recombinant His-tagged hnRNP-H1 from rat. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis, *: statistically significantly different from all other groups (Mann Whitney). For each group, the median 'peak x area' value is indicated.
Figure 5 Prevalence of anti-hnRNP-H1 antibodies in patients with autoimmune connective tissue disorders by performing ELISA using recombinant hnRNP-H1 from rat. SSp: primary Sjogren's syndrome, SLE: systemic lupus erythematosus, MCTD: mixed connective tissue disease, SSc: scleroderma, DM: dermatomyositis, PM: polymyositis, RA: rheumatoid arthritis, *: statistically significantly different from all other groups (Mann Whitney). For each group, the median absorbance value is given.
DETAILED DESCRIPTION OF THE INVENTION
Detailed description of embodiments of the invention
In a preferred embodiment, the invention provides a method of detecting anti-hnRNP-H1 antibodies in a patient suffering from or susceptible to an autoimmune disease such as Sjogren's syndrome.
Preferably, the method of detecting anti-hnRNP-H1 antibodies of the invention includes the step of analyzing a biological sample for the presence of an antibody that specifically binds a hnRNP-H1. Any
suitable biological sample might be used in the method. For example, a biological sample that would normally be expected to contain immunoglobulins might be used. Typically, the sample would take the form of a bodily fluid, such as blood (and fractions thereof, such as serum or plasma), saliva, tears, mucus, and the like. Because immunoassays are known to generally work well with blood or blood fractions, these are preferred. Biological samples can be collected from a subject by any suitable method. For example, a blood sample can be collected using conventional phlebotomy procedures; a saliva sample can be collected by spitting or merely by placing a stick in the mouth and is the preferred patient sample. Blood samples can be used for verification of detection of autoantibodies.
A subject from which a biological sample can be obtained for analysis according to the invention is an animal such as a mammal, e.g. a dog, cat, horse, cow, pig, sheep, goat, primate, rat, or mouse. A preferred subject is a human being, particularly a patient suspected of having or at risk for developing an autoimmune disorder such as Sjogren's syndrome (e.g. an individual suffering from dry eye and/or dry mouth), or a patient with a connective tissue disease (e.g. an individual diagnosed with SLE, rheumatoid arthritis, or scleroderma).
In a preferred embodiment, analysis of a biological sample for the presence of an antibody that specifically binds a hnRNP-H1, an agent to which an anti-hnRNP-H1 antibody specifically binds is used. Such agents might include a native (i.e. naturally occurring) hnRNP-H1 or fragments, mutants or variants thereof; or a non-hnRNP-H1 test reagent such as an anti-idiotypic antibody. A number of different native hnRNP-H1s have been characterized (e.g. amino acid sequenced), including those from human, mouse and rat. Generally, those hnRNP-H1s from the same species as the biological sample are preferred for particular variations of the method of the invention. Nonetheless, due to cross-reactivity, non-species matched assays may also be used. For example, rat hnRNP-H1 can be used to detect human anti-hnRNP-H1 antibodies.
To analyze a biological sample for the presence of an anti-hnRNP-H1 antibody, anti-idiotypic anti- hnRNP-H1 or hnRNP-H1 proteins, peptides, variants or fragments thereof, a test reagent that specifically binds to these proteins, a number of different methods might be used. In general, the sample, or a purified portion thereof, is contacted with the agent under conditions that allow agent- antibody binding. The presence of the formed agent-antibody complex is then detected as an indication that the sample contains an anti-hnRNP-H1 antibody. Methods for detecting antigen (the agent is the antigen in this case)-antibody complexes are well known in the art of immunology, and include techniques that utilize hnRNP-H1-expressing cells as well as non-cellular methods. Cell-based detection methods include techniques such as immunohistochemistry, immunofluorescence microscopy, or flow cytometric analysis. Non-cell based assays include immunosorbent assays (e.g. ELISA and RIA) and immunoprecipitation assays. As one example, hnRNP-H1 is immobilized on a substrate, a human serum sample is placed on the substrate under conditions that would allow binding of anti-hnRNP-H1 antibodies to the immobilized hnRNP-H1. After washing, detectably labeled secondary antibody (e.g. an anti-human immunoglobulin antibody if the biological sample was derived from a human subject) is added to the substrate. The presence of detectable label (e.g. an enzyme,
fluorophore, or radioisotope) on the substrate after washing indicates that the sample contained anti- hnRNP-H1 antibodies. As another example, antibodies contained within a biological sample are immobilized on a substrate, a detectably labeled hnRNP-H1 is then placed on the substrate under conditions that would allow binding of the immobilized anti-hnRNP-H1 antibodies to the hnRNP-H1. The presence of detectable label remaining on the substrate after washing indicates that the sample contained anti-hnRNP-H1 antibodies.
The method of the invention can be carried out as qualitative or quantitative determination. In a qualitative determination, hnRNP-H1 antibody concentrations which are above the so-called cutoff value are classified as positive. The cutoff value can be determined by calibrating the test system with positive and negative control samples. Alternatively, it is also possible to carry out a quantitative determination.
The specific test format is not in general critical. However, a test format can be preferred in which an immune complex comprising a hnRNP-H1 -antigen and the hnRNP-H1 autoantibody to be detected is bound to a solid phase. Alternatively, a competitive test format can be envisaged. Solid phases which can be employed are reaction vessels, microtiter plates, beads, biochips etc. The antigen can be immobilized on the solid phase by adsorptive interactions, covalent bonding or mediated by a high- affinity binding pair (streptavidin/biotin, hapten/anti-hapten antibody). The immobilized test reagent can be employed in a form which is already bound to a solid phase or else be immobilized only during the test.
The method can be carried out as liquid test (e.g. in a reaction vessel) or else as dry test (e.g. on a test strip).
The labeled test reagent may itself have a detectable or signal-emitting group (direct labeling) or be capable of binding to a detectable group (indirect labeling). The labeling group can be selected as desired from all labeling groups known from the prior art for immunological detection methods, for example from enzymes, metal particles or latex particles, and luminescent or fluorescent groups. It is particularly preferred for the labeling group to be selected from enzymes, e.g. peroxidase, β- galactosidase or alkaline phosphatase, and for the method to be carried out in the ELISA format.
Immunological methods to detect anti-hnRNP-H1 antibodies
Single assays:
Dot blot assay
Purified or recombinant hnRNP-H1-antigen is spotted on a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane strips are firstly blocked with blocking reagent. After a washing step to remove unbound proteins, they are incubated with diluted serum of the patient, a positive and a negative control serum. They are further washed with washing buffer and treated with alkaline phosphatase labeled protein A conjugate to bind the captured antibodies. After a final washing step, 5-bromo-4-chloro-3-indolylphosphate/Nitro Blue Tetrazolium (BCIP/NBT) substrate is added to give a
blue colored spot confirming the presence of anti-hnRNP-H1 antibodies. Dot blots assays are a qualitative evaluation method. Several antigens can be tested simultaneously by this method.
Western blot In comparison with the dot blot assay, the purified or recombinant hnRNP-H1-antigen is separated on a one dimensional sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) prior to blotting on a membrane. After electro-transfer of the separated protein on a nitrocellulose or PVDF membrane, the membrane strip is blocked in blocking buffer (BSA or skim milk powder). The membrane is subsequently treated with diluted serum of the patient, anti-human IgG conjugated with horseradish peroxidase (HRP) or a fluorescent dye and a substrate to visualize the bound antibodies. Intermittent washing steps will remove the unbound proteins.
This Western blot assay can be used in combination with other proteins of different molecular weight loaded in the same lane on SDS-PAGE (cfr. line assay). This technique is labor intensive but permits to screen the sera simultaneously for the presence of different auto-antibodies. It's a qualitative method.
ELISA .
An Enzyme Linked Immunosorbent Assay (ELISA) provides a quantitative in vitro assay for the detection of antibodies. Polystyrene microplate strips are coated with purified, biochemically characterized pure or recombinant hnRNP-H1 -antigen as solid phase. If the sample is positive, specific antibodies in the diluted serum sample attach to the antigens coupled to the solid phase. In a second step, the attached antibodies are detected with peroxidase-labeled anti-human antibodies. In a third step, the bound antibodies are visualized using a chromogen/substrate solution which is capable of promoting a color reaction. The intensity of the color produced is proportional to the concentration of antibodies in the serum sample and is measured using a spectrophotometer with filter settings of a suitable wavelength.
FEIA
A Fluoro Enzyme lmmuno Assay (FEIA) provides a quantitative in vitro assay for the detection of antibodies. A carier (e.g. polystyrene microplate strips) is coated with purified, biochemically characterized pure or recombinant antigen (e.g. hnRNP-H1-antigen) as solid phase. If the sample is positive, specific antibodies in the diluted serum sample attach to the antigens coupled to the solid phase. In a second step, the attached antibodies are detected with an enzyme-labeled anti-human antibodies. In a third step, the bound antibodies are visualized using a substrate solution which is capable of promoting a color reaction. The intensity of the color/fluorescence produced is proportional to the concentration of antibodies in the serum sample and is measured using a spectrophotometer with filter settings of a suitable wavelength. The fluorescence of the serum sample is than compared to a standard curve.
Radio lmmuno Assay (RIA)
Radio lmmuno assays are immuno assays in which radioactive isotopes are used as labels for detection. These assays use gamma and beta counters for detection and are highly sensitive. The greatest disadvantage of radio immuno assays is that the amount of use and the kind of radioisotopes are limited by regulations and radioactive wastes are released in large amounts. Therefore, the RIA is becoming less popular as an immunoassay.
Multiplexed immunological assays:
Multiplex platforms allow to screen for autoantibody profiles in human serum samples. In this multiprotein assays, the presence of hnRNP-H1 antibodies is tested simultaneously with other anti- ENA antibodies and quantitatively expressed.
Bead-based assays: Human serum is treated with different sets of color-coded polystyrene beads, each bearing a different antigen (including hnRNP-H1). Individual beads bearing interacting antibodies are detected by flow cytometry. The beads are aligned into single files as they enter a stream of sheath fluid and then enter a flow cell. Once the beads are in single file within the flow cell, each bead is individually interrogated for bead color (analyte) and assay signal strength (fluorescence intensity). This is a fast, reproducible and reliable but expensive technique. Only a limited number of antigens can be screened.
Antigen array: Several antigens, including hnRNP-H1, are printed on nitrocellulose or superaldehyde coated slides. Printed microarray chips are then washed in wash buffer and probed with diluted patient's serum. Afterwards, fluorescently labeled anti-human IgG conjugate is added and the slides are washed again. The fluorescent signal is further quantified by scanning and image analysis software. These antigen arrays allow to simultaneously screen for the presence of an unlimited amount of autoantibodies.
Auto-hnRNP-H1 antibodies for therapy
Autoimmune antibodies or inhibitor(s) of the present invention include polypeptides comprising the epitope of the antibody or biologically active fragment thereof, or polypeptide that is functional in conferring protection in the individual suffering from autoimmune disease, or functionally conserved fragments or amino acid variants thereof. Identification of the epitope is a matter of routine experimentation. Most typically, one would conduct systematic substitutional mutagenesis of the compound molecule while observing for reductions or elimination of cytoprotective or neutralizing activity. In any case, it will be appreciated that due to the size of many of the antibodies, most substitutions will have little effect on binding activity. The great majority of variants will possess at least some cytoprotective or neutralizing activity, particularly if the substitution is conservative. Conservative amino acid substitutions are substitutions from the same class, defined as acidic (Asp, GIu)1 hydroxy- like (Cys, Ser, The), amides (Asn, GIn), basic (His, Lys, Arg), aliphatic-like (Met, He, Leu, VaI, GIy, Ala, Pro), and aromatic (Phe, Tyr, Trp).
Homologous antibody or polypeptide sequences generally will be greater than about 30 percent homologous on an identical amino acid basis, ignoring for the purposes of determining homology any insertions or deletions from the selected molecule in relation to its native sequence. The compounds discussed herein, i.e. autoimmune inhibitors for administration to the patient with autoimmune disease and/or for removal, neutralization or inhibition of the autoimmunogen(s) by extracorporeal immunosorption in accordance with the present invention, also include glycosylation variants as well as unglycosylated forms of the agents, fusions of the agents with heterologous polypeptides, and biologically active fragments of the agents, again so long as the variants possess the requisite neutralizing or cytoprotective activity.
In an embodiment of the invention, treatments involving administration of an autoimmune inhibitor to a patient, and treatments involving the extracorporeal exposure of the patient's fluid to an autoimmune inhibitor, may be performed alone or in combination.
Administered autoimmune inhibitor of the invention binds to, neutralizes and/or inhibits the molecule(s) associated with or causing the autoimmune response in the patient. More specifically, administration of the autoimmune inhibitor to a patient results in suppression of pathological humoral and adaptive immunity in the patient. In other words, in accordance with the method of the present invention, treatment of a patient with the autoimmune inhibitor causes the humoral and adaptive immune response of the patient to be inhibited or neutralized over that which was, or would have been, present in the absence of treatment.
A patient is in need of treatment with an autoimmune inhibitor, when the patient is suffering from an autoimmune disease or when the patient has produced autoantibodies.
The autoimmune inhibitor antibody(ies) is also effective when immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art(Weir et al., "Handbook of experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chap. 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y.(1974)).
For therapeutic purposes, autoantibody gene products may be generated which include proteins that represent functionally equivalent gene products. For example, an equivalent hnRNP-H1 antibody gene product may contain deletions, including internal deletions, additions, including additions yielding fusion proteins, or substitutions of amino acid residues, but that result in a "silent" change, in that the change produces a functionally equivalent auto-hnRNP-H1 antibody gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine,
tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
Alternatively, where alteration of function is desired, deletion or non-conservative alterations can be engineered to produce altered anti-hnRNP-H1 antibody gene products. Such alterations can, for example, alter one or more of the biological functions of the autoantibody gene product. Further, such alterations can be selected so as to generate autoantibody gene products that are better suited for expression, scale up, etc. in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges. This applies to any autoimmune hnRNP-H1 molecule and allelic variants thereof, that are identified in an individual.
The autoantibody gene products, peptide fragments thereof and fusion proteins thereof, of the invention can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors comprising auto-hnRNP-H1 antibody gene product coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook, et a/., 1989, and Ausubel, et a/., 1989. Alternatively, RNA capable of encoding auto-hnRNP-H1 antibody gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, ed., IRL Press, Oxford.
A variety of host-expression vector systems may be utilized to express the autoantibodies, such as anti-hnRNP-H1 gene products. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells that may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the anti- hnRNP-H1 gene product of the invention in situ. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner, et a/., 1987, Methods in Enzymol. 153, 516-544).
In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, JURKAT, Hep2, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138.
A variety of methods can be employed for the diagnostic and prognostic evaluation of Sjogren's syndrome and for the identification of subjects having a predisposition to such autoimmune disorders. Such methods may, for example, detect the presence of hnRNP-H1 gene mutations, or the detection of either over-, under-, or no expression of hnRNP-H1 protein, or mutants.
Mutations at a number of different genetic loci may lead to phenotypes related to autoimmune disorder, structural and synaptic abnormalities. Ideally, the treatment of patients suffering from such disorders will be designed to target the particular genetic loci comprising the mutation mediating the disorder. Genetic polymorphisms have been linked to differences in drug effectiveness. Thus, identification of alterations in hnRNP-H1 molecules, such as, for example, gene or protein can be utilized to optimize therapeutic drug treatments.
In a preferred embodiment, autoimmune related molecule, such as, for example, hnRNP-H1, expression levels, mutations, polymorphisms can be detected by using a microassay of for example, hnRNP-H1 nucleic acid sequences immobilized to a substrate or "gene chip" for detection of hnRNP- H1 molecules (see, e.g. Cronin, ef a/., 1996, Human Mutation 7:244-255). Preferred methods are detailed in the examples which follow.
The level of hnRNP-H1 or any hnRNP-H1 -related molecule gene expression, can also be assayed as described in detail in the examples which follow. Additionally, it is possible to perform hnRNP-H1 gene expression assays "in situ", i.e. directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. For such in situ procedures (see, for example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, N.Y.) standard northern analysis can be performed to determine the level of mRNA expression of the hnRNP-H1 gene.
To assess the efficacy of cell-based gene therapy, in vitro immunoassays can be used. Antibodies directed against hnRNP-H1 gene products may be used in vitro to determine, for example, the level of hnRNP-H1 antibody gene expression achieved in cells genetically engineered to produce such a gene product. In the case of intracellular gene products, such an assessment is done, preferably, using cell lysates or extracts. Such analysis will allow for a determination of the number of transformed cells necessary to achieve therapeutic efficacy in vivo, as well as optimization of the gene replacement protocol.
The tissue or cell type to be analyzed will generally include those that are known, or suspected, to express the hnRNP-H1 gene. The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the hnRNP-H1 gene.
Preferred diagnostic methods for the detection of autoimmune molecules, such as, for example, hnRNP-H1 gene products, conserved variants or peptide fragments thereof, may involve, for example, immunoassays wherein the hnRNP-H1 gene products or conserved variants or peptide fragments are detected by their interaction with an anti-hnRNP-H1 gene product-specific antibody. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
Definitions
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The term "subject" or "patient" refers to any human or animal mammals.
The term "autoimmune disease" (AD) refers to a disease that result from an aberrant immune response of an organism against its own cells and tissues due to a failure of the organism to recognise its own constituent parts (down to the sub-molecular level) as "self. As used herein, "autoimmune disease" is intended to further include autoimmune conditions, syndromes and the like. The term "antigen" as used herein refers to a structure of a macromolecule, typically protein (with or without polysaccharides) or made of proteic composition comprising one or more hapten(s) and/or comprising at least one epitope. An "autoantigen" as used herein refers to a human or animal protein present in the body, which elicits an immune response within the same human or animal body, which may in turn lead to a chain of events, including the synthesis of other autoantigens or autoantibodies. An "autoantibody" is an antibody produced by an autoimmune patient to one or more of his own constituents which are perceived to be antigenic. For example, in SLE autoantibodies are produced to DNA, while in many other types of AD autoantibodies are produced to target cells. Patients suffering from autoimmune diseases including, e.g., rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, systemic lupus erythematosus and others, are in need of treatment in accordance with the present invention. Treatment of patients suffering from these diseases by administration of autoimmune inhibitor and/or removal of compound(s) by extracorporeal immunosorption in accordance with the present invention will alleviate the clinical manifestations of the disease and/or minimize or prevent further deterioration or worsening of the patient's condition. Treatment of a patient at an early stage of an autoimmune disease including, e.g., rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, or others, will minimize or eliminate deterioration of the disease state into a more serious condition. The term "epitope" refers to one or several portions (which may define a conformational epitope) of an antigenic protein which is/are specifically recognised and bound by an antibody or a portion thereof (Fab1, Fab2', etc.) or a receptor presented at the cell surface of a B or T cell lymphocyte, and which is able, by said binding, to induce an immune response. An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids. Methods for determining the
amino acids which make up an epitope include x-ray crystallography, 2 -dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by H. Mario Geysen et al. 1984. Proc. Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506. It is well known to a person skilled in the art that antibodies which recognize and bind an epitope can be monoclonal or polyclonal.
The phrase "specifically (or selectively) binds" to an antibody, when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to marker "X" from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with marker "X" and not with other proteins, except for polymorphic variants and alleles of marker "X". This selection may be achieved by subtracting out antibodies that cross-react with marker "X" molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid- phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. "Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g. a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
"Diagnostic" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 (or 100 when indicated as percentage) minus the (percentage) false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
As used herein, "molecule" is used generically to encompass any vector, antibody, protein, drug and the like which are used in therapy and can be detected in a patient by the methods of the invention. For example, multiple different types of nucleic acid delivery vectors encoding different types of genes which may act together to promote a therapeutic effect, or to increase the efficacy or selectivity of gene transfer and/or gene expression in a cell. The nucleic acid delivery vector may be provided as
naked nucleic acids or in a delivery vehicle associated with one or more molecules for facilitating entry of a nucleic acid into a cell. Suitable delivery vehicles include, but are not limited to: liposomal formulations, polypeptides; polysaccharides; lipopolysaccharides, viral formulations (e.g. including viruses, viral particles, artificial viral envelopes and the like), cell delivery vehicles, and the like.
Detailed description
We identified hnRNP-H1 as a new target of auto-antibodies in patients with primary and secondary Sjogren's syndrome. The sensitivity of anti-hnRNP-H1 antibodies for primary and secondary Sjogren's syndrome was 43%. This was lower than the sensitivity of anti-SSA antibodies which was 85.3% (71.4% in the smaller group of Sjogren's disease patients [n=7] in the group of consecutive samples). The specificity of anti-hnRNP-H1 antibodies (91.7% - 94.4%) tended to be higher than the specificity of anti-SSA antibodies (78.6% - 89%). The low specificity of anti-SSA antibodies is mainly related to the high prevalence of anti-SSA antibodies in patients with SLE. In contrast to anti-SSA antibodies, anti-hnRNP-H1 antibodies were not very much prevalent in patients with SLE. Anti-hnRNP-H1 antibodies are an interesting diagnostic marker in anti-SSA and/or anti-SSB seronegative patients with primary Sjogren's syndrome. We found that 5/11 (45%) anti-SSA/anti-SSB seronegative patients had anti-hnRNP-H1 antibodies.
The results originally obtained by Western blot analysis do not fully correlate with the results obtained by ELISA. This can be due to the conformation of hnRNP-H1 in both assays. In Western blot analysis, the antigen is denatured and reduced, whereas the ELISA technique uses the native recombinant protein. Since the prevalence of anti-hnRNP-H1 antibodies in patients with primary Sjogren's disease is higher in both methods, we can conclude that hnRNP-H1 is a new autoantigen and a valuable additional diagnostic marker for Sjogren's syndrome. We also investigated the presence of anti-hnRNP-H1 antibodies in 188 consecutive antinuclear (anti- cytoplasmic) antibody-positive samples submitted to the clinical laboratory. This approach, in which samples are relatively randomly selected, gives information on the repertoire of diseases that may lead to the production of anti-hnRNP-H1 antibodies. The likelihood ratio of a positive test result was higher for anti-hnRNP-H1 antibodies than for anti-SSA antibodies in the first cohort (n= 246, serum samples from 246 patients with well-defined autoimmune connective tissue disorders) but not in the second cohort (n=188, consecutive serum samples submitted to the clinical laboratory for ANA testing). The likelihood ratio of a negative test result for Sjogren's disease was lower for anti-SSA antibodies than for anti-hnRNP-H1 antibodies. The positive predictive value of anti-hnRNP-H1 for Sjogren's syndrome is comparable to the positive predictive value of anti-SSA for Sjogren's syndrome. The negative predictive value is higher for anti-SSA antibodies than for anti-hnRNP-H1 antibodies. The analysis of the 188 consecutive (random) samples revealed relevant information on the specificity of anti-hnRNP-H1 antibodies and showed that these antibodies can be found, to a lesser extent in a number of other autoimmune diseases. One explanation is that in some of these patients, the detection of hnRNP-H1 antibodies is indicative for the development of secondary Sjogren's syndrome since we have shown (in example 2) that hnRNP-H1 auto-antibodies are an early marker for Sjogren's
syndrome. Also anti-SSA antibodies are found, to a bigger extent compared with hnRNP-H1 antibodies, in a wide range of medical conditions with a possible autoimmune pathogenesis. Anti-hnRNP-H1 antibodies were not only found in samples with a speckled ANA pattern, but also in samples with a homogeneous staining (nuclear dots) pattern. Five of the 18 patients with anti-hnRNP- H1 antibodies received Infliximab, a TNF-α blocking drug, and displayed a homogeneous staining on ANA indirect immunofluorescence analysis. It is known that Infliximab can induce the formation of auto-antibodies, especially anti-dsDNA antibodies. We assumed that in such cases the typical speckled pattern caused by anti-hnRNP-H1 antibodies was masked by other antibodies. The fact that we identified anti-hnRNP-H1 antibodies in patients with symptoms suggestive of Sjogren's syndrome (sicca complaints, hypergammaglobulinemia) but in whom the diagnosis was not (yet) established suggest that the antibodies are an early serologic indicator of primary Sjogren's syndrome.
In the present invention we described hnRNP-H1 as a new target of autoantibodies in patients with Sjogren's syndrome. Therefore, we identified anti-hnRNP-H1 antibodies as a valuable diagnostic marker in Sjogren's syndrome.
The present invention will now be illustrated by means of the following examples which are provided without any limiting intention.
Examples
EΞXAMPLE 1: Materials and Methods Materials
Recombinant his-tagged hnRNP-H1 protein from rat was prepared as described (Fijak M et al., J Pathol. 2005;207: 127-38). Recombinant GST-tagged hnRNP-H1 protein from human was prepared from cDNA according to Devogelaere et al. (Biochem. J. 2007;407:303-311). o
Polyvinylidene difluoride (PVDF) membranes (Hybond-P) and IPG strips (pH-range 3-10, ImmobilineTM DryStrip gels) were from GE Healthcare. Rabbit thymus, goat anti-human IgG, and HPLC grade water were obtained from Sigma-Aldrich. Horseradish peroxidase conjugated rabbit anti- goat IgG was from DakoCytomation. C18 clean-up (ZipTip) was from Millipore Corporation. Purified rabbit anti-hnRNP-H1 antibodies were from Acris Antibodies.
Study population
Several groups of serum samples were included in the study.
A first group consisted of samples obtained from well-defined patients with an autoimmune connective tissue disease (n=246). This included patients with systemic lupus erythematosus (SLE) (n=41 , male/female ratio 5/36, mean age 45 years, range 23-80 years), primary Sjogren's syndrome (n=93, male/female ratio 9/84, mean age 60 years, range 16-94 years), scleroderma (n=39, male/female ratio 10/29, mean age 55 years, range 22-72 years), dermatomyositis (n=15, male/female ratio 6/9, mean
age 48 years, range 25-80 years), polymyositis (n=7, male/female ratio 2/5, mean age 57 years, range 47-70 years), rheumatoid arthritis (RA) (n=42, male/female ratio 13/29, mean age 58 years, range 33- 90 years) and mixed connective tissue disease (MCTD) (n=9, male/female ratio 2/7, mean age 45 years, range 18-63 years). Ten of these patients had secondary Sjogren's syndrome (n=10, male/female ratio 3/7, mean age 54 years, range 34-86 years), four in combination with SLE, two with scleroderma, one with dermatomyositis, two with RA and one with MCTD. The samples were obtained from patients attending the University Hospitals Leuven (Belgium). Seventy seven consecutive patients with primary Sjogren's syndrome were from Rotterdam Erasmus MC, University Medical Center Rotterdam (The Netherlands). The prevalence of anti-nuclear antibody (ANA) positivity in this patient group was 65.9%.
A second group of samples consisted of 188 consecutive serum samples submitted to the clinical laboratory for ANA testing (titer ≥ 160).
Finally, a group of ANA negative serum samples from healthy subjects (n=41 , male/female ratio 4/37, mean age 45 years, range 27-63 years) was included. All patients with primary or secondary Sjogren's syndrome fulfilled the American-European consensus classification criteria (Vitali ef a/., Ann Rheum Dis. 2002;61:554-8). Patients with SLE, scleroderma, poly- and dermatomyositis, RA and MCTD met the classification criteria of the American College of Rheumatology (Hochberg MC, Arthritis Rheum. 1997;40:1725; Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum. 1980;23:581-90 ; Bohan A et al., Medicine (Baltimore) 1977;56:255-86; American College of Rheumatology Subcommittee on Rheumatoid Arthritis Guidelines. Guidelines for the management of rheumatoid arthritis: 2002 update. Arthritis Rheum. 2002;46:328-46; Alarcon-Segovia D and Cardiel MH, J Rheumatol. 1989;16:328-34). The study was approved by the local Ethics Committee.
Antinuclear antibody testing, identification of antibodies to extractable nuclear antigens, counterimmunoelectrophoresis
ANA were determined by indirect immunofluorescence using HEp-2000® cells (Immunoconcepts) with an Axioplan 2 fluorescence microscope (Carl Zeiss Microimaging) and Cytovision 3.6 software (Applied Imaging Corporation). The presence of antibodies to ENAs was assayed by ENA dot blot (BioMedical Diagnostics), UniCaplOO and/or UniCap250 system (Phadia Diagnostics), ANA3 test (anti-SSA 52/anti-SSA 60 status, Euroimmun), or counterimmunoelectrophoresis with rabbit thymus as antigenic source.
Ge/ electrophoresis and Western blotting A salt-based protein extract from rabbit thymus (20 μg) or 300 ng of recombinant hnRNP-H1 were separated by one dimensional SDS-PAGE (12.5%). For the separation of proteins in two dimensions, immobilized pH gradient strips (pH-range 3-10, GE Healthcare) were rehydrated overnight using ~300 μg of protein extract from rabbit thymus. Loaded proteins were first separated by iso-electric focusing and than by SDS-PAGE (12.5%) on a Multiphor Il Electrophoresis System (GE Healthcare) according
to the supplier's instructions. The proteins either underwent Western blotting or were stained by Coomassie Brilliant blue.
For Western blot analysis, polyvinylidene difluoride (PVDF) membranes with electrotransferred proteins (Hybond-P and Novablot apparatus; GE Healthcare) were consecutively treated with 5% (w/v) BSA, human serum (1 :500, overnight), goat anti-human IgG (1 :5,000) and horseradish peroxidase conjugated rabbit anti-goat IgG (1:5,000) with intermittent washings in TBS (3 x 10 min). Protein- antibody interactions were visualized using 0.7 mM 3.3'diamino-benzidinetetrahydrochloride. Blotting signals were scanned using a Typhoon 9400 scanner set on green laser light (532 nm), filter 526 SP, PMT 640V at normal sensitivity and quantified by ImageQuant TL software (both from GE Healthcare). A positive and negative control serum sample were included in each run. The coefficient of variation of the positive control was 23.8% over 13 runs.
Protein identification by MALDI-TOF/TOF
Gel pieces containing the protein of interest were washed with HPLC grade water, dried in a Speed Vac (Savant) and digested overnight at 37 0C with 10 μl of 25 ng/μl trypsin (sequence grade) in 200 mM ammonium bicarbonate. The resulting peptide mixture was subjected to a C18 clean-up (ZipTip) and analyzed by a MALDI-TOF/TOF (Applied Biosystems 4800 Proteomics Analyzer) in the presence of α-cyano 4-hydrocinnamic acid (HPLC grade). Tandem MS data were submitted to the MASCOT search engine (https://www. matrixscience.com) for protein identification using default parameters.
Statistical analysis
Receiver operating characteristics (ROC) analysis, Kruskall-Wallis ANOVA, Mann-Whitney, and Fisher Exact testing was performed using Analyze-lt for Microsoft Excel (version 2.09). Spearman correlation and ordinal regression analysis was performed using SPSS 15.0 for Windows.
EXAMPLE 2: Results
Identification of anti-hnRNP-H1 antibodies
In an attempt to identify new target antigens of ANA we applied an immuno-proteomics approach using serum from a 43-year old patient who had ANA in high titer (>1280) but with no reactivity to SSA, SSB, U1-RNP, Sm, Scl-70, or Jo-1. Counterimmunoelectrophoresis showed a positive precipitation line. Laboratory investigations revealed a polyclonal gammopathy and a positive rheumatoid factor. The patient was diagnosed with an undifferentiated autoimmune connective tissue disease. Primary Sjogren's syndrome was suspected but the patient didn't fulfill the EU-US classification criteria.
Serum from the patient and from a control individual were used for Western blotting after 2D gel separation of rabbit thymus extract. Proteins to which there was reactivity were excised and identified by MALDI-TOF/TOF analysis, and for one of the proteins identified here, a significant score was obtained for the protein hnRNP-H1 from mouse (Mus musculus) mining the SwissProt database (HnηpM, 035737) or Ratsgi from rat (Rattus norvegicus) screening the NCBI database {Hnrnphi, Q8VHV7) (see Fig.1). Alpha-enolase reacted with serum from the patient and from the control,
whereas β-actin, heat shock protein 70 kDa, heterogeneous nuclear ribonucleoprotein (hnRNP)A2/B1, and hnRNP-H1 reacted only with serum from the patient. In subsequent analyses we focused on hnRNP-H1 because this is a common nuclear matrix protein that had not been described as autoantigen in human disease before. The presence of anti-hnRNP-H1 antibodies in the patient's serum was confirmed by 1D SDS-PAGE and Western blotting using recombinant hnRNP-H1 from rat and human. A positive blotting signal was obtained with serum from the patient but not from the control (Fig. 2). HnRNP-HI from rat (Q8VHV7|Q8VHV7_RAT, Swiss-Prot Database) has 99% similarity with human hnRNP-H1 (P31943|HNRH1_HUMAN, Swiss-Prot Database) but lacks 77 N- terminal amino acids. We confirmed that anti-hnRNP-H1 antibodies give a speckled fluorescence pattern on indirect immunofluorescence analysis by two complementary approaches. First, incubating HEp-2 cells with a commercial rabbit anti-hnRNP-H1 antibody resulted in a fine speckled antinuclear antibody pattern (Fig. 3A). Second, pre-absorption of a serum sample with reactivity to hnRNP-H1, using increasing concentrations of recombinant rat hnRNP-H1 resulted in a gradual decrease of the fine speckled fluorescent signal on indirect immunofluorescence analysis (Figs. 3 B-E). Pre-absorption of a serum sample with reactivity to hnRNP-H1 with recombinant rat hnRNP-H1 also resulted in decreased reactivity on Western blotting.
Anti-hnRNP-H1 antibodies are associated with Sjogren's Syndrome In order to examine whether anti-hnRNP-H1 antibodies are associated with Sjogren's syndrome, we tested serum samples from 246 patients with well-defined autoimmune connective tissue disorders for reactivity to hnRNP-H1 in Western blot analysis. The results are shown in Fig. 4. Anti-hnRNP-H1 reactivity was significantly higher in patients with primary Sjogren's syndrome compared to patients with other connective tissue disease (p<0.0001; Kruskall-Wallis ANOVA with Bonferroni adjustments and p=0.0045; Mann-Whitney test). A receiver operating characteristics analysis was performed for anti-hnRNP-H1 antibodies as a marker of primary Sjogren's syndrome. The area under the curve was 0.83 (n=93 patients with primary Sjogren's syndrome vs. 153 diseased controls). The sensitivity of anti-hnRNP-H1 antibodies for primary Sjogren's syndrome was 48.4% when a cutoff ('peak x area' 1.90) was used that corresponded to a specificity of 94.8% in diseased controls. When a cutoff ('peak x area' 0.71) was used that maximized the sum of sensitivity and specificity, then the sensitivity of anti- hnRNP-H1 antibodies for primary Sjogren's syndrome was 74.2% and the specificity 81.7% (Youden index 0.559). In all subsequent analyses, the cutoff based on a specificity of 95% was used. Anti- hnRNP-H1 antibodies were found in 48% (45/93) of patients with primary Sjogren's syndrome, in 2.4% (1/41) of patients with SLE, in 22% (2/9) of patients with MCTD, in 7.7% (3/39) of patients with scleroderma, in 0% (0/15) of patients with dermatomyositis, in 0% (0/7) of patients with polymyositis, in 4.8% (2/42) of patients with RA, and in 5% (2/41) of healthy individuals. Fisher Exact Test indicated a statistically significant difference (p=0.025) between the prevalence of anti-hnRNP-H1 antibodies in patients with primary Sjogren's disease and the prevalence in healthy subjects and in patients with other connective tissue diseases, with the exception of MCTD (p=0.247). This could be due to the small number of patients with MCTD (n = 9) tested. In the diseased control group 8 patients with anti-
hnRNP-M antibodies were identified (1 with SLE, 2 with RA, 2 with MCTD, and 3 with scleroderma) but none of them fulfilled the EU-US criteria of secondary Sjogren's syndrome. For comparison, the sensitivity and specificity of anti-SSA antibodies for primary Sjogren's syndrome in the same patient cohort was 88.2% and 76.3%, respectively. Anti-SSA antibodies were found in 33% of MCTD, in 7.7% of patients with scleroderma, and in 63.4% of patients with SLE. Receiver operating characteristics analysis for anti-hnRNP-H1 antibodies as a marker of both primary and secondary Sjogren's syndrome revealed an area under the curve of 0.80 (n=103 patients with Sjogren's syndrome vs. 143 diseased controls). The sensitivity of anti-hnRNP-H1 antibodies for Sjogren's syndrome was 43.7% and the specificity 94.4%. The area under the curve for anti-SSA antibodies was 0.82. The sensitivity and specificity of anti-SSA antibodies in the same patient group was 85.3% and 78.6%, respectively. The specificity of anti-hnRNP-H1 antibodies for Sjogren's disease was higher than the specificity of anti-SSA antibodies. This is mainly related to the high prevalence of anti-SSA antibodies in patients with SLE. The likelihood ratios were 7.81, 0.60, 3.98, and 0.19 for, respectively, hnRNP-H1 positive, hnRNP-H1 negative, SSA positive, and SSA negative. Eleven out of 93 patients with Sjogren's syndrome were seronegative for anti-SSA (both anti-SSA 52 and anti-SSA 60 kDa) and anti-SSB antibodies. Anti-hnRNP-H1 antibodies were found in 5 of these 11 seronegative patients (45%) (Table 1).
In patients with primary Sjogren's syndrome, we analyzed whether anti-hnRNP-H1 antibodies were correlated with important parameters of Sjogren's disease such as Schirmer's type I test, lip biopsy data (FOCUS score), rheumatoid factor, anti-SSA and anti-SSB antibodies, hypergammaglobulinemia, or cryoglobulinemia. Spearman correlation and multivariate ordinal regression analysis revealed no association with any of these parameters.
Table 1 Anti-hnRNP-H1 antibodies (tested by Western blot) as a function of anti-SSA (52 and 60 kDa) and anti-SSB antibodies in patients with primary Sjogren's syndrome (n=93). FOCUS score of the anti-hnRNP-H1 positive serum samples is indicated. ND means not determined.
Another method for the evaluation of anti-hnRNP-H1 antibodies in a study cohort of 246 serum samples of patients with autoimmune connective tissue diseases and 41 healthy subjects is an ELISA using recombinant His-tagged rat hnRNP-H1. Using this method, the prevalence of anti-hnRNP-H1 antibodies was 22.5% for patients with primary Sjogren's disease. Figure 5 shows the reactivity of patient's serum samples to hnRNP-H1 by performing an ELISA using recombinant His-tagged hnRNP-
H1 from rat. ELISA detected anti-hnRNPH1 antibodies in 2.4% (1/41) of patients with SLE1 in 22% (2/9) of patients with MCTD, in 7.7% (3/39) of patients with scleroderma, in 13.3% (2/15) of patients with dermatomyositis and in 2.5% (1/40) of healthy individuals. No anti-hnRNP-H1 antibodies were detected in patients with RA (0/42) or polymyositis (0/7). A statistically significant difference between the prevalence of anti-hnRNP-H1 antibodies in patients with primary Sjogren's syndrome, healthy controls and diseased controls was found (p=0.0063, Fisher Exact Test).
These experiments also revealed that 36% (4/11 ) of anti-SSA and anti-SSB seronegative patients with primary Sjogren's syndrome have anti-hnRNP-H1 antibodies. Table 2 provides an overview of the results.
Table 2. Anti-hnRNP-H1 antibodies tested by ELISA as a function of anti-SSA and anti-SSB antibodies in patients with primary Sjogren's disorder.
ROC analysis for anti-hnRNP-H1 antibodies as a marker of both primary and secondary Sjogren's syndrome indicated an area of 0.75 (n=103 patients with Sjogren's syndrome vs. 143 diseased controls). The sensitivity of anti-hnRNP-H1 antibodies for Sjogren's syndrome was 19.4% and the specificity 94.4% (cutoff 110, based on 95% specificity for diseased controls).
Anti-hnRNP-H1 antibodies in a routine clinical laboratory setting
Subsequently, we investigated the presence of anti-hnRNP-H1 antibodies (by Western blot analysis) in samples submitted for ANA testing (n=188). This approach may give information on the repertoire of diseases that may lead to the production of anti-hnRNP-H1 antibodies. Anti-hnRNP-H1 antibodies were identified in 18 out of 188 consecutively obtained ANA-positive (titer>160) samples. The fluorescence patterns in these 18 samples were speckled (n=4), homogenous (n=13) and nuclear dots (n=1). Anti-hnRNP-H1 antibodies were present in 3 of 7 patients with Sjogren's disease (primary or secondary). Three of the 18 patients had secondary Sjogren's syndrome (one in combination with SLE and 2 in combination with RA [treated with anti-TNF-α]). The patient with SLE had anti-SSA and anti-SSB antibodies. One of the patients with RA and secondary Sjogren's syndrome had anti-SSA antibodies, whereas the other patient had no anti-SSA antibodies or anti-SSB antibodies. One patient had sicca complaints, Raynaud's phenomenon, hypergammaglobulinemia and cryoglobulinemia. Primary Sjogren's syndrome was assumed in this patient, but he didn't fulfill the EU-US classification criteria. Another patient had undifferentiated connective tissue disease with polyarthritis and autoimmune thyroid disease. Other pathologies included SLE (n=2), ankylosing spondylitis (n=2, both receiving anti-TNF-α), MCTD (n=2), and one each with RA (receiving anti-TNF-α), idiopathic
thrombocytopenic purpura, Wegener's granulomatosis with idiopathic thrombocytopenic purpura, polyarticular juvenile idiopathic arthritis, ulcerative colitis and spondylarthropathy, liver transplantation and no diagnosis. Table 3 provides an overview of the clinical conditions in the 188 patients as well as the anti-hnRNP-H1 and the anti-SSA antibody status. In this patient group, the sensitivity and specificity of anti-hnRNP-H1 for Sjogren's syndrome was 42.9% and 91.7%, respectively. For anti- SSA antibodies, the sensitivity and specificity were, respectively, 71.4% and 89.0%. The likelihood ratios were 5.16, 0.62, 6.46, and 0.32 for, respectively, hnRNP-H1 positive, hnRNP-H1 negative, SSA positive, and SSA negative.
Table 3 Clinical conditions and anti-hnRNP-H1 and anti-SSA antibodies in 188 consecutive samples submitted to the clinical laboratory with an antinuclear antibody titer > 1:160
Medical condition Anti-hnRNP-M (n) Anti-SSA (n) Total (n)
Sjogren's syndrome - primary 0 2 2
Sjogren's syndrome - secondary 3 3 5
Undifferentiated autoimmune disease 2 0 2
Systemic lupus erythematosus 2 16 39
Rheumatoid arthritis 1 0 41
Juvenile arthritis 1 0 4
Spondylitis ankylosans 2 1 19
Mixed connective tissue disease 2 1 5
Dermatomyositis 0 0 1
Diffuse scleroderma 0 1 7
Limited scleroderma 0 0 1
Idiopathic thrombocytopenic purpura 2 0 2
Inflammatory bowel disease 1 0 5
Giant cell arteritis 0 0 2
Autoimmune hepatitis 0 0 3
Autoimmune thyroiditis 0 0 1
Miscellaneous 2 1 49
Total 18 25 188
Miscellaneous: cervicobrachialgia (n=1), chronic urticaria (n=1), chronic fatigue syndrome (n=7), degenerative disease (n=3), diabetes (n=1), fibromyalgia (n=1), no diagnosis (n=9), Goodpasture (n=1), thrombosis (n=1), leucocytoclastic vasculitis (n=1), obesitas (n=1), liver transplantation (n=1), liver dysfunction (n=1), lung fibrosis (n=1), lymphoma (n=1), macrophage activating syndrome (n=1), mitochondrial myopathy (n=1), multiple sclerosis (n=3), nephritic syndrome (n=1), encephalopathy (n=1), cholecystectomy (n=1), pityriasis lichenoides (n=1), pneumonia (n=4), polyarthritis (n=1), polymyalgia rheumatica (n=1), primary biliary cirrhosis (n=1), psoriasis (n=1), psoriatic spondylarthropathy (n=1), paraparesis lower limbs (n=1).
Claims
1. A method for the determination of the predisposition of a patient to develop an autoimmune disease comprising the determination of the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from said patient, the presence of said autoantibodies being indicative for a predisposition to develop said autoimmune disease.
2. The method of claim 1 , wherein said autoimmune disease is Sjogren's syndrome.
3. The method of claims 1 or 2, wherein said determination of the presence of autoantibodies against hnRNP-H1 is determined in an immunoassay.
4. The method of claim 3, wherein said immunoassay employs an antibody-containig, isolated biological sample obtained from said patient.
5. The method of claims 3 or 4, wherein said immunoassay is selected from the group consisting of ELISA, FEIA, western blot, dot blot, bead-based assay, antigen array and Radio lmmuno Assay.
6. The method of any of the claims 1 to 5, wherein the isolated biological sample is a fluid selected from the group consisting of blood, serum, plasma, saliva, tears, mucus and ascites fluid.
7. The method of any of the claims 1 to 6, characterized in that hnRNP-H1 proteins, peptides, variants or fragments thereof are used for determining the presence of said autoantibodies.
8. The method of any of the claims 1 to 7, characterized in that an immobilized hnRNP-H1 -antigen is used for determining the presence of said autoantibodies.
9. A method for detecting hnRNP-H1 antibodies in an isolated biological sample, the method comprising the steps of:
(a) obtaining an isolated biological sample from a patient
(b) analyzing said isolated biological sample for the presence of antibodies against hnRNP- H1.
10. The method of claim 9, wherein the isolated biological sample in step (a) is an antibody-containig sample.
11. The method of claim 9 or 10, wherein the analysis in step (b) is performed using an immunoassay.
12. The method of claim 11, wherein said immunoassay is selected from the group consisting of ELISA, FEIA, western blot, dot blot, bead-based assay, antigen array and Radio lmmuno Assay.
13.The method of any of the claims 9 to 12, wherein said isolated biological sample is a fluid selected from the group consisting of blood, serum, plasma, saliva, tears, mucus and ascites fluid.
14. The method of any of the claims 9 to 13, characterized in that hnRNP-H1 proteins, peptides, variants or fragments thereof are used for determining said hnRNP-H1 antibodies.
15. The method of any of the claims 9 to 14, characterized in that an immobilized hnRNP-H1 antigen is used for determining said hnRNP-H1 antibodies.
16. The method of any of the claims 9 to 15, wherein the presence of antibodies against hnRNP-H1 in said isolated biological sample is indicative for the predisposition of said patient to develop an autoimmune disease.
17. The method of claim 16, wherein said autoimmune disease is Sjogren's syndrome.
18. The method of any of the claims 1 to 17, for predicting responsiveness to a medicament.
19. The use of hnRNP-H1 proteins, peptides, variants or fragments thereof to detect the presence of autoantibodies against hnRNP-H1 in an isolated biological sample derived from a patient.
20. The use according to claim 19 for carrying out the method according to claims 1 to 18.
21. A test kit for detecting the presence of antibodies against hnRNP-H1 in an isolated biological sample.
22. The test kit as claimed in claim 21, additionally comprising a solid phase onto which the antigen is or can be bound.
23. The test kit as claimed in claim 21 or 22, additionally comprising a labeling group which is bound to the antigen or can be bound thereto.
24. The test kit as claimed in any of the claims 21 or 23, additionally comprising at least one other antibody class-specific test reagent.
25. The use of the test kit according to any of the claims 21 to 24 for carrying out the method according to claims 1 to 18.
26. The use of a compound specifically binding to hnRNP-H1 autoantibodies for the production of a medicine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0721160A GB0721160D0 (en) | 2007-10-29 | 2007-10-29 | New method for diagnosing sjogren's syndrome |
| PCT/BE2008/000087 WO2009055880A2 (en) | 2007-10-29 | 2008-10-29 | New method for diagnosing sjogren's syndrome |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP2235540A2 true EP2235540A2 (en) | 2010-10-06 |
Family
ID=38830103
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP08845893A Withdrawn EP2235540A2 (en) | 2007-10-29 | 2008-10-29 | New method for diagnosing sjogren's syndrome |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP2235540A2 (en) |
| GB (1) | GB0721160D0 (en) |
| WO (1) | WO2009055880A2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2936060B1 (en) * | 2008-09-17 | 2010-11-19 | Assist Publ Hopitaux De Paris | METHOD FOR DIAGNOSING SYSTEMIC SCLERODERMY OR PULMONARY ARTERIAL HYPERTENSION |
| GB0915332D0 (en) | 2009-09-02 | 2009-10-07 | Univ Leuven Kath | New methods for diagnosing autoimmune diseases |
| GB2622246A (en) * | 2022-09-08 | 2024-03-13 | Oncimmune Germany Gmbh | Antibody assay |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE60138405D1 (en) * | 2000-10-17 | 2009-05-28 | Bio Rad Laboratories | SCHNE AUTOIMMUNE DISEASES |
| WO2005116652A2 (en) * | 2004-04-16 | 2005-12-08 | Vanderbilt University | Method for in vitro diagnosis of sjörgen’s syndrome and lupus |
-
2007
- 2007-10-29 GB GB0721160A patent/GB0721160D0/en not_active Ceased
-
2008
- 2008-10-29 WO PCT/BE2008/000087 patent/WO2009055880A2/en active Application Filing
- 2008-10-29 EP EP08845893A patent/EP2235540A2/en not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO2009055880A3 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2009055880A3 (en) | 2009-06-04 |
| GB0721160D0 (en) | 2007-12-05 |
| WO2009055880A2 (en) | 2009-05-07 |
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