HK1144015A - Marker sequences for rheumatoid arthritis and use thereof - Google Patents

Description

Marker sequence for rheumatoid arthritis and application thereof

The invention relates to novel marker sequences for rheumatoid arthritis, to the diagnostic use thereof, and to methods for screening potential active substances for rheumatoid arthritis by means of these marker sequences. Furthermore, the invention relates to a diagnostic device, in particular a protein biochip, comprising a marker sequence for rheumatoid arthritis of this type and to the use thereof.

Protein biochips are gaining increasing industrial importance in analysis and diagnosis as well as in drug development. Protein biochips have been established as a screening tool.

Rapid and highly parallel detection of multiple specific binding assay molecules in a single experiment is thereby made possible. To prepare a protein biochip, the desired protein must be obtained. For this purpose, in particular protein expression libraries have been established. High-throughput cloning of defined open reading frames is one possibility (Heyman, J.A., Cornthwaite, J.M., Foncerada, L., Gilmore, J.R., Gontang, E., Hartman, K.J., Hernandez, C.L., Hood, R.L., Hull, H.M., Lee, W.Y., Marcil, R.s., Marsh, E.J., Mudd, K.M., Patino, M.J., Purcell, T.J., Rowland, J.J., Sindici, M.L. and Hoeffl, J.P., (1999), Genome-scale cloning and expression of expression vectors, gene expression of genes, protein, expression of proteins, protein, t, Hudson, j.r., jr., Hartley, j.l., bracho, m.a., Vandenhaute, j., Boulton, s., Endress, g.a., Jenna, s., Chevet, e., papastirolous, v., Tolias, p.p., ptack, j., Snyder, m., Huang, r, Chance, m.r., Lee, h., doucet-stam, l., Hill, d.e., and Vidal, m. (2003) c.elegans orome version 1.1: (ii) experimental version of the genomic and resource for protein-scale protein expression. nat Genet, 34, 35-41; walhout, a.j., Temple, g.f., brachch, m.a., Hartley, j.l., Lorson, m.a., van den Heuvel, s. and Vidal, m. (2000) GATEWAY interactive fastening: application of the closing of large numbers of open reading frames or organisms, methods Enzymol, 328, 575 and 592). However, this type of approach is strongly linked to advances in genome sequencing engineering and annotation of these gene sequences. Moreover, the determination of the expressed sequence may be ambiguous due to the differential splicing process. This problem can be circumvented by the use of cDNA expression libraries (B ü show, K., Cahill, D., Nietfeld, W., Bancroft, D., Scherzinger, E., Lehrach, H., and Walter, G. (1998) A method for a viral protein expression and expression assay, e., C., Lehrach, H., and Walter, C., G., 26, 5007-, protein expr. purif., 20, 372-378). The cDNA for a particular tissue is thus cloned into a bacterial or eukaryotic expression vector, such as, for example, yeast. Vectors for expression are generally characterized in that they carry an inducible promoter, which can be used to control the timing of protein expression. Moreover, the expression vector has a sequence for what is called an affinity epitope or affinity protein which, on the one hand, allows the specific detection of the recombinant fusion protein by means of antibodies directed against said affinity epitope and, on the other hand, makes possible the specific purification by affinity chromatography (IMAC).

For example, gene products from a cDNA expression library derived from human fetal brain tissue in the bacterial expression system Escherichia coli are arrayed in a high density on a membrane and can be successfully screened with different antibodies. It may be shown that the proportion of full-length protein is at least 66%. Furthermore, recombinant proteins from the library can be expressed and purified in a high-throughput manner (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E., and LaBaer, J. (2002) Protein-scale purification of human proteins from bacteria, Proc Natl Acad Sci USA, 99, 2654-. Protein biochips of this type based on cDNA expression libraries are in particular the subject of WO99/57311 and WO 99/57312.

Furthermore, in addition to antigen-presenting protein biochips, Antibody-presenting arrays have been described in the same way (Lal et al (2002) Antibody arrays: An immunological amplification growth technology, DDT, 7, 143-containing 149; Kusnew et al (2003), Antibody microarrays: evaluation of production parameters, Proteomics, 3, 254-containing 264).

However, there is a great need to provide indication-specific diagnostic devices, such as protein biochips.

Marker sequences for rheumatoid arthritis and their diagnostic use, in particular in protein biochip embodiments, and in this respect in assays for active substance screening, are not described in the prior art.

It is therefore an object of the present invention to provide marker sequences and their diagnostic use.

The provision of specific marker sequences allows a reliable diagnosis and classification of patients suffering from rheumatoid arthritis, in particular by means of protein biochips.

The invention therefore relates to the use of marker sequences for rheumatoid arthritis, at least one marker sequence being determined in or from a patient to be examined, the marker sequence being the cDNA selected from the group SEQ1-488, or a protein encoded by it, respectively, or a partial sequence or fragment thereof (hereinafter: marker sequences according to the invention).

By means of a differential screening of samples from healthy test subjects and samples from patients with rheumatoid arthritis, it is possible to identify marker sequences according to the invention.

The term "Rheumatoid Arthritis (RA)" is defined, for example, according to pschmembel, de Gruyter, 261st edition (2007), Berlin. According to the invention, "juvenile congenital arthritis" is likewise covered (ICD-10: M08.-. abbr.: jia. older synonyms: juvenile rheumatoid arthritis, juvenile chronic arthritis, Morbus Still or more generally: childhood rheumatism), which is a collective term (definition, e.g. according to pscharyrembel, de Gruyter, 261) for the major joint diseases (arthritis) in the category of rheumatoid diseases in a plurality of children (adolescents)^stedition (2007), Berlin). This is a polygenic disease which can be diagnosed particularly advantageously by the marker sequence according to the invention, preferably by the marker sequence SEQ 401-488.

In a further embodiment at least 2 to 5 or 10, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences are determined in or from the patient to be examined.

In a particular embodiment of the invention, the marker sequences of SEQ1 to 20 are particularly preferred, the marker sequences SEQ 21 to 50 are preferred and the marker sequences SEQ 51 to 100 are preferred.

In a further embodiment of the invention, the marker sequences SEQ1 to 10 and SEQ 11 to 20, and preferably SEQ 21 to 30, SEQ 31 to 40 or SEQ 41 to 50, respectively, are particularly preferred.

Furthermore, preference is given to the marker sequences SEQ401-488 for the diagnosis of juvenile rheumatoid arthritis, in particular SEQ 401-420, SEQ 421-440, SEQ 441-460 and SEQ 461-488.

In a further embodiment of the invention, the marker sequences according to the invention can likewise bind, complement, fuse or amplify the known biomarkers for this indication as such.

In a preferred embodiment, the determination of the marker sequence is performed outside the human body and the determination is performed in an ex vivo/in vitro diagnosis.

In a further embodiment of the present invention, the present invention therefore relates to the use of marker sequences as diagnostic reagents, wherein at least one marker sequence is a cDNA selected from the group SEQ1 to 488, or a protein encoded respectively thereof, or a partial sequence or fragment thereof, respectively.

Furthermore, the present invention relates to a method for the diagnosis of rheumatoid arthritis, wherein a.) at least one marker sequence selected from the group of cDNA of SEQ1 to 488, or the proteins encoded respectively thereof, or the partial sequences or fragments thereof, respectively, is applied on a solid support; and b.) contacting it with a body fluid or tissue extract of the patient; and c.) performing detection of the interaction between the body fluid or tissue extract and the marker sequence from a).

The invention therefore likewise relates to diagnostic reagents for the diagnosis of rheumatoid arthritis, which are respectively selected from the group SEQ1 to 488, or respectively the proteins encoded thereby, or respectively the partial sequences or fragments thereof.

Detection of this type of interaction can be carried out, for example, by means of probes, in particular by means of antibodies.

The invention therefore likewise relates to the object of providing a diagnostic device or test, in particular a protein biochip, which allows the diagnosis or detection of rheumatoid arthritis.

Furthermore, the invention relates to a method for the stratification, in particular the risk stratification and/or therapy control of patients suffering from rheumatoid arthritis, wherein at least one marker sequence of the cDNA selected from the group SEQ1 to 488 or of the proteins encoded respectively thereof is determined in the patient to be examined.

Furthermore, the stratification of patients suffering from rheumatoid arthritis in a new or established subgroup of rheumatoid arthritis is also covered, as well as an advantageous choice for the clinically developed group of patients for new therapeutic agents. The term treatment control likewise encompasses the assignment of patients to responders (responders) and non-responders (non-responders) to a treatment or course of treatment thereof.

"diagnosis" for the purposes of the present invention means a positive determination by means of the marker sequences according to the invention and assignment to rheumatoid arthritis patients. The term diagnosis encompasses medical diagnosis and examination of this aspect, in particular in vitro diagnosis and laboratory diagnosis, as well as protein and nucleic acid imprinting. Further examination may be necessary to identify and exclude other diseases. The term diagnosis therefore likewise encompasses the differential diagnosis of rheumatoid arthritis by means of the marker sequences according to the invention and the prophylaxis of rheumatoid arthritis.

"grading or therapeutic control" for the purposes of the present invention means that the method according to the invention proposes possible decisions for: the treatment and therapy of a patient, whether hospitalized or not, the use, effect and/or dosage of one or more drugs, the method of treatment or the course of disease or the monitoring of a course of treatment or the etiology or staging of a disease, such as staging to a new or existing subtype, or the differentiation of a disease and its patient.

In a further embodiment of the invention, the term "stratification" specifically covers risk stratification with prognosis of the outcome of an adverse health event.

Within the scope of the present invention, "patient" means any test subject-human or mammal-provided that the test subject is examined for rheumatoid arthritis.

The term "marker sequence" means for the purposes of the present invention that the cDNA or the polypeptide or protein respectively which can be obtained therefrom is significant for rheumatoid arthritis. For example, the cDNA or polypeptide or protein obtainable therefrom, respectively, is capable of exhibiting an interaction (e.g., an antigen (epitope)/antibody (antibody determinant) interaction) with a substance in a body fluid or tissue extract from a patient suffering from rheumatoid arthritis. For the purposes of the present invention, "wherein at least one marker sequence of the cDNA selected from the group SEQ1 to 488 or of the respectively encoded protein or of the respectively partial sequence or fragment thereof, is determined in the patient to be examined" means that the interaction between the body fluid or tissue extract of the patient and the marker sequence according to the invention is detected. Interactions of this type are, for example, the hybridization of binding substances, in particular on at least one marker sequence according to the invention, or, in the case of cDNAs, under selected, in particular stringent, conditions (for example, as are generally defined in J.Sambrook, E.F.Fritsch, T.Maniatis (1989), "Molecular protocols in Molecular Biology", Cold Spring Harbor laboratory Press, Cold Spring Harbor, USA or Ausubel, "Current protocols in Molecular Biology", Green blue Publishing and Wiley Interscience, N.Y. (1989)). One example of stringent hybridization conditions is: hybridization in 4 XSSC at 65 ℃ (optionally in 50% formamide and 4 XSSC at 42 ℃), followed by several wash steps in 0.1 XSSC at 65 ℃ for about 1 hour. An example of less stringent hybridization conditions is hybridization in 4 XSSC at 37 ℃ followed by several wash steps in 1 XSSC at room temperature.

According to the invention, this type of substance is a component of a body fluid, in particular of blood, whole blood, plasma, serum, patient serum, urine, cerebrospinal fluid, synovial fluid or an extract of a patient tissue.

In a further embodiment of the invention, however, the marker sequences according to the invention can be present at a significantly high or low expression rate or concentration which is indicative for rheumatoid arthritis. The relative diseased/healthy expression rate of the marker sequences for rheumatoid arthritis according to the invention is thus determined by means of protein or nucleic acid blotting.

In a further embodiment of the invention, the marker sequence has a recognition signal directed to the substance to be bound (e.g., antibody, nucleic acid). According to the invention, it is preferred that, for proteins, the recognition signal is an epitope and/or an antibody determinant and/or a hapten; and for cDNA, it is the hybridizing or binding region.

The marker sequences according to the invention are the subject of table a and can be identified by means of a respectively referenced database entry (also by means of the internet:http://www.ncbi.nlm.nih.gov/) Is clearly identified (see table a: accession number).

According to the invention, the marker sequences also encompass those modifications of the cDNA sequence and of the corresponding amino acid sequence, such as chemical modifications, for example citrullination, acetylation, phosphorylation, glycosylation or polyadenylation chains, and other modifications known to those of ordinary skill in the art.

In a further embodiment of the invention, partial sequences or fragments of the marker sequences according to the invention are likewise encompassed. In particular those partial sequences which have 95%, 90% identity, in particular 80% or 70% identity, to the marker sequences according to the invention.

In further embodiments, each tag sequence can be present in varying amounts in one or more regions of the solid support. This allows for variation in sensitivity. The regions may each have the entire marker sequence, i.e. a sufficient number of different marker sequences, in particular 2 to 5 or 10 or more, and optionally more nucleic acids and/or proteins, in particular biomarkers. However, at least 96 to 25,000 (numerical) or more are derived from different or identical marker sequences, and further nucleic acids and/or proteins, especially biomarkers, are preferred. Also preferred are more than 2,500, particularly preferred 10,000 or more different or identical marker sequences, and optionally other nucleic acids and/or proteins, in particular biomarkers.

Another object of the invention relates to an arrangement comprising at least one cDNA selected from the group SEQ1 to 488 or a marker sequence of the respectively encoded protein thereof. Preferably, the arrangement comprises at least 2 to 5 or 10, preferably 30 to 50 marker sequences, or 50 to 100 or more marker sequences.

Within the scope of the present invention, "array" is synonymous with "array" and if "array" is used to identify a substance on a marker sequence, this should be understood as an "array" or diagnostic device. In a preferred embodiment, the arrangement is designed such that the marker sequences represented on the arrangement appear on the solid support in the form of a grid. Moreover, those arrangements are preferred which allow a high density arrangement of protein binders and marker sequences to be arranged (spotted). Such an arrangement of high density arrangements is disclosed, for example, in WO99/57311 and WO 99/57312, and can be advantageously used in automated high throughput methods supported by robots.

However, within the scope of the present invention, the term "assay" or diagnostic device equally encompasses those embodiments of devices, such as an ELISA (e.g.individual wells of a microtiter dish are coated with a marker sequence or a combination of marker sequences according to the invention, optionally applied in an robotically supported manner in individual wells of the microtiter dish; the sample is a diagnostic ELISA kit produced by Phadia or a "Searchlight" multiplex ELISA kit produced by Pierce/Thermo Fisher Scientific), a bead-based assay (a spectrally distinguishable bead population is coated with a marker sequence or a combination of marker sequences. a patient sample is incubated with the bead population and bound (self) antibodies are detected by measuring fluorescence by means of a further fluorescently labeled second antibody/detection reagent; i.e.a Borrelia IgG kit or AthenaMultilyte produced by Multimetrix), a line assay (a marker sequence or a combination of marker sequences according to the invention is immobilized on a membrane in an robotically supported manner, it is detected/incubated with a patient sample; examples are "Euroline" produced by Euroimmun AG, western blotting (examples are "Euroline-WB" produced by Euroimmun AG), immunochromatography (e.g., lateral flow immunoassay; the marker sequence or combination of marker sequences is immobilized on a test strip (membranes, US5,714,389, etc.); examples are the "One Step HBsAg" test equipment manufactured by Acon Laboratories), or similar immunological single or multiple detection methods.

The aligned marker sequences are immobilized on a solid support, but are preferably spotted or immobilized or even imprinted on a solid support, i.e., applied in a reproducible manner. One or more marker sequences may appear multiple times in the totality of all marker sequences and be present in different amounts based on a point. Furthermore, the marker sequences can be normalized on a solid support (i.e., internal calibration by serial dilution series of, for example, human globulin as a data normalization and quantitative assessment).

The present invention therefore relates to an assay or protein biochip comprising an arrangement comprising a marker sequence according to the invention.

In a further embodiment, the marker sequence is present as a clone. Clones of this type can be obtained, for example, by means of a cDNA expression library according to the invention (Bussow et al 1998 (supra)). In a preferred embodiment, such an expression library comprising clones is obtained using an expression vector from a cDNA expression library containing cDNA marker sequences. These expression vectors preferably contain an inducible promoter. The induction of expression may be, for example, by an inducer, such as IPTG. Suitable expression vectors are described in Terpe et al (Terpe T Appl Microbiol Biotechnol.2003 Jan; 60 (5): 523-33).

Expression libraries are familiar to the person skilled in the art and can be generated according to standard procedures, for example, Sambrook et al, "Molecular Cloning, A laboratory handbook, second edition (1989), CSH press, Cold Spring Harbor, New York. Tissue-specific (e.g., human tissue, particularly human organs) expression libraries are also preferred. Expression libraries obtainable by exon capture (exon-trapping) are also included according to the invention. One synonym of expression libraries is the expression library (expression bank).

Protein biochips either do not show any redundancy (so-called:libraries) and corresponding expression libraries which can be generated, for example, according to the teachings of WO99/57311 and WO 99/57312, are preferred. These preferred Uniclone libraries have a large portion of the cDNA expression library with completely expressed proteins without defects.

In the context of the present invention, the clone may also be, but is not limited to, a transformed bacterium, a recombinant phage or a transformed mammalian, insect, fungal, yeast or plant cell.

The clones are immobilized, spotted or immobilized on a solid support.

The invention therefore relates to an arrangement in which the marker sequence is present in cloned form.

Furthermore, the tag sequence can be present in various forms of fusion proteins comprising, for example, at least one affinity epitope or "tag". The label may be one such as one comprising c-myc, histidine tag, arginine tag, FLAG, alkaline phosphatase, VS tag, T7 tag or streptomycin tag, HAT tag, NusA, S tag, SBP tag, thioredoxin, DsbA, fusion protein, preferably cellulose binding domain, green fluorescent protein, maltose binding protein, calmodulin binding protein, glutathione S transferase or lacZ.

The marker sequence may also consist of several individual marker sequences. This may include cloning of individual fragments to form one large common fragment, and expression of the combined fragment.

In all embodiments, the term "solid support" encompasses various embodiments, such as filters, membranes, magnetically or fluorophore labeled beads, silica wafers, glass, metals, ceramics, plastics, chips, targets or substrates for mass spectrometry. However, filters according to the invention are preferred.

Also preferred as filters are PVDF, nitrocellulose or nylon (e.g., Immobilon P Millipore, Protran Whatman, Hybond N + Amersham).

In another preferred embodiment of the arrangement according to the invention, the arrangement corresponds to a grid with the dimensions of a microtiter plate (8-12 well strip, 96 well, 384 well or more), a silica wafer, a chip, a target site for mass spectrometry or a matrix.

In a further embodiment, the invention relates to an assay or protein biochip for identifying and characterizing substances for rheumatoid arthritis, characterized in that the arrangement or assay according to the invention is a.) contacted with at least one substance to be tested and b.) the success or failure of the binding is detected.

Furthermore, the present invention relates to a method for identifying and characterizing substances for rheumatoid arthritis, characterized in that the arrangement or assay according to the invention is a.) contacted with at least one substance to be tested and b.) the success or failure of the binding is detected.

The substance to be detected may be any natural or non-natural biomolecule, synthetic chemical molecule, mixture or library of substances.

After the substance to be tested has been contacted with the marker sequence, the success or failure of binding is detected, for example, using commercially available image analysis software (GenePix Pro (Axon Laboratories), Aida (ray test), ScanArray (packard bioscience)).

The visualization of the protein-protein interaction (e.g.protein on a label sequence, as antigen/antibody) or the corresponding "method of detecting binding success" according to the invention can be carried out, for example, in the usual manner using fluorescent labels, biotinylation, radioisotope labels, or colloidal gold or latex particle labels. Detection of the bound antibody is carried out with the aid of a secondary antibody labeled with a commercially available reporter molecule (e.g., Cy, Alexa, Dyomics, FITC or similar fluorescent dye, colloidal gold or latex particles), or with a reporter enzyme, such as alkaline phosphatase, horseradish peroxidase, or the like, and a corresponding colorimetric, fluorescent or chemiluminescent substrate. For example using a microarray laser scanner, a CCD camera or visually.

In a further embodiment, the invention relates to a drug/active substance or prodrug developed for rheumatoid arthritis and obtainable by using the assay or protein biochip according to the invention.

The invention therefore likewise relates to the use of an arrangement according to the invention or of an assay for screening active substances for rheumatoid arthritis.

In a further embodiment, the invention therefore likewise relates to targets for the treatment and therapy of rheumatoid arthritis, which targets are respectively selected from the group SEQ1-488 or the proteins respectively encoded thereby.

In a further embodiment, the invention likewise relates to the use of the marker sequences according to the invention, preferably in an arrangement, as affinity substances for carrying out apheresis or in the broadest sense blood lavage, wherein substances from body fluids of patients suffering from rheumatoid arthritis, such as blood or plasma, are bound to the marker sequences according to the invention and can subsequently be selectively removed from the body fluids.

Examples and figures:

samples of 10 or more patients were individually screened against the cDNA expression library. Multiple rheumatoid arthritis-specific expression clones were identified by comparison with 10 or more healthy samples. The identity of the marker sequence is determined by DNA sequencing.

FIG. 1 shows a differential screen between two protein biochips from one cDNA expression library from a patient and a healthy test subject, respectively. Different clones were detected by means of fluorescent labeling and evaluated by means of bioinformatics.

Table a:

Nr	PRI	login number
			1	A	gi|33519473
2	A	gi|33469975
			3	A	gi|113421166
4	A	gi|113411825
			5	A	gi|55925645
6	A	gi|31341967
			7	A	gi|37537717
8	A	gi|51464299

Nr	PRI	Login number
			9	A	gi|31343485
10	A	gi|73622128
			11	A	gi|22202618
12	A	gi|21956639
			13	A	gi|47894110
14	A	gi|74048536
			15	A	gi|39573729
16	A	gi|113421846
			17	A	gi|34098945
18	A	gi|30583601
			19	A	NM_012292
20	A	NM_004499
			21	B	61064_8_E11
22	B	gi|83716023
			23	B	NM_006796
24	B	gi|11386138
			25	B	gi|21389576
26	B	gi|56676308
			27	B	gi|4503744
28	B	gi|57222567

Nr	PRI	Login number
			29	B	gi|7512821
30	B	NM_000973
			31	B	gi|17149837
32	B	NM_002954
			33	B	gi|22219473
34	B	gi|30583735
			35	B	gi|53733398
36	B	gi|89057118
			37	B	gi|12804481
38	B	NM_003768
			39	B	gi|46391095
40	B	NW_926918
			41	B	gi|89041118
42	B	gi|14424731
			43	B	gi|30410780

44	B	NM_005707
			45	B	gi|4758937
46	B	gi|7661695
			47	B	gi|13559175

44	B	NM_005707
			48	B	gi|83367078
49	B	gi|48146439
			50	B	gi|33591068
51	C	gi|34850060
			52	C	gi|34147654
53	C	gi|62526046
			54	C	gi|52545622
55	C	gi|7512499
			56	C	gi|40255020
57	C	gi|46389548
			58	C	gi|2911264
59	C	gi|15431289
			60	C	61064_8_H12
61	C	gi|13569612
			62	C	gi|38679891
63	C	gi|88943682
			64	C	gi|5381417
65	C	gi|4504982
			66	C	gi|5453690
67	C	NM_020713

44	B	NM_005707
			68	C	gi|21748598
69	C	NM_005354
			70	C	NM_002473
71	C	gi|3287489
			72	C	gi|61656605
73	C	gi|68800242
			74	C	gi|21620021
75	C	gi|20149616
			76	C	gi|40226207
77	C	gi|40807483
			78	C	NM_003475
79	C	gi|61966904
			80	C	NM_001009998
81	C	gi|42490757
			82	C	gi|34335231
83	C	gi|11545906
			84	C	gi|7245833
85	C	gi|7657677
			86	C	NM_000477
87	C	gi|39654744

44	B	NM_005707
			88	C	gi|13938597
89	C	gi|33874730
			90	C	gi|19743569
91	C	gi|37544107
			92	C	gi|51468814
93	C	gi|21739976
			94	C	gi|4758219
95	C	NM_004559
			96	C	gi|5689527
97	C	gi|31077184
			98	C	gi|24308369
99	C	gi|5620310g
			100	C	gi|4507398
101	D	gi|17981697
			102	D	gi|32129198
103	D	gi|6912539
			104	D	gi|89030746
105	D	NM_000386
			106	D	gi|20336766
107	D	gi|16306505

44	B	NM_005707
			108	D	gi|7619703
109	D	gi|253706
			110	D	gi|19913395
111	D	gi|33636763
			112	D	gi|66346709
113	D	gi|38197056
			114	D	gi|29893564
115	D	gi|1362855
			116	D	gi|89057343
117	D	gi|50592995
			118	D	gi|71361681
119	D	gi|32455265
			120	D	gi|10439788
121	D	gi|31092
			122	D	gi|113428396
123	D	gi|7705480
			124	D	gi|5830438
125	D	NT_010194
			126	D	gi|179955
127	D	gi|2547076

44	B	NM_005707
			128	D	gi|4502846
129	D	gi|83641894
			130	D	gi|3642665
131	D	gi|3293553

132	D	NM_003130
			133	D	gi|1134310g3
134	D	gi|34147660
			135	D	gi|85681028
136	D	gi|17572803
			137	D	gi|13124797
138	D	gi|83656780
			139	D	gi|39725676
140	D	gi|19526471
			141	D	gi|13376797
142	D	gi|15214478
			143	D	61064_8_H06
144	D	gi|66346647
			145	D	gi|32879857
146	D	gi|40889757

132	D	NM_003130
			147	D	gi|71772259
148	D	gi|51473210
			149	D	gi|15680208
150	D	gi|16306717
			151	D	gi|4759097
152	D	gi|56550050
			153	D	gi|4506903
154	D	gi|10567816
			155	D	gi|4758985
156	D	gi|16740583
			157	D	gi|1487948
158	D	gi|23238257
			159	D	gi|21758184
160	D	gi|56205191
			161	D	gi|83641890
162	D	gi|17380594
			163	D	NM_001025598
164	D	NM_001024807
			165	D	gi|49456343
166	D	gi|33150630

132	D	NM_003130
			167	D	gi|21595329
168	D	gi|13124696
			169	D	gi|6716561
170	D	gi|25777682
			171	D	gi|18426896
172	D	gi|42544170
			173	D	gi|30584255
174	D	gi|26249286
			175	D	61064_8_C07
176	D	gi|12232414
			177	D	gi|4504618
178	D	gi|39645205
			179	D	NM_004960
180	D	gi|22212941
			181	D	gi|345836
182	D	gi|88999578
			183	D	gi|27807403
184	D	gi|17386088
			185	D	gi|7524353
186	D	gi|5031931

132	D	NM_003130
			187	D	gi|40789265
188	D	gi|32490572
			189	D	gi|14250530
190	D	gi|46249758
			191	D	gi|4507557
192	D	gi|547749
			193	D	gi|62897169
194	D	gi|9651486
			195	D	gi|37182091
196	D	gi|89059027
			197	D	gi|34785019
198	D	NM_005572
			199	D	gi|113428589
200	D	gi|51471030
			201	D	gi|51470970
202	D	gi|20987263
			203	D	gi|13623595
204	D	NM_020967
			205	D	NM_020529
206	D	gi|34784912

132	D	NM_003130
			207	D	gi|38014003
208	D	gi|40807365
			209	D	gi|182118
210	D	gi|60552339
			211	D	gi|33598947
212	D	gi|32401423
			213	D	gi|10434157
214	D	gi|1082338
			215	D	gi|340219
216	D	gi|31542761
			217	D	gi|17149845
218	D	gi|30583065
			219	D	gi|38505154

220	D	gi|19923366
			221	D	gi|15928941
222	D	gi|18426915
			223	D	gi|505108
224	D	gi|34452717
			225	D	gi|6855633

220	D	gi|19923366
			226	D	gi|53729342
227	D	gi|224530
			228	D	gi|6912602
229	D	gi|40789071
			230	D	gi|51706338
231	D	gi|7262378
			232	D	gi|34147665
233	D	NM_002228
			234	D	gi|22713422
235	D	gi|4505904
			236	D	gi|16579885
237	D	gi|47078237
			238	D	gi|3387977
239	D	gi|88972371
			240	D	gi|2981764
241	D	gi|55959290
			242	D	gi|89059359
243	D	gi|32425497
			244	D	gi|31317308
245	D	gi|77404355

220	D	gi|19923366
			246	D	gi|32880093
247	D	gi|12232384
			248	D	gi|38683849
249	D	gi|9966764
			250	D	gi|18390331
251	D	gi|30582607
			252	D	gi|31543190
253	D	gi|55959087
			254	D	gi|7110641
255	D	gi|2632247
			256	D	gi|71594
257	D	gi|46370065
			258	D	gi|339685
259	D	gi|33869643
			260	D	gi|51036581
261	D	gi|10439217
			262	D	gi|39725631
263	D	gi|31563519
			264	D	gi|31542269
265	D	gi|22477334

220	D	gi|19923366
			266	D	gi|13699813
267	D	gi|51493052
			268	D	gi|4503580
269	D	gi|4557839
			270	D	gi|39573730
271	D	gi|89059606
			272	D	gi|31652250
273	D	gi|47519746
			274	D	gi|33244031
275	D	gi|10434039
			276	D	gi|57242773
277	D	gi|21704282
			278	D	gi|11342680
279	D	gi|30584609
			280	D	gi|21739862
281	D	gi|55959475
			282	D	gi|42476191
283	D	gi|34533094
			284	D	gi|15431301
285	D	gi|26986533

220	D	gi|19923366
			286	D	gi|8922332
287	D	gi|40787650
			288	D	gi|9873442
289	D	gi|50086623
			290	D	gi|34147350
291	D	gi|12056467
			292	D	gi|55925607
293	D	gi|38570091
			294	D	gi|29476902
295	D	gi|40796182
			296	D	gi|7770137
297	D	gi|113430465
			298	D	gi|89040669
299	D	gi|10518498
			300	D	gi|34855930
301	D	gi|186696
			302	D	gi|21614499
303	D	gi|3192917
			304	D	gi|32306539
305	D	gi|54607123

220	D	gi|19923366
			306	D	gi|52856410
307	D	gi|33286445

308	D	gi|26344686
			309	D	gi|42716279
310	D	gi|381964
			311	D	gi|46852169
312	D	gi|31874210
			313	D	gi|71565157
314	D	gi|7705475
			315	D	gi|12803375
316	D	gi|113417847
			317	D	gi|14110410
318	D	gi|55957624
			319	D	gi|89027401
320	D	gi|13435438
			321	D	gi|18490263
322	D	gi|4757715
			323	D	gi|12804441
324	D	gi|2134743

308	D	gi|26344686
			325	D	gi|6005923
326	D	gi|6841318
			327	D	gi|12711674
328	D	gi|31563378
			329	D	gi|51173146
330	D	gi|93141017
			331	D	gi|23396512
332	D	gi|55961048
			333	D	gi|18314624
334	D	gi|27552770
			335	D	gi|50345985
336	D	gi|1710248
			337	D	gi|7657441
338	D	gi|40226068
			339	D	gi|42490910
340	D	gi|21307630
			341	D	gi|133254
342	D	gi|340019
			343	D	gi|57997038
344	D	gi|40254816

308	D	gi|26344686
			345	D	gi|27436949
346	D	gi|56789232
			347	D	gi|38257139
348	D	61064_8_A0g
			349	D	gi|13929434
350	D	NM_001012
			351	D	gi|31657179
352	D	gi|16273176
			353	D	gi|14165264
354	D	gi|5123454
			355	D	gi|24234719
356	D	gi|10720282
			357	D	gi|88966845
358	D	NM_014497
			359	D	gi|40795668
360	D	gi|22538467
			361	D	gi|4503179
362	D	gi|68299771
			363	D	gi|62896661
364	D	gi|22027479

308	D	gi|26344686
			365	D	gi|41055203
366	D	gi|4758515
			367	D	gi|21757045
368	D	NM_006086
			369	D	gi|4507284
370	D	gi|4502004
			371	D	gi|51465675
372	D	gi|14249144
			373	D	gi|2276396
374	D	gi|21361525
			375	D	gi|34328690
376	D	gi|13177775
			377	D	gi|13325058
378	D	gi|1903190
			379	D	gi|23111046
380	D	NM_006360
			381	D	gi|7512569
382	D	gi|50843811
			383	D	gi|113423859
384	D	gi|78190466

308	D	gi|26344686
			385	D	gi|7657649
386	D	gi|30583811
			387	D	gi|14150165
388	D	gi|31805540
			389	D	gi|34289
390	D	gi|46249395
			391	D	gi|22137524
392	D	gi|6226705
			393	D	NM_004494
394	D	gi|37552371
			395	D	gi|10241759

396	D	NM_015190
			397	D	gi|40353728
398	D	gi|135412
			399	D	61064_8_F10
400	D	gi|68800343
			401	E	NW_923984
402	E	NM_018442
			403	E	NM_032281

396	D	NM_015190
			404	E	NM_005778
405	E	NM_014859
			406	E	NM_006352
407	E	NM_022088
			408	E	NM_000516
409	E	NM_000237
			410	E	NM_020825
411	E	NM_000076
			412	E	NM_015720
413	E	NM_017596
			414	E	NM_003195
415	E	NM_001280
			416	E	NM_001704
417	E	NM_001686
			418	E	NM_152704
419	E	NT_004350
			420	E	NM_014680
421	E	NM_005801
			422	E	NM_080390
423	E	NT_033903

396	D	NM_015190
			424	E	NM_003025
425	E	NM_006036
			426	E	NM_001551
427	E	NM_004380
			428	E	NM_138559
429	E	NM_006352
			430	E	NM_006428
431	E	NT_029419
			432	E	NW_927628
433	E	NM_006353
			434	E	NM_002154
435	E	NM_003025
			436	E	NM_022359
437	E	NM_032514
			438	E	NW_927195
439	E	NM_012295
			440	E	NW_927628
441	E	NM_006958
			442	E	NM_002013
443	E	NM_198943

396	D	NM_015190
			444	E	NM_002256
445	E	NM_001098
			446	E	NM_005225
447	E	NM_004712
			448	E	NT_010641
449	E	NM_022730
			450	E	NM_000g34
451	E	NM_006590
			452	E	NT_037887
453	E	NM_005736
			454	E	NM_181697
455	E	NM_030g07
			456	E	NM_002613
457	E	NM_002013
			458	E	NM_006373
459	E	NM_000969
			460	E	NM_178159
461	E	NM_024671
			462	E	NW_927762
463	E	NM_007029

396	D	NM_015190
			464	E	XM_937970
465	E	NM_001031735
			466	E	NM_001069
467	E	NM_006841
			468	E	NM_000477
469	E	NM_203346
			470	E	NM_012398
471	E	NM_005851
			472	E	NM_023071
473	E	NT_005612
			474	E	NM_006640
475	E	NM_016300
			476	E	NM_182565
477	E	NT_079595
			478	E	NM_025203
479	E	NM_014593
			480	E	NM_033647
481	E	NM_001098
			482	E	NM_000801
483	E	NM_001032396

484	E	NT_006081
			485	E	NM_018287
486	E	NM_023940
			487	E	NM_002751
488	E	NT_037887

Claims (18)

1. Use of a marker sequence for the diagnosis of rheumatoid arthritis, wherein at least one marker sequence selected from the group consisting of the cDNA of SEQ1-488, or the proteins encoded respectively thereof, or the partial sequences or fragments thereof respectively, is determined on or from a patient to be examined.

2. Use of marker sequences for the diagnosis of rheumatoid arthritis according to claim 1 characterized in that at least 2 to 5 or 10, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences are determined on or from the patient to be examined.

3. Use of a marker sequence for the diagnosis of rheumatoid arthritis according to claim 1, characterized in that SEQ 1-20 or SEQ 21-50 or SEQ 51-100 or SEQ401-488, or the protein encoded respectively thereof, or respectively the partial sequence or fragment thereof, is determined on or from the patient to be examined.

4. Use of a marker sequence for the diagnosis of rheumatoid arthritis according to any of the preceding claims characterized in that said determination is performed by means of in vitro diagnosis.

5. Use of a cDNA or a protein encoded respectively or a partial sequence or fragment thereof, respectively, selected from the group SEQ1-488, as a marker sequence for a diagnostic agent.

6. Use of a marker sequence for the diagnosis of rheumatoid arthritis according to any of the preceding claims characterized in that the marker sequence is applied to a solid support, in particular a filter, a membrane, magnetically or fluorophore labeled beads, a silica wafer, glass, metal, ceramic, plastic, a chip, a target or a substrate for mass spectrometry.

7. Method for diagnosing rheumatoid arthritis, wherein

a.) a cDNA selected from the group SEQ1-488 or a marker sequence for the protein encoded respectively or for a partial sequence or fragment thereof, respectively, is applied to a solid support, and

b.) contacting it with a body fluid or tissue extract of the patient, and

c.) detecting the interaction between the body fluid or tissue extract and the marker sequence from a).

8. Method for fractionation, in particular for risk fractionation and therapy control of patients suffering from rheumatoid arthritis, wherein at least one marker sequence selected from the group consisting of the cDNA of SEQ1 to 488, or the proteins encoded respectively thereof, or the partial sequences or fragments thereof respectively, is determined on or from the patient to be examined.

9. The method according to claim 7, wherein said grading or treatment control covers the decision for: the treatment and therapy of a patient, particularly a hospitalized patient, the use, effect and/or dosage of one or more drugs, the method of treatment or the monitoring of the course of disease or the course of treatment or the etiology or staging of the disease, and the prognosis.

10. An arrangement of tag sequences comprising at least one tag sequence of a cDNA selected from the group of SEQ1 to 488 or of a protein encoded by each thereof.

11. An arrangement according to claim 10, characterized in that it comprises at least 2 to 5 or 10, preferably 30 to 50 marker sequences, or 50 to 100 or more marker sequences.

12. The arrangement according to claim 10, characterized in that the marker sequence is present as a clone.

13. An assay, protein biochip comprising an arrangement according to claim 10, characterized in that the marker sequences are applied to a solid support.

14. Use of an array according to any one of claims 10 to 12 or an assay according to claim 13 for the identification and characterization of rheumatoid arthritis substances, comprising means for detecting the success of binding, characterized in that the array or assay a.) is contacted with at least one substance to be tested and b.) detects the success of binding.

15. Use of an array according to any one of claims 10 to 12 or an assay according to claim 13 for screening for an active substance for rheumatoid arthritis.

16. Diagnostic agent for the diagnosis of rheumatoid arthritis, respectively selected from the group SEQ1-488, or the protein encoded respectively thereof, or the partial sequence or fragment thereof, respectively.

17. A target for rheumatoid arthritis treatment and therapy, respectively selected from the group SEQ1-488, or the protein encoded by each thereof, or the partial sequence or fragment thereof, respectively.

18. Use of a cDNA selected from the group consisting of SEQ1-488, or the proteins encoded respectively thereof, or the partial sequences or fragments thereof, respectively, as an affinity substance for apheresis or blood lavage of a patient suffering from rheumatoid arthritis.