WO1999021011A1

WO1999021011A1 - Type ix collagen antibody and related uses

Info

Publication number: WO1999021011A1
Application number: PCT/US1998/022616
Authority: WO
Inventors: Yang Chunlin
Original assignee: Fibrogen, Inc.
Priority date: 1997-10-23
Filing date: 1998-10-23
Publication date: 1999-04-29
Also published as: AU1276899A

Abstract

Methods directed to detecting or monitoring autoimmune disorders and connective tissue disorders, such as rheumatoid arthritis, by determining the levels of type IX collagen in serum are described. A reagent kit and antibodies useful in carrying out such methods are also presented.

Description

TYPE IX COLLAGEN ANTIBODY AND RELATED USES

1. FIELD OF THE INVENTION

The present invention is directed to antibodies to type IX human collagen and

methods of diagnosing diseases wherein a characteristic of such disease is the abnormal

expression of type IX collagen in relevant body fluids or tissues. More specifically, the present invention is further directed to detecting or monitoring rheumatoid arthritis and other autoimmune diseases wherein the abnormal expression of type IX collagen is

implicated by determining the levels of type IX collagen by way of a type IX collagen

antibody in a patient sample. The present invention is further directed to detecting or

monitoring connective tissue diseases wherein the abnormal expression of type IX collagen is implicated in, for example, chondrosarcoma and endosarcoma. The present

invention is further directed to a reagent kit and antibodies useful in carrying out such

methods.

2. BACKGROUND OF THE INVENTION

Collagen. Collagen is the most abundant component of the extracellular matrix.

The collagen superfamily comprises at least nineteen (19) molecules which are generally

the result of three polypeptide chains containing, in their primary sequence, (-Gly-X-Y-)_n

repeats which allow for the formation of triple helical domains (van der Rest et al., 1991, FASEB J. 5:2814-2823). Characterized by their structure and biological function, specific collagen types arrange within extracellular matrices in precise aggregates,

maintaining a delicate equilibrium in specialized tissues.

The full polypeptide and polynucleotide sequences corresponding to human type IX collagen have been recently reported for each of the molecules comprising the type IX collagen trimer. Mayne, et al, 1993, J. Biol. Chem. 268(13):9381-86; Brewton, et al,

1992, Eur. J. Biochem 205(2):443-9; Brewton, et al, 1991, J. Biol. Chem. 266(8):4752-

57; Zhidkova, et al, 1993, FEBS Lett. 326(l-3):25-28. Type IX collagen is a fibril associated collagen related to types II and XI collagen. Type IX collagen is a

heterotrimer comprised of three different subunits; αl(IX), α2(IX) and α3(IX). The

type IX collagen molecule has three collagenous domains and four non-collagenous

domains.

Diseases And Disorders Associated With Collagen. A number of diseases are

associated with collagen gene expression. In autoimmune disorders, such as rheumatoid arthritis, expression of collagen types IX and XI, as well as collagen type II have been

discovered. In degenerative joint diseases such as osteoarthrosis, collagen expression of

types II and IX have been detected in a model of osteoarthrosis. Lefkoe et al, 1997, J Rheumatol 24(6): 1155-1163. The abnormal expression of type IX collagen have also been observed in other connective tissue diseases which are not "autoimmune" in nature

and which are related to the cartilage. For example, type IX collagen has been detected to

be distinctly localized in cartilaginous tumors in conditions such as enchondroma,

chondrosarcoma, and secondary chondrosarcoma. Kawashima et al, 1993, J Cancer Res Clin Oncol 120:35-40. Moreover, the irregular deposition of type IX collagen in human

cartilaginous tumors has also been observed.

Methods For Diagnosing Autoimmune Disorders. Current methods of

diagnosing and detecting autoimmune disorders typically rely on the onset of symptoms

which characterize the disorder. For example, current methods for diagnosing

rheumatoid arthritis rely upon the presence or absence of seven criteria. Takagishi and Yamamoto, 1992, Nippon Rinsho 50(3):490-494. More recently, studies have been

performed to diagnose rheumatoid arthritis by measuring the presence of collagen type II in cartilage. Fujii, et al, 1992, Int. Orthop 16(3):272-76. These studies have met with

moderate success. For example, according to Fujii, et al, use of antibodies to type II

collagen in enzyme-linked immunosorbent assays resulted in a 22.7% success rate in

detecting rheumatoid arthritis in a patient population known to have such disorder. Studies wherein certain antibody levels from sera are measured have also shown

moderate correlation with disease. Although type IX collagen, in addition to type II

collagen, may be a relevant tool to diagnose rheumatoid arthritis, the extraction of intact

type IX collagen has been difficult to accomplish to date such that antibodies to type IX collagen have not been reported.

No method to date, except for the observation of rheumatoid arthritis "factors"

and symptoms, has provided a means for consistently detecting rheumatoid arthritis.

General Diagnostic Methods For Detecting Diseases And Disorders. Methods

wherein a monoclonal antibody is utilized to diagnose diseases have been described generally in the relevant art. See e.g., United States Patent No. 5,298,393 and United

States Patent No. 4,628,027. Specific diagnostic methods utilizing antibodies to specific markers have also been disclosed. For example, the method of diagnosing diseases, such as cancer, utilizing a monoclonal antibody recognizing glutathione S-transferase is

described by United States Patent No. 5,298,393. Likewise, monoclonal antibodies

which purportedly diagnose fibrosis by recognizing connective tissue proteins,

specifically, collagen types I, II, III, IV, and V, are described generally in U.S. Patent No. 4,628,027 ("027 Patent"). The '027 patent, however, does not disclose antibodies

specific to type IX collagen or their application to the diagnosis of disease.

3. SUMMARY OF THE INVENTION

It has been found that type IX collagen varies in its distribution throughout body

tissues as result of various disease states. The present invention provides for various approaches for detecting and measuring the changes in distribution and levels of type IX collagen, thereby, providing for a diagnostic assessment of the pathological stages of

autoimmune disorders and connective tissue disorders associated with type IX collagen.

Such autoimmune diseases include rheumatoid arthritis. Connective tissue disorders in

which abnormal type IX collagen expression is implicated include ocular disorders, chondrodysplasia. chondrosarcoma, secondary chondrosarcoma, and endochondroma.

Methods. The present invention comprises a means of diagnosing

autoimmune disorders or connective tissue disorders such as rheumatoid arthritis and

osteoarthrosis. Specifically, the present invention provides for the detection and

monitoring of the presence and proliferation of type IX collagen or its subunits in tissues

related to rheumatoid arthritis and osteoarthrosis, e.g., cartilage, and connective tissue

disorders related to ocular diseases, chondrodysplasia, chondrosarcoma, secondary

chondrosarcoma and enchondroma. More specifically, the present invention provides for autoimmune disorders and particularly type IX collagen, subunits, or fragments thereto, by measuring the levels of such collagen in a patient sample, preferably a patient's serum

or synovial fluid.

In one embodiment of the present invention, a method is provided for wherein the presence of type II and/or type XI collagen is measured in addition to type IX collagen, related subunits, and fragments thereto (type IX collagen and subunits, and

fragments thereto are hereinafter defined as "Type IX Collagen"). In another

embodiment of the present invention, patient samples analyzed for the presence of Type

IX Collagen are tissue samples or fluid samples from serum or synovial fluid. Preferably, the method provides for the measurement of Type IX Collagen levels from a serum

sample wherein a comparison is made between the levels of Type IX Collagen in a serum

samples from a patient known or suspected to have an autoimmune disorder and more

particularly rheumatoid arthritis, and the levels of Type IX Collagen in a non-diseased

serum sample. The method provides that higher levels of Type IX Collagen from serum

samples from patients suspected or known to have rheumatoid arthritis to levels in non- diseased serum samples is indicative of the presence of such autoimmune disorder.

Abnormal levels of Type IX Collagen from serum samples of patients suspected or

known to have an autoimmune disorder or connective tissue disorder serve as a

prognostic indicator of such disorder. In addition, the present invention provides for the

measurement of Type IX Collagen levels in synovial fluid samples from patients known

or suspected to have a degenerative joint disease such as osteoarthrosis using similar

markers as described above. The method of the present invention utilizes an antibody or antigen binding fragment thereof, preferably, a monoclonal antibody, which is reactive with and capable

of specifically detecting human Type IX Collagen. In one embodiment of the present

invention, the antibody is human or humanized. The antibodies of the present invention may be used in standard radioimmunoassays or enzyme-linked immunosorbent assays or

other assays which are known to utilize antibodies for measurement of levels of Type IX

Collagen in a serum sample. The method of utilizing an antibody to measure the levels of

Type IX Collagen allows for a non-invasive diagnosis of rheumatoid arthritis, as well as

other autoimmune disorders wherein the abnormal expression of Type IX Collagen is implicated.

The present invention also provides for the specificity and reliability in detecting

and measuring Type IX Collagen in tissue and body fluids for the diagnostic evaluation

of the presence and stages of disorders, and preferably autoimmune or connective tissue disorders. More preferably, the disorder is rheumatoid arthritis. The present invention

also provides for the pathological testing and diagnosing of the malignant catilaginous

tumors associated with diseases such as enchondroma and chondrosarcoma. For

example, the present invention provides for methods of diagnosing the presence of an

autoimmune disorder, for determining the progress of such autoimmune disease in

patients and for serving as a prognostic indicator of the extent of the disease.

Diagnostic Kit. The present invention is further directed to a kit consisting

of antibodies and reagents. In a preferred embodiment, the diagnostic kit comprises

reagents for measuring Type IX Collagen (type IX collagen, subunits thereof, or the individual α chains of type IX collagen) existing in body fluids such as serum. In yet

another preferred embodiment, the kit further comprises antibodies to type II and type XI

collagen. In one embodiment of the present invention, the kit comprises immobilized

antibodies or their chimeras which specifically recognize Type IX Collagen and type II collagen and/or type XI collagen.

The antibodies comprising the present invention may be differentially labeled and

are capable of binding to an antigen component. The kit also comprises the reagents

necessary for the detection of the enzyme-labeled antibody. Other reagents which may be necessary may be added, for example, dissolving agents, cleaning agents and reaction

terminators.

In a preferred embodiment of the invention, the kit is packaged and labeled for

example, in a box or container which includes the necessary elements of the kit, and directions and instructions on the use of such diagnostic kit.

Antibodies. The present invention further comprises antibodies or antigen

binding fragments thereof reactive with and specific for Type IX Collagen. Preferably,

the antibody or antigen binding fragment thereof is to human type IX collagen, subunits,

and fragments thereof and is a monoclonal antibody. Such antibodies may be generated, for example, by obtaining a hybridoma that produces a monoclonal antibody specific to

Type IX Collagen by fusing a myeloma cell with antibody-secreting cells of an animal

immunized with human type IX collagen or the epitopes of the α subunits of type IX

human collagen described below, culturing said hybridoma and/or a cell line arising

therefrom, and harvesting the monoclonal antibody specific to human Type IX Collagen. 4. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (FIG. 1) shows the selection of peptides for immunization: Sequences

underlined were chosen for peptides for immunization of rabbits. Sequences in Italic and

underlined were chosen for peptides used for immunization of goats. FIGURE 1A

corresponds to the sequences for the collagen type IX α(l)IX chain. FIGURE IB

corresponds to the sequences for the collagen type IX α(2)IX chain. FIGURE 1C

corresponds to the sequence for the collagen type IX α(3)IX chain.

Figure 2 (FIG. 2) shows a schematic depiction of collagen type IX.

Figure 3 (FIGS. 3A and 3B) show characterization data related to the synthesized peptides.

Figure 4 (FIG. 4) shows SDS-PAGE of recombinant human collagen type IX.

Figure 5 (FIG. 5) depicts immunoblotting of antisera from rabbits against

individual chains of collagen IX.

5. DETAILED DESCRIPTION OF THE INVENTION

Antibodies. The present invention provides for antibodies specific for Type IX

Collagen. Several methods may be employed for obtaining the monoclonal antibodies of

the present invention. For example, a ready source of monoclonal antibodies may be

obtained from fusion of antibody-secreting cells with non-antibody-secreting myeloma cells resulting in a hybridoma. These hybridomas provide a ready source of monoclonal antibodies.

The hybridoma which produces the monoclonal antibodies of the present

invention, specifically, the monoclonal antibodies recognizing Type IX Collagen may be

produced according to cell fusion techniques as exemplified in publications such as Kohler and Milstein, Nature 256:495-497 (1975) In such technique, animals, for

example, rabbits, monkeys, mice, rats, goats, etc. are immunized with Type IX Collagen (for example, the subunit epitopes at FIG. 1 ) and antibody producing cells from their

spleen, lymph node, etc. are collected and fused with human or animal myeloma cells.

The myeloma cell lines that may be used are described in for example, Monoclonal

Antibodies and T-cell Hybridomas in: J.L. Turk (editor) Research Monographs in Immunology Vol. 3, Elsevier/North Holland Biomedical Press, New York (1981).

Those hybridomas secreting monoclonal antibodies of the present invention may

then be selected from the colonies of hybridomas produced by cell fusion. Selection of

the desired hybridomas can be conducted by techniques such as enzyme- linked- immunosorbent assay (ELISA) or a radioimmnoassay, as described in R. Kennet et al.

Monoclonal Antibodies, hybridomas; a new dimension in biological analyses, pp. 376-

384, Plenum Press, New York (1980), to confirm that the monoclonal antibodies

produced by the hybridomas result in an antigen-antibody reaction with Type IX

Collagen. The hybridomas secreting the desired monoclonal antibody are cloned into individual antibody-producing cell lines by methods described in, for example, G.J.

Hammerling et al, thereby, producing a ready source of monoclonal antibodies specific

for Type IX Collagen. Methods for obtaining human or humanized antibodies may also be used to obtain antibodies of the present invention. Such methods are described in, for example, EP

765172, EP 671951, US 4,574,116, US 4,350,683, US 5,593,822, US 5,422,245, US

4,713,351, EP 589877, US 5,565,332, and EP 616640.

The detection and monitoring of diseases associated with Type DC collagen expression. Although not as prevalent as type II collagen, type IX collagen, as well as

type XI collagen have been shown to be expressed in the cartilage of patients having

certain autoimmune disorders, such as rheumatoid arthritis. To the extent that

rheumatoid arthritis and/or osteoarthrosis can be detected only in an undefined segment

of the relevant patient population by measuring levels of type II collagen, a detection and/or monitoring protocol which measures not only the presence of type II collagen, but

also type IX and/or type XI collagen may be useful. The presence of such collagens may

be detected by use of the antibodies of the present invention. For example, samples from

synovial fluid or serum may be withdrawn from patients suspected of having rheumatoid

arthritis or osteoarthrosis and subjected to immunoassays with antibodies, and preferentially monoclonal antibodies, against Type IX Collagen, as well types II and/or

XI collagen. The presence of abnormal levels of type IX collagen compared to normal

would be a basis for detecting and monitoring the pathological conditions of rheumatoid

arthritis and/or osteoarthrosis. The use of antibodies of the present invention provides for an early and accurate diagnosis of cartilage related autoimmune diseases such as

rheumatoid arthritis or other connective tissue disorders.

The antibodies of the present invention may also be used to diagnose pathological

conditions of autoimmune diseases or connective tissue disorders by histological diagnosis of tissue samples wherein the antibodies against Type IX Collagen detect changes in distribution of Type IX Collagen. For example, it has been demonstrated that

human cartilaginous tumors associated with disorders such as enchondroma and

chondrosarcoma, the uneven distribution of type IX collagen has been detected. Use of

the antibodies specific for Type IX Collagen would allow for detecting changes in type IX collagen distribution in applicable cartilage tissue which could serve as an indicator of

the presence or progressive stage of applicable autoimmune diseases such as rheumatoid

arthritis. Moreover, use of the antibodies or antigen binding fragments of the present

invention may allow for the pathological testing of malignant cartilaginous tumors associated with enchondroma, chondrosarcoma, and other related diseases.

Detection of Type IX Collagen levels may be obtained through immunoassay

methods, for example, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA,

radioimmunoassay or any other known assays which utilize an antibody to detect the presence of a protein marker. The ELISA, sandwhich ELISA and radioimmunoassay

methods are preferred and may, with the monoclonal antibodies of the present invention

be used to detect specifically the presence of Type IX Collagen. In a preferred method of

the invention, serum samples are obtained first from patients suspected or known to have

an autoimmune disorder; wherein said disorder is preferably rheumatoid arthritis. The

serum sample is measured for levels of Type IX Collagen through immunoassay, and

compared with the amount of Type IX Collagen levels in a second non-diseased serum

sample to determine presence or progression of a disorder. The same methods may be

used to monitor the disorder. For example, a first serum sample measured for levels of

Type IX Collagen is obtained from a patient known to have, for example, rheumatoid arthritis, and compared to levels of Type IX Collagen in samples taken subsequent to the

first samples. Such method allows for monitoring the progression of the rheumatoid arthritis.

In the present invention, enzyme-linked immunosorbent assay (ELISA) can be

used to measure levels of Type IX Collagen present in serum samples. For example,

monoclonal antibodies against Type IX Collagen are conjugated to an appropriate enzyme such as horseradish peroxidase, protein ferritin, enzyme alkaline phosphatase, β-

D-galactosidase etc. These enzyme-linked antibody preparations are mixed with the

serum samples which contain unknown amounts of Type IX Collagen. Since the

antibody is specific for Type IX Collagen, antigen-antibody binding occurs after an incubation period, and transferred to a microtiter plate which have been precoated with

Type IX Collagen. Unbound antibodies bind to the antigen absorbed to the walls of the microtiter wells. The initial antigen-antibody complexes are washed out of the wells of

the microtiter plate leaving the enzyme-linked antibodies complexed to the antigen

coating the walls. A substrate is added and enzymatic activity is measured. The method of enzyme-linked immunosorbent assay (ELISA) is described in R. Kennet et al, supra.

In the present invention, sandwich ELISA techniques may also be used to

measure levels of autoantibodies to type IX collagen. More particularly, in autoimmune

diseases such as rheumatoid arthritis wherein a characteristic of the disease is the

production of autoantibodies, use of the monoclonal antibody or antigen binding

fragments thereof reactive with Type IX Collagen, autoantibodies to Type IX Collagen

may be detected using sandwich ELISA techniques. For example, the monoclonal antibody of the present invention or antigen-binding fragment thereof is bound to a solid

phase support, as the first component of a "sandwich-type" assay. A sample containing

human type IX collagen is added to said component wherein an antigen-antibody complex is formed. A second sample of synovial fluid or serum, for example, is contacted to the bound type IX collagen antigen wherein an autoantibody binds to the

type IX collagen antigen. A third antibody conjugated with a labeled enzyme is added

which allows for the detection of the autoantibody specific for type IX collagen. The

techniques of sandwich ELISA are described in, for example, Ausubel et al, Current Protocol of Molecular Biology, Volume 2, pp. 11.2.4-1 1.2.7, Wiley Interscience,

publishers. The detection of such autoantibodies to Type IX Collagen may serve as a prognostic marker for rheumatoid arthritis.

Radioimmunassay may also be used to measure levels of Type IX Collagen. For

example, Type IX Collagen is radioactively labeled and mixed with monoclonal antibodies specific for Type IX Collagen and a serum sample containing an unknown

amount of unlabeled Type IX Collagen. Binding competition between the labeled and

unlabeled Type IX Collagen with the monoclonal antibody occurs. By measuring the

amount of radioactivity of the reaction mixmre, the amount of Type IX Collagen present in the sample can be quantitatively determined. The method of radioimmunoassay is

described further in publications such as US Patent Nos. 4,438,209 and 4,591,573.

The antibodies against Type IX Collagen can be used in combination with

antibodies to detect the related type II and/or type XI collagens in order to better canvas a

patient population. Kits. The present invention also involves reagents and kits comprising such reagent for measuring Type IX Collagen in fluid samples. The diagnostic kit of the invention contains reagents for measuring levels of Type IX Collagen in serum samples.

This reagent kit comprises an antibody, and preferably a monoclonal antibody, which

specifically detects Type IX Collagen bound to a support, and a second antibody, again preferably a monoclonal antibody, which is specific to Type IX Collagen that binds to a

different epitope of the Type IX Collagen molecule. The second is enzyme-labeled. In

addition, the reagent kit of the present invention comprises reagents for detecting the

enzyme-labeled monoclonal antibody. The reagent kit employs immunological methods

in measuring Type IX Collagen in the serum sample, thus allowing for the detection and

monitoring of cartilage related autoimmune diseases such as rheumatoid arthritis. The kit is packaged and labeled, for example, in box or container which includes the necessary

elements of the kit, and directions and instructions on the use of such diagnostic kit.

6. EXAMPLES

Preparation of Antibodies Against Human Collagen IX

Design of Peptides for Immunization. The sensitivity of type IX collagen to

proteinase and its cross-linking to type II collagen make the extraction of intact type IX

collagen from cartilage extremely difficult. Therefore, peptide antibodies were designed and then made. As the collagen domain is a poor immunogen, peptides chosen for

immunization were from non-collagen domains. For αl(IX), one sequence was selected

from NC4 domain and another was chosen from NC2 domain. For α2(IX), two peptides were chosen from NC2 domain with four amino acid residual overlap. For α3(IX) chain,

one was from NC2 and another from the interface between NC2 and Col 2 domain were

selected. The locations of peptides in the collagen IX are shown in FIG. 1. As set forth

at FIG. 2, depicting a schematic representation of collagen type IX, there is no GAG in the NC3 domain of α2(IX) chains.

Peptides Synthesis, Immunization and Sera Collection. Peptides were synthesized

based on the sequences depicted at FIG. 1A-1C using known techniques. One cysteine residue was added in the less immunogenic end of the peptides for conjugating peptides

to the carrier. The peptides were purified by reverse phase C8 column operated by HPLC.

The identity of the peptide was confirmed by Mass Spectrum. One example is shown in

FIG. 3 A and 3B. To obtain the desired immunogenic response to the small peptide, the

peptide was conjugated to a carrier protein. In this example, the carrier used was ovalbumin, one of most commonly used carrier protein with well characterized T-cell determinants.

After pre-bleeding, animals were injected with peptide-carrier conjugates mixed

with Complete Freund's Adjuvant. The animals then were injected every two weeks and

test-bleeds were performed for monitoring the antibody titers. Production bleeds were performed after three boosts.

Antibody Titer and Specificity. Peptide-carrier conjugates were used to test the

antibody titer by ELISA with HRP conjugated second antibodies. The results are

expressed by the reciprocal of the serum dilution that results in an OD492nm of 0.200, as set forth at Table 1, below. Table 1 Antibody titer of antisera against individual chain of human collagen IX from Rabbits α Chain Rabbits Pre-bleed Prod. Bleed αl(IX) 42176 <50 >200,000 αl(IX) 42175 <50 28,400 α2(IX) 42177 <50 >200,000 cc2(IX) 42178 <50 104,500 α3(IX) 42174 <50 31,500 α3(IX) 42173 <50 88,800

The specificity of the antisera were tested by immunoblotting. Heterotrimer of recombinant human collagen type IX and homotrimer of each individual chain expressed in baculovirus system were used. Resolution of cell extracts by polyacrylamide gel

electrophoresis is shown in FIG. 4. The results of immunoblotting using enhanced

chemiluminescence are in FIG. 5 and show that all antisera are specific for recombinant

human collagen type IX, as expressed in a baculovirus system. Antisera against each

individual type IX collagen α chain does not cross-react with other type IX collagen α

chains and all the antisera do not show significant cross-reactivity with host insect cell

protein. The specificity for collagen IX in other expression system as well as in cartilage

could be examined in a similar fashion, if it is needed.

All references cited within the body of the instant specification are hereby

incorporated by reference in their entirety. In addition, the publications listed below are of interest in connection with various aspects of the invention and are incorporated herein

as part of the disclosure:

Claims

CLAIMSThe following is claimed:

1. A method for detecting an autoimmune disorder in a patient suspected of having such disorder comprising:

a. reacting a first sample from said patient with an antibody specific for type

IX collagen or subunits or fragments thereof wherein said patient is suspected of

having the autoimmune disorder;

b. allowing said antibody specific for type IX collagen or subunits or fragments thereof to interact to form an antigen-antibody complex;

c. measuring the amount of said antigen-antibody complex to determine the

amount of said type IX collagen or subunits or fragments thereof present in the

reaction mixture; d. repeating each of steps a, b, and c on a second non-diseased control serum

sample; and

e. comparing the amount of type IX collagen or subunits or fragments

thereof in first sample to the amount of type IX collagen or subunits or fragments

thereof in second sample to determine presence or progression of said autoimmune disorder.

2. The method for detecting the autoimmune disorder according to claim 1 wherein

said antibody specific for type IX collagen or subunits or fragments thereof is enzyme-

labeled and the amount of said antigen-antibody complexes formed in each of the first and second sample mixtures are measured by: a. removing uncomplexed enzyme-labeled antibodies from each of said first and second sample mixtures by allowing said uncomplexed enzyme- labeled antibody-antigen complexes to form on a surface by contacting

said mixtures with such surface to which type IX collagen or subunits or fragments thereof is bound;

b. removing of enzyme-labeled antibody-antigen complexes from said surface; and

c. contacting the enzyme-labeled antibody complexed to said surface-bound

type IX collagen or subunits or fragments thereof with a substrate of the

enzyme, measuring the enzyme activity to determine the amount of type IX collagen or subunits or fragments thereof in the first and second samples.

3. The method according to claim 2 wherein the sample from a patient suspected to

have the autoimmune disorder contains abnormal levels of type IX collagen or subunits

or fragments thereof compared to levels in the control non-diseased sample is indicative

of an autoimmune disorder.

4. The method according to claim 3 wherein said autoimmune disorder is

rheumatoid arthritis or osteoarthrosis.

5. The method of detecting an autoimmune disorder according to claim 1 wherein a

known amount of radiolabeled type IX collagen or subunits or fragments thereof is added

after the antibody specific for said type IX collagen or subunits or fragments has been

added to the first and second samples and the amount of antigen-antibody complexes

formed in each said samples is measured by: a. adding a preparation of anti-immunoglobulin to each mixture to form

immune complexes with the antigen-antibody complexes; and

b. separating the immune complexes from supernatant fractions of the mixtures, measuring the radioactivity of the immune complexes or the supernatant fractions to determine the amount of type IX collagen or

subunits or fragments in the first or second samples.

6. The method according to claim 5 wherein the sample from a patient suspected to

have an autoimmune disorder contains abnormal levels of type IX collagen or subunits or fragments thereof compared to levels in the control non-diseased sample is indicative of an autoimmune disorder.

7. The method according to claim 6 wherein said autoimmune disorder is

rheumatoid arthritis.

8. The method according to claim 5 wherein the sample from a patient suspected or known to have an autoimmnue disorder contains abnormal levels of type IX collagen or

subunits or fragments compared to levels in the control non-diseased sample is predictive

of the prognosis of such autoimmune disorder.

9. The method according to claim 8 wherein said autoimmune disorder is rheumatoid arthritis or osteoarthrosis.

10. The method according to claim 1 wherein said sample is a serum sample or

synovial fluid sample.

11. The method according to claim 1 wherein said antibody specific for type IX

collagen or subunits or fragments thereof is a monoclonal antibody.

12. The method according to claim 1 wherein said antibody specific for type IX collagen or subunits or fragments thereof is a human or humanized antibody.

13. A method for monitoring an autoimmune disorder in a patient known to have such

disorder comprising: a. reacting a first sample from said patient with an antibody specific for type

IX collagen or subunits or fragments thereof wherein said patient is known of having such autoimmune disorder;

b. allowing said antibody specific for type IX collagen or subunits or

fragments thereof to interact to form an antigen-antibody complex; c. measuring the amount of said antigen-antibody complex to determine the amount of said type IX collagen or subunits or fragments thereof present in the

reaction mixture;

d. repeating each of steps a, b, and c on a second sample of said patient taken

subsequent to first sample over a period of time; and e. comparing the amount of type IX collagen or subunits or fragments

thereof in second sample to determine presence or progression of said

autoimmune disorder.

14. The method for monitoring an autoimmune disorder according to claim 13 wherein said antibody specific for type IX collagen or subunit or fragment thereof is

enzyme-labeled and the amount of said antigen-antibody complexes formed in each of the

first and second sample mixtures are measured by: a. removing uncomplexed enzyme-labeled antibodies from each of said first and second sample mixtures by allowing said uncomplexed enzyme-

labeled antibody-antigen complexes to form on a surface by contacting said mixtures with such surface to which type IX collagen or subunit or

fragment thereof is bound;

b. removing of enzyme-labeled antibody-antigen complexes from said surface; and

c. contacting the enzyme-labeled antibody complexed to said surface-bound

type IX collagen or subunit or fragment thereof with a substrate of the

enzyme, measuring the enzyme activity to determine the amount of type

IX collagen or subunit or fragment thereof in the first and second samples.

15. The method according to claim 14 wherein the sample from a patient suspected or known to have an autoimmune disorder contains abnormal levels of type IX collagen or

subunits or fragments thereof compared to levels in the control non-diseased sample is

indicative of said autoimmune disorder.

16. The method according to claim 15 wherein said autoimmune disorder is

rheumatoid arthritis or osteoarthrosis.

17. The method according to claim 14 wherein the sample from a patient suspected or

known to have an autoimmune disorder contains abnormal levels of type IX collagen or

subunits or fragments thereof compared to levels in the control non-diseased serum

sample is predictive of the prognosis of said autoimmune disorder.

18. The method according to claim 17 wherein said autoimmune disorder is

rheumatoid arthritis or osteoarthrosis.

19. The method of monitoring an autoimmune disorder according to claim 13 wherein a known amount of radiolabeled type IX collagen or subunits or fragments thereof is

added after the antibody specific for said type IX collagen or subunits or fragments

thereof has been added to the first and second samples and the amount of antigen- antibody complexes formed in each said samples is measured by:

a. adding a preparation of anti-immunoglobulin to each mixture to form

immune complexes with the antigen-antibody complexes;

b. separating the immune complexes from supernatant fractions of the

mixtures, measuring the radioactivity of the immune complexes or the supernatant fractions to determine the amount of type IX collagen or

subunits or fragments thereof in the first or second samples.

20. The method according to claim 19 wherein the sample from a patient suspected or

known to have an autoimmune disorder contains abnormal levels of type IX collagen or subunits or fragments thereof compared to levels in the control non-diseased sample is indicative of said autoimmune disorder.

21. The method according to claim 20 wherein said autoimmune disorder is

rheumatoid arthritis or osteoarthrosis.

22. The method according to claim 19 wherein the sample from a patient suspected or

predictive of the prognosis of such autoimmune disorder.

23. The method according to claim 22 wherein said autoimmune disorder is rheumatoid arthritis or osteoarthrosis.

24. The method according to claim 13 wherein said sample is a serum sample or

synovial fluid sample.

25. The method according to claim 13 wherein said antibody specific for type IX collagen or subunits or fragments thereof is a monoclonal antibody.

26. The method according to claim 13 wherein said antibody specific for type IX

collagen or subunits or fragments thereof is a human or humanized antibody.

27. A reagent kit comprising: a. a first antibody that specifically binds to collagen type IX or subunits or

fragments thereof bound to a support;

b. a second antibody that specifically binds to collagen type IX or subunits or

fragments thereof that binds to an epitope different from the first antibody wherein said second antibody is enzyme-labeled; and

c. reagents for detecting enzyme-labeled antibody.

28. A reagent kit of Claim 27 which is useful for detecting or monitoring the

progression of an autoimmune disorder.

29. The reagent kit according to claim 27 wherein said autoimmune disorder is rheumatoid arthritis.

30. An antibody that specifically binds to collagen type IX or subunits or fragments

thereof.

31. The antibody of claim 30 wherein said antibody is a monoclonal antibody.

32. The antibody of claim 30 wherein said antibody is a human or humanized antibody.

33. A method for detecting a connective tissue disorder in a patient suspected of

having such disorder comprising:

a. reacting a first sample from said patient with an antibody specific for type IX collagen or subunits or fragments thereof wherein said patient is suspected of having the connective tissue disorder;

b. allowing said antibody specific for type IX collagen or subunits or

fragments thereof to interact to form an antigen-antibody complex;

c. measuring the amount of said antigen-antibody complex to determine the amount of said type IX collagen or subunits or fragments thereof present in the reaction mixture;

d. repeating each of steps a, b, and c on a second non-diseased control serum

sample; and

e. comparing the amount of type IX collagen or subunits or fragments thereof in first sample to the amount of type IX collagen or subunits or fragments

thereof in second sample to determine presence or progression of said connective

tissue disorder.