WO2010085763A1 - Methods for detecting antibodies associated with autoimmune diseases utilizing a three dimensionally heterogeneous peptide antigen - Google Patents

Abstract

A diagnostic method for detecting autoimmune disease is disclosed More particularly, various embodiments comprise systems and methods utilizing three dimensionally heterogeneous peptide antigens with complex structures that bind autoantibodies of at least two different classes, or "isotypes" By diversifying both the antigenic target and the classes of antibodies being detected, sensitivity is enhanced

Description

METHODS FOR DETECTING ANTIBODIES ASSOCIATED WITH

AUTOIMMUNE DISEASES UTILIZING A THREE DIMENSIONALITY

HETEROGENEOUS PEPTIDE ANTIGEN

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Applications 61/205,818, filed on January 23, 2009, and 61/234,227, filed on August 14, 2009, both of which are incorporated herein in their entirety.

TECHNICAL FIELD

[0002] The present invention is in the field of diagnostic methods for detecting autoimmune disease. More particularly, the present invention relates to systems and methods utilizing three dimensionally heterogeneous peptide antigens with complex structures that bind autoantibodies of at least two different classes, or "isotypes". By diversifying both the antigenic target and the classes of antibodies being detected, sensitivity is enhanced.

BACKGROUND OF THE INVENTION

[0003] Detection of antibodies in a biological sample to identify the presence of a disease, infection or an immune reaction is known in the ait. For some indications, such as autoimmune disease, these antibodies are autoantibodies which recognize and complex with self antigens. Because of this, autoimmune disease is a major health risk worldwide. The actual numbers of those individuals that have autoimmune disease are not known due to lack of recorded statistics. However, this number is expected to increase dramatically in the next decade because of a number of factors, including increasing environmental pollution. Today, there are over eighty illnesses caused by autoimmunity and several others believed to be the result of this condition (see Table 1). Approximately 5-7% of all Americans are affected by these diseases and over 75% of those are women. In fact, it is one of the ten leading causes of death in women in all age groups up to 65 years. By comparison, approximately 23.5 million people in the United States suffer from autoimmune disease as compared to 9.0 million that have cancer. [0004] Autoimmune disease arises when the body initiates an immune response against its own tissues and organs. When this occurs, the immune system produces antibodies, also known as autoantibodies, and/or immune cells that target and attack particular cells or tissues of the body. This reaction is called an autoimmune response and is characterized by inflammation and tissue damage. A variety of immunoglobulin types are often involved in the autoimmune response and their presence in the circulation can be an indication of the progression of the disease. For example, in rheumatoid arthritis the first antigen encounter activates naive B cells to proliferate and differentiate into IgM antibody- secreting cells. Upon further contact with T- cells, some B-cells start to produce antibodies of other classes such as IgG.

[0005] Some of the more common autoimmune disorders include rheumatoid arthritis, systemic lupus erythematosus (lupus), and celiac disease. Additional diseases that are believed to be due to autoimmunity include glomerulonephritis, Addison's disease, mixed connective tissue disease, and some cases of infertility. Table 1 lists a number of autoimmune disorders and their conditions, along with known antigenic targets, the sequences of which are reported in the literature. In many cases, the precise peptide sequences of the antigenic epitope associated with the disease is also known.

Table 1 : Autoimmune Disorders

Table 1 : Autoimmune Disorders, cont.

Table 1 Autoimmune Disordeis, cont

[0006] Autoimmune ieactions can be tnggeied in seveial ways They may be triggered when a substance in the body, which is noimally confined to a specific area and hidden fiom the immune system, is released into the bloodstieam oi a normal body substance is altered such as by vii al infection or radiation, oi a foieign substance iesembles a natuial body substance In other cases, the cells that control antibody production, B lymphocytes, malfunction and pioduce abnormal antibodies that attack specific cells of the body In all cases, the underlying problem is similai, the body's immune system becomes misdnected and attacks the veiy oigans it was designed to piotect

[0007] Heiedity may play a iole m some autoimmune disorders In these cases, susceptibility to the disordei, iathei than the disoider itself, is inheiited In susceptible individuals, a vπal infection or tissue damage may trigger the development of the disorder. In other cases, hormonal factors such as estrogen may play a role in the development of autoimmune disease, which may explain why autoimmune disorders are more common among women.

[0008] Symptoms vary depending on the disorder and the part of the body affected. Some autoimmune disorders affect the body systemically such as the blood vessels, cartilage, or skin while others affect particular organs such as the kidneys, lungs, heart, and brain. The resulting inflammation and tissue damage can cause pain, deformed joints, weakness, jaundice, itching, difficulty breathing, accumulation of fluid (edema), delirium, and death. While the severity of the symptoms depend on the disorder and its progression, autoimmune disease is often debilitating, substantially reducing an individual's quality of life.

[0009] Most autoimmune diseases cannot be prevented and to date there are no known cures. While the mechanisms involved in how this disease affects the body are known, its etiology is still unclear, At the simplest level, replacement of the specific secretions of tissues or organs damaged by autoimmune reactions often help. For multisystem autoimmune disease, such as lupus, cortisone derivatives that modify the harmful effects of humoral and cellular autoimmune attack on tissues may be administered that allow the body to reestablish immunologic homeostasis. Cytotoxic immunosuppressive drugs may also be utilized to inhibit the activity of immunologically active cells responsible for autoantibody formation or for cytolytic damage to tissues.

[0010] In autoimmune diseases, early detection and treatment can result in slowing progression of the disease. For example, treating patients who are showing the first signs of MS with beta-interferon-significantly delays the onset of clinically established MS and the occurrence of permanent disabilities, The recent study presented at the Annual European Congress of Rheumatology by Henrike Van Dongen in 2006 suggests that people diagnosed with undifferentiated rheumatoid arthritis could have their disease outlook changed significantly if the proper treatment is given at the proper time. The study concluded that patients receiving methotrexate developed RA more slowly during the observed time than those receiving a placebo. Because of this finding, diagnostic tests that accurately identify autoimmune disease are critical in maintaining a patients existing quality of life. [0011] Current diagnostic tests utilize a peptide fragment of a known antigenic protein that contains the specific epitopic sequence to which the autoantibody of interest is directed. This peptide fragment forms a complex with the autoantibody in solution and is detected by a reporter group. The type of reporter group and the signal it generates depends on the type of assay utilized. A number of patents that disclose a variety of these diagnostic tests are listed in Table 2.

Table 2: Known Diagnostic Tests for Autoimmune Diseases

[0012] One disadvantage of these assays is that they detect only a single antigenic sequence of a protein for which a single autoantibody in the biological sample is directed. If the antibody reacting with this one sequence is not present, or an autoimmune response is not strong enough to be detected, these assays could result in a false negative. Correspondingly, if the detection system is specific for a particular type of antibody class such as IgG and that class of antibody is not produced or is present in such limited quantities that it cannot be easily detected, a false negative would also result. As stated above, the progression of the disease may be a factor in the ability to detect a certain class of antibody, which demonstrates another disadvantage of some of the above assays. In addition, the ability of the autoantibody to bind the antigenic peptide will depend on the three dimensional structure of the antigenic peptide. In many instances, restricting the mobility of the peptide to a less flexible configuration that mimics the binding site of the autoantibody can provide faster and stronger binding. For example, cyclization of the peptides or the formation of multimeric complexes, such as dimers, trimers, tetramers, etc. via intermolecular crosslinking of two or more antigenic peptides can provide the increased rigidity necessary to achieve better results. As discussed elsewhere herein, complex formation results not only in some portion of the peptides being rendered less flexible, but also results in enhancing conformational heterogeneity. In any event, the results obtained are critical for the health care provider to accurately diagnose and initiate treatment for some autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.

[0013] Similar methods as those described for autoimmune disease assay systems have also been applied to assays for other disease states, infections or immune responses in which the presence of an antibody directed against a known antigen associated with the condition will indicate the presence of that condition. The disadvantages observed in autoimmune disease assays also occur in these assays. Because of this, there is a need for an assay to detect autoimmune disease, infections and other immune responses that assists the healthcare provider in making accurate diagnoses to initiate treatment without delay. Since there are no known medications that will eliminate autoimmune disease, slowing the disease progression is critical to maintaining quality of life. Assays that utilize a single antigenic peptide with a homogeneous tertiary structure may not be sufficient to identify patients that do not express antibodies to the sequence or express the antibodies at a level not sufficiently above background to have statistical significance. These assays would result in false negatives. Correspondingly, those assays that detect the presence of peptide autoantibody complexes using antibody isotype-specific reporter groups would result in false negatives if the patient does not express that particular isotype of immunoglobulin. In addition, assays that rely solely on the amino acid sequence of the antigenic peptide without consideration of the secondary, tertiary and possibly quaternary structural configuration of the native protein may promote non-specific binding resulting in false positives, or may result in lack of sensitivity and false negatives. Therefore, test devices are needed that can accurately measure antibodies associated with autoimmune disease to facilitate the diagnosis of autoimmune diseases, reduce the number of false negative and false positive results and expedite initiation of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Size exclusion chromatograms of the peptide wherein the cysteine residues were protected with different pairs of protecting groups that were removed preferentially to allow desired sulfhydryl bridging forming a spectacles shaped peptide.

[0015] Figure 2: Size exclusion chromatograms of (A) the peptide of Figure 1 reduced with DTT showing a primary monomelic peak 3 and multimeric peaks 1 and 2 and (B) the same peptide oxidized showing increased multimeric peaks 1 and 2.

[0016] Figure 3: Enzyme linked immunosorbent assay showing the activity of the peptide in Figure 1 (not normalized for concentration) demonstrating that all forms of the peptide are active.

SUMMARY OF THE INVENTION

[0017] The present invention is an assay for the detection of antibodies in a patient, and more particularly, to detecting autoantibodies in a patient suspected of having an autoimmune disease. The assay comprises the steps of preparing a three dimensionally heterogeneous peptide antigen, adding a biological sample from the patient to the heterogeneous antigen, and measuring the binding of the heterogeneous antigen to antibodies present in the sample. The heterogeneous peptide antigen comprises at least two different antigenic amino acid sequences from at least one protein associated with an autoimmune response in the patient suspected of having an autoimmune disease. The peptide antigen has an amino acid sequence with at least three cysteine residues that can form two or more disulfide linkages, intramolecularly and intermolecularly, resulting in a heterogeneous population of variably constrained configurations of the antigen (Le. a three dimensionally heterogeneous antigen). The presence of the heterogeneous antigen bound to the autoantibodies in the assay indicates the detection of the autoantibodies. [0018] The amino acid sequence of the heterogeneous antigen may also have at least three cysteine residues, or other residues such as lysines, from which at least three covalent linkages from one amino acid to the other may be formed. When the heterogeneous antigen has four cysteine residues, it can form at least two different intramolecular disulfide bonds resulting in a heterogeneous population of variably constrained configurations of the monomer antigen, and several, even hundreds of, different sets of intermolecular disulfide bonds resulting in the formation of polymers. For example, when the heterogeneous antigen contains four or more cysteines, it can form numerous inter-peptide (i.e., between different peptide molecules) antigen disulfide bonds, leading to complex three dimensional structures of dimers, trimers and higher order polymers.

[0019] The assay may be performed on a variety of biological samples including blood, serum, plasma, saliva, tears, synovial fluid and spinal fluid and may be designed to detect a variety of autoimmune diseases such as rheumatoid arthritis, celiac disease, systemic lupus erythematosus, polymyositis and deπnatomyositis, and the other autoimmune diseases listed in Table 1. The assay may also be performed in a variety of formats including enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), chemical-linked immunosorbent assay (CLIA), radioimmuno assay (RIA) and hnmunoblotting.

[0020] In another aspect of the present invention a heterogeneous antigen is provided having at least two antigenic amino acid sequences (i.e., "epitopes") obtained from or homologous to one or more proteins. In the case of rheumatoid arthritis, these proteins may be filaggrin, fibrinogen, collagen, or vimentin. These one or more antigenic sequences may come from the same protein or different proteins. Representative antigenic sequences for detection of rheumatoid arthritis may be selected from ESTRGRSTR (SEQ ID NO: 1), RKRRGSR (SEQ ID NO: 2), RSRRGR (SEQ ID NO: 3), LERRNNRKG (SEQ ID NO: 4), GVRGPRVEXHQS (SEQ ID NO: 5) and YATRSS (SEQ ID NO: 6). These sequences may be identical to the sequences of the selected protein or they may be modified. In rheumatoid arthritis, certain protein antigenic amino acid sequences comprise one or more arginine residues replaced with citrulline or other amino acids or citrulline analogues. In this embodiment, the heterogeneous antigen may comprise antigenic sequences from more than one protein such as filaggrin, fibrinogen, collagen, vimentin and/or PAD-4. [0021] In one embodiment, the heterogeneous antigen may be further modified by inserting a charged amino acid residue adjacent to each cysteine residue. These charged amino acid residues are selected and placed in the sequence of the heterogeneous antigen to assist in (or to hinder) the formation of cysteine disulfide linkages intramolecularly and intermolecularly. More specifically, oppositely charged amino acid residues are placed adjacent to cysteine residues for which disulfide linkages are desired. Correspondingly, identically charged amino acid residues may be positioned adjacent to cysteine residues to reduce their ability to form a disulfide linkage, In addition, a proline residue may be inserted into the heterogeneous antigen between two or more of the antigenic amino acid sequences to provide some structural rigidity.

[0022] In another embodiment of the present invention, the heterogeneous antigen may comprise at least three cysteine residues which may be utilized to influence the three dimensional structure of the heterogeneous antigen itself or for the formation of multimeric complexes containing two or more heterogeneous antigens bound via sulfhydryl bridges. The inclusion of three or more cysteine residues and the choice of neighboring amino acid residues results in a heterogeneous three dimensional structure upon oxidation of the cysteine, such as a mixture of monocyclic, bicyclic, multicyclic, and even more complex "mesh" or "knotted" conformations. Correspondingly, the presence of three or more cysteine residues within the heterogeneous peptide antigen results in complex formation via intermolecular bonding between peptides resulting in dimers, trimers, tetramers and other multimers. Such conformational heterogeneity alone improves sensitivity since some autoantibodies may bind to one conformation and other autoantibodies may bind to a different conformation.

[0023] In one embodiment for the detection of autoantibodies associated with rheumatoid arthritis, a heterogeneous antigen comprises one or more of the sequences X¹EGTRGRTX²RKRRGSRX¹ (SEQ ID NO: 7), X¹EGTRGRTX²RSRRGRX¹ (SEQ ID NO: 8), X¹EGTRGRTRX²LERRNNRKGX¹ (SEQ ID NO: 9), X¹EGTRGRTRX²GVRGPRVERHQX¹ (SEQ ID NO: 10), X¹EGTRGRTR X²YATRSSAX⁵ (SEQ ID NO: 1 1), X¹RKRJlGSRX²RSRRGRXⁱ (SEQ ID NO: 12), X¹RKKRGSRX²LERRNNRKGX' (SEQ ID NO: 13), X¹ RKRRGSRX²G VRGPRVERHQSX¹ (SEQ ID NO: 14), X¹RKRRGSR X²YATRSSAX¹ (SEQ ID NO: 15), X^IRKKRGSRX²ESTRGRTRX¹ (SEQ ID NO: 16), X¹RSRRGRX²LERRNNRKGX¹ (SEQ ID NO: 17), X^!RSRRGRX²GVRGPRVERHQSX¹ (SEQ ID NO: 18), X¹RSRRGRX²YATRSSAX¹ (SEQ ID NO: 19), X¹RSRRGRX²ESTRGRTRX¹ (SEQ ID NO: 20), X¹RSRRGRX²KRRGSRX¹ (SEQ ID NO: 21),

X¹LERRNNRKX²GVRGPRVERHQSX¹ (SEQ ID NO: 22), X ¹LERRNNRKGX²YATR S SAX¹ (SEQ ID NO: 23), X¹ LERRNNRKGX²ESTRGRTRX ' (SEQ ID NO: 24), X¹LERRNNRKGX²RKRRGSRX ' (SEQ ID NO: 25), X¹LERRNNRKGX²RSRRGRX ' (SEQ ID NO: 26), X¹GVRGPRVERHQSX²Y ATRSSAX¹ (SEQ ID NO: 27), X¹GVRGPRVERHQSX²ESTRGRTRX¹ (SEQ ID NO: 28),

X¹GVRGPRVERHQSX²RKRRGSRX¹ (SEQ ID NO: 29), X¹GVRGPRVERHQSX²RSRRGRX ' (SEQ ID NO: 30) and X¹GVRGPR VERHQSX² LERRNNRHGX¹ (SEQ ID NO: 31) wherein one or more of the arginine residues is replaced with citrulline residues; X¹ is -C, C-, CY-, -YC (depending on its orientation) or nothing; X² is -CPC-, -CGGC- or nothing and Y is a charged amino acid residue such as tyrosine. In this example, it should be understood that in all cases, the antigen will include at least three or more cysteine residues, and two or more of the sequences above may be joined together so long as the resulting peptide also has three or more cysteine residues.

[0024] In one embodiment, the sequences above are assembled in a C C C, C

CPC C, or C CPC CPC C configuration , where " '^* is any number of five or more amino acids including an antigenic sequence, C is cysteine and P is proline, such that the formation of conformationally diverse antigenic peptide targets is promoted based on variations in disulfide bridge formation via oxidation.

[0025] In another embodiment, the sequences above are assembled in a — C C C-, —

C C-C C-, -C C-C C-C C- or --C C-C C- configuration, where

" " is any number of Five or more amino acids including an antigenic sequence, and C is cysteine, such that the "formation" of conformationally diverse antigenic peptide targets is promoted based on variations in disulfide bridge formation, '"-" can be any amino acid and amino acid "tails" ("--") are allowed at the ends of the peptides.

[0026] In as yet another embodiment, in addition to including an internal -CPC- moiety, the sequences above are incorporated into a peptide having between 9 and 25 amino acids between the internal -CPC- moiety and external cysteines. [0027] In another embodiment, the covalent crosslinkages may be fonned by chemical modifcation of amino acid side chains by homobifunetional crosslinkers, heterobifunctional crosslinkers, reaction with EDC (l-Ethyl-3(dimethylaminopropyl)carbodiamide hydrochloride) to form a bond between an amino group and a carboxyl group, or other known crosslinking reagents.

[0028] In another aspect of the present invention, an assay for the detection of the increased likelihood of developing rheumatoid arthritis is provided comprising the steps of preparing a three dimensionally heterogeneous peptide antigen, adding a biological sample from a patient to the heterogeneous antigen, and measuring the binding of the heterogeneous antigen to the autoantibodies present in the sample. The heterogeneous peptide antigen comprises at least two antigenic amino acid sequences from at least one of the proteins filaggrin, vimentin, fibrinogen and collagen. The amino acid sequence has at least one citrulline amino acid instead of arginine and at least three cysteine residues that can form two or more disulfide linkages intramolecularly and intermolecuiarly resulting in many different three dimensional configurations of the heterogeneous peptide antigen. The presence of the autoantibodies bound to the heterogeneous antigen indicates the detection of an increased likelihood of the autoimmune disease.

[0029] In accordance with the preceeding summary, one embodiment of the present invention is an immunoassay for detection of antibodies in a subject suspected of having an autoimmune disease comprising the steps of:

[0030] a) preparing a plurality of peptide antigen molecules, wherein the peptide antigen molecules comprise at least two different antigenic amino acid sequences derived from at least one protein associated with an autoimmune disease, and wherein the peptide antigen molecules further comprise at least three cysteine residues;

[0031 ] b) oxidizing the at least three cysteine residues on the peptide antigen molecules to form at least one intermolecular bond between peptide antigen molecules and at least one intramolecular bond within peptide antigen molecules, wherein said oxidizing results in formation of a three dimensionally heterogeneous peptide antigen matrix;

[0032] c) adding a biological sample from the subject to the heterogeneous peptide antigen matrix to form a complex between at least two isotypes of the antibodies and the heterogeneous peptide antigen matrix; and [0033] d) detecting the complex.

[0034] The immunoassay may be a solid phase assay or a liquid phase assay.

[0035] Representative immunoassays include a fluorescent immunosorbent assay (FIA), a chemiluminescent immunosorbent assay (CLIA), a radioimmunoassay (RIA), an enzyme multiplied immunoassay techniques (EMIT), a solid phase radioimmunoassay (SPRIA), and an enzyme linked immunosorbent assay (ELISA).

[0036] The two different antigenic amino acid sequences may be incorporated into one peptide antigen molecule, such that all of the individual peptides in the plurality of peptide antigen molecules have the same amino acid sequence. Alternatively, they may be incorporated into two or more separate antigen molecules, such that the plurality of peptide antigen molecules include two or more species of peptides, each with a different amino acid sequence.

[0037] In one embodiment, the peptide antigen molecules have four cysteine residues, which are oxidized to form two or more intermolecular bonds between peptide antigen molecules and two or more intermolecular bonds within the peptide antigen molecules.

[0038] The oxidizing step may be performed by simply exposing the cysteine residues to air..

[0039] In one embodiment, the biological sample is serum or plasma.

[0040] In an alternative embodiment, the antibody isotopes being detected in the immunoassay are IgG and IgA.

[0041] The detecting step may be accomplished using a fluorescer, an enzyme, a chemiluminescer, a photos ensitizer or suspendable particles.

[0042] The subject may be suspected of having systemic lupus erythematosus, celiac disease,

Grave's disease, scleroderma, rheumatoid arthritis or type I diabetes mellitus.

[0043] The heterogeneous peptide antigen may have a sequence selected from the group consisting of SEQ ID NO. 138, SEQ ID NO. 139, SEQ ID NO. 140, SEQ ID NO. 141, SEQ ID

NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 145, SEQ ID NO. 146, SEQ ID NO.

147, SEQ ID NO. 148, SEQ ID NO. 161, SEQ ID NO. 162, SEQ ID NO. 163, SEQ ID NO. 164,

SEQ ID NO. 165, SEQ ID NO. 166, SEQ ID NO. 167, SEQ ID NO. 186, SEQ ID NO. 187, SEQ

ID NO. 188, SEQ ID NO. 189, SEQ ID NO. 190, SEQ ID NO. 191, SEQ ID NO. 192, SEQ ID

NO. 193, and SEQ ID NO. 194.

[0044] In yet another aspect of the present invention, there is provided a method of enhancing sensitivity of an immunoassay for detection of antibodies in a subject suspected of having an autoimmune disease by using a three dimensionally heterogeneous peptide antigen matrix as a target of the antibodies, wherein the peptide antigen matrix is prepared using the steps of:

[0045] a) preparing a plurality of peptide antigen molecules, wherein the peptide antigen molecules comprise at least two different antigenic amino acid sequences derived from at least one protein associated with an autoimmune disease, and wherein the peptide antigen molecules further comprise at least three cysteine residues; and

[0046] b) oxidizing the at least three cysteine residues on the peptide antigen molecules to form at least one intermolecular bond between peptide antigen molecules and at least one intramolecular bond within peptide antigen molecules, wherein said oxidizing results in foπnation of a three dimensionally heterogeneous peptide antigen matrix.

[0047] Other aspects of the invention are found throughout the specification and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0048] In the description that follows, a number of terms used in the field of molecular biology, immunology and medicine are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following non-limiting definitions are provided. AU references cited herein are incorporated by reference.

[0049] When the terms "one," "a," or "an" are used in this disclosure, they mean "at least one" or "one or more," unless otherwise indicated.

[0050] The term "antibody" refers to immunoglobulin molecule that is capable of binding an epitope or antigenic determinant. The term "antibody" includes whole antibodies and antigen- binding fragments thereof, including single-chain antibodies. Such antibodies include human antigen binding antibody and antibody fragments, including, but not limited to, Fab, Fab' and F(ab')₂, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a V₁ or V_n domain. The antibodies may be from any animal origin such as for example mammals including human, murine, rabbit, goat, guinea pig, camel, horse and the like.

[0051] The term "antigen'^" or ''antigenic determinant'^* or ''antigenic amino acid sequence" refers to a molecule or portion of a molecule capable of being bound by an antibody. An antigen, antigenic determinant or antigenic amino acid sequence may be additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes.

[0052] The term "autoantibody^"' refers to an immunoglobulin, antigen specific B cell surface receptor (surface immunoglobulin), directed against self-protein, carbohydrate or nucleic acid.

[0053] The term "control'^" or "control sample'^" refers to one or more biological samples, such as a serum sample, taken from at least one healthy donor.

[0054] The term "heterogeneous peptide antigen" has a dual meaning with respect to the present invention. In the first meaning, this term refers to the fact that two or more noncontiguous different antigenic amino acid sequences from a single protein or from more than one protein are synthesized into a single polypeptide. The antigenic amino acid sequences that comprise the heterogeneous peptide antigen are sometimes between 5 and 50 amino acids in length, or 5 and 30 amino acids in length, and even between 5 and 25 amino acids in length. In the second meaning, this term refers to the intramolecular and intermolecular bonding between side chains of one or multiple reactive amino acid residues, such as cysteine, within the antigen amino acid sequence or between one or more antigens which result in a heterogeneous population of structures that are variably constrained configurations (i.e. multiple cyclic or shaped configurations of a single antigen as well as dimer, trimer, tetramer and other multimer configurations between two or more antigens). Stated differently, the antigenic substance used in one embodiment of the diagnostic assays of the present invention has a homogeneous primary structure (i.e., the antigen is a collection of molecules, each of which has the same amino acid sequence), but they are allowed to form complex tertiary structures via covalent bond formation. Such an assay format enhances specificity. [0055] The term "intramolecularly" refers to the ability of amino acid residues within the sequence of a single peptide molecule to form covalent bonds. Such intramolecular binding may be by sulfhydryl bridging between cysteine residues within the amino acid sequence of the peptide molecule resulting in cyclization of the peptide forming cyclic structures.

[0056] The term "intermolecular" refers to the ability to form covalent bonds between the amino acid residues of two or more peptide molecules. Such intermolecular bonds may be formed by foπning sulfhydryl bridges between cysteine residues via oxidation resulting in two, three, four or more heterogeneous peptide antigens forming dimeric, trimeric, tetrameric or niultimeric complexes respectively. The intermolecular bonds may also be formed by chemical crosslinking between amino groups and carboxyl groups by addition of EDC, or by addition of bifunctional crosslinkers.

[0057] The term "patient^*' or "subject" as used herein refers to a variety of animal species that a sample may be obtained from for the detection of the presence of an autoimmune disease. These include but are not limited to mammals, birds, reptiles and fish. More specifically, the patient or subject is a mammal and most specifically, the mammal is a human. The patient may also be suffering from an infectious disease, cancer, a vaccine response, or some other state wherein measuring the antibody response is useful.

[0058] The term "biological sample" as used herein refers to any sample that may be obtained or prepared from a patient that would contain a detectable amount of autoantibodies if the patient were to have an autoimmune disease. For example, this could include biological samples such as serum, plasma, saliva, tears, blood, vaginal secretion, semen or homogenized tissue.

[0059] The phrase "a protein responsible for the autoimmune response" as used herein refers to a protein or proteins, which are recognized by antibodies in a subject.

[0060] The phrase "amino acid substitution" as used herein refers to the replacement or conversion of an amino acid residue in a sequence with another molecule. The molecule may be another natural or non-natural amino acid, an organic molecule or other chemical moiety, In replacement, one amino acid in a sequence being synthesized may be replaced with another amino acid. For example, replacing leucine with isoleucine which would be a conservative substitution wherein one hydrophobic amino acid is replaced with another hydrophobic amino acid. Another conservative substitution would be replacing lysine with serine. In conversion, one amino acid residue in a sequence is converted to another amino acid. For example, a glutamine may be converted by deamination to a glutamic acid, or an arginine residue converted by deiminase to a citrulline residue.

[0061] The present invention is an assay for the detection of antibodies in a patient, and more particularly, to detecting autoantibodies in a patient suspected of having an autoimmune disease. The assay comprises the steps of preparing a three dimensionally heterogeneous peptide antigen, adding a biological sample from the patient to the heterogeneous antigen, and measuring the binding of the heterogeneous antigen to antibodies present in the sample. The heterogeneous peptide antigen comprises at least two different antigenic amino acid sequences from at least one protein associated with an autoimmune response in the patient suspected of having an autoimmune disease. The peptide antigen has an amino acid sequence with at least three cysteine residues that can form two or more disulfide linkages, intramolecularly and intermolecularly, resulting in a heterogeneous population of variably constrained configurations of the antigen (i.e. a three dimensionally heterogeneous antigen). The presence of the heterogeneous antigen bound to the autoantibodies in the assay indicates the detection of the autoantibodies.

Autoimmune Disease

[0062] The human immune system protects the body against infection either by generating antibodies against foreign substances or by producing cytotoxic T cells that have receptors that recognize the surfaces of invading or infected cells. These antibodies, abbreviated Ig, are proteins that are found in blood or other bodily fluids of vertebrates. They are typically made of basic structural units — each with two large heavy chains and two small light chains — to form, for example, monomers with one basic unit, dimers with two units or pentamers with five units. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, IgM, IgA, IgG, IgD and IgE, which perform different roles, and help direct the appropriate immune response to each different type of foreign molecule they encounter.

[0063] Although the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures to exist. This region is known as the hypervariable region. Each of these variants can bind to a different target, known as an antigen. This huge diversity of antibodies allows the immune system to recognize an equally wide diversity of antigens. The unique part of the antigen recognized by an antibody is called an epitope. These epitopes bind with their antibody in a highly specific interaction, called induced fit, that allow antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organism. Recognition of an antigen by an antibody tags it for attack by other parts of the immune system. Antibodies can also neutralize targets directly by, for example, binding to a part of a pathogen that it needs to cause an infection,

[0064] The large and diverse population of antibodies is generated by random combinations of a set of gene segments that encode different antigen binding sites (or paratopes), followed by random mutations in this area of the antibody gene, which create further diversity. Selection of antibodies with high affinity for the pathogen driving the immune response results in an increased production of reactive antibodies. Antibody genes also re-organize in a process called class switching that changes the base of the heavy chain to another, creating a different isotype of the antibody that retains the antigen specific variable region. Class switching allows a single antibody reactivity to be used by several different parts of the immune system. Production of antibodies is the main function of the humoral immune system.

[0065] Each antibody class is structurally adapted for a particular biological activity and functions best at a different site in the body. This is why there are several genes for the constant region of the H chain.

[0066] IgM is a large protein (macroglobuljn) made up of five identical monomelic subunits held together by a small protein (J chain) and largely confined to the circulation. It is also present in the monomelic form on the surface of most B lymphocytes. The secreted form is predominant in early immune responses to most antigens and provides an effective first-line defense against bacteraemia. Although each subunit has a low affinity for antigen, its pentameric structure gives the whole molecule a relatively high avidity. The affinity is a measure of the strength of binding of a single binding unit to an epitope, whereas the avidity is the strength of binding of the entire antibody molecule with its multiple binding sites to several identical epitopes of an antigen. The multiple binding sites of IgM allow it to attach to several identical epitopes on different particulate antigens simultaneously, leading to agglutination. Blood group antibodies are mainly of this class.

[0067] IgG is the predominant immunoglobulin in the blood stream and tissue fluids, where it combats microorganisms and their toxins. There are four subclasses of IgG, all monomelic

IgG antibodies generally have a higher affinity for antigens than IgM. As well as being able to pass from blood to tissues, IgG is the only class of antibody to cross the placenta and this provides the newborn child with useful protection against infections to which the mother is immune.

[0068] IgA exists in both monomeric and dimeric forms. It is monomeric in the circulation and there are two subclasses. In its dimeric form it provides the primary defense against "local'^* infections owing to its abundance in saliva, tears, bronchial secretions, nasal mucosa, prostatic fluid, vaginal secretions and mucous secretions of the small intestine.

[0069] IgE is present as a monomer at very low levels in the circulation but provides protection for external body surfaces, especially mucosal surfaces by recruiting antimicrobial cells and agents. Mast cells have high-affinity receptors for the Fc region of IgE. The IgE- mediated degranulation of the mast cell, on exposure to its antigen, enhances the acute inflammatory response by recruiting a variety of cells into the site of infection. Levels of this antibody are increased during woπn infections and this class of antibody may have evolved for protection against such intruders. On the negative side, IgE is responsible for the symptoms of atopic allergy.

[0070] IgD is present predominantly as a monomer, together with IgM, on the surface of most B lymphocytes. Its function is at present unclear but it probably acts as an antigen receptor for the control of lymphocyte activation and suppression. It is thought to be absent from memory cells.

[0071] Thus, each antibody class/subclass, through its Fc region carries out a particular function. In some cases, antibodies of different classes may be expressed at different stages of an infection or disease even thought they have the same biological activity, e.g. IgM and IgG in fixing complement.

[0072] Autoimmune disease occurs when the immune system malfunctions, interpreting the body's own macromolecules or tissues as foreign and producing autoantibodies or immune cells that target and attack particular macromolecules, cells or tissues of the body. As with a normal immune response against a foreign molecule, the autoimmune response also produces different classes at different stages of an autoimmune disease. The symptoms affect almost every human organ system, including the nervous, gastrointestinal, endocrine, circulatory systems, as well as connective tissues and eyes. They include more than 80 widely varied chronic illnesses that affect 5-7% of the population, about two thirds of which are women. As a group they are the least understood diseases. It is suspected that these diseases have genetic pre-disposition, but are triggered by an infection.

[0073] Detecting and identifying autoantibodies in these diseases have yielded valuable information for both diagnosis and treatment of autoimmune diseases. Among the most popular diagnostic strategies is utilizing peptides that comprise specific antigenic sequences to capture the autoantibodies. These sequences are identified from the proteins that elicit the autoimmune responses. For example, antigenic sequences of the protein filaggrin have been identified and used in assays to detect the presence of autoantibodies in the serum of patients suspected of having rheumatoid arthritis.

Heterogeneous Antigen

[0074] The heterogeneous antigen of the present invention comprises at least two antigenic sequences from proteins recognized by autoantibodies generated in autoimmune disease. This feature is important, because not al! patients suffering from a particular autoimmune disease have circulating antibodies to the same exact antigenic epitope, and in many cases, their autoantibodies are directed towards epitopes on entirely different proteins.

[0075] In addition, the heterogeneous peptide antigen may also have the same antigenic sequence repeated two or more times, but it is still considered heterogeneous herein, because the multiple crosslinks may put the two epitopes into many different configurations, even if they have the same primary sequence. [0076] These proteins will vary depending on the autoimmune disease. For example the proteins filaggrin, vimentin, collagen, fibrinogen and PAD-4 have been identified in rheumatoid arthritis and antigenic sequences of these proteins may be utilized to identify the presence of this disease. Since there are over 80 autoimmune diseases that have been identified, heterogeneous antigens may be prepared for a variety of autoimmune diseases from antigenic sequences of the protein or proteins that elicit autoantibody responses. These sequences vary in length and can be identified utilizing routine techniques known in the art. One such technique digests the protein into fragments and another prepares synthetic overlapping peptides of the protein sequence to determine which of the sequences bind specifically to the autoantibody of interest.

[0077] It should be understood by one of skill in the art that while rheumatoid arthritis is presented as a model assay herein, other combinations of known antigenic sequences could be incoiporated into a heterogeneous antigenic target according to the teachings herein and easily screened against known positive autoimmune sera to select a heterogeneous antigen with the desired sensitivity.

[0078] The two antigenic sequences in the heterogeneous peptide antigen range in length from about 5 to about 30 amino acids, or more particularly from about 6 to about 25. The heterogeneous antigen comprises at least two antigenic sequences and may range in length from about 9 to about 50 amino acid residues, but may have as many as 120 residues. Its size is based on the number and length of the antigenic sequences as well as the increased immunospecificity which are often afforded by presenting smaller regions. Typically, a heterogeneous peptide antigen contains no more than about 100, preferably no more than about 70 and usually no more than about 40 amino acid residues. By incorporating more than two antigenic sequences into the heterogeneous peptide antigen, with at least 14 amino acids, such as between 14 and 18 each, between any given three cysteine residues, formation of a three dimensionally diverse antigen population can be accomplished thus enhancing sensitivity.

[0079] The antigenic sequences may have the identical sequence of a portion of the protein from which it was obtained, or it may be a modified sequence. These modified sequences may be functionally or immunologically equivalent variants that occur as natural biological variations (e.g., allelic variants, orthologs, splice variants or post-translational variants), or they may be novel sequences. They may be prepared using standard modification techniques such as for example site-directed or random mutagenesis or chemical synthesis such as replacing one amino acid with another, which preserves or enhances the immunological character and structure.

[0080] Post-translational modifications occur during the life of the protein and are considered "permissible substitutions" of amino acids that enhance the binding of the antigenic sequence to the autoantibody. In rheumatoid arthritis, fϊlaggrin is post translationally modified by deiminase that converts arginine residues to citrulline and in celiac disease deaminase converts glutaniine to glutamic acid in gliadin. These are two types of permissible amino acid substitutions that may be used to modify the antigenic sequences to enhance binding. While all post-translational modifications of proteins that may be bound by autoantibodies are not known, it is anticipated that one skilled in the art would make such permissible substitutions within antigenic sequences once such post-transϊational modifications for that particular protein are identified to determine if enhanced binding can be achieved.

[0081] Other modifications, within the scope of the present invention, provide for certain advantages in the use of the heterogeneous peptide antigen specifically in its ability to mimic the antigenic site of the protein and affinity for the autoantibody of interest. Such modifications include variants of the antigenic sequences. The term "variant" includes any antigenic sequence having an amino acid sequence in which one or more residues have been inserted, deleted or substituted with another residue.

[0082] Further, it has been observed that peptides containing at least three cysteine residues which are oxidized to form a heterogeneous peptide antigen exhibit enhanced binding of autoantibodies present in patient's serum or plasma. The cysteine could be a naturally occurring amino acid within the protein sequence from which it was obtained or may be added to the sequence during synthesis. This was an unexpected observation that is not thought to be associated with intramolecular binding alone when only two cysteine residues are present in the peptide sequence. However, it was this observation that led to the addition of three or more cysteine residues being incorporated into the peptide sequence.

[0083] Addition variants may include N- or C -terminal fusions, as well as intrasequence insertion, of single or multiple amino acids. This may also include the addition of cysteine residues at the terminal ends, or internally to the sequence, of the heterogeneous peptide antigen. The cysteine residues may be present in the antigenic sequences because they are part of the native protein sequence, added at the ends of the antigenic sequences or inserted between the antigenic sequences. These cysteine residues join to form disulfide linkages upon oxidation within the heterogeneous peptide antigen or between two or more heterogeneous peptide antigen molecules.

[0084] The number of conformations that may be formed by the heterogeneous peptide antigen will depend on the number of cysteine residues within the sequence and its degree of oxidation or crosslinking. Controlled or specific disulfide linkage foπnation intramolecularly may be accomplished using protecting groups that may be selectively removed thus allowing only those cysteine residues that are unblocked to form disulfide linkages (B. Hargittai and G. Barany J. Peptide Res. 54:468-479, 1999). It is also hypothesized that preferential disulfide linkages may be formed by inserting charged amino acid residues adjacent to the cysteine residues. Where foπnation of the disulfide linkage is preferred, oppositely charged amino acids are placed adjacent to the cysteine residues desired to form a linkage. Correspondingly, where certain cysteine residues are flanked by identically charged amino acids, it is anticipated that disulfide formation will not be favored. In addition, dimer, trimer, tetramer and multimer complex formation, or intermolecular sulfhydryl bridge formation between two or more heterogeneous peptide antigens may be controlled by the oxidizing conditions and antigen concentration. Alternatively, the number of conformations may be dependent on other forms of intra- and inter-molecular covalent crosslinking that are formed within a heterogeneous antigen and between heterogeneous antigens.

[0085] It has been observed unexpectedly that when there are three or more cysteine residues within the heterogeneous peptide sequence that various disulfide bridge formations, both intramolecularly and intermolecularly, result in the formation of a heterogeneous population of variably constrained conformations that provides an enhanced signal over a single-shaped (e.g., circular) confomier in a three dimensionally homogeneous version of the same peptide sequence. It is believed that this heterogeneous population of confoπners provides the antigenic sequences in a variety of structural configurations for more efficient binding of the autoantibodies. In addition, the complex size and shape of the antigen may allow a greater mass to bind to the solid phase than a linear or circular peptide, and may lift the antigenic sites higher above the solid phase than a linear form, thus making them more available to the antibodies. All of these may contribute to the increased immunoreactivity of a heterogeneous antigen over a linear or cyclic peptide.

[0086] In some cases, the unmodified antigenic amino acid sequence of a known protein provides optimal binding to antibodies in an immunoassay. However, sometimes variations of the known sequence may create antigens having greater binding affinity than the native antigen. Deletion variants may be intrasequence or may be truncations from the N- or C-termini. For example, it has been observed that in the filaggrin antigenic sequences, certain amino acids positioned within two to three amino acids residues of citrulline when removed do not affect the binding affinity of the antigenic sequence for its autoantibody.

[0087] In contrast, substantial modifications in the functional and/or chemical characteristics of the antigenic sequences may be accomplished by selecting amino acid substitutions that differ significantly in their effects on the structure (secondary, tertiary, and/or quaternary), in their charge or hydrophobicity of the antigenic sequence. For example, certain of the amino acid residues provide rigidity to the heterogeneous antigen based on its position within sequence. The amino acid proline acts to restrict the conformation of peptides. It has been hypothesized that there is enhanced autoantibody binding resulting in an increased signal when a proline is inserted between the antigenic sequences of the heterogeneous antigen.

[0088] Amino acid sequence modifications accomplished by chemical and biological synthetic methods are well known in the art. However, it should be noted that the heterogeneous peptide sequence should still contain at least two antigenic sequences with at least five amino acids each derived from the protein target or targets of interest.

[0089] Preferred antigenic amino sequences for the preparation of the heterogeneous antigen for the detection of rheumatoid arthritis are: ESTRGRSTR (SEQ ID NO: 1), RKRRGSR (SEQ ID NO: 2), RSRRGR (SEQ ID NO: 3), LERRNNRKG (SEQ ID NO:4), GVRGPRVEXHQS (SEQ ID NO: 5) and YATRSS (SEQ ID NO: 6) wherein one of more of the arginine residues is replaced with a citmlline residue. A heterogeneous antigenic sequence for the detection of rheumatoid arthritis comprises one or more of these antigenic sequences. More particularly the heterogeneous antigen comprises one or more of the following combined antigenic amino acid sequences: X^SEGTRGRTX²RKRRGSRX¹ (SEQ ID NO: 7), X¹EGTRGRTX²RSRRGRX¹ (SEQ ID NO: 8), X¹ EGTRGRTRX²LERRNNRKGX ' (SEQ ID NO: 9),

X¹EGTRGRTRX²GVRGPRVERHQX¹ (SEQ ID NO: 10), X¹EGTRGRTR X²YATRSSAX¹ (SEQ ID NO: 11), X¹RKRRGSRX²RSRRGRX¹ (SEQ ID NO: 12),,

X¹RKKRGSRX²LERRNNRKGX^! (SEQ ID NO: 13), X¹RKRRGSRX²GVRGPR VERHQSX¹ (SEQ ID NO: 14), X¹RKRRGSR X²YATRSSAX¹ (SEQ ID NO: 15),

XⁱRKKRGSRX²ESTRGRTRX^l (SEQ ID NO: 16), X¹RSRRGRX²LERRNNRKGX¹ (SEQ ID NO: 17), X¹RSRRGRX²GVRGPRVERHQSX¹ (SEQ ID NO: 18), X¹RSRRGRX²YATRSSAX¹ (SEQ ID NO: 19), X¹RSRRGRX²ESTRGRTRX ' (SEQ ID NO: 20), X⁸RSRRGRX²KRRGSRX¹ (SEQ ID NO: 21), X¹LERRNNRKX²GVRGPRVERHQSX¹ (SEQ ID NO: 22), X¹LERRNNRKGX²Y ATRSSAX¹ (SEQ ID NO: 23), X¹ LERRNNRKGX²E STRGRTRX¹ (SEQ ID NO: 24), X^ILERRNNRKGX²RKRRGSRX¹ (SEQ ID NO: 25),

X¹ LERRNNRKGX²RS RRGRX¹ (SEQ ID NO: 26), X¹GVRGPRVERHQSX²YATRSSAX¹ (SEQ ID NO: 27), X¹GVRGPRVERHQSX²ESTRGRTRX^I (SEQ ID NO: 28), X¹GVRGPRVERHQSX²RKRRGSRX¹ (SEQ ID NO: 29), X¹GVRGPRVERHQSX²RSRRGRX¹ (SEQ ID NO: 30), and X¹GVRGPRVERHQSX²LERRNNRHGX¹ (SEQ ID NO: 31) wherein one or more of the arginine residues is replaced with citrulline residues; X¹ is -C, C-. CY-, -YC (depending on its orientation) or nothing; X² is -CPC- or -CGGC- and Y is a charged amino acid residue. In this example, it should be understood that in all cases, the antigen will include at least three or more cysteine residues, and two or more of the sequences above may be joined together so long as the resulting peptide also has three or more cysteine residues.

[0090] In one embodiment, the sequences above are assembled in a C C C, C

CPC- -C, CE RCPCR EC, C CGGC-— -C or C— -CPC-- CPC C configuration, where " " is any number of five or more amino acids including an antigenic sequence, such that the formation of conformationally diverse antigenic targets is promoted based on variations in disulfide bridge formation intramolecular Iy and intermolecularly.

[0091] In another embodiment, the sequences above are assembled in a --C C C-, —

C C-C- -C-, -C C-C C-C- — C- or -C C-C C- configuration, where "- — " is any number of five or more amino acids including an antigenic sequence, and C is cysteine, such that the "formation" of conformational^ diverse antigenic peptide targets is promoted based on variations in disulfide bridge formation, and amino acid "tails" (^■'--") are allowed at the ends of the peptides.

[0092] In as yet another embodiment, in addition to including an internal -CPC- moiety, the sequences above are incorporated into a peptide having between 9 and 25 amino acids between the internal -CPC- moiety and external cysteines.

[0093] The heterogeneous antigen may be prepared using recombinant nucleic acid methodologies well known in the art. A DNA segment coding for a heterogeneous antigen of this invention can be synthesized by chemical techniques, for example the phosphotriester method of Matteucci et al, J. Am. Chem. Soc, 103:3185, (1981). The DNA segment can then be ligated into an expression vector, and a host transformed therewith can be used to produce the polypeptide. See, for example, Current Protocols In Molecular Biology, Ausubel et al, eds., John Wiley & Sons, New York, N.Y.; U.S. Patent 4,237,224, 4,356,270, 4,468,464, 4,683,195 and 4,889,818.

[0094] The recombinant expression vectors capable of expressing a heterogeneous antigen and methods of their use for producing a heterogeneous antigen are contemplated as pail of the present invention.

[0095] The heterogeneous antigen can also be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am. Chem. Soc, 85:2149-2154 (1963). Other synthesis techniques may be found, for example, in M. Bodanszky et al, Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) as well as in other reference works known to those skilled in the art. A summary of synthesis techniques may be found in J. Stuart and J. D. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, 111., 3d Ed., Neurath, H. et al, Eds., p. 104-237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in such syntheses will be found in the above texts as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). [0096] In general, those synthetic methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing polypeptide chain. Normally,-the amino group of the C-terminal amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine or cysteine.

[0097] Using a solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl group. The protecting group of the amino group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final heterogeneous antigen. To control the cyclization of the heterogeneous antigen having multiple cysteine residue, such as four cysteine residues, different protecting groups may be used that can be removed independently allowing only the desired cystienes to be reactive for disulfide linkage formation (B. Hargittai and G. Barany J. Peptide Res. 54:468-479, 1999 incorporated herein by reference).

[0098] Derivatives of the heterogeneous antigen may be by chemical or biological modification, including protein post-translation modification, such as acylation (i.e., acetylation or formylation), biotinylation, carboxylation, deamination, glutathionylation, glycosylatϊon, lipidation (i.e., farnesylation, geranylgeranylation,prenylation, myristoylation, palmitoylation, or stearoylation), methylation, nitrosylation, phosphorylation, sulphation, fucosylation, and ubiquitination. Additionally, derivatives may be prepared through crosslinking with disulfide bond formation as well as other crosslinking reagents such as EDC (1-Ethyl- 3(dimethylaminopropyl)carbodiamide hydrochloride) which forms cross linkages between amino groups and carboxyl groups. Alternatively, many different types of homo-bifunctional and hetero-bifunctional crosslinking reagents are commercially available and known to those skilled in the art. [0099] Complex formation of antigen following synthesis into dimers, trimers, tetramers and other multimers by sulfhydryl bridge formation between two or more heterogeneous antigens (i.e. intermolecular crosslinking) may be controlled by the oxidizing conditions and antigen concentration. In particular, oxidation by bubbling oxygen through a solution containing the heterogeneous antigen will result in aggressive sulfhydryl bridge formation between local cysteine residues as compared to keeping the solution in an air-tight container. Correspondingly, the concentration of the antigen in solution can also tend to regulate the size of complex formation, wherein greater concentration will tend to produce a larger population of multimers having higher molecular weight complexes than lower concentrations, which tend to produce smaller populations for multimers having lower molecular weights.

Diagnostic Assays

[00100] Useful solid and liquid phase assay methods known in the art may be utilized with the present invention. While the particular assay described is an ELISA, the present invention is not specifically limited to this type of assay.

[00101] Other well known immunoassays that may be adapted to detect the level of autoantibodies in a sample which react with the heterogeneous antigen include fluorescent immunosorbent assay (FIA), chemiluminescent immunosorbent assay (CLIA), radioimmuno assay (RIA), enzyme multiplied immunoassay techniques (EMIT), solid phase radioimmunoassay (SPRIA), immunoblotting, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, etc. Any assay method that results in a signal imparted by the reaction of autoantibodies with a heterogeneous antigen of this invention is considered. Each of those assay methods can employ detecting techniques in which an indicating means is utilized to signal the immunoreaction, and thereby the binding of an autoantibody to be detected with a heterogeneous antigen of this invention. Such means may be either a mixture of two isotype-specific detecting reagents or a detecting reagent capable of recognizing two or more antibody isotypes. For a review of the different immunoassays which may be used, see: The Immunoassay Handbook, David Wild, ed., Stockton Press, New York, 1994. A competitive immunoassay with solid phase separation or an immunometric assay for antibody testing is particularly suitable for use in the present invention. See, The Immunoassay Handbook, chapter 2 and Maggio, Enzyme Immunoassay, CRC Press, Cleveland, Ohio (1981); and in Goldman, Fluorescent Antibody Methods, Academic Press, New York, N.Y. ( 1980).

[00102] One general assay method deteπnines the presence, and preferably the amount, of autoantibodies in a biological fluid sample in three steps. First, the biological fluid sample is admixed with a heterogeneous peptide antigen to form an immunoreaction admixture. The antigen is preferably operatively linked to a solid support such that the immunoreaction admixture has both a liquid phase and a solid phase. Next, the immunoreaction admixture is maintained under biological assay conditions for a time period, typically predetermined, sufficient to form a heterogeneous antigen-autoantibody complex in the solid phase. In heterogeneous assay formats, the reactants are usually separated after the maintenance period, typically by washing and retaining the solid-phase. In the last step, the presence and preferably the amount of complex formed in the second step and thereby the presence or amount of autoantibodies in the biological fluid sample is then determined.

[00103] Biological assay conditions are those conditions that are able to sustain the biological activity of the immunochemical reagents of this invention and the autoantibody sought to be assayed. Those conditions include a temperature range of about 4° C to about 45° C, a pH value range of about 5 to about 9 and an ionic strength varying from that of distilled water to that of about one molar sodium chloride. Methods for optimizing such conditions are well known in the art.

[00104] In another embodiment, the labeled specific binding agent is a labeled antiimmunoglobulin antibody. One or a mixture of two or more antiimmunoglobulin isotypes, such as anti-IgA anti-IgM and anti-IgG are labeled and utilized. Similar embodiments include using labeled Protein A, which can bind both IgG and IgA, or labeled Jacalin, a lectinn that can bind IgA, and labeled protein G, a bacterial protein that can bind IgG. It has been reported that about 1 to 2 percent of individuals suffering from rheumatoid arthritis who do not express IgG autoantibodies to citrullinated antigens do express IgA antibodies to citrullinated antigens; consequently those assay systems that use other antiimmunoglobulin antibodies cannot detect these individuals. The present invention incoiporates the use of multiple isotypes of antiimmunoglobulin antibodies to provide signals where one isotype of immunoglobulin is not produced by the patient or to enhance the signal obtained by the binding of two or more bound immunoglobulin isotypes.

[00105] In one embodiment the label can be a fluorescent agent chemically bound to the antibodies or antigens to form a fiuorochrome that is a useful immuno fluorescent tracer. Suitable fluorescent labeling agents include for example fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylarnine-l-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRlTC), lissamine, rhodamine 8200 sulfonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et al, eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incoiporated herein by reference.

[00106] In other embodiments, the labeling group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like, ϊn such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (imtmmoreactanf) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as 2,2'-azino-di-(3- ethyl-benzthiazoline-G-sulfonic acid) (ABTS) or (3, 3\ 5, 5' tetramethyl benzidine (TMB).

[00107] Radioactive elements may also be used as labeling agents. Exemplary radio-labeling agents are those that produce gamma ray emissions such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr. Particularly preferred is ^"I. Another group of useful labeling agents are those elements such as ^{1 1}C, ¹⁸F, ¹⁵O and ¹³N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such ^πIn or ³ H.

[00108] The linking of labels to proteins, more specifically antibodies, is well known in the ait. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et ah, Meth. Enzymol. 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et ah, Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et ah, Biotech., 3:889-894 (1984), and U.S. Patent 4,493,795.

[00109] In one exemplary embodiment of the invention, the diagnostic assay is an ELISA. In the ELISA, a heterogeneous antigen is immobilized on a solid support directly or indirectly. An aliquot of a biological sample, such as serum, from a subject suspected of having an autoimmune disease is added to the solid support and allowed to incubate with the heterogeneous antigen on the solid phase. An enzyme-labeled secondary antibody that recognizes a constant region in the autoantibodies present in the sample which have reacted with the heterogeneous antigen is added. When the subject is a human, this secondary antibody is an anti-human immunoglobulin. The secondary antibody is specific for IgA, IgG, or IgM heavy chain constant regions may be employed. In the present invention, one, two or more, labeled secondary antibodies are utilized to identify the presence of one, two or more isotypes of the autoantibodies present in the biological sample. After separating the solid support from the liquid phase, a colorimetric reagent is added to the solid support. The reaction of the enzyme label with the reagent produces a detectable visible color that can be quantitated. The presence of this signal indicates the presence of the autoantibodies and therefore the presence of the disease. A more in depth description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D. P. Sites et ah, published by Lange Medical Publications of Los Altos, Calif, in 1982 and in U.S. Patents 3,654,090; 3,850,752 and 4,016,043, which are all incoiporated herein by reference.

[001 10] The heterogeneous antigen is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and peptides well known to those skilled in the art may be used.

[00111] Useful solid matrices are also well known in the art for preparing a solid support containing a reagent affixed thereto. Such materials are water insoluble and include the cross- linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ.); agarose; microparticles such as latex particles, glass or silicon chips or polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, 111.; polyvinyl chloride, polystyrene, cross-linked polyacryl amide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.

[00112] Suitable methods for immobilizing the heterogeneous antigen on solid matrices include ionic, hydrophobic, covalent interactions and the like. The solid matrix can be chosen for its intrinsic ability to attract and immobilize the heterogeneous antigen or it may retain an additional molecule, which has the ability to attract and immobilize the heterogeneous antigen. This additional molecule may be a charged substance that is oppositely charged with respect to the heterogeneous antigen itself or a charged substance conjugated to the heterogeneous antigen. Alternatively, the molecule can be any specific binding member, which is immobilized upon (attached to) the solid matrix and which has the ability to immobilize the heterogeneous antigen through a specific binding reaction. The molecule enables the indirect binding of the heterogeneous antigen to a solid matrix before the performance of the assay or during the performance of the assay.

[00113] The signal producing system is made up of one or more components, at least one of which is a label, which generate a detectable signal that relates to the amount of bound and/or unbound label. The label is a molecule that produces or which may be induced to produce a signal. Examples of labels include fluorescein, enzymes, chemiluminescers, photosensitizers or suspendable particles. The signal is detected and may be measured by detecting enzyme activity, luminescence or light absorbance. Radiolabels may also be used and levels of radioactivity detected and measured using a scintillation counter.

[00114] Examples of enzymes which may be used to label the anti-human immunoglobulin include β-D-galactosidase, horseradish peroxidase, alkaline phosphatase, and glucose-6- phosphate dehydrogenase ("G6PDH"). Examples of fluorescein which may be used to label the anti-human immunoglobulin include fluorescein, isothiocyanate, rhodamines, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, and Alexa Fluor® dyes (that is, sulfonated courmarin, rhodamine, xanthene, and cyanine dyes). Chemiluminescers include e.g., isoluminol. For example, the anti-human immunoglobulin may be enzyme labeled with either horseradish peroxidase or alkaline phosphatase.

[001 15] Enzymes may be covalently linked to anti-human immunoglobulins for use in the methods of the present invention using well known methods. Conjugation methods for preparing these labeled anti-human immunoglobulins, are well known in the art. For example, alkaline phosphatase and horseradish peroxidase may be conjugated to antibodies using glutaraldehyde. Horseradish peroxidase may also be conjugated using the periodate method. Commercial kits for enzyme conjugating antibodies are widely available. Enzyme conjugated anti-human specific antibodies are also available from multiple commercial sources.

[001 16] Biotin labeled antibodies may be used as an alternative to enzyme linked antibodies. In such cases, bound antibody would be detected using commercially available streptavidin- horseradish peroxidase detection systems.

[001 17] Enzyme labeled antibodies produce different signal sources, depending on the substrate. Signal generation involves the addition of substrate to the reaction mixture. Common peroxidase substrates include ABTS (2,2'-azinobis(emylbenzothiazoline-6-sulfonate)), OPD (O- phenylenediamine) and TMB (3,3', 5,5'-tetramethylbenzidine). These substrates require the presence of hydrogen peroxide, p-nitrophenyl phospate is a commonly used alkaline phosphatase substrate. During an incubation period, the enzyme gradually converts a proportion of the substrate to its end product. At the end of the incubation period, a stopping reagent is added which stops enzyme activity. Signal strength is determined by measuring optical density, usually via spectrophotometer.

[001 18] Alkaline phosphatase labeled antibodies may also be measured by fluorometry. Thus in the immunoassays of the present invention, the substrate 4-methylumbelliferyl phosphate (4- UMP) may be used. Alkaline phosphatase dephosphorylated 4-UMP to form A- methylumbelliferone (4-MU), the fluorophore. Incident light is at 365 nm and emitted light is at 448 nm.

[00119] The amount of color, fluorescence, luminescence, or radioactivity present in the reaction (depending on the signal producing system used) is proportionate to the amount of autoantibodies in a sample which react with the heterogeneous antigen. Quantification of optical density may be performed using spectrophotometric or fluorometric methods, including flow cytometers. Quantification of radiolabel signal may be performed using scintillation counting.

[00120] In another exemplary embodiment, the assay is a competitive immunoassay, which employs one or more labeled antibodies that binds to the same antigenic sequences of the heterogeneous antigen as the autoantibodies. In the assay, these labeled antibodies and the autoantibodies in a sample compete for binding the heterogeneous antigen. Typically, a constant amount of a labeled antibody is incubated with different concentrations of a sample from a subject. These labeled antibodies may be monoclonal or polyclonal.

[00121] As described herein above, the antibody may be labeled with a fluoresces enzyme, chemiluminescer, photosensitizer, suspendable particles, or radioisotope. After incubation, bound labeled antibodies are separated from free antibodies. Depending on the signal producing system used and if necessary, an appropriate substrate with which the labeled antibody reacts is added and allowed to incubate. The signal generated by the sample is then measured. A decrease in optical density or radioactivity from before and after addition of the serum sample or between experimental and control samples, is indicative that autoantibodies in the sample have bound to the heterogeneous antigen.

[00122] In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Patents 5,885,530, 4,981,785, 6,159,750, and 5,358,691 , each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a prognosis based on the presence or absence of a series of proteins corresponding to autoimmune or chronic inflammatory disease markers is utilized.

[00123] Since the diagnostic assay of the present invention is used for autoantibody detections, it should be understood that the antibodies can be directed to any antigenic sequences of the heterogeneous antigen. These antigenic sequences are normally derived from proteins recognized as being responsible for or associated with the autoimmune response for which the autoantibodies are being generated. Furthermore, these autoantibodies may be of any variety or isotype as described elsewhere herein. EXAMPLES

[00124] The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the preferred embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes (for carrying out the invention that are obvious to persons of skill in the art) are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to he incorporated herein by reference.

EXAMPLE 1 Preparation of a heterogeneous antigen

[00125] The amino acid sequence of (pro)filaggrin, vimentin, fibrinogen α, fibrinogen β and fibrinogen γ were all obtained from the National Center for Biotechnology (NCBI) website, protein section. The accession version numbers are: (pro)filaggrin, NPJ302004.1 GI:60097902; vimentin, NP_003371.2 01:62414289; fibrinogen α, NP_000499.1 Gl:4503689; fibrinogen β, NP 005132.2 GI:70906435; fibrinogen γ, NP_000500.2 GI:70906437. These sequences were used as the basis for production of synthetic peptides. The equipment used to produce the peptide is any standard model peptide synthesizer including the ACT 396, 348, 496, RANIN SYMPHONY or ABI 433 A. HPLC models used for the purification of the synthesized peptide can include the BIOCAD 60, BIOCAD 700E, BECKMAN 24karat, PHARMACIA AktaPurifier 10 or Varian Galaxie. The final amino acid composition of the peptides was confirmed by mass spectrometry.

[00126] Synthesized peptides were solubilized in distilled water. Preliminary studies showed that each of the peptides bound to plastic ELISA plates.

[00127] Antigenic sequences of the proteins where arginines were replaced by citrullines during synthesis were identified by their affinity for binding autoantibodies directed against these peptides. X represents citrulline in all of the following peptide sequences. [00128] CHQCHQESTXGRSRGRCGRSGS (SEQ ID NO: 32)

[00129] CQESRTRKXXGSRVSQDRDC (SEQ ID NO: 33)

[00130] CHQCHQESTXGXSRGRCGRSGS (SEQ ID NO: 34)

[00131] CQESXTRKXXGSRVSQDRDC (SEQ ID NO: 35)

[00132] CTIHAHPGSXXGGRHGYHH (SEQ ID NO: 36)

[00133] CGVYATXSSAVRLRSSVPGC (SEQ ID NO: 37)

[00134] CGGGVXGPXVVERHQSACKC (SEQ ID NO: 38)

[00135] CSTRSVSSSSYXXMFGGPGC (SEQ ID NO: 39)

[00136] CGVYATRSSAVXLRSSVPGC (SEQ ID NO: 40)

[00137] CRKRPSSLEXXNNRKGNKGC (SEQ ID NO: 41)

[00138] Reactive sequences that were identified were further modified by combining them into a single peptide to determine if increased reactivity could be observed. Representative examples are shown below. The sequences below with three cysteine residues were more reactive than the ones with two cysteine residues following oxidation.

[00139] CHQCHQESTXGXSRKXXGSRCGSXXGGPXVVE (SEQ ID NO: 42)

[00140] CHQESTXGXSRKXXGSRCGEXNRLNXLLXKLL (SEQ ID NO: 43)

[00141] CHEXNSTXGXSRGCGRKXXGSRLNXLLXKLL (SEQ ID NO: 44)

[00142] CHQCHQESTXGRKXXGSCGSXXGG (SEQ ID NO: 45)

[00143] CPGSXXGGRKXXGSRGPXVVEC (SEQ ID NO: 46)

[00144] The antigenic sequences of further interest were synthesized as a heterogeneous peptide antigen (shown below) having four cystiene residues, one on the N and C terminal ends of the peptide and two internal to the heterogeneous peptide antigen about centrally located having two antigenic sequences on either side of the central cysteine residues. To allow for ease of intramolecular crosslinking, spacer amino acids were placed between the two centrally located cysteine residues to reduce any steric hinderence affects that could result in diminished disulfide linkage formation because of their close proximity. The spacer amino acid selected was glycine. Representative samples are shown below.

[00145] CQESTXGXSRKXXGSRCGGCRSXXGRYATXSSAQC (SEQ ID NO: 47) [00146] CESTXGXSRKXXGSRCGCRLERXNNXKGEC (SEQ ID NO: 48) [00147] Still other antigenic sequences with varying degrees of citrulline substitution and inclusion of cysteines are as follows:

[00148] CSHQESTRGRSRGXSGRSGS (SEQ ID NO: 49)

[00149] CSHQESTXGRSRGRSGXSGS (SEQ ID NO: 50)

[00150] CTIHAHPGSXXGGRHGYHH (SEQ ID NO: 51)

[00151] CHQCHQESTXGRSRGRCGXSGS (SEQ ID NO: 52)

[00152] CGHGQASSAVRDSGHXGYS (SEQ ID NO: 53)

[00153] CHSTSQEGQDTIHGHXGS (SEQ ID NO: 54)

[00154] CGKTVETXDGQVINETSQH (SEQ ID NO: 55)

[00155] CMSTXSVSSSSYXXMFGGPG (SEQ ID NO: 56)

[00156] CGPGTASXPSSSXSYVTTS (SEQ ID NO: 57)

[00157] CSYVTTSTRTYSLGSALRPS (SEQ ID NO: 58)

[00158] CSTSXSLYASSPGGVYATXS (SEQ ID NO: 59)

[00159] CATRSSAVXLXSSVPGVRLL (SEQ ID NO: 60)

[00160] CDTHSKXTLLIKTVETXDGQC (SEQ ID NO: 61 )

[00161] CTTSTXTYSLGSALXPSTSC (SEQ ID NO: 62)

[00162] CGGGKXXXXXXXKGGGC (SEQ ID NO: 63)

[00163] CTHSTKXGHAKSRPVRDC (SEQ ID NO: 64)

[00164] CTHSTKXGHAKSXPVRDC (SEQ ID NO: 65)

[00165] CTHSTKRGHAKSXPVXDC (SEQ ID NO: 66)

[00166] CGGGVXGPRVVERHQSACKC (SEQ ID NO: 67)

[00167] CGGGVRGPXVVEXHQSACKC (SEQ ID NO: 68)

[00168] CSTRSVSSSSYRXMFGGPGC (SEQ ID NO: 69)

[00169] CSTRSVSSSSYXRMFGGPGC (SEQ ID NO: 70)

[00170] CQGSRHQQAXDSSRHSTSQC (SEQ ID NO: 71)

[00171] CKTVIEYXSQKTSXLPIIDC (SEQ ID NO: 72)

[00172] CHHGSNXXQRRGSNSSDRKC (SEQ ID NO: 73)

[00173] CHHGSNRRQXXGSNSSDRKC (SEQ ID NO: 74)

[00174] CHHGSNRXQXRGSNSSDRKC (SEQ ID NO: 75)

[00175] CLSEAANXNNDALXQAKQEC (SEQ ID NO: 76)

[00176] CEEEMRELXXQVDQLTNDKC (SEQ ID NO: 77)

[00177] CDNKQEENKENXKXPSSLEC (SEQ ID NO: 78)

[00178] CRKRPSSLEXXNNRKGNKGC (SEQ ID NO: 79)

[00179] CGRTRTSTGXXQGSHHEQAC (SEQ ID NO: 80)

[00180] CQESRTXKXXGSRVSQDRDC (SEQ ID NO: 81)

[00181] CQESRTRKXXGSXVSQDRDC (SEQ ID NO: 82)

[00182] CQESXTXKXXGSXVSQDXDC (SEQ ID NO: 83)

[00183] CGVYATXSSAVXLRSSVPGC (SEQ ID NO: 84)

[00184] CGVYATXSSAVXLXSSVPGC (SEQ ID NO: 85)

[00185] CGGGVXGPRVVEXHQSACKC (SEQ ID NO: 86)

[00186] CHQESTXGRSRKXXGSRVSC (SEQ ID NO: 87)

[00187] CHQESTXGRSXTRKXXGSRVSQC (SEQ ID NO: 88)

[00188] CHQCHQESTXGRSRGRSGRSGS (SEQ ID NO: 89)

[00189] CQESRTRKXXGSRVSQDRD (SEQ ID NO: 90)

[00190] CQECRTRKXXGSRVCQDRDC (SEQ ID NO: 91) [00191 ] CHQCHQESTXGXSRGRCGRSGS (SEQ ID NO: 34)

[00192] CRREKVQRRHXXXNSGTDGC (SEQ ID NO: 92)

[00193] CGPXGEXGFPGEXGSPGAQC (SEQ ID NO: 93)

[00194] CGPRGERGFPGERGSPGAQC (SEQ ID NO: 94)

[00195] CSRTRKXXGSRVYATXSSAVRC (SEQ ID NO: 95)

[00196] CGVYATXSSAVRGSXXGGRHGC (SEQ ID NO: 96)

[00197] CAHPGSXXGGRKXXGSRVSC (SEQ ID NO: 97)

[00198] CPGSXXGGRKXXGSRGPXVVEC (SEQ ID NO: 46)

[00199] CHQCHQESTXGRSRGRCGRKXXGS (SEQ ID NO: 98)

[00200] CGSTXGRKXXGSXXGGTXSSGC (SEQ ID NO: 99)

[00201] CHQESTXGXSRKXXGSRCGSXXGGR (SEQ ID NO: 100)

[00202] CQESTXGXSRKXXGSRCGSXXGRYATXSSA (SEQ ID NO: 101)

[00203] LKKLLXLLXKLLXLLXXLLKL (SEQ ID NO: 102)

[00204] LKKLLXLLKXLLXLLXKLLKL (SEQ lD NO: 103)

[00205] LKNLGXLLKXLVXLLXRLLRLLXXLLKL (SEQ ID NO: 104)

[00206] CRKXPSSLEXXNNXKGNKGC (SEQ ID NO: 105)

[00207] CRKRPSSLEXRNNXKGNKGC (SEQ ID NO: 106)

[00208] CRKXPSSLERXNNXKGNKGC (SEQ ID NO: 107)

[00209] EGPVVVXPLTVXDIQKT (SEQ ID NO: 108)

[00210] THRIPLVDXLXHPMNXQDQ (SEQ ID NO: 109)

[0021 1 ] TALDTHXWAXDNHXGP (SEQ ID NO: 110)

[00212] SSAVXLXSSVPGVXLLQD (SEQ ID NO: 1 11)

[00213] SSAVRLXRSVPGVXLLQD (SEQ ID NO: 112)

[00214] GDXGGQXGAXGFPGEXGS (SEQ ID NO: 1 13)

[00215] GGGVXGPRVVEXHQSAK (SEQ ID NO: 1 14)

[00216] CGVXGPRVVEXHQSC (SEQ ID NO: 1 15)

[00217] CGGGVXGPRVEXHQSACKC (SEQ ID NO: 1 16)

[00218] CESTXGXSRKXXGSRCGSXXGR (SEQ ID NO: 117)

[00219] CESTXGXSRKXXGSRLEXXΗNXKGC (SEQ ID NO: 118)

[00220] CGSTTTTXXSCSKT (SEQ ID NO: 119)

[00221] HSTKXGHAKSXPVXDCDD (SEQ ID NO: 120)

[00222] SSAVSDXGHXGSSGSQ (SEQ ID NO: 121)

[00223] TENTXLGDNXKXLSEXLEEK (SEQ ID NO: 122)

[00224] IGHXQASSAVXDSGHXGSSG (SEQ ID NO: 123)

[00225] LSEAANXNNDALXQAKQE (SEQ ID NO: 124)

[00226] PLQPEQPFPGGPEQLPQFEE (SEQ ID NO: 125)

[00227] CPEQLPQFEEGGPLQPEQPFPC (SEQ ID NO: 126)

[00228] SHQCHQESTXGXSRGRSGRSGS (SEQ ID NO: 127)

[00229] CHQESTXGXSRGRCGRSGS (SEQ ID NO: 128)

[00230] CHQCHQESTXGXSRGRC (SEQ ID NO: 129)

[00231] CHQESTXGXSRGRC (SEQ ID NO: 130)

[00232] CQESTXGXSRGC (SEQ ID NO : 131 )

[00233] CESTXGXSRC (SEQ ID NO: 132)

[00234] CGSTXGXSRC (SEQ ID NO: 133)

[00235] CHQESTXGXSRKXXGSRCG (SEQ ID NO: 134)

[00236] CESTXGXSRKXXGSRCC (SEQ ID NO: 135) [00237] CHQESTXGXSRKXXGSRCGVXGPREXHQS(SEQ ID NO: 136) [00238] CHQSRKXXGSRSTXGXSRCG (SEQ ID NO: 137)

[00239] Additional exemplary heterogeneous peptide antigens are as follows, with the exception of SEQ ID NOs: 141 and 144 which had only two cysteines (140 and 143, respectively):

[00240] CQESTXGXSRKXXGSRCGGCREXNRLNXLLXKLQC (SEQ ID NO: 138) [00241] CQESTXGXSRKXXGSRCGGCRGVXGPRVEXHQSQC (SEQ ID NO: 139) [00242] CESTXGXSRKXXGSRCPCRSXXGRYATXSSAEC (SEQ ID NO: 140)

[00243] CESTXGXSRKXXGSRSXXGRYATXSSARC (SEQ ID NO: 141)

[00244] CESTXGXSRKXXGSRCPCRLERXNNXKGEC (SEQ ID NO: 142)

[00245] CESTXGXSRKXXGSRCGCRLERXNNXKGSXXGRYATXSSAEC

(SEQ ID NO: 143) [00246] CESTXGXSRKXXGSRLERXNNXKGSXXGRYATXSSARC

(SEQ ID NO: 144)

[00247] CESTXGXSRKXXGSRERXNNXKGSXXGRYATXSSARC (SEQ ID NO: 145) [00248] CESTXGXSRKXXGSRLERXNNXKGSXXGRGVXGPRVEXHQSRC

(SEQ ID NO: 146) [00249] CESTXGXSRKXXGSRCPCRERXNNXKGSXXGRGVXGPRVEXHQSEC

(SEQ ID NO: 147) [00250] CKPGSXXGGRKXXGSRGPXVVECPCELERXNNXKGSVEXHQSKC

(SEQ ID NO: 148)

[00251] Still other antigenic sequences that were tested are as follows:

[00252] CLKKLLXLLXKLLXLLXXLLKLC (SEQ ID NO: 149)

[00253] CEGPVVVXPLTVXDIQKC (SEQ ID NO: 150)

[00254] CTHRIPLVDXLXHPMNXQDQC (SEQ ID NO: 151)

[00255] CTALDTHXWAXDNHXGPC (SEQ ID NO: 152)

[00256] CKSAVXLXSSVPGVXLLQDC (SEQ ID NO: 153)

[00257] CKSAVRLXRSVPGVXLLQDC (SEQ ID NO: 154)

[00258] CGDXGGQXGAXGFPGEXGSC (SEQ ID NO: 155)

[00259] CHSTKXGHAKSXPVXDCDDC (SEQ ID NO: 156)

[00260] CKSAVSDXGHXGSSGSEC (SEQ ID NO: 157)

[00261] CTENTXLGDNXKXLSEXLEEKC (SEQ ID NO: 158)

[00262] CIGHXQASSAVXDSGHXGSSGC (SEQ ID NO: 159)

[00263] CTHXIPLVDXLRHPMNXQDQC (SEQ ID NO: 160)

[00264] The below represent additional examples of heterogeneous peptide antigens:

[00265] CESTSGSSRKSSGSRCPCRSSSGRYATSSSAEC (SEQ ID NO: 161)

[00266] CESTXGXSRKXXGSRCECRSXXGRYATXSSAEC (SEQ ID NO: 162)

[00267] CESTXGXSRKXXGSRCECGSXXGGSATXSSAEC (SEQ ID NO: 163) [00268] ECESTXGXSRKXXGSRCECRSXXGRYATXSSAECE (SEQ ID NO: 164)

[00269] CRSTXGXSRKXXGSRCEEPEECGSXXGGYATXSSARC (SEQ ID NO: 165)

[00270] EECESTXGXSRKXXGSRCPCRSXXGRYATXSSAECEE (SEQ ID NO: 166)

[00271] EECESTXGXSRKXXGSRCPCRGSXXGGSATXSSAECE (SEQ ID NO: 167)

[00272] Other exemplary embodiments of antigenic sequences are as follows:

[00273] CPEQLPQFEEGGPLQPEQPFPC (SEQ ID NO: 168)

[00274] CPEQLPQFEEGGPLQPEQPFPC (SEQ ID NO: 169)

[00275] CRSXXGRYATXSSAEC (SEQ ID NO: 170)

[00276] CRSXXGRSATXS SAEC (SEQ ID NO: 171 )

[00277] CRSXXGRATXSSAEC (SEQ ID NO: 172)

[00278] CRSXXGGYATXSSAEC (SEQ ID NO: 173)

[00279] CGSXXGRYATXSSAEC (SEQ ID NO: 174)

[00280] CGSXXGGYATXSSAEC (SEQ ID NO: 175)

[00281 ] CGSXXGGSATXSSAEC (SEQ ID NO: 176)

[00282] CGSXXGGSATXSSEC (SEQ ID NO: 177)

[00283] CRGSXXGGSATXSSEC (SEQ ID NO: 178)

[00284] CRGSXXGGSATXSSECEE (SEQ ID NO: 179)

[00285] CRSXXGGSATXSSEC (SEQ ID NO: 180)

[00286] EECGSXXGGSATXSSARC (SEQ ID NO: 181)

[00287] EECGSXXGGSATXSSRC (SEQ ID NO: 182)

[00288] CESTXGXSRGRKXXGSRC (SEQ ID NO: 183)

[00289] CESTXGXSRERKXXGSRC (SEQ ID NO: 184)

[00290] EEECESTXGXSRKXXGSRC (SEQ ID NO: 185)

[00291] The above can be assembled into heterogeneous peptide antigens as follows, except for SEQ ID NOs 189 and 191, which only had two cysteine residues, and were found to be less reactive than their three cysteine residue counterpart (SEQ ID NO: 190):

[00292] CPESTXGXSRKXXGSRCPCRSXXGRYATXSSAPC (SEQ ID NO: 186)

[00293] CPESTXGXSRKXXGSRCPCRSXXGRYATXSSAPCPPKRHKRHKRH

(SEQ ID NO: 187)

[00294] CESTXGXSRKXXGSRCPCRSXXGRYATXSSAECPPKKHKKHKKHKK

(SEQ ID NO: 188)

[00295] CPEQLPQFEEGGPLQPEQPFPCPPKRHKRHKRKH (SEQ lD NO: 189)

[00296] CPEQLPQFEEGGPLQPEQPFPCPPCKRHKRHKRKHC (SEQ ID NO: 190)

[00297] CPEQLPQFEEGGPLQPEQPFPCPPKKHKKHKKHKKKH (SEQ ID NO: 191)

[00298] CPSTXGXSRKXXGSRCPCRSXXGRYATXSSAPC (SEQ lD NO: 189)

[00299] CPESGXGXSRKXXGSRCPCRSXXGRYATXSSAPC (SEQ ID NO: 192)

[00300] ECPSTXGXSRKXXGSRCPCRSXXGRYATXSSAPCE (SEQ ID NO: 193)

[00301] ECESTXGXSRKXXGSRCPCRSXXGRYATXSSAECE (SEQ ID NO: 194) [00302] Other antigenic sequences that were tested for inclusion in heterogeneous peptide antigens are as follows:

[00303] CHQCHQESTXGXSRGRC (SEQ ID NO: 195)

[00304] CHQCHQESTXSXSRGRC (SEQ ID NO: 196)

[00305] CHQCHQESTXAXSRGRC (SEQ ID NO: 197)

[00306] CHQCHQESSXGXSRGRC (SEQ ID NO: 198)

[00307] CHQCHQESTXGXTRGRC (SEQ ID NO: 199)

[00308] CHQCHQESSXGXTRGRC (SEQ ID NO: 200)

[00309] CHQCHQESTXGXGRGRC (SEQ ID NO: 201)

[00310] CHQCHQESGXGXSRGRC (SEQ ID NO: 202)

[00311] CHQCHQESGXGXGRGRC (SEQ ID NO: 203)

[00312] CSRGRSGRSGSC (SEQ ID NO: 204)

[00313] CSXGRSGXSGSC (SEQ ID NO: 205)

[00314] CSRGXSGRSGSC (SEQ ID NO: 206)

[00315] CSXGXSGRSGSC (SEQ ID NO: 207)

[00316] CSRGRSGXSGSC (SEQ ID NO: 208)

[00317] CSRGXSGXSGSC (SEQ ID NO: 209)

[00318] CSXGXSGXSGSC (SEQ ID NO: 210)

[00319] CSXGRSGXSGSC (SEQ ID NO: 21 1)

[00320] CHQCHQESTXGXTRGRC (SEQ ID NO: 212)

[00321] CHQCHQESTXGXGrGRC (SEQ ID NO: 213)

[00322] CHQCHQEGTXGXTRGRC (SEQ ID NO: 214)

[00323] CHQCHQETTXGXTRGRC (SEQ ID NO: 215)

[00324] CHQCHQEATXGXTRGRC (SEQ ID NO: 216)

[00325] CHQCHQEGTXGXGRGRC (SEQ ID NO: 217)

[00326] CHQCHQETTXGXGRGRC (SEQ ID NO: 218)

[00327] CHQCHQEATXGXGRGRC (SEQ ID NO: 219)

[00328] CHQCHQEETXGXGRGRC (SEQ ID NO: 220)

[00329] CHQCHQSXGRSGXSGSC (SEQ ID NO: 221)

[00330] The above sequences are assembled into heterogeneous peptide antigens as follows:

[00331 ] CESSXGXTRKXXGSRCPCRSXXGRYATXSSAEC (SEQ ID NO: 222)

[00332] CEGTXGXTRKXXGSRCPCRSXXGRYATXSSAEC (SEQ ID NO: 223)

[00333] CEATXGXGRKXXGSRCPCRSXXGRYATXSSAEC (SEQ ID NO: 224)

[00334] CEGTXGXTRKXXGSRCPCRSXXGRYATXSSAEC (SEQ ID NO: 225)

[00335] CEGTXGXTRKXXGSRCPCRSXXGRYATXSSAECC (SEQ ID NO: 226)

[00336] CCEGTXGXTRKXXGSRCPCRSXXGRYATXSSAEC (SEQ ID NO: 227)

[00337] CEGTXGXTRKXXGSRCPCRSXXGRYATXSSAECPPKKHKKHKKHGGFW

(SEQ ID NO: 228)

[00338] The antigenic sequences (with two cysteines) and the heterogeneous peptide antigens above (with three or more cysteines) were first oxidized and screened against know positive autoimmune sera from rheumatoid arthritis patients, and it was found that with the same antigenic epitopes, constructing heterogeneous peptide antigens with three or more cysteines improved sensitivity.

[00339] As described above, it was also hypothesized that by inserting charged amino acid residues adjacent to the cystiene residues, disulfide linkage formation by the cysteines could be improved. Consequently, oppositely charged amino acid residues were placed adjacent to the cysteine residues for which disulfide linkages were desired, while simultaneously limiting their formation where like charged amino acid residues were present next to cystiene residues.

[00340] Further modification of the heterogeneous peptide antigen was performed by inserting an amino acid between the internal cysteine residues that restrained the peptide structurally to determine if increased reactivity could be observed. The amino acid selected was proline.

[00341] The heterogeneous peptide antigen was then intramolecularly and intermolcecularly crosslinked following synthesis by dissolving the peptide in buffer above pH 8 and stirring to mix the solution with air. No further purification of the heterogeneous antigen was performed following the crosslinking procedure. Crosslinking of the heterogeneous antigen results in a population of unique conformations including linear, looped (both single and multiple loop conformations) as well as complexes of the monomeric antigen into dimers, trimers, tetramers, and multimers, forming a mesh. It is speculated that this population of configurations yields the heterogeneous antigen in a multitude of three dimensional conformations for binding the autoantibody more quickly and with greater affinity.

EXAMPLE 2 Analysis of Heterogeneous Peptide Antigens

A. Polyacrylamide Gel Electrophoresis

[00342] Four heterogeneous peptide antigens were synthesized as provided in Example 1 and run on SDS polyacrylamide gel electrophoresis (PAGE). The first peptide (Antigen 1, also referred to as the unmodified peptide), contained at least four cysteine residues and was subsequently tested in both reduced (Antigen 2), minimal sulfhydryl bridging (Antigen 3), and oxidized (Antigen 4), having increased sylfhydryl bridging, forms. Its primary sequence can be represented as:

C C-C C where C is cysteine and "-"is any number of any amino acid, including at least two of the above mentioned antigenic sequences.

[00343] Antigen 1 was synthesized by standard peptide chemistry. The fully oxidized monomer of the antigen with 4 cysteine residues has a number of conformations based on the different intramolecular disulfide bridges that can be formed. The oxidized forms are generally more compact or shaped than the linear monomer. This heterogeneous peptide antigen can also form polymers through inteπnolecular disulfide bridges, which yields dimers, trimers and higher polymers of very complex "mesh" conformation.

[00344] To more clearly describe this conformation it is beneficial to label the four cysteine residues in the sequence 1, 2, 3 and 4 in the order in which they appear. The peptide would be depicted as:

[00345] Cl-- C2--C3- — C4

[00346] In this case, Cl, C2, C3 and C4 are cysteine residues numbered from left to right, or N-terminus of the antigen to the C-terminus with"-" represents any number of both natural and unnatural amino acids,

[00347] ϊn the reduced foπn there are no disulfide bridges formed and the antigen has a linear structure. In the fully oxidized forms of the individual monomer antigen, only three sets of disulfide bonds, and therefore 3 different structures, are possible. One conformation has disulfide bridges between C land C2 and also C3 and C4 (the "spectacle" structure). Another possible conformation has disulfide bridges between C land C4 and also C2 and C3 (the "circle within a circle" structure). The third conformation has disulfide bridges between C land C3 and also C2 and C4 (the "pretzel" structure).

[00348] Shorthand for the above 3 structures is: [00349] Cl -C2, C3-C4 ("spectacle" structure)

[00350] C1-C4, C2-C3 ("circle within a circle" structure)

[00351 ] C1-C3, C2-C4 ("pretzel" structure)

[00352] The second peptide (Antigen 2, called '"linear'^") was synthesized having the same sequence as the first peptide however; the four cysteine residues within the amino acid sequence had been replaced with serine residues preventing intramolecular and intermolecular sulfhydryl bridging.

[00353] The third peptide, (Antigen 3, called "spectacle") was prepared having the same sequence as the first peptide but configured into a spectacle conformation. Antigen 3 was forced into a "'spectacle" conformation by selective deprotection of the 4 cysteines. Cysteine residues 1 and 2 were protected by a different group than cysteine residues 3 and 4, and the protecting groups were removed sequentially, constraining the available disulfide bond formation. In the spectacle confirmation sulfhydryl bridging occurs between cysteine residues 1 and 2 and residues 3 and 4. It is the structure C1-C2, C3-C4 above.

[00354] The oxidized peptide was prepared as follows; a 1 mg/ml solution at basic pH was made and 5OuL was placed on parafilm open to the air on a benchtop for one hour. The solution was recovered and the volume was increased to 50 uL with the same buffered solution. This volume was then added to the gel. The reduced peptide was prepared by adding lOuL of a 100 mM solution of dithiotheitol (DTT) to 5OuL of a 1 mg/mL solution of peptide and incubating the mixture for 1 hour.

[00355] A heterogeneous antigen with 4 cysteine residues can form 60 different sets of disulfide bonds in a fully oxidized dimer. This means the dimer can have 60 different structures. The cysteine residues in one heterogeneous peptide can be labeled ACl , AC2, AC3 and AC4. The cysteines in an identical heterogeneous peptide will be identified as BCl, BC2, BC3 and BC4. The letters A and B are used to identify the cysteines in the two identical peptides, not to deniark 2 peptides of different amino acid sequences. The 60 possible sets of disulfide bridges in a completely oxidized dimer of a heterogeneous peptide with 4 cysteines are described below. [00356] Fifteen of the possible disulfide bonded structures are written below. There are 15 more structures when the initial disulfide bond is AC1-BC2, 15 more when the initial bond is AC1-BC3, and 15 more when the initial bond is AC1-BC4.

[00357] ACl -BCl , AC2-AC3, AC4-BC2, BC3-BC4

[00358] ACl -BCl , AC2-AC3, AC4-BC3, BC2-BC4

[00359] AC 1 -BC 1 , AC2- AC3 , AC4-BC4, BC2-BC3

[00360] ACl-BCl, AC2-AC4, AC3-BC2, BC3-BC4

[00361] ACl-BCl, AC2-AC4, AC3-BC3, BC2-BC4

[00362] AC 1 -BC 1 , AC2-AC4, AC3-BC4, BC2-BC3

[00363] ACl-BCl, AC2-BC2, AC3-AC4, BC3-BC4

[00364] ACl-BCl, AC2-BC2, AC3-BC3, AC4-BC4

[00365] ACl-BCl , AC2-BC2, AC3-BC4, AC4-BC3

[00366] ACl-BCl, AC2-BC3, AC3-AC4, BC2-BC4

[00367] ACl -BCl , AC2-BC3, AC3-BC2, AC4-BC4

[00368] ACl-BCl, AC2-BC3, AC3-BC4, AC4-BC2

[00369] ACl-BCl , AC2-BC4, AC3-AC4, BC2-BC3

[00370] ACl-BCl, AC2-BC4, AC3-BC2, BC3-BC4

[00371] ACl-BCl, AC2-BC4, AC3-BC3, AC4-BC2

[00372] None of the dimers above are as simple as a cyclic structure. They all have complex inter- and intra-molecular disulfide bonds, forming mesh-like structures. If one counts partially oxidized dimers the number of possible dimeric structures increases. There are a very large number of possible structures in trimers and higher order polymers.

[00373] The forth peptide (Antigen 4, called "cyclic") with a different amino acid sequence than Antigen 1 and containing just 2 end cysteine residues was also synthesized and analyzed by non-reducing SDS PAGE. An antigen with just 2 cysteine residues is an example of a non- heterogeneous antigen since it has less than 3 cysteine ressidues and can only form linear or cyclic structures. It can be represented as:

[00374] Cl- — C2 [00375] -Cl- — C2--

[00376] C is cysteine and "-" is any number of any amino acids, natural or unnatural. The cyclic structures formed upon oxidation include monocyclic monomers, as well as monocyclic dimers, etc., but are not heterogeneous because only cyclic structures are formed with very little conformational heterogeneity.

[00377] A fully oxidized dimer of an antigen with 2 cysteines can form only 2 sets of disulfide bonds. Peptide A has cysteine residues ACl and AC2, while peptide B has cysteine residues BCl and BC2. Since in this example antigens A and B have exactly the same amino acid sequences, the letters are being used to describe the different possible inter- and intra-molecular disulfide bonds. As with the monomer, only a cyclic structure is possible in a fully oxidized dimer of an antigen with 2 cysteine residues.

[00378] In the fully oxidized dimer of a non-hetrogeneous peptide with 2 cysteine residues the 2 possible structures are listed below.

[00379] AC 1 -BC 1 , AC2-BC2 [00380] AC1-BC2, AC2-BC1.

[00381] Both of the structures above are cyclic. No intra-molecular disulfide bonds are possible in dimers, trimers or higher ordered structures of peptides containing 2 cysteine residues. Only inter-molecular disulfide bonds are possible.

[00382] If the peptides are not fully oxidized, only a linear structure is possible. In fact, even in trimers, tetramers and higher order polymers, only a cyclic structure or a linear structure are possible.

[00383] In non-reducing PAGE, peptides move in order of their radius of gyration. Peptides with a larger radius of gyration run slower (higher up on the gel) than peptides with a smaller radius of gyration. The radius of gyration is related to the size and shape of the peptide. Because no reducing agent is present, disulfide bonds are preserved, allowing peptides to retain any shape held in place by disulfide bonds, and to retain any polymers formed by intermolecular disulfide bonds.

[00384] A linear peptide runs slower on a non-reducing SDS PAGE than the same peptide that has shape caused by intramolecular disulfide bonds because the linear peptide has a larger radius of gyration. [00385] Five ug/lane of each of the four peptide solutions was added to SDS PAGE: a peptide with a "linear" conformation (Antigen 2), wherein the cysteine residues were replaced with serine and having a molecular weight of 3,758 g/mol. It ran as a linear peptide since there are no cysteine residues present to create intramolecular or intermolecular bonds.

[00386] The migration of Antigen 3, the "spectacle" antigen, having molecular weight 3,822 g/mol, made by sequential deprotection of the cysteine residues, runs predominantly as a single band ahead of Antigen 2. This indicates that Antigen 3 has a more compact structure than Antigen 2. A minor higher band was interpreted to be a dimer of Antigen 3 since it is significantly larger than Antigen 2, the linear form of the antigen. The difference in molecular weight of 64 g/mol between the two antigens comes about because 4 cysteines are 63 g/mol heavier than 4 serines.

[00387] The migration of Antigen 1 , the foπn of the heterogeneous antigen oxidized at a relatively high concentration, runs as three resolved bands on the gel as well as unresolved high molecular weight staining material and other staining material between the resolved bands. The lowest band is interpreted as having a molecular weight of 3,822 g/mol and being a compact structure made up of the three possible oxidized forms of the monomer described above, because it runs at nearly the same rate as Antigen 3. A second lowest band is interpreted to be a reduced, linear monomer, because it runs at nearly the same rate as Antigen 2, the linear antigen. A third band is interpreted to be a mixture of the possible dimers described above. The smear of staining material above the third band is inteipreted to be a mixture of the many possible structures formed by trimers, tetramers and higher order polymers. Distinct bands are not observed because there is such a wide range of structures of each polymer, no individual bands are resolved.

[00388] The migration of the oxidized form of Antigen 4, the "cyclic" conformation, the monomer having a molecular weight of 2,340 g/mol, runs as at least twelve resolvable bands on PAGE. The lowest band is interpreted to be the oxidized cyclic monomer, since it is the fastest migrating band on this gel. The second lowest band is interpreted as the cyclic dimer, since it is running near the linear foπn of the significantly larger antigen. There does not appear to be the linear monomer of this antigen on this gel. The third and higher bands are inteipreted to be the cyclic trimer, cyclic tetramer, cyclic pentamer and so on up to at least a polymer of twelve units. These are well resolved because only cyclic structures are possible in the oxidized form of Antigen 4.

[00389] These results demonstrate that the oxidized form of Antigen 1 is a three-dimensional heterogeneous mixture of monomers and multimers.

B. Size Exclusion Column Chromatography (SEC)

[00390] The four antigens in Example 2 were passed through a molecular sieve column. Antigen 3, the spectacle conformation of the peptide, eluted at about 12.9mL (Figure 1). Antigen I₅ the unmodified peptide reduced with DTT, eluted at about 11 ,8mL (Figure 2A), and the oxidized form of the unmodified peptide, Antigen 4, eluted at about 12.8mL (Figure 2B).

[00391] The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously, or near simultaneously, particles of the same size should elute together. Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of this range and is where molecules are too large to be trapped in the stationary phase. The permeation limit defines the molecular weight at the lower end of the range of separation and is where molecules of a small enough size can penetrate into the pores of the stationary phase completely. This results in larger molecules passing through the column matrix faster and eluting more quickly that those of permeable molecular weight which are caught up in the column matrix and are eluted later in time.

[00392] Each of the chromatograms show primary elution peaks in the range of 11 ,8mL to 12.8mL. Taken in conjunction with the PAGE results for the oxidized and reduced forms of the unmodified peptide, it is believed that these peaks are complex and consist of a number of intramolecularly bound species having similar three dimensional configurations allowing them to elute at slightly different solvent volumes. [00393] The peptide treated with DTT, Antigen 1 , is generally linear and elutes at 1 1.8mL (Figure 2A). The chromatogram obtained from the peptide, peptide 6, immediately following rehydration eluted at 11.9 mL indicating that it too is generally linear since its elution profile is substantially similar to that of the DTT treated peptide. The oxidized form of the peptide is presumed to contain sulfhydryl bridging and elutes at 12.8 mL (Figure 2B). The primary peaks at 1 1.9mL and 12,8 mL are believed to contain a number of conformation structures resulting from the formation of sulfhydryl bridging within the heterogeneous antigen. During oxidation, substantial amounts of dimer, trimer and higher order polymers are made, and elute ahead of peak 3. We interpret Peak 1 to contain oxidized tetramer and higher intermolecularly crosslinked polymers, which are very complex, eluting at or near the exclusion limit of this column. We interpret Peak 2 to be predominantly comprised of oxidized dimers and trimers of the heterogeneous peptide. The peak at 12.8 mL presumably containing substantially more of these conformers than the peak at 11.9 mL. These conformations include the spectacle conformation, the pretzel conformation and the double looped configuration. More specifically, if the cysteine residues are labeled 1 , 2, 3, and 4 in the order in which they appear in the heterogeneous antigen sequence, the spectacles conformation would result from the sulfhydryl bridging of cysteine residues 1 to 2 and 3 to 4, the pretzel conformation would be between 1 to 3 and 2 to 4 and the double looped conformation would be 1 to 4 and 2 to 3. These conformers are identical in molecular weight and not substantially different enough in three dimensional configurations to be clearly separated into peaks on SEC. Consequently, they appear as a single primary peak.

[00394] When considering interactions between cysteine residues within the peptide and between peptides, under oxidizing conditions those cysteine residues that are closest interact (closest neighbor interactions). Depending on concentration, the closest neighbor may be a cysteine residue of an adjacent molecule as opposed to a cysteine residue within the amino acid sequence of the peptide itself. Consequently, disulfide bridging is promoted under oxidizing conditions and multimer complex formation results when the effective concentration brings antigens into such proximity that disulfide bond formation occurs between neighboring molecules as opposed to intramolecular! y.

[00395] In each of these chromatograms, a large complex peak elutes prior to the major peak. Upon further analysis, it appears that this peak may be comprised of a population of complexes formed by disulfide bridging between independent peptide molecules. While unexpected, it is believed that these peaks represent dimers eluting around 12.8 niL, trimers eluting at about 10.3mL, tetramers and possibly other multimers of the peptide eluting prior to 10.OmL (Figure 1). When considering interactions between cysteine residues within the peptide it is speculated that under oxidizing conditions those cysteine residues that are closest interact (closest neighbor interactions).

[00396] The spectacle form of the peptide showed peaks eluting following the primary peak. These peaks are smaller than the peptide and are believed to be failed sequences (Figure 1).

[00397] The major property of a heterogeneous peptide antigen is the large number of different polymers a single antigen with 3 or more cysteine residues can produce, particularly when compared to an antigenwith 2 cysteine residues that can form only cyclic or linear structures. The elution profile using SEC confirms the results seen on PAGE.

C. Enzyme-Linked Immunosorbent Assays (ELISAs)

[00398] Enzyme-linked immunosorbent assays were performed on the unmodified peptide and peptides 1 and 3. Saturating amounts of the heterogeneous peptide antigen is bound to the surface of a microwell plate. Pre-diluted controls and diluted patient samples are added to separate wells, allowing any IgG and/or IgA antibodies present to bind to the immobilized antigen. Unbound sample is washed away and an enzyme labeled anti-human IgG/IgA conjugate is added to each well. A second incubation allows the enzyme labeled anti-human IgG and/or IgA to bind to any patient antibodies that have become attached to the microwells. After washing away any unbound enzyme labeled anti-human IgG/IgA, the remaining enzyme activity is measured by adding a chromogenic substrate and measuring the intensity of the color that develops. The assay can be evaluated spectrophotometrically by measuring and comparing the color intensity that develops in the patient wells with the color in the control wells. Both the peptide containing serine residues, peptide 1 , (linearized) and the spectacle form of the peptide, peptide 3, showed reduced activity for the RA sera tested when compared to the activity of the unmodified peptide. Peptide 1 showed the least activity having positive results for 26 of the 66 rheumatoid arthritis patient serum tested. Correspondingly peptide 3 was positive for 40 of the 66 rheumatoid arthritis patient sera tested wherein all those that were positive for peptide 1 were positive for peptide 3. The unmodified peptide showed the most positive results capturing 80% of the rheumatoid arthritis patient sera, 53 out of the 66 tested. The PAGE indicated that there are a number of possible configurations of the peptide as well as complexes made up of two or more peptide bound via cystine residue bonds present in solution. It is because of this that we believe the unmodified peptide demonstrates increased activity over the two isolate configurations of the peptide, the linear and spectacles forms. This reactivity seems to indicate that it is this population of the peptide that is able to more broadly capture the wide variety of autoantibodies resulting from rheumatoid arthritis. It is also believed that the multimeric complexes of the peptide may provide a larger structural profile to present the antigen, increasing the distance of the antigen from the surface, in a configuration or configurations more acceptable to binding antibody.

[00399] Figure 3 shows the Optical Densities (ODs) from the RA patients run on the three ELISAs. They are sorted from highest to lowest ODs on the oxidized foπn of the antigen. Antibodies from the RA patients bound more strongly to Antigen 1 , the oxidized heterogeneous peptide, than to either Antigen 3, the "spectacle" foπn, or Antigen 2, the linear form, as measured by the higher ODs on the ELISA using Antigen 1. The average OD of all samples was 1.66 OD for the oxidized peptide, 1.02 OD for the "spectacle" form of the antigen, and 0.58 OD for the linear form. Samples reacted more strongly with the "spectacle" form than the linear form, showing that even some structure increases immuno reactivity.

[00400] The oxidized heterogeneous peptide also showed higher immunoreactivity than the "spectacle" foπn or the linear form as measured by sensitivity of detection of antibodies in patients with RA. Using 0.4 OD as the cutoff between positive and negative, a typical cutoff for this type of ELISA, 51 of the 66 RA patients tested were positive on the oxidized foπn, 36 were positive on the "spectacle" form, and only 24 were positive on the linear form. With sensitivity as the measurement, the "spectacle" form again showed higher immunoreactivity than the linear foπn. [00401] There are several possible explanations for the greater imrrmnoreactivity on the oxidized heterogeneous antigen than the "'spectacle" form or the linear form. They may all contribute somewhat.

[00402] At least some of the added immunoreactivity comes from the added structural complexity of the oxidized heterogeneous peptide compared to the other foπns. This interpretation is supported by the observation that binding of antibodies from RA patients on the 3 antigens is not proportional but the relative reactivity is different with different individuals. For example, two samples are strong reactors at 2.80 and 2,38 OD on the oxidized form, only 0.84 and 0.59 OD on the "spectacle" form, and negative at 0.17 and 0.07 OD on the linear form. Two other samples have similar high reactivity on the oxidized form at 2,36 OD, while there is still good reactivity on the "spectacle" form of 1.19 and 1.04 OD, and still positive reactivity on the linear form with 0.48 and 0.56 OD.

[00403] The most straightforward interpretation of the above results is that the oxidized form contains at least some epitopes that are not present on the "spectacle" or linear form because the relative reactivities of the antigens are different for different patient samples.

[00404] Another possible reason that the oxidized form is more immunoreactive than the other forms is that there is more mass of antigen bound to the ELISA plate. It is possible that a polymer of many antigens together binds to the plate better than individual antigens.

[00405] Another possible reason that the oxidized form may be more immunoreactive than the other forms is that part of the antigen may be raised up above the ELISA plate, thus preventing steric hindrance of the reactive site by the ELISA plate, giving more access of the antigen to the antibodies in solution.

[00406] Another possible reason that the oxidized form may be more immunoreactive than the other foπns is that there is greater surface area on the oxidized heterogeneous antigen bound to the plate than when the other forms of the antigen are bound to the plate.

[00407] Other reasons for increased immunoreactivity may be possible, and all the reasons may contribute something to the observed increased immunoreactivity of the oxidized form compared to the other forms. [00408] These same reasons may be the cause of increased immunoreactivity of heterogeneous antigens in other types of immunoassays as well, when the antigen may be bound to a latex bead, a nylon membrane, or any of several other solid phases.

* * Φ Φ Ψ

[00409] The examples set forth above are provided to give those of ordinary skill in the ait with a complete disclosure and description of how to make and use the preferred embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes (for carrying out the invention that are obvious to persons of skill in the art) are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to he incorporated herein by reference. [00410]

Claims

CLAIMSWe claim:

1. An immunoassay for detection of antibodies in a subject suspected of having an autoimmune disease comprising the steps of: a) preparing a plurality of peptide antigen molecules, wherein the peptide antigen molecules comprise at least two different antigenic amino acid sequences derived from at least one protein associated with an autoimmune disease, and wherein the peptide antigen molecules further comprise at least three cysteine residues; b) oxidizing the at least three cysteine residues on the peptide antigen molecules to form at least one intermolecular bond between peptide antigen molecules and at least one intramolecular bond within peptide antigen molecules, wherein said oxidizing results in formation of a tlnee dimensionally heterogeneous peptide antigen matrix; c) adding a biological sample from the subject to the heterogeneous peptide antigen matrix to form a complex between at least two isotypes of the antibodies and the heterogeneous peptide antigen matrix; and d) detecting the complex.

2. The immunoassay according to claim 1, wherein the immunoassay is selected from the group consisting of a solid phase assay or a liquid phase assay.

3. The immunoassay according to claim I₃ wherein the immunoassay is selected from the group consisting of a fluorescent immunosorbent assay (FIA), a chemiluminescent immunosorbent assay (CLIA), a radioimmunoassay (RlA), an enzyme multiplied immunoassay techniques (EMIT), a solid phase radioimmunoassay (SPRIA), and an enzyme linked immunosorbent assay (ELISA),

4. The immunoassay according to claim 2, wherein the immunoassay is an enzyme linked immunosorbent assay (ELlSA).

5. The immunoassay according to claim 1 , wherein the at least two different antigenic amino acid sequences are in one peptide antigen molecule.

6. The immunoassay according to claim 1, wherein the at least two different antigenic amino acid sequences are in more than one peptide antigen molecule.

7. The immunoassay according to claim 1, wherein the peptide antigen molecules further comprise four cysteine residues.

8. The immunoassay according to claim 7, wherein four cysteine residues on the peptide antigen molecule are oxidized forming two or more intermolecular bonds between peptide antigen molecules and two or more intermolecular bonds within the peptide antigen molecules.

9. The immunoassay according to claim 1, wherein the oxidizing step is performed by exposing the at least three cysteine residues to air.

10. The immunoassay according to claim 1 , wherein the biological sample is serum or plasma.

11. The immunoassay according to claim 1, wherein the at least two isotypes of the antibodies are IgG and IgA.

12. The immunoassay according to claim 1, wherein detecting the complex is by using a label selected from the group consisting of a fiuorescer, an enzyme, a chemiluminescer, a photosensitize!' and suspendable particles.

13. The immunoassay according to claim 1, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, celiac disease, Grave's disease, scleroderma, rheumatoid arthritis and type I diabetes mellitus.

14. The immunoassay according to claim 1, wherein heterogeneous peptide antigen is the sequence selected from the group consisting SEQ ID NO. 138, SEQ ID NO. 139, SEQ ID NO. 140, SEQ ID NO. 141 , SEQ lD NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 145, SEQ ID NO. 146, SEQ ID NO. 147, SEQ ID NO. 148, SEQ ID NO. 161, SEQ ID NO. 162, SEQ ID NO. 163, SEQ ID NO. 164, SEQ ID NO. 165, SEQ ID NO. 166, SEQ ID NO. 167, SEQ ID NO. 186, SEQ ID NO. 187, SEQ ID NO. 188, SEQ ID NO. 189, SEQ ID NO. 190, SEQ ID NO. 191, SEQ ID NO. 192, SEQ ID NO. 193, and SEQ ID NO. 194.

15. A method of enhancing sensitivity of an immunoassay for detection of antibodies in a subject suspected of having an autoimmune disease by using a three dimensionally heterogeneous peptide antigen matrix as a target of the antibodies, wherein the peptide antigen matrix is prepared using the steps of: a) preparing a plurality of peptide antigen molecules, wherein the peptide antigen molecules comprise at least two different antigenic amino acid sequences derived from at least one protein associated with an autoimmune disease, and wherein the peptide antigen molecules further comprise at least three cysteine residues; and b) oxidizing the at least three cysteine residues on the peptide antigen molecules to form at least one intermolecular bond between peptide antigen molecules and at least one intramolecular bond within peptide antigen molecules, wherein said oxidizing results in formation of a three dimensionally heterogeneous peptide antigen matrix.