WO2007120311A2

WO2007120311A2 - Detection of soluble adiponectin receptor peptides and use in diagnostics and therapeutics

Info

Publication number: WO2007120311A2
Application number: PCT/US2006/061555
Authority: WO
Inventors: Michael J. Pugia
Original assignee: Siemens Healthcare Diagnostics Inc.
Priority date: 2005-12-07
Filing date: 2006-12-04
Publication date: 2007-10-25
Also published as: US20130149720A1; WO2007120311A3; ES2463451T3; CA2636129A1; US8093017B2; JP4927093B2; EP1954312A4; BRPI0619503A2; CA2636129C; AU2006342086A1; US8846346B2; EP1954312A2; US20130196353A1; KR20080075005A; NO20083044L; NO345378B1; JP2009518653A; US20100143958A1; US8632990B2; EP1954312B1

Abstract

The present invention relates to soluble C-terminal fragments of the adiponectin receptor and their use in the diagnosis and management of disorders.

Description

DETECTION OF SOLUBLE ADiPONECTIN RECEPTOR PEPTIDES ANT) USE IN

DIAGNOSTICS AND THERAPEUTICS

CROSS REFERENCE TO RELATED APPLICATION

|000l f This application claims the benefits of U. S Provisional Application No. 60/748,305 filed December 7. 2005.

FIELD

[0002| The present invention relates to soluble C-terminal fragments of the adiponectm receptor and their use in the diagnosis and management of disorders.

BACKGROUND OF TBE INVENTION

[0003] Obesity with chronic inflammation has a large and growing population. This population clearly has a high cardiovascular and diabetes risk and frequently develops metabolic syndrome with insulin resistance. Recently adiponectm and other adipokines have been discovered as fat cell hormones thai control glucose metabolism. Both type and location of fat cells are important. Obesity produces additional adipocytes which secrete adiponectin into the biood helping muscle eel! metabolism of fats and glucose. Some overweight patients become insulin resistant. In this case, adipocytes stop producing adiponectin. Levels of adiponectm in the blood are decreased under conditions of obesity, insulin resistance arid Type 2 diabetes. Methods exist for measuring adiponectin levels in subjects for the prognosis of these and other disease states. Measurement of adiponectin lev els, however, has proven to be a weak indicator of disease. A need exists for better methods of monitoring disease states associated with abnormal adipocyte activity. The present invention provides this and other needs.

SUMMARY

[0004] The present inventors have discovered, that C terminal fragments of the adiponectin receptor are soluble and can be detected in bodily fluids. Accordingly, the present invention provides, among other thing, the fragments, methods of detecting them, methods of using them, and antibodies capable of binding to them. [0005] Methods foi detecting fragmentation of an adiponectm receptor m a biological fluid sample obtained from a subject can comprise the steps of assa> ing for the presence or absence of at least one soluble t -teimmal fragment of the adφonectin receptor in certain embodiments the total concentration of C -terminal fragments in a biological sample is determined

[0006[ Methods for detecting the lev el of expression of an adiponectm receptor in a subject are prm ided herein Ihese methods can comprise the steps of determining the lex el of at least one O-teiminal fragment of the adiponectm receptor m a biological ftmd sample and correlating the lex el of the O-teimnial fragment xuth the lex el of expression of the adiponectm receptor In certain embodiments, the total cυncentiation of C-terminaJ fragments in a biological sample is determined

[0007 j Methods for detecting the lex el of expression of adiponectm in a subject are provided herein These methods can compose the steps of determining the lev el of at least one C-termuial fragment of the adipυnectin receptor m a biological fluid sample aid con elating the le\ el of the O -terminal fragment with the le\ e! of expression of adiponectm In certain embodiments, the total concent tali on of 0-termmal fragments in a biological sample is determined

[0008| Methods for detei mining piogression of a condition, onset of a condition. t 1 diagnosis, υr efficacx of treatment, / c . the responsiv eness of and indiv idual to theiap} with a particular d tug are encompassed b\ the present inx ention Preferahh the condition v\ til be one associated with abnormal fragmentation patterns of an adiponectm receptor These methods can compose the steps of determining the lev el of at least one O-ierminai fragment of the adiponeclm receptor in a biological fluid sample and correlating the level of the (^'-terminal fragment vxUh progression of the condition, onset of the condition, or efficacv of the treatment In certain embodiments, the total concentration of C -terminal fragments m a biological sample is determined

[QQΘ9J in the methods of the present im en U on one ov more (/ c at least one) soluble C- terminal fragment of the adiponectm receptor can be detected For example, an\ combination of fragments 1 to 22 of ΛdipoRl and or AdipoR2 can be detected In certain embodiments, fragments 1 to 22 of AdipoRJ and oi AdipoR2 can be detected and differentiated ITS their masses Aecotdmgh . the present inv ention prm ide methods of determining the lev el of fragments liaurm, for example, masses of from about ! kDa to about 3 ld)a. including, for example, a mass of about 2 kDa { e g . fragments represented b\ SEQ ID NOS 3, 12-22. 25. and 34-44) or fragments haung masses of from about 3 5 to about 4 2 iDa. including for example a mass of about 3.9 kDa (e.g.. fragments represented by SEQ ID NOS. K 2, 4- J 1 , 23. 24 and 26- 33). These size Fragments are typically present as monomers. The present invention also provides methods of determining the level of fragments having, for example, masses of from about 2 kDA to about 6 kDA. including for example a mass of about 4 kDa or masses of about 7 kDa to about 8,4 kDa, including for example, a niass of about 7.8 kDa. These size fragments are typically present as dimers.

100101 to the methods of the present invention, one or more soluble C-terminal fragment of the adiponectin receptor (e.g., SBQ ID NOS. i-44) can be detected when bound to a carrier protein. For example, any combination of fragments i to 22 of AdipoR !. and/or AdipoR2 can be detected when attached to a carrier protein. Accordingly, in certain embodiments, the present invention provide methods of determining the level of fragments having masses of about 4.5-6.9, 7-8.2, y-1 L 13-15, 17-19. 27-29, or 30-34, kDa. In certain embodiments, the carrier protein is adiponectin, including adiponectin fragments. In certain embodiments, the combined adiponectin receptor fragment with bound adiponectin has a mass of about 3-5, 4-8, 7-1 L i 3- 17, 22-26 or 28-32 kDa. The present invention provides methods of detecting these fragments.

[00111 ^'Hie present invention also provides polypeptides that are substantially identical to fragments having {he sequences of SEQ ID NOs: 1 to 44 and the nucleic acid sequences that correspond to these fragments. Antibodies that specifically bind to at least one of the C-terminal fragments of the adiponectin receptor provided herein are also included.

[0OJ 2f The present invention provides a kit for use in determining treatment strategy for an individual with any of the disorders described herein comprising a means for detecting at least one of the fragments described herein: and optionally instructions for use and interpretation of the kit results. The kit can also comprise, for example, a means for obtaining a biological sample from an individual.

DETAILED DESCRfPTiON OF iLLUSIIlATIVE EMBODIMENTS

I. Introduction

[0013] The present inventors have discovered, that C terminal fragments of the adiponectin receptor are soluble and can be detected in bodily fluids. Moreover, the present inventors have observed that the presence or absence of certain soluble fragments of the adiponectm receptor in bodily fluids is predictive of disease and that the level, i.e.. concentration, of total soluble adiponectin receptor fragments in the bodily fluid is predictive of disease. [0014] It is to be understood that the invention described herein is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that {he terminology used herein is for {he purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular tonus "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a ceil^" includes a combination of two or more cells, and the like.

[0015| TIi e term "about^" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of. ±2U% or ±10%, more preferably ±5%, even more preferably ±1%. and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

ϊl. AcHponectin Receptor and Fragments thereof

[00!6| The adiponectin receptor ΪS a transmembrane receptor thai was first described by Yamauchi et a!.. (Nature. 2003, 423(694.1). 762-9) and has several types. Three adiponectin receptor types have been identified, adiponectin receptor 1 (also referred to as AdipoRi), adiponectin receptor 2 (also referred to as AdipoRl) and adiponectin receptor 3 {also referred to as AdipoR3). Adiponectin receptors specifically bind to and are modulated by adiponectin. an adipocyre-derived factor that plays a significant role in lipid and glucose metabolism in the muscle and liver.

[GG 17] The nucleic acid and amino acid sequence of human adiponectin receptors 1 and 2 are accessible in public databases (e.g., see Gen bank accession numbers NM 015999. AK222503. AK025085. AK222503. NM_02455i . Q96A54, and Q86V24) and are provided herein. The nucleic acid and amino acid sequences of human adiponectin receptor 3 is provided in U.S. Publication No. 20050032166, incorporated herein by reference in its entirety. Jt will be understood that the term adiponectin receptor, as used herein, not only encompasses adiponectin receptors having the sequences described herein but also includes, for example, naturally- occurring truncated forms of an adiponectin receptor, naturally-occurring variant forms (e.g , alternatively spliced forms), conservatively modified variants, and naturally-occurring allelic variants. fOOlS] "Conservatively modified variants'^* applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences Because of the degeneracy of the genetic code, a large number of funetionaih identical nucleic acids encode any given protein For instance, the codons CCA, GCC. GCG and GCU aJϊ encode the amino acid alaiine. Thus, at e\ er>- position where an alanine is specified fay a codon, the codon cao be altered Io any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent \ ariations.^" which are one species of consen ativ ely modified \ ariations. Even, nucleic acid sequence herem which encodes a polypeptide also describes every possible silent \ anation of the nucleic acid. One of skill will recogni/e that each codon in a nucieic acid (except AUG. which is ordinarily the onh codon for methionine, and TGG. which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[00I9J As to amino acid sequences, one of skill will recogni/e that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conserv ativ ely modified v ariant^" where the alteration results in the substitution of an amino acid with a chemically similar amino acid Conserv ativ e substitution tables prm iding functional!} similar amino acids are Λ\ell known m the art. Such conseπ atn eiy modified \ ariants are in addition to and do not exclude polymorphic \ ariants. interspecies homologs, and alleles of the im entϊon

[0020J The follow ing eight groups each contain amino acids that are conservative substitutions for one another: i) Alanine (A). CJK cine (CJ). 2} Aspartic acid (D). Glutamic acid (E): 3) Asparagine (N). Glutamine (Q); 4) Argimne (R). Lysine (K), 5) Isoleucme (I ), Leucine (L). Methionine (M), Valine (V): 6) Phenylalanine CF), Tyrosine (Y), Tryptophan (W): 7) Serine (S), Threonine (T): and S) Cysteine (C), Methionine (M) (see. e.g.. Creighton. Proteins (1S>H4».

|0021| A particular nucleic acid sequence also implicitly encompasses "'splice v ariants ^" Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice \ariant of mat nucleic acid "Splice variants.^" as the name suggests, are products of alternate e splicing of a gene After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucieic acid splice products encode different polypeptides. Mechanisms for the production of splice \ ariants \ an^*, but include alternate splicing of exons Alternate polypeptides derived from the same nucleic acid by read- through transcription are also encompassed b> this definition. Am products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. JOO22J The phrase "soluble C terminal fragments of lhe adiponectin receptor" refers to fragments from the C terminus of the adiponectin receptor that break off from the adiponectin receptor and are soluble in bodily fluids, A variety of bodily fluids can be used in practicing the methods of the invention including, for example, blood, serum, plasma, urine, salivary fluid, ascite fluid and the like.

[0023] Adiponectin is well known in the art as a hormone secreted by adipocytes having msulin-sensiti zing, antiatherogenic, and antiinflammatory properties. Levels of adiponeciin are decreased under certain conditions, including obesity, insulin resistance and diabetes. The activity of adiponectin is mediated by its .receptors. Adiponectin can exist as a full-length or as a smaller globular fragment. There are four distinct regions of adiponeciin. The first is a short signal sequence that targets the hormone for secretion outside the cell, next is a short region that varies between species; the third is a region with similarity to collagenous proteins; and the last^" is a globular domain. The predicted monomelic mass for adiponectin is 26 kDa with a range of from about 17 to about 33 kDa. Oligomer formation of adiponectin depends on disulfide bond formation mediated by an interna! cysteine residue. Adiponectin exists in a wide range of multimer complexes in plasma and combines via its collagen domain to create 3 major oligomeric forms: a low, middle and high molecular weight form. Serine proteases such as elastase and trypsin have multiple sites for cleaving adiponectm. A release of globular adiponectin at an average molecular weight of about 16 kDa is known to occur in patients. The remaining non-globular adiponectin has an average .molecular weight of 10 kDA. The cleavage of adiponectin by a trypsin type serine protease can occur, for example, at amino acid 101 causmg a 16.5 kDA globular adiponectin or by an elestase type serine protease at amino acid 108 causing a 15.8 kDa globular adiponectin. There are multiple potential cleavage sites in amino acid 88 to 108 causing a mass range for globular adiponectin of between about 17.8 kDa to about 9.7 kϋa. Additional protease cleavage of non-globular adiponectin can produce fragments as small as 3 kDa.

|0024j Without wishing to be bound by theory, it is believed that the c-terminal tail of the adiponectin receptor acts to capture full length adiponectin. The binding is believed to occur between the non-globular portion of the adiponectin protein and the adiponectin tail binding domain of the adiponectin receptor. After cleavage by the protease, the non-globular adiponectin is believed to remain bound to the c-terniinaS region of the adiponectin .receptor. The freed globular adiponectin is thought to interact with another region on the receptor to cause further activation, ϊn the absence of non-globular adiponectin, binding to the c-lerminal is not believed to occur. [0025] The present inventors have discovered that the CMerminal portion of the adiponeclin receptor fragments off the receptor and is present in bodily fluid. The presence or level of non-globuiar adiponectin can impact the fragmentation pattern for the c-temiinal of the adiponectin receptor. The present inventors have detected fragments of adiponectin receptor i and 2 in bodily fluid. The observation and conclusion that the adiponectin receptor can be detected in biological fluid and provide a reliable and practical indicator of disease states is particularly surprising given the fact that the adiponectin receptor is an integral membrane protein. Il is also surprising that certain fragments tend to be absent in disease and that increase in the total number or concentration of receptor fragments occurs in disease slates, given that adiponectin levels decreases with disease.

[0026J The present invention provides, inter aha, adiponectin receptor fragments 1 to 22 (SEQ ID NOS' 1 -22) of AdipoRl . Fragment 1 of AdipoR I has 34 amino acids corresponding to amino acids 361 to 375 on AdipoRl . Amino acids I -14 is the serine protease cleavage domain; ammo acids 15-22 is the adipoR2-hke domain: and amino acids 23-34 are the adiponectin binding domain. The sequence of fragment 1 of AdipoR i is vivvaaafvh fygvsnlqef rygleggctd dtl! (SEQ ID NO: i) This fragment can be further fragmented at any amino acid, and, in particular, at any amino acid within the serine protease cleavage domain, adipoRl -like domain, or adiponectin binding domain. Certain key fragments present in bodily fluid are fragment 2 with a sequence of Iwaaafvh fyg\ snlqef rygieggctd dtl! (SEQ ID NO:2) and fragment 3 with a sequence of snlqef rygleggctd dill (SEQ ID NO^"3) but at least the following fragments can be found: vvaaafvh fygvsnlqef rygleggctd dtl! (SEQ ID NQ:4), vaaafvh fygvsnlqef rygleggctd dtll (SEQ ID NO:5), aaalvh fygvsnlqef rygieggctd dtli (SEQ ID NO:6). aafvh fygvstilqef rygleggctd dtl! (SEQ ID NO: 7). afvh fygvsnlqef rygleggctd dtl! (SEQ ID NO:8), fvh fygvsnlqef rygieggctd dtll (SEC⁾ ID NO: 9). vh fygvsnlqef rygleggctd dtll (SEQ ID NO: 10). h fygvsnlqef rygleggctd dtll (SEQ ID NO: 1 I k fygvsnlqef rygleggctd dtll (SEQ ΪD NO: 12), ygvsnlqef ryyieggctd dtil (SEQ ID NO: 13), gvsnϊqef rygleggctd dill (SBQ ID NO: ! 4). vsπlqef rygleggctd dtil (SEQ ID NO: 15). nlqef rygieggctd dtll (SEQ ID NO: 16), Iqef rygleggctd dtll (SEQ ID NO: 17). qef rygleggcid dill (SEQ ID NO: 18), ef rygleggctd dtll (SEQ JD NO: 19), f rygleggctd dtil (SEQ I D N0:20), rygleggctd dtil (SEQ ID N0:2i). and ygleggctd dtil (SEQ ID NO:22).

[002?| The present invention provides, mler alia, adiponectin receptor fragments i to 22 (SEQ ID NOS:23~44) of AdipoR2. Fragment !. of AdipoR2 has 34 amino acids corresponding to amino acids 353 to 386 on AdipoR2. Amino acids ϊ -14 is the serine protease cleavage domain: amino acids 15-22 is the ariipoR2-like domain: and amino acids 23-34 are the adiponectin binding domain. The sequence of fragment i of AdipoR2 is ifv vagafvh fhgvsnlqef rfmigggcse edal (SEQ ID NO:23). This fragment can be further fragmented at any amino acid, and. in particular, at any amino acid within the serine protease cleavage domain, adipoR:2~like domain, or adiponectin binding domain. Hie key fragments present in bodily fluid are fragment 2 with a sequence of vagafvh fhgvsniqef rfmigggcse edal (SEQ ID NO:24) and fragment 3 with a sequence of sπlqef rfinigggcse edaf (SEQ ID NO:25) but at least the following fragments can also be found: fvvagafvh fhgvsniqef rfmigggcse eda! (SEQ ID NO:26). vvagafvh fhgvsniqef rfrmgggcse edal (SEQ ID NO:27), agafvh fhgvsniqef rfmigggcse edal (SEQ ΪD N():28), gafvh fhgvsniqef rfmigggcse edai (SEQ ID NO:29), afvh fhgvsniqef rfmigggcse edal (SEQ ID NO:30). fvh fhgvsniqef rfmigggcse edal (SEQ ID NO.31 ), vh fhgvsniqef rfmigggcse edal (SEQ ID NO: 32). h fhgvsniqef rfmigggcse eda! (SEQ ID NQ;33), fligvsniqef rfmigggcse edal (SBQ ID NO.34). hgvsniqef rfmigggcse edal (SEQ ID NO:35), gvsnlqef rfmigggcse eda! (SEQ ID NO:36X vsnlqef rfmigggcse edal (SEQ ID NO: 37). nlqef rfmigggcse edai (SEQ FD NO.38), Iqef rfmigggcse edal (SEQ ID NO: 39), qef rfmigggcse edal (SBQ ID NO:40), ef rfmigggcse edal (SEQ ID N0:41), f rfmigggcse edal (SEQ ID NO:42). rfmigggcse edal (SEQ ΪD NO:43). and fπiigggcse edal (SEQ ΪD NO.44).

[0028{ to certain instances, the adiponectin receptor present in the body does not have the exact sequence as described herein but is present as a naturally occurring variant form. For example, the adiponectin receptors can substitute at least up to 5% or even up to 10% of their amino acids without having a loss of function. Accordingly, at least a couple of the amino acids in SEQ ID NOS 1 to 44 can be substituted with other amino acids Accordingly, the present invention encompasses nol only fragments 1 -22 of AdipoR I and AdipoR2 but also fragments having substantial identity to the fragments described herein. Substantia! identity is described herein as having about 75% or 80% or greater identity to the fragments. Accordingly, the fragments can have about 80%. about 81 %, about 82%. about 83%, about 84%, about 85%, about 86%. about 87%. about 88%, about 89%, about 90%_s about 91%. about 92%, about 93%, about 94%. about 95%, about 96%, about 97%. or about 98% to SEQ ID NQS 1 to 44,

|0029j Percent identity can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide sequence m the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optima! alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions m the window of comparison and mulϋpiving the resuit by 100 to yield the percentage of sequence identity. Identify is evaluated using any of {he v ariety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to. TBLASTN, BLASTP, FASFA. TFASTA. CLUS FALW. FASTDB, the disclosures of which are incorporated b> reference m their entireties Pearson, et a! . Proc, Nati, Acad Set U S. A.. 85 2444-2448, 1988; Ahschul. et at.. J. MoI Biol.. 215. 403410. 1990: Thompson, et a!.. Nucleic Acids Res , 22: 4673-4680, 1994; Higgins, et al.. Meth. Fn/ymol . 266- 383402, i W6; Altschul, et aL Nature Genetics. 3- 266-272. 1993; Brutiag. et al.. Cornp. App Biosci . 6: 237-24 IWO

[0030] The terms "polypeptide.^{" "}peptide^" and ^"protein^"' are used interchangeably herein to refer Io a poh mer of amino acid residues. The terms appK to amino acid polymers in w hich one or more amino acid residue JS an artificial chemical mimetic of a corresponding naturally occurring amino acid, as veil as to naturally occurring amino acid polymers and non- naturally occumng amino acid polymer.

[0031 j As used herein, the term "polynucleotide"^' means a polymeric form of nucleotides of at least about 10 bases or base pairs in length, either ribonucleotides or deoxynucieotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA

[ΘΘ32J The adiponectin receptor fragments described herein can dimerive through {he cysteine ammo acid present near the c terminal of the fragment (position 28 m SHQ fϋ NO.1 and 16). Accordingly, these fragments can be present in the bodily fluid as dimers.

[0033J Several of the ammo acids present m SEQ ID NOS 1-44 are potential sites for post-translational modification For example, the glycine present in the fragments (position 26 in SEQ ID NO; 1 and 23) is a potential N-ra> ristov lotion site, the argimne (position 21 in SEQ IO NO' 1 and 23) is a potential N-glyean site and the serine (position 15 in SEQ ID NO. I and 23) is a potential O-glyean site Accordingly , the fragments may have additional mass due to post- transiationai modifications

[0034J One aspect of the present invention is the provision of the fragments described herein Accordingly, the present im entton pro\ ides isolated fragments ha\ ing substantial identity to SEQ ID NOS 1 -44. Isolated fragments are {hose that have been purified from a biological source or have been prepared by recombinant or synthetic methods Methods of doing so are well known m the art and are thus, not described herein.

[00351 Another aspect of the present in\ ention is the detection of the fragments described herein in a biological fluid sample [0036] The present im entois hax e disxtn es ed thai fragments of the adiponectm receptor, a transmembrane receptor, can be detected in biological fluid sample b\ assas ing for the presence of a c-teimmai region υf the receptor in the biological fluid

[0037J It lias also been disco\ ercd that the !e\ ei of expression of the adiponectin receptor m tissue can be determined b\ dctei mining the lex el of at least one C -tei ramal fragment of the adiponectin receptor m a biological fluid sample and comparing the lc\el of the at least one € -terminal fragment to the Sex el of the same fragment in a cormol sample

[0038] it has aiso been discos cred that the lex ei of expression of adipυnectm m a subject can be determined b\ determining the lex el of at least one C -terminal fragment of the adipυnectin ieceptor in a biological fluid sample and compaiing the le\ el of the at least one C- termmal fragment to the !e\ ei of the same fiagment in a coπtiol sample

|0039f Flagmen ts of the adiponectin ieceptoi can be found in the bodih fluids_* of diseased and non-diseased indn iduaSs the presence or lex el of adiponectin, hcmex cr, can impact the fragmentation pattern for the c-teπiiinal of the adiponectin receptor The le\ els and h pe of adiponectin in a subject can also impact the lev els of adiponecim ieceptoi present in the subject

[0040] The present in\ entors hax e disctn ered that in normal subjects {/ e , non-diseased) ha\ ing normal levels of full -length adiponectin. Saiger adiponectsn teceptor fragments are found in the biological fluids, i c fragments that ate 2^ to 34 ammo acid in length {ι c SFQ TD 1NOS L 2 4-1 1. 23, 24, and/oi 26-33 ) These fragments aϊe IΛ pj calls unbound Mans of these larger fragments are absent or present in ssgnϊficantlv loxser lc\ eK m subjects suffeπng from adiponectm related disease (J e . fragments ase ptesent in diseased patients at lev els 2\. 5\, I Ov 20\, 5Ox. 100\ less, oi more than J 00\ less than in non-diseased patients) B\ unbound fragments. H ΪS meant fragments that are not bound to earner protein, / c adiponectin

[00411 The present im entors ha\ e discox ered that in diseased subjects, smaller adipυnectm fiagrnents are found m the biological fluids i e fiagments> that are about B to 24 amino acids HI length O e , SF,Q ID NOS 3, 12-22 2\ and or 34-44j liiese fragments can be bound or unbound These fragments are also found m norma! subjects but general!) not at the same levels as in diseased subjects (ι e . fragments are present m diseased patients at lex els 1 5\, 2\, 2 5\. 4\, 5x, 6\. 7\. 8\ ⁽>\, or 10\ more than in πυn-diseased patients)

[00421 Tl^lύ present im entors hase found that m both notmal and diseased subjects, c- temiinai adiponectin receptor fragments bound to adiponectin are obsen ed These bound receptor fragments can be the larger or snmllet fragments In mam cases these fragments were bound to the non-globular portion of adiponectin whether partially fragmented or full length The leu'1 of bound adiponectin receptor fragments is increased in subjects with disease.

(øø43| The presence and/or levels of the unbound and bound fragments and smaller and larger f ragmen Is can. according!} . be used to determine the level of expression of adiponectm and the adiponectm receptor in an individual as well as disease states in indi\ idυals

ill. Detection of soluble C terminal fragments of the adiponectin receptor

[0044J The present invention provides methods for assaying for the presence or absence and/or determining the lex el of at least one soluble C terminal fragment of the adiponectin receptor in bodily fluid. The phrase "determining the level^" means detecting the presence or absence of an analy te in a sample or quantifying the amount in relative or absolute terms. A relative amount could be. for example, high, medium or low. Art absolute amount could reflect the measured strength of a signal or {he translation of this signal strength into another quantitative format, such as micrograms ml.

[004SJ The C terminal fragments can be detected by any suitable method Detection paradigms that can be employed include, for example, optical methods, electrochemical methods (λ oltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g.. multipolar resonance spectroscopy. Optical methods include, for example, colorimetric assays, electron impedance spectroscopy, microscope , both coiifoca! and rton- confocal. detection of fluorescence, luminescence, chemiluminescence. absorbance, reflectance, iransmittance. and birefringence or refraeth e index, (e g. , surface pi as man resonance, eihpsontetry. a resonant mirror method, a grating coupler \sa\ eguide method or interferomelry).

[0046J In certain preferred embodiments, the level of expression, including presence or absence of at least one soluble C terminal fragment of the adiponectin receptor is assayed by an immunoassay Those skilled in the art are aware that, in its broadest context, an ^"Immunoassay ^"' comprises incubating a test sample with one or more immuiiointeractive molecules specific for a target for a time and under conditions sufficient for binding thereto and detecting said binding. As used herein, the term "target ^* refers to the analvle which a probe is designed to bind In certain preferred embodiments, the iramυrioinieractn e molecule Λ\ ill he an an Ii body Conditions for incubating an antibody with a test sample vary, depending upon the format employed in the assay, the detection methods employed and the type and nature of the antibody molecule used m the assay. Those skilled in the art will recogni/e that any one of the commonly av ailable immunological assay formats, for example radioimmunoassay , en^ me-linked immunosorbent assays (HLlSA). immuno-tubimeiπc, immunonephrometπc. magnetic imrnuno particle separation, immunochroniatography, irnmuno-rnicroflυdic, nnniuno-centrifugal, diffusion-based Oucbterlom . rocket gel Immunoelectrophoresis or in situ immunoassay can be readily adapted Io the present purpose.

[0047J Immunoassays are useful in the quantification of at least one soluble C terminal fragment of the adiponecfin receptor in a test sample, in particular to determine whether the lev el of the at least one soluble C terminal fragment is altered compared to normal lex els detectable in non-diseased mdi\ jdυals As a consequence, such an immunoassay ;s of particular use in determining whether a patient may ha\ e a disease or predisposition to disease The immunoassay can have other uses as well, such as, for example, use in the monitoring of disease progression or monitoring of response to therapeutic mten entions. The in\ ention described herein extends to all such uses of immunointeractπ e molecules and diagnostic assa> s which require said immunoassays for their performance.

[(MMSj By way of example only, in certain embodiments, an antibody raised against the fragment is immobilised onto a solid substrate to form a first complex and a biological test sample from a patient is brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody- secondary complex, a second antibody labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allow mg sufficient time for the formation of a tertian¹ complex Am un reacted material is washed aw as . and the presence of the tertian¹ complex is determined by observation of a signal produced by the reporter molecule The results can either be qualitativ e, b\ simple observ ation of the visible signal or may be quantitated by comparison with a control sample containing know a amounts of hapten. Variations of this assay include a simultaneous assay, in winch both sample and labelled antibody are added simultaneously to the bound antibody, or a re\ erse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound aniibodv . These techniques are well known to those skilled in the art. and the possibility of v ariations will be readily apparent.

|0049j By "reporter molecule^", as used in the present specification, is meant a molecule which, by its chemical nature, produces an analytically identifiable signal which allows the detection of antigen-bound antibod\ . Detection can be either qualitative or quantitativ e. The most commonly used reporter molecule in this ts pe of assav are either colored latex particles, metal particles, ert/% mes, fluorophores or radionuclide containing molecules (i.e. radioisotopes)

[005Oj The solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, poh acrylaimde, n\ lon.mtrocellulose_* polystyrene. pol> v im 1 chloride or poh propylene. The solid supports may be in the form of strips, cassettes, tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The bindin ^■>gg processes are well-known in the an and generally consist of cross-linking covaiemly binding or physically adsorbing the molecule Io {he insoluble carrier.

[0051 J A variety of immunoassay formats, including, for example, competitive and noncompetitive immunoassay formats, antigen capture assays and two-antibody sandwich assays can be used in the methods of the invention (Self and Cook, Curr. Opin. Biofechnoi. 7:60-65 {1996}). ϊn an antigen capture assay, antibody is bound to a solid phase, and sample is added such that a soluble adiponectm receptor C terminal fragment antigen is bound by the antibody. The antibody can be specific for one or two or more of the soluble C terminal fragments. After unbound proteins are removed by washing, {he amount of bound antigen can be quantitated, if desired, using, for example, a radioassay (Harfow and Lane, Λnnbixiies A laboratory Manual Cold Spring Harbor Laboratory; New York. 1988)). Immunoassays can be performed under conditions of antibody excess, or as antigen competitions, to quantitate the amount of antigen and, thus, determine a level of soluble adiponectin receptor C terminal fragments.

[0052| Enzyme-linked immunosorbent assays (BIiSAs ) can be useful in certain methods of the invention In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of giutarakiehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include, for example, horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydroK sis by the corresponding enzyme, of a detectable color change. It is also possible to employ fluorogenic substrates, for example, which yield a fluorescent product An enzyme such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-gaiactosidase or urease can be linked, for example, to an ami-adiponectin receptor C terminal fragment or to a secondary antibody for use in a method of the invention. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tεtramethylbenzidme (TMB), which yields a soluble product in {he presence of hydrogen peroxide that is detectable at 450 nm. Other convenient enzyme-linked systems include, for example, the alkaline phosphatase detection system, which can be used, for example, with the chromogenic substrate p-nilrophenyl phosphate to yield a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with, for example, the chromogenic substrate o~nitτophenyϊ~β~D~ galactopyranoside (ONPG) to yield a soluble product detectable at 410 nm. or a urease detection system can be used with, for example, a substrate such as urea-bromocresol purple (Sigma Immunochemicals. St. Louis. Mo ). Useful enzyme-linked primary and secondary antibodies can. be obtained from a number of commercial sources such as Jackson Ϊmmuno-Research (West Grove, Pa).

[0053J In certain embodiments, the soluble C terminal fragments can be detected and measured using chernilurnhtescent detection For example, in certain embodiments, adiponeetin receptor C terminal fragment specific antibodies are used to capture the fragments present in the biological sample and a antibody specific for the specific antibodies and labeled with an chemilυniinescent label is used to detect the fragments present in the sample. Any chemiluminescent iabel and detection system can be used in the present methods. Chemiiuniinescent secondary antibodies can be obtained commercially from various sources such as Amersham. Methods of detecting chemiluminescent secondary antibodies are known in the art and are not discussed herein in detail.

[0054] Fluorescent detection also can be useful for detecting the adipcmectin receptor fragments in certain methods of the invention. Useful fluorochromes include, for example, DAPI. fluorescein, ianthanide metals, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-- phycoerythrin, rhodamine, Texas red and Iissamine Fluorescein or rhodamine labeled α2-MG-. HA-. TiMP-! - orYK.L~4ϋ~speeific binding agents such as anti-α2-MO. anti-HA. an ti -Tl MlM , or anti-YKL-40 antibodies, or fluorescein- or rhodarnine-labeled secondary antibodies can be useful in the invention. Useful fluorescent antibodies can be obtained commercially, for example, from Tag o Immunologicals {Burlingame. Calif.) as described further below. Fluorescent compounds, can be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the tluorochronie-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. føøSSj Radioimmunoassays (RIAs) also can be useful in certain methods of the invention. Such assays are well known in the art. Radioimmunoassays can be performed, for example, with ¹²³l-labe1ed primary or secondary antibody (Hariow and Lane, supra. 1988).

[0056] A signal from a detectable reagent can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation, such as a gamma counter for detection of ¹^l, or a ttuororneter to detect fluorescence in the presence of light of a certain wavelength. Where an enzyme-linked assay is used. quantitative analysis of the amount of soluble adiponectin receptor fragments can be performed using a spectrophotometer such as an EMAX Microplate Reader {Molecular Devices; Menio Park. Calif.) in accordance with the manufacturer's instructions. Hie assays of the invention can be automated or performed robotically, if desired, and that the signal from multiple samples can be detected simultaneously.

(0057| The methods of the invention also encompass the use of capillary electrophoresis based immunoassays (CEIA), which can be automated, if desired. Immunoassays also can be used in conjunction with laser-induced fluorescence as described, for example, in Schmalzmg and Nashaheh, Electrophoresis 18:2184-93 ( 1997). and Bao, ./ Chromatogr. B, BiomeJ ScL 699:463-80 (1997). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome irnnmπosensors. also can be used to detect soluble C terminal adiponectin fragments or to determine a level of C terminal adiponectin fragments according to certain methods of the invention (Ron gen et a^'L J. Immunol. Methods 204: 105- 133 (1997)).

[0058J Sandwich enzyme immunoassays also can be useful in certain methods of the invention. In a two-antibody sandwich assay, a first antibody is bound to a solid support, and the antigen is allowed to bind to the first antibody. Hie amount of soluble C terminal adiponectin fragments can be quantitated by measuring lhe amount of a second antibody that hinds to it.

[0059| Quantitative western blotting also can be used Io determine a level of soluble C terminal adiponectin fragments in a method of the invention. Western blots can be quantitated by well known methods such as scanning densitometry-. As an example, protein samples are electrophoresed on 10% SDS-PAGE Laemmii gels. Primary murine monoclonal antibodies are reacted with the blot, and antibody binding confirmed to be linear using a preliminary slot blot experiment. Goat anti-mouse horseradish peroxidase-coupled antibodies (BioRad) are used as the secondary antibody, and signal detection performed using chemi luminescence, for example, with the Renaissance chemi luminescence kit (New England Nuclear: Boston, Mass.) according to the manufacturer's instructions. Autoradiographs of the blots are analyzed using a scanning densitometer {Molecular Dynamics; Sunnyvale, Calif.) and normalized to a positive control. Values are reported, for example, as a ratio between the actual value to the positive control (densitometric index). Such methods are well known in the art as described, for example, in Parra et aL J. Vase. Surg. 28:669-675 {1998}.

[0060] Levels of adiponectin receptor fragments can also be determined using protein microarrays. Methods of producing protein microarrays that may be adapted for detecting levels of protein in a clinical sample are described in the ail (see for example of Xiao et ai. (2005) MoI Cell Endocrinol ; 230(1 -2):95- 10; Protein Microarrays (2004) Mark Schena (Ed) Jones & BartSett Publishers, inc.). U.S. patent Pub. 2003/0153013 describes methods of defecting proteins, e.g. antigens or antibodies, by immobilizing antibodies in a protein microarray on a membrane and contacting the microarray with detection proteins which can bind to the proteins to form protein complexes. Similarly, U.S. patent Pub. 2004/0038428 describes methods of constructing protein microarrays.

(006! J In certain preferred embodiments, a sample is analyzed by means of a biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound {here.

[0062] Protein biochips are biochips adapted for the capture of peptides. Many protein biochips are described in the art. These include, for example, protein biochips produced by Ciphergen Biosyslems, inc. (Fremont, CA), Packard BioScienee Company (Menden CT). Zyomyx (Hayward. CA). Phylos (Lexington, MA) and Biacore (Uppsala, Sweden). Examples of such protein biochips are described in the following patents or published patent applications: U.S. Patent No. 6,225,047; PC T International Publication No. WO 99/51773; U.S. Patent No. 6329,209, PCT International Publication No. WO 00/56934 and U.S. Patent No. 5,242.828. incorporated herein by reference in their entirety and for all purposes.

[0063{ for use herein, lhe assay methods can involve capturing the C-tennhtal adiponectin receptor fragments onto a solid substrate. Typically they will be captured using a bϊoxpeαfie capture reagent such as an antibody and, m particular, an antibody used in an immunoassay. Biospecifie capture reagents include those molecules that bind a target analyte with an affinity of, for example, at least H)^"9 M, 10^"™ M, H)^'" M or ]Q^~yz M. These molecules also can be captured with non-specific methods, such as chromatographic materials.

[0064| In certain embodiments of the present invention, at least one C terminal fragment of the adiponectin receptor will be detected by mass spectrometry Examples of mass spectrometers are time-of-fiight magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these.

[006SJ A preferred mass spectrometric technique for use in the invention is "Surface Enhanced Laser Desorptioπ and Ionization" or "SELDI,^'' as described, for example, in U S. Patents No. 5,719,060 and No. 6,225,047. both to Hutchens and Yip, each of which is incorporated herein by reference in its entirety and for all purposes. Tins refers to a method of desorptioπ/ionizatiori gas phase ion spectrometry (e.g... laser desorption/ionizalϊon mass spectrometry) in which an analyte is captured on the surface of a SELDl probe that engages the probe interface of the mass spectrometer.

[0066| One version of SELDI is called "affinity mass spectrometry.^" This version involves the use of probes comprising of an absorbent surface (an "affinity mass spectrometry probe"). In this context, "probe" refers to a device adapted to engage a probe interface and to present an analyle to ionizing energy for ionization and introduction into a mass spectrometer. A probe typically includes a solid substrate, either flexible or rigid, that has a sample-presenting surface, on which an analyte is presented Io the source of ionizing energy.

[QQ67J Another version of SELDI is Surface-Enhanced Neat Desorptiort ("SEND^"), which involves the use of probes comprising energy absorbing molecules attached to the probe surface (^"'SEND probe^"). The phrase ^"Energy absorbing molecules^"' (EAM) denotes molecules that are capable of absorbing energy from a laser desorption/ionization source and, thereafter, contributing to desorption and ionization of analyte molecules in contact therewith. The EAM category includes molecules used in M ALDL frequently referred to as ^"matrix,^"' and is exemplified by cinnaniie acid derivatives, sinapinic acid (SPA), cyano-hydroxy-cinnamic acid (CHCA) and dihydroxy benzoic acid, ferulic acid, and hydroxyaceto-phenone derivativ es. In certain embodiments, the energy-absorbing molecule is incorporated into a linear or cross-linked polymer, e.g.. a poiymethaeiylate. For example, the composition can be a co-polymer of a- cyano-4-methacryloyloxycinnamic acid and aery late. In another embodiment, the composition is a co-polymer of a-cyano-4-methae.tΛloyloxyci.nnarnie acid, aery JaIe and 3-( lri-ethoxy)silyi propyl methacryϊate. In another embodiment, the composition is a co-polymer of a-cvano-4- methacϊvloylowcinnamic acid and octadecylmethacryiate (^''C¹ I S SEND'^"). SEND is further described in Ii. S. Patent No. 6,124,137, incorporated herein by reference in its entirety and for all purposes.

[0068] A "selective surface'^* can be used to capture the fragments for SELDl analysis. The selective surface has an ^''adsorbent.^" also called a ^'■binding moietv ^" or "capture reagent" attached to the surface. An "adsorbent^"' or "capture reagent" or "binding moiety/^* can be any material capable of binding an analyte. The capture reagent can be attached directly to the substrate of the selective surface, or the substrate can be a "reactive surface^"' that carries a "reactive moiety^'* that is capable of binding the capture reagent, e.g.. through a reaction forming a covaleiit or coordinate covalent bond. Epoxide and carbodi imidazole are useful reactive moieties to covalently bind polypeptide capture reagents such as antibodies or cellular receptors. Nitπloacetic acid and iminodiacetic acid are useful reactive moieties that function as chelating agents to bind metal ions that interact non-covalently with histidme containing peptides.

[00691 fa^* certain embodiments, the adsorbent used io capture the C-terminaϊ adiponectin receptor fragments comprises a biospecific capture reagent. A "biospecific adsorbent^"' refers to an adsorbent that binds to an analyte with an affinity of at least 10^*9 M, IO^"K1 M, 10^*" M or 10^*L; M. The preferred biospecific capture reagent is an antibody or a binding fragment thereof. This includes intact immunoglobulins and the variants arid portions of them well known in the art such as. Fab^" fragments, F(ab)^" 2 fragments, and scFv proteins. Other biospecific capture reagents include affibodies (Affibody, Teknikrmgen 30, floor 6. Box 700 04. Stockholm SE- 10044, Sweden, US Pat No; 5,831.012; see also Surface Logix, Inc., 50 Soldiers Held Place. Brighton, MA 02135 and Hodneland, C. D, et al_t 2002, Proc Natl. Acad. Sci. 99: 5048-5052)

[0070J The fragments of the present invention can be captured on chromatographic adsorbents. ^"Chromatographic adsorbent^" refers to an adsorbent material typically used in chromatography. Chromatographic adsorbents include, for example, nitrocellulose membranes, ion exchange materials, metal chelators (e.g.. nitriloacetie acid or iminodiacetic acid), immobilized metal chelates, hydrophobic interaction adsorbents, hydrophiiic interaction adsorbents, dyes, simple biomolecuies (t;.g.. .nucleotides, ammo acids, simple sugars and fatty acids) and mixed mode adsorbents (e.g.. hydrophobic attraction/electrostatic repulsion adsorbents),

|0071] In certain embodiments, a substrate with an adsorbent is contacted with the sample, e.g., patient serum, for a period of time sufficient to allow the target analvtes that may be present to bind to the adsorbent. After an incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used; preferably, aqueous solutions are employed. The extent to which molecules remain bound can be manipulated by adjusting the stringency of the wash. The eiution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of ehaotropism, detergent strength, and temperature. Unless the probe has both SEAC and SEND properties, an energy absorbing molecule then is applied to the substrate with the bound target analy tes.

[0072] The biomolecules bound to the substrates can be detected in a gas phase ion spectrometer such as a time-of-flight mass spectrometer. The target analvtes can be ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The detector then translates information of the detected ions into .mass-to-charge ratios. Detection of a target analyte typically will involve detection of signal intensity. Thus, both the quantity and mass of the target anaiyte can be determined.

[0073| In another mass spectrometry method, the target analvtes can be first captured on a chromatographic resin having chromatographic properties that bind the target analy tes, e.g., an antibody or antibodies, in the present example, this can include an immuno-chromatographic resm that comprises antibodies that bind C-ierminal adiponectin receptor fragments. Unbound material can be w ashed from lhe resin Then the target analy tes can be eluted from the resin Finally, the eluted target analytes can be detected by MALDi or by SELDl

(0074| Analysis of anah les b> time-of- flight mass spectrometry generates a time-of - ftight spectrum The n^'πie-of~ftight spectrum ultimate!} analyzed lypicaily does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses This reduces noise and increases dynamic range. This time-of-fiight data is then subject to data processing.

J0075] Data generated b> desorption and detection of target analytes can be ana!} /ed ^■with the use of a programmable digital computer. The computer program analyzes the data Io indicate the number of proteins detected, aid optionally the strength of the signal and the determined molecular mass for each target anaiyte detected Data analysis can include steps of determining signal strength of a target anaiyte and removing data deviating from a predetermined statistical distribution. For example, the observed peaks can be normalized, by calculating the height of each peak reiafh e to some reference. The reference can be background noise generated fay the instrument and chemicals such as the energy absorbing molecule which is set as zero in Lhe scale

[0076J Analysis generally im olves the identification of peals in the spectrum that represent signal from an anah te. Peak selection can be done \ usually, but software is a\ ailabie that can automate the detection of peaks. In general, this software functions by identify mg signals ha\ ing a signal-lo-noise ratio afatπ e a selected threshold and labeling the mass of the peak at the centroid of the peak signal In one useful application many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra. One v ersion of this software clusters all peaks appealing JΠ the various spectra wUhin a defined mass range, and assigns a mass (M/Z) to all the peaks that are near the mid-point of the mass (MfZ) cluster

|0077| Software used to analyze the data can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a peak in a signal that corresponds to a large, anaiyte according Io the presenl invention. The softw are also can subject the data regarding observed target anaiyte peaks to classification tree or ANN analysis, to determine whether a target anaiyte peak or combination of target anah te peaks is present that indicates cardiovascular disease status. Analysis of the data may be ^"keyed^" to a variety of parameters thai are obtained, either directly or indirectly , from the mass spectrometric anah sis of the sample. These parameters include, but are not limited to. the presence or absence of one or more peaks, the shape of a peak or group of peaks, the height of one or more peaks, the log of the height of one or more peaks, and other arithmetic manipulations of peak height data.

- 10 - l\\ Antibodies

[007${ This in\ ention further provides antibodies that specifically bind to the C -terminal fragments of the adiponeciin receptor Methods of making antibodies having binding specifier^ to select peptides are w ell km_π\n m the art For example, such antibodies can be selected by immunizing an animal with the target molecule, generating antibodies, and testing the antibodies to identify whether a particular antiboφ binds with the target molecule. Antibodies that bind with {he target molecule can be selected. For example, one can generate monoclonal antibodies against these molecules

[0079 j Hie phrase "specifically binds to^" refers to a binding reaction which is determinative of the presence of a target in the presence of a heterogeneous population of other biologies. Thus, under designated assay conditions, the specified binding region bind preferentially to a particular target and do not bind in a significant amount to other components present in a test sample Specific hmding to a target under such conditions can require a binding moiety that is selected for its specificity for a particular target. A \ ariety of assas formats can be used to select binding regions that are specifically reacti\ e \x itli a particular artaly te Typically a specific or seiectn e reaction will he at least twice background signal or noise and more typically more than IO times background.

[00S0| The term "antibody" is used m the broadest sense and specifically covers monoclonal antibodies. poK clonal antibodies, antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, chimeric, single-chain, and humanized antibodies, as well as antibody fragments (c.#. Fab, F(ab^')?- and F\ ). so long as the> exhibit the desired biological activity Antibodies can he labeled for use m biological assays (c.#,- radioisotope labels, fluorescent labels) to aid in detection of the antibody.

[00811 Antibodies can be labeled/conjugated to reporter molecules for use m biological assays (t\# , radioisotope labels, fluorescent labels) to aid m detection of the fragments described herein.

[ΘΘ82J The term "monoclonal antibody^"' as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e.. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts Monoclonal antibodies are htghSs specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations Λλhich t\ picaSK include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybiϊdoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal^"' indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohier, et a!. , Nature, 256: 495. 1975, or can be made by recombinant DNA methods (see. e.g... U.S. Pat. No. 4,816,567. Cabiliy, el al.). The "monoclonal antibodies^*' can also be isolated .from phage antibody libraries using the techniques described hi Clackson, et al , 624-628, 1991 : Marks, el ai... I MoI Biol, 222: 581-597, 199K for example.

[0083J The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or Sight chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous Io corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (Cabilly, et aL. siφm: Morrison et al.. Proc. Nail Acad.. Sa. U.S.A., 81 : 6851 -6855, 1984).

[0084J Monoclonal antibodies can be obtained by various techniques familiar to those skilled in the art Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma ceil (see, Ivobler, el al., Eur. J Immunol.. 6: 51 1 -519, 1976). Alternative methods of immortal i/.au^'ou include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the arl. Colonies arising from single immortalized ceils are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such ceils can be enhanced by v arious techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one can isolate DNA sequences which encode a monoclonal antibody or a binding Fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by H use, et al.. Science. 246; 1275-1281, 1989.

[00S5| Monoclonal antibodies and poly clonal sera can be collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support Typically, polyclonal antisera with a titer of 10⁴ or greater are selected and tested for their cross reactivity against using a competitive binding immunoassay. Specific polyclonal antisera arid monoclonal antibodies will usually bind with a Kd of at least about 0. ϊ mM, more usually at least about 1 μJVL preferably at least about 0. \ μM or better, and most preferably, 0.0! μM or better.

(0086| '^"Humanized^" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab\ F(ab^'); or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For {he most pan, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary -determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances. Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at leas! one. and typically two. variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are {hose of a human immunoglobulin sequence. Hie humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc). typically that of a human immunoglobulin. For further details, see Jones, el ai. Nature, 321; 522-525. 1986; Reichmann, et ai . Nature, 332: 323-329. 1988; Presta, Cnrr. Op. Struct. Biol , 2: 593-596. 1.992. The humanized antibody includes a Primatized™ antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest,

[0087] A number of immunogens comprising portions of the fragments described herein can be used to produce antibodies specifically reactive with the fragments. For example, a fragment of the present invention, can be isolated using techniques known in the art. Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Alternatively , a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Naturally occurring protein can also be used either in pure or impure form. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated, for subsequent use in immunoassays to measure the protein. [0088] Methods of production of polyclonal antibodies are known Io those of skill in lhe art. An inbred strain of mice (e.g._ BALB/C mice) or rabbits is immunized with the protein using a standard adjuvant such as Freund^'s adjuvant, and a standard immunization protocol. The animal's immune response to the imniunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the beta subunits. When appropriately high titers of antibody to the immunogen are obtained, biood is collected from {he animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Hariow & Lane, supra),

[0089] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty, et al . Nature, 348: 552-554, 1990; Clackson, et al . Nature, 352: 624-628. 1991 ; Mails, et aL J. MoI Biol., 222; 581-597, J 991. describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (ΏM range) human antibodies by chain shuffling (Mark, et a!., Bio-Technology, 10; 779-783. 1992), as well as combinatorial infection and in viva recombination as a strategy for constructing very large phage libraries (Waterhouse, et al , Nuc. Acids. Res., 21 : 2265-2266, 1993). Thus, these ^"techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[0090J In certain embodiments, the antibodies are selected to distinguish between one fragment of C-tεrmhial adiponectin receptor and another, that is, the antibodies are selected that specifically bind to one form, but not another, under the same assay conditions,

[0091| Accordingly, the present invention provides an antibody that specifically binds to an epitope of an adiporteclin receptor fragment having SEQ I^ ^Q: ^ ■ ^ certain embodiments, the antibody will specifically bind to a region of SEQ ID NO; 1 that^" is outside of the adiponectin bmding domain, .i.e., the antibody will specifically bind to an epitope within resides 1 -22 of SEQ ID NO: 1. in certain embodiments, the antibody will specifically bind to an epitope within resides 1-14, 2-14, 2-14. 3-14, 4-14, 5-14. 6-1.4, 7-14, 8-14. 9-14. 10-14, 14-22 or within residues 23-34 of SEQ FD NO: 1 , In certain embodiments, the antibody will bind to an epitope present on one of SEQ ID NO: 1 , 2, X 4,, S. 6. 7. 8, 9, U), 1 1 , 12. I 3. 14, 15, 16, 17. 18. 19, 20, 21 , or 22. in certain embodiments, the antibody will specifically bind to a region of SEQ ID NOS: 1-12 that is outside of the adiponectin binding domain

[0092J The present invention also provides an antibody that specifically binds to an epitope of an adiponectin receptor fragment havmg SEQ ID NO:23. ϊn certain embodiments, the antibody will specifically bind to a region of SEQ ID NO;23 that is outside of the adiponectin binding domain, .i.e., the antibody will specifically bind to an epitope within resides 1-22 of SEQ ID NO:23. Jn certain embodiments, {he antibody will specifically bind to an epitope within resides 1-14. 2- 14. 2-14, 3-14, 4-14, 5-14. 6-14, 7-14. 8- 14, 9-14, 10-14. 14-22 or within 23-34 residues of SBQ ID NO: 23. In certain embodiments, the antibody will bind to an epitope present on one of SEQ ID NO: 23, 24. 25. 26, 27, 28, 29, 30. 3 L 32, 33, 34, 35. 36. 37, 38, 39, 40. 41. 42. 43, or 44. in certain embodiments, the antibody will specifically bind {o a region of SEQ ID NOS: 23-44 that is outside of the adiponectin binding domain.

V. Correlation of adiponectin receptor fragments with disease states

J00931 For certain of the methods described herein, the level of at least one soluble adiponectin receptor fragment is determined in different patient samples for which either diagnosis or prognosis information is desired, to provide profiles. A profile of a particular sample is essentially a "fingerprint'^* of the stale of the sample, A normal state can be distinguished from a disease state, and within disease states, different prognosis states (good or poor long term survival prospects, for example) can be determined. Diagnosis can be done or confirmed by comparing patient samples with the known profiles. By assessing the evolution of soluble adiponectin receptor fragments different limes during disease progression, the stage of disease can be determined as well as the likely prognosis.

[ΘΘ94| A principle of diagnostic testing is the correlation of the results of a procedure with particular clinical parameters The correlation necessarily involves a comparison between two or more groups distinguished by the clinical parameter. A clinical parameter could be. for example, presence or absence of disease, risk of disease, stage of disease, severity of disease. class of disease or response to treatment of disease Accordingly, the diagnostician uses this correlation to qualify the status of a subject with respect to the clinical parameter. Thai is, the diagnostics at* uses the results of a procedure on a subject to classify or diagnose a subject status with respect to a clinical parameter, the confidence of the diagnosis/classification being related to the classifying or splitting power of the signs or symptoms used in the test

[0095J The methods described herein for quali fying or quantifying soluble adiponectin receptor fragments provide information which can be correlated with pathological conditions, predisposition to disease, therapeutic monitoring, risk stratification, among others.

[0096J The present methods are particularly useful for diagnosing conditions, evaluating whether certain drugs will have a desired effect, and determining prognoses. The present methods can be used for early detection of diseases as well as for the optimization of treatment protocols. Preferably the condition, i.e.. disease state, will be one associated with abnormal fragmentation patterns of an adiponeclin receptor.

(0097| For use herein, '^"diagnosing a condition^" refers to determining whether or not a subject has an increased likelihood of having a specified condition. Tests that are used to diagnose a condition, such as the assays described herein, in certain instances, may not be able to diagnose a condition on their own but^" are used in combination with other tests to diagnose a condition. Accordingly ^"'diagnosing a condition^"' is meant to include any methods thai aJso aids in the diagnosis of a condition.

[00981 to certain embodiments, the invention provides methods for monitoring fhε progression of disease states in a patient. The method typically comprise the steps of providing a first biological sample from {he patient, preferably a urine, biood plasma, blood serum and/or whole blood sample, measuring at least one soluble adiponeetin receptor fragment in a firs I biological sample at a first time point; providing a second biological sample from the patient, measuring the soluble receptor fragment in the second biological sample at a second time point, and determining progression of the disease state in the patient based upon the change m amount of adiponeetin receptor fragment or based upon a comparison to measurements from a control population. By measuring the soluble receptor fragments in a patient sample over time, a clinician will be able to determine whether the disease state has worsened or improved. A clinician can therefore utilize these measurements for tailoring treatment appropriately. Methods for monitoring the progression of disease states comprising determining level of at least one soluble C terminal fragment can be combined with other tests to monitor progression of the disease state.

[0099] The present inventors have discovered that subjects having an adipocyte imbalance have different patterns of adiponeetin receptor fragments in blood than do normal subjects. The present invention thus provides methods of determining whether a subject has an adipocyte imbalance by determining the levels of at least one adiponeetin receptor fragment in a bodily fluid sample from the subject.

[0 JOOj For example, in order to determine whether a subject has an adipocyte imbalance, once could determine the levels of the fragment described herein. In certain embodiments, the absence or presence of only very low levels of certain fragments, i.e.. fragments that are 25 to 34 amino acid in length {i.e.. SEQ ID NOS- L 2, 4-1 1 , 23, 24, and 26- 33} and generally unbound will be indicative of an increased likelihood of having adipocyte imbalance. Conversely, the presence of normal levels of these fragments will be indicative of a normal adipocyte balance. In certain embodiments, the presence of increased amounts of certain smaller fragments, i.e., unbound fragments that are 13 to 24 amino acids in length (i.e.. SEQ ID NOS: 3, 12-22. 25, and 34-44) will be indicative of an increased likelihood of having adipocyte imbalance. Conversely, the presence of normal levels of these fragments will be indicative of a norma! adipocyte balance. The presence of increased total levels of adiponectin receptor fragments, i.e.. total concentration of adiponectin receptor fragments, will be indicative of a respective likelihood of having adipocyte imbalance.

[0101] As Wood levels of adiponectin decrease, the percentage of patients with disease increases. In patients with blood levels of adiponectin of less than about 4.0 μg/niL, the number of patients diagnosed with metabolic syndrome increases strikingly and the risk for coronary artery disease increases as well. For example, a subject having blood levels of adiponectin of less than or equal to about 4.0 μg/m.L has an increased chance of having coronary artery disease as compared to a subject having blood levels of adiponectin of greater than 4 0 μg/mL (odds ratio is greater than 3.0 for men and women or greaser than 1.7 in men and greater than S O in women) The term adiponectin refers to total adiponectin measured including monomers of lull length, globular and non-globular portions as was as oiigmers of adiponectin. Thresholds can be adjusted for specific assays able to measure individual forms.

[0102J By measuring the levels of these fragments in a biological fluid sample obtained from a subject at different time points, it can be determined whether the adipocyte imbalance ΪS improving or worsening. Similarly, by measuring the levels of these fragments before and after therapeutic intervention, it can be determined whether the therapy is effective.

[0103] Adiponectin is an adipocyte implicated in a number of disease states, including, for example, obesity, insulin resistance, ty pe II diabetes, metabolic syndrome, dysKpidemia. cardiovascular disease, and hypertension. For use herein, a subject that has hypoadiponectinemia has reduced plasma adiponectin concentrations as compared to normal subjects. Subjects having hypoadiponectinemia can be identified using the present methods. The present methods can be used to determine onset of hypoadiponectinemia. progression of hypoadiponectinemia.. and/or efficacy of treatment of hypoadiponectinemia in a subject. Similarly, the present methods can be used to determine onset of a condition characterized by hypoadiponectinemia,. progression of a condition characterized by hypoadiponectinemia, and/or efficacy of treatment of a condition characterized by hypoadiponectinemia in a subject.

[01041 For example, in order to determine whether a subject has hypoadiponectinemia, one can determine the levels of the Fragment described herein. In certain embodiments, me absence or presence of increased levels of certain fragments, i.e.. generally unbound fragments that are about 13 to 24 amino acids m length (i.e.. SEQ ID NOS; 3. 12-22. 25, and/or 34-44) will be indicative of an increased likelihood of having hypoadiponectmemia In certain embodiments, the presence of decreased amounts of certain larger fragments, / e . generally unbound fragments that are 25 to 34 amino acids in length {i.e., SHQ f D NOS: ID NOS: 1, 2. 4- 1 L 23. 24. and/or 26-33} will be indicative of an increased likelihood of having bypoadiponeetinemia. l^~he presence of increased lev els of iota! adiponeclm receptor fragments, unbound or bound to carrier protein, i.e., adiponectin, will generally be indicativ e of a respectix e likelihood of

mg hypoadiponectinemia.

[0105| By measuring the levels of these f ragmen Is in a biological fluid sample taken from a subject at different time points, ii can be determined whether the hypoadipoπectineroia is improving or worsening. Similarly.

measuring the levels of these fragments before aid after therapeutic interv ention, it can be determined whether the therapv is effectiv e.

|0 J06J Normal insulin sensitivity results when insulin causes fat ceil to produce adiponectin. Full length adiponectiii aggregates into mul timers, typically called LMU^', MMW and HMW forms Adiponetin interacts with the adiponectin receptor 2 in the li\ er and adiponectin receptor I in the muscle to stop glucose production and cause gl\ coK sis and fatty acid oxidation The adiponectin receptor 1 reacts with a cleaved form of adiponectiii called globular adiponectin where as adiponectin receptor 2 reacts to full length adiponectin Globular adiponectin was recently shown by others Io form by action of blood elasla&e.

[0I07J Insulin resistance occurs when adipocytes become hypertropic and produce less adiponeclin in response to insulin in this state, the cells become more apoptotic and cell division slows. As a result plasma adiponectin levels decreases insulin levels rise in an effort to cause cells, to release more adiponectin Hov\ e\ er as the iosυi in resistance Λ\ orsens more msuli α and less adiponectin is produced. The lesser adiponectin results m less glycolysis and fatty acid oxidation in muscle and prevents liv er glucose production from stopping. følθSj For use herein, insulm resistance refers to a decrease in an individual in the biological action of insulin in v ivo as assessed by the rate of disposal of glucose from the bloodstream (e g . into insultn-sertshtve tissue, such as muscle, fat and liv er).

[0109{ Diabetes mellitus is defined as chronic hy perglycemia due to defective insulin secretion and/or action The tv\ o major classifications of the disease are

pe L which involv es pancreatic beta-celi destruction, usuailv by an autoimmune process, and type IL impaired physiological effectiveness of insulin, i.e. , insulin resistance Diabetes nielli t us is often first diagnosed by the demonstration of hyperglycemia through the use of random or fasting plasma glucose determinations, or by an oral glucose tolerance test. Glucose tolerance tests do not measure insulin resistance. [0110] Once diabetes is diagnosed, assays for insulin and C-peptide can be used to differentiate between type 1 and type 11 diabetes, and among t\ pe 11 diabetes, to distinguish those w ho require insulin treatment from those who can be managed with changes in diet and exercise patterns It is difficult to distinguish those needing insulin treatment from borderline cases who can be managed with changes in diet and exercise alone.

[O J I I j Insulin is a polypeptide hormone released by pancreatic beta cells to reduce blood glucose levels b\ promoting cellular uptake of glucose and suppressing endogenous glucose. The immediate precursor of insulin is promsulin (MW. M kDa). a single-chain poK peptide consisting of 86 amino acids w ilh three disul fide bridges ProteoK tic clea\ age produces insulin (MW. 6 kDa) which consists of 51 amino acids in two chains joined by two disulfide bridges, and the connecting peptide (C-peptide; MW. 3 kDak a single polypeptide chain containing 31 amino acids fiquimolar amounts of insulin and C-peptide are then secreted into circulation. Circulating C-peptide concentrations are approximately 5~ to 10-fold higher than those of insulin as a result of the much longer half-hfe of C -peptide. C-peptide is therefore a measure of the body^'s natural insulin production and can be measured in the presence of intravenous synthetic insulin

[0112] The gold standard for measurement of insulin resistance is the glucose clamp method (.VJ \ alue) Io measure glucose infusion rate (GlR) adjusted b\ insulin infusion rate (HR) to maintain a blood glucose !e\ el, A second common measurement is the fasting glucose and insulin (HOMA-IR), It has been reported that M \ aiue (as determined by glucose clamp method. a gold standard) correlated with blood lex els of adiponectin show that adiponectin can be an indicator for insulin resistance An additional correlation is the measurement of the fasting glucose and insulin blood lev els corrected by adiponectin (FBS x Fi Rl ^'AND} ( fasting blood glucose x fasting insulin leu'l/adiponectin)

[0Ϊ 13J The present methods can. be use to identify subjects having insulin resistance. Further, the present methods can be used to determine the sev erity of insulin resistance in diabetic subjects and Io recommend the appropriate treatment.

[01141 Fc*⁵" example, in order to determine whether a subject has insulin resistance, one can determine the lev els of the fragment described herein, In certain embodiments, the absence or presence of decreased lev els of certain fragments, i.e.. fragments that are 25 to 34 amino acid \n length, and that are generally unbound, {i.e.. SEQ ID NOS. 1 , 2, 4-1 L 23, 24. and or 26-33) will be indicatix e of an increased likelihood of having insulin resistance Com erseK . the presence of normal levels of these fragments w ill be indicate e of a normal state, In certain embodiments, the presence of increased leλ els of certain smaller fragments, i.e . unbound fragments that are 13 Io 24 amino acids in length (i.e., SEQ ID NOS: 3. 12-22, 25, and/or 34-44) will be indicative of an increased likelihood of having insulin resistance. Conversely, the presence of normal levels of these fragments will be indicative of a normal stale. An increase in total concentration of adiponeclϊn receptors fragments (bound or unbound) to carrier protein, i.e., adiponectin, is generally indicative of an increased likelihood of having insulin resistance.

[O J ISj By measuring {he levels of these fragments in a biological fluid sample taken from a subject at different time points, il can be determined whether the insυhn resistance is improving or worsening. Similarly, by measuring the levels of these fragments before and after therapeutic intervention, it can be determined whether the therapy is effective.

|O1Ϊ6| Metabolic syndrome has been associated with reduced plasma adiponectin levels and can be monitored using the methods of the present invention. Metabolic syndrome, also known as syndrome X₅ is a cluster of risk factors that is blamed for the excess cardiovascular disease morbidity among overweight and obese patients and patients with type 2 diabetes melhtus.

[011?| Both the World Health Organization and National Cholesterol Education Program - Adult Treatment Patent (NCEP-ATP III) have set forth diagnostic criteria for metabolic syndrome. For use in the present invention, metabolic syndrome is defined by {lie WfK) diagnostic criteria as provided below (Darwin Deen, American Family Physician, 69( 12} (2004) 2875-2882).

*— Insulin resistance is identified by type 2 diabetes melhtus or impaired fasting glucose.

(01 ISj The present inventors hav e found that the level of sol ubϊe adiponecti a receptor fragments in bodily fluid is an indicator of metabolic syndrome in a subject. Accordingly, the present method can be use to identify subjects hav sng metabolic s> ndrome These methods can be used in combination with am one of the other diagnostic criteria for identify ing metabolic S} ndrome

[Ol I9J Foi example. »1 υrdei to deteimine whether a subject has metabolic ssndiomc. one can determine the le\ els of the fragment described herein ϊn certain embodiments, the absence or presence of decreased lev els of certain fragments. / e , fragments {hat are 2^ to 34 ammu acid m length (i e SEQ ID NOS K 2, 4- 1 L 23. 24. and. or 25-33). and that are general!) unbound vuil be indicatn e of an increased likelihood of ha\ ing metabolic ssndiomc Com erseK . the presence of noimal levels of these fragments \\ι\\ be indicative of a normal state In certain embodiments, the piesence of increased amounts of certain smaller fragments. / c fiagraents that aie 13 to 24 amino acids in length (/ c , SFQ ID NOS 3. 12-22. 25. and or 34-44} w ill be mώcatjx e of an mci eased 1 ikehhood of bin ing metaboli c s> mis ome An increase m total concentration of adiponeetni receptor fragments, unbound or bound to carrier protein. / c , adiponectin. is generalh mdicatn e υf an increased hkehhoυd of hav ing metabolic s\ ndrome

[0120| Acute Coronars S-S ndrornes (ACS) has been applied to a gioup of coionan disorders that result from ischemic insult to the heart 4cute cotonan s\ ndiome is defined as a \ ascular blockage of greater than 60" o b\ angiograpb e\ aluation \\jth our \Mthout a cardiac condition

[0121 J Hie present ira entors ha\ e found that the le\ el of s>oi iible adφonectin receptor ltaginerits in bodiK fiind ι.s an indicator of \ a&cular blockage m a subject AccordingK , the present method can be use to identify subjects ha\ sng a \ ascular blockage These methods can be used in combination w ith an\ one of the othei diagnostic criteria for identif> i tig \ ascuiai blockages

[0122J For example, m order to determine \\ hether a subject has a \ ascular blockage, one can determine the ie\ els of the fragment described herein hi certain embodiments, the absence or pre&enee of decieascd lev els of certain fragments. ; e , fiagraents that aie 25 to 34 amino acid in length (/ e SFQ H> NOS J 2. 4-1 1. 23. 24 and or 26-33 ). and that are generalh unbound v\ ill be mdseatn e of an increased likelihood of ha\ mg \ ascular blockage Com erseh . the presence of normal Se\ els of these fragments v\ ill be i ndicati \ e of a normal state In certain embodiments, the pjesence of increased ainoiuits of ceitαin smaller fjagments. i c fragments that are 1 ^"S to 24 amino acids m length U v SEQ ID NOS 3. 12-22 25 and or 34-44} will be indicam e of an increased likelihood of ha\ ing \ ascular blockage Com erseh , the presence of noitnal lev els of these fraϋments will be indicativ e of a normal state An mαease in total concentration of adiponectin icccptor fragments unbound or bound to earner pjotein. ; e , adiponectin. is general!) indicatn e of an increased likelihood of hav ing a \ ascular blockage

(0123J Λ cardiac condition, also known as a cardiov ascular disease condition, generaiK means disease which iesults from a caidio\ asculai insufficiency . including, but not limited to corcmars heart disease (which further includes im ocatdial infarction and angina pectoi is) or coronarv arters disease, stroke, congenital heart failure and congcsm e heart failure, congenita! heart failure, and high blood pressure Ooionan heart disease also includes m\ ocardiaJ infarction and angma pectoris Caidiυ\ a-scuiai diseases arc geneiaih characterized In an unpaired suppK of blood to the heart ov othei target organs "Heart fajlute" refers Io an abnormality of cardiac function w here the heart does not pump blood at the rate needed for the requirements of metaboh/ing tissues The heart failure can be caused b\ a numbet of factors including ischemic, congenital, rheumatic, or idiopathic forms

JO 124j Coronarv heart disease (CHD) is caused b> a thickening of the inside w alls of the coronarx arteries I his thickening, called atherosclerosis, narrows the space through w hich blood can flow , decteasing and sometimes complctels cutting off the suppK of o\\ gen and nutrients to the heart Atherosclerosis usualK occurs when a pet son has high lev els of cholesterol in the blood Cholesterol and fat. circulating m the blood, build up on the w alls of the arteπes The buildup naπows the arteues and can slow ot block the flow of blood When the le\ el of cholesterol in the blood is high theie is a greater chance that it v\ ill be deposited onto the arters \\ alls This piocess begms in most people during childhood and the teenage s eais, and worsens as thex get older

[0 J25| OongetΛn e heart failure or (Cl IF) is a progiessπ e pathologic state where the heart ss increasingK unable to supph adequate casdiac output (the x oluntc of blood pumped by the heart ox er time) to dch\ er the o\> genated blood to peripheral tissues As CHF progresses, stiuctural and hemody namic damages occur While these damages ha\ e a \ attet> υf mauifestahons, one characteiistic s> mptorn is \ entπcular h\ pcrtϊophx Cl IF is a common end result of a numbet of various cardiac disordeis

|0126| M\ ocardial infarction general!) results from atherosclerosis of the coronan arteries, often with superimposed cotonan thrombosis It ma\ be dn ided into two major h pes transmural infarcts, ni which m\ ocardial necrosis »n oh es the full thickness of the \ onlπcuJai ^■w all and subendocardial (nonltansmuial) mfatcts, in which the necrosis sm ohes the sυbendocardium, the intramural m\ ocardium, or both, without extending all the wa> through the \ entnculai wall to the epjcaidium M> ocatdιal infarction is UiθΛ\n to cause both a change m hemod^y namic effects and an alteration in structure in the damaged and health} /ones of the heart. Thus, for example, myocardial infarction reduces the maximum cardiac output and the stroke \ olume of the heart Also associated with myocardial infarction is a stimulation of the DNA synthesis occurring in the interstice as well as an increase in {he formation of collagen in the areas of the heart not affected.

|0 J 27 j Angina pectoris ("angina") is a recurring pain or discomfort tn the chest that happens when some part of the heart does not receix e enough blood, it is a common symptom of coronary heart disease (CHD). which occurs when \ essels that earn blood to the heart become narrowed and blocked due to atherosclerosis.

[01281 The diagnosis and monitoring of all of these diseases b\ the present methods is encompassed by the present im ention.

[0129J For example, the present im entors have found that the le\ el of soluble adiponeclin receptor fragments in bodily fluid is an indicator of whether a subject, particularly a subject already suffering from arteriosclerosis, is likely to develop or ha\ e congestiv e heart failure, myocardial infarction, or ischemia. Accordingly, {he present method can be use to identify subjects having congesth e heart failure, myocardial infarction, or ischemia These methods can be used in combination with any one of the other diagnostic criteria for identifying these conditions

[013Oj For example, in order to determine whether a subject has or is hkeh to develop congestive heart failure, myocardial infarction, or ischemia, one can determine the levels of the fragments described herein In certain embodiments, the absence or presence of decreased lev els of certain fragments, i c , fragments that are 25 to 34 amino acid in length (/ L^J . SEQ ID NOS: 1 , 2. 4-1 K 23. 24. and/or 26-33). and thai are generally unbound will be indicative of an increased likelihood of having congestive heart failure, myocardial infarction, or ischemia. Conv ersely, the presence of normal lev els of these fragments will be indicativ e of a normal state In certain embodiments, the presence of increased amounts of certain smaller fragments, i.e., unbound fragments that are 13 to 24 amino acids in length (/ e., SEQ ΪD NOS 3. 12-22. 25. and.'or 34-44} will be iiidicatix e of an increased likelihood of having congestn e heart failure. m\ ocardial infarction, or ischemia Conv ersely, the presence of normal levels of these fragments will be indicativ e of a normal slate. An increase in total concentration of adiponectin receptor fragments unbound or bound to carrier protein, i.e., adiponectin. is generally indicative of an increased likelihood of hav ing congesiix e heart failure. m\ ocardiaS infarction, or ischemia

[013IJ Similarly, the present methods can be used to identify subjects ha\ ing h\ pertemion. obesity, hpidemia. or inflammation. These methods can be used in combination with any one of the other diagnostic criteria for identifying these conditions. In all of these conditions, in certain embodiments, the absence or presence of decreased levels of certain fragments, i.e., fragments that are 25 to 34 amino acid in length (i.e., SEQ ID NOS: SEQ ID NOS: i, 2, 4-1 i , 23, 24. and/or 26-33), and that are generally unbound will be indicative of an increased likelihood of having the condition. Conversely, the presence of normal levels of these fragments will be indicative of a norma! state. In certain embodiments, the presence of increased amounts of certain smaller fragments, Le . unbound fragments that are 13 to 24 amino acids in length (i.e., SEQ ID NOS: 3, 12-22. 24, and/or 34-44) will be indicative of having the condition. Conversely, the presence of normal levels of these fragments will he indicative of a normal state. An increase in total concentration of adiponectin receptor fragments unbound or bound to carrier protein, i.e., adipoϊieetin. is generally indicative of an increased likelihood of having the condition.

[01.321 The present invention provides diagnostic, prognostic find therapeutic methods using the specific measurement of at least one fragment described herein. The methods involve first providing a measurement of the adiponectin receptor fragment and then correlating the measurement with a disease state. By correlating the measurement, one is able to qualify the subject status with respect to the particular clinical parameter in question, in a preferred embodiment, the measurement is made by affinity mass spectrometry as discussed above.

J0133 J The power of a diagnostic test to correctly predict status is commonly measured as the sensitivity of the assay, the specificity of the assay or the area under a receiver operated characteristic (^"1ROC) curve. Sensitivity is the percentage of true positives that are predicted by a test to be positive, while specificity is the percentage of true negatives that are predicted by a test to be negative. An ROC curve provides the sensitivity of a test as a function of 1-specificity. The greater the area under the ROC curv e, the more powerful the predictiv e value of the test. Other useful measures of the utility of a test are positive predictive value and negative predictive value. Positive predictive value is the percentage of actual positives that test as positive. Negativ e predictive value is the percentage of actual negatives that test as negative.

[0134} Thee fragments described herein, individually, or. in combination, are useful in aiding in the determination of a disease status. In certain embodiments, first, the selected biomarker, i.e., particular fragment, is measured in a subject sample using the methods described herein, e.g., capture on a SELDl biochip followed by detection by mass spectrometry. Then, the measurement is compared with a diagnostic amount or cutoff that distinguishes one diagnostic parameter from another, e.g. , a positive insulin resistance parameter from a negative insulin resistance parameter. The diagnostic amount represents a measured amount of a biomarker above which or below which a subject is classified as having a particular disease. For example, if the fiagnient is up-segulatcd compared to noimal in the disease state then a measured amount abo\ e the diagnostic cutoff pro\ ides a diagnosis of disease AJternatix ei> , if the bionwkei is down-ieguiaied in the disease, {hen a measured amount below the diagnostic cutoff prm ides a diagnosis of the disease As is well undeistood m tiie art. b\ adjusting the particular diagnostic cutoff used in an assax one can increase sensitsx itx or specsficm of the diagnostic assa\ depending on the preference of the diagnostician

[0135 j In some embodiments, the meie pie^ence oi absence of a particular fragment without quantify nvj the amount of the fiagiπent, is useful and can he coπelated xuth a piobabie diagnosis of disease i e msuhn resistance Thus a delected presence or absence, respectu eh of these maikers in a subject can indicate tiiat the subject has a highei piøbabilit\ of ha\«ig insulin resistance

|0 J36J In cei lain embodiments of the methods of qualifs mg disease status, the methods_* further compnsc managing subject treatment based on the status Such management describes the actions of the ph> siciars or clinician subsequent to deteimming disease status ^or example if a pln sitian makes a diagnosis of disease, then a cemun tieatment iegimen vuil he followed tar example fot mam people, cardiov ascular heait disease is managed with hfesix Ie changes and medications Others with sex ere cardifn ascular heart disease ma\ need surgeπ, In am case, once catώo^ ascuSai heart disease de\ elops. it ieι|uues lifelong management Altematn eh , a diagnosis of no coionan heaii disease status or othei cardu>\ ascukir disease staius might be follow ed with no treatment If the diagnostic lest gn es an inconclusu e resυli on concerning coronan heait disease status, fusthei tests ma\ be called for

[013?1 \\ hile indn idua! biomaikers ate useful diagnostic markets, it has heen found that a combination of hionwkαs can pro ide gi eater predictive value of a paϋieulai status than single maskers alone Speαficalh the detection of a phirahrΛ of markers in a sample can increase the peiceiitage of taie posit* \e and tiue negatn e diagnoses and deci eases the peicentage of false positn e oi false negatπ e diagnoses Accordingh m certain embodiments the piesent methods im oh e detecting a plurality of the fragments described herein

JO I38j Accordmgh . in one aspect, this in\ ention pso\ ides a method for disco\ enng patterns of adiponechn ieceptoi fiagments, which patterns correlate with a clinical paiantetei of interest

[0139| In certain embodiments the piesent m\ ention prox ides methods for nieaMirmg the icsponse to theiap\ compnsing the steps of piox sdmg a fiist biological sample, piefeiabix a Ui me and o* blood plasma sample, measuring the amount of at least one soluble adiponectin ieceptor fjagment in the fusi biological sample at a first time point. p*o\ iding a second biological sample from the patient, measuring the fragment in the second biological sample at a second time point and determining response in the patient based upon the change in the amount of the fragment or based upon a comparison to a control population. The subject may be a positive responder. poor responded or non-responder. For use herein, a positive responder. is a subject who positively responds to treatment, i.e., a subject who experiences success in amelioration of the condition, including any objective or subjective parameter such as abatement; remission: diminishing of symptoms or making the condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject^'s physical or menial well-being. A positive responder is one in which any toxic or detrimental side effects of the biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-responder is a subject who doesn^'t respond to the treatment or doesn^'t respond to a satisfactory level. A poor responder is a subject who responds to treatment but not at the level of the positive responder.

[014Oj In certain embodiments wherein the disease state is insulin resistance or another condition related to insulin resistance such as diabetes or metabolic syndrome, the therapeutic treatment generally comprises the step of administering an effective amount of one or more insulin sensitizing pharmaceuticals. Insulin sensitizing pharmaceuticals are known in the art and include, for example. PPAR agonists such as a thia/.ohdinedione (also referred to as a TZD); or PPAR gamma partial agonists, also known as selective PPAR gamma modulators (SPPARM's) . PPAR alpha-gamma dual partial agonists (selective PPAR alpha-gamma dual selective modulators}' and PPAR pan-agonists. PPAR gamma agonists that have a TZD structure include pioglita/.one. rosighta/.one, ciglmvone. darglita/.oue. englita/.one. balaglita/one, isagUta/one, troglitazone, netoghtazone. MCC-555. and BRL-4%53. Other PPAR gamma agonists, some of which have a TZD structure, include CLX-0921 , 5-BTZD, OW-0207, LO- i 00641 , LY-300512. NN-2344, LY 818. GW-677954. GW-7282, and T- 131. PPAR alpha/yamma dual agonists that exhibit both alpha and gamma agouism and can be used to treat type 2 diabetes and to reduce lipids PPAR alpha/gamma agonists include KRP-297 (MK-0767), muraglitazar (BMS-298585), farglitazar, ragaglitazar. tesaglitazar (AZ-242), JT-501. GW-2570, GI-262579, CLX-0940, GW- 1536. GW ! 929, GW 2433, L-79M49, LR-90, SB-219994, LY-578. LY-4655608. LSN-862. LY- 510929. and LY-929.

[01411 The methods described herein can be used to determine whether a patient is likely to be a responder to treatment with any drug used to treat type 2 diabetes or insulin resistance including, for example, a bϊgυanide (e.g. metformin): a sulfonylurea; another chemical class of insulin secretogogue other than a sulfonylurea, such as a meglitinide; insulin (which can be formulated for subcutaneous or intramuscular injection, or in a formulation for avoiding the need for injection, such as oral, buccal, or nasal): a DP-IV inhibitor; a PTP-I B inhibitor; a GLP- 1 aialog; a gh cogen phosphorylase inhibitor; a glucagon receptor antagonist; a hydi'øx> sterol dehydrogenase (HSD- I ) inhibitor; a ylucoLinase activator, or a TZD or non-TZD PPAR gamma agonist; or any combination of treatment thereof

JO 142 j Hie methods described herein can be used to determine whether a patient is IiIeK to be a responded to treatment with any drug that can be used to treat obesm in an obese patient who also has type 2 diabetes or msuiin resistance, including, for example, ibutramme. orϋstat. phentermtne. an Mc4r I agonist, cannabinoid receptor 1 (CB-I } antagonist/im erse agonist, a 33 adrenergic agonist: or a TZD or non-TZD PPAR gamma agonist; or am combination of treatment thereof

|0 J43J The methods described herein can be used to determine whether a patient is a responder to treatment with any drug used to reduce total cholesterol or LDL-cbolesteroi and/or raise HDL-cholesteroI. including, for example, an HMG-CoA reductase inhibitor (lox astalin. sim\ astatin. rosuvastatin. pravastatin, fhn astatin. atorvaslatm, m astatiπ. pi tauss latin. ZD-4522. and other stating): niacin; a cholesterol absorption inhibitor (e/etmiibe); a CETP inhibitor (torcetrapib): a PPAR alpha agonist (fenofibrate, gemfibri/ol. cioilbrate, or be/afibrale); an ACAT inhibitor (av asjrmbe), an anti-o.xidanl (probυcol); or a bile acsd sequestrant (cholestyramiiie). or a TZD or non-TZD PPAR gamma agonist; or am combination of treatment thereof

[0144{ In certain embodiments, the le\ el of adiponectin receptor fragments is determined before treatment beg»i& and then after treatment has proceeded for a time long enough for the changes in the level or patterns of the fragments to reflect whether the patient will respond to treatment. After treatment has proceeded, a patient who is a likely responder to the therapeutic will ha\e increased els of certain fragments, i.e., fragments that are 25 to 34 amino acid in length {t.c , SEQ ID NOS; I . 2. 4- I I , 23. 24. and/or 26-33) and that are generally unbound, and decreased amounts of certain smaller fragments, i.e.. fragments that are 13 to 24 amino acids in length {i.e., SEQ ID NOS' 3. 12-22, 24, and/or 34-44) In certain embodiments, the differences will be ob&erv ed within four weeU after treatment commences, preferably within two weeks after treatment commences, and most preferably ^■within one week after treatment commences. VI, Additional biβmarkers

JO I45j In certain embodiments, assessment of one or more additional markers are combined to increase the predϊcm e \ alue of the analysis in comparison to that obtained from measurement of adiponeclin receptor fragments alone. For example one or more markers for the disease state, i.e.. adipocyte imbalance, insulin resistance, diabetes, metabolic syndrome, acute coronary syndrome (i e , vascular blockage), cardiovascular heart disease, stroke, congenita! heart failure, congests e heart failure, hypertension, angina.

ocardial infarction, ischemia, atherosclerosis, obesits . lipidemia. or inflammation, can be measured along with adiponectm receptor fragments to enhance the predict! \ e \ alue of the described methods, Biornarkers that can be used in combination with the present methods include for example, adipocyte factors, for example, adiponectin. ieplin. visfatin. klotho. glucagoπ-like peptide- 1 (GLP-I). DDPlV. resistin. ghrelin. AMP-activaled protein kinase (AMPKl StrtK PFAR agonists. ARN T (aryJ hy drocarbon receptor nuclear translocator), HlFl B, C¹ -peptide, Fo\a2. insulin, or glucose, including fragments, peptides and \ anants thereof and/or inflammation markers, for example. RBF--k C- reactive protein (CRP), resisϋπ, MCP- K IL-6. TNF-α. IL- I beta, PAI-I . bikυnm. auto-immune factors, autoantibodies to glutamic acid, islet cell auto-antibodies, insulin auto-antibodies. autoantibodies to 1L-2. autoantibodies KJ I A-2. incretins, and other auto-immune factors and fragments, peptides or \ anants thereof

[0146] The methods described herein can be used in combination with any other tests that w ill aid in the diagnosis of a disease, determination of progression of a disease, or determination of efficacy of treatment of a disease,

[0147J In certain embodiments, adiponectm levels will also be measured in the subject Methods of measuring adφonecttn and correlating adiponectm levels with disease slates are known in the art, see for example, U.S. Patent No. 6,461 ,821. U S Publication Nos. Us2OO5UO540⁽>5 and US2OO50O48565. and International Publication Numbers WO2004086040. WO2OO5046734. WO2005O38457. and WO2004O22596. each of which is incorporated herein by reference in its entirety and for all purposes. For use herein, the term adiponeclin includes variants thereof ha\ ing adiponectin activity. Jn certain embodiments, she amount of RHaI adiponectm. the amount of ion molecular weight, the amount of high molecular Λ\ eight adiponectm or the ratio between these numbers will he used in combination with the methods of the present invention. According!}', the present methods can include the step of measuring the lex el of adiponectin (total adiponectin, high molecular weight adtponeciin. low molecular weight adiponectin, or other forms of adiponectin. including fragments, and \ anants thereof) in a biological sample from a subject and correlating the amount with the presence of a disease state. with progression of disease, or efficacy of treat mem. Reduced amounts of adiponeclm are indicativ e of a disease as well as a smaller ratio of high molecular weight adiponectin to total or low molecular weight adiponeetm.

[0I48J In certain embodiments, ieptm levels will be measured m the subject. Methods of measuring leptin. including variants thereof, and correlating leptin levels with disease states are known in {he art (See, for example. Gorden and Gavriiov a, Current Opinon in Pharmacology. (2003) 3'655-65'λ incorporated herem b> reference in its entirety and for all purposes).

[0149] In certain embodiments, brain natriuretic peptide (FJNP) levels can be measured to aid in the diagnosis or progression of \ ascular blockage and cardiovascular disease. Methods of measuring BNP ie\ els and correlating them with disease states are known in the art. See. for example. Frank Peacock, Cleveland Clinic Journal of Medicine (2002). 69(3) 243-25 ! . incorporated herein by reference in its entirety and for all purposes

[015Oj Hie present methods can be used to identify subjects having inflammation and certain diseases characterized by excessive inflammation. These methods can be used in combination with known methods of determining levels of inflammation in a subject

[015IJ In certain embodiments, bikunin and/or uristatin levels will be measured in the subject. Bikunin represents the inhibitory light chain of the inter-α-lr> psin inhibitor protein. It is a protease inhibitor, known to be elev ated in the urine of patients with inflammatory diseases and is considered an acute phase protein For use herein the term bikunm includes \ ariants thereof having bikunin activity Uristalin is a trypsin inhibitor present in urine that is increased in most patients with bacterial or viral infections and in mam Λ\ ith inflammatory disorders. Methods of measuring bikunin or uristalin, including variants thereof, and correlating bikunin or uristatin levels with disease states are known in the an Uristatin is a trypsin inhibitor present in urine that is increased in most patients with bacterial or \ iral infections and m mam with inflammatory disorders (Pugia and Lett, CUn Chem Lab Med 2005 43( 1 ): I - 16. International Publication No. WO200504022. each of which incorporated herem by reference m its entirety and for all purposes)

[0J52| In certain embodiments. C-reactiv e protein lev els will be measured in the subject. Methods of measuring C -reactiv e protein, including v ariants thereof, and correlating C- reacUve protein iev els w ith disease states are known in the art For example C-reacth e protein in present in the blood serum during episodes of acute inflammation or infection. CRP levels of about 1 iBg dL is usually considered high for C¹RP and most infections and inflammations result in CRP lev els above 1 * > my/dJL For use herem the term C-reacti ve protein includes v ariants thereof having C-reactive protein activity. {Pυgia and Lott, Clin. Chem Lab Med 2005 43(1): 1 - 16, incorporated herein by reference in its entirely and for ail purposes).

(0153J In certain embodiments, a white blond cell count can be performed in combination with the methods described herein. Methods of measuring white biood cells and correlating white biood cell levels with disease stales are known in the art. White biood cell (WBC) count or the measure of white biood cells in the biood. is a reliable and widely used marker thai reflects inflammation throughout the body. WBC" count is aiso linked to other chronic conditions, including cardiovascular disease, hypertension and diabetes.

[01541 IΓΪ certain embodiments, fasting glucose, glucose tolerance measurements, and/or insulin aid giucagon-stimulated C -peptide levels will be measured in the subject. Methods of measuring insulin and C -peptide, including valiants thereof, and correlating insulin and C- peptide levels with disease states are known in the art. For example, giueagon-sfimuϊafed C- peplide levels greater than about 1.8 ng/mL have been reported to identify type 2 diabetics who could be managed without insulin treatment. Typically. 3.0 ng/mL is used as an upper limit indicative of hyperinsulinemia or insulin resistance, In contrast, levels less than about 0.5 ng/mL reportedly identify type J patients requiring insulin treatment due to hypoinsuliriemsa The normal reference range for normal adults is 0.5 - 2 ng/mL,

|01551 In embodiments wherein one or more markers are used in combination with adiponectin receptor fragments to increase the predictive value of the analysis, the level of the additional markers can be measured in the same biological sample from the subject or in another, which can be of the same type or of a different type. For example, the level of adiponectin receptor fragments can be measured in a sample of blood plasma, while the level of an additional marker, can be measured in the same sample of plasma, a different sample of plasma, or in a sample of serum or urine from the subject.

VH. Additional disease states

[01561 Adiponecthi is involved in many process and pathways in the body. Accordingly, the detection of the fragmentation pattern of adiponectin receptor fragments can be used to determine the onset, monitor progression and/or determine the efficacy of drug treatment for many disease states. In particular, the detection of soluble adiponectin receptor fragments can be used in combination with other diagnostic methods arid tools for determining the onset, monitoring progression arid/or determining the efficacy of drug treatment for many disease states. Ia particular, the detection of soluble adiponectin receptor fragments can be associated vαth angiogenic, atherogenic, aid macrophages transformation of cells, Adiponectin receptor I is upregulated by binding to LXR nuclear receptors which are activ ated by fatty acids and LXR receptors are integral to macrophage transformation Adiponectin receptor i expression has also shown Io be increased duraig monocyte transformation

[0IS7J Accordingly, inflammatory diseases, i t\, disease triggered by cellular or non- cellular mediators of the immune system or tissues causing the inflammation of body tissues and subsequently producing an acute or chronic inflammatory condition, can be detected and monitored using the present methods. Examples of such disease, include, for example. hypersensιliut> of type I- IV. for example, hypersensitivity disease of the lung including asthma, atopic diseases, allegic rhinitis or conjuncth itis. angioederoa of the lids, hereditary agioedema, antireceptor hypersensitiv ity reactions and autoimmune diseases, Hashimoto^'s thyroiditis. systemic lupus erythematosus. Goodpasture^'s syndrome, pemphigus, myasthenia

Grave^'s and Raynaud^'s disease, rheumatoid arthritis, psoriasis, Crohn^'s disease, scleroderma, mixed connectiv e tissue disease, polymyositis, sarcoidosis, urinary tract infection. IgA nephropathy. glomerulonephritis, acute or chronic host graft reactions

[015S| Cancers can also be detected and monitored using the present methods. Cancer refers to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of alϊected cells to spread locally or through the bloodstream and lymphatic system to other parts of the

(i.e.. metastasize) as well as am of a number of characteristic structural and/or molecular features. The term cancer includes, but is not limited to. cancers of the female reproductiv e organs including, but not limited to. o\ arian cancer. cer\ ical cancer and uterine cancer; lung cancer; breast cancer: renal cell carcinoma; Hodgkin's h mphoma: Noα-Hodgkin's lymphoma: cancers of the gemtouπnan s> stem including, but not limited to. kidney cancer, prostate cancer, bladder cancer, and urethral cancer; cancers of the head and neck; liver cancer; cancers of the gastrointestinal system including, but not limited to, stomach cancer, esophageal cancer, small bowel cancer or colon cancer; cancers of the bihar> tree; pancreatic cancer; cancers of the male reproductive system including, hut not limited to. testicular cancer. Gestational trophoblastic disease; cancers of the endocrine s\ stem including, but not limited to, thyroid cancer, parathyroid cancer, adrenal gland cancer, carcinoid tumors, insulinomas and PNET tumors; sarcomas, including but not limited to,

ing^'s sarcoma osteosarcoma, iiposarcoma. leiomyosarcoma, and rhabdomyosarcoma; mesotheliomas, cancers of the skin, melanomas; cancers of the central nerx ous system; pediatric cancers, and cancers of the hematopoietic system including, but not limited to all forms of leukemia. myekκK splastic syndromes. m> eloproliferatn e disorders and multiple eloraa. VIIl. Kits

|0159] For use in the applications described or suggested above, kits are also provided by the invention. Such kits can. for example, comprise a earner means being compartmentalized to receive in close confinement one or more container means such as strips, cassettes, microfluidie chips, vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in she method. For example, one of the container means can comprise a probe that is or can be detectabh labeled. Such probe can be an antibody or polynucleotide specific for a soluble C-lerπύnal receptor fragment.

[01.6Of In addition, the kits can include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g.. magnetic discs, tapes, cartridges, chips, and the like), optical media (e.g., CD ROM), and the like. Such media can include addresses to internet sites thai provide such instructional materials.

[0161 f The kit can also comprise, for example, a means for obtaining a biological sample from an individual. Means for obtaining biological samples from individuals are well known in the art, e.g., catheters, syringes, and the like, and are not discussed herein in detail. fO162] The following Exemplary Embodiments of specific aspects for earning out the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

EXAMPLES

.Example 1 - Detection of C-tercninal fragments in diabetic individuals

[0163] One hundred and sixteen patients were assessed by medical history. Fifty had no history of diabetes or metabolic risk factors (lipids, hypertension, obesity) were not diagnosed as metabolic syndrome according to WHO definition. Sixty nine had a history of diabetes either Type 1 or Type2. lnsuhn resistance was further assessed in ail patients by glucose measurement, c-peptide and hemoglobin AI c.

[01.64 J Adiponeetin, c-peptide, insulin, and HMW Adiponectin were measured using commercial ELiSA kit. HbAIc was measured with the DCA 2000+ instrument (Bas er) and glucose by the YSf. The following AdψoRl ELISA assay was used Io measure all C-teπmnal fragments whether bound or unbound. |0165] The materials for the ELISA assay of €-terrainal fragment AdipoRl included microliter plates (Costar PN 3690, high binding;), Tris Buffered Saline (IBS) (Pierce Product Number 28376).. Adiponeciin receptor 1 (Adipolli) peptide (peptides 16-34) (Phoenix Pharmaceuticals, Inc., Product Number 001 -44 ). Super Block in TBS (Pierce Product Number 37535), TBS/TW - Tris Buffered Saline containing 0.05% Tween 20 (Tween 20 - Pierce Product Number - P8341), Rabbit anii-AdipoRl antibody ( Phoenix Pharmaceuticals, inc.. Product^' Number G-OO 1-44), ALP-Goal anti-rabbil ϊgG (Sigma Product Number A 3687), I-Slep PNPP (Pierce Product Number 37621 ) and 2N NaOH.

[01661 Reagents were prepared as follows, A stock solution of AdipoRl peptide (Phoenix Pharmaceuticals, Inc. Product Number 001 -44) was prepared by dissolved ϊOOug peptide m IGGuL 60% Acetomtriie containing 0.1% TFA as directed.. This was further diluted this LO mg/mL solution to 10 mL with nanopure water araing aa 10 ug/niL stock solution. This solution was aiiqυoied 50OuL per vial is being stored frozen at - 70⁰C. A 0. 1.0 ug/mL AdipoR 1 peptide in TBS was used to coat plates and was prepared by adding l OOtiL of 10 ug/mL AdipoRl peptide in TBS (A above) to 990OuL of TBS and mixing well. A stock solution of Rabbit artti- AdipoRl (Phoenix Pharmaceuticals, lnc , Product Number G-OO 1-44) was prepared by dissolved 20OuG antibody in 20OuL nanopure water as directed. This makes a ! .0 mg/mL antibody solution. Aliquot into 5OuL aliqouts and store fro/.en at -7O⁰C. A solution of 6.0 ug/raL Rabbit an Ii -AdipoRl in Super Blocker made by adding 18.OuL of stock anti-AdipoRl into 2982uL of Super Blocker and mix well A solution of 3 75 ug/mL Rabbit anti-AdipoRi in Super Blocker (used to dilute plasma samples 5-fold: change concentration for other dilutions) was made by adding 56.25uL of stock anti-AchpoRl (C) to ] 5.0GGuL of Super Blocker and mixing well. A 1 /2000 dilution of ALP-Goat anti-rabbit IgG was made by adding 7.5 uL ALP-Goat anti-rabbit fgG (Sigma. Product Number A 3687) into 15.OmL Superblocker and mixing well.

|0167| Preparation of calibrators were done usmg AdipoRl peptide in superblocker containing 3.Gug/mL Rabbit anti- AdipoRl to achieve AdipoRl peptide concentrations of 5.0, 2 5₅ 1.25, 6.25, 0.312. 0.156. 0 078 and 0 ug/nil,

10168] The method for AdipoRl ElJSA Assay was done by coating the micortiter plate with 50 uL/weli of O. l Oug/mL AdipoRl peptide in TBS, and store at 4°C for minimum of 72 hours, removing the coated microliter plate from the refrigerator, and emptying the plate and wash the 3 times with 20OuLMeIi TBS. This w as followed by adding 15OuL of Super Block buffer {Pierce PN 37535) to each well and shaking the plate for 30min at 25⁰C. The plate was emptied and wash 5 tunes with TBS/TW. This is followed by the addition of the prepared calibrators containing 5000, 2500, 1250, 625, 312. 156, 78, and O ng/mL AdipoR l peptide or of samples diluted 5-fold with blocker buffer AU samples and eahbators contained 3.0ug ^'niL Rabbit anti-AdipoR 1 The samples or calibrates were added as5θuL/w ell and incubated cn emight at 5^ftC in a refrigerator. This was followed by emptying tiie plate and wash 5 times with TBS/TW.

|0 J69J The J /2000 dilution of ALP-Goat anti-rabbit IgG m Super Blocker was added at 50uLAveil Io all the wells in the ELiSA Template. This was incubated for 2 hours at 25°C on the Jitterbug Shaker at shaker setting #2 The plate was empties and wash the 5 times with TBS/TW 50ul. of t-Step PNPP (Pierce PN 37621 ) was added to each well. The plate was incubated for JH) rain at25"C on the Jitterbug Shaker 25uL of 2N NaOH was added to each well to stop the en/yme reaction. The plate was allowed to stand at least 5 min before reading at 405nm.A fit calibrator data to a standard cuπ e and calculate unknowns. (Single phase exponential decay usually gives best fit.) was done to calculate the v alues in the sample.

[0170{ Adiponectin decreases with type 2 diabetes Adiponectin was unchanged with type 1 diabetes. Adiponectin was higher in normal controls and Type 1 patients compared to TΛ pe 2 (see Table 2 K Adiponectin decreased and then increased with HbAIc All differences are small and not very significant with T-\ alues below 1.4 (Probability of <-W% significance) Adiponectin differences w ere not predictive of BMl {body mass index).

[0171 j Use of a ratio of HMW Adφonecti a' total impren es differences in populations and maintains the same trends seen with adiponectin The ratio increase with disease. HMW.' total adiponectin decreases with high HbAI c (See Table 3). Again the.

pe t diagnosis h less correlated with higher HMW /^'total adiponectin than a Type 2 diagnosis However Type 1 diabetes diagnosis ga\ e a higher ratio than normal controls.

[0172{ The total levels of soluble C terminal AdipoRl increases with diabetes pathology (e g. type 2 diabetes or insulin resistance) These differences are much more significant than for adiponectin or the HMW ratio (T \ aiue > 3 tf. Probability of >99 9% significance) (See Table 4). Surprising!}'. AdipoRl increases with Type I diabetes indicating receptor is also related to Type 1 These patients w ouid also be expected to suffer from adipocyte in balance, but also ha\ e beta cell loss AdipoRl increases more with higher HbAI c that adiponeciin CH era! I. AdipoRl h more sensith e than adiponectin and the I LMW ratio. The combination of AdipoRl and adiponectin in a mathematical relationship was better than adiponectin alone at predicting diabetes pathology The combination of AdipoRl and c-peptide in a mathematical relationship w as also was better than c-peptide alone at predicting diabetes pathology.

Example 2 - Detection of Oterminal fragments in individuals having metabolic syndromes and other cardiovascular and coronary disorders

[0173] Another group of 188 patients were fully characterized for cardiovascular conditions and risk by various diagnostic test and angiography Normals ( n^::::l 13) were considered those without metabolic syndrome, diabetes, acute coronary¹ syndrome (ACS), AMI or CHF. Patients out of IHH group w ere placed into affected groups for metabolic syndrome. inflammatory markers, ACS. AMI and CHF, hypertension, obesity, lipidemia, inflammatory response and ami-inflammatør> response. Acute coronary syndrome was defined as blockage "^■60*! o by angiograph ev aluation vviih or without acute cardiac condition. Metabolic syndrome was defined by insulin resistant or more than two metabolic risk factors by WHO definition. insulin resistant was accessed by diagnosis and diabetic medication. Metabolic risk factors include hypertension, lipidemia and obesity. Obesity was assessed b> bod\ mass index (BMI). Hypertension was assessed by blood pressure or medication. Lipidemia was assessed by iipid ratio or Iipid lowering medication Inflammation v\a& access by while blood ceil count or CRP. Antiinflammatory status was access by immunoassay for urinary trypsin inhibitors m blood and urme (Bikumn and Urislatm immunoassay measurements K All patients were additional assessed by medical history and medication ami characterized into affected groups accordingly.

[0174{ Adiponectin, and HMW Adiponeetin were measured using commercial FUSA kit. Cardiac markers were measures using the Centaur instrument (Bayer). The AdipoRl ELlSA assay described in Example 1 was used to measure all C- terminal fragments whether bound or unbound

[0175) Adiponectin decreases with ASC and metabolic syndrome but the significance of the \ ai ties w ere less than expected for ⁽⁾⁽⁾. V% certain < See Table 5 K Adiponectsn was i ncreased with CIiF and Ml which would interferes with {he assessment Adiponectin was also not \ ery correlated with inflammatory status

[0176[ The total levels of soluble AdipoR l in serum increases with ASC and metabolic s> ndrome and the significance of the \ a! ties were highh significant ( ^<■>^<■> 9°-o certain) and much more significant than that observ ed for adiponectin (See Table 6). Surprising AdipoRl increases as conditions become more acute and as the inflammatory and anti-inflammatory response increase. AdipoRl further predicted for metabolic syndrome Soluble AdipoRl was also found in urme and plasma to correlate with metabolic syndrome.

(I includes patients with AMI and CI lF

(2) Significance is 99.9% prob or 0.0! two tail when T value is greater than >3.8

includes AMI and CIiF>2.4 is approximately 99% prob or 0.0i two tail. >3.8 is approximate!}' 99.9% prob or 0.001 two tail Example 3 — Elucidating biochemical pathway

[01??| ^"Normal insulin sensitivity results when insulin causes fat cell to produce adiponectin Full length adiponectin aggregates into muUimers, ts picaJls culled LMW, MMW and HMW forms Adiponelin interacts with the adiponectiti receptor 2 in {he li\ er and adiponeclm receptor i in the muscle to stop glucose production and cause glycolysis and fatt\ acid oxidation. The adiponectin receptor 1 reacts with a cleaved form of adiponectin called globular adiponeclm where as adiponeclm receptor 2 reacts Io full length adiponectin Globular adiponectin was recently shown by others to form by action of blood elastase.

[0178] Insulin resistance occurs when adipocytes become h\ pertropic and produce less adiponectin in response to insulin. In this state, the cells become more apoptotic and cell di\ ision slows As a result plasma adiponectin le\ els decreases Insulin le\ els rise in an effort to cause cells to release more aώponectm How e\ er as the msuhn resistance w orsens more insulin and less adiponectin is produced. The lesser adiponeclin results in less glycolysis and fatty acid oxidation in muscle and prex ents Ih er glucose production from slopping

J0 I79J It was confirmed that inflammatory elastase and white blood cells are significant!;_* derated in diabetes patients. A renew of the literature agreed thai that both man made insulin and natural msuhn increase while blood ceils in diabetics As eiastase increases in inflammation, a higher percentage of globular adiponeclin is produced. The lack of muliiroers causes less action on the liver It was confirmed that anti-inflammatory protease inhibitors (Uri and Bik) were significant!} elev ated in diabetes. These inhi bitors are formed b> elastase and were recently shown in our cell models to induce by pertropic apopfosis in normal cell lines.

[0 JSOj Hie receptor fragments after elastase exposure was proposed as mechanism for the formation of the soluble fragment in patient samples. This was tested iissng affinity mass spectroscopy with a polyclonal antibody for the AdipoRl C terminal and patient samples

[01811 The AdipoRl \x as confirmed bs mass spectra to be in blood v\ ith a mass of 34, 28-2^<Λ I¹M S. I 5-1.V>.5-9.O. 7 ^<-U> <\6 5,5 2. 4 0 to 3 H and i to 2 kDa This data confirmed that 1) AdipoRi fragments, were found in patients and controls 2} AdipoRl fragments form dimmers and 3 } AdipoRl fragments were bound to adiponectin. The last was proven by repeating the affinity mass spectroscopy with a polyclonal aniibods for the adiponectin

[0 J82{ The lack of mυitimer adiponectin during insulin resistance was further proposed to the potential cause of differences in fragmentation patterns between patients and normals. Indeed a disappearance of the 3.9 and 7.8 masses forms occurred in diabetics but were present in all normals to the data below, all 5 patient lacked these masses and all 5 normal had the 3901 and 78! 4 Da masses (see graph data). Therefore, different masses thought to be due to exposure of proteolytic cleave sites when there is availability of mui timers for binding.

Example 4 - Preparation of monoclonal antibodies

[0183 j BALB/c mice were immunized with 100 μg/mouse of synthetic AdipoR 1 ^■peptide immuoogen composition. After one month, ocular bleeds were taken from each mouse and titered by EUSA against the immunogen to assess the immune response. The mice shoving the best response were boosted by injection of K)O μg/mouse with the immunogen. After four days, mice were sacrificed and their spleens used for fusion according to the method of Kohl er and Milstein, Nature 256:495 (1975). The spleenoeytes were fused with SP2-0 Ag 14 myeloma cells using PEG (polyethylene gl> col) solution with a ratio of spleenocyles to .Myeloma cells of 5: 1 aid plated into % well plates using 50% PEG/HAT growth media. After 7- 10 days of incubation at 37 degrees Celsius, fusion cultures were monitored for growth by feeding every 3-4 days utilizing the HAT (hypoxanthine. amrnopterin, thymidine) selection method followed by suhcuHuring with HAT growth media.

(O184| After 2-3 weeks, the wells having hybridoma colony growth were tested by ELISA to determine which growths produced an antibody immune response to the peptide. The 96 well plate cultures were tested with the uristatin peptide at 1 ug/mL coated plates. After coating plates overnight at 2-8^'''C, all plates were washed and blocked. Cell culture supernatants were then applied 1 OOμl/weli for one hour at room temperature. After washing plates. Goat anti- mouse IgG Horse Radish Peroxidase at 1 :2000 dilution was applied at IQQuLΛveil for one hour. Plates were washed once again followed by OPD (o-phenylene diamine dihydrochioride) substrate and read at 490nm on a Spectra Max plate reader,

[018Sj The colonies giving a positive response were transferred to 24 well plates for further expansion and retesling to verify the positive results The colonies testing positive were further expanded in six well plates in iscove's Modified Duibεcco's Medium (IMDM) with 10% Fetal Bovine Serum (FBS). After expansion, the colonies were frozen at - 70⁰C and then transferred to liquid nitrogen for long-term storage. Based on ELISA results using the purified peptide, various clones were further expanded in IMDM. 1.0% FBS and frozen down.

Example 5 - Characterization of Monoclonal Antibodies with SELDl |0186] A method of measuring the specific adipoR! fragments in patient samples was done using monoclonal antibodies and rabbis polyclonal antibodies were tested with soluble AdipoR standards and patients' plasmas on chip surfaces. The binding was estimated by Surface-Enhanced Laser Desorptioπ/Ionization (SELDI) analysis on a SELDl PBS Il time of flight mass spectrometer (Ciphergen, Fremont, California) to determine the mass to charge ratios Cm//) for the proteins binding to the antibodies. Ten plasma specimens from patients were tested further: fivs patients were positive for diabetes: five patients were negative for diabetes. Binding was measured on two types of surfaces (PS2U and RS100) using a standard incubation procedure The signal for each .mass measurement was compared to the background .noise to obtain the signal to noise ratios (S/N). Only masses with S/N ratios greater than 10 were accepted.

|0187 f The SELDf procedure was as follows' Three microliter of 50 mmol/L NaHCO_;; CpH 8.0} was added to each spot on the protein chip and covered with a plate (i.e. a bioprocessor) to form sample wells followed by the addition of 1 μL antibody (i nig/iriL) to each spot and. incubated at room temperature for 2 hours with shaking in a controlled-humidity chamber. The solution from each spot at that time was washed twice with 5μL of washing buffer (phosphate buffered saline (PBS) + 0,5% Triton detergent^"). The unbound sites were blocked with 5μL of either 2 rag/roL BSA (bovine serum albumin) or 1 mol/L ethanolanune. After incubation at room temperature the BSA or eihanolamine was discarded and the spots were washed twice with 5μL of washing buffer (PBS -'- 0.5% Triton). Five μL of PBS was added to each spot and the chips were placed into the bioprocessor. An additional S OμL PBS as well as 10 μL of the sample to be tested (or PBS as a control) were added to each well, followed by shaking the sealed wells at 4⁰C for 18 hours. The wells were then washed with washing buffer and PBS and again shaken at room temperature for 2 min. The wells were rinsed twice with 300μL of deionized water saturated with sinapimc acid: this serves as an energy-absorbing molecule during protonalion of proteins bound to the antibodies. The latter are attached to the surface of the chips. The chips containing the antibody-bound specimens were analyzed for bi.nd.ing .mass using the SELDI mass spectrometer according to the manufacturer's instructions.

Example 6 - Soluble C Terminal Fragments in Diabetics

[01 SSj Table 6 shows the results of multiple determinations for five normal patients for the detection of an adiponectin receptor fragment mass of 7812 and five diabetics not having the same mass. A similar separation was found for a mass of 3901. These two masses are present in normal subjects but absent or present in very Sow levels, i.e., decreased levels in subjects having the disease conditions provided herein.

j01S9| Table 7 shows the results of multiple determinations for five normal and diabetic patients for the detection of adiporteclin receptor fragments having masses of 4 5-6.9, 7-8.2. 9- I L 13-15, 1 7- 19, 27-29, or 30-34 and live diabetics not having the same masses. .

Example 7 - Serine proteases

(0I90J Trypsin family serine proteases are increased during inflammation and include trypsin, chymotrypsirt, kalϋkrein. piasmirt, complement D. thrombin, and Factors IX a, Xa, XIa and XlIa. All have trv piase primary affinity cleaving Arg-Xaa or Ly s-Xaa. Additional rry psin family serine proteases released by immune cells include elastase, granzyme (A, B, H, M). tryptase 2 and mast celi proteases I . The key elastase homologies including cathepsin G. proteinase 3. azurocidin and mycolobastin have Val-Xaa > Ala-Xaa cleaving affinity , Granzymes A and K have tπ ptase cleaving affinity. Gran/yme B has aspase cleaving affinity for Asp-Xaa. Granzyme M has metase cleaving affinity for Met-Xaa or Leu-Xaa Giarm me H and Mast cell protease 1 have ehymase cleaving affinity for cleaving Phe-Xaa, Tyr-Xaa. or Trp- Xaa. [0191 ] Anal ysi s of fragmen tali on patterns for adi ponectin and adi poneeti π receptor fragments were determined using trypsin and elastase as example inflammatory proteases. The fragments {hat are 29 to 34 ammo acid in length {i.e.. SEQ ID NOS: K 2, 4-1 L 16, 17, aid/or 19- 26 ) were predicted by eiastase cleavage. The fragments thai are 20 to 25 amino acids in length (i.e., SEQ ID NOS; 3. 12-15, 18, and/or 27- 30) were predicted by general trypsin family serine proteases or gran zy me.

Example 8 - Setting sensitivity and specificity

[01.92| The samples are divided between normal and abnormal. Results are collected for all and the observed adipoRl values are judged against an assigned adipoRJ threshold. Hie threshold is the value below which all results are considered normal and above which results are considered positive. The threshold is varied from a low number to a high number and the predictive value of the result is calculated, using the .number of true positives, false positive, true negativ es and false negatives found. The sensitivity (TP/TP-t-FP) and specificity (TN/TN÷FN) are calculated for each threshold tested. The threshold with the highest sensitivity and specificity- gives the best predictive value, (100% would be idea!).

Example & ~ Adiponectin receptor fragment thresholds

|0193J The following is an example of setting threshold using the patients and methods shown in example 1. ϊn the table below, the threshold above which AdipoRl result was considered positive is varied from 15 to 21 ug/mL, In this example, a higher value is considered a positive. The number of true negatives, or correctly identified patients without diagnosed diabetes, is calculated along with the number of false positives, false negatives and true positive. Ideally, an assay would have no false positives or 100% specificity and no false negatives or 100% sensitivity. As can be seen from the data, the threshold of 15 was better for sensitivity while the threshold of 21 was better for specificity. The threshold and range are dependent on the fragment detected aid analytical method used. In this example the total assay range was 5 to 30 ug/mL or approximately 6X. Accordingly, the concentration units and range varied with the fragment detected and analytical method used (SELDl vs ELISA). For example, for the fragment tested in Example 6 and Table 7, the difference between normals and diabetic was often KiOX. As expected, the concentration for one specific fragment was less than the concentration of all fragments. The type of sample used, whether urine, plasma or serum also impacted the concentrations of fragment. Urine and serum had fragment concentration about 10 fold lower than plasma. Once an assay and fragment is selected, lhe thresholds are adjusted Io best achieve {he clinical agreement desired, using the methods shown.

Example (0 ~ Use of psmeJs for adipocyte imbalance assays

[0194] The following is an example of using additional and related biomarkers with the AdipoRI result to improve the prediction of diabetic disorder. The diabetic and normal patients and methods shown in example J are used. In the table below, three analyte are compared for their ability to detect diabetes. The thresholds used offered comparable TN. As expected, ADIPORi detected more true positives than the other anaiyes.

[0195| Twenty fι\z of the sixty -nine diabetics were Type 1. The anatytes were also compared for the ability to be positive for type 1 diabetes. Only 4 of the 25 type 1 diabetics had an abnormally low adiponectin C-peptide uses two thresholds, one for abnormally low and one for abnormally high levels, Ai abnormally low c-peptide. indicates a lack of insulin and as expected 23 of the 25 type 1 diabetic had abnormally low c-pepude. Type 1 diabetics also often had AdipoRI fragments. Few tv pe 1 diabetics had abnormally high c-peptide. [0196] These analy tes detect di fferent patients. This is likely explained by differences in the pathology, as the each analyle measures a different pan of the imbalance. For example, il is believed thai the lack of insuim impacting c-peptide is due to the islet cells whereas the lack of adiponectin is due to the adipocytes failing to produce the hormone. The presence of adipoR 1 fragments in this example is believed to be due to muscle ceil shedding the receptor from over use.

J01971 This data demonstrates that the combination of the analytes together could be better than any one alone. Tn the table below, the simplest of relationships is tested by considering any one of the panel positive to mean that the result is ab.nor.mal. Accordingly ail analy tes must be negative to be considered a normal result. As described above, thresholds are adjusted to achieve the best results.

[0198{ The highest number of true positives was obtained for the combined use of c- peptide, adiponectin and adipoR 1. The highest number of true negatives was obtained for the combined use of c-pepude and adipoR 1 or by the use c-peptide. adiponectiti and adφoRΪ . In both cases, the numbers of true negatives were comparable to the adipoR 1.

Example H - Soluble Adiponectin Receptor I Levels In Plasma of CAD Patents

J01991 The following is an example of using additional and related biomarkers with the AdipoR i result to improve the prediction of cardiovascular disorder. The cardiovascular disorder and normal patients and methods shown in example 2 are used. Affect patients were those with ACS by angiograph or high risk by meeting the definition of metabolic syndrome. Patients with pre-existing cardiovascular conditions such AMI and CHF were excluded as a diagnosis would already be made by the TaI or BNP assays or other diagnostic assessments.

[02001 Adiponectin receptor 1 soluble C terminal fragments were measured by ELISA as shown in Example 1 , The results correlated well with degrees of vascular blockage (Table 1 1 } Rtsk of cardiovascular disorders was also assessed by additional marker for pro and anti- inflammatory response and adiponectin. Ajπalyfes for pro and aπti -inflammatory response were compared to the adiporl . Abnormal AdipoR results were more likely present in patients with vascular blockage than adiponectin, uristatin. bikunin, WBC or CRP. The higher sensitivity supports a diagnostic correlation of adiponectin receptor 1 for vascular blockage due to atherosclerosis

[02011 The use of panels of pro and anti-inflammatory response and adipocyte markers were compared for there ability to detect cardiovascular disorders. The highest number of true positive was obtained for the combined use of Bikunin. uristatin, CRP, WBC, adiponectm and adipoRi. The highest number of true negative was obtained for the combined use of adiponectin and adipoR i.

|02021 Each recited range includes all combinations and sub-combmahons of ranges, as well as specific numerals contained ^"therein,

[02031 All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[0204J Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in Sight of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is Claimed'

1. A method for detecting fragmentation of an adiponectin receptor in a subject comprising the step of assaying for the presence or absence of at least one soluble C- terminal fragment of the adiponeetin receptor in a biological fluid sample obtained from the subject,

2. The method of claim 1 wherein the at least one C -terminal .fragment is bound to carrier protein.

3. The method of claim 2 wherein the carrier protein is adiponectin.

4. The method of claim 1 wherein the at least one C -terminal fragment of the adiponectin receptor is detected by an antibody

5. The method of claim 2 wherein the at least one C-terminai fragment bound to carrier protein is detected by an antibody.

6. The method of claim J wherein the step of assaying for the presence of at least one C- terminal fragment of the adiponectin receptor in the biological fluid comprises contacting the sample with a binding agent specific for the at least one C-termina! fragment of the adiponectin receptor.

7. The method of claim 6 wherein the binding agent comprises an antibody.

8. The method of claim 7 wherein the antibody is labeled with a reporter molecule,

9. The method of claim 6 w herein the step of assaying for the presence of at least one C- terniina! fragment of the adiponectin receptor in the biological fluid further comprises contacting the sample with a second binding agent specific for the first binding agent.

10. The method of claim 9 wherein the second binding a«ent is an antibody

1 1. The method of claim 10 wherein the second binding agent is labeled with a reporter molecule

- 62 -

12. The method of claim 2 wherein the step of assaying for the presence of at least one C- terminal fragment of the adiponectin receptor in the biological fluid comprises contacting the sample with a binding agent specific for the carrier protein bound to the at least one adiponectin receptor fragment and the method further comprises the step of determining whether the carrier protein is hound to the at least one adiponeclin receptor fragment.

13. The method of claim J wherein the bodily fluid is blood plasma or whole blood.

14. The method of claim 1 wherein the bodily fluid is urine

15. A method of detecting the level of expression of an adiporteclin receptor in a subject comprising the step of determining the level of at least one C-terminal fragment of the adiponectin receptor in a biological fluid sample and correlating the level of the at least one C-termmal fragment with the level of expression of the adiponectin receptor.

16. A method of detecting the level of expression of adiponectin in a subject comprising the step of determining the level of at least one C-terminal fragment of the adiponectin receptor in a biological fluid sample and correlating the level of the at least one C-terminai fragment with the level of expression of adiponectin.

1 7. A method for determining progression of a condition, onset of a condition, or efficacy of treatment of a condition characterized by an adipocyte imbalance in a subject comprising determining the level of at least one soluble C-terminal fragment of the adiponectin receptor present m a bodily fluid sample obtained from the subject and correlating the level with progression of the condition, onset of the condition, or efficacy of treatment of the condition.

18. The method of claim 17 wherein the at least one soluble C-terminal fragment is bound to carrier protein.

19. The method of claim 17 wherein the at least one soluble C -terminal fragment is unbound.

- 63 -

20. The method of claim 17 further comprising determining the level of adiponectin in a biological sample obtained from the subject and correlating the level of adiponectin with progression of the condition, onset of the condition, or efficacy of treatment of the condition.

21. The method of claim 17 further comprising determining the level of bikunin in a biological sample obtained from the subject and correlating the level of bikunin with progression of the condition, onset of the condition, or efficacy of treatment of the condition.

22. The method of claim 17 further comprising determining the level of C-reactive protein in a biological sample obtained from the subject and correlating the level of C~reactive protein with progression of the condition, onset of the condition, or efficacy of treatment of the condition.

23. The method of claim 17 further comprising determining the level of white blood cells in a biological sample obtained from the subject and correlating the level of white blood cells with progression of the condition, onset of the condition, or efficacy of treatment of the condition.

24. The method of claim 17 further comprising determining the level of c-peptide in a biological sample obtained from the subject and correlating the level of c-peptide with progression of the condition, onset of the condition, or efficacy of treatment of the condition.

25. The method of claim 17 wherein the method is for determining onset of the condition characterized by an adipocyte imbalance.

26. The method of claim 17 wherein the method is for determining progression of the condition characterized by an adipocyte imbalance.

27. The method of claim 17 wherein the method is for determining efficacy of treatment of the condition characterized by an adipocyte imbalance.

- 64 -

28. The method of claim 27 wherein the treatment is administration of a PPAR gamma agonist.

29, The method of claim 17 wherein the condition is metabolic syndrome.

30, The .method of claim 1.7 wherein the condition is vascular blockage.

31. The method of claim 17 wherein the condition is diabetes type 1 or diabetes type II.

33. The method of claim 17 wherein the condition is cardiovascular disease.

34. The method of claim 33 wherein the cardiovascular disease is congestive heart failure, acute myocardial .infarction, coronary artery disease, atherosclerosis, or ischemia.

35. The method of claim 17 wherein the condition is insulin resistance.

36. A method for determining whether a. subject having arteriosclerosis is likely to develop cardiovascular disease comprising determining ύw level of at least one soluble C -terminal fragment of lhe adiponeciin receptor present in a biological fluid sample obtained from the subject and correlating the level with the likelihood of development of cardiovascular disease.

37. The method of claim 36 wherein the cardiovascular disease is congestive heart failure, acute myocardial infarction, or ischemia.

- 65 -