CA2156935C - Methods of detecting collagen degradation in vivo - Google Patents

Abstract

Methods of determining collagen degradation in vivo, by quantitating the concentration of a peptide in a body fluid, the peptide being a C-terminal type II
collagen telopeptibe containing a hydroxylysyl pyridinoline cross-link or a type III
collagen telopeptide containing a hydroxylysyl pyridinoline crops-link.
Suitable methods include immunometric assay, fluorometric assays, and electrochemical titrations for quantitation. The structures of specific peptides having cross-links and kits for quantitating these peptides in a body fluid are described.

Description

METHODS OF DETECTING COLLAGEN DEGRADATION IN VIVO
This invention was made with U. S. government support under grants AR37318 and AR36794 awarded by the National Institutes of Health. The U. S. government has certain rights in the invention. This application is a divisional application of Serial No. 2,031,265 filed on November 30th, 1990.
The present invention relates to methods for detecting and monitoring collagen degradation in vivo. More specifically, it relates to methods for quantitating cross-linked telopeptides produced in vivo upon degradation of collagen types II and III.
Three known classes of collagens have been described to date. The class I collagens, subdivided into types I, II, III, V, and XI, are known to form fibrils. These collagens are all synthesized as procollagen molecules, made up of N-terminal and C-terminal propeptides, which are attached to the core collagen molecule. After removal of the propeptides, which occurs naturally in vivo during collagen synthesis, the remaining core of the collagen molecule consists largely of a triple-helical domain having terminal telopeptide sequences which are nontriple-helical. These telopeptide sequences have an important function as sites of intermolecular cross-linking of collagen fibrils extracellularly.
The present invention relates to methods of detecting collagen degradation based on assaying for particular cross-linked telopeptides produced in vivo upon collagen degradation.

In the past, assays have been developed for monitoring degradation of collagen in vivo by measuring various biochemical markers, some of which have been degradation products of collagen. For example, bone turnover associated with Paget's disease has been monitored by measuring small peptides - la -containing hydroxyproline, which ere excreted in the urine following degradation of bone collagen. Russell et al., Metab. Eons Dis. and Ret. Rsa. 4 and 5, 255-(1981); and Singer, F.R., et al., Metabolic Hone Disease, Vol. I1 (ads.
Avioli, L.Y.
and Kane, S.M.), 489-678 (1978), Academic Press, New York.
Other researchers have measured the crosa-iinking compound pyrid)noline in urine sa an index of collagen degradation in joint disease. Bee, for background and for example, Wu and Eyre, Btochemi'try, Z3e1850 (1984); 8laek et al., Annals of the Rheumatic Diseases, 48:841-844 (18891; Robins et al.; Annals off' the Rheumatic Diseases, 45:969-993 (198B); and Seibel et al., The Journal of Rheumatology, 16:984 (1989). In contrast to the present invention, some prior researohers have hydrolyzed peptides from body fluids and then looked for the presence of individual hydroxypyridlnlum residues. None of these researchers have reported measuring a teiopeptfde containing a cross-link that is naturally produced tn vivo upon oollegen degradation, as in the present invention.
U.K. Patent application GB 2,206,843 reports that the degradation of type III
collagen in the body is quantitatively determined by measuring the coneentratlon of an N-terminal telopoptide from type Ill collagen in a body fluid. Irt this reference, It is reported that cross-linked teIopeptide regions are not desirable. In fact, this reference reports that it is necessary to use a non-cross-linked source of ZO collagen to obtain the telopeptlde. The peptides of the present invention are all cross-linked. Collagen cross-links are discussed In greater detail below, under the heading "Collagen Cross-Linking."
There are a number of reports indicating that collagen degradation can be measured by quantitating certain procollagen peptides. The present invention involves telopeptides rather than propeptides, the two being distinguished by their location in the collagen molecule and the timing of their oleavage in vivo.
See U.S. Patent 4,504,587; U.S. Patent 4,312,853; Pterard et al., Analytical Biochemistry 141:129-I3B (1984); Niemela, Clin. Chem., 31/8:1301-1304 (1985);
and Rohde et al., European Journal of Clinical Inveattgation, 9:451-459 (I979).
U.9. Patent 4,7T8,T88 relates to a method of determining changes occurring in artieular cartilage involving quantifying proteoglycan monomer or antigenic fragments thereof in a synovial tluld sample. This patent does not relate to deteoting cross-linked telopeptides derived from degraded collagen.
Dodge, J. C(in. Invest., 83:847-881 (1981) discloses methods for analyzing type II collagen degradation utilizing a polyclonal antiserum that specifically reacts with unwound alpha-chains and cyanagen bromide-derived peptides of human and bovine type II eollagens. The peptides involved are not cross-linked telopeptides as in the present invention.

Amino acid sequences of human type III collagen, human proal(IT) collagen, and the entire preproal(III) chain of human type III collagen and corresponding cDNA clones have been investigated and determined by several groups of researchers. See Loidl et al., Nucleic Acids Research 12:938.3-9394 (1984); Sangiorgi et al., Nucleic Acids Research, 1.3:2207-2225 (1985); Baldwin et al., Bioclrem. J., 262:521-528 (1989); and Ala-Kokko et al., Biochem. J., 260:509-516 (1989).
None of these references specifies the structures of particular telopeptide degradation products that could be measured to determine the amount of degraded fibrillar collagen in vivo.
In spite of the above-described background information, there remains a need for effective and simple assays for determining collagen degradation in vivo. Such assays could be used to detect and monitor disease states in humans, such as osteoarth ritis (type II collagen degradation), and various inflammatory disorders, such as vasculitis syndrome (type III collagen degradation).
2Q Assays for type I collagen degradation can be utilized to detect and assess bone resorption in vivo.
Detection of bone resorption may be a factor of interest in monitoring and detecting diseases such as osteoporosis.
Osteoporosis is the most common bone disease in man. Primary osteoporosis, with increased susceptibility to fractures, results from a progressive net loss of skeletal bone mass. It is estimated to affect 15-20 million individuals in the United States. Its basis is an age-dependent imbalance in bone remodeling, i.e., lI1 the rates of synthesis and degradation of tooue tissue.
About 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured periptreral bones. Twelve to 200 of the hip fractures are fatal because they cause severe trauma and bleeding, and half of tt~e surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $7 billion annually (Barnes, O.M., Science, 236:914 (1987)).
Osteoporosis is most common in postmenopausal women who, on average, lose 15°s of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, no method in available for measuring bone resorption rates in patients or normal human subjects. A
2.0 major difficulty in monitoring the disease is the lack of a specific assay for measuring bone resorption rates.
- 3a -Methods for assessing bone mass often rely on measuring whole-body calcium by neutron activation analysis or mineral mass in a given bone by photon absorption techniques. These measurements can give only long-term impressions of whether bone mass is decreasing. Measuring calcium balances by comparing intake wtth output is tedious, unreliable and can only indirectly appraise whether bone mineral is being lost over the long term. Other methods currently available for assessing decreased bone mass and altered bone metabolism include quantitative scanning radiometry at selected bone looations (wrist, calcaneus, etc.) and histomorphometry of Iliac crest biopsies. The former provides a crude measure of the bone mineral content at a specific site in a single bone.
Hlstomorphometry gives a semi-quantitative assessment of the balance between newly deposited bone seams and resorbing surfaces.
A urinary assay for the whole-body output of degrsded bone in 24 hours would be much more useful. Mineral studies (e.g., calcium balance) cannot do this ; 5 reliably or easily. Since bone resorptlon involves degradation of the mineral and the organic matrix, a specific biochemical marker for newly degraded bone products in body fluids would be the (deal Index. Several potential organic irldiQes have been tested. For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein In bone and all other connective 2p tissues, is excreted 1n urine. its excretion rate is known to be increased in certain conditions, notably Paget's disease, a metabolic bone disocder in which bone turnover is greatly increased, es pointed out above. For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation. Singer, F.R., et al. (1978), cited hereinabove.
25 U.S. Patent No. 3,600,132 discloses a prncess foe determination of hydroxyproline in body fluids such as serum, urine, Lumbar fluid and other intercellular fluids in order to monitor deviations in collagen metabolism. (n particular, this inventor notes that in pathologic conditions such as Pager's disease, Marfan'a syndrome, osteogenesis imperfecta, neoplastic growth in 30 collagen tissues and in various forms of dwarfism, increased collagen anabolism or catabolism ae m~aaurvd by hydroxyproline content in biological fluids can be determined. This inventor measures hydroxyproline by oxidizing it to a pyrrole compound with hydrogen peroxide end N-ehloro-p-toluenesulphonamide followed by colorimetric determination in p-dlmethyl-amino-benzaldehyde.
35 In the case of Paget's disease, the increased urinary hydroxyprvline probably comes largely from bone degradation; hydroxyproline, however, generally cannot be used as a specific index. Much of the hydroxyproltnc In urine may come from new collagen synthesis (considerable amounts of the newly made protein are degraded and excreted without ever becoming incorporated into tissue fabric), and from turnover of certain blood proteins as well as other proteins that contain hydroxyproline. Furthermore, about 80Q6 of the free hydroxyproline derived from protein degradation la metabolized in the liver and never appears in the urine Klviriko, K.I. Int. Rsv. Connect. ?tssua Res. 5s93 (1970), and Welss, P.H. and Klein, L., J. Clin. lnvaat. 48t 1 (1969).
Hydroxylyaine and its glycoside derivatives, both peculiar to collagenous proteins, have been aonaidered to be more accurate than hydroxypfoline as markers of collagen degradation. However, for the same reasons described above for hydsoxyproline, hydroxylysine and !ts glycosides are probably equally non-specific markers of bone resorption. Krane, S.M. and Simon, L.S. Develop.
Btochsm., 22:185 (1981).
In addition to amino acids unique to collagen, various non-eollagenous 1 S proteins of bone matrix such as osteocaleln, or theft breakdown products, have formsd the basis of immunoassays aimed at measuring bone metabolism. Price, P.A. et nl. J. Clip. Invsst., 68s 878 (1980), and Gundberg, C.M. et al., Meth.
Enzymol., 10T:518 (198!). However, it Is now clear that Donrderived non collagenous prote(ns, though potentially a useful index o! bone metabolic activity are unlikely, on their own, to provide quantitative measures of bone resorption.
The concentration In serum of osteocalein, for example, fluctuates quite widely both normally and in metabolic bone disease. its concentration is elevated in states of high skeletal turnover but ft 1s unclear whether this results from increased synthesis or degradation of bone. Krane, S.M., et al., Develop.
Biochsm., 2Zs185 (1981), Prlee, P.A. et al., J. Clip. invest., 66e878 (1980);
and C3undberg, C.M. et al., Moth Enrymol., 107:516 (1984).
Collagen Croae-Linking The polymers of most genetic types of vertebrate oollagen require the formation of aldehyde-mediated cross-links for normal function. Collagen alde 3~ hydes are derived from a few specific lysine or hydroxylysine side-chains by the action of lyayl oxidnse. Various dl-, tri- and t~trafunetionel cross-linking amino acids are formed by the spontaneous intra- and intermolecular reactions of these aldehydes within the newly formed collagen polymers; the type of cross-linklng residue varies specifically with tissue type (see Eyre, D.R. et al., Ann. Rev.
Btochsm., 5:717-748 (1984)).
Two basic pathways of cross-linking can be differentiated for the banded ($?nm repeat) fibrillar collagens, one based on lysine aldehydes, the other on 215693a -s-hydroxylysine aldehydes. The lysine aldehyde pathway dominates in adult skin, cornea, sclera, and rat tall tendon and also frequently occurs in other soft connective tissues. The hydroxylysine aldehyde pathway dominates in bone, cartilage, ligement, most tendons and most internal eonnectivt tissues of the body, Eyre, D.R. et al. (1974) vide supra. The operating pathway is governed by whether lysine residues are hydroxylated fn the telopeptide sites where aldehyde residues will later be formed by lysyl oxidase (Barnes, M.J. et al., Biochem.
J., 139t4fi1 (1974)).
The chemical structures) of the mature cross-linking amino acids on the t0 lysine aldehyde pathway are unknown, but hydroxypyridinium residues have been identified as mature products on the hydroxylysine aldehyde route. On both pathways and in most tissues the intermediate, borohydride-reducible crass-linking residues disappear as the newly formed collagen matures, suggesting that they ace relatively short-lived intermediates (Bailey, A.J. et al., FEBS Lett., 16s8s (1971)). Exceptions are bone and dentin, where the reducible residues persist in appreciable concentration throughout life, In part apparently because the rapid mineralization of the newsy made collagen fibrils inhibits further spontaneous cross-linking interactions (Eyre, D.R., In: The Chemistry and Biology of ldineralized Connective Tissues, (Veil, A. ed.) pp. 51-65 (1981), Elsevier, New York, and Waiters, C. et al., Calc. Tip. IntL, 35:401-405 (1983)).
Two chemical forms of 3-hydroxypyridinium cross-link have been identified (Formula 1 and II). Both compounds are naturally fluoresetnt, with the same characteristic excitation and emission spectra (Fujimoto, D. et al. Biochem.
Biophys. Ras. Common., 78:1124 (1977), and Eyre, D.R., Develop. Biochem., 22:50 1981)). These amino acids can be resoived and assayed directly in tissue hydrolysates with good sensitivity using reverse phase HPLC and fluorescence detection. Eyre, D.R. et al., Analyta. Bioctt8m., 137:380-388 (1984). It should be noted that the present Invention involves quantitating particular peptides rather then amino acids.

215~~35 -7_ f~X
CXp-CB-XXZ g2~,~ CNI-CA-NHZ
_ CA-CXt CAZ Oil Cfl--CNZ-wCH2 Ol( AOOC ~ ~ f100C ~
+~ I+~
x i~r A
I=
o ix~ iX
(xz I xz CH Cll / \ / \
l~ N COOK A? N C00g In growing anima), it has been reported that these mature cross-links may be concentrated more in an unmineralized fraotfon of bone collagen than in the mineralized collagen (Bsnes, A.J., et al., Biochem. Biophys. Rea. Commun., 20 113s1975 (1983). However, other studies on young bovine or adult human bone do not support this concept, Eyre, D.R., Ini The Chemistry and Biology of Mineralized Tissues (Butler, W.T. ed.) p. 105 (1985), l:bsco Media Inc., Htrmingham, Alabama.
The presanee of oollagen hydroxypyrldinfum cross-links in human urine was lirst reported by Gunja-Smith and Boucek (Gunja-Smith, Z. and Boucek, R.J., 25 Btocherrr d., 197s759-782 (1981)) using lengthy isolation procedures for peptides and conventtonal amino acid analysis. At that time, they were aware only of the HP form of the erona-link. Robins (Robins, S.P., Biochem J., 209:619-620 (19821 has reported an enzyme-linked,mmunoassey to measure HP in urine, having raised polyelonal antibodies to the free amino aold conjugated to bovine serum nlbumin.
30 This assay is Intended to provide an index for monitoring increased Joint destruc-tion that occurs with arthritis diseases and is based, according to Robins, on the finding that pyridinoline is mueh,more prevalent !n cartilage than in bone colla-gen.

In more recent work involving enzyme-linked immunoassay, Robins reports that lysyl pyridinoline is unreactive toward antiserum to pyridinoline covalently linked to bovine serum albumin (Robins et al., Ann Rheum. Diseases, 45:969-973 (1986)). Robins' urinary index or cartilage destruction is based on the discovery that hydroxylysyl pyridinoline, derived primarily from cartilage, is found in urine at concentrations proportional to the rate of joint cartilage resorption (i.e., degradation). In principle, this finder. could bP_ USP_d to measure whole body cartilage loss;
however, no information on bone resorption would be available.
A need therefore exists for a method that allows the measurement of whole-body bone resorption rates in humans.
The most useful such method would be one that could be applied to body fluids, esper_ially urine. The method should be sensitive, i.e., quantifiable down to 1 picomole and rapidly measure 24-hour bone resorption rates so that the progress of various therapies (e. g., estrogen) can be assessed.
The present invention is based on the discovery of the presence of particular cross-linked telopeptides in body fluids of patients and normal human subjects. These telopeptides are produced in vivo during collagen degradation arrd remodeling. The term "telopeptides" is used in a broad sense herein to mean cross-linked peptides craving sequences that are associated with the telopeptide region of, e.g., type II and type III collagens and which may have cross-linked to them a residue or peptide associated with the collagen triple-helical domain. Generally, the telopeptides disclosed herein _ g _ will have fewer amino acid residues than the entire telopeptide domains of type II and type III collagens.
Typically, tire t,elopepl:ides of floe present invention will comprise two peptides linked by a pyridinium cross-link and further linked by a pyridinium cross-link to a residue or peptide of the collagen triple-helical domain. Having disclosed the structures of these telopeptides herein, it will he appreciated by one of ordinary skill in the art that they may also be produced other than vivo, e.g., synthetically.
These peptides will generally be provided in purified form, e.g., substantially free of impurities, particularly other peptides.
The present invention also relates to methods for determining in vivo degradation of type II and type III
collagens. The methods involve quantitating in a fluid the concentration of particular telopeptide that have a 3-Irydroxypyridinium cross-link and that are derived from collagen degradation. The methods disclosed in the present invention are analogous to those for determining the - 8a -__ ._ __ _ ___ _g_ absolute rate of bone reaorption in vfvo. Those methods involved quantitatiri6 in a body fluid the concentration of telopeptides having a 3-hydroxypyridinium eross-link derived from bone collagen reaorption.
In a representative assay, the patient's body fluid is contacted with an immunologicai binding partner apenific to a telopeptide having a 3-hydroxypyridiniurn eroea-link derived from type II or type lII collagen. The body fluid may be used as is or purified prior to the contacting step. This purification step may be accomplished using a number of standard procedures, including cartridge adsorption and elution, mol~cutar sieve chromatography, dialysis, ion exchange, alumlna chromatography, hydroxyapatIte chromatography, and combinations thereof.
ether representative embodiments of quantitating the eoncentretion of peptide fragments having a 3-hydrozypyridfniurn cross-link in a body fluid include eleetroehemical titration, natural fluorescence sp~etroacopy, and ultraviolet absorbanoe. Eleotroohemical tltretion may be conducted directly upon a body fluid without further purification. However, when this is not possible due to excessive quantities of contaminating substances, the body fluid is first purified prior to the electrochemical titration step. Suitable methods for purification prior to electrochemioal detection Include dialysis, ion ezehange chromatography, alumina chromatography, molecular sieve chromatography, hydroxyapatite chromatography and ion exchange absorption and elution.
Fluorornetria measurement of a body fluid containing a 3-hydroxypyridinium cross-link is an alternative way of quantitatlng collagen degradation (and, hence, bone resorption, if type I peptides are quantitated). Ths fluoromatric assay can be conducted directly on a body fluid without further purification. However, for certain body fluids, particularly urine, it is preferred that purification of the body fluid be conducted prior to the fluorometric assay. Th(s purification stee consist, of dialyzing an aliquot of a body fluid such as urine against an aqueous solution thereby producing partially purtfled peptide fragment9 retained within the nondfffusate (retentate). The nondiffusate is then lyophilized, dissolved in an ion pairing solution and adsorbed onto an affinity chromatography column. The chromatography column la washed with a volume o! ton pairing solution and, thereafter, the peptide fragments are eluted from the column with an eluting solution. These purified peptide fragments may then be hydrolyzed and the hydrolysate resolved ehromatographically. Chromatographic resolution may be conducted by either high-performance liquid chromatography or microbore high performance liquid chromatography.

The invention includes peptides having structures identical to peptides derived from collagen degradation, substantially free from other human peptides, which may be obtained from a body fluid. The peptides contain at least one 3-hydroxypyridinium cross-link, in particular, a lysyl pyridinoline cross-link or a h yclroxylysyl pyridinoline cross-link, and are derived from tile telopeptide region of type II
or type III collagen linked to one or more residues from a triple-helical domain, typically by the action of endogenous proteases and/or peptidases.
The structures of the type II and type III
l:elopeptides are disclosed below. Information on the type I
1:p1011epr:~C~P_S i.S alSO i11C1UdP_d.
Another aspect of the present invention involves assays for the peptides described herein in which the pyridinium rings are intact arid cleaved. Since it is suspected that some cleavage of pyridinium rings occurs in vivo, assays that detect both intact and cleaved pyridinium rings may lead to more accurate assessments of collagen degradation. In connection with this aspect of the present invention, specific binding partners to the individual peptides containing intact or cleaved pyridinium rings, may be employed in the assays. Individual specific binding partners that recognize both types of peptides (both intact and cleaved pyrid~inium ring containing peptides) may be employed.
Alternatively, specific binding partners that discriminate between peptides containing the intact pyridinium ring and those in which the pyridinium ring is cleaved, could also be used.
Structure of Cross-Licked Telopeptides Derived from Type I Collagen A specific telopeptide having a 3-hydroxypyridinium cross-link derived from the N-terminal (amino-terminal) telopeptide domain oL bone type I collagen has the following amino acid sequence:
FORriULA III
Asp-Glu-K-Ser-Thr-Gly-Gly Gln-Tyr-Asp-Gly-K-Gly-Val-Gly K
where K
K
K
is liydroxylysyl pyridinoline or lysyl pyridinoline, and Gln is glutamine or pyrrolidine carboxylic acid.
The invention also encompasses a peptide containing at least one 3-hydroxypyridinium cross-link derived from the C-terminal (carboxy-terminal) telopeptide domain of bone type I collagen. These C-terminal telopeptide sequences are cross-linked with either lysyl pyridinoline or hydroxylysyl pyridinoline. An example of such a peptide sequence is represented by the formula:
- loa -21~693~
FoRriuLA zv Asp-Gly-Gln-Fiyp-Gly-Ala H yp-Glu-Gly-Lys Gly-Asp-Ala-Gly-Ala-K-Gly-Asp Glu-K-Ala-His-Asp-Gly-Gly-Arg Glu-K-Ala-His-Asp-Gly-Gly-Arg where K
K
K
is hydroxylysyl or lysyl pyridinoline.
The inventor has also discovered evidence of two additional type I collagen telopeptides in body fluids, having 7_0 tire following structures:
FORriULA V
Gly-Glu-Hyp Gly-Asp-Ala-Gly-Ala-K-Gly-Asp Glu-K-Ala-His-Asp-Gly-Gly-Arg Glu-K-Ala-His-Asp-Gly-Gly-Arg 30 and 2~~6935 FORMULA VI
K
Glu-K-Ala-His-Asp-Gly-Gly-Arg Glu-K-Ala-His-Asp-Gly-Gly-Arg These telopeptides may also be quantitated in body fluids in accordance with the invention. The compounds of formula VI
are used as assays, kits and methods of the invention of the parent application which also concerns binding partners to such compounds and cells which produce such binding partners.
Structure of a Cross-Linked Telo eptide Derived from Type II
Collagen A specific telopeptide having a hydroxylysyl pyridinoline cross-link derived from the C-terminal telopeptide domain of type II collagen has the following amino acid sequence (referred to hereinbelow as the core peptide structure):
FORMULA VII

Glu-Hyl-Gly-Pro-Asp al(II)C-telopeptide Glu-iyl-Gly-Pro-Asp al(II)C-telopeptide Gly-Val-Hyl al(II)helical domain wherein the cross-linking residue depleted as Hyl-Hyl-Hyl is hydroxylysyl pyridinoline (HP), a natural 3-hydroxypyridinium residue present in mature collagen fibrils of various tissues.
Amino-terminal telopeptides from type II collagen have not been detected in body fluids, and it is suspected that potential peptides derived from the N-terminal telopeptide region of type II collagen are substantially degraded in vivo, perhaps all the way to the free HP cross-linking amino acid.
Structure of Cross-Linked Telopeptides Derived from Type III
Collagen By analogy to the above disclosure, cross-linked peptides that are derived from proteolysis of human type III
collagen may be present in body fluids. These peptides have a core structure embodied in the following parent structures:
FORMULA VTII
Gln-Tyr-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val al(III)N-telopeptide Gln-Tyr-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val al(III)N-telopeptide Gly-Ala-Ala-Gly-Ile-Hyl-Gly-His-Arg al(III)helical domain - 12a -E04 EE2 EE82 McCarthyTetrault ili'_0i?0 11:06 014 and FORMULA tR
Gly-tle-Gly-G1y-G1u-Hyl-Ala-G1y-Gly-Phe-A1a al(III)C-telopeptide G1y-Ile-G1y-Gly-G1u-HI1-A1a-G1y-Gly-Phe-Ala al(III)C-telopeptide G1y-Phe-Pro-G1y-Met-Hyl-G1y-His-Arg Q1(III} helical domain where K
I
K
K
i~ hydroxylysyl or lysyl pyridinollnt, and Gln is ~lutamine or pyrrolidine carboxylic acid.
A likely cross-linked pepttde derived from type IIl collagen in body fluids has the core struoture:
PORMULA 1~
Asp-Val-Hyl-Ser-Gly-Vat Asp-Va1-Hyl-Ser-Gly-Vdl Hlyl chat is derived from two ol(IIl)H-telopeptide domnlna linked to an hydroxylysyl pyridlnoline residue (Hyl-Hyl-Hyl).
A second possible lraQmeni of the C-telopepttde cross-linking domain, based on the collegen types I and II peptldea observed In urine, has the core structure:

Glu-Hyi-A1a-G1y-Gly-Phe Glu-Hlyl-Ala-Gly-Gly-Phe Hy i E04 662 E682 McCarth~JTet~ault ili30i90 11:06 015 Smaller and larger versions (differing by one to thrte amino acids on each component chain) of these two peptides corresponding to the parent sequences shown above (FORlHULAE VIII and I?Q may also be present end measurable in body tlulds. Analogous smaller and larger versions of each of the peptides disclosed herein form pest of the present invention as well.
The Invention Qenerally includes all specific binding partners to the peptides described herein. "Specific binding partners" era molecules that are capable of binding to the peptides of the prexnt Invention. Included within this farm are fmmunologieal binding partners, such as antibodies (monoclonal and polyalonal), antigen-binding fragments of antibodies (e.g., Feb and F(ab72 tragmenb), singte-chain antigen-binding molecules, and the like, whether made by hybridoma or rDNA technologies.
The Invention includes fused cell hybrids (hybridomas) that produce monoclonal antibodies specific for the above-dtscrlbed collagen peptides having 3-hydroxypyridtnlum cross-links (both with an intact pyridinium ring and one that has been cleeved).
The invention further Includes monoclonal antibodies produoed by the fused cell hybrids, and those antibodies Ins will as binding fragments thereof, e.g., Fab) coupled to a detectable marker. Examples of dettetable markers include enzymes, ohromophores, fluorophores, coenzymes, enzyme inhibitors, cnemiluminesoent materials, paramagnetic metals, spin labels, and radioisotopes.
Such specific binding partners may alternatively be coupled to one member of a ligand-binding partner complex (a.g., avldin-biotin), in whioh case the detectable marker can be supplied bound to the complementary member of the complex.
c5 The invention aLo includes test kits useful for quantitating the amount of peptides having 3-hydroxypyridinium cross-links derived from collagen degradation in a body fluid. The kits may include a specific binding partner to a peptide derived from degraded oollagen as disclosed herein. The specittc binding partner of the test kits may be coupled to a detectable marker or a member of a ligand-binding partner complex, as described show.
FIQURE 1 1t a depiction of type lI collagen and s proposal for the source of telopeptides. It is not established whether the two talopeptldes shown come from one collagen molecule as depicted In F1QURE 1 or from two collagen molecules.
~5 FIGURE 2 shown relative fluorescence (297 nm exoitation= 390 nm emission) versus fraction number (4 ml), obtained dur(ng moleoular sieve chromatographic puriflcatton of cross-Linked telopepttdes. Cross-linked type I1 collagen telopepttdes are contained In the fractions deaignatsd II.

FIGURE 3A shows relative fluorescence (330nm excitation, 390nm emission) versus elution time of fractions during ion exchange HPLC (DEAF-SPW). Cross-linked type II collagen telopeptides are contained in the fraction designated IV.
FIGURE 3B shows absorbance (220nm) versus elution time in minutes for the same S chromatogram.
FIGURE 4A shows relative fluorescence (297nm excitation, 390nm emission) versus elution time of fractions during reverse phase HPLC. Cross-linked type II
collagen telopeptides are eluted as indicated. The fractions indicated by the bar (-) show evidence by sequence and composition analysis of the peptides indicated that retain or have lost the gly (G) and pro (P) residues.
FIGURE 4B shows absorbance (220nm) as a function of elution time during reverse phase HPLC.
FIGURE 5 compares the concentration of HP and LP in both cortical and cancellous human bone with age.
FIGURE 6 depicts the cross-link molar ratios of HP to LP as a function of age.
FIGURE 7A shows relative fluorescence (297nm excitation, >370nm emission) as a function of elution volume during reverse phase HPLC separation of cross-linked type I collagen N-telopeptides.
FIGURE 7B shows relative fluorescence (297nm excitation, >370nm emission) versus elution volume during reverse phase HPLC separation of cross-linked type I
collagen C-telopeptides.
FIGURE 8A shows relative fluorescence (297nm excitation, >380nm emission) as a function of elution time for the cross-linked type I collagen telopeptides.
FIGURE 8B shows relative fluorescence (297nm excitation, >380nm emission) as a function of elution time for the cross-linked type I collagen telopeptides.
FIGURE 9 shows results of binding experiments with the representative monoclonal antibody HB 10611 and: the P1 peptide (Formula III herein, open squares); an a2 (I) N-telopeptide (QYDGKGVGC, solid diamonds); and an al (I) N-telopeptide (YDEKSTGGC, solid squares).
FIGURE 10 shows a portion of the structure of the N-telopeptide region of decalcified human bone collagen. The F1 peptide (Formula III) is enclosed in a box; it contains an epitope that correlates with bone resorption.

''15695 ape II Collagen Telopeptldes The core peptide structure of the type 1I collagen peptides may be found in body fluids as a component of larger peptides that bear additional amino acids or S amino acid sequerrees on one or mare ends of the three peptide sequencts Joined by the HP residue. FIGURE 1 shows hog type ft collagen telopeptides, which are linked to a triple-helical sequence, may be produced in vtvo lrom a human source using the proteolytle enzymes pepsin and trypatn. Smaller tra~mente that have lost amino acids from the core peptide structure, particularly from the helical sequence, may also occur in booty fluids. Generally, additions or deletions of amino acids from the core peptide structure will involve from 1 to about 3 amino acids. Additional amino ealds will Qenerally be determined by the type It collagen telopeptide sequence that occurs naturally in vivo. As examples, peptides having the following structural FORMULA Xll Glu-Hyl-Gly-Pro-Asp-Pro-Leu Glu-Hyl-Gly-Pro-Asp G1y-Va1-Hy1 and FORMULA XIII
Glu-Hyl-Gly-Pro-Asp-Pro Glu-Hyl-Gly-Pro-Asp Gly-yal-Hyl can b~ isolated ehromato~raDhioally from urine, and another of strueturts PORMULA ZIV
Glu-Hyl-Gly-Pro-ASp Glu-Hyl-Gly-Pro-Asp Val-Hlyl ~ms93~

may also be isolated. In addition, glycasylated variants of the core structure and its larger and smaller variants may occur in which a galactose residue or a glucosyl galactose residue are attached to the side chain hydroxyl group of the HP
- cross-linking residue. Each peak in the graph shown in Figures IA and lH may correspond to a cross-linked fragment of particular structure that may bt quantttated for purposes of the present invention.
These atructuree are consistent with their site of origin in human type lI
cohagen ilbrils at a molecular cross-linking site formed between two al(II) C-telopeptides and residue B7 of a triple-helical domain, the known sequences about which area FORIiDLA JCV

Gly-Leu-Gly-Pro-Arg-Glu-Hyl-G1y-Pro-Asp-Pro-Leu Human nl(II) Gly-Leu-Gly-Pro-Arg-Glu-Hyl-Gly-Pro-AsD-Pro-Leu Human al(II) Gly-Leu-Pro-Gly-Val-Hyl-Gly-His-Arg Human al(II) The isolated peptide fragments represent the products of proteolytic degradation of type lI collagen fibrils wfthtn the body. The core structure containing the HP residue is relatively resLatant to further proteolysis and provides a quantitative measure of the amount of type 11 collagen degraded.
Collagen type U fa present In hyaline cartilage of Joints in the adult skeleton. Quantitation of the collagen type 11 telopeptides in a body fluid, for example by way of a monoclonal antibody that recognizes an epltop; in the peptide structure, would provide a quantitative measure of whole-body cartilage dast~uetion oc remodeling. In a preferred embodiment, the present invention involves an assay for cartilage tissue degradation fn humans based on quantifying the urinary ezoretfon rate of at least one member of this family of telopeptides.
Sueh an assay could be used, for example, to:
(1) screen adult human subjects for those individuals having abnormally high rates of cartilage destruction as an early diagnostic indicator of osteoarthrltisi (2) monitor the effects of pottntial antlarthrltle drug's on cartilage metabolism In osteoarthritie and rheumatoid arthritic patlentsi or ~ms~J~

(3) monitor the progress of degenerative joint disease in patients with osteoarthritis and rheumatoid arthritis and their responses to various therapeutic interventions.
Osteoarthritia is a degenerative disease of the artioulat(ng cartilages of joints. In its early stages ft is largely non-inflammatory (l.e. distinct iron rheumatoid arthritis). It is not a single disease but represents the later stages of joint failure that may cesult from various factors (e.g. genetic predisposition, meehanteal overuaage, jotnt malformation or a prior injury, eta.). Destruction of joint articular cartilage Is the central progressive feature of oateoarthritis. The incidence of asteoarthritts, based on rndiographio surveys, ranges from 496 in the 18-24 year age group to 8596 in the T6-T9 year age group. At present the disease can only be die.gnoaed by pain e0d radiographic or other Imaging signs of advanced cartilage erosion.
The assays disclosed above may be used to deteot early evidenoe of accelerated cartilage degradation fn mildly symptomatic patients, to monitor disease progress In more advanced patients, and as a means of monitoring the effects of drugs or other therapies.
In normal young adults (with mature skeletons) there is probably very little degradation of cartilage collagen. A test that could measure fragments of cartilage collagen fn the urine (and tn the blood and joint fluid) would be very useful for judging the "health" of cartilage in the whole body and in individual Joints. The type it collagen-specific peptide assays described above will accomplish this. In the long term, such an assay could become a routine diagnostic aerean for spotting those individuals whose joints are wearing away.
They could be targeted early on for preventative therapy, for example, by the nezt generation of so-aallad chondroprotectlve drugs now being evaluated by the major pharmaceutical companies who are all actively seeking better agents to treat oeteoarthritis.
Other diseases In which joint cartilage is destroyed include: rheumatoid arthcftts, juvenile rheumatoid arthritis, ankyloaing apondylltts, psoriatfe arthritis, Relter's syndrome, relapsing polychondritia, the low back pain syndrome, and ocher infectious corms of arthritis. The type II collagen-specific assays described hccein could be used to diagnose and monitor these diseases and evaluate their response to therapy, as disclosed above in conntetton with oeteoarthritis.

~e lII Collagen Telo eptidcs As pointed out above, human type III collagen telopeptides that may be present in body fluids are expected to have a core structure embodied in the following parent structures:
FORb~IULA V>T1 Gln-Tyr-Ser-Tyr-Asp-V31-Hyl-Ser-Gly-Val nl(II1)N-telopeptide Gin-Tyr-Ser-Tyr-Asp-Val-Hyl-Ser-Gly-Val al(III)N-telopeptide G1y-A1a-Ala-Gly-Iie-Hyl-Gly-His-Arg al(III) helical domain and FORI4iUl(.A IR
Gly-I1e-Gly-Gly-Glu-Hyl-Ala-Gly-Gly-Phe-A1a a1(III)C-telopeptide 1$ Gly-Ile-Gly-Gly-Glu-Hlyl-Ala-Gly-Gly-Phe-Ala al(III)C-telopeptide Gly-Phe-Pro-Gly-Met-Hyl-Gly-His-Arg al(III) helical domain wherein Hy 1 Hy 1 Hyl Is hydroxylyayl pyridinollne.
By analogy to the type II peptides, the type II1 collagen peptides may occur In glycosylated for m s of the core structure. For exa m plc, galaetose residues or glucosylgalactose residues m ay be attached to the core structure, e.g. by w ay of hydroxyl eroupa.
The cross-linking residue of the type iIt collagen peptides Is depleted as a 3-hydroxypyridinium residue, hydroxylysyl pyridinoline. The type fI
telopeptlde structures have been found to primarily have hydroxylyayl pyridinoline cross-linking residues. However, whereas the type II collagen peptides are derived from the N-terminal telopeptide region of type I! collagen, the type III collagen peptides may be derived from either the N-terminal or the C-terminal of type lII
collagen, as long as at least one cross-linking residue is present.
Type III collagen Is present In many connective tissues in association with type I collagen. It Is especially concentrated In vascular walls, in the skin and in, for example, the synovial membranes of joints where its accelerated turnover might be observed in inflammatory joint diseases such as rheumatoid arthritis.

A specific assay for type !II collagen degradation by quantitating cross-linked type III collagen peptides as disclosed above, can be used for detecting, diagnosing, and monitoring various inflammatory disorders, possibly with particular application to the vaseulitia syndromes. In conjunotion with assays for measuring bone type i and cartilage tppe II collagen degradation rates, such an essay could be used as a differential diagnostic tool for patients with various degenerative and Inflammatory disorders that result in cvnnentive tissue destruction or pathological processes.
Isolation of Type II and Type III Collagen Telopeptides General Procedure:
Urine is collected form a normal adolescent during a rapid phase of skeletal growth. Using a sequence of chromatographic steps that include but are not limited to, adsorption on selective aertridges of a hydraphobie Interaction support and an ion-exchange support and molecular sieve, ion-exchange and reverse-phase HPLC column chromatography steps, individual peptides are isolated. The eross tinked peptides containing HP (and LP) residues are d~teettd during column chromatography by their natural fluorescence (Ex max 299 nm ~ pH 4, Bx max 330 nm, > pH 6; Em max 390 nm). An exemplary isolation procedure is provided In the Example below.
Specific Example:
Fresh urine (at 4°C) diluted 5 times with water and adjusted to 296 {v/v) trifluoroacetie acid, passed through a C-18 hydrophobia binding cartridge (waters TM
25 C-18 9ep-pak prewetted with 8096 (v/v) aeetonitrile then washed with water).
Retained peptides were washed with water then eluted with 3 ml of 2096 {v/v) eaetonitrile, and this eluent was adjusted to 0,05 M NH4HC03, 1096 (v/v) aeetonitrile by addition of an equal volume of 0.1 M NH4HC03. This solution was TM
passed through a QMA-Sep-pak (Waters), which was washed with 10 ml of O.1M NeCi, 2096 (vlv) acetonitrile followed by 10 ml of water and the p~ptldo~
were then eluted with 3 ml of 196 (v/v) trifluoroaeetl~ acid and dried by Spesd-VacM
(Savant).
Peptides were fractionated in three chromatographic steps. The first step TM
was rooleeular sieve chromatography on a column of Hio-Gel P-10 (Bio Rad Labs, 2.5 cm X 90 cm) eluted by 1096 (v/v) acetic acid, monitoring the effluent far HP
fluorescence as shown in FIGURE 2. 1n FIGURE 2, the Y-axis is the relative fluorescence emi3sion at 390nm (29T nm eROitation), and the X-axis is the fraction number. The fraction size rues 4 ml. The fractions indicated as 1I are enriched in the cross-linked collagen type II telopeptides. The cross-linked collagen type I
telopeptides are contained in the fractions indicated as III and IY. Fractiens spanning pool II (enriched in the type II collagen cross-linked peptides) were 5 combined; freeze-dried and fractionated by ien-exchange column chromatography on a DEAE-HPLC column (TSK-DEAE-SPW, 7.5mm X 7.Smm, Hlo-Rad Labs), equilibrated with 0.02 M Tcis/IiCI, 1096 (v/v) acetonitrile, pH T.S and eluted with a gradient of 0-0.5M NaCl in the same buffer as shown in FIGURL 2.
FIQURE 3A plots relative fluorescence emission at 390nm (330 nm 10 excitation) versus elution time. The cross-linked collagen type II
telopeptides are found primarily in the segment indicated as IV. FIGURE 3f3 plots absorbance at 220nm as a function of elution tims in minutes. Pool IV contains the type II
collagen arose-linked peptides. individual peptides were then resolved from pool IV by reverse phase HPLC on a C-18 column (Aquapore RP-300,M25em IC d.Bmm, 15 Srownlee Lebs), eluting with a grgdient of 0-309b (v/v) aeetonitrile in 0.196 (vlv) trifluoroacetie acid. F1QURE 4A shows a plot of relative fluorescence intensity at 390 nm (29? nm excitation) as a function of elution time. The peaks associated with particular peptides are indicated in FIGURE 4A. FIGURE ~1H shows the relative absorbanee at 220nm as a function of time.
20 Cross-linked peptide fragments of type Ill collagen containing HP eross-iinking residues may be isolated by a similar combination of steps from the urine of normal growing subjects or, for example, from the urine of patients with inflammatory disorders of the vaseulature.
Typo i Collas~,en Telo aptides 25 This aspect of the invention is based on the discovery that both lysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) peptide fragments (i.e., telopeptides, as used herein) derived from reabsorbed bone collagen are excreted in the urine without being metabolized. The Invention is also based on the discovery that no other eonn~ctive tissues contain significant levels of LP
and 30 that the ratio of HP to LP In mature bone collagen remains relatively constant over a person's lifetime.
FIGURE 5 compares the concentration of HP and LP in both cortical and caneellous human bone with age. It is observed that the eoncentratton of HP
plus LP cross-links in bane collagen reaohes a maximum by age 10 to 15 years and 35 remains reasonably constant throughout adult life. Furthermore, the ratio of HP
to LP, shown in FIGURE 6, shows little change throughout life, remaining constant at about 3.5 to 1. These baseline data demonstrate that the 3-hydroxypyTidinium cross-links fn bone collagen remains relatively constant and therefore that body fluids derived from bone collagen degradation will contain 3-hydroxyoyridinium cross-linked peptide fragments at concentrations proportional to the absolute rate of bone resorption.
Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen, the method for determining the absolute rate of bone reaorptlon, in its simplest form, is based on quantitating the concentration of peptide fragments containing 3~hydroxypyridinlum cross-links and preferably lysyl pyridinoline (LF) cross-links in a body fluid.
As used in this description and in the appended claims pith respect to type I, It, or III telopeptides, by "quantttattng" la meant measuring by any suitable means, including but not limited to spectrophotometrte, gravimetrte, volumetric, coulometric, immunornetric, potenttometric, or amperometrlc means the concentration of peptide fragments containing 3-hydroxypyridinium cross-links in an aliquot of a body fluid. Suitable body fluids include urine, serum, and synovlal fluid. The preferred body fluid is urine.
Since the concentration of urinary peptides will decrease as the volume of urine increases, it Is further preferred that when urine !s the body fluid selected, the aliquot assayed be from a combined pool of urine collected over a fixed period ZD of time, for example, 24 hours. in this way, the absolute rate of bone resorptton or collagen degradation is calculated for a 24 hour period. Alternatively, urinary peptides may be measured as a ratio relative to a marker substance found in urine such as ereatinine. In this way the urinary index of collagen degradation and bone resorption would remain independent of urine volume.
In one embodiment of the present invention, monoclonal or polyclonal anti-bodies ere produced which are specific to the peptide fragments containing lysyl pyridlnoiine cross-links found in a body fluid such as urine. Type 1 telopeptlde fragments may be isolated from a body fluid of any patient, however, it is preferred that these peptides are isolated from patients with Paget's disease or 3D from rapidly growing adolescents, due to their high concentration of type 1 pepttde fragment. Type lI and type III telopeptldes may be isolated from a Dody fluid of any patient but may be more easily obtained from patients suffering from diseases involving type II or type III collagen degradation or from rapidly growing adolescents.

Isolation of Type ( Collagen Tslopeptidea Urine from patients with active Paget's disease is dialyzed in reduced porosity dialysis tubing (<3,500 mol. wt. cut off Spectropore) at 4°C
for 48h to remove bulk solutes. Under these conditions the peptides of interest ere largely retained. The freeze-dried non-diffusate is then eluted (200 mg aliquots) from a -TM
column (90.cm x 2.b em) of Blo-eel P2 (200-400 mesh) in 1096 acetic acid at room temperature. A region of elfluent that ~ombinea the cross-linked peptides la defined by measuring the fluoreacenae of collected fractions at 297 nm exoltation/395 ntn emission, and this pool is freeze-dried. Further resolution of TM
this materiel is obtained on a column of 8to-Oel P-d (200-400 mesh, 90 cm x 2.5 em) eluted in 1096 acetic acid.
Two contiguous bastion pools are defined by monitoring the fluorescence of the eluant above. Tha earlier fraction is enriched in peptide fragments having two amino acid sequences that derive from the C-terminal teloDeptide domain of the 0l(1) chain of bone type 1 coilagen linked to a third sequence derived from the triple-helical body of bone type I collagen. These three peptide sequences are cross-linked with 3-hydroxypyridinium. The overlapping later fraction is enriched in peptide fragments having an amino acid sequence that is derived from the N-terminal telopeptida domain of bone type 1 collagen linked through a 3-hydroxy pyrldinium cross-links.
Individual peptides are then resolved from each of the two fractions obtained above by ion-exchange HPLC on a TSK DEAE-S-PW column (Bio Rad 9.5 cm z 7.5 mm) eluting with a gradient of NaCl (0-0.2M) in 0.02M Tris-HCI, pH 7.5 containing 10916 (v/v) a~etonitrUe. The N-terminal telopeptide-based and C-terminal telopeptide-based cross-ltnked peptides elute in a series of 3-4 peaks o! fluorescence between 0.08M and O.iSM NaCI. The C-terminal telopeptlde-based cross-linked peptides elute first es a series of fluorescent peaks, and the major and minor N-terminal telopeptide-based cross-linked peptides elute towards the end of the gradient ae oharaeteristic peaks. Each of these is collected, freeze-dried and ehromatographed on a C-18 revera~ phase HPLC column (vydae 218TP54, 25 em x 4.6 mm) eluted with a gradient (0-1096) of eaetonitrile:
n-propanol (3:1 v/v) in O.O1M trifluoroacetic acid. About 100-600 a g of individual peptide fragments containing 3-hydroxypyridinlum arose-links can be Isolated by this procedure from a single 24h collection of Paget's urine.
Amino acid compositions of the major isolated peptides confirmed purity and molecular sizes by the whole number stoieMometcy of recovered amino acids.
N-terminal sequence analysis by Edman degradation confirmed the bade core ~15~935 structures corresponding to the sequences of the known cross-linking sites in type I collagen and from the matching amino acid eomposittons. The N-terminal telopeptide sequence of the a2(I) chain was blocked from sequencing analysts due presumably to the known cycllzation of the N-terminal glutamine to pyrrolldone carborylic acid.
A typical elution profile of N-terminal telopeptidee obtained by the above procedure is shown in FIGURE ?A. The major peptide fragment obtained has an amino acid composition: (Aax)z(Glx)2(Gly)SVaI-Tyr-Sar-Thr, whtre Asx is the amino acid Asp or Asn and Glx i9 the amino acid Gln or Giu. The sequence of this peptide is represented by Formula III below.
The C-terminal telopeptide-based cross-linked peptides resolved Dy reverse phase HPLC as described above arc shown in FIGURE 78. As can be seen from this figure, these peptides are further resolved into a series of C-terminal telopeptides each containing the 3-hydroxypyrfdi.nium cross-links. The major 1 ~ peptide, shown in FIGURE 7H, was analyzed as described above and was found to have the amino acid composition: (Asp)5(Glu)4(Gly)10(His)Z(Arg)2(Hyp)2(Ala)6.
The sequence of this peptide is represented by formula IV below. It is believed that the other C-terminal telopeptlde-based cross-linked peptides appearing as minor peaks In FIGURE 7B represent additions and deletions of amino acids to the structure shown in Formula IV. Any of the peptides contained within these minor peaks are suitable for use as immunogena as described below.
FORMULA iII
Asp-Glu-K-Ser-Thr-Gly-G1y Gln-Tyr-Asp-Gly-i-Gly-11a1-Gly K
FORMOLA IV
Asp-G1y-Gin-Hyp-G1y-A1a Hyp-Glu-Gly-Lys Gly-ASp-A1a-Gly-Ala-K-Gly-Asp G1u-K-Ala-H1s-Asp-Gly-Gly-Arg 3~
G1u-K-Ala-His-Asp-G1y-G1y-Arg ~1~693~

Tt(1D illTl.l V
Hyp-61u-G1y G1y-Asp-A1a-Gly-Ala=i-Gly-Asp G1u-K-A1a-His-Asp-G1y-G1y-Ar9 G1u-K-A1a-His-Asp-Gty-Gly-Arg and FORMULA VI
K
Glu-K-Ald-His-Asp-Gly-Gly-Arg Glu-K-Ala-His-Asp-Gly-Gly-Arg where K
K
K
represents the HP or LP cross-links and Gln represents glutamine or pyrrolidone carboxylic acid.
Equivalents of the peptides represented by the above structures, in terms of their presence in a body fluid due to collagen degradation, Include those Cases where there is some variation in the peptide structure. Examples of such variation include 1-3 amino said additions to the N and C termini as well as 1-terminal amino acid deletions. For example, a peptide corresponding to Formula tit, but having a tyrosine residue attached to the amino terminus of the N-terminal aspartate residue has been detected In relatively minor quantities In human urine. Smaller peptide fragments of the molecule represented by Formula IV derived from bone resorption are especially evident In urine. These are found in the minor peaks of the C-terminal telopeptide fraction seen in Figure 7H
and can be identified by amino acid composition and sequence analysis.
Examples of Procedures for Quantitating Peptides A. lmmunological Procedure For Quantitating Peotides lmmunological binding partners capable of specifically binding to peptide fragments derived from bone collagen obtained from a physiological fluid can be prepared by methods well known in the art. The preferred method for isolating these peptide fragments Is described above. By irnmunological binding partners as 21~6~3~
used herein is meant antibodies and antibody fragments capable of binding to a telopeptide.
Both monoclonal and polyclonal antibodies specifically binding the peptides disclosed here in and their equivalents are prepared by methods known in the art. For example, Campbell, A. M. Laboratory Techniques in Biochemistry and Molecular Biolocty, Vol. 13 (1986). Elsevier. It is possible to produce antibodies to the above peptides or their equivalents as isolated. However, because the molecular weights of these peptide fragments are generally less than 5,000, it is preferred that the hapten be conjugated to a carrier molecule. Suitable carrier molecules include, but are not limited to, bovine serum albumin, ovalbumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). Preferred carriers are thyroglobulin and KLH.
It is well known in the art that the orientation of the hapten, as it is bound to the carrier protein, is of critical importance to the specificity of the antiserum.
Furthermore, not all hapten-protein conjugates are equally successful immunogens. The selection of a protocol for binding the particular hapten to the carrier protein therefore depends on the amino acid sequence of the urinary peptide.
fragments selected. For example, if the peptide represented by Formula III is selected, a preferred protocol involves coupling this hapten to keyhole limpet hemocyanin (KLH), or other suitable carrier, with glutaraldehyde. An alternative protocol is to couple the peptides to KLH with a carbodiimide.
These protocols help to ensure that the preferred epitope 215~9~5 (discussed below under the heading "Characteristics of a Preferred epitope") are presented to the primed vertebrate antibody producing cells (e. g., B lymphocytes).
Other peptides, depending on the source, may require different binding protocols. Accordingly, a number of binding agents may be suitably employed. These include, but are not limited to, carbodiimides, glutaraldehyde, mixed anhydrides, as well as both homobifunctional and heterobifunctional reagents (see for example the Pierce 1986-87 catalog, Pierce Chemical Co., Rockford, IL). Preferred binding agents include carbodiimides and heterobifunctional reagents such as m-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS).
Methods for binding the hapten to the carrier molecule are known in the art. See for example, Chard, T., Laboratory Techniaues in Biochemistry and Molecular Biology, Vol. 6 (1987) Partz Elsevier, N.Y..
Either monoclonal or polyclonal antibodies to the hapten-carrier molecule immunogen can be produced. However, it is preferred that monoclonal antibodies - 26a -. ~ _ __ 21~693~

(MAb) be prepared. For this reason it is preferred that immunization be carried out in the mouse. Immunization protocols for the moue usually include an adjuvant. Examples of suitable protocols arc described by Chard, T. (1987) olds supra. Spleen calls from the immunized mouse are harvested and homogenised and thereafter fused with cancer cello in the presence of polyethylene glycol to produce a fused cell hybrid which produces monoclonal antibodies apecif is to peptide fragments derived from collagen. Examples of ouch peptides are represented by the formulas given above. Suitable cancer cells include myeloma, hepatoma, carcinoma, and sarcoma cells. Detailed deacriptiona of this procedure, including screening protocols, protocols for growing selected hybrid cells and harvesting monoclonal antibodies produced by the selected hybrid cells are provided tn Galfre, G. and Milstein, C., Meth. Enzymol., 731 (1981). A
preferred prelimtnnry screening protocol Involves the use of peptide fragments derived from bone collagen resorption and containing 3-hydroxypyridinium cross-links in a solid 1 S phase radtolmmunoasaay. A specific example describing a preferred monoclonal antibody is provided below.
The monoclonal antibodies or other immunologicnl binding partners used In connection with the present are preferably specific for a particular type of collagen telopeptide. For example, assays for the type II or type III collagen degradation telopeptides should preferably be able to distinguish between the type I, type II, and type III peptides. However, In some cases, such selectivity will not be neaeasary, for example, if it is known that a patient is not suffering degradation of one type of collagen but is suspected of suffering degradation from the assayed type of collagen. Because of the differences In amino acid sequences between the type I, type II, and type III families of telopeptides, cross-reactivity should not occur to a significant degree. Indeed, hybridomas can be selected for during the screening of splenocyte fusion clones that produce monoclonal antibodies specific for the cross-linked telopeptide of interest (and lack affinity for those of the other two collagen types). Based on the differences in sequence of the isolated peptide structures, such specificity is entirely feasible.
Peptide fragments of the parent types I, II and IlI collagens, suitable for such hybridoma screening, can be prepared from human bone, cartilage and other tissues end used to screen clones from mice Immunized appropriately with the individual cross-linked peptide antigens isolated from body fluid.

. _ 215693 __ __ __ __ ___ Immunologieal binding partners, especially monoclonal antibodies, produced by the above procedures, or equivalent procedures, are employed In various immunometric assays to quantitate the concentration of the peptides having 3-hydroxypyridinium cross-links described above. These immunometric assays preferably comprise a monoclonal antibody or antibody fragment coupled to a detectable marker. Examples of suitable detectable marker: include but are not limited to: enzymes, coenzymes, enzyme inhibitor:, ohromophorea, fluorophores, chemiluminescent materials, paramagnetic metals, spin labels, .and radionuelides.
Examples of standard immunometric methods suitable for quantitating the telopeptides include, but are not limited to, enzyme linked immunoaorbent assay (ELIBA) (lngvall, E., M~th En,rymol., TO (1981)), radio-immunoassay (RIA), and "sandwich" immunoradiometric away (IRMA).
In its simplest form, these lmmunometric methods can be used to determine the absolute rate of bone resorptton or collagen degradation by simply contacting a body fluid with the immunological binding partner specific to a collagen telopeptfde having a 3-hydroxypyridinium cross-link.
It is preferred that the immunometrie assays described above be conducted directly on untreated body fluids (e.g. urine, blood, strum, or synovial fluid).
Occasionally, however, contaminating substances may interfere with the assay necessitating partial purification of the body fluid. Partial puritfeatton procedures include, but are not limited to, cartridge adsorption and elution, mole-cular sieve chromatography, dlalysi~, ion exchange, elumina chromatography, hydroxyapatite chromatography and combinations thereof.
Test kits, suitable for use in accordance with the present invention, contain specific binding partners such as monoclonal antibodies prepared as described above, that apeaifically bind to peptide fragments derived from collagen degradation found in a body fluid. It is preferred that the specific binding partners of this test kit be coupled to a detectable marker of the type described above. Test kits contalntng a panel of two ar more speclfia binding partners, particularly immunologieal binding partners, are also contemplated. Each immunological binding part~tar in such a test kit will preferably not cross-react substantially with a telopeptide derived from another type of collagen. For example, an irnmunological binding partner that binds specifically with a type lI
collagen telopeptide should preferably not cross-react with either a type I or type III collagen telopeptide. A small degree (e.g., 5-1096) of cross-reactivity may be tolerable. Other test kits may contain a first speclfte Dlnding partner to a collagen-derived telopeptide having a cross-link containing a pyridinium ring (which may be OH-substituted), and a second specific binding partner to a telopeptide having the same structure as the first telopeptide except that the pyridinium ring has been cleaved, such as photolytically.
(1) Monoclonal Antibody Production S The following is an example of preparation of a monoclonal antibody against a peptide immunogen based on Formula III above.
A fraction enriched in the peptide of Formula III (indicative of bone collagen degradation) was prepared from adolescent human urine using reverse phase and molecular sieve chromatography. The peptide was conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde using standard procedures. Mice (Balb/c) were immunized subcutaneously with this conjugate (50-70 fig), first in complete Freund's adjuvant, then boosted (25 ~tg) at 3 weekly intervals in incomplete Freund's adjuvant intraperitoneally. After test bleeds had shown a high titer against the Formula III peptide (referred to herein as P1) conjugated to bovine serum albumin (BSA) using an ELISA format, selected mice were boosted with a low dose (S pg) of the immunogen in sterile PBS Intravenously. Three days later, cells from the spleens of individual mice were fused with mouse myeloma cells using standard hybridoma technology. The supernatants of hybridoma clones growing in individual wells of 96-well plates were screened for reactive monoclonal antibodies, initially using a crude P1 preparation conjugated to BSA. After formal cloning by limiting dilution, the antibodies produced by individual hybridomas were characterized against a panel of screening antigens using ELISA analysis. These antigens were the P1 (Formula III) and P2 (Formula VII) peptides conjugated to BSA. An inhibition assay was used in which P1 conjugated to BSA
was plated out in the plastic walls, and antibody was pre-incubated with a solution of the potential antigen. A secondary antibody (goat anti-mouse IgG conjugated to horseradish peroxidase, HRP) was used for color development using an appropriate substrate. A
desirable monoclonal antibody with high binding affinity for the P1 peptide was identified.
When used as an ascites fluid preparation, the antibody worked in an inhibition assay with optimal color yield at 2 million-fold dilution (which indicates a binding constant in the range of 10-9 to 10-11 M-1, most likely about 10'10 M-1). In an ELISA format, the antibody was able to detect and measure P 1 present in normal human urine without any concentration or clean-up steps. The hybridoma that produces this preferred monoclonal antibody has been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, under accession number HB 10611. This hybridoma is designated below as 1H11; the monoclonal antibody it produces is designated below as MAb-1H11.

21~6~~5 Sandwich assays were also shown to work using the P1-specific monoclonal antibody and a polyclonal antle~rum rained in rabbits egainat conjugated P1.
Either P1-specific monoclonal antibodies, polyelonal antiserum, binding fragments thereof, or the like can be used to bind specifically to P1 from urine, in a detectable manner using3tandard ELISA and other immunoaaaay protocols.
(II) Charaeteriatiea of a Preferred Epitope The epitope recognized by the antibody MAb-1H11 is embodied in the structure of P 1. The epitope is raco~nlzed in pure P 1 and in certain larger peptides that contained the P1 structure (e.g., P1 attached to a tyrosine residue via the N-terminal aspartate residue of P1). The epitope includes chemical features of both of the two telopeptide sequences embodied in the structure of peptide P1. Peptides synthesized to match the human al (I) and a2 (I) N-telopeptide sequences, with the addition of a C-terminal cyateine for coupling to bovine serum albumin (i.e., YDEK9TGGC and 6~YDGKGVaC), were riot recognized 1 S by MAb-1H11. Thin was shown by ELI$A using the free peptides competing against plated-out P1 (see FIGURE 9) or directly as binding partners conjugated to HSA and plated out. Referring to FIGURE 9, the absorbanee at a = 450 nm of a detectable marker Is plotted aQainat the concentration of fret P1 peptide. As the amount of free P1 inereasee, the amount of det~etable marker bound to immobilized (plated-out) Pl diminishes. In comparison, the a2(I) and al(I) N-telopeptides demonstrate little if any significant competitive binding with MAb-1H11.
In addition, a larger form of P1 bearing a tyrosine residue on the N-terminal aspartie acid was recovered Prom urine by affinity binding to MAb-1H11, but in lower yield than P1. Other slightly iarger peptides bearing the P1 epitope were also recovered but In even smaller amounts.
The antibody was not seleetlv~ for the nature of the cross-link in P1, i.e., whether hydroxylysyl pyridinoline (HP) or lysyl pyridinoline (LP). Hoth HP-containing and LP-containing forma were bound, apparently with equal affinity, judging by the analysis of peptides isolated Crom urine by an affinity column consisting of MAb-1H11 coupled to agaroee.
The free cross-linking amino acids, HP and LP, either made by acid hydrolysis from bone colleQen or as present naturally in urine were not recognized by MAb-1H11. After photolytie opening of the 3-pyTidinol ring in peptide P1 with UV light (long UV wavelengths), speeifiQ antibody binding was also unaffected, presumably because the individual peptides remained cross-linked to each other.
The epitope recognized by MAb-1H11, therefore, fs made up of at least a combination of chemical and conformational features embodied i.n the two telopepti.de sequences shown boxed in FIGURE 10, t:oget:lrer with sterir_ features imposed by the trivalent cross-linking amino acid that links them. The a2 (I) N telopeptide sequence, QYDGK, is a particularly significant part of the epitope.
Tlre fact that the epitope recognized by MAb-1H11 does not depend on an intact pyridiniurn ring is an unexpected discovery. If ring-opening occurs either in vivo or even in vitro under routine handling conditions, as appears likely, .
then a quantitative assay of the subject peptides) having ini.~ct-. pyr:idi.ni.~.rm rings will underestimate the amount of_ bone resort>taon. Preliminary ol:rservations indicate that degradation of pyridinium rings in the subject peptides appears to occur particularly in urine and/or in urine samples, even if refrigerated. Accordingly, an assay based on tine present disclosure i_s expected to be comparatively more accurate. Two embodiments are envisioned: a single specific binding partner is employed that recognizes both closed and open-ringed embodiments of the targeted peptides) or two sper_ific binding partners are employed, which differentiate between tire closed-and open-ringed epitopes, respectively.
Specific binding partners that discriminate between open and closed ring forms of the targeted peptides may be obtained by incorporating an appropriate screening step into the standard procedures for obtaining such specific binding partners. For example, to obtain a monoclonal antibody that binds specifically to an open ring from of the P1 peptide, a library -31 a-of candidate monoclonal antibodies can be screened for their ability to bind to P1 having an opened pyridinoline ring (e.g., by ultraviolet light irradiation) and their inability to bind to P1 having an intact pyridinoline ring.
Recent results have shown the following:
Using conditions that had been shown to completely destroy HP and LP (as evidenced by loss of fluorescence of characteristic fluorescent peaks on RP-HPLC) either as the free amino acids or insoluble peptides and intact protein chains, a preparation of PI
was irradiated (long wavelength setting - Mineralight UV SL-25 lamp, Ultra-Violet Products, Inc., San Gabriel, California).
This solution was assayed for binding to mAB 1H11, using a control solution of exactly the same material not irradiated. The results of an ELISA with 1H11 showed essentially no loss of binding to P1, implying that ring cleavage had not affected the epitope significantly. Under the conditions of UV Irradiation (pH9), cleavage of a single bond to open the ring rather than a double cleavage to eliminate the ring nitrogen and its side-arm would be expected.
Further experiments showed that the epitope resides in human bone collagen but is exposed and bound by MAb-1H11 only after extensive proteolysis. Thus, peptides produced from decalcified human bone collagen by bacterial collagenase were bound by MAb-1 H 11 and shown to be derived from the N-telopeptide to helix site shown in FIGURE 10. One form contained the hexapeptide GIKGHR (in place of the non-telopeptide K arm in P1), which is clearly derived from al (I) residues 928-933. Another form embodied an equivalent but distinct hexapeptide that was derived from the a2 (I) chain. Fragments of human bone collagen solubilized by pepsin, CNBr, or trypsin were not recognized by MAb-1H11, either in an ELISA format when used as competitive inhibitors or on a Western blot after SDA-polyacrylamide electrophoresis, indicating that these solubilizing agents do not produce the epitope recognized by MAb-1 H 11.
B. Electrochemical Procedure For Assaying For Peptides An alternative procedure for assaying for the above-described peptides consists of measuring a physical property of the peptides having 3-hydroxypyridinium cross-links.
One such physical property relies upon electrochemical detection. This method consists of injecting an aliquot of a body fluid, such as urine, into an electrochemical detector poised at a redox potential suitable for detection of peptides containing the 3-hydroxypyridinium ring. The 3-hydroxypyridinium ring, being a phenol, is subject to reversible oxidation and tl~erefor_e the electrochem.ir_al detector (e. g., l4odel 5100A
Coulochem sold by Esa - 31b -45 Wlggins Ave., Bedford, MA) is a highly desirable instrument suitable for quantitating the concentration of the present peptides. Two basic forms of electrochemical detector are currently commercially available= amperometrie (e.g., HioAnalytical Systems) and coulometrie (ESA, lnc., Bedford, MA 01730).
S Both are suitable for use in accordance with the present invention, however, the latter system is inherently more sensitlva and therefore preferred since complete oxidation or reduction of the analyzed molecule in the column effluent is achieved. In addition, screening or guard electrodes can be placed "upstream"
from the analytlcnl electrode to selectively oxidize or reduce interfering substances thereby greatly improving selectivity. Essentially, the voltage of the analytfeal electrode is tuned to the redox potential of the sample molecule, and one or more pretreatment dells are set to destroy interferents in the sample.
In a preferred essay method, a standud ourrent/voltage curve is established for standard peptides containing lysyl pyridlnoline or hydroxylysyl pyrldinoline in order to determine the proper voltage to set for optimal sensitivity. This voltage is then modified depending upon the body fluid, to mlntmize Interference from contaminants and optimize sensitivity. Electroehernieel detectors, and the optimum conditions for their use art known to those skilled in the art.
Complex mixtures of body fluids can olten be directly analyzed with the eleetrachemlcal detector w(thout interference. Accordingly, for most patients no pretreatment of the body fluid is necessary. In same cases however, interfering compounds may reduce the reliability of the measurements. In such cases, pretreatment of the body fluid (e.g., urine) may be necesanry.
Accordingly, in an alternative embodiment of the invention, a body fluid is first purified prior to electrochemically tltratfng the purified peptide fragments.
The purification step may be conducted in a variety of ways including but not limited to dialyei3, ion exchange chromatography, alumina chromatography, hydroxyapatite chromatography, molecular sieve chromatography, or combinations thereof. In a protected purification protocol, a measured aliquot (25 ml) of a 24 hour urine sample is dialy2ed in reduced porosity dialysis tubing to remove the bulk of contaminating fluorescent solutes. The non-diffusate is then lyophilized, redlssolved in 196 heptafluorobutyrie acid (HFHA1, an ion pairing solution, and the peptides adsorbed on a Waters Sep-1?ak C-18 cartridge. This cartridge is then washed with 5 ml of 196 HFBA, and then eluted with 3 mi of 5096 methanol in HFHA.
Another preferred method of puriftcatfon consists of adsorbing a measured aliquot of urine onto an ion-exchange adsorption filter and eluting the adsorption filter with a buffered eluting solution. The eluate fractions containing peptide fragments having 3-hydroxypyridinium cross-links are then collected to be assayed.
Still another preferred method of purification employs molecular sieve chromatography. Fvr example, an nH
aliquot of urine is applied to a Bio-Gel-P2 or Sephadex G-20 column and the fraction eluting in the 1000-5000 Dalton range is collected. It will be obvious to those skilled in the art that a combination of the above methods may be used.to purify or partially purify urine or other body fluids in order to isolate the peptide fragments having 3-hydroxypyridinium cross-links. The purified or partially purified peptide fragments obtained by the above procedures may be subjected to additional purification procedures, further processed or assayed directly in the partially purified state. Additional purification procedures include resolving partially purified peptide fragments employing high performance liquid chromatography (HPLC) or microbore HPLC when increased sensitivity is desired. These peptides may.then be quantitated by electrochemical titration.
A preferred electrochemical titration protocol consists of tuning the redox potential of the detecting cell Of the electrochemical detector (Coulocl~emMl~todel 5100A) for maximum signal with pure HP. The detector is then used to monitor the effluent from a C-18 HPLC column used to resolve the partially purified peptides.
C. Fluorometric Procedure For Quantitatinq Peptides An alternative preferred method for quantitating the z156~3~
COIIGPIrtratlOn of peptides having 3-hydroxypyridinium cross-links as described herein i.s to measure tl~e characteristic natural fluorescence Uf these peptides. For those body fluids containing few naturally occurring fluorescent materials other tl~ac~ the 3-lrydroxypyridinium cross-links, fluorometric assay may be r_onducted directly without further purification of the uody fluid. In this Case, the peptides are resolved by FiPLC
and the natural fluorescence of the HP and LP amino acid residues is measured at 395 nm upon excitation at 297 nm, esse«tially as described by Eyre, D.R., et al., Analyte, B Lowlrem. 13'7 : 38U ( 1984 ) .
It is preferred, in accordance with the present lrlVP_Iltion, that the fluorometric assay be conducted on urine.
()rive, however, usually contains substantial amounts of ~iaturally occurring fluorescent contaminants that must be removed prior to conducting the fluorometric assay.
llccordingly, urine samples are first - 33a -r~
~I~~~3 partially purified as described above for electrochemical detection. This partially purified urine sample can then be fluorometrically assayed as described above.
Alternatively, the HP and LP cross-linked peptides in the partially purified urine samples or other body fluids can be hydrolyzed in 6M HCl at about 108°C
for approximately 24 hours as described by Eyre, et al. (1984) vide auk. This process hydrolyzes the amino acids connected to the lysine precursors of "tripeptide"
HP
and LP cross-links, producing the free HP and LP amino acids represented by Formulae I and lt. These small "tripeptides" are then resolved by the techniques described above, preferably by HPLC, and the natural fluorescence is measured (Ex 297 nm, Ex 390 nm).
Optionally, the body fluid (preferably urine) is passed directly through a C-reverse phase affinity cartridge after adding acetonltrtle/methanol 5 to 1096 V/V.
The non-retentatt is adjusted to 0.05-O.lOM with a cationic ion-pairing agent such as tetrabutyl ammonium hydroxide and passed through a second C-18 revere phase cartridge. The washed retentate, containing fluorescent peptides, from this second cartridge is eluted with acetonitrile:water (or methanol:water), dried and fluoresoent peptides are analyzed by reverse phase HPLC or microbore HPLC
using an anionic ion-pairing agent such as O.O1M trifluoroncttie said !n the eluant.
FIGURE 8A displays tht elution profile resolved by reverse phase HPLC of natural fluorescence for a hydrolysate of peptide fragments from normal human urine. Measurement of the integrated area within the envelope of a given component is used to determine the concentration of that component within the sample. The ratio of HP:LP found in normal human urine and urine from patients having Paget's dia~ase, FIGURE 88, are both approximately 4.5:1. This is slightly higher than the 4:1 ratio found In bone itself (Eyre, et al., 1984). The higher ratio found in urine indioatea that a portion of the HP traction in urine may come from source' other than bone, such as the diet, or other sources of collagen degradation, i.e., cartilage catabolism. it fs for this reason that it is preferred that LP which derives only from bone be used to provide an absolute Index of Done resorption. However, Sn the absence of excessive cartilage degradation such as in rheumatoid arthritis or in cases where bone is rapidly being absorbed, HP or a combination of HP plus LP may be used as an index of bone resorption.
While the invention has bets described in conjunction with preferred embodi ments, one of ordinary skill after reading t"~e foregoing specification will be able to effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein. Renee, the invention can be practiced In ways other than those specifically described herein, It is therefore intended that the 21~693~

protection granted by Letters Patent hereon be limited only by the appended claims and equivalents thereof.

Claims (21)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a method of analyzing a body fluid sample for the presence or concentration of a peptide indicative of a physiological condition of collagen degradation, comprising the steps of contacting the body fluid sample with an immunological binding partner which binds to the peptide, detecting binding of the immunological binding partner in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting the body fluid sample with an immunological binding partner which binds to both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the amino-terminal telopeptide domain of type I collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link selected from among hydroxylysyl pyridinoline and lysyl pyridinoline, and Gln is glutamine or pyrrolidine carboxylic acid, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, and correlating any detected binding to degradation of type I collagen in vivo.

2. An immunological binding partner which binds to both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the amino-terminal telopeptide domain of type I collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link selected from among hydroxylysyl pyridinoline and lysyl pyridinoline, and Gln is glutamine or pyrrolidine carboxylic acid, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved.

3. In a method of analyzing a body fluid sample for the presence or concentration of a peptide indicative of a physiological condition of collagen degradation, comprising the steps of contacting the body fluid sample with an immunological binding partner which binds to the peptide, detecting binding of the immunological binding partner in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting the body fluid sample with an immunological binding partner which binds to both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the carboxy-terminal telopeptide domain of type II collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, and correlating any detected binding to degradation of type II collagen in vivo.

4. An immunological binding partner which binds to both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the carboxy-terminal telopeptide domain of type II collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved.

5. In a method of analyzing a body fluid sample for the presence or concentration of a peptide indicative of a physiological condition of collagen degradation, comprising the steps of contacting the body fluid sample with an immunological binding partner which binds to the peptide, detecting binding of the immunological binding partner in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting the body fluid sample with an immunological binding partner which binds to both:

(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the amino-terminal telopeptide domain of type III collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, and correlating any detected binding to degradation of type III collagen in vivo.

6. An immunological binding partner which binds to both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the amino-terminal telopeptide domain of type III collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved.

7. In a method of analyzing a body fluid sample for the presence or concentration of a peptide indicative of a physiological condition of collagen degradation, comprising the steps of contacting the body fluid sample with an immunological binding partner which binds to the peptide, detecting binding of the immunological binding partner in the body fluid sample, and correlating any detected binding to the physiological condition, the improvement comprising contacting the body fluid sample with an immunological binding partner which binds to both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the carboxy-terminal telopeptide domain of type III collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, and correlating any detected binding to degradation of type III collagen in vivo.

8. An immunological binding partner which binds to both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the carboxy-terminal telopeptide domain of type III collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved.

9. Use of the immunological binding partner according to claim 2, 4, 6 or 8, to diagnose a physiological condition associated with collagen degradation.

10. A diagnostic composition comprising the immunological binding partner according to claim 2, 4, 6, or 8 and a diluent or carrier.

11. A diagnostic kit comprising:
(a) one or more containers containing the diagnostic composition according to claim 10; and (b) instructions for use.

12. A method for diagnosing a physiological condition of collagen degradation, comprising the steps of:
(a) contacting a body fluid sample with the immunological binding partner according to claim 2, 4, 6 or 8;
(b) detecting binding of the immunological binding partner in the body fluid sample to said peptide; and (c) correlating any detected binding to the physiological condition;
wherein the presence or concentration of said peptide is indicative of said physiological condition of collagen degradation.

13. The method according to claim 12, wherein said body fluid is selected from the group consisting of urine, serum or synovial fluid.

14. The immunological binding partner according to claim 2, 4, 6 or 8, further comprising a detectable marker.

15. The immunological binding partner according to claim 2, 4, 6 or 8, wherein said immunological binding partner is a monoclonal antibody.

16. The immunological binding partner according to claim 2, 4, 6 or 8, wherein said immunological binding partner is a polyclonal antibody.

17. An assay for measuring collagen degradation, comprising determining in a sample of body fluid the presence or concentration of both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the amino-terminal telopeptide domain of type I collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link selected from among hydroxylysyl pyridinoline and lysyl pyridinoline, and Gln is glutamine or pyrrolidine carboxylic acid, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, wherein the presence of said first peptide and said second peptide is indicative of type I
collagen degradation.

18. An assay for measuring collagen degradation, comprising determining in a sample of body fluid the presence or concentration of both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the carboxy-terminal telopeptide domain of type II collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, wherein the presence of said first peptide and said second peptide is indicative of type II
collagen degradation.

19. An assay for measuring collagen degradation, comprising determining in a sample of body fluid the presence or concentration of both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the amino-terminal telopeptide domain of type III collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, wherein the presence of said first peptide and said second peptide is indicative of type III
collagen degradation.

20. An assay for measuring collagen degradation, comprising determining in a sample of body fluid the presence or concentration of both:
(i) a first peptide containing a 3-hydroxypyridinium cross-link derived from the carboxy-terminal telopeptide domain of type III collagen, the first peptide comprising wherein is a 3-hydroxypyridinium cross-link of hydroxylysyl pyridinoline, and (ii) a second peptide identical to the first peptide except that the pyridinium ring of the 3-hydroxypyridinium cross-link is cleaved, wherein the presence of said first peptide and said second peptide is indicative of type III
collagen degradation.

21. The assay according to any one of claims 17 to 20, wherein the step of determining in a sample of body fluid the presence or concentration of both said first peptide and said second peptide comprises contacting the body fluid with an immunological binding partner which binds to both said first peptide and said second peptide.