Estimation of the fragmentation pattern of collagen in body fluids and the diagnosis of disorders associated with the metabolism of collagen

The fragmentation pattern of collagen, especially of type 1, as reflected in breakdown products of collagen in a body fluid such as serum or urine is estimated by measuring the levels of such breakdown products using two or more distinct immunoassays. The results may be combined into a numerical index diagnostic of one or more pathological conditions or patient types.

The present invention relates to a method of estimating the fragmentation
 pattern of collagen in body fluids. The invention further relates to
 analytical systems to be used when determining the collagen fragmentation
 pattern. Still further, the invention relates to the use of the above
 methods to diagnose and characterise the presence of disorders associated
 with the metabolism of bone.
 Diseases of bone, among these osteoporosis, are becoming an increasing
 burden to society. The total cost in the USA in 1992 of osteoporosis
 related injuries alone is estimated to be at least USD 10 billion (Riggs,
 New England Journal of Medicine, 327:620-627 (1992)).
 Osteoporosis as well as a number of other diseases of bone are
 characterised by an increased rate of bone loss when compared to the rate
 of loss in a healthy population. The rate of loss has been shown to be
 highly correlated to the future fracture risk (Christiansen et al.,
 Prediction of future fracture risk. In: Christiansen et al., eds,
 Proceedings 1993. Fourth International Symposium on Osteoporosis, Hong
 Kong. Osteopress Aps 1993; pp. 52-54). Therefore the rate of loss is an
 important parameter to estimate for the diagnosis of such diseases.
 In order to assess the rate of loss the estimation of the rate of bone
 resorption plays a key role. Even though the rate of loss is the net
 difference between the bone formation and bone resorption rates, markers
 of the bone resorption alone have proved to be good estimates of the rate
 of loss (Bonde et al. "Immunoassay for Quantifying Type 1 Collagen
 Degradation Products in Urine Evaluated" Clin. Chem. 40/11, 2022-2025
 (1994)--Endocrinology and Metabolism. The estimate of bone loss is
 improved, however, by including also markers of bone formation (Qvist et
 al. American Society of Bone and Mineral Research, Abstract # B 419,
 Kansas City, 1994).
 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, hydroxyproline, an amino acid largely restricted to collagen,
 and the principal structural protein in bone and all other connective
 tissues, is excreted in urine. Its excretion rate is known to be increased
 in certain conditions, notably Paget's disease, a metabolic bone disorder
 in which bone turnover is greatly increased, as discussed further below.
 For this reason, urinary hydroxyproline has been used extensively as an
 amino acid marker for collagen degradation; Singer, F. R. et al.,
 Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.),
 489-575 (1978), Academic Press, New York.
 U.S. Pat. No. 3,600,132 discloses a process for the determination of
 hydroxyproline in body fluids such as serum, urine, lumbar fluid and other
 intercellular fluids in order to monitor deviations in collagen
 metabolism. The Patent states that hydroxyproline correlates with
 increased collagen anabolism or catabolism associated with pathological
 conditions such as Paget's disease, Marfan's syndrome, osteogenesis
 imperfecta, neoplastic growth in collagen tissues and in various forms of
 dwarfism.
 Bone resorption associated with Paget's disease has also been monitored by
 measuring small peptides containing hydroxyproline, which are excreted in
 the urine following degradation of bone collagen; Russell et al., Metab.
 Bone Dis. and Rel. Res. 4 and 5, 2250262 (1981), and Singer, F. R., et
 al., supra.
 In the case of Paget's disease, the increased urinary hydroxyproline
 probably comes largely from bone degradation; hydroxproline, however,
 generally cannot be used as a specific index for bone degradation. Much of
 the hydroxyproline 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 80% of the free hydroxyproline derived from protein
 degradation is metabolised in the liver and never appears in the urine.
 Kiviriko, K. I., Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss, P.
 H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyproline is a good
 marker for osteoporosis as it is specific for collagen in bones even if it
 is not specific for bone resorption, but it is troublesome to handle.
 Hydroxylysine and its glycoside derivatives, both peculiar to collagenous
 proteins, have been considered to be more accurate than hydroxyproline as
 markers of collagen degradation. However, for the same reasons described
 above for hydroxyproline, hydroxylysine and its glycosides are probably
 equally non-specific markers of bone resorption; Krane, S. M. and Simon,
 L. S., Develop. Biochem. 22:185 (1981).
 Other researchers have measured the cross-linking compound
 3-hydroxypyridinium in urine as an index of collagen degradation in joint
 diseases. See, for back-ground and as examples, Wu and Eyre, Biochemistry,
 23:1850 (1984): Black et al., Annals of the Rheumatic Diseases, 45:969-973
 (1986); and Seibel et al., The Journal of Dermatology, 16:964 (1989). In
 contrast to the present invention, these prior researchers have hydrolysed
 peptides from body fluids and then looked for the presence of free
 3-hydroxypyridinium residues.
 Assays for determination of the degradation of type I, II, and III collagen
 are disclosed in EP-0394296 and U.S. Pat. No. 4,973,666 and U.S. Pat. No.
 5,140,103. However, these Patents are restricted to collagen fragments
 containing the cross-linker 3-hydroxypyridinium. Furthermore, the above
 mentioned assays require tedious and complicated purifications from urine
 of collagen fragments containing 3-hydroxypyridinium to be used for the
 production of antibodies and for antigens in the assays.
 At present very few clinical data using the approach described in U.S. Pat.
 No. 4,973,666 and U.S. Pat. No. 5,140,103 are available. Particularly, no
 data concerning the correlation between the urinary concentration (as
 determined by methods described in the above mentioned patents) of
 3-hydroxypyridinium containing telopeptides of type I collagen and the
 actual bone loss (as determined by repeated measurements by bone
 densiometry) have been published. The presence of 3-hydroxypyridinium
 containing telopeptides in urine requires the proper formation in bone
 tissue of this specific cross-linking structure at various times before
 the bone resorbing process. Very little information on these processes is
 available and it would be desirable to avoid this dependence on the
 correct formation of the cross-linking structure.
 GB Patent Application No. 2205643 reports that the degradation of type III
 collagen in the body can be quantitatively determined by measuring the
 concentration of an N-terminal telopeptide from type III collagen in a
 body fluid. This method uses antibodies generated to N-terminal
 telopeptides released by bacterial collagenase degradation of type III
 collagen, said telopeptides being labelled and used in the assay.
 Schroter-Kermani et al., Immunol. Invest. 19:475-491 (1990) describe
 immunological measurement systems based on CNBr fragments of collagen type
 I and II. Use is made of pepsin-solubilised collagen, leaving the
 telopeptides in the tissue (cf. the above mentioned GB Patent Application
 No. 2205643).
 The development of a monoclonal antibody raised against pepsin-solubilised
 type I collagen is described in Werkmeister et al., Eur. J. Biochem.
 1987:439-443 (1990). The antibody is used for immunohistochemical staining
 of tissue segments and for measuring the collagen content in cell
 cultures. The measurements are not carried out on body fluids.
 EP Patent Application No. 0505210 describes the development of antibody
 reagents by immunisation with purified cross-linked C-terminal
 telopeptides from type I collagen. The immunogen is prepared by
 solubilising human bone collagen with bacterial collagenase. The
 antibodies thus prepared are able to react with both cross-linked and
 non-cross-linked telopeptides, and cross-linkers other than pyridinoline.
 International Patent Application No. WO 91/09114 discloses certain
 synthetic peptides which are used to promote cellular adhesion to a solid
 substrate. The use of the synthetic peptides as immunological reagents is
 not mentioned.
 There are a number of reports indicating that collagen degradation can be
 measured by quantitating certain collagen propeptides. Propepcides are
 distinguished from telopeptides and the alpha-helical region of the
 collagen core by their location in the procollagen molecule and the timing
 of their cleavage in vivo; see U.S. Pat. No. 4,504,587; U.S. Pat. No.
 4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984);
 Niemela, Clin. Chem. 31/8:1301-1304 (1985); and Rohde et al., European
 Journal of Clinical Investigation, 9:451-459 (1979).
 EP Patent Application No. 0298210 and No. 0339443 both describe
 immunological determination of procollagen peptide type III and fragments
 thereof. Further, a method based on the measurement of procollagen is
 disclosed in EP Patent Application No. 0465104.
 The use of synthetic peptides with sequences derived from type IX collagen
 for the development of immunological reagents is disclosed in PCT Patent
 Application No. WO90/08195. Likewise the application describes the use of
 the antibodies thus produced for the determination of type IX collagen
 fragments in body fluids.
 U.S. Pat. No. 4,778,768 relates to a method of determining changes
 occurring in articular cartilage involving quantifying proteogylcan
 monomers or antigenic fragmemts thereof in a
 Dodge, J. Clin Invest 83:647-661 (1981) discloses methods for analysing
 type II collagen degradation utilising a polyclonal antiserum that
 specifically reacts with unwound alpha-chains and cyanogen bromide-derived
 peptides of human and bovine type II collagens. The degradation products
 of collagen were not detected in a body fluid, but histochemically by
 staining of cell cultures, i.e. by "in situ" detection.
 WO94/03813 describes a competitive immunoassay for detecting collagen or
 collagen fragments in a sample wherein a binding partner containing a
 synthetic linear peptide corresponding to the non-helical C-terminal or
 N-terminal domain of collagen is incubated with an antibody to the linear
 synthetic peptide and the sample, and wherein the binding of the antibody
 to the binding partner is determined.
 WO95/08115 relates to assay methods in which collagen fragments in a body
 fluid are determined by reaction with an antibody which is reactive with a
 synthetic peptide. The assay may be a competition assay in which the
 sample and such a peptide compete for an antibody, possibly a polyclonal
 antibody raised against fragments of collagen obtained by collagenase
 degradation of collagen. Alternatively, it may be an assay in which an
 antibody, possibly a monoclonal antibody, is used which has been raised
 against such a synthetic peptide.
 One particular peptide fragment which we have found in body fluid,
 particularly urine, is of the formula:
 ##STR1##
 In the above formula, K--K--K represents cross-link which may for instance
 be a hydroxypyridinium cross-link but may be any naturally occurring
 cross-link and specifically any of those discussed in Last et al. Int. J.
 Biochem. Vol. 22, No. 6, 559-564, 1990.
 A larger peptide fragment including the above smaller fragment is reported
 in EP 0394296.
 In one bone resorption assay (CrossLaps.TM.) described in WO95/08115,
 fragments of type I collagen containing a specific 8 amino acid sequence
 of the C telopeptide of type 1 collagen are quantitated (see also Bonde et
 al., Immunoassay for quanti-fying type I collagen degradation products in
 urine evaluated, Clin. Chem. 40/11, 2022-2025 (1994)--Endocrinology and
 Metabolism.
 Another bone resorption assay (described in WO94/038113) relates to all
 fragments containing a pyridinoline structure and two peptide chains of
 the N-telopeptides of type I collagen (see also Hanson et al., A specific
 immunoassay for monitoring human bone resorption: quantitation of type 1
 collagen cross-linked N-telopeptides in urine, Journal of Bone and Mineral
 Research, Vol. 7, Number 11, 1992). We believe the fragments reactive in
 both these assays to have a considerable variation in respect of their
 size and their content of crosslinking molecules (e.g. pyridinoline,
 Ehrlich chromogen, and pyrrole structures).
 Various studies have been done comparing the results obtained using one
 prior art assay with those of another such assay on the same samples. The
 purpose of these studies has been to establish the reliability of these
 assays as measures of the rate of bone resorption, see for instance
 Garnero et al, Journal of Clinical Endocrinology and Metabolism, 70, No.3,
 780-785.
 Whilst studies of this type look for significance in the similarities of
 the results given by different assays, we believe that they fail to
 appreciate the valuable information regarding the origin and causes of
 bone resorption in individual patients which can be revealed by the
 differences in these results.
 The exact fragmentation pattern of type I collagen in vivo is not yet fully
 elucidated. It has been shown, however, that the fragmentation pattern of
 type I collagen as measured by the pattern of reactivity in gelfiltration
 techniques is significantly different in women receiving one
 anti-resorptive therapy when compared to women receiving another (Garnero
 et al. American Society of Bone and Mineral Research, Abstract 134, Kansas
 City, 1994). It has also been shown that the fragmentation pattern varies
 significantly in untreated women (unpublished observations).
 We have now further established that these differences in fragmentation
 patterns are reflected in differences in results obtained using different
 immunological assays for bone collagen degradation products.
 The present invention now provides a method of estimating the fragmentation
 pattern of collagen, preferably type 1 collagen, in a body fluid,
 comprising subjecting a sample of said body fluid to at least two distinct
 immunological assays, each of which measures the amount of a respective
 population of collagen breakdown products in said sample and comparing the
 results of the said measurements.
 Thus, in a diagnosis situation, one may aim at measurements of the
 fragments of type I collagen which put more emphasis on those which may be
 of "pathological" nature and put less emphasis on the fragments generated
 in a "healthy" renewal of the skeleton.
 It will be understood that the population of breakdown products detected by
 the respective assays may overlap. Indeed, one of said populations may be
 a sub-population which is wholly within the other said population.
 The use of a comparison, e.g. by forming a ratio, between the concentration
 of specific fragments and the concentration of other fragments or the sum
 of fragments is highly relevant in this context as a high rate of bone
 resorption can probably occur in a "healthy" renewal of the skeleton,
 provided of course that the rate of bone formation also is high. As an
 example one assay could be used for measurement of the sum of degradation
 products whereas another would preferentially detect molecules generated
 during "normal" or "regular" collagen degradation. By creating the index
 between the two assays one will indirectly have information about the
 amount of collagen fragments generated by "pathological" collagen
 degradation, e.g. by bone metastasis. A parallel diagnostic relation
 exists in the area of estimating the risk of atherosclerosis. In this case
 the total cholesterol and subfractions of cholesterol in form of HDL and
 LDL are measured.
 According to the invention, the results may preferably be compared by
 combining them mathematically to form a numerical index, e.g. by taking
 their ratio.
 A ratio formed between the concentration of the fragments measured in two
 independent immunological assays of bone resorption, provides an index
 which is dependent on the fragmentation pattern of type I collagen and
 which therefore can be used for diagnostic purposes in relation to
 disorders associated to the metabolism of collagen.
 A numerical index derived from two or more assays may be linked to a
 particular identified pattern of fragmentation if desired by separating
 collagen fragments in the sample, e.g. by HPLC or by gel-filtration, and
 measuring the amounts of peptide in specific fractions, optionally
 identifying the peptides in question. However, this is not necessary to
 the practice of the invention.
 One may simply associate particular numerical index results with particular
 patient types. This may be done by subjecting a range of samples of known
 type to selected pairs or larger multiples of assays and building up a
 data-base of results. One may then identify the fragmentation pattern of
 an unknown sample as being typical of a particular class of sample
 previously tested. The term "patient type" embraces both healthy patients
 of different age and/or sex and patients with one or more pathological or
 abnormal conditions.
 Preferably in accordance with the invention each of said populations of
 breakdown products comprises breakdown products of telopeptides of type I
 collagen.
 Preferably at least one said population is of breakdown products containing
 peptides comprising one or more of the following amino acid sequences of
 human type 1 collagen:
 Asp-Glu-Lys-Ser-Thr-Gly-Gly--SEQ. ID. No.2
 Glu-Lys-Ala-His-Asp-Gly-Gly-Arg--SEQ. ID. No.3
 Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly--SEQ. ID. No.4
 Gly-Met-Lys-Gly-His-Arg--SEQ. ID. No.5
 Gly-Ile-Lys-Gly-His-Arg--SEQ. ID. No.6
 Gly-Phe-Lys-Gly-Ile-Arg--SEQ. ID. No.7
 Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn--SEQ. ID. No.8
 One such population may contain peptides comprising one or more of the
 following amino acid sequences of human type II collagen:
 Glu-Lys-Gly-Pro-Asp--SEQ. ID. No.9
 Gly-Val-Lys--SEQ. ID. No.10
 Pro-Gly-Val-Lys-Gly--SEQ. ID. No.11
 Pro-Gly-Pro-Lys-Gly-Glu--SEQ. ID. No.12
 Gly-Gln-Lys-Gly-Glu-Pro--SEQ. ID. No.13
 or Gly-Asp-Ile-Lys-Asp-Ile-Val--SEQ. ID. No. 14
 or one or more of the following amino acid sequences of human collagen type
 III:
 Asp-Val-Lys-Ser-Gly-Val--SEQ. ID. No. 15
 Glu-Lys-Ala-Gly-Gly-Phe-Ala--SEQ. ID. No. 16
 Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg--SEQ. ID. No. 17
 or Gly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg--SEQ. ID. No. 18
 Similar sequences with isoaspartic acid replacing aspartic acid may be
 detected.
 Preferably, one of said assays measures the amount of population of
 breakdown products characterised by containing isoaspartic acid.
 Said population may comprise or consist of breakdown products containing
 one or more peptides of the sequence EKAH*GGR (SEQ. ID No. 19), wherein *
 is isoaspartic acid and K is part of a collagen cross-link or is lysine.
 One of the assays may involve determining the amount of the peptide of
 formula 2 (below) present in said body fluid:
 ##STR2##
 wherein K--K--K is any naturally occurring cross-link and * is isoaspartic
 acid, or of one or more peptides incorporating an epitope present in the
 peptide of formula 2 which contains isoasparcic acid.
 Said determination may be carried out using an immunological binding
 partner specific for an isoaspartic acid containing species present in the
 sample during the procedure.
 The immunological binding partner may be an antibody raised against a
 linear peptide corresponding to a sequence within collagen with
 isoaspartic acid substituting in said amino acid sequence for aspartic
 acid in said collagen sequence. It may be an antibody raised against a
 fragment of collagen, selected for its affinity for such an isoaspartic
 acid containing peptide.
 One of the assays may preferably measure a population of breakdown products
 containing peptides related to those detected in one of the other assay by
 the presence of aspartic acid in place of isoaspartic acid.
 The invention includes a kit for use in estimating the fragmentation
 pattern of collagen type 1 in a body fluid, comprising an immunological
 binding partner for a first population of collagen type I breakdown
 products, an immunological binding partner for a second population of
 collagen type 1 breakdown products and optionally one or more assay kit
 ingredients selected from buffers, wash solutions, synthetic peptides,
 anti-idiotype antibodies, antibody-enzyme conjugates, substrates for
 antibody-enzyme conjugates, body fluid control samples, standard solutions
 and enzyme conjugate reaction stopping solutions.
 In accordance with a particularly preferred practice of the invention, we
 have found that specific fragmentation patterns of type I collagen, as
 determined in urine samples of gelfiltration techniques, can be estimated
 by forming a ratio between results obtained using two immunological
 assays, both assays being assays of bone resorption and both measuring
 degradation products of type I collagen. A first of the assays is based on
 a polyclonal antibody and is described in Bonde et al., Immunoassay for
 quantifying type I collagen degradation products in urine evaluated, Clin.
 Chem. 40/11, 2022-2025 (1994)--Endocrinology and Metabolism. The second
 assay is based on a monoclonal antibody and is described in Fledelius et
 al., American Society of Bone and Mineral Research, Abstract C 344, Kansas
 City, 1994). Both of these assays are also described in WO 95/08115.
 It is observed, using gelfiltration experiments on urine samples, that the
 degradation of type I collagen varies from individual to individual not
 only in a quantitative manner but also in a qualitative manner. In order
 to express the qualitative differences in the degradation of type I
 collagen in a quantitative manner, a ratio may be formed between the
 results obtained in each of the above mentioned assays detecting different
 degradation fragment of type I collagen.
 This ratio can be used for distinguishing between urine samples giving
 identical readings in one or other assay (see Table 1), and therefore has
 utility for diagnostic purposes.
 It is contemplated that the method of forming the relevant ratio between
 assays of bone resorption will be used to diagnose disorders of the
 metabolism of collagen analogously to the diagnosis and estimation of risk
 of atherosclerosis, namely by measuring the total cholesterol and
 subfractions (HDL, LDL) and by forming the relevant ratios between the
 subfractions and the total cholesterol.
 In brief the assays referred to above are based on an immobilised synthetic
 peptide with an amino acid sequence found in a part of the C-terminal
 telopeptide of the .alpha.I chain of type I collagen
 (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg=8AA SEQ. ID. No. 3).
 To produce the polyclonal antibody used in the first assay, rabbits were
 immunised with collagenase treated collagen and antibody serums reactive
 with 8AA were selected.
 To produce the monoclonal antibody of the second assay rabbits were
 immunised with 8AA conjugated to BSA using a two step carbodimide
 procedure.
 For coating of microtiter plates and 8AA peptide was conjugated to
 thyroglobulin using glutaraldehyde (Soinila S, Mpitsos G J, Soinila J.
 Immunchistochemistry of encephalins: Model studies on hapten-carrier
 conjugates and fixation methods. J. Hitochem Cytochem 1992:2:231-9).
 During incubation of samples with these antibodies a competition takes
 place between the immobilised peptide and the breakdown products of type I
 collagen in urine. As the content of the peptide in the solution
 increases, less antibody will bind to the immobilised peptide leading to a
 decreasing optical density.
 Surprisingly it has been found that whilst the monoclonal antibody
 successfully detects peptides in urine containing all or some of the 8AA
 sequence, the polyclonal antibody under assay conditions selectively
 detects in urine peptides containing all or some of an analogous amino
 acid sequence in which isoaspartic acid replaces aspartic acid in the 8AA
 sequence (iso-8AA). In place of such a polyclonal antibody one may
 therefore use a polyclonal antibody selected for reactivity with the
 iso-8AA peptide or a monoclonal antibody raised against iso-8AA.
 Thus, we have now discovered that a proportion of the peptide fragments in
 body fluid are related to peptides of equivalent formula, e.g. peptides of
 formula 1, by their replacement of aspartic acid in the formula by
 isoaspartic acid.
 The isomerization of aspartic acid has been reported previously to be a
 spontaneous reaction occurring under physiological conditions.
 See for instance Brennan et al. Protein Science 1993, 2, 331-338, Galletti
 et al., Biochem. J. 1995, 306, 313-325, Lowenson et al., Blood Cells 1988,
 14, 103-117 and Oliya et al., Pharmaceutical Research, Vol. 11, No. 5,
 1994, p.751.
 The above discovery indicates that this isomerization also occurs in
 bone-issue and the extent of isomerization is expected therefore to be
 marker for the age of the bone tissue concerned.
 Furthermore, the presence in such bone peptide fragments of the
 isomerization provides confirmation that the peptide fragments indeed
 derive from bone degradation and not some other source such as the
 degradation of newly formed collagen never incorporated into bone.
 Preferably, therefore one of the assays is carried out using an
 immunological binding partner specific for an isoaspartic acid containing
 species present in the sample during the procedure, preferably said
 peptide of formula 2 or a peptide incorporating an epitope present in the
 peptide of formula 2 which contains isoaspartic acid.
 The immunological binding partner may be a monoclonal or polyclonal
 antibody. By the requirement that the immunological binding partner be
 specific for the isoaspartic acid containing species is meant that the
 immunological binding partner distinguishes between said species and the
 analogous spartic acid containing species to an extent useful in the
 assay.
 Suitable immunological binding partners also include fragments of
 antibodies capable of binding the same antigenic determinant including
 Fab, Fab' and F(ab').sub.2, fragments.
 Preferably, the immunological binding partner is an antibody raised against
 a linear peptide, preferably a synthetic peptide, corresponding to a
 sequence within collagen with isoaspartic acid substituting in said amino
 acid sequence for aspartic acid in said collagen sequence
 Each assay may take many forms including ELISA, RIA, or IRMA, procedures
 for which are too well known to warrant description here.
 In an ELISA of this type, the synthetic peptide may be immobilised on a
 solid support. A sample may be incubated with a polyclonal antibody or
 monoclonal antibody for the synthetic peptide in contact with the solid
 support and after washing, a peroxidase-conjugated (revealing) antibody
 may be added. After further incubation, a peroxidase substrate solution is
 added. By competition, peptides in the sample reactive with the antibody
 inhibit the peroxidase reaction.
 Where the synthetic peptide is used to raise a monoclonal immunological
 binding partner, the synthetic peptide need not be a competing agent in
 the assay. For instance, collagenase treated collagen may be purified and
 immobilised onto the solid support and an ELISA may be carried out using a
 monoclonal antibody.
 Antibodies may be prepared which are respectively selective for one or more
 aspartic acid containing peptides and for their isoaspartic acid
 containing analogues. It is then possible to carry out an assay for both
 variants of the peptide or peptides. The relative amount of isoaspartic
 acid will provide an indication of the age of the bone which is being
 broken down. Accordingly, the invention provides a method of obtaining
 information regarding collagen resorption in a patient, comprising
 measuring in a body fluid the relative amounts of at least one aspartic
 acid containing peptide derived from collagen and a corresponding
 isoaspartic acid containing peptide.
 The invention may be applied both to humans and to animals.
 Suitable body fluids include, e.g. human, urine, blood, serum, plasma and
 synovial fluid. It is contemplated that the method may also be used e.g.
 on saliva and sweat. The body fluid may be used as it is, or it may be
 purified prior to the contacting step. This purification step may be
 accomplished using a number of standard procedures, including, but not
 limited to, cartridge adsorption and elution, molecular sieve
 chromatography, dialysis, ion exchange, alumina chromarography,
 hydroxyapatite chromatography, and combinations thereof.

In preferred embodiments of methods according to the invention, one or both
 assays are performed by an inhibition ELISA (enzyme linked immunosorbent
 assay) by contacting a sample with a synthetic peptide having a sequence
 derived from collagen and with an antibody, which is immunoreactive with
 the synthetic peptide. The synthetic peptide is immobilised on a solid
 support. The antibody may be raised against the synthetic peptide or
 raised against collagen degradation products and screened for by use of
 such a synthetic peptide.
 The preparation of synthetic peptides may be performed according to
 procedures well known in the art, e.g. by solid-phase peptide synthesis
 techniques commonly described as "Merrifield synthesis". Also classical
 solution phase techniques may be used. Sequences of interest include
 potential sites for collagen cross-linking (see for example Kuhn, K., in
 Immunochemistry of the extracellular matrix, 1:1-29(1982), Eyre, D. R.,
 Ann. Rev. Biochem. 53:717-48 (1984), or U.S. Pat. No. 5,140,103). Examples
 of such peptides sequences are given above.
 Regarding the synthetic peptides, it is possible to omit (or add) one or
 more amino acid residues from (or to) the crosslinkable site sequences
 without substantial loss of the ability to (a) raise antibodies
 recognising the isoaspartic acid analogue of the corresponding native
 collagen fragment or (b) inhibit the binding of such antibodies to the
 said analogue of the native fragment. It is possible to use longer
 collagen fragments and/or chimeric peptides to raise the antibodies and,
 in principle, it is not necessary to use the same peptide as the immunogen
 and the competitor in a competition assay.
 The methods for preparation of both monoclonal and polyclonal antibodies
 are well known in the art. For example, see Campbell, A. M., Laboratory
 Techniques in Biochemistry and Molecular Biology, Vol. 12 (1986). It is
 possible to produce antibodies to synthetic peptides by immunisation.
 However, because of the relatively small molecular weight of these
 compounds it is preferred that the hapten be conjugated to a carrier
 molecule. Suitable carrier molecules include, but are not limited to,
 bovine serum albumin, thyroglobulin, ovalbumin, tetanus toxoid, and
 keyhole limpet hemocyanin. The preferred carrier is bovine serum albumin.
 To present the hapten in its most immunogenic form to the antibody
 producing cells of the immunised animal a number of alternative coupling
 protocols can be used. Suitable procedures include, but are not limited
 to, glutaraldehyde carbodiimide, and periodate. Preferred binding agents
 are glutaraldehyde and carbodiimide.
 The preparation of antibodies may be carried out by conventional techniques
 including immunisation with collagen fragments or synthetic peptides
 conjugated to a carrier. To improve the immunogenicity it is preferred
 that the immunogen be mixed with an adjuvant before injection. Examples of
 adjuvants include, but are not limited to, alulminium hydroxide, Freund's
 adjuvant, and immune-stimulating complexes (ISCOMs). ISCOMs can be made
 according to the method described by Morein, B. et al., Nature 308:457-460
 (1984).
 Either monoclonal or polyclonal antibodies to the hapten on carrier
 molecule can be produced. For the production of monoclonal antibodies it
 is preferred that mice are immunised. Spleen cells from the immunised
 mouse are harvested, homogenised, and thereafter fused with cancer cells
 in the presence of polyethylene glycol to produce a cell hybrid which
 produces monoclonal antibodies specific for peptide fragments derived from
 collagen. Suitable cancer cells include, but are not limited to, myeloma,
 hepatoma, carcinoma, and sarcoma cells. Detailed descriptions of the
 production of monoclonal antibodies are provided in Goding, J. W., in
 Monoclonal Antibodies: Principles and Practice, (1986). A preferred
 preliminary screening protocol comprises the use of synthetic peptides
 conjugated to a carrier and coated on to the solid surface of a microtiter
 plate.
 For the preparation of polyclonal antibodies, which are reactive with
 peptide fragments derived from collagen, different animal species can be
 immunised. Suitable species include, but are not limited to, chicken,
 rabbit and goat. Chicken and rabbit are preferred.
 Antibodies so produced may be screened for suitability for use according to
 the invention by testing for reactivity with an isoaspartic acid
 containing synthetic peptide of appropriate sequence.
 Antibody fragments are prepared by methods known in the art (see E.
 Ishikawa. Journal of Immunoassay 3:209-327 (1983)).
 Accordingly, by utilisation of an immunoassay with the antibodies prepared
 as above it is possible to assay a biological fluid sample without prior
 fractionation or hydrolysis. The specificity for the desired collagen in
 the biological fluid may be supplied by the antibody in combination with
 the use of a synthetic peptide (against which the antibody was raised or
 in any event with which the antibody is immunochemically reactive) in the
 assay construction.
 As an alternative the immunoassay may be performed using a monoclonal
 antibody. The basic idea of this assay design is to shift the specificity
 of the assay from the antigen (synthetic peptide to collagen) to the
 antibody (from rabbit antiserum to monoclonal antibody). Using this
 construction the assay does not need to make further use of a synthetic
 peptide. This version of the immunoassay is suitably performed by
 incubating the patient sample or a standard solution with a
 peroxidase-conjugated antibody solution in a microtiter plate pre-coated
 with purified collagenase-treated collagen. After washing, the wells of
 the plate are incubated in the dark with a substrate solution. The colour
 reaction is stopped by the addition of a stopping solution, and finally
 the absorbance is measured.
 The immunoassays themselves may be conducted using any procedure selected
 from the variety of standard assay protocols generally known in the art.
 As it is generally understood, the assay is constructed so as to rely on
 the interaction between the specific immunological binding partner and the
 desired analyte for specificity and to utilise some means to detect the
 complex formed by the analyte and the immunological binding partner. The
 immunological binding partner may be complexed to a solid support and used
 as a capture immunological binding partner for the analyte. This protocol
 may be run in a direct form, wherein the formation of
 analyte-immunological binding partner complex is detected, e.g. by a
 fluorescent, radioactive or enzymatic label, or it may be run in a
 competitive format wherein a labelled standard competes with the analyte
 for the immunological binding partner. The format may also be constructed
 as an agglutination assay or the complex may be precipitated by addition
 of a suitable precipitant to the reaction mixture. The specific design of
 the immunoassay protocol is open to a wide variety of choice, and the
 number of clinical assay devices and protocols available in the art is
 multitudinous. For a variety of such protocols, see U.S. Pat. No.
 5,001,225.
 The antibodies and revealing reagents for the conduct of an immunoassay
 using standard detection protocols, for example radioisotope labelling,
 fluorescent labelling or ELISA, either in a direct or competitive format,
 may conveniently be supplied as kits which include the necessary
 components and instructions for the assay. In one embodiment of the
 invention such a kit includes a microtiter plate coated with a relevant
 synthetic peptide, standard solutions for preparation of a standard curve,
 a urine or other body fluid control for quality testing of the analytical
 run, rabbit antibodies reactive with the above mentioned synthetic
 peptide, anti-rabbit immunoglobulins conjugated to peroxidase, a substrate
 solution, a stopping solution, a washing buffer and an instruction manual.
 Since immunoassays can be constructed using antibodies and specific
 synthetic peptides, the ratios of the corresponding collagen fragment
 sequences in an appropriate biological fluid can be determined as well as
 their individual levels and their total. Thus, the assay can be designed
 to include antibodies which will result in determination of several
 isoaspartic acid containing peptides and optionally the native peptide
 sequences or determination of a single isoaspartic acid containing peptide
 sequence and a corresponding or different native peptide sequence, or any
 desired combination thereof.
 The following examples are intended to illustrate, but not to limit the
 invention.
 General
 For the practical performance of the assays described in the following
 examples, 15 .mu.l Standard or unknown sample was pipetted in duplicates
 into the appropriate wells in the pre-coated ELISA plate. Then 100 .mu.l
 Antibody Solution was added to each well, the plate was covered with
 sealing tape and incubated at room temperature for 60 min on a shaking
 device. All the following procedures were also carried out at room
 temperature. After incubation the plates were washed three times with
 diluted Washing Buffer.
 Peroxide conjugated Antibody (HRP-conjugated goat antibodies to rabbit IgG,
 100 .mu.l/well) was added and the sealed wells were incubated 60 min on a
 shaking device. Following another washing procedure, 100 .mu.l of TMB
 Substrate Solution was added to all wells which were sealed and incubated
 for 15 min. The enzyme reaction stopped after 15 min by addition of 100
 .mu.l Stopping Solution. The optical density was read in an ELISA-reader
 at 450 nm.
 A calibration curve was constructed on a log-linear graph paper by plotting
 the mean absorbances of the five standards (0.1-5.0 .mu.g/ml). The
 concentration of equivalents to the synthetic peptide
 (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg; SEQ. ID No. 3) in each patient specimen
 were determined by interpolation on the calibration curve.
 The pipetting scheme as well as the incubations and washing steps for the
 assay employing the monoclonal antibody (ASbAY) was the same as for the
 assay employing the polyclonal antibody.
 EXAMPLE 1
 Gelfiltration of Urine From Children and Adult Women
 One urine sample from a child (age: 11 years) and one urine sample from a
 women (age: 53 years) were applied to respective gelfiltration columns
 0.75 ml of urine was injected into the identical 900.times.10 mm columns
 containing 58 ml of G25 Sephadex superfine gel (Pharmacia, Uppsala,
 Sweden.). Elution was performed at a 0.22 ml/min flow rate, using a 25
 mol/l phosphate buffer of pH 7.4 and was monitored with a 280 nm UV
 detector. The collected fractions (1.6-1.8 ml per fraction) were analysed
 in the CrossLaps.TM. ELISA (Bonde et al., Clin. Chem. 40/11, 2022-2025
 (1994)--Endocrinology and Metabolism).
 As can be seen from FIG. 1, the urine from the child of 11 years shows only
 one major peak after approximately 56 ml. When looking at the elution
 profile from urine from the woman (age: 53 years), two distinct peaks are
 observed (first peak at 53 ml and peak 2 at 64 ml). These observations
 indicate that there is a difference in the distribution of the fragments
 of type I collagen between the subjects. It appears that the urine from
 the child is deficient in the smaller fragments as this urine is lacking
 the second peak found at 64 ml in the urine of the adult woman.
 EXAMPLE 2
 Immunoassays on Urine Samples From Children and Adult Women
 Urine samples from 8 women (age 23-56) and 8 children (age 8-12) were
 analysed in the two immunoassays, one polyclonal and one monoclonal
 described above. Table 1 shows the results of each urine sample.
 Furthermore, it gives the ratio of the values obtained in the two systems
 on one sample. The values from the children all are in the range
 0.82-1.12, whereas the values for the adult women are in the range
 0.14-0.25. Each value given in the table is based on three independent
 tests in the two assays.
 TABLE 1
 Poly Mono Ratio
 Sample ID (.mu.g/ml) (.mu.g/ml) (mono/poly)
 Child #1 4.14 4.05 0.98
 Child #2 8.12 8.87 1.09
 Child #3 3.22 3.28 1.02
 Child #4 1.23 1.09 0.89
 Child #5 3.40 2.79 0.82
 Child #6 2.12 1.90 0.86
 Child #7 1.45 1.51 1.04
 Child #8 6.03 5.49 0.91
 Woman #1 4.14 0.79 0.19
 Woman #2 6.12 0.98 0.16
 Woman #3 1.88 0.47 0.25
 Woman #4 5.22 0.83 0.16
 Woman #5 2.76 0.66 0.24
 Woman #6 5.04 0.71 0.14
 Woman #7 7.45 1.42 0.19
 Woman #8 4.76 0.86 0.18
 EXAMPLE 3
 Paget's Disease
 The same two immunoassays were used on samples from known Paget's disease
 patients and control. The results in Table 2 below show that the differing
 fragmentation patterns produce ratios that enable the samples to be
 distinguished.
 TABLE 2
 CONTROLS CROSSLAPS MABA7 RATIO
 SAMPLE No. (MG/L) (MG/L) MABA7/CROSS
 1 4.14 0.79 0.19
 2 6.12 0.98 0.16
 3 1.88 0.47 0.25
 4 5.22 0.83 0.16
 5 2.76 0.66 0.24
 6 5.04 0.71 0.14
 7 7.45 1.42 0.19
 8 4.76 0.86 0.18
 MEAN 0.19
 PATIENTS CROSSLAPS MABA7 RATIO
 SAMPLE No. (MG/L) (MG/L) MABA7/CROSS
 1 0.79 0.63 0.80
 2 26.37 49.96 1.89
 3 12.24 4.61 0.38
 4 1.97 2.97 1.51
 5 24.33 78.74 3.24
 6 5.53 9.95 1.80
 MEAN 1.60
 CROSSLAPS = polyclonal
 MABA7 = monoclonal
 EXAMPLE 4
 Breast Cancer With Secondary Bone Metastasis
 The assays used in Example 3 were run on urine samples from patients
 suffering from breast cancer with secondary bone metastasis and on healthy
 patient controls. The ratio of the results for the healthy population was
 found to be from 0.1 to 0.4 whereas 50% of the samples from bone
 metastasis patients had a ratio of greater than 0.4. The results are shown
 in FIGS. 3 and 4.