AU682188B2 – Method to detect bone and other connective tissue disorders in humans and animals
– Google Patents
AU682188B2 – Method to detect bone and other connective tissue disorders in humans and animals
– Google Patents
Method to detect bone and other connective tissue disorders in humans and animals
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Publication number
AU682188B2
AU682188B2
AU68648/94A
AU6864894A
AU682188B2
AU 682188 B2
AU682188 B2
AU 682188B2
AU 68648/94 A
AU68648/94 A
AU 68648/94A
AU 6864894 A
AU6864894 A
AU 6864894A
AU 682188 B2
AU682188 B2
AU 682188B2
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Prior art keywords
free
pyd
dpd
crosslinks
bone
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1989-12-30
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AU6864894A
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S P Robins
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Rowett Research Institute
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Rowett Research Institute
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1989-12-30
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1994-07-21
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1997-09-25
1994-07-21
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1994-11-24
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1997-09-25
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1997-09-25
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patent/AU682188B2/en
2010-12-28
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Classifications
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
G—PHYSICS
G01—MEASURING; TESTING
G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 – G01N31/00
G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
G—PHYSICS
G01—MEASURING; TESTING
G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
G01N2800/00—Detection or diagnosis of diseases
G01N2800/10—Musculoskeletal or connective tissue disorders
G01N2800/108—Osteoporosis
Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
Y10S436/00—Chemistry: analytical and immunological testing
Y10S436/811—Test for named disease, body condition or organ function
Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
Y10S436/00—Chemistry: analytical and immunological testing
Y10S436/811—Test for named disease, body condition or organ function
Y10S436/813—Cancer
Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
Y10S436/00—Chemistry: analytical and immunological testing
Y10S436/815—Test for named compound or class of compounds
Abstract
The invention is directed to antibodies which are specifically immunoreactive with forms of crosslinks which occur free in biological fluids. Methods to assess connective tissue, especially bone, metabolism in disease or to monitor therapy are also disclosed. Such methods comprise assessing the levels of native free collagen-derived crosslinks in biological fluids, especially urine. The method can be enhanced by concomitantly determining the levels of an indicator of bone formation in biological fluids of the same individual and assessing the differences between the degradation marker and the formation indicator.
Description
8~1~68~1181 11 1Y r II-r 1
AUSTRALIA
Patents Act 1990 THE ROWETT RESEARCH INSTITUTE
ORIGINAL
COMPLETE SPECIFICATION STANDARD PATENT 0* off.
0:0.
000.0′, 5
S
Invention Title: Method to detect bone and other connective tissue disorders in humans and animals The following statement is a full description of this invention including the best method of performing it known to us:-
III
ICU-sl METHOD TO DETECT BONE AND OTHER CONNECTIVE TISSUE DISORDERS IN HUMANS AND ANIMALS Technical Field The invention relates to methods of diagnosis in medical and veterinary contexts. More specifically, it concerns methods to assess bone and other connective tissue metabolism by detecting free crosslinks formed by collagen degradation in biological fluids, such as urine.
.e Background Art The association of collagen as a major structural material in a multiplicity of tissues, including bone, cartilage, skin, tendons, dentine and various soft tissues is well known. It is also known 20 that the fiber structure of collagen is stabilized by crosslinking. The presence of the fluorescent pyridinium ring system as a non-reducible crosslink in collagen was reported by Fujimoto, et al., J Biochem (1978) 83:863-867. The Fujimoto paper reported isolation of a S 25 fluorescent peptide from pronase digestion of bovine Achilles tendon collagen. The isolated hydrolyzed pyridinoline (Pyd) was thought to contain three residues of hydroxylysine and it was recognized that, prior to hydrolysis, peptide fragments were attached to the pyridinoline moiety. Further work on characterization was conducted by Gunja-Smith, et al., Biochem J (1981) 197:759-762, using hydrolyzed urine, and advantage was taken of the presence of the pyridinoline in urine by Robins, Biochem J (1982) 207:617-620, who linked
I
-2pyridinoline obtained from hydrolyzed urine to a carrier to raise antibodies. The antibodies were then employed in an immunoassay to determine the concentration of pyridinoline in hydrolyzed urine. The procedure was stated by Robins as useful to provide an index of the degradation of certain forms of mature collagen by analysis of physiological fluids.
In all of the foregoing, hydrolyzates were employed to obtain total pyridinoline since much of the crosslink retained peptide extensions of the hydroxylysyl residues responsible for its formation. Thus, in order to obtain a homogenous preparation containing the pyridinium ring, a preliminary hydrolysis step was necessary.
By 1982, it was established that there were two pathways of crosslink formation depending on whether lysine or hydroxylysine residues were present in the telopeptides from which these crosslinks were derived (Robins, in “Collagen in Health and Disease” (1982) 20 Weiss, et al., eds., pages 160-178, Churchill Livingstone, Edinburgh). This was stated to result in a specificity of crosslinking whereby in soft tissues, such as skin, reducible aldimine linkages are formed from oxidized lysyl residues, whereas in cartilage and bone S 25 these bonds, initially formed from hydroxylysine aldehydes, undergo a spontaneous rearrangement to more stable 3xoimine crosslinks. These bonds undergo further reaction to form 3-hydroxy-pyridinium crosslinks. The stable crosslinking pyridinoline analog involving lysine rather than hydroxylysine in the helix portion was identified and quantified by Ogawa, et al., Biochem Biophys Res Commune (1982) 107:1251-1257; Eyre, et al., Anal Biochem (1984) 137:380-388, and designated deoxypyridinoline (Dpd). This material was then believed i I I -3to be restricted to bone collagen, although amounts vary between species.
Further work by Robins, Biochem J (1983) 215:167-173, provided evidence for the existence of glycosylated pyridinoline in bone. Robins proposed a structure which showed the derivation of the ring from three residues of hydroxylysine and also showed that alkali hydrolyzates of collagen provided an O-galactosyl derivative substituted at the sidechain hydroxy group.
As this material was extremely labile to mild acid treatment, this material would not have been present in .samples of hydrolyzed tissue or body fluid.
Fujimoto, et al., J Bicchem (1983) 94:1133-1136, chromatographed unhydrolyzed urine samples 15 and showed that the 3-hydroxypyridinium ring portion was present in substantial proportion as the “free” form, the three hydroxylysyl-derived residues which composed it did not contain further peptide extensions.
On amino acid analysis, whereas pyridinoline isolated 20 from an acid hydrolyzate of collagen gave an asymmetric peak, “free” urinary pyridinoline gave a symmetric peak.
The authors concluded this to be due to isomerization by epimerization of the hydroxylysine moiety of the pyridinoline system during hydrolysis. In addition, 25 relationship of levels of total pyridinoline (after hydrolysis) to age was determined by these workers as a ratio to creatinine levels. It was found that the ratio was high in the urine of children but decreased with age until growth ceases. It was further found that this ratio is relatively constant in adults, but increases slightly in old age. The authors speculate that this may correspond to the loss of bone mass observed in old age.
Attempts were also made to characterize the above-mentioned peptide extensions. Robins, et I II I Ir ~a -L C~ I I -4al., Biochem J (1983) 215:175-182, proposed that in cartilage-derived type II collagen, the pyridinoline links two C-terminal telopeptide chains with a single chain of the helical peptide. An additional pyridinoline crosslink, with the ring derivatized to other peptides, was thought to link two N-terminal non-helical peptides with a third chain in the helical portion of the molecule. The studies were conducted by isolating the fluorescent pyridinoline crosslinks from tissues by specific cleavage with CNBr, thus preserving peptide sequences as extensions of the hydroxylysyl residues forming the ring. The crosslink was localized in the .collagen fibers by determining the amino acid sequences of these extensions.
S* 15 In a paper similar in approach to that of Robins (supra), Wu, et al., Biochemistry (1984) 23:1850-1857, conducted CNBr cleavage of mature cartilage and determined the sequence of the peptide extension residues of the hydroxylysyl participants in the 20 pyridinium ring. Their conclusions were similar to those of Robins.
Robins, et al., Biochim Biophys Acta (1987) 914:233-239, used CNBr digestion of bone derived collagen to localize the crosslinks in the type I collagen structure. These authors concluded that the proportions of the crosslink derived from lysine and that derived from hydroxylysine were present in the same proportions in each of the isolated peptide forms. They also concluded that this showed that these two crosslink analogs occupy the same loci in the collagen fiber and that the form apparently derived from one lysyl participant appears to arise through incomplete hydroxylation of the appropriate lysine residues in the helix. Amino acid analysis indicated that the crosslinks I I LL
~II_
must be situated at two locations involving both the Nand C- terminal telopeptide regions.
Henkel, et al., Eur J Biochem (1987) 165:427-436, determined the amino acid sequences associated with the crosslinks in type I collagen isolated from aorta. These sequences are different from those obtained for type II collagen. Similar results were found by Eyre, et al., FEBS (1987) 2:337-341, who demonstrated that the crosslinks from type IX and type II collagens displayed distinctive peptides attached to the pyridinoline crosslinks.
PCT application W089/04491 to Washington Research Foundation proposes a urinary assay for measuring bone resorption by detection in urine of the specific crosslinks, characterized by their peptide extensions, associated with bone collagen. The assay relies on quantifying the concentration of peptides in a body fluid where the peptide fragments having a pyridinium crosslink are derived from bone collagen resorption. Two specific entities having peptide extensions presumed to be associated with bone collagen are described. These are obtained from the urine of patients suffering from Paget’s disease, a disease known to involve high rates of bone formation and destruction.
Macek, et al., Z Rheumatol (1987) :46:237-240, proposed an assay for osteoarthrosis which -ernds upon the peptides associated with the crosslinks from collagen breakdown. In this approach, the urine sample was size-separated for peptides of molecular weight greater than 10 kd, which peptides were then separated by HPLC using a fluorescence detector to determine those fractions containing the fluorescence due to the pyridinium ring. The spectra obtained from patients with osteoarthrosis were compared to those from I~ 1 i I C RBI~IIII I IP -6healthy patients, and it was easily demonstrable that the multitude of fluorescent peaks associated with the diseased condition was absent from the healthy counterpart. Furthermore, urine from the same diseased patient two weeks after total endoprosthesis of the diseased hip, thereby decreasing the products of osteoarthrosis, gave a spectrum of fluorescent peaks which more closely resembled that of normals.
Furthermore, the osteoarthrosis spectrum was readily distinguished from that obtained from patients with rheumatoid arthritis. The closer resemblance of the rheumatoid arthritis spectrum to that of the spectrum from normal controls was attributed by the authors to the higher activity of proteases in rheumatoid arthritis.
15 This was presumed to digest collagen structures into smaller fragments not detectable in their system.
Study of the elevated levels of total 3hydroxypyridinium ring crosslinks in hydrolyzed urine of patients with rheumatoid arthritis has also been suggested as a method to diagnose this disease by Black, et al., Annals of Rheumatic Diseases (1989) 48:641- 644. The levels of “hydrolyzed” crosslink for patients with rheumatoid arthritis (expressed as a ratio of this compound to creatinine) were elevated by a factor of 5 as 25 compared to controls. In this method, crosslinks derived from hydroxylysine were distinguishable from those derived from lysine; only the hydroxylysine-derived crosslinks were measurably increased. In a more extensive study using hydrolyzed urines, Seibel et al., J Rheumatol (1989 16:964-970, showed significant increases in the excretion of bone-specific crosslinks relative to controls in both rheumatoid and osteoarthritis, but the most marked increases for hydroxylysine-derived pyridinium were in patients with rheumatoid arthritis.
II I II PPBPI~YI~B~PI; -7- While measures related to the presence of collagen-derived crosslinks have been used as indices of the degradation of specific collagen types, including that of bone, conversely, efforts have been made to identify markers of bone formation. Delmas, et al., J Bone Mineral Res (1986) 1:333-337, used the level of GLA- protein in serum as a marker for bone formation in children; the same group, Brown, et al., used a similar assay to assess bone formation in post-menopausal osteoporosis (Lancet (19b4) 1091-1093.
There are many conditions in humans and animals which are characterized by a high level of bone resorption and by an abnormal balance between bone formation and bone resorption. Among the best known of 15 these are osteoporosis and Paget’s disease. However, abnormalities in bone metabolism occur in a variety of other conditions including the progress of benign and malignant tumors of the bone and metastatic cancers which have been transferred to bone cells from, for example, prostate or breast initial tumors. Other conditions include osteopetrosis, osteomalacial diseases, rickets, abnormal growth in children, renal osteodystrophy, and a drug-induced osteopenia. Irregularities in bone metabolism are also often side effects of thyroid treatments and thyroid conditions per se, such as primary hypothyroidism and thyrotoxicosis as well as Cushing’s disease. It would be useful to have a diagnostic which readily recognizes a subject’s condition as an irregularity in bone metabolism, even without defining the precise syndrome from among the possible choices, such as those listed here. Additional tests within the sphere of known bone diseases can be performed once it is established that this is the subset of problems from which diagnosis will emerge.
r~IIPI~PII~I~ l–pll -8- The invention provides just such a screening test, which is general for bone metabolism abnormalities.
Disclosure of the Invention The invention provides a straightforward, and noninvasive, if desired, test to identify subjects who have conditions which are characterized by abnormalities in the formation and resorption of bone and the balance between them. The test is based on the quantitation of tp) native free pyridinoline or deoxypyridinoline crossiinks derived from collagen degradation which are present in Sbiological fluids such as serum and urine. The test is specifically directed to either or both of the forms of crosslinks which occur in such fluids in forms 15 independent of additional amino acid sequence associated with the condensed lysyl or hydroxylysyl residues which constitute the collagen-derived crosslinks.
Accordingly, in one aspect, the invention is directed to a method to diagnose the presence of 20 disorders associated with bone metabolism abnormalities, which method comprises assessing the level of native free 9 crosslinks in a biological fluid of the subject. This level is then compared with the level of the native free crosslinks in normal subjects. Elevated levels of native 25 free crosslinks indicate the presence of such S” abnormalities.
This method can be fine-tuned by assessing the level of these degradation products in comparison with indicators of bone formation. Additional information as to the condition of the subject can be obtained if it is found that the difference between the level of bone resorption, as characterized by the presence of native free crosslinks in the biological fluid, and the level of bone formation, as characterized by the level of the 1–M CCBBRII~ I1ILo~arroaa~Rla~l~llrra~—— -9indicator, is the same or different from that of normal subjects. In general, those suffering from disorders which deplete the skeletal structure are characterized by larger differences between the resorption and formation rates, where resorption predominates.
Thus, a further aspect of the invention is directed to a method to diagnose the presence of the above-mentioned metabolism abnormalities which comprises comparing the levels of an indicator of bone formation in a biological fluid with the level of native free crosslinks in a biclogical fluid from the same individual and comparing the difference between these levels and the differences found for normal subjects. Elevated differences between bone resorption and bone formation 15 indicate problems in maintaining skeletal integrity.
It has been found by the inventor herein that antibodies which bind to hydrolyzed free crosslinks obtained from tissues or biological fluids by treatment with acids are not cross-reactive with native free S 20 crosslinks–either those which contain a lysyl sidechain or those with a hydroxylysyl sidechain. However, antibodies may be prepared which are specific for these free crosslinks. These antibodies may not be cross-reactive with the hydrolyzed forms; for purposes of assessing biological samples directly, this does not o matter, as the hydrolyzed forms are not present. These antibodies may be prepared, if desired, so as to distinguish between the lysyl and hydroxylysyl sidechaincontaining native free crosslink forms. Based on previous experience with polyclonal antibodies against hydrolyzed pyridinoline, the antibodies are likely to distinguish the free forms from the native peptidecontaining forms.
i I 4 Accordingly, another aspect of the invention is directed to antibodies specifically immunoreactive with the native free crosslinks or with either the lysyl or hydroxylysyl forms of native fee crosslinks, or with the glycosylated forms thereof.
Another aspect of the invention is a method to identify subsets of arthritic disease by determining the breakdown of other connective tissues, including cartilage, which method comprises determining the ratio of hydroxylysyl sidechain crosslinks to lysyl sidechain crosslinks (Pyd/Dpd) in a biological fluid of a subject and comparing said ratio to that in normal 10 controls, wherein an increase in said ratio in said subjects over normal controls indicates cartilage breakdown in said subject.
Another aspect is a method of determining the presence of an indicator of connective tissue formation which, in combination with free crosslink levels, provides an assessment of the subject’s metabolic state.
Still another aspect provides a kit for immunoassay determination of the amount or concentration of native free crosslinks in a biological fluid, said crosslinks A ing determinabl, as total free crosslinks or those selected .I from N-Pyd and N-Dpd. The kit includes a set of containers at least one of which contains an antibody or fragment thereof specifically immunoreactive 20 with native free total crosslinks or N-Pyd or N-Dpd and at least one of which contains an additional reagent for conduct of the immunoassay such as a label along with instructions for the conduct of the assay. Preferably, the biological fluid is a serum or urine. The free crosslinks may then be determined as total native free crosslinks. However, the free crosslinks can be determined individually as lysyl sidechain crosslinks (N-Dpd) or as hydroxylysyl sidechain crosslinks (N-Pyd), or as glycosylated Pyd, or any combination of these. The kit may further include a container an antibody of fragment thereof specifically immunoreactive with one or more bone formation indicators.
V I -11crosslinks can be determined individually as I sidechain crosslinks (Dpd) ur a xylysyl sidechain crosslinks (P s glycosylated Pyd, or any nation of these.
In still another aspect, the invention is directed to the use of the assay kits containing the antibodies of the invention or fragments thereof as specific reagents for the crosslinks to be detected.
Brief Description of the Drawings Figure 1 shows a chromatographic trace of pyridinoline obtained from an acid hydrolyzate superimposed on a trace of the pyridinoline obtained without hydrolysis from urine. The figure further compares the o 15 elution pattern as determined by fluorescence with the elution pattern as determined by reaction with antipyridinoline antibody prepared from hydrolyzate.
Modes of Carrying Out the Invention The invention provides an improvement over the presently available methods to diagnose bone disorders or other diseases characterized by abnormalities in collagen metabolism. The invention method utilizes variations in the levels of collagen-derived pyridinium crosslinks in 25 biological fluid as an index of these abnormalities.
*o Prior art methods have involved the hydrolysis of a sample, typically urine, to provide analyte in the form of hydrolyzed crosslinks, free of peptide sidechains, which can then be quantitated in an immunoassay using antibodies raised with respect to the hydrolyzed crosslinks. While this method provides useful information, the preliminary hydrolysis required prevents the assay from becoming a simple clinical assay run directly on an untreated biological sample.
I
~CLI_- -12- It has been found, by the inventors herein, that antibodies raised with respect to the hydrolyzed forms of the pyridinium crosslink do not cross-react either with the free crosslinks present in urine or other biological fluids, or with these crosslinks conjugated to peptides prior to hydrolysis. Thus, the antibodies presently available in the art cannot be used directly with an untreated biological sample.
The present invention overcomes this disadvantage by providing reagents which can be reacted directly with the biological sample to determine the crosslinks present in free form as the diastereomer present prior to hydrolysis. As shown in the examples below, direct measurement of these free and unhydrolyzed crosslinks provides data which are comparable to those obtainable only through the presently available, more complex assay.
Some background information as to the crosslink structures involved will be useful: Nature of the Crosslinks The abbreviations Dpd and Pyd will be used herein to denote the two known forms of the isolated crosslink itself. Pyd or pyridinoline refers to crosslinks formed *wherein the ring N is from the E amino group of an hydroxylsyl residue; Dpd or deoxypyridinoline refers to crosslinks formed wherein the ring N is from the e amino group of a lysyl residue. (Various methods of denoting these variations have been used; for example, HP has been used to designate the “hydroxylsyl” form, and LP has been used to refer to the “lysyl” form.) Specifically, Dpd is believed to represent compounds of the formula:
I
-13-
CHCH-NHCOOH
HOOCH
1
NCHCHCH
2 O 0H I
CH
2
CH,
CH,
CHNH
2
COOH
and Pyd is believed to describe compounds of the formula: 00 CHCHNL4H, COO H 15 OOCH,-NCHCH,CH~ OH
CHOH
CH,
It is seen that both forms of crossliniks are 25 1,4,5 trisubstituted 3-hydroxypyridinium residues. Pyd has zt free hydroxyl group on the sidechain which can be glycosylated, and it is known to be glycosylated in some tissues. The glycosylation is labile to acid, and also to base, but to a lesser degree. Pyd has been shown to occur as Gal-Pyd; the inventor herein has also demonstrated the presence of Glc.Gal-Pyd in urine (see PCT application WO 89/00715). These forms of free Pyd have the acetals
I
-14-
CHOH
HO O
OH
CHOH CHOH HO and OH0
O
OH OH OH HO OH conjugated to the sidechain hydroxyl, respectively.
It is seen that Dpd contains three chiral centers–those of the three a-amino positions in the sidechains. Pyd contains four such centers, as there is an additional chiral center at the sidechain hydroxyl position. Presumably, in the unhydrolyzed samples, whether derivatized further to peptides or not, the three a-amino groups are derived from the natively occurring 15 L-enantiomers, and the OH is in a configuration also determined by the biological system.
As set forth in the Background section above, a substantial proportion of the crosslinks present in urine (about 40% in adults) is in the form of “free” 26 crosslinks–i.e., there are no peptide chains conjugated to the Pyd, glycosylated Pyd, or Dpd structures shown above, even before hydrolysis of the sample is conducted.
Thus, by “free” crosslink is meant compounds of the formulas shown above.
25 It is noted that with respect to Pyd and Dpd, the chirality of the chiral centers is not specified.
Thus, “free,” refers to these crosslinks, whether or not they have been subjected to hydrolysis conditions. The present work demonstrates that these “free” crosslinks differ in chirality when obtained in their “native” form, as compared to their “hydrolyzed” form. As used herein, “native free” crosslinks refers to Dpd or Pyd or its glycosylated forms as they occur in free form in the biological sample; “hydrolyzed free” crosslinks refers to -11M I_ I_ these structures as they occur in hydrolysates. Of course, as the glycosidic bond is labile to the hydrolysis conditions, “hydrolyzed free” crosslinks will not contain sugars.
As the native free crosslinks are the product of the biological system, it is assumed that the biologically favored chirality occurs at all three or four chiral centers. Presumably the three chiral centers represented by the a-amino groups of the sidechains are in the L configuration, as in the naturally occurring amino acid, and the chirality of the carbon containing the sidechain hydroxyl in Pyd is also representative of a single configuration. This is confirmed by the results shown in Figure 1, in which the dotted line represents 15 the result of ion-exchange chromatography on sulfonated polystyrene beads (7 j) equilibrated with sodium citrate performed with the previously isolated Pyd in its native free form. As seen in Figure 1, the Pyd isolated directly from urine elutes at a single peak. This is consistent with the presence of only a single diastereomer.
After hydrolysis, however, the hydrolyzed free Pyd elutes as a mixture, shown by the solid line in Figure 1. This is consistent with racemization at the 25 chiral centers to obtain a mixture of diastereomers which no lcr.ger exhibit identical chromatographic behavior.
Similar results are obtained comparing native free Dpd with hydrolyzed free Dpd.
The “native free” crosslinks thus differ from hydrolyzed free forms of crosslinks. It appears that during conventional acid hydrolysis racemization occurs which changes the configuration of some of the molecules.
However, enhancement of the yield of total “native free” crosslinks in the biological sample could also be
I
I
-16obtained by proteolytic treatment of total native Dpd and Pyd to liberate the “native free” crosslink form. In addition, the crosslinks per se are identical across species, and other species besides human could be utilized to prepare native free crosslink standards for use in the assay system or for use as immunogens. In particular, porcine urine contains high amounts of native free crosslinks. Any source of the biologically important diastereomer could be used.
It has been shown by the inventor herein that the antibodies raised against the free Pyd which is generated as the result of hydrolysis–i.e., wherein the immunogen is obtained by treating the biological fluid or tissue in concentrated acid so as to destroy peptide 15 linkages and separating Pyd from Dpd–show little or no cross-reactivity with native free forms of either Dpd or Pyd. Furthermore, antibodies raised against the Pyd formed from the hydrolyzate cross-react only slightly with Dpd thus formed. Antibodies raised against Pyd from 20 an acid hydrolyzate of bone or cartilage do cross-react with the crosslink in urine after acid hydrolysis.
~A typical set of results is shown in Table 1.
Table 1 presents the results of an ELISA assay using antiserum obtained by immunization with the Pyd hydrolyzate isolated from bone. The ELISA uses this o* hydrolyzate as antigen, and the results are given in terms of the ability of the candidate crosslink to inhibit the binding of the hydrolyzate antigen to the antiserum. Using this criterion, antibodies which were obtained by immunization of rabbits against Pyd isolated from an acid hydrolyzate of cartilage or bone were only cross-reactive with Pyd in its native free form from urine (U-Pyd) although completely cross-reactive with Pyd after hydrolysis in acid of the purified native, free
~I~
-17crosslink isolated from urine. These antibodies, further, were 20% cross-reactive with Dpd isolated from the same bone hydrolyzate and were less than 1% cross-reactive with Dpd in its native free form from urine (U-Dpd); about 70% of the reactivity with these antibodies was recovered after acid hydrolysis of the native free form (Table 1).
Table 1 pmol required for 50% Cross inhibition reaction 15 Pyd from hydrolyzate 1.6 100 of bone Free Pyd from urine (U-Pyd) 29.6 U-Pyd from urine 1.5 107′ hydrolyzed in acid 2 Dpd from hydrolyzate 8.1 of bone Free Dpd from urine (U-Dpd) >260 <1 U-Dpd from urine 11.5 14 hydrolyzed in acid This is further shown Figure 1, which, as stated above, presents the result of ion-exchange chromatography on sulfonated polystyrene beads (7 A) equilibrated with sodium citrate. The elution patterns for free Pyd and hydrolyzate were determined by fluorescence. Antibodies raised against are to react significantly only ii -18the hydrolyzate. discrepancy reactivity two major peaks attributable differing immunogenicity these fractions.
Preparation Native Free Crosslinks prepared native crosslink either a total fraction or, preferably, each component this fraction. Gross separation pyridinium linkage its "free" forms fragments containing protein can be achieved, example, method Fujimoto, J Biochem (1983) 94:1133-1136 (supra). In preparation, concentrate 9* applied Sephadex G-10 column pyridinium-containing fractions eluted. eluate then phosphocellulose citrate, eluted salt. rather simple procedure results crosslinks single peak. As sample not subjected hydrolysis conditions peak contains Dpd :i 20 forms, but also glycosylated including Gal-Pyd Glc.Gal-Pyd described above. Further conveniently conducted standard methods, example using ion exchange or HPLC. Typical protocols found, Black, et al., Anal (1988) 169:197-203; Seibel, Rheumatol.(1989) 16:964-970.
Antibody preparation conventional techniques injection mixture individual components conjugated carrier into suitable mammalian subjects such rabbits mice according immunological generally known art. materials carriers BSA I e 'Ic~c- -19tetanus toxoid conjugation methods enhance immunogenicity. Sera titrated determine antibody formation respect immunogen. If desired, spleen cell peripheral blood lymphocytes may harvested immortalized produce cultures cells capable continuous production monoclonal antibodies immunoreactive desired component.
These preparations have enhanced specificity components.
Thus, polyclonal antisera obtained which specifically form occurring biological fluids, particular urine. By "specifically immunoreactive" meant that serum forming complexes fluid sufficiently greater affinity comparison other permit determination an immunoassay. Some portion antiserum crossreact having peptide chains attached; assays standardized do thus crossreact, standardizing account crossreactivity.
The availability routine obtain :monoclonal permits reproducible reproduction specificity.
Thus, utilizing screening utilizes criterion ability supernatant immunoreact with, Pyd, fail peptides, reliable source obtained. Conversely, it advantageous use, assessment samples, cocktails unique specificities so all determined.
Immortalized lines secrete cultured vitro practical quantities monoclonals Such culture now available commercial scale. addition, injected somewhat cruder isolated ascites *fluid. purified if immunogen ligand.
It should noted while clear collagen-derived failed importance whether converse true, since present unhydrolyzed samples. Thus, procedures assure absence crossreactivity unnecessary.
Conduzt Immunoassays Accordingly, utilization immunoassay above possible assay without prior fractionation hydrolysis. both supplied preparation.
The immunoassays themselves variety in
-II~
-21the understood, constructed rely interaction between specific analyte utilize some means detect complex formed antibody. itself immunologically reactive fragment thereof Fab, Fab', F(ab') 2 fragments. complexed solid support used capture analyte.
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