WO1998008098A2 - Assaying d-amino acids in body fluids - Google Patents
Assaying d-amino acids in body fluids Download PDFInfo
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- WO1998008098A2 WO1998008098A2 PCT/EP1997/004372 EP9704372W WO9808098A2 WO 1998008098 A2 WO1998008098 A2 WO 1998008098A2 EP 9704372 W EP9704372 W EP 9704372W WO 9808098 A2 WO9808098 A2 WO 9808098A2
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- sequence
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- 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
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- 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
Definitions
- the present invention relates to the assaying of collagen or other protein degradation products and materials useful tnerefor.
- Osteoporosis is the most common bone disease in humans. Primary osteoporosis, accompanied by increased suscepti- bility to fractures, results from a progressive reduction in skeletal bone mass. It is estimated to affect 15-20 million individuals m the USA alone. Its basis is an age-dependant imbalance in bone remodelling, .e. in the rates of formation and resorption of bone tissue. In the USA 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 peripheral bones. Between 12 and 20% of the hip fractures are fatal because they cause severe trauma and bleeding, ana half of the surviving patients require nursing home care.
- Osteoporosis is most common in postmenopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrhe c women athletes . Despite tne major, and growing, social and economic consequences of osteoporosis, the availability of reliable assays for measuring bone resorption rates in patients or in healthy subjects is very limited.
- Collagen type I accounts for more than 90% of the organic matrix of bone. Therefore, in principle, it is possible to estimate the rate of bone resorption by monitoring the degradation of collagen type I. Likewise, a number of other disease states involving connective tissue can be monitored by determining the degradation of collagen.
- Examples are collagen type II degradation associated with rheumatoid arthritis and osteoarthritis and collagen type III degradation in vasculitis syndrome.
- Type I, II, and III collagens are all formed in the organism as procollagen molecules, comprising N-terminal and C-termmal propeptide sequences, which are attached to the core collagen molecules.
- procollagen molecules comprising N-terminal and C-termmal propeptide sequences, which are attached to the core collagen molecules.
- tne remaining core of the collagen molecules consists largely of a triple-helical domain having terminal telopeptide sequences which are non-triple-helical .
- These telopeptide sequences have an important function as sites of intermolecular cross-linking of collagen fibrils extra- cellularly.
- the alpha-helical region also includes crosslinkable sites. Intermolecular cross-links provide collagen fibrils with biomechanical stability.
- crosslinks are initiated by modification of lysine and hydroxylysme residues to the corresponding aldehydes.
- Several of these residues located on adjacent chains of collagen will spon-taneously form different termolecular cross-links.
- the exact position of the sites for cross- linking on collagen telopeptides and from the helical region has been previously described. See, for example, Kuhn, K., in Immunochemistry of the extracellular matrix, 1:1-29, CRC Press, Inc., Boca Raton, Florida (1982), Eyre, D.R., Ann. Rev. Biochem., 53:717-48 (1984) or US Patent No. 5140103 and 5455179.
- ammo acid sequences of some potential sites for cross-linking in type I, II, and III collagen are given in Table 1 below.
- fibrous proteins collagen and elastin
- the fibrous proteins are cross- linked by a unique mechanism based on aldehyde formation from lysine or hydroxylysme side chains.
- Four homologous loci of cross-linking are evident in molecules of type I, II and III collagens (for review see Kuhn, K. , in Immunochemistry of the extracellular matrix, 1:1-29 (1982)).
- Two are aldehyde sites, one in each telopeptide region.
- the other two sites are hydroxylysme symmetrically placed at about 90 residues from each end of the molecule.
- These latter sites in the helical region align and react with telopeptide aldehydes in adjacent molecules.
- 3-hydroxypyr ⁇ d ⁇ n ⁇ um residues are the mature cross-link coming from hydroxylysine-derived aldehydes.
- the mature cross-linking residues of the other pathway i.e. from aldehyde formation of lysine residues, are however, still unknown.
- the two 3-hydroxypyr ⁇ d ⁇ n ⁇ um cross-links have been found to be hydroxylysyl pyridinolme (also known simply as “pyridinolme”) and lysyl pyridinolme (also known as “deoxypyridmol e” ) .
- hydroxylysyl pyridinolme also known simply as "pyridinolme”
- lysyl pyridinolme also known as “deoxypyridmol e”
- These cross-linking compounds are naturally fluorescent.
- Some hydroxylysyl pyridinolme cross-link are found to be glycosylated as discussed for instance EP-A-0424428.
- hydroxyprolme 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 hydroxyprolme 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. US Patent No.
- 3600132 discloses a process for the determination of hydroxyprolme in body fluids such as serum, urine, lumbar fluid and other intercellular fluids in order to monitor deviations m collagen metabolism.
- body fluids such as serum, urine, lumbar fluid and other intercellular fluids
- hydroxyprolme 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 hydroxyprolme, 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 .
- hydroxyprolme In the case of Paget 's disease, the increased urinary hydroxyprolme probably comes largely from bone degradation; hydroxyprolme, however, generally cannot be used as a specific index for bone ⁇ egradation. Much of the hydroxyprolme 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 hydroxyprolme.
- Hydroxyprolme 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. Hydroxylysme and its glycoside derivatives, both peculiar to collagenous proteins, have been considered to be more accurate than hydroxyprolme as markers of collagen degradation.
- telopeptide degradation of type III collagen m the body can be quantitatively determined by measuring the concentration of an N- termmal telopeptide from type III collagen in a body fluid.
- This method uses antibodies generated to N-terminal telo- peptides released by bacterial collagenase degradation of type III collagen, said telopeptides being labelled and used in the assay.
- EP Patent Application No. 0505210 describes the development of antibody reagents by immunisation with purified cross-linked C-termmal telopeptides from type I collagen.
- the lmmunogen 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-lmked telopeptides, and cross-linkers other than pyridinolme .
- 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 lmmunological reagents is not mentioned .
- Propeptides are distinguished from telopeptides and alpha-helical region of the collagen core by their location in the procollagen molecule and the timing of their cleavage in vivo; see US Patent No. 4504587; US Patent No. 4312853; 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 lmmunological determination of procollagen pept de type III and fragments thereof. Further, a method based on the measurement of procollagen is disclosed in EP Patent Application No. 0465104.
- US Patent No. 4778768 relates to a method of determining changes occurring in articular cartilage involving quantifying proteoglycan monomers or antigenic fragments thereof m a synovial fluid sample.
- WO94/03813 describes a competitive immunoassay for detecting collagen or collagen fragments m a sample wherein a binding partner containing a synthetic linear peptide corresponding to the non-helical C-termmal 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.
- it may be an assay in which an antibody, possibly a monoclonal antibody, is used which has been raised against such a synthetic peptide.
- 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 the above referenced paper of Last et al.
- a larger peptide fragment including the above smaller fragment s reported in EP 0394296 and the above fragment is reported in WO 91/08478.
- the isomerization has the effect of transferring that part of the pepti ⁇ e chain which runs downstream of the aspartic acid residue in the carboxy terminus direction from the alpha carboxylic acid of the aspartic acid to which it s bonded via a peptide bond in the normal protein to the side chain carboxylic acid in a non-peptide amide bond, as shown below:
- the non-peptide bonded aspartic acid residue is termed "isoaspartic acid”.
- the present invention now provides m a first aspect a method of measurement of the rate of degradation of a body protein such as collagen, e.g. from bone, comprising determining the amount of one or more D-amino acid containing species in a body fluid by the reaction of said species with an lmmunological binding partner capable of distinguishing said D-amino acid containing species from the corresponding L-amino acid containing species.
- the D-amino acid containing species (peptide analogues) in question may be characteristic of type I, type II or type III collagen, but preferably are characteristic of type I collagen.
- the D-amino acid contained in said species may preferably be D-aspartic acid, D-asparagme, D-glutamic acid or D-glutamine and may be bonded via a normal peptide bond or in its iso-form.
- such a method determines the amount of one or more specific D-aspartic acid or D-isoaspartic acid containing peptide analogues present in said body fluid.
- the method determines the amount of a peptide analogue of formula 2 (below) present in said body fluid:
- D* is D-aspartic ac d or D- lsoaspartic acid, or of one or more peptide analogues incorporating an epitope present in a peptide analogue of formula 2 which contains D-aspartic ac d or D-isoaspartic acid.
- EKAH GGR is SEQ.ID.No.l in the attached sequence listing .
- K-K-K is a cross-link such as a hydroxypyridinium cross-lmk which may be pyridinolme (which may be glycosylated or non-glycosylated) or deoxy- pyridinoline, or any other collagen cross link.
- said determination is carried out using an lmmunological binding partner specific for a D-isoaspartic acid containing species present in the sample during the procedure, preferably said isomerized peptide analogue of formula 2 or a isomerized peptide incorporating an epitope present in the isomerized peptide analogue of formula 2 wnich contains D-isoaspartic acid.
- an lmmunological binding partner specific for a D-isoaspartic acid containing species present in the sample during the procedure preferably said isomerized peptide analogue of formula 2 or a isomerized peptide incorporating an epitope present in the isomerized peptide analogue of formula 2 wnich contains D-isoaspartic acid.
- the lmmunological binding partner may be a monoclonal or polyclonal antibody.
- the lmmunological binding partner be specific for tne D-optically active amino acid containing species is meant that the lmmunological binding partner distinguishes between said species and the analogous L-amino acid or L-isoamino acid containing species to an extent useful in the assay.
- Suitable lmmunological binding partners also include fragments of antibodies capable of binding the same antigemc determinant including Fab, Fab' and F(ab') . agments .
- the lmmunological binding partner is an antibody raised against a linear D-peptide analogue or -.somerized pepti ⁇ e analogue, preferably a synthetic D- peptide analogue or isomerized peptide analogue, corresponding to a sequence within collagen witn a D-amino acid, e.g. D-aspartic acid or D-isoaspartic acid substituting in said am o acid sequence for the corresponding L-amino acid, e.g. aspartic acid, in said collagen protein sequence.
- a D-amino acid e.g. D-aspartic acid or D-isoaspartic acid
- the assay may take many forms including but not limited to heterogeneous assays e.g. ELISA and, RIA and homogeneous assays, e.g. turbidimetric assays, procedures for which are too well known to warrant description here.
- heterogeneous assays e.g. ELISA and, RIA
- homogeneous assays e.g. turbidimetric assays, procedures for which are too well known to warrant description here.
- the invention includes the use in an assay for collagen derived peptides or isomerized peptides of a synthetic peptide analogue or isomerized peptide analogue having an ammo acid sequence corresponding to a sequence within collagen with a D-amino acid, e.g. D- aspartic acid or D-isoaspartic acid, substituting in said ammo acid sequence for the corresponding L-am o acid, e.g. L-aspartic acid, said collagen protein sequence.
- a D-amino acid e.g. D- aspartic acid or D-isoaspartic acid
- the said synthetic peptide analogue or isomerized peptide analogue may be used to compete for an lmmunological binding partner with one or more D-form peptide analogue or isomerised peptide analogues in the sample .
- the D-form synthetic peptide analogue or pepti ⁇ e isomer analogue may be immobilised on a solid support.
- a sample may be incubated with a polyclonal antibo ⁇ y reactive with the synthetic peptide analogue or peptide isomer analogue in contact with the solid support and after washing, a peroxidase-con ⁇ ugated (revealing) antibody may be added. After further incubation, a peroxidase substrate solution is added.
- D- form peptide analogue or peptide isomer analogue in the sample reactive with the antibody inhibits the peroxidase reaction .
- the D-form synthetic peptide analogue or peptide isomer analogue may be used to raise a monoclonal lmmunological binding partner.
- the syntnetic peptide analogue or isomerized peptide analogue need not then be a competing agent in the assay.
- collagenase treated collagen may be purified and immobilised onto the solid support and an ELISA may be carried out using a monoclonal antibody.
- the invention includes an antibody, preferably a monoclonal antibody, specific for an amino acid sequence corresponding to a sequence within a protein, e.g. collagen, with a D-amino acid, e.g. D-aspartic acid or isoaspartic acid, substituting in said am o acid sequence for the corresponding L-amino acid, e.g. aspartic acid, in said protein, e.g. collagen, sequence.
- a monoclonal antibody specific for an amino acid sequence corresponding to a sequence within a protein, e.g. collagen
- a D-amino acid e.g. D-aspartic acid or isoaspartic acid
- the antibody is specific for a peptide analogue sequence or an isomerized peptide analogue sequence including the sequence EKAHD*GGR or EKAHiD ⁇ GGR (SEQ. ID. o .1 ) an epitope included in either sequence and containing D*, wherein D* is D-aspartic acid or D-isoaspartic acid.
- this aspect of the invention includes an antibody, preferably a monoclonal antibody, reactive with an epitope containing, contained in, or constituted by the peptide isomer sequence EKAHD*GGR or EKAH ⁇ D*GGR, (SEQ. ID. No.1) , wherein ID* is D-isoaspartic acid and D* is D-aspartic acid.
- EKAHD*GGR or EKAH ⁇ D*GGR SEQ. ID. No.1
- ID* is D-isoaspartic acid
- D* D-aspartic acid
- the invention provides an antibody, preferably a monoclonal antibody, raised against a peptide analogue or peptide-isomer analogue having an am o acid sequence corresponding to a sequence withm a protein, e.g. collagen, with a D-amino acid, e.g. D-aspartic acid or D- lsoaspartic acid, substituting in said amino acid sequence for the corresponding L-amino acid, e.g. L-aspartic acid in said collagen protein sequence.
- a monoclonal antibody raised against a peptide analogue or peptide-isomer analogue having an am o acid sequence corresponding to a sequence withm a protein, e.g. collagen, with a D-amino acid, e.g. D-aspartic acid or D- lsoaspartic acid, substituting in said amino acid sequence for the corresponding L-amino acid, e.g. L-aspartic
- the invention includes cell lines producing monoclonal antibodies according to the third or fourth aspects of the invention .
- the invention also includes antibodies according to the third or fourth aspects of the invention coupled to a detectable marker.
- detectable markers include, but are not limited to , enzymes, chromophores, fluorophores, coenzymes, enzyme inhibitors, chemilummescent materials, paramagnetic materials, spin labels, radio-isotopes, nucleic acid or nucleic acid analogue sequences.
- the invention includes the use m an assay for collagen or other protein derived peptides of an antibody specific for an ammo acid sequence corresponding to a sequence with the protein, (e.g. collagen) with a D- amino acid, e.g. D-aspartic acid or D-isoaspartic acid, substituting in said amino acid sequence for the corresponding L-ammo acid, e.g. aspartic acid, in said protein (e.g. collagen) sequence to obtain information regarding the amount of D-aspartic acid or D-isoaspartic acid containing peptide analogue or peptide isomer analogue in said body fluid.
- a sequence with the protein e.g. collagen
- a D- amino acid e.g. D-aspartic acid or D-isoaspartic acid
- the invention includes a synthetic peptide isomer having an amino acid sequence corresponding to a sequence withm collagen with a D-ammo acid, e.g. D- aspartic acid or D-isoaspartic acid, substituting said ammo acid sequence for the corresponding L-ammo acid, e.g. L-aspartic acid, m said collagen protein sequence, preferably in at least the substantial absence of the corresponding all L-peptide.
- a glyc e residue adjacent the D- ammo acid residue site in the native peptide form of the ammo acid sequence as an adjacent glycme facilitates the racemisation of aspartic acid and other relevant ammo acids .
- Antibodies may be prepared which are respectively selective for one or more L-aspartic acid containing peptides and for their D-aspartic acid or D-isoaspartic acid containing analogues. It is then possible to carry out an assay for both D and L variants of the peptide or peptides.
- the invention provides a method of obtaining information regarding collagen resorption a patient, comprising measuring in a body fluid the relative amounts of at least one L-ammo acid, e.g. L-aspartic acid, containing peptide derived from collagen and a corresponding D-amino acid, e.g. D-aspartic acid or D-isoaspartic acid containing peptide analogue.
- L-ammo acid e.g. L-aspartic acid
- D-aspartic acid or D-isoaspartic acid containing peptide analogue e.g. D-aspartic acid or D-isoaspartic acid containing peptide analogue.
- kits useful in the methods described above.
- Such kits may comprise an antibody according to the third or fourth aspect of the invention, or similarly specific antibody fragment, preferably in combination with any one or more of:- a synthetic peptide analogue containing a D-amino acid such as D-aspartic acid or D-isoaspartic acid reactive with the antibody, an antibody-enzyme conjugate and/or a substrate therefor, an enzyme conjugate-substrate reaction stopping composition, or a wash solution.
- Suitable body fluids include, human or animal urine, olood, serum, plasma and synovial fluid. It is contemplated that the method may also be used e.g. on saliva and sweat.
- Tne body fluid may oe 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 chromatography, hydroxyapatite chroma- tography, and combinations thereof.
- the peptide used in the Examples below is (in its ⁇ L form), EKAHDGGR (SEQ. ID. No.1 ) (Glu . Lys .Ala . His .Asp . Gl . Gly . Arg) and in body fluids such as urine may be present as part of a variety of larger peptides which may include crosslinks of the various types formed in collagen. It derives from the non-helical C-termmal part of type I collagen.
- FIG 1 shows the results obtained in Example 1 depicted graphically.
- Figure 2 shows the correlation between measurements of ⁇ L and ⁇ D forms of the peptide m urine samples.
- the assaying of type I, II or III collagen fragments n urine or other body fluid is performed by an inhibition ELISA (enzyme linked immunosorbent assay) by metering off a sample of urine or other body fluid, contacting the sample with a synthetic peptide D-analogue having a sequence derived from collagen and with an antibody, which is immunoreactive with the synthetic peptide D-analogue.
- the synthetic peptide D-analogue is immobilised on a solid support.
- the antibody may be raised against the synthetic peptide D-analogue or may be raised against collagen degradation products.
- the preparation of synthetic peptides and peptide D- analogues 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., Immunochemistry of the extracellular matrix, 1:1-29(1982), Eyre, D.R., Ann. Rev. Biochem. 53:717-48 (1984), or US Patent No. 5140103). Examples of such peptides sequences are given in Table 1 below.
- the conventional peptide synthesis method applied to aspartic acid containing peptides, but using D-aspartic acid may produce a mixture of peptide (with normal peptide bonded aspartic acid ⁇ D ) and peptide analogue with isomerization of the bonding to the aspartic acid ( ⁇ D ) .
- synthetic peptide analogues it is possible to omit (or add) one or more amino acid residues from (or to) the crosslmkable site sequences without substantial loss of the ability to (a) raise antibodies recognising the ⁇ D or ⁇ D analogue of the corresponding native collagen fragment or (b) inhibit the binding of such anti- bodies to the said analogue of the native fragment. It is possible to use longer collagen fragments and/or chimeric peptide analogues to raise the antibodies and, in principle, it is not necessary to use the same peptide analogue as the immunogen and the competitor in a competition assay.
- telopeptide analogues N C ⁇ l (II) N-term. Gly-Asp-Ile-Lys-Asp-Ile-Val - SEQ. ID. No.6 ⁇ l (II) C-term. Glu-Lys-Gly-Pro-Asp - SEQ. ID. No.7
- Suitable carrier molecules include, but are not limited to, bovine serum albumin, tnyroglobulin, ovalbum , tetanus toxoid, and keyhole limpet hemocyamn.
- the preferred carrier is bovine serum albumin.
- Suitable procedures include, but are not limited to, glutaraldehyde, carbodnmide, 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 containing natural racemisation and optionally isomerization or synthetic D-form peptide analogues conjugated to a carrier.
- the immunogen be mixed with an adjuvant before injection.
- adjuvants include, but are not limited to, aluminium 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-camer molecule can be produced.
- 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 isomerized peptide fragments derived from collagen.
- Suitable cancer cells include, but are not limited to, myeloma, hepatoma, carcinoma, and sarcoma cells.
- a preferred preliminary screening protocol comprises the use of synthetic D- peptide analogue conjugated to a carrier and coated on to the solid surface of a microtitre plate.
- Suitable animal 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 a D-amino acid containing synthetic peptide analogue of appropriate sequence.
- Antibody fragments are prepared by methods known in the art (see E. Ishikawa. Journal of Immunoassay 3:209-327 (1983) ) . Conduct of Immunoassays
- an immunoassay with the antibodies prepared as above it is possible to assay a bio- logical fluid sample without prior fractionation or hydrolysis.
- the specificity for the desired collagen fragments m the biological fluid may be supplied by the antibody in combination with the use of a synthetic D- peptide analogue (against which the antibody was raised or m any event with which the antibody is immunochemically reactive) the assay construction.
- 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 analogue of 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 analogue.
- 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 precoated 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 under- stood, the assay is constructed so as to rely on the interaction between the specific lmmunological binding partner and the desired analyte for specificity and to utilise some means to detect the complex formed by the analyte and the lmmunological binding partner.
- the lmmunological binding partner may be complexed to a solid support and used as a capture lmmunological 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.
- 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 avail-able m the art is multitudinous. For a variety of such protocols, see US. Patent No. 5001225.
- a homogeneous assay format may be used in which for instance latex particles are conjugated to the peptide or isomerised peptide and the sample and the particles compete to bind the antibody. Specific agglutination of the particles by antibody produces a change which is optically detectable as a change in scattering or absorbance and which is inhibited by crosslinks in the sample.
- 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.
- such a kit includes a microtiter plate coated with a relevant synthetic peptide D-analogue, standard solutions for preparation of standard curve, a body fluid (e.g. urine) control for quality testing of the analytical run, rabbit antibodies reactive with the above mentioned synthetic peptide D-analogue, anti-rabbit immunoglobulins conjugated to peroxidase, a substrate solution, a stopping solution, a washing buffer and an instruction manual.
- immunoassays can be constructed using antibodies and specific synthetic peptide D-analogues, the ratios of the corresponding collagen fragment sequences in an approp ⁇ ate biological fluid can be determined as well as their individual levels and their total.
- the assay can be designed to include antibodies which will result in determination of several peptide D-analogues and optionally the native peptide sequences or determination of a single D- amino acid containing peptide analogue sequence, or any desired combination thereof.
- bone metabolic balance is advantageously determined by the substantially simultaneous determination of a marker of the formation of bone in the same or other appropriate biological fluid from the same individual.
- substantially simultaneous means the same day, preferably within 4 hours.
- markers include osteocalcm (also Known as bone GLA protein of BGP) , propeptides of procollagen type I, bpne alkaline phosphatase and total alkaline phosphatase. Suitable methods for the determination of these markers can be found, for example, in Delmas, P.D., et al., J. Bone Mm. Res. (1986) 1:333-337.
- the assay of the present invention which provides an index to determination of the metabolic status of tissues, which generate collagen-derived peptides and peptide analogues when degradation occurs, is useful in a variety of contexts.
- the assays are methods to assess an abnormal condition of a subject by indicating, for example, excessive bone resorption. This may show the presence of an osteo- porotic condition or the metastatic progress of a malignancy. Other conditions characterised by excessive bone resorption include Page ' s disease and hyper- parathyroidism. Likewise, a number of other disease states involving connective tissue may be monitored by determination of the degradation of collagen.
- Examples are collagen type II degradation associated with rheumatoid arthritis and osteoarthritis and collagen type III degradation in vascuiitis syndrome. Since the condition of the subject can be monitored continuously, application of these assays can also be used to monitor the progress of tnerapy administered to treat these or other conditions. Further, the assays can be used as a measure of toxicity, since the administration of toxic substances often results tissue degradation.
- the assays may be applied in any situation wherein the metabolic condition of collagen tissues can be used as an index of the condition, treatment, or effect of substances directly a ⁇ ministered to the subject or to which the subject is exposed in the environment.
- ⁇ L CROSSLAPS RIA This assay employs in RIA format a monoclonal antibody raised against the peptide EKAHDGGR (all L- and normally peptide bonded) .
- ⁇ L CROSSLAPS ELISA This assay in ELISA format utilises a rabbit antiserum produced by immunisation with bacterial collagenase treated bone collagen (CTC) and spontaneously having essentially no reactivity to the peptide EKAHDGGR in its normal ⁇ L form and essentially no reactivity to the ⁇ D analogue of said peptide.
- EKAHDGGR SEQ. ID. No .1
- EKAH ⁇ D DGGR EKAHDGGR
- One antiserum was selected that had the greatest specificity for this peptide analogue.
- the ELISA format used was as follows :-
- the polyclonal ELISA is based on an immobilised synthetic peptide analogue with an amino acid sequence of eight am o acids (8AA) characteristic of an isomerised part of the C-telopeptide of the ⁇ l-cha of type I collagen (Glu- Lys-Ala-His-Asp-Gly-Gly-Arg-) (SEQ . ID . o.1 ) in L or D form.
- 8AA amino acid sequence of eight am o acids
- a 25 ⁇ l sample or standard is added to each well of a formula 1 antigen-coated microplate, followed by lOO ⁇ l of antiserum raised against collagenase treated type 1 collagen.
- the plates are incubated for I hour at room temperature under agitation and washed five times with a washing buffer.
- a goat anti-rabbit immunoglobulin G horseradish peroxidase conjugate (lOO ⁇ l) is added to each well. After incubation for 1 hour at room temperature, plates are washed five times as before.
- the enzyme substrate (lOO ⁇ l/well) is added, and after 30 minutes of incubation in the dark, the reaction is stopped by adding lOO ⁇ l 0.18M H.SO,.
- the optical density of 450 nm is measured with a microplate reader. Duplicate measurements are performed for each sample, and tne data are expressed as nanograms per mol creatinine (Cr) , measured by a standard colorimetnc technique .
- Varying concentrations of the peptide EKAHDGGR (SEQ. ID. No.1) and its analogues EKAH ⁇ L DGGR and EKAH ⁇ D DGGR were formulated as samples and run in ⁇ D CROSSLAPS ELISA as described above. The results are shown in Figure 1. Cross reactivity is calculated based on the concentration of sample needed to inhibit 50% of the signal generating binding to the plate m the assay. Compared to ⁇ D , the cross-reactivity to both ⁇ and ⁇ L is below 0.5%, showing chat the assay is highly specific for the ⁇ D form of the isomerised sequence which compared to the natural sequence nas undergone both lsome ⁇ sation and racemisation.
- MOLECULE TYPE peptide (in) HYPOTHETICAL: NO (lv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
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JP10510349A JP2000516721A (en) | 1996-08-22 | 1997-08-12 | Method for measuring D-amino acids in body fluids |
US09/242,721 US6300083B1 (en) | 1996-08-22 | 1997-08-12 | Assaying D-amino acids in body fluids |
AU39435/97A AU3943597A (en) | 1996-08-22 | 1997-08-12 | Assaying d-amino acids in body fluids |
EP97936704A EP0922228A2 (en) | 1996-08-22 | 1997-08-12 | Assaying d-amino acids in body fluids |
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WO1998026286A2 (en) * | 1996-12-09 | 1998-06-18 | Osteometer Biotech A/S | Sandwich assays for collagen type i fragments |
WO1999020300A1 (en) * | 1997-10-22 | 1999-04-29 | The Regents Of The University Of Minnesota | Inhibition of tumor cell adhesion to type iv collagen |
WO2001013110A2 (en) * | 1999-08-17 | 2001-02-22 | Osteometer Biotech A/S | Detection of isomerised epitopes in autoimmune diseases |
US6300083B1 (en) | 1996-08-22 | 2001-10-09 | Osteometer Biotech A/S | Assaying D-amino acids in body fluids |
WO2002013844A2 (en) * | 2000-08-16 | 2002-02-21 | Osteometer Biotech A/S | Specific autoimmune reactions against isomerised/optically inverted epitopes: application for treatment of autoimmune diseases |
US7354723B2 (en) * | 1999-11-26 | 2008-04-08 | Nordic Bioscience Dagnostics A/S | Assay of isomerised and/or optically inverted proteins and protein fragments |
US8128928B2 (en) | 2002-03-12 | 2012-03-06 | Wyeth Llc | Humanized antibodies that recognize beta amyloid peptide |
US8642044B2 (en) | 1997-12-02 | 2014-02-04 | Janssen Alzheimer Immunotherapy | Prevention and treatment of amyloidogenic disease |
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GB9928052D0 (en) * | 1999-11-26 | 2000-01-26 | Osteometer Biotech As | Assay of isomerised and/or optically inverted proteins and protein fragments |
US20050124071A1 (en) * | 2003-09-30 | 2005-06-09 | Kraus Virginia B. | Methods and compositions for diagnosing musculoskeletal, arthritic and joint disorders by biomarker dating |
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US6300083B1 (en) | 1996-08-22 | 2001-10-09 | Osteometer Biotech A/S | Assaying D-amino acids in body fluids |
WO1998026286A2 (en) * | 1996-12-09 | 1998-06-18 | Osteometer Biotech A/S | Sandwich assays for collagen type i fragments |
WO1998026286A3 (en) * | 1996-12-09 | 1998-08-13 | Osteometer Biotech As | Sandwich assays for collagen type i fragments |
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WO1999020300A1 (en) * | 1997-10-22 | 1999-04-29 | The Regents Of The University Of Minnesota | Inhibition of tumor cell adhesion to type iv collagen |
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WO2001013110A3 (en) * | 1999-08-17 | 2001-06-21 | Osteometer Biotech As | Detection of isomerised epitopes in autoimmune diseases |
US7354723B2 (en) * | 1999-11-26 | 2008-04-08 | Nordic Bioscience Dagnostics A/S | Assay of isomerised and/or optically inverted proteins and protein fragments |
WO2002013844A3 (en) * | 2000-08-16 | 2002-05-30 | Osteometer Biotech As | Specific autoimmune reactions against isomerised/optically inverted epitopes: application for treatment of autoimmune diseases |
WO2002013844A2 (en) * | 2000-08-16 | 2002-02-21 | Osteometer Biotech A/S | Specific autoimmune reactions against isomerised/optically inverted epitopes: application for treatment of autoimmune diseases |
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US9067981B1 (en) | 2008-10-30 | 2015-06-30 | Janssen Sciences Ireland Uc | Hybrid amyloid-beta antibodies |
Also Published As
Publication number | Publication date |
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GB9617616D0 (en) | 1996-10-02 |
WO1998008098A3 (en) | 1998-04-30 |
JP2000516721A (en) | 2000-12-12 |
US6300083B1 (en) | 2001-10-09 |
AU3943597A (en) | 1998-03-06 |
EP0922228A2 (en) | 1999-06-16 |
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