[go: up one dir, main page]

WO1998007032A1 - Method and kit for assessing bone resorption - Google Patents

Method and kit for assessing bone resorption Download PDF

Info

Publication number
WO1998007032A1
WO1998007032A1 PCT/US1997/014434 US9714434W WO9807032A1 WO 1998007032 A1 WO1998007032 A1 WO 1998007032A1 US 9714434 W US9714434 W US 9714434W WO 9807032 A1 WO9807032 A1 WO 9807032A1
Authority
WO
WIPO (PCT)
Prior art keywords
tgf
level
bone
latent
resoφtion
Prior art date
Application number
PCT/US1997/014434
Other languages
French (fr)
Inventor
David J. Grainger
Original Assignee
Metra Biosystems, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metra Biosystems, Inc. filed Critical Metra Biosystems, Inc.
Priority to AU41520/97A priority Critical patent/AU4152097A/en
Publication of WO1998007032A1 publication Critical patent/WO1998007032A1/en

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/495Transforming growth factor [TGF]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention relates to methods for detecting screening for and monitoring bone metabolism abnormalities, particularly those relating to elevated bone reso ⁇ tion
  • the invention also relates to reagents and kits for use in such methods
  • a variety of disease conditions in humans are characterized by an elevated level of bone reso ⁇ tion or by an abnormal imbalance between bone reso ⁇ tion and bone formation.
  • these conditions are osteoporosis, osteoarthritis. hyperthyroidism, hype ⁇ arathyroidism, osteomalacia, osteosarcomas, and Paget's disease (Epstein, 1986; Van
  • Osteoporosis is particularly problematic, affecting upwards of twenty million people in the United States alone. Osteoporosis is characterized by pathologically low bone density, often first presenting clinically with fractures. Both the deposition of bone, which is mediated by osteoblasts, and bone reso ⁇ tion, which is mediated by osteoclasts, occur in an ongoing recycling process of the bone matrix throughout the lifetime of humans.
  • agents have been found to affect the rate of deposition of inorganic matrix by cultured osteoblasts, either by affecting the rate of proliferation of these cells or by increasing their differentiation status. Similarly, agents have been identified which modulate the rate of reso ⁇ tion by osteoclasts in bone explant cultures. The in vivo roles of many of these agents remain to be clarified, and the signalling pathways which mediate deposition and reso ⁇ tion are not well understood.
  • a number of serum and/or urinary substances have been proposed as possible indicators for bone degradation.
  • Exemplary substances which have been studied include hydroxyproline, tartrate-resistant acid phosphatase, galactosyl hydroxylysine, various collagen peptides, and peptide-free pyridinium species (e.g. , Taylor, 1994).
  • the invention includes a method of assessing the status of bone reso ⁇ tion in a mammalian subject, based on measuring the level of total TGF-B3, or a selected subform thereof, in a body fluid sample from the subject.
  • a level of total TGF-B3 in the sample is determined.
  • the determined level is compared with a predetermined threshold or level characteristic of normal subjects, wherein a determined level that is above a level characteristic of normal subjects is an indication that the subject has a bone reso ⁇ tion disorder.
  • a level below a selected threshold which may be different from the first threshold, may indicate that the subject's level of bone reso ⁇ tion is within normal limits.
  • the invention includes a method of assessing the status of bone reso ⁇ tion in a mammalian subject
  • the levels of (1) non-latent TGF- ⁇ and (n) the sum of small latent TGF- ⁇ plus mature TGF- ⁇ 1 dimer are determined in a body fluid from the subject
  • the determined levels are compared with each other, where a determined ratio of non-latent TGF- ⁇ versus small latent TGF- ⁇ plus mature ⁇ l dimer (non-latent/(small latent TGF- ⁇ + mature ⁇ l)) above a selected threshold is an indication that a bone reso ⁇ tion disorder may be present in the subject
  • a ratio below a selected threshold which may be different from the first threshold, may be used as an indication that the subject's level of bone reso ⁇ tion is within normal limits
  • the methods of the invention are useful in the context of detecting or aiding in the diagnosis of a variety of disorders associated with bone metabolism abnormalities (bone reso ⁇ tion disorders), such as osteoporosis, osteoarth ⁇ tis, hype ⁇ arathyroidism rheumatoid arthritis, Paget's disease, or a malignant tumor or metastatic cancer in bone
  • bone reso ⁇ tion disorders such as osteoporosis, osteoarth ⁇ tis, hype ⁇ arathyroidism rheumatoid arthritis, Paget's disease, or a malignant tumor or metastatic cancer in bone
  • the methods of the invention may be used as a screening tool to identify subjects from the general population who are at increased risk for elevated bone loss or bone fracture, where a level, ratio or difference of TGF- ⁇ species above a selected threshold is an indication of increased risk of developing a bone reso ⁇ tion disorder, such as osteoporosis, or of suffering a bone fracture
  • the methods of the invention may also be used in combination with other diagnostic methods, such as radiographic techniques for measuring bone density, to further define a patient's condition That is, if an elevated level, ratio, or difference of an analyte or analytes of the invention is detected, indicating the presence of a bone reso ⁇ tion disorder or an increased risk for such a disorder or bone fracture, the subject is then further tested using an independent technique, such as lumbar spine bone mineral density (LS-BMD) measurements, to further characterize the health status of the subject
  • LS-BMD lumbar spine bone mineral density
  • the methods of the invention may also include determining the level of an indicator of bone formation, such as bone alkaline phosphatase, tartrate resistant acid phosphatase (TRAP), Type HI procollagen, or the like, to assess the balance between bone formation and bone reso ⁇ tion in the subject
  • an indicator of bone formation such as bone alkaline phosphatase, tartrate resistant acid phosphatase (TRAP), Type HI procollagen, or the like
  • the invention includes a method of monitoring bone degradation in a subject, for detecting changes in the bone degradation status of the subject over time
  • the level, ratio or difference of TGF- ⁇ species discussed above is measured at least twice in a body fluid from the subject over a period of time, where the measured level, ratio or difference is used to assess whether bone reso ⁇ tion has increased, decreased, or stayed the same
  • the invention includes a substantially pure OP-TGF- ⁇ polypeptide having an apparent molecular weight of between 10 and 15 kDa, as measured by gel filtration chromatography under non-denaturing conditions, and which is characterized by the elution and
  • TGF- ⁇ activity properties shown in Figs 3B and detailed in Example 7
  • the polypeptide is particularly useful as a standard cahbrant in the OP-TGF- ⁇ assay above
  • the analytes of the invention are measured by any analytical method capable of quantitatively measuring the analyte(s) of interest without significant interference from other components in the sample fluid
  • suitable methods may include chromatographic, electrophoretic, and immunoassay methods, and combinations thereof
  • the analyte(s) are measured by immunoassay techiques using polyclonal or monoclonal antibodies, or art-recognized equivalents thereof, which are specific for the analyte(s) of interest
  • the invention also contemplates kits and reagents for use in carrying out the methods described above
  • Fig 1A illustrates an assay format for measuring small latent TGF- ⁇ species plus mature ⁇ l dimer
  • Fig. IB illustrates an assay format for measuring non-latent TGF- ⁇ species
  • Figs 2A and 2B show serum TGF- ⁇ 3 levels measured in normal (2A) and osteoporotic subjects (2B), Fig 3A shows chromatograms of TGF- ⁇ -contaimng fractions (assay in accordance with
  • Fig 4 shows serum TGF- ⁇ 3 levels measured in four groups of patients (only individuals with TGF-B3 concentrations > 0 3 ng/mL are shown) group 1 28 women with normal bone density, group 2 24 osteoporotic women presenting with vertebral fracture, group 3 1 1 osteoporotic women with a family history of osteoporosis, and group 4 26 osteoporotic patients with osteoporosis of the hip
  • body fluid includes any human body fluid suitable for diagnostic assay for assessing the level of bone reso ⁇ tion in accordance with the present invention
  • body fluids include whole blood and blood fractions, such as serum, plasma, and platelet-poor plasma, urine, saliva, sweat, synovial fluid, tear fluid, cerebral spinal fluid, and liquid extracts of bone tissues (e g , bone biopsy)
  • the body fluid is a blood fraction or urine, and more preferably is serum or plasma
  • TGF- ⁇ and active, mature TGF- ⁇ refer to the homodime ⁇ c form of TGF- ⁇ comprising ammo acid residues 279 to 390 of the pre-pro polypeptide form of TGF- ⁇
  • active TGF- ⁇ or “non-latent TGF- ⁇ ” encompasses monome ⁇ c and homodime ⁇ c forms of TGF- ⁇ comprising amino acid residues 279 to 390 of the pre-pro polypeptide form of TGF- ⁇ , or a subset thereof, and which is not complexed with LAP or LTBP
  • “Latent-associated peptide” and “LAP” refer to a polypeptide chain spanning residues 30 to 278 of the pre-pro polypeptide form of TGF- ⁇
  • Distal latent TGF- ⁇ refers to a 1 10 kDa dimer containing a mature TGF- ⁇ dimer complexed noncovalently with two LAP chains
  • Plate large latent complex refers to a 220-240 kDa complex containing a mature TGF- ⁇ dimer, two LAP chains, and an LTBP chain
  • Latent TGF- ⁇ binding protein and LTBP refer to the 130 kDa polypeptide which associates covalently with latent TGF- ⁇ to form the 220-240 kDa large latent complex, as described in Miyazono et al (1988)
  • Latent TGF- ⁇ and “latent forms of TGF- ⁇ ” refer to proteins or protein complexes which contain a mature TGF- ⁇ dimer and at least one other associated polypeptide, and which are unable to interact with cellular surface receptors for TGF- ⁇
  • Exemplary latent forms of TGF- ⁇ include small latent TGF- ⁇ and the large latent complex
  • TGF- ⁇ isoform refers to mature TGF- ⁇ 1 , ⁇ 2, 153, ⁇ 4, and/or ⁇ 5, a polypeptide derived therefrom, or a polypeptide having greater than 95 % homology therewith
  • total TGF- ⁇ 3 The phrases “total TGF- ⁇ 3" .
  • total TGF- ⁇ 3 species and “total TGF- ⁇ 3 antigens” refer to the total amount of mature TGF-153 species (and fragments thereof) measurable after release of any latent proteins from the TGF- ⁇ 3 species present in the sample
  • total TGF- ⁇ 3 can be prepared by treating the sample with acidic urea, as described below
  • proteolyt-ic treatments may be used, for example, to selectively destroy associated structure of the TGF-B3 polypeptides to be measured
  • test subject is intended to have its conventional meaning, and includes man, horse, cow, sheep, rabbit, dog, cat, rat, and mouse, for example
  • test subject is a human subject
  • TGF- ⁇ Transforming growth factor- ⁇
  • TGF- ⁇ is a plu ⁇ potent cytokme which has been found to regulate cell growth in a variety of tissues Reported activities include suppression of thymocytes and lymphocytes, stimulation or inhibition of osteoblast function, mediation of the formation of extracellular matrices, and more generally, inhibition or stimulation of proliferation and differentiation of a host of cell types (e g , Sporn et al , 1992, 1993, Ammann et al , 1992) TGF- ⁇ has also been proposed for use in wound repair (Derynck et al , 1989), for promoting bone growth (Ammann et al , 1992), and for immunosuppression and septic shock (Burnier et al , 1991 ) To date, at least five isoforms of TGF- ⁇ have been identified in mammals, designated
  • TGF- ⁇ l through TGF- ⁇ 5 Of these, only TGF- ⁇ l , ⁇ 2, and ⁇ 3 have been found in humans The five isoforms share high sequence similarities and overlapping biological activities, although some differences in activity have been reported (R&D Systems 1994 Catalog, Minneapolis, MN, pp 4-6) In humans, TGF- ⁇ l is found in highest concentrations m platelets and bone, TGF-B2 is found in bone and other tissues, and TGF-B3 is found mostly in cells of esenchymal origin Properties of the TGF- ⁇ proteins have been reviewed (Sporn et al , 1992, 1993)
  • TGF- ⁇ is synthesized as a 390 residue pre pro polypeptide which dime ⁇ zes to form a homodimer
  • the pre-pro form contains a 29 residue signal sequence which is cleaved mtracellularly to form a pro-TGF- ⁇ l dimer of 261 -residue chains
  • pro-TGF- ⁇ l is further cleaved to produce a 1 10 kDa nicked dimer in which the polypeptide chains of the dimer have been cleaved between Arg278 and Ala279
  • the newly created C-terminal chains, spanning residues 279 to 390 from the pre- pro polypeptide, constitute the polypeptide chains
  • Additional proteins may associate with latent TGF- ⁇ , either covalently or non-covalently to form additional forms of TGF- ⁇
  • a complex has been isolated from human platelets containing LAP linked to an additional 130 kDa protein (known as the latent TGF- ⁇ binding protein, or LTBP) by a single disulphide bond (Miyazono et al , 1988, 1991)
  • This 220-240 kDa complex which includes a mature (25 kDa) TGF- ⁇ dimer, two associated LAP chains, and an LTBP chain, has been termed the "large latent complex '
  • Active, mature TGF- ⁇ can be formed in vitro by exposing latent TGF- ⁇ or the large latent complex to strong acid, strong base, or chaotropic agents Such conditions presumably disrupt the non-covalent interactions between the mature TGF- ⁇ dimer core and the non-covalently attached polypeptides (LAP and LTBP) to produce active TGF- ⁇ Formation of active TGF- ⁇ in vivo may occur through the action of proteases which degrade the LAP but not the TGF- ⁇ core Plasmm has been implicated as the physiological activator under some circumstances (Grainger et al , 1994, 1995), but other proteases may also be involved (Kojima et al , 1993 Dennis et al , 1991) At present, the actual in vivo mechanism for forming active TGF- ⁇ from its latent forms is not known III Reso ⁇ tion Assay Methods
  • the present invention is directed generally to a method of assessing the status of bone reso ⁇ tion in a subject, based on the measurement of certain TGF- ⁇ species in a body fluid, particularly serum, plasma or urine
  • a measured level, ratio, or difference, of the selected TGF- ⁇ species that is at or above a selected threshold value is an indication that an elevated level of bone resorption is present in the subject
  • the method may also be configured such that a measured level, ratio or difference, of the selected TGF- ⁇ species that is at or below a selected threshold value can be used as an indication that bone reso ⁇ tion is within normal limits
  • the level of true positives detected must be weighed against the number of false positives as well as false negatives For example, a higher threshold will lead to a lower rate of false positives, but at the expense of reduced
  • the method of the invention may be used in a monitoring mode, where at least two measurements are made in body fluid samples from a subject at different times, and the change in the measured level, if any, is used to determine whether the level of bone reso ⁇ tion has increased, decreased, or remained the same
  • the invention may also be used in prognostic applications, for predicting the likelihood or risk of developing a bone reso ⁇ tion abnormality such as discussed above, or for predicting the likelihood or risk of suffering a bone fracture
  • an increased risk may be indicated by a level of selected TGF- ⁇ species that is above a selected threshold, where the threshold has been selected on the basis of clinical trials, for example, or by a level which is a selected percentage above a baseline level established for a particular individual
  • the methods of the invention may be used in a screening capacity for the general public, or may be targeted to certain patients for whom such testing is deemed appropriate
  • the present reso ⁇ tion assessment methods may be used in conjunction with other medical techniques, such as bone density measuring methods or diagnostic assays based on other markers
  • TGF- ⁇ -containing species may be detected using antibodies which are lmmunoreactive with one or more TGF- ⁇ species, allowing highly sensitive detection of low concentrations of the species of interest by conventional immunoassay techniques
  • Antibodies raised against mature TGF- ⁇ l , ⁇ 2, or ⁇ 3, and having high binding affinity for TGF- ⁇ l are useful for detecting the OP-TGF- ⁇ protein of the present invention
  • Such antibodies are readily available from commercial sources (e g , R&D Systems. Minneapolis. MN, R&D Systems, Oxford, United Kingdom, Genzyme Diagnostics, Cambridge, MA) or may be prepared by standard methods (e g , Harlow, 1988)
  • Section A describes an aspect of the invention based on measuring total TGF-B3, or a subform thereof, such as active ⁇ 3 dimer
  • TGF-B3 species were not known to be present in significant amounts in plasma or serum
  • Section B describes an aspect of the invention based on measuring the levels of (I) non- latent TGF- ⁇ and (u) the sum of small latent TGF- ⁇ plus mature TGF- ⁇ l dimer are determined in a body fluid from the subject The ratio or difference of these measured levels is used as an indicator of the level of bone reso ⁇ tion in the subject
  • Section C describes an aspect of the invention based on the measurement of a novel protein designated herein as OP-TGF- ⁇
  • the level of total TGF- ⁇ 3, or alternatively, mature TGF- ⁇ 3 dimer, in a body fluid is measured, where a measured level above a selected threshold level is an indication that a bone metabolism abnormality associated with elevated bone resorption may be present in the subject Similarly, a level below a selected threshold, which may be different from the first threshold, may indicate that the subject's level of bone reso ⁇ tion is within normal limits
  • TGF-B3 may be measured using any analytical technique available in the art, and is conveniently measured using immunoassay techniques
  • Example 4 describes an exemplary assay format which is useful in this aspect of the invention
  • the assay includes an immobilized TGF- ⁇ Type II receptor such as described in Example 1 , for binding the TGF- ⁇ 3 dimer to the support for subsequent detection
  • the assay further includes an antibody which is specific for the ⁇ l isoforms of TGF, to bind to and sequester the ⁇ l isoforms, to prevent ⁇ l monomer or dimer from competing with ⁇ 3 dimer for binding to the immobilized receptor This allows low levels of TGF- ⁇ 3 to be detected even in the presence of a high concentration of TGF- ⁇ l species, when the binding partner is cross reactive with both ⁇ 1 and ⁇ 3 isoforms
  • the serum samples are treated with acidic urea to release the active, dime ⁇ c forms of TGF- ⁇ 3 from their latent forms
  • the samples are mixed with a neutralization solution and then incubated with a ⁇ l-specific antibody (BDA19) to sequester ⁇ l species in the samples
  • BDA19 ⁇ l-specific antibody
  • the samples are then contacted with the immobilized TGF- ⁇ receptors to bind the TGF-B3 to the solid support
  • Primary, non-immobilized antibody is then added to bind to the immobilized TGF- ⁇ 3, followed by addition of peroxidase-labeled antibody for detection
  • the non-latent forms of TGF-B3 can be measured without treating the sample to convert latent to non-latent forms
  • the subjects with low bone density exhibited a mean TGF-B3 level of 6 4 .+ . 0 6 ng/mL, compared with 2 7 +_ 0 6 ng/mL for the control group
  • more than half of the control individuals showed no detectable TGF-B3
  • all but two of the osteoporosis subjects showed detectable TGF-B3 levels greater than about 2 ng/mL
  • Fig 4 shows additional data illustrating the usefulness of the method
  • Serum samples were collected from 28 women with normal bone density (group 1) and 61 women with osteoporosis (groups 2-4) Serum TGF- ⁇ 3 levels were measured as described in Example 4 Results ( 1 ) Of the 28 women with normal bone density, 5 had detectable serum TGF-B3 levels ( > 0 1 ng/mL), but only one had a level greater than 2 ng/mL (2 2 ng/mL) (2) Of the 61 osteoporotic women (groups 2-4 together).
  • Sample Storage Serum samples containing TGF- ⁇ 3 can be subjected to repeated freeze-thaw cycles with only a slight reduction in the measured amount of TGF-B3 Thus, repeated freeze-thawing of human serum samples does not significantly affect TGF- ⁇ 3 measurements However, it is preferable to store samples at -20°C until measurement of TGF- ⁇ 3, without thawing until testing
  • the applicant has found that red blood cells contain about 800 molecules of TGF- ⁇ 3 per cell, equivalent to about 100 ng of TGF- ⁇ 3 per mL of blood Thus, hemolysis should be avoided to obtain an accurate measure of serum TGF-B3 species
  • the degree of hemolysis in a test sample is less than 2% , e g , when a threshold of 2 ng/mL is used to discriminate between normal and "diseased" or at- ⁇ sk subjects This level of hemolysis can be readily detected visually by eye from the red color of the sample, indicating that another sample should probably be taken from the subject
  • hemolysis is generally not a significant problem
  • Studies by the applicant have shown that hemolysis of at least 3 2 % (as measured by assaying serum hemoglobin concentration after drawing blood through a fine gauge needle a variable number of times prior to clotting and collection of serum) is generally
  • TGF- ⁇ 3 Levels and Drug Treatment There is some variation in the effects of hormone-mimetic drugs on TGF- ⁇ 3 levels measured in women For example, prophyllactic hormone replacement therapy (HRT) was found to lead to mildly increased levels of TGF-B3 during treatment
  • HRT prophyllactic hormone replacement therapy
  • Serum and Plasma The assay described in Example 4 detected the same levels of total TGF- ⁇ 3 in serum and platelet-poor plasma prepared from the same blood samples Thus, testing either serum or plasma samples should provide similar results
  • TGF- ⁇ + mature ⁇ l dimer are measured, and either the ratio (i) (n) or the difference (l - n) is used as an indicator of the level of bone reso ⁇ tion in the subject
  • the measurement of non-latent TGF- ⁇ , and the sum of small latent TGF- ⁇ + mature ⁇ l dimer, may be carried out using any suitable analytical approaches known in the art for measuring these species
  • non-latent TGF- ⁇ can be measured by immunoassay using lmmunological binding partners which are lmmunospecific for both desired TGF- ⁇ species, and which do not significantly react with the large latent complex
  • the assay may be homogeneous or heterogeneous, and may utilize a direct (e g , sandwich) or competitive format in accordance with assay configurations well known in the art (e g , Harlow)
  • FIG. 1 A One suitable assay format for measuring non-latent TGF- ⁇ species is illustrated in Figure 1 A In this assay, an immunological binding partner, 12a, 12b, 12c, 12d, here shown as a type
  • TGF- ⁇ receptor is immobilized on a solid support 40, for binding to non-latent monomenc and dimeric forms of TGF- ⁇ , particularly those containing TGF- ⁇ l and TGF- ⁇ 3 chains
  • the assay format also includes antibodies 30a, 30b, for binding to the target TGF- ⁇ species, and reporter-labeled antibodies 32a, 32b, for binding to the primary antibodies 30a, 30b to produce a measurable signal
  • the sample to be tested may contain any or all of the following TGF- ⁇ species (0 a monomenc form of TGF- ⁇ ( 14 in the figure), such as the OP-TGF- ⁇ protein described further below, (n) a mature TGF- ⁇ dimer 16, (in) small latent TGF- ⁇ 20, which contains a TGF- ⁇ dimer 16 and two LAP chains 18, and/or (IV) a large latent complex 24 which contains a mature TGF- ⁇ dimer 16, two LAP chains 18, and an LTPB chain 22
  • TGF- ⁇ species a monomenc form of TGF- ⁇ ( 14 in the figure), such as the OP-TGF- ⁇ protein described further below, (n) a mature TGF- ⁇ dimer 16, (in) small latent TGF- ⁇ 20, which contains a TGF- ⁇ dimer 16 and two LAP chains 18, and/or (IV) a large latent complex 24 which contains a mature TGF- ⁇ dimer 16, two LAP chains 18, and an LTPB chain 22
  • TGF- ⁇ monomenc species 14 is bound by immobilized receptor 12a
  • the immobilized TGF- ⁇ monomer is then detected by binding to a first antibody 30a which binds directly to monomer 14, followed by the binding of reporter- labeled antibody 32a
  • the TGF- ⁇ is then detected by virtue of the reporter-labeled antibody immobilized on the solid support
  • TGF- ⁇ dimer 16 is likewise detected when bound to immobilized receptor 12b, by virtue of binding with primary antibody 30b which in turn is bound by reporter labeled antibody 32b Since receptor 12c does not bind latent forms of TGF- ⁇ , small latent form 20 and large latent form 24 remain free from the support, and thus cannot serve to anchor a reporter labeled antibody 32 to the support Details of an exemplary assay format in accordance with the above are provided in Examples 1 and 2
  • the primary antibody 30a does not bind significantly to the latent forms of TGF- ⁇ , it is not important whether the primary antibody 30a can bind the latent forms as well as the non-latent forms, provided the primary and reporter-labeled antibodies are present in excess relative to the TGF- ⁇ which could potentially be bound
  • immobilized binding partner 12 can be selected to bind to both non-latent as well as latent forms of TGF- ⁇
  • the specificity of the assay for the non- latent forms is achieved by selecting the primary antibody (or other suitable binding partner) to be lmmunospecific for the non-latent forms
  • the immobilized binding partner 12 can be an antibody which binds with TGF- ⁇ species 14, 16, 20, and 24, and antibody 30 could be replaced with a type II TGF- ⁇ receptor which recognizes only non-latent TGF- ⁇ forms
  • the sum of small latent TGF- ⁇ + mature ⁇ l dimer can be measured using an assay format such as illustrated in Fig IB and detailed in Example 3 In the format of Fig I B, receptor 12a from Fig 1A has been replaced with an immunological binding partner 42 (shown schematically as an antibody) which can bind mature ⁇ l dimer (16) and small latent TGF- ⁇ (20), but not monomer 14 or large latent complex 24 Dimer 16 and small latent form 14 may
  • Reporter R is any entity which is effective to produce a detectable signal for the pu ⁇ ose of detecting or quantitating the TGF- ⁇ analyte of interest
  • exemplary reporters include enzymes, such as alkaline phosphatase and horse radish peroxidase, radioactive labels such as l25 I, spin labels, etc
  • binding partner 30 does not bind significantly with monomer 14 or large latent complex 24 m order to avoid competition between species 14 and 24 and species 16 and 20 for binding to binding partner 30 Otherwise, there might be an insufficient amount of binding partner 30 available to bind with and detect species 14 and 16
  • blood samples were collected from 39 women with osteoporosis and 41 women having normal bone density The samples were tested using the non-latent TGF- ⁇ assay of Example 3, and the small latent + mature TGF- ⁇ dimer assay described in Example 2 The results were as follows
  • the average level of small latent TGF + mature dimer was 7 0 _+ 1 6 ng/mL, whereas the average for the osteoporosis subjects was 4 3 +_ 0 8 ng/mL
  • the average level was 5 4 +_ 1 6 ng/mL
  • the average was 10 7 +_ 2 7 ng/mL
  • the average ratio [non- latent] /[small latent + mature dimer) was about 0 8
  • the average ratio for the osteoporosis subjects was about 2 5
  • only 10-15 % of the women with normal bone density had a ratio > 1 0, whereas > 80% of the osteoporosis subjects had such a ratio ( > 1 0)
  • results show that the measured ratio provides a useful indication of the status of bone reso ⁇ tion in human subjects Furthermore, the results show how a threshold can be selected to discriminate between subjects likely to have elevated bone reso ⁇ tion levels, and subjects who are either healthy or whose bone reso ⁇ tion status is uncertain For example, subjects showing ratios greater than 1 0, as just discussed, would be likely candidates for elevated bone reso ⁇ tion levels, although some false positives would also occur Conversely, a ratio of less than 1 0 would be a likely indication that the subject has normal bone reso ⁇ tion, although false negatives on the order of 10-20% might occur It will be appreciated that other threshold ratios could be selected to improve the accuracy of the assay, depending on the desired level of tolerance for false positives or false negatives
  • the difference between the levels of non-latent TGF- ⁇ and small latent TGF- ⁇ + mature ⁇ l dimer is determined, where a difference that is above a selected threshold is an indication that a bone reso ⁇ tion disorder may be present in the subject, and a difference that is below a selected threshold may be used as an indication of normal bone reso ⁇ tion
  • the level of a protein designated OP-TGF- ⁇ in a body fluid is measured, where a measured level above a selected threshold level is an indication that a bone metabolism abnormality associated with elevated bone reso ⁇ tion may be present in the subject
  • Example 7 describes a study showing certain physical properties of the TGF- ⁇ -OP protein of the invention
  • serum samples were obtained from two normal, healthy male subjects (45 and 54 years old, respectively), and from a woman diagnosed with osteoporosis Each sample was applied to an anion exchange column and fractionated using a sodium chloride salt gradient in 10 itiM sodium phosphate, pH 6 8
  • One mL fractions were collected and assayed for TGF- ⁇ l -containing proteins using a commercial ELISA kit (Quantikine ELISA kit, R&D Systems), after an initial acidic urea pre-treatment step to release mature TGF- ⁇ from latent forms
  • the two healthy subjects showed qualitatively similar profiles (Fig 3A) Specifically, the sample from the 45 year old subject showed TGF-prote peaks at elution volumes of about 12, 24, and 30 mL, corresponding to the mature TGF- ⁇ dimer (A), the platelet large latent complex (P), and the small latent form (L), respectively In addition, this sample showed a peak at about 19 mL, which appears to correspond to a previously uncharac- te ⁇ zed form of TGF- ⁇ The sample from the 54 year old showed peaks at about 24 mL and 30 mL, corresponding to the large latent complex and small latent complex, respectively, but only a weak peak at 14 mL for the mature dimer form
  • the osteoporotic subject on the other hand, exhibited a large new peak eluting at about 35 L (indicated by arrow and labeled OP-TGF- ⁇ ), in addition to the peaks typically observed in normal subjects
  • the new peak constitutes a new form of TGF- ⁇ which is clearly distinguishable from other known forms of TGF- ⁇ , particularly mature TGF- ⁇ , small latent TGF- ⁇ , and the 220-240 kDa large latent complex
  • the OP-TGF- ⁇ protein When chromatographed on a gel filtration column, the OP-TGF- ⁇ protein exhibits an elution volume consistent with a molecular weight of about 10 to 15 kDa, suggesting that OP- TGF- ⁇ consists of a mature TGF- ⁇ chain in monomenc form, or a truncated TGF- ⁇ monomer
  • studies conducted in support of the invention indicate that the OP-TGF- ⁇ protein is recognized by (I) a type II TGF- ⁇ receptor and (n) a antibodies which recognized mature TGF- ⁇ , but that the protein is not bound by antibodies which recognize latent forms of TGF- ⁇ , consistent with the OP-TGF- ⁇ protein being a mature TGF- ⁇ monomer
  • the invention includes a method of purifying an OP-TGF- ⁇ polypeptide from a liquid sample, comprising the steps of (I) contacting the liquid sample with a solid support having immobilized thereon a binding partner which is lmmunospecific for mature TGF- ⁇ l and OP-TGF- ⁇ , under conditions effective to specifically bind the polypeptide to the immobilized
  • the purification method is conducted using an immobilized binding partner which is immunospecific for OP-TGF- ⁇ . to the exclusion of mature dimeric TGF- ⁇ l or any latent form thereof, such that the OP-TGF- ⁇ polypeptide is eluted in substantially pure form
  • the invention includes an immunological binding partner which is immunospecific for OP-TGF- ⁇ , and which does not bind mature dimeric TGF- ⁇ or any latent form thereof
  • BDA19 antibody is a chicken polyclonal IgY antiserum
  • BDA47 is an affinity-purified rabbit polyclonal IgG antibody
  • BDA19 binds the mature dimer and small latent forms of TGF- ⁇ l
  • TGF- ⁇ 3 BDA47 binds both the mature dimer and small latent forms of TGF- ⁇ l
  • TGF-B3 BDA48 is a polyclonal antiserum specific for the B3 isoform of TGF
  • TGF- ⁇ l and TGF- ⁇ 3 were obtained from R&D Systems or Amersham International
  • peripheral venous blood samples (10 L) were collected from human subjects and transferred immediately to tubes containing 1 1 mL of sterile 3 8% (w/v) t ⁇ sodium citrate in MilhQ water at room temperature
  • the samples were cent ⁇ fuged to remove red blood cells (250 x g, 15 mm)
  • Apyrase (Sigma) was added to the resulting platelet-rich plasma to a final concentration of 100 mg/L, to prevent platelet degranulation PMSF (1 mmol/L) and aprotinm (1 mg/L) were added to prevent proteolytic activation or degradation of TGF- ⁇
  • the samples were then cent ⁇ fuged (700 x g, 15 mm), and the supernatant platelet-poor plasma was separated from the platelet pellet
  • the platelet-poor plasma was kept at room temperature until assay by ELISA within 2 h of preparation or was stored frozen in 0 5-mL aliquots at -80°C
  • peripheral venous blood samples from human subjects were dispensed immediately into polypropylene tubes and allowed to clot at room temperature for 2 h
  • the collected samples were centrifuged (1000 x g, 4 min), after which the serum was removed and either stored on ice until assay within 2 h or stored at -80 °C for later assay
  • the extracellular domain of the TGF- ⁇ type II receptor was amplified from the vector H2 3FF, as described in Lin et al ( 1992)
  • the vector DNA was linearized with NotI, precipitated, and resuspended at 10 ng/ ⁇ L
  • Amplification by polymerase chain reaction (PCR) was carried out in a 50 ⁇ L reaction volume containing vector DNA (2 5 ⁇ L), lOx TAQ buffer (5 ⁇ L) (LKB Pharmacia, Uppsala, Sweden), 250 ng ofeach ohgonucleotide primer (5'-GAATTCCCATGGGTCGGGGGCTGCTC and GAATTCGTCAGGATTGCTGGTGTT), TAQ polymerase ( 1 U) and a mixture of dAI P, dTTP, dCTP and dGTP to give a final concentration of 200 ⁇ M for each nucleotide
  • the sample was overlaid with 50 ⁇ L paraffin oil -The reaction was carried out using a thermal cycler for 30 cycles (denaturing at 94°C for 1 min, annealing at 55 °C for 2 min, and elongation at 72 °C for 2
  • the 450 bp product was purified by eiectrophoresis in low gel temperature agarose, digested with EcoRI, and cloned into the glutathione-S-transferase fusion vector pGEX 2T (LKB Pharmacia) Vectors carrying inserts in the required orientation were identified by plasmid mapping The sequence of the insert was checked by subcloning the 450 bp EcoRI fragment from the chosen clone (pGTIC) into Bluesc ⁇ pt KS + , followed by double strand sequencing The sequence showed a single base change (C to A at position + 13 from the initiation codon) compared with the published sequence (Lin et al , 1992), which introduces a Leu to Met mutation in the expressed receptor protein
  • the plates were washed with a wash buffer consisting of TBS, 3 % FAF-BSA, and 0 1 % Triton X-100, and incubated with 20 ⁇ L detection antibody (BDA47) at 1 ⁇ g/mL in wash buffer for 1 h
  • the plates were rinsed with wash buffer (3 3 mm) and incubated with an antibody against rabbit IgG conjugated to horseradish peroxidase at 1 2500 dilution in wash buffer for 1 h
  • wash buffer 3 x 3 min
  • the plates were incubated for 15 min with o-phenylenediamine according to the manufacturer's instructions (Sigma Chem Co )
  • the reaction was stopped by addition of an equal volume of 3 M HC1, and the absorbance values were read on an ELISA platereader (Titerteck Multiscan, Flow Laboratories, High Wycombe, UK) within 15 min of stopping the reaction
  • TGF-B3 stock recombinant human TGF-B3 (2 ⁇ g) was dissolved in 4 mM HC1 (400 ⁇ l) containing 1 mg/mL fatty acid-free bovine serum albumin (Sigma, A-6003) to give a final concentration of 5 ⁇ g/mL
  • an initial standard solution of TGF-153 was prepared by adding 2 ⁇ l of TGF- ⁇ 3 stock (5 ⁇ g/mL) to an Eppendorf tube containing 100 ⁇ l BDA19 working solution (40 ⁇ g/mL BDA19) and 300 ⁇ l RD6M diluent, giving a final BDA19 concentration of 10 ⁇ g/mL and a final TGF- ⁇ 3 concentration of 25 ng/mL
  • this initial TGF- ⁇ 3/BDA19 solution was diluted serially 1 1 into six successive Eppendorf tubes each containing 150 ⁇ l RD6M diluent and 50 ⁇ l working BDA19 solution
  • the seven TGF-B3 standard tubes thus contained 10 ⁇ g/mL BDA19 and from 25 ng/mL to 0 39 ng/mL TGF- ⁇ 3
  • an additional tube containing 150 ⁇ l RD6M diluent and 50 ⁇ l working BDA19 solution was also prepared without TGF-B3
  • TGF- ⁇ 3/BDA19 and TGF-free control were each loaded into separate wells (200 ⁇ l each) in a Quantikine ELISA strip (R&D Systems)
  • 75 ⁇ l neutralisation buffer and 75 ⁇ l BDA19 working solution The contents of the sample tubes were mixed, and 200 ⁇ l ahquots of each were transferred to separate wells on the ELISA strip
  • the strips were covered with adhesive film and incubated for 1 hour at room temperature on a microtitre plate shaker (500 ⁇ ) After incubation, the wells were aspirated by inversion and washed 3 times with wash buffer (R&D Systems, 1 25 dilution) To ensure liquid removal, the strips were blotted on a clean paper towel between wash steps
  • 50 ⁇ l BDA48 working solution 100 ⁇ g/mL
  • Serum samples were obtained as described in the Material and Methods section from two normal, healthy male subjects and (45 and 54 years old, respectively), and from a female patient diagnosed with osteoporosis
  • Each sample (1 L) was diluted 1 3 with 10 mM sodium phosphate, pH 6 8, and then applied to a 1-mL ResourceQ disposable MonoQ column (Pharmacia) on an FPLC system (Pharmacia) equilibrated in 10 mM sodium phosphate pH 6 8 (buffer A)
  • the column was washed with buffer A (flow rate 1 mL/min) until the absorbance at 280 nm had returned to baseline
  • TGF- ⁇ proteins were assayed for TGF- ⁇ proteins as follows A quots of the fractions were diluted 1 1 with 2 5 M acetic acid containing 10 M urea for 10 mn at room temperature to release mature 25-kDa TGF- ⁇ 1 dimer Samples were then neutralized by adding the same volume again of 2 7 M NaOH/1 M HEPES Neutralized samples were assayed immediately for TGF- ⁇ l using a Quantikine ELISA kit (R&D Systems) in accordance with the manufacturer's instructions Fractions containing TGF- ⁇ (typically fractions 20 to 29) were combined, diluted 1 4 with 200 mM sodium phosphate pH 6 8, and concentrated to 200 ⁇ L using an Amicon cell with a 3,000 kDa cut-off membrane The concentrated sample was then loaded onto a Superose 12 gel filtration column (Pharmacia) equilibrated in 200 mM sodium phosphate pH 6 8 on an FPLC system Fractions (0 5 mL) were

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The present invention is directed generally to a method of assessing the status of bone resorption in a subject, based on the measurement of TGF-β3 species in a body fluid, particularly serum. A measured level of the selected TGF-β3 species that is at or above a selected threshold value is useful as an indication that an elevated level of bone resorption is present in the subject. A measured level of the selected TGF-β3 species that is at or below a selected threshold value may be used as an indication that bone resorption is within normal limits. Also contemplated are a kit and reagents for use in the method.

Description

Method and Kit for Assessing Bone Resorption
This application claims priority to U S Provisional Patent Application No 60/024,084 filed August 16, 1996, which is incorporated herein by reference
Field of the Invention
The present invention relates to methods for detecting screening for and monitoring bone metabolism abnormalities, particularly those relating to elevated bone resoφtion The invention also relates to reagents and kits for use in such methods
References
Am ann, J A , et al , U S Pat No 5, 158,934 issued Oct 27 1992 Burhna, A , et al , Chn Chim Acta 226 141 (1994)
Burnier, J P , et al , U S Pat No 5,061 ,786 issued Oct 29, 1991
Dennis, P A , and Rifkm, D B , Proc Natl Acad Sci USA 88 580-584 (1991)
Derynck, R M A , et al , U S Pat No 4,886,747 issued Dec 12, 1989
Epstein, E , et al , C n Lab Med 84 695 (1986) Garnero P , et al , J Chn Endocπnol Metab 77 1046 (1993)
Grainger, D J , et al , Nature 370 460-462 ( 1994)
Grainger, D J , et al , Chn Chim Acta 235 1 1 ( 1995a)
Harlow, E , and Lane, D , Antibodies A Laboratory Manual. Cold Spring Harbor Laboratory, 1988 Koji a, S , et al , J Cell Biol 121 439-448 (1993)
Lin, H Y , et al , CeJi 68 775 (1992)
Miyazono, K , et al , J Biol Chem 263 2407-2417 1988)
Miyazono, K , et al , EMBO J 10 1091-1101 (1991)
Smith, D B , et al , Gene 67 31 (1988) Sporn, M B , and Roberts, A B , J Cell Biol 1 19 1017 ( 1992)
Sporn, M B , and Roberts, A B , Growth Factors 8 1 (1993)
Taylor, A K , et al , Rheum Pis Chn N Amer 20 589 (1994)
Van Straalen, J P , et al , Chn Chim Acta 201 27 (1991)
Wakefield, L M , et al , J Biol Chem 263 7646-7654 (1988) Background of the Invention
A variety of disease conditions in humans are characterized by an elevated level of bone resoφtion or by an abnormal imbalance between bone resoφtion and bone formation. Among the more common of these conditions are osteoporosis, osteoarthritis. hyperthyroidism, hypeφarathyroidism, osteomalacia, osteosarcomas, and Paget's disease (Epstein, 1986; Van
Straalen, 1991 ; Garnero, 1993; Burlina, 1994).
Osteoporosis is particularly problematic, affecting upwards of twenty million people in the United States alone. Osteoporosis is characterized by pathologically low bone density, often first presenting clinically with fractures. Both the deposition of bone, which is mediated by osteoblasts, and bone resoφtion, which is mediated by osteoclasts, occur in an ongoing recycling process of the bone matrix throughout the lifetime of humans.
A large number of agents have been found to affect the rate of deposition of inorganic matrix by cultured osteoblasts, either by affecting the rate of proliferation of these cells or by increasing their differentiation status. Similarly, agents have been identified which modulate the rate of resoφtion by osteoclasts in bone explant cultures. The in vivo roles of many of these agents remain to be clarified, and the signalling pathways which mediate deposition and resoφtion are not well understood.
A number of serum and/or urinary substances have been proposed as possible indicators for bone degradation. Exemplary substances which have been studied include hydroxyproline, tartrate-resistant acid phosphatase, galactosyl hydroxylysine, various collagen peptides, and peptide-free pyridinium species (e.g. , Taylor, 1994). Although considerable efforts have been made to develop such indicators for clinical application, there remains a great need to identify additional markers for the diagnosis and prognosis of bone disorders.
Summary of the Invention
In a first aspect, the invention includes a method of assessing the status of bone resoφtion in a mammalian subject, based on measuring the level of total TGF-B3, or a selected subform thereof, in a body fluid sample from the subject. In the method, a level of total TGF-B3 in the sample is determined. The determined level is compared with a predetermined threshold or level characteristic of normal subjects, wherein a determined level that is above a level characteristic of normal subjects is an indication that the subject has a bone resoφtion disorder. Similarly, a level below a selected threshold, which may be different from the first threshold, may indicate that the subject's level of bone resoφtion is within normal limits. In a second aspect, the invention includes a method of assessing the status of bone resoφtion in a mammalian subject In the method, the levels of (1) non-latent TGF-β and (n) the sum of small latent TGF-β plus mature TGF-β 1 dimer are determined in a body fluid from the subject The determined levels are compared with each other, where a determined ratio of non-latent TGF-β versus small latent TGF-β plus mature β l dimer (non-latent/(small latent TGF-β + mature βl)) above a selected threshold is an indication that a bone resoφtion disorder may be present in the subject Similarly, a ratio below a selected threshold, which may be different from the first threshold, may be used as an indication that the subject's level of bone resoφtion is within normal limits In an alternative embodiment relating to the second aspect of the invention, the difference between the levels of non-latent TGF-β and small latent TGF-β -I- mature β l dimer is determined, where a difference that is above a selected threshold is an indication that a bone resoφtion disorder may be present in the subject, and a difference that is below a selected threshold may be used as an indication of normal bone resoφtion In a third aspect, the invention includes a method of assessing the status of bone resoφtion in a mammalian subject, based on measuring the level of a previously unknown TGF-β protein species, designated herein as OP-TGF-β, in a body fluid from the subject A measured level of OP-TGF-β which is above a selected threshold level is an indication that a bone resoφtion disorder associated with elevated bone resoφtion may be present in the subject Similarly, a level below a selected threshold, which may be different from the first threshold, may indicate that the subject's level of bone resoφtion is within normal limits
The methods of the invention are useful in the context of detecting or aiding in the diagnosis of a variety of disorders associated with bone metabolism abnormalities (bone resoφtion disorders), such as osteoporosis, osteoarthπtis, hypeφarathyroidism rheumatoid arthritis, Paget's disease, or a malignant tumor or metastatic cancer in bone
The methods of the invention may be used as a screening tool to identify subjects from the general population who are at increased risk for elevated bone loss or bone fracture, where a level, ratio or difference of TGF-β species above a selected threshold is an indication of increased risk of developing a bone resoφtion disorder, such as osteoporosis, or of suffering a bone fracture
The methods of the invention may also be used in combination with other diagnostic methods, such as radiographic techniques for measuring bone density, to further define a patient's condition That is, if an elevated level, ratio, or difference of an analyte or analytes of the invention is detected, indicating the presence of a bone resoφtion disorder or an increased risk for such a disorder or bone fracture, the subject is then further tested using an independent technique, such as lumbar spine bone mineral density (LS-BMD) measurements, to further characterize the health status of the subject
The methods of the invention may also include determining the level of an indicator of bone formation, such as bone alkaline phosphatase, tartrate resistant acid phosphatase (TRAP), Type HI procollagen, or the like, to assess the balance between bone formation and bone resoφtion in the subject
In a related aspect, the invention includes a method of monitoring bone degradation in a subject, for detecting changes in the bone degradation status of the subject over time The level, ratio or difference of TGF-β species discussed above is measured at least twice in a body fluid from the subject over a period of time, where the measured level, ratio or difference is used to assess whether bone resoφtion has increased, decreased, or stayed the same
In another aspect, the invention includes a substantially pure OP-TGF-β polypeptide having an apparent molecular weight of between 10 and 15 kDa, as measured by gel filtration chromatography under non-denaturing conditions, and which is characterized by the elution and
TGF-β activity properties shown in Figs 3B and detailed in Example 7 The polypeptide is particularly useful as a standard cahbrant in the OP-TGF-β assay above
The analytes of the invention are measured by any analytical method capable of quantitatively measuring the analyte(s) of interest without significant interference from other components in the sample fluid For this purpose, suitable methods may include chromatographic, electrophoretic, and immunoassay methods, and combinations thereof Preferably, the analyte(s) are measured by immunoassay techiques using polyclonal or monoclonal antibodies, or art-recognized equivalents thereof, which are specific for the analyte(s) of interest The invention also contemplates kits and reagents for use in carrying out the methods described above
These and other objects and features of the invention will become more apparent from the following detailed description in light of the accompanying drawings
Detailed Description of the Drawings
Fig 1A illustrates an assay format for measuring small latent TGF-β species plus mature βl dimer; Fig. IB illustrates an assay format for measuring non-latent TGF-β species,
Figs 2A and 2B show serum TGF-β3 levels measured in normal (2A) and osteoporotic subjects (2B), Fig 3A shows chromatograms of TGF-β-contaimng fractions (assay in accordance with
Fig IB) obtained from anion exchange chromatography of partially purified serum samples from two healthy subjects (A = mature TGF-β dimer, P = platelet large latent TGF-β, L = small latent TGF-β), Fig 3B shows a similarly obtained chromatogram from a subject suffering from chronic osteoporosis, and
Fig 4 shows serum TGF-β3 levels measured in four groups of patients (only individuals with TGF-B3 concentrations > 0 3 ng/mL are shown) group 1 28 women with normal bone density, group 2 24 osteoporotic women presenting with vertebral fracture, group 3 1 1 osteoporotic women with a family history of osteoporosis, and group 4 26 osteoporotic patients with osteoporosis of the hip
Detailed Description of the Invention
I Definitions
As used herein, the terms below are intended to have the following meanings unless indicated otherwise
The term "body fluid" includes any human body fluid suitable for diagnostic assay for assessing the level of bone resoφtion in accordance with the present invention Exemplary body fluids include whole blood and blood fractions, such as serum, plasma, and platelet-poor plasma, urine, saliva, sweat, synovial fluid, tear fluid, cerebral spinal fluid, and liquid extracts of bone tissues (e g , bone biopsy) Preferably, the body fluid is a blood fraction or urine, and more preferably is serum or plasma
"Mature TGF-β" and "active, mature TGF-β" refer to the homodimeπc form of TGF-β comprising ammo acid residues 279 to 390 of the pre-pro polypeptide form of TGF-β
"Active TGF-β" or "non-latent TGF-β" encompasses monomeπc and homodimeπc forms of TGF-β comprising amino acid residues 279 to 390 of the pre-pro polypeptide form of TGF- β, or a subset thereof, and which is not complexed with LAP or LTBP
"Latent-associated peptide" and "LAP" refer to a polypeptide chain spanning residues 30 to 278 of the pre-pro polypeptide form of TGF-β
"Small latent TGF-β" refers to a 1 10 kDa dimer containing a mature TGF-β dimer complexed noncovalently with two LAP chains
"Platelet large latent complex", "large latent complex", and "LLC" refer to a 220-240 kDa complex containing a mature TGF-β dimer, two LAP chains, and an LTBP chain "Latent TGF-β binding protein" and "LTBP" refer to the 130 kDa polypeptide which associates covalently with latent TGF-β to form the 220-240 kDa large latent complex, as described in Miyazono et al (1988)
"Latent TGF-β" and "latent forms of TGF-β" refer to proteins or protein complexes which contain a mature TGF-β dimer and at least one other associated polypeptide, and which are unable to interact with cellular surface receptors for TGF-β Exemplary latent forms of TGF-β include small latent TGF-β and the large latent complex
"TGF-β isoform" refers to mature TGF-β 1 , β2, 153, β4, and/or β5, a polypeptide derived therefrom, or a polypeptide having greater than 95 % homology therewith The phrases "total TGF-β3" . "total TGF-β3 species" , and "total TGF-β3 antigens" refer to the total amount of mature TGF-153 species (and fragments thereof) measurable after release of any latent proteins from the TGF-β3 species present in the sample These species may be prepared by any appropriate treatment of the sample, as necessary For serum or plasma samples, for example, total TGF-β3 can be prepared by treating the sample with acidic urea, as described below Alternatively, proteolyt-ic treatments may be used, for example, to selectively destroy associated structure of the TGF-B3 polypeptides to be measured
The term "mammal" is intended to have its conventional meaning, and includes man, horse, cow, sheep, rabbit, dog, cat, rat, and mouse, for example Preferably, the test subject is a human subject
II TGF-β Proteins Background
Transforming growth factor-β (TGF-β) is a pluπpotent cytokme which has been found to regulate cell growth in a variety of tissues Reported activities include suppression of thymocytes and lymphocytes, stimulation or inhibition of osteoblast function, mediation of the formation of extracellular matrices, and more generally, inhibition or stimulation of proliferation and differentiation of a host of cell types (e g , Sporn et al , 1992, 1993, Ammann et al , 1992) TGF-β has also been proposed for use in wound repair (Derynck et al , 1989), for promoting bone growth (Ammann et al , 1992), and for immunosuppression and septic shock (Burnier et al , 1991 ) To date, at least five isoforms of TGF-β have been identified in mammals, designated
TGF-βl through TGF-β5 Of these, only TGF-βl , β2, and β3 have been found in humans The five isoforms share high sequence similarities and overlapping biological activities, although some differences in activity have been reported (R&D Systems 1994 Catalog, Minneapolis, MN, pp 4-6) In humans, TGF-βl is found in highest concentrations m platelets and bone, TGF-B2 is found in bone and other tissues, and TGF-B3 is found mostly in cells of esenchymal origin Properties of the TGF-β proteins have been reviewed (Sporn et al , 1992, 1993)
For human TGF-βl , B2, and β3, the biologically active form is a 25 kDa dimer containing two identical 112 residue polypeptide chains linked by disulphide bonds The synthesis of active TGF-β in vivo has been reported in the literature to involve the following steps Initially, TGF-β is synthesized as a 390 residue pre pro polypeptide which dimeπzes to form a homodimer The pre-pro form contains a 29 residue signal sequence which is cleaved mtracellularly to form a pro-TGF-βl dimer of 261 -residue chains Prior to secretion from the cell, pro-TGF-β l is further cleaved to produce a 1 10 kDa nicked dimer in which the polypeptide chains of the dimer have been cleaved between Arg278 and Ala279 The newly created C-terminal chains, spanning residues 279 to 390 from the pre- pro polypeptide, constitute the polypeptide chains that eventually form active, mature TGF-β The newly created N-terminal chains, spanning residues 30 to 278, are referred to as ' latency- associated peptides" (LAPs) (Wakefield et al , 1988) The secreted 1 10 kDa LAP-TGF dimer is referred to as "latent TGF-β" (or small latent TGF-β) since it is unable to interact with cellular surface receptors for TGF-β and has no known biological activity
Additional proteins may associate with latent TGF-β, either covalently or non-covalently to form additional forms of TGF-β For example, a complex has been isolated from human platelets containing LAP linked to an additional 130 kDa protein (known as the latent TGF-β binding protein, or LTBP) by a single disulphide bond (Miyazono et al , 1988, 1991) This 220-240 kDa complex, which includes a mature (25 kDa) TGF-β dimer, two associated LAP chains, and an LTBP chain, has been termed the "large latent complex '
Active, mature TGF-β can be formed in vitro by exposing latent TGF-β or the large latent complex to strong acid, strong base, or chaotropic agents Such conditions presumably disrupt the non-covalent interactions between the mature TGF-β dimer core and the non-covalently attached polypeptides (LAP and LTBP) to produce active TGF-β Formation of active TGF-β in vivo may occur through the action of proteases which degrade the LAP but not the TGF-β core Plasmm has been implicated as the physiological activator under some circumstances (Grainger et al , 1994, 1995), but other proteases may also be involved (Kojima et al , 1993 Dennis et al , 1991) At present, the actual in vivo mechanism for forming active TGF-β from its latent forms is not known III Resoφtion Assay Methods
The present invention is directed generally to a method of assessing the status of bone resoφtion in a subject, based on the measurement of certain TGF-β species in a body fluid, particularly serum, plasma or urine In one general embodiment, a measured level, ratio, or difference, of the selected TGF-β species that is at or above a selected threshold value is an indication that an elevated level of bone resorption is present in the subject The method may also be configured such that a measured level, ratio or difference, of the selected TGF-β species that is at or below a selected threshold value can be used as an indication that bone resoφtion is within normal limits In selecting the particular threshold to be used to discriminate between test subjects who appear to have an elevated level of bone resoφtion versus those who appear to be healthy or whose bone resoφtion status is uncertain, the level of true positives detected must be weighed against the number of false positives as well as false negatives For example, a higher threshold will lead to a lower rate of false positives, but at the expense of reduced sensitivity in detecting true positives Similarly, a lower threshold will lead to detection of more true positives, but at the expense of an increased proportion of false positives as well It will be appreciated that similar principles will apply when the method is used to verify normal bone resoφtion status on the basis of TGF-β levels falling below a selected level The exact threshold used in the method will depend on empirical studies of control and diseased subjects, and on the level of sensitivity and specificity desired for the particular diagnostic or prognostic application
The method of the invention may be used in a monitoring mode, where at least two measurements are made in body fluid samples from a subject at different times, and the change in the measured level, if any, is used to determine whether the level of bone resoφtion has increased, decreased, or remained the same
The invention may also be used in prognostic applications, for predicting the likelihood or risk of developing a bone resoφtion abnormality such as discussed above, or for predicting the likelihood or risk of suffering a bone fracture For such uses, an increased risk may be indicated by a level of selected TGF-β species that is above a selected threshold, where the threshold has been selected on the basis of clinical trials, for example, or by a level which is a selected percentage above a baseline level established for a particular individual
In other embodiments, the methods of the invention may be used in a screening capacity for the general public, or may be targeted to certain patients for whom such testing is deemed appropriate In addition, the present resoφtion assessment methods may be used in conjunction with other medical techniques, such as bone density measuring methods or diagnostic assays based on other markers
A number of methods have been described for detecting or measuring various forms of TGF-β For example, TGF-β-containing species may be detected using antibodies which are lmmunoreactive with one or more TGF-β species, allowing highly sensitive detection of low concentrations of the species of interest by conventional immunoassay techniques Antibodies raised against mature TGF-βl , β2, or β3, and having high binding affinity for TGF-βl , are useful for detecting the OP-TGF-β protein of the present invention Such antibodies are readily available from commercial sources (e g , R&D Systems. Minneapolis. MN, R&D Systems, Oxford, United Kingdom, Genzyme Diagnostics, Cambridge, MA) or may be prepared by standard methods (e g , Harlow, 1988)
Section A below describes an aspect of the invention based on measuring total TGF-B3, or a subform thereof, such as active β3 dimer The applicant notes that prior to the present invention, TGF-B3 species were not known to be present in significant amounts in plasma or serum Section B describes an aspect of the invention based on measuring the levels of (I) non- latent TGF-β and (u) the sum of small latent TGF-β plus mature TGF-βl dimer are determined in a body fluid from the subject The ratio or difference of these measured levels is used as an indicator of the level of bone resoφtion in the subject Section C describes an aspect of the invention based on the measurement of a novel protein designated herein as OP-TGF-β
A TGF-β3-Based Methods
In this aspect of the invention, the level of total TGF-β3, or alternatively, mature TGF-β3 dimer, in a body fluid is measured, where a measured level above a selected threshold level is an indication that a bone metabolism abnormality associated with elevated bone resorption may be present in the subject Similarly, a level below a selected threshold, which may be different from the first threshold, may indicate that the subject's level of bone resoφtion is within normal limits
TGF-B3 may be measured using any analytical technique available in the art, and is conveniently measured using immunoassay techniques Example 4 describes an exemplary assay format which is useful in this aspect of the invention The assay includes an immobilized TGF-β Type II receptor such as described in Example 1 , for binding the TGF-β3 dimer to the support for subsequent detection The assay further includes an antibody which is specific for the βl isoforms of TGF, to bind to and sequester the βl isoforms, to prevent βl monomer or dimer from competing with β3 dimer for binding to the immobilized receptor This allows low levels of TGF-β3 to be detected even in the presence of a high concentration of TGF-βl species, when the binding partner is cross reactive with both β 1 and β3 isoforms
In practicing the assay of Example 4, for measurement of total TGF-β3 species, the serum samples are treated with acidic urea to release the active, dimeπc forms of TGF-β3 from their latent forms After incubation, the samples are mixed with a neutralization solution and then incubated with a βl-specific antibody (BDA19) to sequester βl species in the samples The samples are then contacted with the immobilized TGF-β receptors to bind the TGF-B3 to the solid support Primary, non-immobilized antibody is then added to bind to the immobilized TGF-β3, followed by addition of peroxidase-labeled antibody for detection
Alternatively, the non-latent forms of TGF-B3 can be measured without treating the sample to convert latent to non-latent forms
The utility of the TGF-β3 assay is illustrated in Examples 5 and 6 With reference to Example 5, total TGF-β3 was tested for in 10 healthy individuals 24 to 54 years in age No TGF-B3 was detected in any of the plasma or serum samples from these subjects However, low levels of TGF-β3 ( = 2 ng/mL) were detected in the platelet samples from most of the subjects, suggesting that perhaps platelet degranulation should be avoided when assaying blood or serum for TGF-B3 More recent studies by the applicant have shown that human platelets do not contain TGF-β3, suggesting that the observation of TGF-β3 in platelets mentioned above was artifactual
In the study described in Example 6, the levels of total TGF-β3 were measured in subjects showing normal bone densities (n=29) and in osteoporotic subjects with bone densities more than 2 standard deviations below the population mean (n = 25) The results are shown in Figs 2A and 2B None of the subjects had undergone, or were undergoing, hormone replacement therapy
As can be seen, the subjects with low bone density exhibited a mean TGF-B3 level of 6 4 .+. 0 6 ng/mL, compared with 2 7 +_ 0 6 ng/mL for the control group In addition, more than half of the control individuals showed no detectable TGF-B3, whereas all but two of the osteoporosis subjects showed detectable TGF-B3 levels greater than about 2 ng/mL These results show that the presence of TGF-B3 is an excellent indicator of the presence of elevated bone resoφtion
Fig 4 shows additional data illustrating the usefulness of the method Serum samples were collected from 28 women with normal bone density (group 1) and 61 women with osteoporosis (groups 2-4) Serum TGF-β3 levels were measured as described in Example 4 Results ( 1 ) Of the 28 women with normal bone density, 5 had detectable serum TGF-B3 levels ( > 0 1 ng/mL), but only one had a level greater than 2 ng/mL (2 2 ng/mL) (2) Of the 61 osteoporotic women (groups 2-4 together). 35 had TGF-β3 levels higher than 2 ng/mL, and 41 had detectable TGF-β3 levels (3) Of the 24 patients with vertebral fracture (group 2), 8 showed TGF-β3 levels greater than 2 ng/mL (4) Of the 1 1 osteoporotic women with a family history of osteoporosis (group 3), 8 had detectable TGF-B3 levels and 6 had levels greater than 2 ng/mL (5) The prevalence of elevated TGF-β3 was even greater in the osteoporotic patients with osteoporosis of the hip (group 4), where 21 of 26 patients had TGF-B3 levels greater than 2 ng/mL Further studies conducted in support of the invention showed the following
1 Sample Storage Serum samples containing TGF-β3 can be subjected to repeated freeze-thaw cycles with only a slight reduction in the measured amount of TGF-B3 Thus, repeated freeze-thawing of human serum samples does not significantly affect TGF-β3 measurements However, it is preferable to store samples at -20°C until measurement of TGF- β3, without thawing until testing
2 Effects of Hemolvsis While platelets (a significant source of TGF-βl) generally do not contain TGF-B3, the applicant has found that red blood cells contain about 800 molecules of TGF-β3 per cell, equivalent to about 100 ng of TGF-β3 per mL of blood Thus, hemolysis should be avoided to obtain an accurate measure of serum TGF-B3 species Preferably, the degree of hemolysis in a test sample is less than 2% , e g , when a threshold of 2 ng/mL is used to discriminate between normal and "diseased" or at-πsk subjects This level of hemolysis can be readily detected visually by eye from the red color of the sample, indicating that another sample should probably be taken from the subject However under normal laboratory blood collection conditions, hemolysis is generally not a significant problem Studies by the applicant have shown that hemolysis of at least 3 2 % (as measured by assaying serum hemoglobin concentration after drawing blood through a fine gauge needle a variable number of times prior to clotting and collection of serum) is generally necessary to produce a measured serum TGF-β3 level above 2 ng/mL However, such high hemolysis levels are easily avoided in ordinary clinical sample collection 3 TGF-βl and TGF-B2 Unlike TGF-β3, TGF-βl and TGF-β2 levels are not elevated in osteoporotic subjects Thus, TGF-β3 is a relatively specific TGF-β lsoform for bone degradation, especially for osteoporosis
4 TGF-β3 Levels and Drug Treatment There is some variation in the effects of hormone-mimetic drugs on TGF-β3 levels measured in women For example, prophyllactic hormone replacement therapy (HRT) was found to lead to mildly increased levels of TGF-B3 during treatment
Treatment of two 28-year old healthy pre-menopausal women with the contraceptive pill femodene (75μg gestodene, 30 μg ethynyl estradiol) led to mild suppression of serum TGF-β3 levels during the period when the pills were taken (three consecutive weeks out of a four week cycle), and return to normal during the one week of the cycle when the pills were not taken
Serum levels of TGF-B3 in 15 men (aged 47-63) who had angiographically-proven coronary artery disease, and who were treated with anti-estrogen tamoxifen for 10 days did not show any changes in serum TGF-B3 levels during treatment These observations suggest that the test subject should be asked it he/she is under any medication in case the medication alters the subject's level of TGF-B3 in a manner that prevents diagnostic inteφretation of the measured TGF-B3 level In particular, HRT may produce false positives due to elevation of TGF-β3 caused by HRT, so that an elevated TGF-β3 levels might not be diagnostic of elevated bone resoφtion If a normal TGF-β3 level is observed in an HRT subject, this may indicate that the subject's bone resoφtion level is normal If the subject has ceased HRT before testing, elevated TGF-B3 levels are more likely to indicate elevated bone resoφtion and the presence of a bone resoφtion disorder However, treatment with femodene or tamoxifen does not appear to interfere with diagnostic inteφretation of the TGF-β3 assay
5 TGF-B3 Levels During Menstruation In healthy premenopausal women with low levels of serum TGF-B3, there was no significant fluctuation in TGF-β3 levels during the normal menstrual cycle (n= 3, age 23 to 28, less than 2 ng/mL in all cases)
6 Serum and Plasma The assay described in Example 4 detected the same levels of total TGF-β3 in serum and platelet-poor plasma prepared from the same blood samples Thus, testing either serum or plasma samples should provide similar results
B Assays of Non-Latent TGF-β and Latent TGF-β + Mature βl
In this aspect of the invention, the levels of (l) non-latent TGF-β and (n) small latent
TGF-β + mature βl dimer are measured, and either the ratio (i) (n) or the difference (l - n) is used as an indicator of the level of bone resoφtion in the subject The measurement of non-latent TGF-β, and the sum of small latent TGF-β + mature βl dimer, may be carried out using any suitable analytical approaches known in the art for measuring these species
Conveniently, non-latent TGF-β can be measured by immunoassay using lmmunological binding partners which are lmmunospecific for both desired TGF-β species, and which do not significantly react with the large latent complex The assay may be homogeneous or heterogeneous, and may utilize a direct (e g , sandwich) or competitive format in accordance with assay configurations well known in the art (e g , Harlow)
One suitable assay format for measuring non-latent TGF-β species is illustrated in Figure 1 A In this assay, an immunological binding partner, 12a, 12b, 12c, 12d, here shown as a type
11 TGF-β receptor, is immobilized on a solid support 40, for binding to non-latent monomenc and dimeric forms of TGF-β, particularly those containing TGF-β l and TGF-β3 chains The assay format also includes antibodies 30a, 30b, for binding to the target TGF-β species, and reporter-labeled antibodies 32a, 32b, for binding to the primary antibodies 30a, 30b to produce a measurable signal
The sample to be tested may contain any or all of the following TGF-β species (0 a monomenc form of TGF-β ( 14 in the figure), such as the OP-TGF-β protein described further below, (n) a mature TGF-β dimer 16, (in) small latent TGF-β 20, which contains a TGF-β dimer 16 and two LAP chains 18, and/or (IV) a large latent complex 24 which contains a mature TGF-β dimer 16, two LAP chains 18, and an LTPB chain 22
With continued reference to Fig 1A, TGF-β monomenc species 14 is bound by immobilized receptor 12a The immobilized TGF-β monomer is then detected by binding to a first antibody 30a which binds directly to monomer 14, followed by the binding of reporter- labeled antibody 32a The TGF-β is then detected by virtue of the reporter-labeled antibody immobilized on the solid support
TGF-β dimer 16 is likewise detected when bound to immobilized receptor 12b, by virtue of binding with primary antibody 30b which in turn is bound by reporter labeled antibody 32b Since receptor 12c does not bind latent forms of TGF-β, small latent form 20 and large latent form 24 remain free from the support, and thus cannot serve to anchor a reporter labeled antibody 32 to the support Details of an exemplary assay format in accordance with the above are provided in Examples 1 and 2
It will be appreciated with reference to Fig 1A that when the immobilized binding partner
12 does not bind significantly to the latent forms of TGF-β, it is not important whether the primary antibody 30a can bind the latent forms as well as the non-latent forms, provided the primary and reporter-labeled antibodies are present in excess relative to the TGF-β which could potentially be bound
Alternatively, immobilized binding partner 12 can be selected to bind to both non-latent as well as latent forms of TGF-β In this embodiment, the specificity of the assay for the non- latent forms is achieved by selecting the primary antibody (or other suitable binding partner) to be lmmunospecific for the non-latent forms For example, the immobilized binding partner 12 can be an antibody which binds with TGF-β species 14, 16, 20, and 24, and antibody 30 could be replaced with a type II TGF-β receptor which recognizes only non-latent TGF-β forms The sum of small latent TGF-β + mature β l dimer can be measured using an assay format such as illustrated in Fig IB and detailed in Example 3 In the format of Fig I B, receptor 12a from Fig 1A has been replaced with an immunological binding partner 42 (shown schematically as an antibody) which can bind mature βl dimer (16) and small latent TGF-β (20), but not monomer 14 or large latent complex 24 Dimer 16 and small latent form 14 may then be detected using a pair of antibodies 30, 32 as in Fig 1 A, where antibody 30 is capable of binding at least species 14 and 16 to immobilize a reporter moiety (R) for detection
Reporter R is any entity which is effective to produce a detectable signal for the puφose of detecting or quantitating the TGF-β analyte of interest Exemplary reporters include enzymes, such as alkaline phosphatase and horse radish peroxidase, radioactive labels such as l25I, spin labels, etc
Preferably, binding partner 30 does not bind significantly with monomer 14 or large latent complex 24 m order to avoid competition between species 14 and 24 and species 16 and 20 for binding to binding partner 30 Otherwise, there might be an insufficient amount of binding partner 30 available to bind with and detect species 14 and 16 In studies conducted in support of the invention, blood samples were collected from 39 women with osteoporosis and 41 women having normal bone density The samples were tested using the non-latent TGF-β assay of Example 3, and the small latent + mature TGF-β dimer assay described in Example 2 The results were as follows
In the control group, the average level of small latent TGF + mature dimer was 7 0 _+ 1 6 ng/mL, whereas the average for the osteoporosis subjects was 4 3 +_ 0 8 ng/mL However this difference was not statistically significant (tied p = 0 09, Mann Whitney U-test) For non-latent TGF-β, the trend was reversed* in control subjects, the average level was 5 4 +_ 1 6 ng/mL, whereas for the osteoporosis subjects the average was 10 7 +_ 2 7 ng/mL Thus, in the control group, the average ratio [non- latent] /[small latent + mature dimer) was about 0 8, whereas the average ratio for the osteoporosis subjects was about 2 5 Moreover, only 10-15 % of the women with normal bone density had a ratio > 1 0, whereas > 80% of the osteoporosis subjects had such a ratio ( > 1 0)
These results show that the measured ratio provides a useful indication of the status of bone resoφtion in human subjects Furthermore, the results show how a threshold can be selected to discriminate between subjects likely to have elevated bone resoφtion levels, and subjects who are either healthy or whose bone resoφtion status is uncertain For example, subjects showing ratios greater than 1 0, as just discussed, would be likely candidates for elevated bone resoφtion levels, although some false positives would also occur Conversely, a ratio of less than 1 0 would be a likely indication that the subject has normal bone resoφtion, although false negatives on the order of 10-20% might occur It will be appreciated that other threshold ratios could be selected to improve the accuracy of the assay, depending on the desired level of tolerance for false positives or false negatives
In an alternative embodiment, the difference between the levels of non-latent TGF-β and small latent TGF-β + mature βl dimer is determined, where a difference that is above a selected threshold is an indication that a bone resoφtion disorder may be present in the subject, and a difference that is below a selected threshold may be used as an indication of normal bone resoφtion
C TGF-β-OP Protein Assay
In this aspect of the invention, the level of a protein designated OP-TGF-β in a body fluid is measured, where a measured level above a selected threshold level is an indication that a bone metabolism abnormality associated with elevated bone resoφtion may be present in the subject Example 7 describes a study showing certain physical properties of the TGF-β-OP protein of the invention In brief, serum samples were obtained from two normal, healthy male subjects (45 and 54 years old, respectively), and from a woman diagnosed with osteoporosis Each sample was applied to an anion exchange column and fractionated using a sodium chloride salt gradient in 10 itiM sodium phosphate, pH 6 8 One mL fractions were collected and assayed for TGF-βl -containing proteins using a commercial ELISA kit (Quantikine ELISA kit, R&D Systems), after an initial acidic urea pre-treatment step to release mature TGF-β from latent forms
Fractions containing TGF-β proteins (which eluted as a broad peak) were combined, concentrated, and loaded on a gel filtration column Fractions (0 5 L) were collected and assayed for TGF-β proteins as with the preceding anion exchange column The anion-exchange chromatograms are shown in Figs 3A and 3B
As can be seen, the two healthy subjects showed qualitatively similar profiles (Fig 3A) Specifically, the sample from the 45 year old subject showed TGF-prote peaks at elution volumes of about 12, 24, and 30 mL, corresponding to the mature TGF-β dimer (A), the platelet large latent complex (P), and the small latent form (L), respectively In addition, this sample showed a peak at about 19 mL, which appears to correspond to a previously uncharac- teπzed form of TGF-β The sample from the 54 year old showed peaks at about 24 mL and 30 mL, corresponding to the large latent complex and small latent complex, respectively, but only a weak peak at 14 mL for the mature dimer form
The osteoporotic subject, on the other hand, exhibited a large new peak eluting at about 35 L (indicated by arrow and labeled OP-TGF-β), in addition to the peaks typically observed in normal subjects The new peak constitutes a new form of TGF-β which is clearly distinguishable from other known forms of TGF-β, particularly mature TGF-β, small latent TGF-β, and the 220-240 kDa large latent complex
When chromatographed on a gel filtration column, the OP-TGF-β protein exhibits an elution volume consistent with a molecular weight of about 10 to 15 kDa, suggesting that OP- TGF-β consists of a mature TGF-β chain in monomenc form, or a truncated TGF-β monomer In addition, studies conducted in support of the invention indicate that the OP-TGF-β protein is recognized by (I) a type II TGF-β receptor and (n) a antibodies which recognized mature TGF-β, but that the protein is not bound by antibodies which recognize latent forms of TGF-β, consistent with the OP-TGF-β protein being a mature TGF-β monomer
The foregoing results do not rule out the possibility that the OP-TGF-β protein is a TGF-β monomer or dimer complexed with one or more additional polypeptides, or is a truncated form of mature TGF-β dimer Furthermore, it is possible that the migration of OP-TGF-β through the gel filtration matrix is retarded due to transient interactions with the matrix, giving rise to an anomolously low apparent molecular weight However, the OP-TGF-β protein is clearly different from mature TGF-β and the small and large latent complexes, as demonstrated by the chromatogram shown in Fig 3B In another embodiment, the invention includes a method of purifying an OP-TGF-β polypeptide from a liquid sample, comprising the steps of (I) contacting the liquid sample with a solid support having immobilized thereon a binding partner which is lmmunospecific for mature TGF-βl and OP-TGF-β, under conditions effective to specifically bind the polypeptide to the immobilized binding partner, (n) washing the support to remove non-specifically bound materials, and (in) after the washing step, eluting bound polypeptide from support Where the sample to be contacted with the support contains both mature TGF-β and OP-TGF-β, the polypeptide eluted from the support may be further purified, e g , by gel filtration or C- 1 reversed phase HPLC, to resolve the OP-TGF-β from mature TGF-β and produce substantially pure OP-TGF-β Alternatively, where the liquid sample contacted with the support contains OP-TGF-β, but not any other TGF-β species bound significantly by the immobilized binding partner, the OP-TGF-β polypeptide eluted from the support is substantially pure
In a related embodiment, the purification method is conducted using an immobilized binding partner which is immunospecific for OP-TGF-β. to the exclusion of mature dimeric TGF-βl or any latent form thereof, such that the OP-TGF-β polypeptide is eluted in substantially pure form In this regard, the invention includes an immunological binding partner which is immunospecific for OP-TGF-β, and which does not bind mature dimeric TGF-β or any latent form thereof
The invention can be further understood from the following examples, which are intended to illustrate but not in any way limit the scope of the invention
EXAMPLES Materials and Methods
Goat anti-rabbit IgG coupled to horseradish peroxidase was obtained from Sigma Chemical Co (Poole, UK) or Jackson Laboratories (Cat No 705-035-147) Antibodies BD A 19, BDA47 and BDA48 were obtained from R&D Systems (Oxford, United Kingdom) The BDA19 antibody is a chicken polyclonal IgY antiserum BDA47 is an affinity-purified rabbit polyclonal IgG antibody BDA19 binds the mature dimer and small latent forms of TGF-βl , but not TGF-β3 BDA47 binds both the mature dimer and small latent forms of TGF-βl and TGF-B3 BDA48 is a polyclonal antiserum specific for the B3 isoform of TGF
Recombmant human TGF-βl and TGF-β3 were obtained from R&D Systems or Amersham International
To prepare platelet-poor plasma, peripheral venous blood samples (10 L) were collected from human subjects and transferred immediately to tubes containing 1 1 mL of sterile 3 8% (w/v) tπsodium citrate in MilhQ water at room temperature The samples were centπfuged to remove red blood cells (250 x g, 15 mm) Apyrase (Sigma) was added to the resulting platelet-rich plasma to a final concentration of 100 mg/L, to prevent platelet degranulation PMSF (1 mmol/L) and aprotinm (1 mg/L) were added to prevent proteolytic activation or degradation of TGF-β The samples were then centπfuged (700 x g, 15 mm), and the supernatant platelet-poor plasma was separated from the platelet pellet The platelet-poor plasma was kept at room temperature until assay by ELISA within 2 h of preparation or was stored frozen in 0 5-mL aliquots at -80°C
For serum preparation, peripheral venous blood samples from human subjects were dispensed immediately into polypropylene tubes and allowed to clot at room temperature for 2 h The collected samples were centrifuged (1000 x g, 4 min), after which the serum was removed and either stored on ice until assay within 2 h or stored at -80 °C for later assay
Example 1 Extracellular Domain of TGF-β Type II Receptor
A Cloning of Extracellular Domain of TGF-β Type II Receptor
The extracellular domain of the TGF-β type II receptor was amplified from the vector H2 3FF, as described in Lin et al ( 1992)
In brief, the vector DNA was linearized with NotI, precipitated, and resuspended at 10 ng/μL Amplification by polymerase chain reaction (PCR) was carried out in a 50 μL reaction volume containing vector DNA (2 5 μL), lOx TAQ buffer (5 μL) (LKB Pharmacia, Uppsala, Sweden), 250 ng ofeach ohgonucleotide primer (5'-GAATTCCCATGGGTCGGGGGCTGCTC and GAATTCGTCAGGATTGCTGGTGTT), TAQ polymerase ( 1 U) and a mixture of dAI P, dTTP, dCTP and dGTP to give a final concentration of 200 μM for each nucleotide The sample was overlaid with 50 μL paraffin oil -The reaction was carried out using a thermal cycler for 30 cycles (denaturing at 94°C for 1 min, annealing at 55 °C for 2 min, and elongation at 72 °C for 2 min)
The 450 bp product was purified by eiectrophoresis in low gel temperature agarose, digested with EcoRI, and cloned into the glutathione-S-transferase fusion vector pGEX 2T (LKB Pharmacia) Vectors carrying inserts in the required orientation were identified by plasmid mapping The sequence of the insert was checked by subcloning the 450 bp EcoRI fragment from the chosen clone (pGTIC) into Bluescπpt KS + , followed by double strand sequencing The sequence showed a single base change (C to A at position + 13 from the initiation codon) compared with the published sequence (Lin et al , 1992), which introduces a Leu to Met mutation in the expressed receptor protein
B Purification of Expressed Receptor
An overnight culture of Escherichia coll TGI containing pGTIC was diluted 1 100 into fresh 2YT medium (500 mL) containing 270 μM ampicillin and grown to an OD^o of 0 5 Production of receptor protein was induced by addition of 1 mM isopropylthiogalactoside and the cells were harvested 5 h later by centπfugation The cells were resuspended in 50 mL phosphate-buffered saline (PBS, 150 mM NaCl, 6 M sodium phosphate pH 7 3) containing TRITON X-100 and 1 mM PMSF and lysed by sonication for 5 min The lysate was centπfuged (10,000 x g, 5 min) and the receptor protein was purified from the supernatant by the one-step purification method of Smith and Johnson ( 1988) The purified protein was greater than 95 % pure, as judged by chromatography on a Superdex 200 HR column in 20 mM ammonium bicarbonate, pH 8 0
Example 2
Immunoassay for Latent TGF-β + Mature TGF-βl Dimer MAXISORP 96-well microtiter plates (Gibco, Uxbπdge, UK) were incubated with 50 μL BDA19 anti-TGF-β chicken IgY (40 g/mL) diluted in Tπs-buffered saline (TBS, 137 mM NaCl, 50 mM Tπs-HCl, pH 7 4) and shaking the plates until dry by evaporation at room temperature (approx 12 h) The plates were washed 3 3 min with PBS, blocked with 350 μL 3 % FAF-BSA in TBS for 1 h, washed 3 x 3 min with TBS, and incubated for 2 h with 100 μL of test samples or dilutions of TGF-β stock solution for calibration Standard TGF-β 1 stock solutions were diluted in TBS to concentrations between 0 4 pM and 4000 pM
The plates were washed with a wash buffer consisting of TBS, 3 % FAF-BSA, and 0 1 % Triton X-100, and incubated with 20 μL detection antibody (BDA47) at 1 μg/mL in wash buffer for 1 h The plates were rinsed with wash buffer (3 3 mm) and incubated with an antibody against rabbit IgG conjugated to horseradish peroxidase at 1 2500 dilution in wash buffer for 1 h After washing with wash buffer (3 x 3 min), the plates were incubated for 15 min with o-phenylenediamine according to the manufacturer's instructions (Sigma Chem Co ) The reaction was stopped by addition of an equal volume of 3 M HC1, and the absorbance values were read on an ELISA platereader (Titerteck Multiscan, Flow Laboratories, High Wycombe, UK) within 15 min of stopping the reaction A scheme for this assay is provided in Fig IB
Example 3
Immunoassay for Non-Latent TGF-β This immunoassay was performed in the same way as the assay in the preceding example, except that the microtiter plates were coated with purified TGF-β receptor from Example 1 (20 μL per well of receptor m TBS, 50 μg/mL) instead of BDA 19, and the BDA47 antibody was used at 5 μg/mL instead of 1 μg/mL A scheme for the assay is provided in Fig IB Example 4 Assay for TGF-β3 For the assays below, the following components from a Quantikine ELISA kit obtained from R&D Systems were used (a) microtitre plate strips coated with TGF-β receptor, (b) wash buffer 25X concentrate, (c) RD6M diluent, and (d) stop solution (3 M HC1) A Solutions and Reagents The following solutions were prepared
1 BDA19 stock 5 mg/mL BDA19 (R&D Systems) in phosphate buffered saline pH 7 4 (PBS)
2 BDA 19 working solution BDA19 stock diluted 1 125 in RD6M to a final BDA19 concentration of 40 μg/mL
3 BDA48 stock 5 mg/mL BDA48 (R&D Systems) in PBS, pH 7 4
4 BDA48 working solution BDA48 stock diluted 1 50 in RD6M to give a final BDA19 concentration of 100 μg/mL
5 Anti-goat IgG peroxidase conjugate working solution prepared by 1 2000 dilution of == 1 mg/mL conjugate stock obtained from Sigma (cat no A-4174) into RD6M to give a final conjugate concentration of = 500 ng/mL, or prepared by 1 5000 dilution of a 0 5 mg/mL stock of anti-goat IgG peroxidase conjugate from Jackson Laboratories into RD6M to give a final conjugate concentration of = 100 ng/mL
6 TGF-B3 stock recombinant human TGF-B3 (2 μg) was dissolved in 4 mM HC1 (400 μl) containing 1 mg/mL fatty acid-free bovine serum albumin (Sigma, A-6003) to give a final concentration of 5 μg/mL
7 Activation buffer 1 25 M acetic acid/5 M urea in water
8 Neutralisation buffer 1 37 M NaOH/0 5 M HEPES in water
B Samples and Controls Frozen serum samples were thawed at room temperature prior to assay Eppendorf tubes (1 5 mL) were loaded each with 75 μl of activation buffer followed by 75 μl of thawed sample The contents of the tubes were mixed by pipette and then incubated for 10 minutes at room temperature
In the meantime, an initial standard solution of TGF-153 was prepared by adding 2 μl of TGF-β3 stock (5 μg/mL) to an Eppendorf tube containing 100 μl BDA19 working solution (40 μg/mL BDA19) and 300 μl RD6M diluent, giving a final BDA19 concentration of 10 μg/mL and a final TGF-β3 concentration of 25 ng/mL After thorough mixing, this initial TGF- β3/BDA19 solution was diluted serially 1 1 into six successive Eppendorf tubes each containing 150 μl RD6M diluent and 50 μl working BDA19 solution The seven TGF-B3 standard tubes thus contained 10 μg/mL BDA19 and from 25 ng/mL to 0 39 ng/mL TGF-β3 As a control, an additional tube containing 150 μl RD6M diluent and 50 μl working BDA19 solution was also prepared without TGF-B3
C Assay The seven TGF-β3/BDA19 and TGF-free control were each loaded into separate wells (200 μl each) in a Quantikine ELISA strip (R&D Systems) To each acid-treated sample tube (after the above 10 minute incubation) was added 75 μl neutralisation buffer and 75 μl BDA19 working solution The contents of the sample tubes were mixed, and 200 μl ahquots of each were transferred to separate wells on the ELISA strip The strips were covered with adhesive film and incubated for 1 hour at room temperature on a microtitre plate shaker (500 φ ) After incubation, the wells were aspirated by inversion and washed 3 times with wash buffer (R&D Systems, 1 25 dilution) To ensure liquid removal, the strips were blotted on a clean paper towel between wash steps To each well was then added 50 μl BDA48 working solution (100 μg/mL), and the strips were again covered with adhesive film and incubated for 1 hour at room temperature with shaking After incubation, the wells were aspirated and washed three times as before 1 o each well was then added 200 μl of anti-goat IgG peroxidase conjugate working solution, and the wells were sealed and incubated for 30 minutes at room temperature with shaking
After incubation, the wells were aspirated and washed four times as before To each well was then added 200 μl of peroxidase substrate solution (K-BLUE substrate solution containing TMB with stabilized peroxide, ELISA Technologies), and the strips were incubated at room temperature for exactly 12 minutes without shaking The peroxidase reactions were stopped by adding 50 μl stop solution (3 M HC1), and the absorbance of each well was measured within 30 minutes at 450 nm using a microtitre plate reader
Example 5
Assay for TGF-B3 in Blood Fractions from Healthy Subiects Serum, plasma and platelet samples were prepared from 10 healthy individuals consisting of 7 males and 3 females and ranging from 24 to 54 years in age The samples were then assayed for TGF-B3 as described in Example 4 No TGF-β3 was detected in any of the plasma or serum samples from these subjects However, low levels of TGF-β3 ( = 2 ng/mL) were detected in the platelet samples from most of the subjects Example 6 Assay for TGF-B3 in Normal and Osteoporosis Subiects Serum samples were prepared from 25 osteoporosis patients identified by their having age- corrected lumbar spine bone densities more than 2 standard deviations below the population mean, and from 29 control individuals showing normal bone densities (within 1 standard deviation of the age-corrected mean) The subjects were women from 46 and 67 years in age The serum samples were assayed for TGF-β3 as described in Example 4 The results are shown in Figs 2A and 2B As can be seen, the subjects with low bone density exhibited a mean TGF-β3 level of 6 4 +_ 0 6 ng/mL (n = 25), compared with 2 7 +_ 0 6 ng/mL for the control group In addition, more than half of the control individuals showed no detectable TGF β3, whereas all but two of the osteoporosis subjects showed detectable TGF-β3 levels greater than about 2 ng/mL
Example 7 Chromatographic Measurement of OP-TGF-β
Serum samples were obtained as described in the Material and Methods section from two normal, healthy male subjects and (45 and 54 years old, respectively), and from a female patient diagnosed with osteoporosis Each sample (1 L) was diluted 1 3 with 10 mM sodium phosphate, pH 6 8, and then applied to a 1-mL ResourceQ disposable MonoQ column (Pharmacia) on an FPLC system (Pharmacia) equilibrated in 10 mM sodium phosphate pH 6 8 (buffer A) After the sample had been loaded on the column, the column was washed with buffer A (flow rate 1 mL/min) until the absorbance at 280 nm had returned to baseline Bound proteins were gradient-eluted using increasing amounts of 10 mM sodium phosphate pH 6 8 containing 1 5 M NaCl (buffer B) using the following gradient program flow rate = 1 mL/min, 1 L per fraction collected, 100% buffer A for the first 5 mL, 0 to 33% buffer B over the next 30 L, constant 33% buffer B over the next 5 mL, 33 % to 100% buffer B over the next 10 mL, 100% buffer B for the next 5 mL, and re-equilibration in buffer A before the next run
The collected fractions were assayed for TGF-β proteins as follows A quots of the fractions were diluted 1 1 with 2 5 M acetic acid containing 10 M urea for 10 mn at room temperature to release mature 25-kDa TGF-β 1 dimer Samples were then neutralized by adding the same volume again of 2 7 M NaOH/1 M HEPES Neutralized samples were assayed immediately for TGF-βl using a Quantikine ELISA kit (R&D Systems) in accordance with the manufacturer's instructions Fractions containing TGF-β (typically fractions 20 to 29) were combined, diluted 1 4 with 200 mM sodium phosphate pH 6 8, and concentrated to 200 μL using an Amicon cell with a 3,000 kDa cut-off membrane The concentrated sample was then loaded onto a Superose 12 gel filtration column (Pharmacia) equilibrated in 200 mM sodium phosphate pH 6 8 on an FPLC system Fractions (0 5 mL) were collected from the time the sample was loaded (0 mL) until 25 mL of buffer had been passed through the column Insulin, which was used as a marker protein ( 1 1 kDa), eluted in an elution volume of 19 mL The collected fractions were assayed for TGF-β 1 proteins as for the MonoQ column The chromatograms obtained with the MonoQ column are shown in Figs 3A-3B for the normal and diseased samples, respectively
Although the invention has been described with reference to particular embodiments and illustrations, it will be appreciated that various modifications can be made without departing from the spirit of the invention

Claims

1 A method of screening for or monitoring the level of bone resoφtion in a human subject comprising, determining a level of total TGF-β3 in a body fluid sample obtained from a human subject, and comparing the level determined with a predetermined level characteristic ot normal subjects, wherein a determined level that is above a level characteristic of normal subjects is an indication that the subject has a bone resoφtion disorder
2 The method of claim 1 , wherein the bone resoφtion disorder is related to osteoporosis, hypeφarathyroidism, rheumatoid arthritis, osteoarthntis, Paget's disease, a malignant bone tumor, or metastatic cancer in bone
3 The method of claim 2, wherein the bone resoφtion disorder is osteoporosis
4 A method in accordance with any preceding claim, wherein the level of TGF-1.3 is measured using TGF-β3-specιfιc antibodies
5 The method of claim 4, wherein the antibodies are monoclonal antibodies
6 The method of claim 4, wherein the antibodies are polyclonal antibodies
7 The method of any preceding claim, wherein the body fluid is a blood fraction or urine
8 The method of claim 7, wherein the body fluid is serum or plasma
9 A method of assessing a human patient's risk of contracting a bone resoφtion disorder comprising determining a level of total TGF-β3 in a body fluid sample obtained from a human subject, and comparing the level determined with a predetermined level characteristic of normal subjects, wherein a determined level that is above a level characteristic of normal subjects is an indication of increased risk of contracting a bone resoφtion disorder
10 The method of claim 9, wherein the bone resorption disorder is related to osteoporosis, hypeφarathyroidism, rheumatoid arthritis, osteoarthntis, Paget's disease, a malignant bone tumor, or metastatic cancer in bone
1 1 The method of claim 10, wherein the bone resoφtion disorder is osteoporosis
12 A method in accordance with any of claims 9 to 1 1 , wherein the level of TGF B3 is measured using a TGF-β3-specιfιc antibody
13 The method of claim 12, wherein the antibody is a monoclonal antibody
14 The method of claim 12, wherein the antibody is a polyclonal antibody
15 The method of any of claims 9 to 14, wherein the body fluid is a blood fraction or urine
16 The method of claim 15, wherein the body fluid is serum or plasma
17 A method of assessing a human subject's risk of suffering a bone fracture, comprising determining a level of total TGF-β3 in a body fluid sample obtained from a human subject, and comparing the level determined with a predetermined level characteristic of normal subjects, wherein a determined level that is above a level characteristic of normal subjects is an indication of increased risk of bone fracture
PCT/US1997/014434 1996-08-16 1997-08-15 Method and kit for assessing bone resorption WO1998007032A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41520/97A AU4152097A (en) 1996-08-16 1997-08-15 Method and kit for assessing bone resorption

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2408496P 1996-08-16 1996-08-16
US60/024,084 1996-08-16

Publications (1)

Publication Number Publication Date
WO1998007032A1 true WO1998007032A1 (en) 1998-02-19

Family

ID=21818792

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/014434 WO1998007032A1 (en) 1996-08-16 1997-08-15 Method and kit for assessing bone resorption

Country Status (2)

Country Link
AU (1) AU4152097A (en)
WO (1) WO1998007032A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000013024A1 (en) * 1998-08-26 2000-03-09 Medvet Science Pty Ltd. Predictive assessment of certain skeletal disorders
US6300534B1 (en) 1998-07-01 2001-10-09 Nippon Petrochemicals Company, Limited Process for producing dehydrogenated compounds of m-ethyldiphenylalkane

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5262319A (en) * 1985-04-19 1993-11-16 Oncogene Science, Inc. Method for obtaining bone marrow free of tumor cells using transforming growth factor β3
US5393739A (en) * 1990-11-30 1995-02-28 Celtrix Pharmaceuticals, Inc. Use of bone morphogenetic protein in synergistic combination with TGF-β for bone repair

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5262319A (en) * 1985-04-19 1993-11-16 Oncogene Science, Inc. Method for obtaining bone marrow free of tumor cells using transforming growth factor β3
US5393739A (en) * 1990-11-30 1995-02-28 Celtrix Pharmaceuticals, Inc. Use of bone morphogenetic protein in synergistic combination with TGF-β for bone repair

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6300534B1 (en) 1998-07-01 2001-10-09 Nippon Petrochemicals Company, Limited Process for producing dehydrogenated compounds of m-ethyldiphenylalkane
WO2000013024A1 (en) * 1998-08-26 2000-03-09 Medvet Science Pty Ltd. Predictive assessment of certain skeletal disorders

Also Published As

Publication number Publication date
AU4152097A (en) 1998-03-06

Similar Documents

Publication Publication Date Title
JP3387745B2 (en) MET proto-oncogene as liver growth factor receptor
EP0742902B1 (en) A method of assaying collagen fragments in body fluids, a test kit and means for carrying out the method and use of the method to diagnose the presence of disorders associated with the metabolism of collagen
EP1151307B1 (en) Methods for differentiating and monitoring parathyroid and bone status related diseases
Palosuo et al. Purification of filaggrin from human epidermis and measurement of antifilaggrin autoantibodies in sera from patients with rheumatoid arthritis by an enzyme-linked immunosorbent assay
Muller et al. Autoantibodies reacting with poly (ADP-ribose) and with a zinc-finger functional domain of poly (ADP-ribose) polymerase involved in the recognition of damaged DNA
Liu et al. Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation
WO1998028006A1 (en) Diagnosis and treatment of pathological pregnancies
IL201186A (en) Method of determining risk of scoliosis
US20030059859A1 (en) Netrin receptors
EP0820598B1 (en) Assaying protein fragments in body fluids
US20030119058A1 (en) Method of assaying type III collagen degradation
Chia et al. Direct binding of F‐actin to ponticulin, an Integral plasma membrane glycoprotein
Klein et al. A microtiter well assay system to measure insulin activation of insulin receptor kinase in intact human mononuclear cells: decreased insulin effect in cells from patients with NIDDM
US8663944B2 (en) Cartilage intermediate layer protein 2 C1 and its use to differentiate osteoarthritis from rheumatoid arthritis and non-disease conditions
AU672808B2 (en) Method of determining metastatic potential of tumor cells
US7479278B2 (en) Troponin I polypeptide fragments and uses thereof
JPH0678788A (en) Method of testing bone fragility and osteoporosis for danger of fracture
CN108139404B (en) Antibody specifically recognizing and binding to REIC/Dkk-3 protein of active structure, and monitoring of cancer therapy using the anti-REIC/Dkk-3 antibody
US20100297147A1 (en) Compositions and methods for modulating tlr14 activity
US6107047A (en) Assaying protein fragments in body fluids
WO1998007032A1 (en) Method and kit for assessing bone resorption
Hildebrand et al. Characterization and identification of heparin-induced nonopioid-binding sites for beta-endorphin in human plasma.
Matsuda et al. Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease
JP5852433B2 (en) Determination method of arteriosclerosis by sortilin
WO1993024836A1 (en) Screening method for identifying women at increased risk for preterm delivery

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA CN CZ HU JP KP KR MX NO NZ RU SE SG US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998510076

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA