Note: Descriptions are shown in the official language in which they were submitted.
WO94/1~72 PCT~S931L~21
~ 21~2~
SERUM PYRIDINIUM CROSSLINKS ASSAY
1. Field of the Invention
The present invention relates to a m~thod for
assessing bone collagen breakdown in a hur.lan subject,
by assaying the level of peptide-free pyridinium
crosslinks in a human blood fluid sample.
2. References
Black, D., et al., Anal. Biochem. 169:197-203
(1988).
Black, D., et al., Annais of Rheumatic Diseases
15 48:641-644 (1989).
Brown, J.P., et al., Lancet 1091-1093 (1984).
Campbell, A., Monoclonal Antibody and
Immunosensor Technology, Elsevier (1991).
Daniloff, Y., et aL., Connect. Tissue. Res.
20 27:187 (1992).
Eyre, D.R., et al., Anal. Biochem. 137:380-388
(1984).
Eyre, D.R., et al., FEBS 2:337-341 (1987).
Fujimoto, D., et al., J. Biochem. 83:863-867
25 (1978).
Fujimoto, D., et al., J. Biochem. 94:167-173
(1983).
Gosling, J., Clin. Chem. 36(8):1408 (1990).
Gunja-Smith, Z., et al., Biochem. J. 197:759-762
30 (1981).
Harlow, E., et al., Antibodies: A Laboratory
Manual, Cold Spring Harbor Lab (1988).
Henkel, W., et al., Eur. J. Biochem. 165:427-436
(1987).
Macek, J., et al., Z. Rheumatol. 46:237-240
(1987).
WO94/1~72 PCT~S93/~21 ~
21~ 2~4
Ogawa, T., et al., Biochem. Biophys. Res.
Commune. 107:1251-1257 (1982).
Robins, S.P., Bioc~em J. 207:617-620 (1982a).
Robins, S.P., in "Collagen in Health and
Disease" (Weiss, J.B., et al., eds.) pp. 160-178,
Churchill Livingstone, Edinburgh (1982b).
Robins, S.P., Biochem. J. 215:167-173 (1983).
Robins, S.P., et al., Ann. Rheumatic Dis.
45:969-973 (1986).
Robins, S.P., et al., Biochim. Biophys. Acta.
914:233-239 (1987).
Segel, I., Biochemical Calculations, John Wiley
and Sons, (1976).
Seibel, et al., J. Rhe~matol 16:964-970 (1989).
Wong, S.S., ChemistrY cf Protein Coniuqation and
Cross-Linkinq, CRC Press, Boca Raton, Florida (1991).
3. Backqround of the Invention
There are a variety of conditions in humans
which are characterized by a high level of bone
resorption and by an abnormal balance between bone
formation and bone resorption. Among the more common
of these are osteoporosis, Paget's disease, and
conditions related to the progress of benign and
malignant tumors of the bone and metastatic cancers
which have been transferred to bone cells from, for
example, prostate or breast initial tumors. Other
conditions which are associated with changes in
collagen metabolism include osteomalacial diseases,
rickets, abnormal growth in children, renal
osteodystrophy, and a drug-induced osteopenia.
Irregularities in bone metabolism are often side
effects of thyroid treatments and thyroid conditions
per se, such as primary hypothyroidism and
thyrotoxicosis as well as Cushing's disease.
~ WO94/1~72 2 ~ 5 ~ 2 3 ~ PCT~S93/L~21
It has been recognized that disorders of bone
resorption or other conditions characterized by an
abnormal balance between bone formation and bone
resorption can be detected by altered levels of
pyridinium crosslinks in urine (Robins, 1982b; Macek;
Black). The crosslinks take the form of compounds
containing a central 3-hydroxy pyridinium ring in
which the ring nitrogen is derived from the epsilon
amino group of lysine or hydroxylysine (Fujimoto,
1978; Robins, 1982a; Gunja-Smith; Ogawa; Eyre).
The pyridinium crosslink compounds found in
urine can be grouped into four generai classes: (1)
native free crosslinks having a molecular weight of
about 400 daltons (Fujimoto), (2) glycosylated
crosslinks and crosslink peptide forms having a
molecular weight of between about 550 and 1,000
daltons (Robins, 1983), (3) crosslink peptide forms
having a molecular weight between 1,000 and 3,500
daltons (Robins, 1983, 1984, 1987; Henkel; Eyre), and
(4) crosslink peptide forms having a molecular weight
greater than 3,500 daltons. In normal adults, these
forms account for about 38~ (1), 40% !2), 15% (3),
and 7~ (4) of total urinary crosslinks (Daniloff).
About 80% of the free crosslinks in normal adult
urine is pyridinoline (or Pyd), whose ring nitrogen
is derived from a hydroxylysine residue, and about
20~, deoxypyridinoline, or Dpd, whose ring nitrogen
is derived from a lysine residue. This ratio of
Pyd/Dpd applies roughly to the other three classes of
crosslinks in urine. The higher molecular weight
crosslinks can be converted to free crosslinks by
acid hydrolysis (Fujimoto, 1978).
Methods for measuring pyridinium crosslinks in
urine have been proposed. One of these methods
involves the measurement of total hydrolysed Pyd,
CA21 51 234
i.e., Pyd produced by extensive hydrolysis of urinary crosslinks, by
quantitating the hydrolysed Pyd peak separated by HPLC (Fujimoto, 1983).
The relationship between total hydrolysed Pyd to age was determined by
these workers as a ratio to total hydrolysed Pyd/creatinine, where creatinine
level is used to normalize crosslink levels to urine concentration and skeletal
mass. It was found that this ratio is high in the urine of children, and
relatively constant throughout adulthood, increasing slightly in old age. The
authors speculate that this may correspond to the loss of bone mass
observed in old age.
Studies on the elevated levels of total crosslinks in hydrolyzed urine of
patients with rheumatoid arthritis has been suggested as a method to
diagnose this disease (Black, 1989). The levels of total hydrolyzed
crosslinks for patients with rheumatoid arthritis (expressed as a ratio of totalcrosslinks measured by HPLC to creatinine) were elevated by a factor of 5
as compared to controls. However, only total hydrolysed Pyd, but not total
hydrolysed Dpd, showed a measurable increase.
In a more extensive study using hydrolyzed urines, Seibel et a/.
showed significant increases in the excretion of bone-specific total
hydrolysed Pyd and Dpd crosslinks relative to controls in both rheumatoid
and osteoarthritis. The most marked increases for total hydrolysed Pyd were
in patients with rheumatoid arthritis (Seibel).
21~1234
4a
Paterson et al. (Br. J Cancer (1991) 64:884-886)
report that total hydrolyzed Pyd and Dpd measured in
hydrolyzed urine may be useful ~or detecting the
spread of metastatic cancer to bone.
Assay methods, such as those just noted, which
involve HPLC quantitation of crosslinks ~rom hydrolzed
samples are relatively ~ime-consuming and expensive to
carry out, and may not be practical for widespread
screening or monitoring therapy in bone-metabolism
disorders.
5~,a~5~ -
~A215 1 234
Immunoassays have also been proposed for measuring urinary
crosslinks. U.S. Patent Nos. 4,973,66 and 5,140,103 and International
Patent Application No. W0 01/08478 disclose an assay for measuring bone
resorption by detection in urine of certain peptide-linked pyridinium species
associated with bone collagen. These are obtained from the urine of
patients suffering from Paget's disease, a disease known to involve high
rates of bone formation and destruction. The assay relies on
immunospecific binding of crosslink compounds containing the specific
peptide fragment or extension with an antibody prepared against the
crosslink peptide. It is not clear whether and how the concentration of
crosslink peptide being assayed relates to total urinary crosslinks.
Robins has described a technique for measuring pyridinoline in urine
by the use of an antibody specific to hydrolysed Pyd (Robins, 1986). The
method has the limitation that the antibody was found to be specific for the
hydrolized form of Pyd, requiring that the urine sample being tested first be
treated under hydrolytic conditions. The hydrolytic treatment increases the
time and expense of the assay, and precludes measurements of other native
pyridinium crosslinks.
PCT International Publication No. W0 91/10141 discloses a method
of assessing bone collagen degradation in human subjects by measuring the
level of native, peptide-free pyridinoline or deoxypyridinoline in non-
hydrolyzed urine samples. The method represents an improvement over
earlier methods based on analysis of urine samples because it avoids the
hydrolytic sample pretreatment employed previously.
W094/1~72 PCT~S93/12321
21~1~3~
It would be desirable to assay bone collagen
degradation levels in a human subject by measuring
the level of pyridinium crosslink species in a blood
fluid sample. Such an assay could be integrated into
an automated clinical system designed to assay a
variety of serum analytes. The approach also has the
advantage that the measured levels would not have to
be corrected for variation in sample volume, e.g., by
determining a ratio of deoxypyridinoline/creatinine.
4. Summary of the Invention
The present invention includes, in one aspect, a
method of screening for the presence of a bone
resorption disorder in a human subject. In the
method, a blood-fluid sample is obtained from a
subject and is reacted with an antibody which is
capable of reacting immunospecifically with
pyridinium crosslinks selected from the group
consisting of native free pyridinoline, native free
deoxypyridinoline, or both native free pyridinoline
and deoxypyridinoline, to form an immunocomplex
between the antibody and such pyridinium crosslinks
in the sample. The amount of immunocomplex formed is
measured to determine the concentration of the
selected pyridinium crosslinks in the sample. The
subject is indicated as having such a bone resorption
disorder if the determined concentration is above (i)
5 nM native free pyridinoline, (ii) 1 nM native free
deoxpyridinoline, or (ii) 6 nM combined native free
pyridinoline and dexoypyridinoline. The blood fluid
sample may be serum or plasma, for example.
The bone resorption disorders that are screened
for by the method include disorders that are
characterized by elevated levels of hydrolysed
pyridinoline crosslinks in the urine.
~ WO94/1~72 21 512 ~ ~ PCT~S93/12321
The antibody in the method can be a monoclonal
antibody or polyclonal antibody, and preferably has a
binding constant with respect to the selected
pyridinium crosslinks of at least 5 x 107/molar.
In one embodiment, the antibody used in the
method has a ratio of reactivity toward the selected
pyridinium crosslinks and urinary pyridinium peptides
larger than 1,000 daltons in molecular weight, of
greater than about 5:1. In related embodiments, the
antibody has a ratio of reactivity toward the
selected pyridinium crosslinks and urinary pyridinium
peptides larger than 1,000 daltons in molecular
weight, of greater than about 10:1, and more
preferably, greater than 20:1.
In a specific embodiment, the antibody is
specific for native free pyridinoline, and has a
ratio of reactivity toward native free pyridinoline
and native free deoxypyridinoline of greater than
about 5:1. In a second embodiment, the monoclonal
antibody is specific for native free deoxypyri-
dinoline, and has a ratio of reactivity toward native
free deoxypyridinoline and native fr e pyridinoline
of greater than about 25:1. In another embodiment,
the monoclonal antibody is specific for both native
free pyridinoline and native free deoxypyridinoline
and has a ratio of reactivity toward native free
pyridinoline and native free deoxypyridinoline of
between about 2:1 and 1:2.
More generally, the ratio of reactivity toward
native free pyridinoline and native free deoxy-
pyridinoline can be from greater than 5:1 to less
than 1:25, including all ratios in between.
In one preferred embodiment, the sample is
contacted, in the presence of the antibody, with a
WO94/1~72 PCT~S931L~21
215~ 234
selected pyridinium crosslink-coated solid support
effective to compete with such selected crosslinks in
the sample for binding to the antibody. The amount
of immunocomplex formed between the antibody and the
selected pyridinium crosslinks in the sample is
measured indirectly, by measuring the amount of
antibody bound to the solid support.
In another preferred embodiment, the sample is
contacted, in the presence of exogenous reporter-
labeled or reporter-labelable selected pyridinium
crosslinks, with a solid-phase support that is coated
with a selected amount of pyridinium crosslinks,
under conditions such that the selected pyridinium
crosslinks from the sample compete with the exogenous
crosslinks for binding to the immobilized antibody on
the support.
The method may be used for monitoring treatment
of a condition associated with an elevated level of
collagen degradation, by monitoring changes in the
concentration of the selected pyridinium crosslinks
during such treatment.
In a related aspect, the invention includes a
method of monitoring the status of a human cancer
which involves or has the potential to progress to a
metastatic condition which involves abnormalities in
bone resorption rates. In the method, a blood-fluid
sample is obtained from a subject and reacted with an
antibody capable of reacting immunospecifically with
pyridinium crosslinks selected from the group
consisting of native free pyridinoline, native free
deoxypyridinoline, or both native free pyridinoline
and deoxypyridinoline, to form an immunocomplex
between the antibody and such pyridinium crosslinks
in the sample. The amount of immunocomplex formed is
measured to determine the concentration of the
I
2 3 ~
selected pyridinium c_osslinks in the sample. An
abnor~ality in bone resorption rzte associated with
the cancer, suggesting possible metastisis in bone,
is indicated if the determined pyridinium crosslinks
concentration is above (i) 5 nM native free
pyridinoline, (ii) 1 nM free deox~yridinoline, and
(ii) 6 nM combined pyridinoline and
deo~ypyridinoline. _-
The method may be used for monitoring treatment
of a cancer which is characterized by involvement of
collagen de~radation, by monitoring changes in the
selected pyridinium crosslinks concentration during
treatment of the cancer.
In another aspect, the invention includes a
method of assaying bone collagen breakdown lev~ls in
a human subject. In the method, a blood-rluid sample
is obtained from a subject and is reacted with an
antibody which is capzble of reacting
immunospecifically with pyridinium crosslinks
selected from the group consisting of native free
pyridinoline, native free deoxypyridinoLine, or both
native free pyridinoline and deoxypyridinoline, and
where the antibody hzs a binding constant for said
selected crosslinks of at least lO~/molzr, to form an
immunocomplex between .he antibody and such
pyridinium crosslinks in the sample. The amount of
immunocomplex formed is measured to determine the
concen~ration of the selected pyridinium crosslinks
in the sample. The subject is indicated as having
suc~ a bone resorption disorder if the determined
concentration is above (i) 5 n~ native free
pyrLdinoline, (ii) 1 nM native free deoxpyridinoline,
o_ (ii) 6 nM combined native free pyridinoline and
deoxypyridinoline.
. .~ ... ~
WO94/14072 PCT~S93/12321
2~S 1 234
For the methods above, it will be appreciated
that lower threshold values can be used in order to
increase the likelihood of identifying individuals
with elevated levels of bone degradation -- for
example, for detecting native free pyridinoline, a
value of about 3 nM; for native free deoxypyri-
dinoline, a value of about 0.5 nM, and for detecting
the combined concentration of native free pyri-
dinoline and deoxypyridinoline, a value of about
3.5 nM.
In another embodiment, the invention includes an
antibody having an affinity constant with respect to
the above selected pyridinium crosslinks of at least
lO8/molar. In one embodiment, the antibody has a
ratio of reactivity toward the selected pyridinium
crosslinks and urinary pyridinium peptides larger
than l,OOO daltons in molecular weight, of greater
than about 3:l, and preferably greater than about
5:l.
In another aspect, the invention includes a
diagnostic kit for use in the methods above. The
method includes an antibody such as described above,
and detection means for detecting the amount of
immunocomplex formed by reaction of the antibody with
the selected pyridinium crosslinks from a blood fluid
sample.
For detecting native free pyridinoline, the kit
for use in the method is preferably effective to
detect a blood pyridinoline concentration ("threshold
concentration") of at least about l nM, and more
preferably, at least 0.5 nM. For detecting native
free deoxypyridininoline, the kit is preferably
effective to detect a blood deoxypyridinoline
concentration of at least about O.l nM, and more
W094/1~72 PCT~S93/L~21
2151234
preferably, at least about 0.05 nM. For detecting
the combined concentration of pyridinoline and
deoxypyridinoline, the threshold concentration is
preferably 1.1 nM, and more preferably, 0.6 nM.
In one embodiment, the detection sensitivity of
the kit allows detection of the selected pyridinium
crosslinks concentration in a selected range, for
example, 1-10 nM for pyridinoline, or 0.1-10 nM for
deoxypyridinoline.
These and other objects and features of the
invention will become more fully apparent when the
following detailed description of the invention is
read in conjunction with the accompanying drawings.
Brief DescriPtion of the Drawinaqs
Figs. lA-lC illustrate steps in practicing one
embodiment of the invention;
Fig. 2 is a titration curve for an antibody
suitable for detecting pyridinoline (N-Pyd) in a
blood fluid sample;
Fig. 3 is a titration curve for an antibody
suitable for detecting deoxypyridinoline (N-Dpd) in a
blood fluid sample;
Fig. 4 shows concentrations of N-Dpd measured in
serum samples from normal (control) subjects
(column 1), patients with primary hyperparathyroidism
(column 2), osteomalacia (column 3), calcium
metabolism disorder (column 4), and osteoporosis
(column 5);
Fig. 5 shows concentrations of serum Pyd
measured in accordance with the present invention in
healthy control patients and in cancer patients.
W094/1~72 PCT~S93/L~21 ~
2 1 ~
Detailed Description of the Invention
I. Definitions
As used herein, the terms below have the
following definitions:
"Pyd" or "pyridinoline" or "free pyridinoline"
refers to the crosslink compound shown at I below,
where the ring N is derived from the ~ amino group of
a hydroxylysyl residue, and "Dpd" or "deoxy-
pyridinoline" or "free deoxypyridinoline" refers to
the crosslink compound shown at II below, where the
ring nitrogen is derived from ~ amino group of a
lysyl residue.
~T2~R ~rOn~ C~2OEN~2Coo~
5 ~ 0E II ~ OE
1 2 1 2
C~o~ C~2
~2 C52
C52 C~2
~2~r~n~7 ~3r~n~
"Free crosslinks" refers to either compounds I
or II or a mixture of the two, i.e., free of any
peptide or glycosyl group attachments.
~Glycosylated pyridinoline~ or "glyco-Pyd"
refers to glycosylated forms of compound I, wherein
glycosyl groups are covalently bound to the aliphati~
hydroxyl group of Pyd. Two glyco-Pyd crosslinks
which have been identified are Gal-Pyd and Glc Gal-
Pyd, which contain the acetals shown at III and IV
below, respectively.
WO94/1~72 PCT~S93/1~21
21~1~3~
13 C~2~
III ~ IV ~O ~ ~ ¦
o~
0~
"Pyd-peptides" or ''pyridinoline-peptidesll refers
to peptide-derivatized forms of compound I, in which
one or more of the three amino acid residues in the
compound is linked via a peptide linkage to
additional amino acid residues. Similarly, "Dpd-
peptides" or "deoxypyridinoline-peptides" refers to
peptide-derivatized forms of compound II, in which
one or more of the three amino acid residues in the
compound is linked via a peptide linkage to
additional amino acid residues.
"Pyridinium-peptides" refers to a mixture of
Pyd-peptides and Dpd-peptides.
"Pyd-peptides having a molecular weight greater
than lOoO daltons~ or "Pyd-peptides (MW>1000)" refers
to Pyd-peptides retained by a dialysis membrane
having a 1,000 molecular weight cutoff.
"Pyd crosslinks" refers to the pyridinium
crosslinks which contain compound I either in free or
peptide-derivatized form. Pyd crosslinks include
Pyd, glyco-Pyd and Pyd-peptides. Similarly, "Dpd
crosslinks" refers to the pyridinium crosslinks which
contain compound II either in free or peptide-
derivatized form. "Dpd crosslinks" include Dpd and
Dpd-peptides.
"Pyridinium crosslinks" refers to pyridinium
crosslinks which contain compounds I and/or II in
free and/or peptide-linked form.
"Total Pyd" or "T-Pyd" refers to total
hydrolysed Pyd produced by hydrolyzing Pyd crosslinks
WO94/1~72 PCT~S93/1~21 ~
~15~2~
14
to Pyd. Similarly, "Total Dpd" or "T-Dpd" refers to
total hydrolysed Dpd produced by hydrolyzing Dpd
crosslinks to Dpd.
"Hydrolysed-Pyd" of "H-Pyd" refers to Pyd
produced by hydrolysing Pyd crosslinks in 6N HCl at
110C for 16 hours. Similarly, "Hydrolysed-Dpd" of
"H-Dpd" refers to Dpd produced by hydrolysing Dpd
crosslinks in 6N HCl at 110C for 16 hours.
"Native Pyd" of "N-Pyd" refers to Pyd which has
not been subjected to hydrolytic conditions.
Similarly, "Native Dpd" of "N-Dpd" refers to Dpd
which has not been subjected to hydrolytic
conditions.
"Native free" or "native, peptide-free" refers
to a pyridinium compound having structure I or II (or
both) shown above, and which has not been subjected
to hydrolytic conditions.
"Blood fluid" refers to cell-free fluid and
fractions thereof, obtained from blood, e.g., serum
or plasma.
"Limit of detection;~ refers to a concentration
of the selected pyridinium crosslinks that can be
distinguished from a negative sample (i.e., a sample
lacking the selected pyridinium crosslinks). More
specifically, the limit of detection is the selected
concentration of the selected pyridinium crosslinks
which gives rise to a signal that is different by at
least two standard deviations from the signal
observed for a negative sample. Thus a limit of
detection of native free deoxypyridinoline of about
0.05 nM implies the ability to detect concentrations
of native free deoxypyridinoline of at least 0.05 nM.
The actual limit of detection may be lower than the
specified limit, e.g., in the present example, an
WO94/1~72 ~ ~ 51~ ~ ~ PCT~S93/L~21
assay with a specified limit of detection of 0.05 nM
may have an actual limit of detection of 0.02 nM.
"Detection sensitivity" refers to the range of
analyte concentrations which can be reliably measured
in a given assay procedure. ThUs, a detection
sensitivity which allows detection of native free
pyridinoline concentrations in the range 0.1-10 nM
means that the assay procedure can detect
concentrations of analyte of 0.1 n~, and can detect
lo differences in the analyte concentration in the range
0.1-10 nM. The actual range of detection for the
assay may be broader than the spec fied range, e.g.,
in the present example, an assay method having a
detection sensitivity in the range 0.1-10 nM may be
able to detect and distinguish analyte concentrations
in the range 0.05-20 nM.
II. PreParation of AntibodY Reaqent
This section describes the production of
monoclonal and polyclonal antibodies ("antibody
reagent") which are specific for selected native free
pyridinium crosslinks (either N-Py~, N-Dpd, or both).
In one embodiment, the antibodies have a ratio of
reactivity toward the selected native free pyridinium
crosslink and urinary pyridinium peptides larger than
1,ooo daltons in molecular weight, of greater than
about 3:1, and preferably greater than about 5:1.
In a specific embodiment, where the antibody is
for binding native free pyridinoline, the antibody
preferably has a ratio of reactivity toward native
free pyridinoline and native free deoxypyridinoline
of greater than about 5:1, preferably greater than
about 20:1, and more preferably greater than about
100: 1.
215 ~ ~34
In another specific embodiment, where the
zntibody is for binding native free deoxypyri-
dinoline, the antibody preferably has a ratio of
reactivity toward native free deoxypy_idinoline and
native free pyridinoline of greater than about 5:1,
preferably greater than about 25:1, and more
preferably greater than about 100:1.
In a third specific embodiment, where the
antibody is fcr binding both native free pyridinoline
and native free deoxypyridinoline, the antibody
preferably has a ratio of reactivity toward native
free pyridinoline and native free deoxypyridinoline
of between abcut 2:1 and 1:2.
The antikody reagent of the invention preferably
has a binding affinity constant for the selected
pyridinium species (N-Pyd or N-Dpd) of greater than
about 5 x 10~/molar.
~. Immunoaen
The immunogen used in producing the antibody
reagent is Dpd or Pyd conjugated to a carrier
molecule, typically a carrier protein such as keyhole
limpet hemocyanin (KLH).
The Pyd can be native Pyd (N-~yd) or hydrolyzed
Pyd (H-Pyd). Likewise, the Dpd can be native Dpd (N-
Dpd) or hydrolyzed Dpd (H-Dpd). For obtaining N-Dpd
or N-Pyd, gross separation of N-Dpd or N-Pyd from
other pyridinium compounds in urine can be achieved
by fractionation of urine, as described in Example 2.
Briefly,~a concentrate of urine is applied to a
Sephadex G-10 colu~n, and the total pyridinium-
contzining fractions are eluted. The eluate is then
applied to 2 column of phosphocellulosé eauilibrated
-~ith sodium citrate, and eluted with salt, yielding
_W094/1~72 PCT~S93/L~21
~ 2~51~
free crosslinks in a single peak. As the sample is
not subjected to hydrolysis conditions, the peak
contains not only the N-Dpd and N-Pyd forms ("free
crosslinks"), but also glyco-Pyd, including Gal-Pyd
and Glc-Gal-Pyd as described above. Further
purification is then conveniently conducted by
standard methods, for example, using ion exchange on
sulfonated polystyrene beads, or HPLC. Typical
protocols for this separation are found, for example,
in Black, et al., 1988, Seibel, et al., 1989, and
detailed in Example 2.
Alternatively, hydrolyzed Pyd or Dpd can be
produced by acid hydrolysis of pyridinium crosslinks
in bone collagen or urine, purified as described in
Black et al., 1988, for example.
Coupling of Pyd or Dpd to a carrier protein is
by standard coupling methods, typically using a
bifunctional coupling agent which forms, at one
coupling end, an amide linkage to one of the free
carboxyl groups of Pyd or Dpd, and at the other
coupling end an amide or ester or disulfide linkage
to the carrier protein, according to standard
methods.
Alternatively, in a preferred embodiment, the
Pyd or Dpd can be directly coupled to the protein,
e.g., in the presence of a water-soluble carboxyl
activating agent such as EDC (l-t3-dimethyl-
aminopropyl)-3-ethylcarbodiimide), also according to
well known methods. The latter approach is
illustrated in Example 3, which describes the
coupling of Dpd to keyhole limpet hemocyanin (KLH) by
EDC activation. General coupling reactions for
derivatizing a carrier protein with a peptide antigen
are given in Harlow (1988), pp. 77-87, and in Wong
(1991).
WO94/1~72 PCT~S93/1~21 ~
2~23'~ .
18
B. Monoclonal AntibodY Reagent
To prepare a monoclonal antibody reagent, the
immunogen described above is used to immunize an
animal, such as a mouse, from which antigen-specific
S lymphocytes can be obtained for immortalization. One
animal that has been found suitable is the
"autoimmune" MRL/MpJ-lpr mouse available from Jackson
Laboratory (Bar Harbor, MN).
Where an antibody which is specific for N-Pyd is
desired, a Pyd-immunogen is typically used.
Likewise, where an antibody which is specific for N-
Dpd is desired, a Dpd-immunogen is typically used.
An antibody which recognizes both Pyd and Dpd may be
obtained using a Pyd-immunogen or a Dpd-immunogen.
B.l N-Pyd Monoclonal Antibody. For producing a
monoclonal antibody reagent which is specific for N-
Pyd, mice can be immunized using a series of
injections of H-Pyd-KLH immunogen, as outlined in
Example 4. About 8 weeks after initial immunization,
spleen cells are harvested and fused with a
P3X63Ag8.653 myeloma cell line. Selection for
successful fusion products can be performed in HAT in
conditioned S-DMEM medium, according to published
methods (see, generally, Harlow, pp. 196-212).
Successful fusion products are then screened for
immunoreactivity with N-Pyd, using a competitive
immunoassay format similar to that described in
Example 8. Cell lines which show high affinity
binding to N-Pyd are subcloned by limiting dilution
and further screened for production of antibodies
with high binding affinity for N-Pyd. One subcloned
cell line obtained by the procedure above and which
gave high antibody affinity for N-Pyd is designated
herein as Mab-XXV-3G6-3B11-lA10. Samples of this
WO94/1~72 2 ~ 3 ~ PCT~S93/L~21
19
cell line have been deposited with the American Type
Culture Collection, 12301 Parklawn Dr., Rockville MD
20852), and have been assigned ATCC No. HB11089.
To produce the antibody reagent, the hybridoma
cell line is grown in a suitable medium (Harlow, pp.
247-270), such as Dulbecco's modified Eagle's medium
(DMEM) supplemented as described in the Materials and
Methods section below. Monoclonal antibodies
("Mabs") are harvested from the medium and can be
concentrated and stored according to published
methods (Harlow pp. 271-318).
As noted above, an important feature of the
present invention is the specificity of the antibody
reagent for N-Dpd and N-Pyd relative to larger
molecular weight pyridinium crosslinks in urine. The
relative specificity of the antibody reagent for N-
Pyd, N-Dpd, and other urinary pyridinium crosslinks
can be determined by a competitive binding assays for
N-Pyd, as detailed in Example 10.
Briefly, various purified crosslink samples,
including N-Pyd and N-Dpd, as well as an amino acid
mixture containing the 20 common amino acids in
equimolar amounts (150 ~M each), are reacted with a
limiting amount of the antibody reagent over a solid-
phase support having attached N-Pyd under conditions
in which the pyridinium crosslinks in the sample
compete with the support-bound N-Pyd for binding to
the antibody. The extent of binding of antibody to
the solid-support provides a measure of the relative
reactivities of the sample crosslinks for the
antibody reagent.
In accordance with the procedure outlined in
Example lo, the levels of binding of N-Pyd, N-Dpd,
Pyd-peptides (MW>l,OoO), and an amino acid mixture
(150 ~M each of the common 20 amino acids), to
WO94/1~72 ~ PCT~S93/L~21
21~123~
monoclonal antibodies from cell line Pyd XXV-3G6-
3Bll-lA10 were ~m; ned. The apparent Pyd
concentration of each sample was determined using
standard curves established using purified N-Pyd.
The percent reactivity of each sample was calculated
as a ratio of apparent concentration (measured using
the N-Pyd standard curve above) to total Pyd
crosslink concentration in the sample determined by
HPLC for total H-Pyd (times 100), or to total Dpd-
crosslink concentration as determined by ~PLC fortotal H-Dpd (times 100) in the case of the N-Dpd
sample. The results are shown in Table 1, where
reactivity with N-Pyd has been defined as 100%.
15Table 1
Cross-Reacti~ity of N-Pyd Monoclonal Antibody
N-Pyd 100%
N-Dpd 16%
Pyd-Peptide (>1000) <1%
20Amino Acid Mixture (150 ~M) <1%
As seen, the monoclonal antibody reagent is
highly selective for N-Pyd relative to N-Dpd, showing
a ratio of reactivity toward native free pyridinoline
and native free deoxypyridinoline that is greater
than about 3:1, and in the present case, greater than
5:1. The reagent is also selective for N-Pyd over
the pyridinium-peptide forms tested (quantitated for
total Pyd content), showing a ratio of reactivity
toward pyridinoline peptides larger than 1,000
daltons in molecular weight, of greater than about
100:1. In addition, the reagent shows minimal cross
reactivity (<1%) with the amino acid mixture tested.
~ WO94/1~72 2 ~ 51 2 3 4 PCT~S93/12321
More generally, the Mab reagent which is
specific for N-Pyd has a reactivity toward native
free pyridinoline (N-Pyd) and Pyd-peptides (MW>1000),
of greater than 5:1, preferably greater than 10:1,
more preferably greater than 25:1, and in the present
case, greater than 100:1, as measured by the above
assay.
B.2 N-Dpd Monoclonal AntibGdy. For producing a
monoclonal antibody reagent which is specific for N-
Dpd, the above procedure for obtaining N-Pyd Mabs can
be used, except that Dpd-KLH is used as immunogen,
and immunoreactivity screening is done with an assay
for N-Dpd. One subcloned cell line obtained by this
procedure, and which gave high antibody affinity for
N-Dpd, is designated herein as Mab-Dpd-II-7B6-lF4-
lH11 (see Example 5).
The antibody reagent is prepared from the
hybridoma cell line and stored by the same general
procedures described above for N-Pyd Mabs.
The relative specificity of the antibody reagent
for N-Dpd, N-Pyd, and other urinary pyridinium
crosslinks can be determined by tne approach
described above (section B.1), but using a solid-
phase support having attached N-Dpd.
In accordance with the procedure outlined in
Example 10, the levels of binding of N-Dpd, N-Pyd,
Dpd-peptides (MW>1,000), and an amino acid mixture
(150 ~M each of the common 20 amino acids), to
monoclonal antibodies from cell line Mab-Dpd-II-7B6-
lF4-lH11 were examined. The apparent Dpd
concentration of each sample was determined using
standard curves established using purified N-Dpd.
The percent reactivity of each sample was calculated
as a ratio of apparent N-Dpd concentration (measured
W094/1~72 PCT~S93/L~21 ~
2 ~ 3 ~ -
using the N-Dpd standard curve above) to total Dpd-
crosslink concentration in the sample determined by
HPLC for total H-Dpd (times 100), or to total Pyd-
crosslink concentration as determined by HPLC for
total H-Pyd (times 100) in the case of the N-Pyd
sample. The results are shown in Table 2, where
reactivity with N-Dpd has been defined as 100%.
Table 2
Cro~-Re~ctivity of N-Dpd Monoclonal Anti~ody
N-Dpd 100%
N-Pyd <1%
Dpd-Peptide (>1000) 13%
Amino Acid Mixture (150 ~M) <1%
As seen, the monoclonal antibody reagent is
highly selective for N-Dpd relative to N-Pyd, showing
a ratio of reactivity toward native free
deoxypyridinoline and native free pyridinoline that
is greater than about 100:1. The reagent is also
selective for N-Dpd over the pyridinium-peptide forms
tested (quantitated for Dpd content), showing a ratio
of reactivity toward deoxypyridinoline peptides
larger than 1,000 daltons in molecular weight, that
~5 is greater than about 3:1, and preferably, greater
than about 5:1. In addition, the reagent shows
minimal cross reactivity (<1%) with the amino acid
mixture tested.
More generally, the Mab reagent which is
specific for Dpd has a reactivity toward native
pyridinoline (N-Dpd) and Dpd-peptides (MW>1000), of
greater than about 5:1, preferably greater than 10:1,
more preferably greater than 25:1, and in the present
WO94/1~72 ~ 3~ PCT~S93/L~21
case, greater than 100:1, as measured by the above
assay.
B.3 Monoclonal Antibodies Which Bind N-Pyd and
N-Dpd With Comparable Affinities. For producing a
monoclonal antibody reagent which binds N-Pyd and N-
Dpd with comparable affinity, the procedures
described above for obtaining N-Pyd Mabs and N-Dpd
Mabs can be used. The immunogen may be Pyd-KLH or
Dpd-KLH, and immunoreactivity screening is done with
separate assays for N-Pyd and N-~pd. One subcloned
cell line obtained by the procedure above, using H-
Dpd-KLH as immunogen, and which gave high antibody
affinity for both N-Dpd and N-Pyd, is designated
herein as Mab Pyd/Dpd-V-6H2-2H4-lE4 (see Example 6).
The antibody reagent is prepared from the
hybridoma cell line and stored by the same general
procedures described above for the N-Pyd Mabs.
The relative specificity of the antibody reagent
for N-Dpd, N-Pyd, and other urinary pyridinium
crosslinks can be determined by the procedure
described above (sections B.1 and B.2). In the
present case, for antibodies produced using the
Pyd/Dpd-V-6H2-2H4-lE4 cell line, the percent
reactivity of each sample was calculated as a ratio
of apparent N-Dpd concentration (measured using the
N-Dpd standard curve above) to total Dpd crosslink
concentration in the sample determined by HPLC for
total H-Dpd (times 100), or to total Pyd crosslink
concentration determined by HPLC for total H-Pyd in
the case of the N-Pyd sample. The results are shown
in Table 3, where reactivity with N-Dpd has been
defined as 100%.
WO94/1~72 PCT~S931L~21 ~
21~3~
Table 3
Cross-Re~ctivity of Pyd/Dpd Monoclon~l Antibody
N-Dpd 100%
N-Pyd 102%
Dpd-Peptide (>1000) 1%
Pyd-Peptide (>1000) 11%
Amino Acid Mixture (150 ~M) 5%
As seen, the monoclonal antibody reagent
recognizes N-Dpd and N-Pyd with comparable
affinities, with a cross-reactivity ratio close to
l:1. The reagent is also selective for N-Dpd over
the pyridinium-peptide forms tested (both Pyd and Dpd
peptides), showing a ratio of reactivity toward
pyridinium peptides larger than 1,000 daltons in
molecular weight, of greater than about 3:1, and in
the present case, greater than 9:1. In addition, the
reagent shows minimal cross reactivity (5%) with the
amino acid mixture tested.
More generally, the Pyd/Dpd-specific Mab reagent
has a reactivity toward native free pyridinoline (N-
Pyd) and native free deoxypyridinoline (N-Dpd) of
between about 2:1 and 1:2.
C. Polyclonal Antibodies
Polyclonal antibody preparation is by
conventional techniques, including injection of the
immunogen into suitable mammalian subjects, such as
rabbits or mice, according to immunological protocols
generally known in the art, e.g., Harlow, pp. 93-115.
Typically, rabbits are injected subcutaneously with
the immunogen in an adjuvant, and booster immuniz-
ations are given by subcutaneous or intramuscular
WO94/1~72 PCT~S93/L~21
injection every 2-3 weeks; mice may be injected
intraperitoneally according to a similar schedule.
Blood is collected at intervals, e.g. 1-2 weeks after
each immunization injection. Antisera may be
titrated to determine antibody formation with respect
to N-Pyd or N-Dpd, according to standard immuno-
precipitation methods (Harlow, pp. 423-470). Details
of one method for producing polyclonal antibodies in
rabbits are given in Example 11.
The binding affinity constant for polyclonal
antisera can be determined by known methods (e.g., by
Scatchard analysis using an immunoprecipitation or
ELISA assay; see Campbell, Segel), and represents an
average binding affinity constant value for the
antibodies in the antisera which are specific against
the selected pyridinium species. Polyclonal
antibodies obtained from rabbit VI-8 have a binding
constant for N-Pyd of about 1 x 1o8, as determined by
Scatchard analysis.
The relative binding specificity of the antibody
reagent for the selected pyridinium species and for
other pyridinium crosslinks can be determined by a
competitive binding assay such as described above and
detailed in Example 14. Table 4 shows the relative
binding specificities of anti-Pyd antiserum obtained
from rabbit VI-8, where reactivity with N-Pyd has
been defined as 100%.
Table 4
Cro~s Reactivity of N-Pyd Polyclonal Antibody
N-Pyd 100%
N-Dpd <10%
Pyd-Peptide (MW>1000) <5%
J~ 2~51~3~
26
Cross Reactivity of N-Pyd Polyclonal Antibody
Amino Acid Mixture -12%
As seen, the antibody reagent is specific for N-
Pyd, showing less than 10~ cross-reactivity with N-
Dpd, less than 5% cross-reactivity wi~h Pyd-pe~tides
(MW>1000), ar.d moderate (-12%) cross-reactivity with
the amino acid mixture. In accordance with one
aspect of t~e invention, the polyclonal antibody
reagent has a reactivity toward a selected native
free pyridinium species (N-Pyd, N-Pyd, or both) and
urinary pyridinium pep~ides larger than 1,000 daltons
in molecular weight, of greater than 3:1, and
preferably greater than about 5:1, as measured by the
above antigen-competition assay.
III. Immunoassav Kit
In another aspect, the invention includes a
diagnostic kit for use in assaying bone collagen
deqradation levels in a human subject. The kit
includes an antibody reagent of the type described in
~he section above, which preferably has 2 binding
constant for native free deoxypyridinoline of greater
than about 5 x 10'/molar, and more preferably,
greater than 8 x 108/molar. The kit may also include
detection means for detecting the amount of
immur.ocomplex formed by reaction of the antibody
reagent with seLected pyridinium crosslinks, where
the detection ~eans is effective to measure the level
of the selected crossl~nks in a ~lood fluid sample.
For the purpose of illustration, a specific
embodiment of such a kit, for measurLng N-Pyd in a
sample, is shown at 10 in Figs. lA-lC. A solid-phase
~0~0S
_ WO94/1~72 PCT~S93/L~21
3 ~
27
support 12 in the kit has a surface to which the
binding agent can be adsorbed or chemically attached.
A variety of glass and polymer resin supports having
chemically derivatizable groups, or surfaces
effective in protein adsorption are available. In
one preferred embodiment, the kit provides 96 assay
wells in a microtitre plate, where the well surfaces
form the solid-phase support surfaces in the kit.
The binding agent in kit 10 is N-Pyd, indicated
by Pyd(N) molecules in the figures, such as at 16.
The binding agent is attached to the solid phase, in
this case, each of the wells in a 96-well microtitre
plate, by first adsorbing an porcine serum albumin-
biotin complex, such as complex 18 in Fig. lA, to the
well surfaces, then attaching an N-Pyd-streptavidin
complex, such as complex 20, to the adsorbed biotin.
The antibody reagent in the kit is indicated at
22 in Figs. lB and lC, and includes the polyclonal or
monoclonal reagent described in the section above.
As shown in Fig. lB, pyridinium crosslinks in a
sample, such as the N-Pyd crosslink indicated at 26,
competes with surface-bound N-Pyd for binding to the
antibody reagent. The immunocomplex formed by
reaction of the antibody reagent with sample
crosslinks is indicated at 28 in this figure.
The detection reagent (detection means) in the
kit is a reporter-labeled second antibody, indicated
at 24 in Fig. lC, which is effective to bind to
antibody reagent which is itself bound to N-Pyd
attached to the solid support. Reporter-labeled
antibodies, such as enzyme-labeled antibodies, are
commercially available or readily constructed
(Harlow, pp. 319-358) for a variety of reporter
moieties. One preferred enzyme in an enzyme-labeled
antibody is alkaline phosphatase, which can react
2 1 ~
with a D-ni~rophenylphosphate substrate to produce a
colored product having a strong absorption peak at
~05 nm.
The reporter-labeled second antibody is
typically an anti-IgG antibody, such as an anti-
rabbit IgG antibody, where the polyclonal antibody
reagent in the kit is obtained from i~munized
rabbits, or an anti-mouse IgG antibody, where the
zntibody reagent is a mouse monoclonal antibody.
Here, the antibody reagent (which is immunoreactive
with N-Pyd as above) is "reporter-labelable", since
the antibody reagent can become labeled by reaction
with the re?orter-labeled second antibody. Other
instances o' a reporter-labelable antibody reagent
lS include a biotin- or streptavidin-labeled antibody
which can be reacted with a reporter-labeled
streptavidin or biotin-labeled partner for detection
purposes.
In an ~lternative embodiment, the detection
reagent can be the anti-Pyd antibody reagent itself,
labeled with a reporter, such as an enzyme.
The de~ection means in the kit may also includ~
necessary substrates or the like needed for detection
of the reporter in the reporter-labeled antibody.
In an alternative kit embodiment, the binding
agent attached to the support is an anti-Pyd antibody
reagen~ such as described in Section II. The
an~ibody may be at~ached to the solid support by a
variety of known methods, including chemical
derivatization or high-affinity binding of the
antibody by support-bound protein A or anti-IgG
antibody, according to standard methods. The kit may
additionally include a pyridinoline reagent
which is effective to compete with native free
3 5 pyridinoline in a sample for binding to the
~ 21S~3~
29
antibody reagent on the support. For detection
purposes, the pyridinoline reagent may include a
re~orter-label attached covalently to
pyridinoline (i.e., the reagent can be a
reporter-l2beled pyridinoline). Preparation and
use of an exemplary kit having this format zre
illustrated in Examples 4~-~3.
Alternatively, the pyridinoline reagent may
be reporter-label2ble, in that the pyridinoline --
reagent can include Pyd conjugated to an agent suchas biotin or streptavidin, for example, for
r~cognition by a corresponding reporter-labéled
streptavidin or biotin molecule.
In another general embodiment, the kit is
1~ designed for a homogenous assay in which sample
pyridinoline can be detected directly in
solution.
It can be appreciated that the kit of ~he
nvention can be adapted to a number of othe_ assay
formats, including formats based on radiotracers,
coupled enzymes, fluorescence, chemiluminescence, or
ar EMIT configuration (Gosling), for exam~le.
Thus, in another preferred embodime~t, the
detection means in the kit includes a radioactive
reporter group effective to produce a radioactive
signal in proportion to the amount of immunocomplex
formed by reaction of ~he antibody reagent with
native free Pyd.
While the kit is illustrated above for assay of
N-Pyd, it can be zppreciated that a similar format
can be useà where the ki~ is for measurement of N-
Dpd, using an N-Dpd specicic zntibody reagent, or for
measurement of the sum of N-~yd and ~-Dpd, using an
antibody reagent which binàs N-~yd and N-Dpd with
3 5 comparable a~f inities .
1-- 2 ~ 3 4
For detec.ing N-~yd, the kit hzs a limit of
detec.lon for N-PydAl ~M or less, preferably 0.5 nM,
and more preferably 0.2 nM. Fig. 2 shows an N-~yd
ti~ration curve carried out with the immunoassay
~ormat desc-ibed in Example 13, using polyclonal
antiserum obtained from rabbit VI-8 characterized in
Table 4. As may be seen from the ~igure, the kit
affords a sensitivity of about 0.2 nM, while also
providing reliable measurements of N-Pyd extending
beyond 10 ~.
For detecting N-Dpd, the kit preferably has a
limit of detection for ~-Dpd of 0.1 nM or less,
preferably ~.05 nM, and more preferably 0.02 nM.
Fig. 3 shows an N-Dpd titration curve carried out
with the im~unoassay format described in Example 9,
using monoclonal antibodies obtained from the
hybriàoma cell line 13D4 noted above. As can be seen
from the figure, the kit affords a limit of detection
of about 0.02-0.05 nM, while also providing reliable
mezsure~ents of N-Dpd extending up to about 10 nM.
It will be appreciated that the limit of
detec~ion in the kit can be selected such that only
pyridinium crosslinks levels in a range considered to
be above normal are detected, while those faLling
wi~hin generally normal leveLs are not detected in
the assay.
IV. Immuno2ss2v Method
The present invention provides a method of
ass2ying bone collagen breakdown levels in a human
subject, as outlined in the section above entitled
'~Su~ary of the Invention."
The blood fluid sa~ple is preferably pretreated
to ~emove potentially interfering substances, prior
to assay o. the s2mp!~. SUch pretreatment may be
_
W094/1~72 2 ~ 3~ PCT~S93/L~21
31
accomplished by trichloroacetic acid precipitation,
wherein the sample is mixed 10:1 with 50~
trichloroacetic acid, and then centrifuged to remove
the precipitate. Alternatively, the sample may be
passed through a protein A column or contacted with
Staphylococcus aureus cells (e.g., PANSORBIN cells,
available from Calbiochem, San Diego, CA) to remove
immunoglobulins and the like. In a preferred
pretreatment step, the sample is filtered to remove
sample components having a molecular weight of
greater than about 30 kDa. Such filtration may be
accomplished by centrifugation using a Centricon-30
filtration device (Amicon, Mass.).
As indicated in Section III above, the reaction
of sample with the antibody reagent may be carried
out in a solid-phase format, using a variety of
configurations, or in a homogeneous assay format.
For illustrative purposes, the immunoassay
method will be described with particular reference to
an assay format for assaying native free
deoxypyridinoline in serum, in accordance with
Example 9, wherein the solid support has surface-
attached anti-Dpd antibodies, and exogenous enymze-
labeled deoxypyridinoline capable of competing with
native free deoxypyridinoline from the sample for
binding to the support-bound anti-Dpd antibody. It
will be appreciated how the method can be adapted to
other solid-phase or homogeneous assay formats.
In an exemplary embodiment of the method, a
known volume, e.g., 100 ~1, of the filtered or
precipitated serum sample is added to an anti-Dpd-
antibody-coated solid support, e.g., the wells in a
microtitre plate prepared as in Example 8. Sample
addition is followed by addition of a known volume,
typically 50-200 ~1, of reporter-labeled Dpd at a
W094/1~72 PCT~S93/L~21 ~
~1234
known dilution. In Example 9, the reporter-labeled
Dpd is an alkaline phosphatase-Dpd conjugate, i.e.,
enzyme-labeled Dpd. The mixture on the solid support
surface is then incubated, preferably under
conditions effective to achieve equilibrium for
binding of the anti-Dpd antibody with sample Dpd and
enzyme-labeled Dpd. In the method detailed in
Example 9, the incuba~ion is overnight at 4C.
After incubation, the support is again washed to
remove non-specifically bound material, and the level
of enzyme bound to the support is determined by
addition of enzyme substrate, with spectrophotometric
determination of converted substrate. Details are
given in Example 9.
In a typical assay, N-Dpd standards containing
increasing concentrations of N-Dpd are added in
duplicate to some of the wells, for purposes of
generating an N-Dpd concentration standard curve. Up
to 40 samples are then added in duplicate to
remaining wells, and the wells are then assay as
above. The standard curve is used for determining
pyridinium crosslink values for the samples in terms
of N-Dpd concentrations.
V. ApPlications
The immunoassay method, antibody reagent, and
kit of the invention, described above, are useful in
assaying the level of collagen breakdown activity in
a human subject.
In general, the invention is useful in detecting
increased blood levels of Pyd and Dpd associated with
bone collagen breakdown conditions in general. Such
conditions may include osteoporosis, Paget's disease,
hypothyroidism, osteoarthritis, and rheumatoid
VO94/1~72 ~ 3 ~ PCT~S93/L~21
arthritis, for example. Other conditions involving
increased native free pyridinium crosslinks levels
include various forms of metastatic cancer which
become established in bone tissue or which otherwise
alter bone metabolism.
Use of the method of the invention for detecting
elevated levels of serum Dpd is illustrated in Fig.
4, which shows N-Dpd levels measured in serum samples
from healthy (control) patients (column 1), and in
serum samples from patients with primary
hyperparathyroidism (column 2), osteomalacia (column
3), calcium metabolism disorder (column 4), and
osteoporosis (column 5). The study was carried out
using the assay protocol described in Example 9, with
antibodies from hybridoma cell line 13D4 (see Table 2
above).
As can be seen from the Figure, N-Dpd levels in
the control group were between about 0.2 and 0.5 nM,
with an average level (+ standard deviation) of 0.33
+ 0.07 nM. The primary hyperparathyroidism group
showed levels between about 0.4 and 1.4 nM, with one
patient showing a level of abovt 4.2 nM (mean = 1.4
nM). The osteomalacia group showed levels between
about 0.5 and 2.6 nM (mean 1.2 nM); the calcium
metabolism disorder group showed levels between about
0.4 and 1.4 nM (mean 0.9 nM); and the osteoporosis
group showed levels between about 0.3 and 1.1 nM
(mean 0.6 nM).
The Fig. 4 data from the diseased groups as a
whole are consistent with increased collagen
breakdown in these patients. The results show that
serum N-Dpd levels above about 0.8 nM, and more
preferably, above about 0.5 nM, are a useful
indicator of increased collagen breakdown in such
patients.
WO94/1~72 PCT~S93/L~21 ~
2 3 ~ `
Figure 5 compares N-Pyd levels measured in serum
samples from a group of healthy patients (group 1)
with levels measured in samples from a group of
cancer patients with established or suspected bone
metastases. The study was carried out using the
assay protocol described in Example 13, with
antibodies from rabbit VI-8 characterized above in
Table 4. As can be seen from the Figure, N-Pyd
levels in the control group were between about 1 and
lo 3 nM, with an average level (+ standard deviation) of
1.7 + 0.4 nM. The cancer group, on the other hand,
showed levels betweer. about 2 and 13 nM, with one
patient showing a level of about 23 nM, consistent
with increased collagen breakdown in these patients.
The results show that serum N-Pyd levels above about
5 nM, more preferably above about 3 nM, is a useful
indicator of increased collagen breakdown in such
patients.
From the foregoing, it can be appreciated how
the objects of the invention are met. By employing a
blood fluid sample, the assay method allows
measurement of native free pyridinium crosslink
levels to be integrated with other clinical tests
with blood samples. The approach also avoids the
correction for variation in sample volume typically
needed where the sample is a urine sample (e.g.,
determination of urinary creatinine). The assay
utilizes an antibody reagent, and can thus be adapted
to a number of convenient and rapid assay formats,
such as described above. The invention can be used
both for detecting increased collagen degradation in
a patient, and also for monitoring the course of
therapy of a variety of collagen-pathology states.
~WO94/1~72 PCT~S93/L~21
-- 21~ 23~
The following examples illustrate methods of
producing antibody reagents and assay methods in
accordance with the invention. The examples are
intended to illustrate, but in no way limit, the
scope of the invention.
EXAMPLES
Materials and Methods
Female autoimmune MRL/MpJ-lpr mice were
purchased from the Jackson Laboratory, Bar Harbor,
Maine.
Mouse non-secreting P3X63Ag8.653 myeloma cells,
and mouse monocyte-macrophage cell lines P388Dl(IL-1)
and J774A.l were purchased from American Type Culture
Collection (ATCC), Rockville, Maryland.
Adjuvant Ribi and Ribi(CWS) were purchased from
RIBI Immunochem Research, Inc., Hamilton, Montana.
50% PEG 1500 (polyethylene glycol 1500, 50% (w:v) in
water) was purchased from Boehringer Mannheim,
Indianapolis, Indiana. HAT and HT were purchased
from Sigma Chemical Company, St. Louis, Missouri.
Dulbecco's Modified Eagle Medium (DMEM), NCTC-
109, and gentamicin were purchased from Gibco, Grand
Island, New York. Fetal clone bovine serum was from
Hyclone Laboratories, Inc., Logan, Utah. Oxaloacetic
acid and insulin were from Sigma Chemical Company.
S-DMEM was formulated as follows, where the
percentages indicate final volume percentages in the
final medium: DMEM (80%), NCTC-109 (10%), fetal
clone bovine serum (10%), oxaloacetic acid (1 mM), L-
glutamine (2 mM), gentamicin (50 ~g/ml) and insulin
(10 ~g/ml).
For preparation of conditioned media, mouse
monocyte cell lines P388Dl (IL-l), or
interchangeably, cell line J774A.l, were grown in S-
WO94/1~72 PCT~S93/L~21
36
DMEM medium, with a 1:4 split twice a week. Every 3days, tissue culture supernatants were filtered
through a 0.2 micron filter and then supplemented
with 4 mM L-glutamine. The resultant concentrated
conditioned media were used as 20% supplement for S-
DMEM to raise hybridoma cells.
Unless stated otherwise, PBS is defined as a
buffer containing 0.01 M phosphate and 150 mM NaCl,
pH 7.
Example 1
HPLC Measurement of Crosslinks
HPLC analysis for Pyd and Dpd was done
essentially as described in Black (1988). Briefly,
urine samples were adjusted with butanol and glacial
acetic acid to 4:1:1 (v:v:v) mixture and applied onto
CF1 cellulose (Whatman) cartridge, followed by a wash
with 4:1:1 (butanol:acetic acid:water) solution.
Only free crosslinks were retained. The free
crosslinks were eluted from CF1 cellulose with water.
Eluted material was analyzed on a C18 reverse phase
column (Rainin, C18-80-200-C3) using a water-
acetonitrile (3-17% in 10 minutes) gradient delivered
at 1 ml/minute and monitoring fluorescence at 295 nm
of excitation, 395 nm of emission. Mobile phase
contained 0.1% HFBA.
Total urinary crosslinks were measured by
hydrolyzing a urine _ample in HCl (6N) at 110 C for
16 hours, followed by the CF1 pretreatment and HPLC
analysis as above. HPLC separation yielded
hydrolysed Pyd and Dpd fractions, from which T-Pyd
and T-Dpd were quantitated.
2~ 3~
ExzmDle 2
Purification of Crosslinks
Hum2n urine was filtered through 3000 daLton
molecular cut off filter (Filton Co.) applyins 40 psi
of back pressure. The filtrate was then lyophilized
and recons'ituted to 1/20 of the original volume with
0.2 M ace~ic acid.
Concentrated urine was then applie~ onto
Sephadex G-10 2.6 x 95 cm column equili~rated with
0.2 M acetic acid. Elution from the column materlal
was analyzed for free Pyd and Dpd as described above.
rhe free crosslink containing fractions were pooled
together, adjusted to pH 2.0 and applied onto 1 x 18
cm cation exchan~e column (Lacarte Co., UK) and
equilibrated with 0.1 M sodium citrate ~H 4.2.
Glyco-Pyd, Pyd and Dpd were coeluted thereafter
from the ion exchange column with 0.1 M sodium
citrate pH 4.2. Collected fractions were analyzed-
for the presence of crosslin~s by HPLC analysis as
above. Fractions containing specific crosslinks
(glyco-Pyd, Pyd and Dpd) were pooled toaether and
applied onto 2.5 x 10 cm reverse phase C18 column
(Waters) which W2S subsequently developed with 2-20%
gradient of acetonitrile containing 0.1~ HFBA.
Separated fractions (glyco-Pyd, Pyd and Dpd) were
collected and concentrated by lyophilization. Dry
r~sidue was reconstituted in 0.2 M acetic acid and
stored at 4C. Purity of the final material WZ5
measured by gravimetric and elementary analysis.
Urinary crosslink-peptides were prepared by
exhaustive dialysis o_ human urine USLng 1000 D
~olecuLar weight cut oCf dialysis membranes (Spectra-
Por). The T-Pyd and T-Dpd c.osslink content of the
peptide fractions was de~ermined by hydrolyzing
~i -~ '
_ W094/1~72 PCT~S93/L~21
2~ 51~3~
38
peptide samples with 6N HCl at 110C for 16 hours
followed by HPLC analysis for Pyd and Dpd.
Preparative amounts of H-Pyd and H-Dpd were
obtained from hydrolyzed powdered bovine or sheep
bone as described by Black et al. (1988).
ExamPle 3
Preparation of Immunogens
The following procedures illustrates how
immunogens can be prepared for obtaining monoclonal
or polyclonal antibodies against native free
pyridinoline, native free deoxypyridinoline, or both.
The procedures in A and B below are described with
respect to Pyd-immunogens; Dpd-immunogens are
prepared the same way, but using Dpd instead of Pyd.
A. PYd-BSA Immunoqen
To a 3.1 ml solution consisting of 9 mg of
bovine serum albumin (BSA) and 3.8 mg of Pyd in 0.1 M
MES pH 5.0 was added an 0.88 ml aqueous solution
containing 88 mg of EDC. The mixture reacted for
four hours at room temperature then was exhaustively
dialyzed versus phosphate buffered saline pH 7.0
(PBS). W and fluorescence measurements indicated
5.8 moles of pyridinoline substituted per mole of
albumin.
B. Pvd-KLH Immunogen
To a solution of dried H-Pyd (6 mg) in water
adjusted to pH 5 + 0.5 (200 ~1) was added 2 ml of a
10 mg/ml solution of keyhole limpet hemocyanin (KLH)
in PBS. To the mixture was added 30 mg solid 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (EDC,
Pierce), and ten minutes later, another 30 mg of EDC,
and the reaction was allowed to proceed for 4 h at
~ W094/1~72 PCT~S93tL~21
. .
39
room temperature. The reaction mixture was then
exhaustively dialyzed versus PBS, after which the
Pyd-KLH immunogen was collected and stored.
Example 4
PreParation of Anti-PYd Monoclonal Antibodies
A. Immunization Protocol
Female 5-week-old autoimmune MRL/MpJ-lpr mice
were immunized using the protocol below:
Table 5
Immunization Protoco~ for Pyd Mice
Immuniz- Days from T ogen I Inject.
15ationFusionInjected (~g)Adjuvant Uode
1 60 100 Ribi ipZ
2 46 100 Ribi ip
3 32 100 Ribi ip
4 18 100 Ribi ip
4 200 ~~ iV3
IAdjuvant and antigen were suspended in Hank's
balanced salt solution
2Intraperitoneal
3Intravenous
On the day of fusion, the immunized mouse was
sacrificed by CO2 gas, and the spleen was excised
from the mouse and placed in a culture dish
containing 5 ml of serum-free DMEM medium preheated
to 37C. Following removal of adipose tissue
attached to the spleen, the spleen was washed with 5
ml of serum-free DMEM medium. The spleen was then
cut into small pieces which were placed in a cell
homogenizer containing 7 ml of serum-free DMEM
WO94/1~72 PCT~S93/L~21 ~
1 2'3 ~
medium, and the cells were homogenized to form a cell
suspension.
B. Fusion Protocol
The following steps were performed at room
temperature.
The spleen cell suspension (-2 x 108 cells in
serum-free DMEM me~ium) and log-phase P3X63Ag8.653
myeloma cells (-7 ~ 107 cells in serum-free DMEM
lo medium) were centrifuged independently at 400xg for
10 min. The resultant cell pellets were suspended
together in serum-~ree DMEM medium (10 ml) in a 50 mL
centrifuge tube and then centrifuged at 400xg for 10
min. The supernatant was removed completely, and the
centrifuge tube wa tapped to loosen the cell pellet.
For cell fusion, a solution of 50% PEG 1500 (4
ml) was added dropwise to the tube with gentle mixing
by pipette over a 90 second period. Next, serum-free
DMEM (4 ml) was added dropwise over 1 min. S-DMEM
(40 ml) was then added over 2 min with gentle mixing,
after which the mixture was mixed by pipette for an
additional 2.5 min. The resultant mixture was
centrifuged at 400xg for 10 min. After thorough
removal of the supernatant, the cells were suspended
in 320 ml of HAT in 20% P388D1-conditioned S-DMEM
medium. The cell suspension was plated in 16 96-well
tissue culture plates, 200 ~l/well, and the plates
were then incubated at 37C in an atmosphere
containing 7% CO2. The cell mixtures were fed at day
3 and day 7 by removing 100 ~l/well of old medium and
adding 150 ~l/well of either HAT medium (day 3) or HT
medium (day 7). The wells were ready to screen 7 to
10 days after fusion.
WO94/1~72 PCT~S93/L~21
41
C. Screeninq Hybridomas for Production of Anti-N-
Pyd Monoclonal Antibodies
Successful fusion products were screened for
immunoreactivity using the N-Pyd immunoassay format
described in Examples 12 and 13. Cell lines which
showed high affinity binding to N-Pyd were subcloned
by limiting dilution and further screened for
production of antibodies with high binding affinity
for N-Pyd. One of the subcloned cell lines which
gave high antibody affinity for N-Pyd is designated
herein as Mab Pyd-XXV-3G6-3B11-lA10. The specificity
of antibodies produced by this cell line is shown in
Table 1 above.
Example 5
Preparation of Anti-DPd Monoclonal Antibodies
Anti-Dpd monoclonal antibodies were prepared by
the procedure described in Example 4, using Dpd-KLH
immunogen prepared as in Example 3. The mouse
immunization procedure was the same as in Example 4,
except that Ribi(CWS) was used as adjuvant instead of
Ribi, and 75 ~g Dpd-immunogen per mouse was used in
the fourth immunization step (18 days from fusion)
instead of 100 ~g.
Successful fusion products were screened for
immunoreactivity using the N-Dpd immunoassay format
described in Example 9. Cell lines which showed high
affinity binding to N-Dpd were subcloned by limiting
dilution and further screened for production of
antibodies with high binding affinity for N-Dpd. One
of the subcloned cell lines which gave high antibody
affinity for N-Dpd is designated herein as Mab Dpd-
II-7B6-lF4-lH11. The specificity of antibodies
produced by this cell line is shown in Table 2 above.
WO94/1~72 PCT~S93/L~21
2~51 ~3~
42
ExamPle 6
Preparation of Monoclonal Antibodies
S~ecific for Both N-Pvd and N-DPd
Monoclonal antibodies specific for both N-Pyd
and N-Dpd were prepared by the procedure in Example
5, using H-Dpd-KLH (Example 3) as immunogen.
Successful fusion products were screened for
immunoreactivity using the N-Dpd immunoassay format
described in Example 9. Cell lines which showed high
affinity binding to N-Dpd were subcloned by limiting
dilution and further screened for production of
antibodies with high binding affinity for both N-Pyd
and N-Dpd. One of the subcloned cell lines which
gave high antibody affinity for both N-Pyd and N-Dpd
is designated herein as Mab Pyd/Dpd-V-6H2-2H4-lE4.
The specificity of antibodies produced by this cell
line is shown in Table 3 above.
Example 7
Alkaline PhosPhatase-Dpd Coniuqate
Alkaline phosphatase-H-Dpd conjugate was
prepared using bis (sulfosuccinimidyl)suberate (BSSS)
as a coupling agent. In brief, 425 ~L of a 7.1 mg/mL
solution of alkaline phosphatase (AP, 3 mg, 140,000
MW, ~ = 0.963 mg/mL~~cm~~) (Biozyme Laboratories, San
Diego, CA) dialyzed the previous night at 4C in PBS
was mixed with 24 ~L of an 11 mg/mL solution (0.27
mg) of H-Dpd (~ = 4933 M-lcm~l) in 0.1 M phosphate
buffer, pH 7.5, and the volume of the resultant
mixture was adjusted to 500 ~L with PBS.
To the mixture was then added 0.61 mg of BSSS
(Pierce, Rockford, IL) dissolved in 50 ~L DMSO
(dimethylsulfoxide). The reaction vessel was covered
to exclude light, and the coupling reaction was
allowed to proceed at room temperature for 2 hours.
~ 2 ~ 3 !~
43
The reaction wzs then auenched by adding ~oO ~l of 10
~M glycine (in 0.1 M phosphate buffe- pH 7.5) and
allowing the mixture to incubate for another 2 hours
at room temperature, covered from light. The
quenched reaction mixture was then dialyzed a~ainst
four changes of PBS (2 L each, at four hour
intervals) at 4C in darkness.
The stoichiometry of Dpd to AP in the dialyzate
was determined spectrophotometrically ~y measuring
the absorbances at 326 nm and 280 nm. The ratio of
Dpd to A~ was typically from 1:1 to 2:1. The
enzymatic activity of the AP-H-Dpd conj~gate was
determined a5 a percent of the activity of native AP
in a standard A~ assay.
Exam~le 8
Pre3aration of Anti-D~d-Antibodv Coated ?lates
96-well ELISA plates were coated as follows.
200 l~1 of a solution containing 3 /~g/ml rabbit ~nti-
mouse IgG in PBS containing 0.05% NaN3 were added toeach well, and the plates were incu~ated 18-24 hours
at room temperature. After the incubation, the
plates were washed 3x with 300 ~l~ er weLl of wash
buffer (PBS containing 0.3% Tween 20). After
as~iration of the wells in the final wash, 150 ~l of
capture solution containing lO0 ~ phosphate, 150 mM
NaCl, 0.05% Tween-20, 0.05% NaN3, 0.1% bovine serum
albumin, and 10 ng/ml of mouse anti-Dpd monoclonal
zntibody (13~4), pH 7, was added. to e~ch well. The
plates were incubated for 18-24 hours at roo~
temperature. After the incubation w~s co~plete, each
well W2S wzshed 3x with wzsh buffer 2S above. After
final aspiralion of the wells, 2So ~l of a preserv-
ative solution containing 10% sucrose, lO0 mM
' 2~12~
44
phosphate, 150 mM NaCl, and 0.05% NaN3 (pH 7) were
added to each well, and the plates were incuba~ed for
1 hour at room temperature. The preservative
solution was then removed by aspiration, and the
plates were placed at 37C, < 10% humidity, for 18-24
hours. The coated plates were sealed in foil with a
dessicant packet and stored at room temperature.
Exam~le 9
Immunoassav for Serum Dpd
N-Dpd s.andard solutions and serum samples were
tested in duplicate. The standard solutions
typically consis.ed of 0, 0.05, 0.1, 0.2, 0.4, 1.0,
3.0, and 9.0 nM N-Dpd in 10 mM PBS containins 0.05%
NaN3 and 10 mg/~L bovine serum albumin.
To a 400 /~l aliquo. of each standard solution or
serum sample wzs added 40 ~l of 50% v:v aqueous
trichloroacetic acid (TCA), and the resultant
mixtures were vortexed briefly and centrifuged at
10,000 rpm for 5 minutes. The supernatant W2S
collected (300 ~l) from each centrifuge tube, and the
pH was adiusted to pH 7.0 + 0.5 with 3N NaO~.
To each well of an anti-Dpd-antibody-coated
plate from Example 8 was added 100 ul of TCA-
precipitated standard or serum sample, and 50 ~l of
Dpd-alkaline phosphatase solution (containing -7~
ng/mL Dpd-AP conjugate, 100 mM PBS, 0.7% bovine serum
albu~in, 0~9% sucrose, 7 mM Tris, 0.15 mM MgCl"
0.05% Tween-20, and 0.05% azide). After incubation
of the plates overnight at 4C, the wells were washed
with P~S con~aining and 0.05% NaN3 and 0.05% Tween-
20.
Dpd-alkaline phosphatase conjugate retaLned in
each welL was assayed by adding to each well 150 ~L
-... .
WO94/1~72 ~ PCT~S93/L~21
; of substrate solution (p-nitrophenyl phosphate, 2
mg/mL in 1 M diethanolamine, pH 10, containing 1 mM
MgCl), incubating at room temperature for one hour,
stopping the enzymic reaction by addition of 50 ~L 3
N NaOH, and reading the optical density of the well
at 405 nm using a Vmax reader (Molecular Devices
Corp.). The Dpd crosslink concentration for each
serum sample was determined by comparison with a
standard curve constructed with the N-Dpd standard
solutions.
Example 10
Bindinq SelectivitY of AntibodY Reaqent
N-Pyd, N-Dpd, and pyridinium-peptides (MW>1000)
were isolated from urine samples as described above.
Aliquots of the pyridinium-peptide fraction were
hydrolysed to convert the crosslinks in the fraction
to H-Pyd and H-Dpd. The concentrations of Pyd in the
N-Pyd and H-Pyd preparations, of Dpd in the N-Dpd and
H-Dpd preparations, and of Dpd in the pyridinium-
peptide preparation, were determined by HPLC, as in
Example 1. In addition, an amino acid solution
containing an equimolar mixtu~e of the 20 common
amino acids, 150 ~M each in PBS, was prepared.
Aliquots (50 ~l) of the native crosslink
preparations and the amino acid mixture were added in
duplicate to anti-Dpd-antibody-coated microtitre
wells (Example 8), and each well was assayed for N-
Dpd as in Example 9, except that 100 ~L Dpd-AP
conjugate solution was used rather than 50 ~L. The
optical density readings (405 nm) from duplicate
samples were averaged, and from these values, the
apparent N-Dpd concentration of each sample was
determined using a standard curve established with
purified N-Dpd. The percent reactivity of each
WO94/1~72 PCT~S93/1~21 ~
2~5~23~
46
sample was calculated as a ratio of apparent N-Dpd
concentration (measured using the N-Dpd standard
curve above) to total Dpd crosslink concentration in
the sample determined by HPLC for total H-Dpd (times
lO0). The relative reactivity determined for
purified N-Dpd was arbitrarily set at lO0~, and the
reactivities of the other crosslink preparations (and
the amino acid mixture) were expressed as a
percentage of lO0. Results obtained with this assay
are shown in Table 2 above.
Exam~le ll
Pre~aration of Anti-PYridinoline Antiserum
New Zealand white rabbits (a total of 59) for
immunization were divided into eight groups according
to immunization protocol, as indicated below in Table
6. The immunization dose was 200 ~g of Pyd-BSA
(Example 3A), low-hapten Pyd-BSA immunogen (prepared
as in Example 3A for Pyd-BSA, but with a lower
Pyd:BSA stoichiometry), or Pyd-KLH (Example 3B), in
l.0 ml PBS mixed with l.0 ml of Ribi adjuvant (Ribi
ImmunoChemical Research, Inc.). Initial immunization
was by subcutaneous injections at multiple sites, and
subsequent booster immunizations were given at three
week intervals intramuscularly. Antiserum was
collected lO days after each immunization.
-
47
Table 6
Group ~' Rabbits Rabbits Kept Carrier
I 4 1 BSA
II 10 0 BSA
III 10 2 BSA
IV 5 1 BSA
V 5 2 BSA
VI 10 1 BSA
VII 5 0Low Hapten BSA
VIII 10 1 KLH
Total 59 8
Upon collection, each antiserum was tested for
Pyd binding affinity using the assay format described
in Example~3. In brief, binding of anti-Pyd
antibodies from the serum to Pyd immobilized on a
solid support was detected using an alkaline
phosphatase-labeled goat anti-rabbit I~G antibody
reagent.
Immunized animals were kept if their antisera
satisfied the followlng criteria, defined further in
the following paragraph: AA < 20%, Pyd-peptide <
10~, titer > 5000, and a 0 to 25 nM Pyd signal
separation of > 10% of total modulated signal.
Profiles of the most strongly reactive antisera
zre shown in Table 7 below, as measured using the
2ssay format described in Example~3. The first
column inàicates the immunization program from which
the rabbit antiserum came. The second column
indicates the bleeds which were pooled for analysis.
The column mar~ed "titer" indicates thé dilution of
each antiserum necessary to achieve an optical
~ W094/1~72 21 5 1 2 ~ 4 PCT~S93/1~21
48
density reading of 1.2 to 1.6 with a Pyd-negative
sample (no Pyd present) in the immunoassay. The
column marked "AA" shows the cross-reactivity of each
antiserum with the amino acid mixture described in
Example 7. The column marked "Pyd-pep >1000 MW"
shows the cross-reactivity of each antiserum with
Pyd-peptides (>1000 MW). The last column shows the
separation between signals for O and 25 nM Pyd
samples as a fraction of the total modulated signal.
Table 7
Rabbit Pyd-pep. Sens.
# Bleed~ Titer AA >1000 MM 25 nM
I-3 21-28200Kl 2% 4.6% 18%
15III--3 11--18 20K 16% 8.3% 37%
III-5 11-1852K 1% 8.1% 13%
IV-4 4-14 84K 4% 4.9% 10%
V-3 4-14 22K 18% 4.0% 15%
V-4 11-149700 15% 5.2% 29%
20 VI-8 2-11 30R 10% 0.6% 61%
VIII-4 3-10 34K -0% 3.4~ 11%
IK = X 1000.
As can be seen, rabbits III-3, V-4, and VI-8
showed significant modulation of signal from O to 25
nM N-Pyd. The serum with highest activity (VI-8) was
selected for use in the N-Pyd assays described
herein.
Example 12
Preparation of Pyd-Coated MicroPlates
W094/1~72 PCT~S93/L~21
21~12~
49
Biotin-labeled ovalbumin and a streptavidin-Pyd
conjugate were utilized in the microplate coating.
Biotinylation of ovalbumin was carried out by adding
10 mg of biotin-X-2,4-dinitrophenol-X-L-lysine,
succinimidyl ester (Molecular Probes) in 400
microliters of dimethylformamide to a 10 ml solution
of PBS containing lS0 mg of ovalbumin. The mixture
was allowed to react for two hours at room
temperature, followed by G25 column chromatography.
lo Spectrophotometric analysis indicated two biotins
substituted per mole of ovalbumin.
Conjugation of H-Pyd to streptavidin was
accomplished by coupling a thiolated streptavidin to
H-Pyd via the coupling agent, SMCC. Thiolated
streptavidin was prepared by reaction with N-
succinimidyl-3-(2-pyridylthio)proprionate (SPDP,
Pierce) as follows. To a 0.75 ml solution of 5 mg of
streptavidin in PBS was added 21 uL of
dimethylformamide containing 260 ug of SPDP. The
mixture was allowed to react for one hour at room
temperature, and then was dialysed against PBS. The
SPDP-labeled streptavidin was reduced by the addition
of dithiothreitol to a final concentration of 10 mM.
After incubation for one hour at room temperature,
the thiolated streptavidin was purified on a G-25
column.
To form H-Pyd-streptavidin, a solution
containing 180 ug of succinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate (SMCC,
Pierce) in dimethylformamide (4 ul) was added to a
solution containing 0.5 mg thiolated streptavidin and
50 ug of H-Pyd in 100 ~l of PBS. The mixture was
allowed to react for 3 hours at room temperature and
then was dialysed versus PBS. Spectrophotometric
analysis of the resultant Pyd-streptavidin indicated
-
~ 21S ~ 23~
between l and 2 equivalents of pyrLdinoline
bound pe- equivalent of strepta~idin.
Each of the wells in a 96-well ELISA plate were
coated with with N-~yd as follows. To each well was
added 150 microliters of biotin-ovalbumin solution at
3.8 ug/ml in PBS, followed by an overnight incubation
at 2-8C. The microplates were washed with PBS and
blocked by adding 200 ul of ovalbumin at 1 mg/mL with
an overnight incubation at room temper2ture. T~e
microplates were then twice washed with PBS. T~e
streptavidin-Pyd conjugate is immobilized via t~e
streptavidin mediated binding to biotin. 150 ul of a
solution containing strepta~idin-Pyd at 100 ug/~.l in
PBS was added to each well of the biotin-ovalbumin
coated microplate. After a one hour incubation at
room temperature, the plates are twice washed with
PBS. Residual liquid was removed from the microplate
by drying overnight in a convection oven at 370C.
Exam~le 13
Immunoassay for Pyd Using Polyclonal Antibody Reagent
The following immunoassay was performed using
rabbit polyclonal antibody VI-8 characterized in
Tables 4 and 7 above, and the N-Pyd-coated microtiter
plate described in Example 12.
N-Pyd standard solutions and ~lood serum samples
were tested in duplicate. The standard solutions
consisted of 0 nM, 0.2 nM, 0.6 nM, 2.0 nM, 6.0 nM,
and 2~ nM N-Pyd in assay bufrer (0.05~ NaN3, 0.05%
Tween 20, and 0.1% BS~ in 100 mM sodium phosp~zte
containing 150 mM NaCl, pH 7). Serum samples were
fil~ered through a Centricon-30 filter devLce
(A~icon, Mass.) prior to assay.
Following the addition of sample or standard (25
~l/well), 125 ul/weLL of VI-8 antiserum diluted
.. . , ,_ .
~ WO94/1~72 21~1~ 3 4 PCT~S93/L~21
20,000-fold in assay buffer was added, and the assay
plate was incubated at 4C overnight. After the
plate was washed 3 times with 300 /~l/well of wash
buffer, 150 ~l/well of goat anti-rabbit IgG-alkaline
phosphatase conjugate (1:1000 dilution in assay
buffer) was added, and the plate was incubated at
room temperature for 1 h. The wells were then washed
3 times with wash buffer.
To each well was added 150 uL of enzyme
substrate solution (2 mg/mL of p-nitrophenylphosphate
(Sigma) in 1.0 M diethanolamine, pH 9.8, containing 1
mM MgCl2). Following a 1 hour incubation at room
temperature, 50 ~l of 3.0 N NaOH was added to each
well to stop the enzymatic reaction. The optical
density at 405 nm was then measured with a Vmax
reader (Molecular Devices Corp.).
The optical density readings (405 nm) from
duplicate samples were averaged, and the averaged
readings from the N-Pyd standards were used to
construct a standard curve of OD reading vs. N-Pyd
concentration. From this curve, the free N-Pyd
crosslink concentration in each serum sample was
determined.
25ExamPle 14
Bindinq Selectively of Polyclonal Antibody Reaqent
N-Pyd, N-Dpd, and pyridinium-peptides (MW>1000)
were isolated from urine samples as described above.
Aliquots of the pyridinium preparations were
hydrolysed to convert the crosslinks in the fractions
to H-Pyd and H-Dpd. The concentrations of Pyd in the
N-Pyd and H-Pyd preparations, of Dpd in the N-Dpd and
H-Dpd preparations, and of Pyd in the pyridinium-
peptide preparation, were determined by HPLC, as in
W094/1~72 PCT~S93/1~21 ~
3 ll
52
Example l. In addition, an amino acid solution
containing an equimolar mixture of the 20 common
amino acids, 150 ~M each in PBS, was prepared.
Aliquots (50 ~l) of the native crosslink
preparations and the amino acid mixture were added in
duplicate to Pyd-coated microtitre wells, and each
well was assayed for pyridinoline as in Example 13.
The optical density readings t405 nm) from duplicate
samples were averaged, and from these values, the
apparent N-Pyd concentration of each sample was
determined using a standard curve established with
purified N-Pyd. The percent reactivity of each
sample was calculated as a ratio of apparent
concentration (measured using the N-Pyd standard
curve above) to total Pyd crosslink concentration in
the sample determined by HPLC for total H-Pyd (times
lO0). The relative reactivity determined for
purified N-Pyd was arbitrarily set at 100%, and the
reactivities of the other crosslink preparations (and
the amino acid mixture) were expressed as a
percentage of lO0. Results obtained with this assay
are shown in Tables 4 and 7 above.
Although the invention has been described with
respect to particular embodiments, it will be
appreciated that various changes and modifications
can be made without departing from the invention.
