Note: Descriptions are shown in the official language in which they were submitted.
-
21~749B
BOEHRINGER MANNHEIM GMBH 3699/OB
Imm~no~ y for the detection of collagen or collagen
fragments
The invention concerns an immunoassay for the detection
of collagen or collagen fragments in a sample using at
least one antibody which recognizes a synthetic linear
peptide which corresponds to a sequence of the non-
helical C-terminal or N-terminal region of collagen in
which the sample is preferably denatured.
Collagen is an important structural protein in the
connective tissue of the skin, cartilage and bone. 11
types are known which are each composed of three chains.
Each type is composed of 1 - 3 different chains which
are denoted al, a2 and a3 (E. Miller et al., in Methods
in Enzymology 144, Structural and Contractile Proteins,
ed. L. Cunningham, Academic Press Inc. 1987, p. 3 - 41).
A characteristic property of the mature collagen of
certain tissues such as bone or cartilage in particular,
is the cross-linking of adjacent fibres by hydroxylysyl-
pyridinoline or lysylpyridinoline (D. Fujimoto et al.,
J. Biochem 83 (1978), 863 - 867; D. Eyre et al., Ann.
Rev. Biochem 53 (1984), 717 - 748 and D. Eyre, Methods
in Enzymology 144 (1987), 115 - 139). These cross-links
can be utilized as chemical markers for the specific
detection of collagen (Z. Gunja-Smith et al., Biochem.
J. 197 (1981), 759 - 762). When extracellular collagen
is degraded, hydroxylysylpyridinoline or lysyl-
pyridinoline derivatives which contain peptide side
chains or free pyridinoline derivatives with lysyl or
hydroxylysyl residues as described in WO 91/10141 pass
into body fluids such as blood or urine. The detection
-- 21674gB
-- 2
of these compounds in body fluids is therefore an
indicator for the degradation of extracellular collagen
such as that which for example occurs in osteoporosis
and as a result of bone tissue tumours. Monoclonal
antibodies which were obtained by immunization with
appropriately cross-linked collagen fragments which can
be isolated from the urine were described in WO 89/12824
for the detection of such hydroxylysylpyridinolines or
lysylpyridinolines with peptide side chains. Also in the
method described in WO 91/08478 the collagen test is by
means of an antibody to natural, i.e. generated in vivo,
cross-linked degradation products of collagen.
A disadvantage of these peptides isolated from natural
sources is that there is no reliable source for a
reproducible production of the antigens or binding
partners in the test. A further disadvantage of the
peptides isolated from natural sources is the risk of
contamination with infectious material.
Defined antigens can for example be obtained by chemical
synthesis of a peptide which corresponds to an epitope
of the antigen. If small peptides with a molecular
weight of about 700 - 1500 D are used for this, then it
is necessary to bind them to a carrier molecule in order
to obtain an antigen with an immunogenic effect. In this
process the structure of the epitope must not be changed
by binding to the carrier molecule. Therefore coupling
to the carrier molecule has previously been carried out
at the ends of the peptide chain at an adequate distance
from the presumed epitope region (Laboratory Technics in
Biochemistry and Molecular Biology, Synthetic
Polypeptides as Antigens, Editors R.H. Burdon and P.H.
van Knippenberg, Elsevier, Amsterdam, New York, Oxford
1988, pages 95 - 100).
2167~g6
-- 3
A problem in the chemical synthesis of a defined antigen
that corresponds to a natural degradation product of
cross-linked collagen is the hydroxylysylpyridinoline or
lysylpyridinoline structure resulting from the cross-
linking the chemical synthesis of which is very
complicated.
The object of the invention was therefore to provide a
defined antigen for the production of antibodies to
collagen or collagen fragments, for use as a specific
binding partner of the antibody to collagen or collagen
fragments in a competitive immunoassay and as a standard
material for establishing a standard or calibration
curve in a competitive immunoassay for the detection of
collagen or collagen fragments.
Previously it has always been assumed that in order to
detect collagen or collagen degradation products in a
sample it is necessary to detect the cross-linked
structures per se or so-called cross-linked peptides
which result from the cross-linking of hydroxylysyl or
lysyl residues since this hydroxylysylpyridinoline or
lysylpyridinoline structure is characteristic for
collagen. Examples of such methods of detection are
described in WO 89/12824, WO 91/08478, WO 89/04491 and
W0 91/10141.
It has now been surprisingly found that the use of a
defined antigen, a binding partner or a standard
material containing a synthetic linear peptide which
corresponds to a sequence of the non-helical linear
C-terminal or N-terminal region of collagen is
sufficient for achieving the aforementioned object. The
advantages of using synthetic linear peptides as binding
2iS7~9~
-- 4
partners in immunoassays, as a standard material or as
an immunogen in the antibody production are that these
peptides, in contrast to peptides from natural sources,
can be produced reproducibly with an exactly defined
structure. Moreover an immunoassay in which such short
synthetic peptides are used is less susceptible to
interference.
The invention therefore concerns a competitive
immunoassay for the detection of collagen or collagen
fragments in a sample which is characterized in that a
binding partner containing a synthetic linear peptide
which corresponds to a sequence of the non-helical C-
terminal or N-terminal region of collagen is incubated
with an antibody capable of binding the synthetic linear
peptide and the sample, and the binding of the antibody
to the binding partner is determined in a suitable
manner.
It has proven to be particularly advantageous to
denature the sample or rather the collagen or collagen
fragments present in this sample and thus improve the
accessibility of the epitopes to the antibody binding.
The most suitable method is to pretreat the sample
before incubation with the antibody with a denaturing
agent for proteins which are known to a person skilled
in the art. In order to limit or completely avoid
interference of the immunological reaction between the
sample and the specific antibodies, the denatured sample
is preferably additionally diluted before incubation
with the antibody. All agents known to a person skilled
in the art for this purpose are suitable as denaturing
agents. Potassium thiocyanate (KSCN) at a concentration
of 2 - 6 M and tetradecyltriethylammonium bromide (TTAB)
at a concentration of 0.5 - 2 M have proven to be
21fi74g~
-- 5 --
particularly suitable for the denaturation.
The introduction of a denaturation step of the sample
enables antibodies which only bind negligibly to non-
denatured samples to be used in addition i.e. they bind
a denatured sample very well. Therefore as a result of
the introduction of this denaturing step more antibodies
are available for a diagnostic test for collagen or
collagen fragments.
The invention in addition concerns a standard material
for establishing a standard or calibration curve in a
competitive immunoassay for the detection of collagen or
collagen fragments characterized in that it contains an
antigen that contains a synthetic peptide which
corresponds to a sequence of the non-helical C-terminal
or N-terminal region of collagen.
The invention in addition likewise concerns an antigen
for the production of antibodies to collagen or collagen
fragments which contains a synthetic linear peptide
corresponding to a sequence of the non-helical C-
terminal or N-terminal region of collagen and the
antibodies produced using this antigen.
All consecutive amino acid sequences of the non-helical
C-terminal or N-terminal region of collagen are suitable
as synthetic linear peptides. These regions are known
from Chu et al., Nature 310, 337-340 (1984), Click et
al., Biochemistry 9, 4699-4706 (1970), Morgan et al., J.
Biol. Chem. 245, 5042-5048 (1970) and Bernard et al.,
Biochemistry 22, 5213-5223 (1983). Peptides comprising 5
to 25 amino acids are preferably used and especially
those comprising 8 to 20 amino acids. In this connection
~167~96
it is not necessary that the sequence comprises the
cross-linking region. However, it can indeed also
overlap this region. However, in no case is a
hydroxylysylpyridinoline or lysylpyridinoline cross-link
present in the synthetic peptide. Synthetic peptides
from the C-terminal region of collagen have proven to be
particularly suitable since the non-helical C-terminal
region is larger than the non-helical N-terminal region
of collagen. Thus more potential epitopes are available
in this region than in the N-terminal region. Peptides
having the sequence shown in SEQ ID NO. 1, 2, 3 or 4
from the C-terminal region of the al chain of collagen
are particularly suitable.
The concentration of degradation products of collagen is
an important diagnostic marker for the extent of
osteolysis. The synthetic linear peptides enable a
competitive immunoassay to be carried out for the
detection of collagen or collagen fragments. It has
surprisingly turned out that these peptides compete very
well with the collagen fragments that occur in natural
samples such as plasma, serum or urine for antibodies to
these collagen fragments and thus enable a competitive
test. Such antibodies to collagen or collagen
degradation products are commercially available for
example in the telopeptide ICTP [125I] radioimmunoassay
kit from the Orion Diagnostica Company Finland. They
can, however, be preferably produced according to the
invention using the synthetic linear peptide.
For an application in a competitive immunoassay, the
synthetic linear peptide can be used directly as a
binding partner which is bound to a solid phase or it
can be coupled to a second component. Coupling to the
second component is preferably achieved via the
~ ~ ~ 7 4 ~ ~
-- 7 --
N-terminal and C-terminal amino acids of the linear
peptide. Optionally a spacer can in addition be inserted
between the peptide and the second component. The second
component can for example serve for coupling the peptide
indirectly to a solid phase. Examples of this are known
to a person skilled in the art. The peptide is
preferably coupled to bovine serum albumin and the
coupling product is adsorptively bound to a solid phase
such as a plastic tube. The peptide can also be bound
covalently to biotin. It is then attached to the solid
phase by means of binding to avidin or streptavidin
which itself has been bound to the solid phase. The
second component can also serve as a carrier for several
peptides for example in a competitive turbidimetric
inhibition immunoassay (TINIA) in which several peptides
are for example coupled to albumin, immunoglobulin,
B-galactosidase, to polymers such as polylysine or to
dextran molecules such as those described in EP-A-O 545
350 or to particles such as latex. Preferably 30 to 40
peptide molecules are coupled per carrier molecule. The
peptide can also be coupled to a component which
represents a label. Examples of all these test variants
are known to a person skilled in the art.
In the test procedure the antibody can be incubated
simultaneously or sequentially with the sample and with
the binding partner which contains the synthetic linear
peptide. Subsequently the amount of bound or non-bound
antibody is determined in the usual manner. For example
the antibody used can itself be labelled and this label
serves directly as a measure of the bound or unbound
antibody. It is also possible to use a second labelled
antibody which is directed towards the bound or unbound
antibody such as towards the Fc part of this antibody.
Agglutination tests such as TINIA or FPIA (fluorescence
2167496
-- 8 --
polarisation immunoassay) (W. Dandliker et al., J. Exp.
Med. 122 (1965), 1029), EMIT (enzyme multiplied
immunoassay) (Gunzer et al., Kontakte III (1980), 3 -
11) and CEDIA techn;ques (Henderson et al., Clinical
Chemistry 32 (1986), 1637 - 1641) can for example serve
as competitive test variants for determining the amount
of bound or unbound antibody. The peptides according to
the invention have proven to be particularly suitable
for use as defined binding partners which compete with
the sample for binding to the antibodies. The synthetic
linear peptides with the sequences shown in SEQ ID N0 1,
2, 3 or 4 are particularly preferred.
After determining the extent of antibody binding to the
binding partner, which is a measure of the amount of
antigen in the sample, it is possible to determine the
exact amount of antigen in the sample in the usual
manner by comparison with a standard treated in the same
manner.
Collagen degradation products isolated from natural
materials can be used as a standard. However, these are
characterized by a certain intrinsic variability. An
antigen containing the synthetic linear peptide
according to the invention has proven to be more
suitable as a standard material. In this case the
antigen of the standard can either be composed solely of
this peptide or of this peptide coupled to a suitable
carrier which for example serves to improve the water-
solubility of the peptide. In order to produce the
standard material comprising peptide and carrier, the
linear peptide which corresponds to a sequence of the
non-helical C-terminal or N-terminal region of collagen
is synthesized and bound to the carrier molecule via its
N-terminal or C-terminal amino acid by suitable coupling
~167496
methods. One or several peptides can be bound per
carrier molecule. Optionally the coupling can be
achieved via a spacer. For certain purposes, such as for
agglutination tests, it may be advantageous to bind
several peptides according to the invention with
different sequences to a carrier molecule especially if
polyclonal antibodies are used in the test that were not
produced with the aid of the antigen according to the
invention and thus usually recognize several epitopes.
The already known antibodies to collagen degradation
products can be used as antibodies in the competitive
immunoassay. Antibodies which have been obtained with
the aid of an antigen containing the linear synthetic
peptide according to the invention are especially
suitable.
For the immunization the linear synthetic peptides
corresponding to one or several sequences of the non-
helical C-terminal or N-terminal region of collagen are
preferably bound to a suitable carrier protein such as
keyhole limpet haemocyanin, bovine serum albumin or
edestin.
In order to produce these antigens or immunogens the
linear peptides are firstly chemically synthesized in
the usual manner. Subsequently the synthetic peptides
are coupled to the aforementioned carrier proteins via
the N-terminal amino group using maleinimidohexanoic
acid N-hydroxysuccinimide ester. It has surprisingly
turned out that synthetic linear peptides having the
sequence shown in SEQ ID NO 1, 2, 3 or 4 are
particularly suitable for the production of antibodies
that are suitable for a competitive test procedure.
2167496
-- 10 --
The antigens according to the invention containing a
synthetic linear peptide that corresponds to a sequence
of the non-helical C-terminal or N-terminal region of
collagen enable antibodies to be obtained which not only
recognize the peptide according to the invention but
also the degradation products of collagen occurring in
body fluids.
The invention therefore in addition concerns a process
for the production of antibodies to collagen or collagen
fragments by immunization with an antigen according to
the invention and isolation of the desired antibody from
the serum of the immunized animals by known methods. The
desired antibody is preferably isolated by means of
immunosorption to a peptide having the sequence shown in
SEQ ID NO 1, 2, 3 or 4 coupled to a carrier protein and
preferably to sepharose.
A preferred subject matter of the invention is a process
for the production of monoclonal antibodies to collagen
or collagen fragments by immunization with an antigen
according to the invention, immortalization of the
spleen cells of the immunized animals, cloning those
immortalized spleen cells which produce the desired
antibody and isolation of the antibody from the cloned
cells or from the culture supernatant of these cells.
The immunization is carried out in animals that are
usually used for this; mice or rabbits are preferably
used.
The spleen cells of the immunized animals are
15749~
immortalized by methods familiar to a person skilled in
the art such as e.g. the hybridoma technique (Kohler and
Milstein, Nature 256 (1975), 495 - 497) or by
transformation with the Epstein-Barr virus (EBV
transformation). In order to detect those immortalized
cells which produce the desired antibody, a sample of
the culture supernatant is incubated in a conventional
immunoassay with the antigen according to the invention
used for the immunization and it is examined whether an
antibody binds to this antigen.
The invention furthermore concerns the polyclonal and
monoclonal antibodies that can be obtained by the
process according to the invention.
These polyclonal and monoclonal antibodies not only
react with the hapten according to the invention used
for the immunization but also react well with collagen
and with the natural degradation products of collagen
found in body fluids. Collagen or fragments thereof
present in the sample are preferably denatured which in
most cases considerably improves the binding of the
antibodies according to the invention.
The antibodies according to the invention can therefore
be used in test procedures for the determination of
collagen or collagen fragments.
Therefore the invention furthermore concerns an
immunoassay for the detection of collagen or collagen
fragments in a sample using at least one antibody that
recognizes a synthetic linear peptide which corresponds
to a sequence of the non-helical C-terminal or N-
terminal region of collagen which is characterized in
- 12 - 21~749~
that the sample is denatured. The competitive
immunoassay described above in which the linear peptides
according to the invention are additionally used has
proven to be most suitable. The use of antibodies
according to the invention is not limited at all to a
competitive immunoassay. The antibodies can be also used
in other test formats such as a sandwich immunoassay.
Antibodies of the state of the art can for example be
used as a second antibody in this immunoassay. One only
needs to take care that the two antibodies do not
compete with one another for the same binding site.
The invention therefore also concerns the use of a
polyclonal or monoclonal antibody according to the
invention for the determination of osteolysis by
incubating the antibody with a tissue sample and
determining the collagen degradation product binding to
this antibody.
The invention furthermore concerns a test combination
for the detection of collagen or collagen fragments in a
sample containing a protein denaturing agent in a first
reagent and separate from this in a second reagent an
antibody which recognizes a synthetic linear peptide
that corresponds to a sequence of the non-helical C-
terminal or N-terminal region of collagen. The reagents
are either present in the form of aqueous, preferably
buffered, solutions in which case all common buffers
known to a person skilled in the art that do not
interfere with the immunological reaction can be used as
the buffer or in the form of dry, preferably
lyophilized, mixtures that can be reconstituted by the
addition of a suitable solvent such as water.
- 13 - ~1~749~
It can also contain other common test additives such as
substances that reduce interference, proteins or
detergents. The test combination also preferably
additionally contains a synthetic linear peptide that
corresponds to a sequence of the non-helical C-terminal
or N-terminal region of collagen as a binding partner of
the antibody. The synthetic linear peptide can either be
coupled directly to a carrier or be coupled to a second
component which mediates binding to the solid phase. The
peptide can also be coupled to a component which
represents a label.
Furthermore a standard for establishing a standard or
calibration curve may be present in a third reagent
which contains an antigen containing a synthetic peptide
that corresponds to a sequence of the non-helical C-
terminal or N-terminal region of collagen.
The invention is elucidated in more detail by the
following examples in conjunction with the sequence
protocols.
EQ ID NO 1 shows the sequence of a peptide according
to the invention composed of 9 amino acids
in which Xaa denotes an arbitrary amino
acid.
EQ ID NO 2 shows the sequence of a peptide according
to the invention composed of 16 amino
acids.
- 14 - ~1674~
EQ ID NO 3 shows the sequence of a peptide according
to the invention composed of 10 amino
acids.
EQ ID NO 4 shows the sequence of a peptide according
to the invention composed of 13 amino
acids.
- 15 _ 216~96
Example
Peptide syntheses
The peptides having a partial sequence of the amino acid
sequence of collagen as shown in the sequence protocols
SEQ ID NO 2 and 3 are synthesized by means of fluorenyl-
methyloxycarbonyl (Fmoc) solid phase peptide synthesis
on a a) Labortec SP 640 peptide synthesizer or b)
Zinsser analytic SMPS 350 peptide synthesizer.
a) Production of acetyl-Ser-Ala-Gly-Phe-Asp-Phe-Ser-
Phe-Leu-Pro-Gln-Pro-Pro-Gln-Glu-Lys-Amid (SEQ ID N0
2)
4.0 equivalents of each of the following Fmoc amino
acid derivatives are used in the stated sequence:
Lys with a tert. butyloxycarbonyl protecting group
Glu with a tert. butyl ester protecting group
Gln without a side chain protecting group
Pro without a side chain protecting group
Pro without a side chain protecting group
Gln without a side chain protecting group
Pro without a side chain protecting group
Leu without a side chain protecting group
Phe without a side chain protecting group
Ser with a tert. butyl ether protecting group
Phe without a side chain protecting group
Asp with a tert. butyl ester protecting group
Phe without a side chain protecting group
Gly without a side chain protecting group
Ala without a side chain protecting group
-
~1674g6
- 16 -
acetyl acetic anhydride
The amino acids or amino acid derivatives are dissolved
in N-methylpyrrolidone.
The peptide is synthesized on 3 g 4-(2',4'-dimethoxy-
phenyl-Fmoc-aminomethyl)-phenoxy resin (Tetrahedron
Letters 28 (1987), 2107) with a loading of 0.87 mmol/g
(JACS 95 (1973), 1328). The coupling reactions are
carried out for 60 minutes using 4.4 equivalents in
relation to the Fmoc amino acid derivatives of
dicyclohexylcarbodiimide and 4.8 equivalents N-hydroxy-
benzotriazol in dimethylformamide as the reaction
medium. The coupling yield is monitored by means of a
Kaiser test (Anal. Biochem. 34 (1970), 595) on the
synthesis resin washed with isopropanol. If this does
not show a complete conversion, the conversion is
completed by recoupling under the conditions stated
above. After each step in the synthesis the Fmoc group
is cleaved within 20 minutes using 20 % piperidine in
dimethylformamide. The resin loading is determined by
means of the W absorbance of the released fulvene group
after each piperidine treatment. The loading is still
0.68 mmol/g after the synthesis.
The peptide is released from the synthesis resin and the
acid-labile protecting groups are cleaved with 80 ml
trifluoroacetic acid, 5 ml ethanedithiol, 2.5 g phenol,
2.5 ml m-cresol and 5 ml water within 60 minutes at room
temperature.
The reaction solution is subsequently concentrated in a
vacuum. The residue is taken up in diisopropyl ether,
stirred vigorously for 1 - 2 hours and then filtered.
21~7~9~
- 17 -
The material is then pre-purified by means of gel
permeation chromatography on Sephadex G15 using 0.5 %
acetic acid as the eluting agent. The crude material
obtained is subsequently filtered and isolated within
120 minutes by means of preparative HPLC on Nucleosil
RP18 (column 40 mm x 250 mm 300 A, 5 ~m) using a
gradient of 100 % buffer A (water, 0.1 % trifluoroacetic
acid) to 100 % buffer B (60 % acetonitrile, 40 % water,
0.1 % trifluoroacetic acid). The identity of the eluted
material is determined by means of fast-atom-
bombardment-mass spectrometry (FAB-MS).
b) Synthe~i~ of Ala-Gly-Phe-A~p-Phe-8er-Phe-Leu-Pro-Gln
~EQ ID NO 3)
The peptide was synthesized on 30 mg 4-(2',4'-
dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin SA 5030
from the Advanced Chemtech Company with a loading of
0.47 mmol/g. 140 ~mol of each of the following Fmoc
amino acid derivatives was coupled twice together in
each case with 140 ~mol l-hydroxybenzotriazol in
dimethyl-formamide DMF and 10 ~mol N,N-diisopropyl-
carbodiimide in DMF to the solid phase-bound peptide to
be constructed:
Glu with a trityl protecting group
Ser with a tert. butyl protecting group
Asp with a tert. butyl protecting group
Pro
Leu
Phe > each without a side chain protecting group
Gly
Ala
2167496
- 18 -
The coupling times were 30 and 40 minutes. The cleavage
time was 20 minutes and was carried out with a solution
of 50 % piperidine in DMF. The washing steps were
carried out eight times after each reaction step using
DMF. The peptide was released by treating the resin,
that had been filtered free of solvent and washed with
dichloromethane and methanol, with 1 ml of a solution of
sO % trifluoroacetic acid, 3 % thioanisol, 3 % ethande-
dithiol and 3 % thiocresol within 20 minutes and 140
minutes. The product was precipitated by addition of 15
ml cold diisopropyl ether to the pooled filtrate and
isolated by filtration. The residue was dissolved in
50 % acetic acid and lyophilized. 8 mg white
lyophilisate with a purity of 79 % according to HPLC was
obtained. The identity was confirmed by means of FAB
mass spectroscopy.
The peptide with the sequence SEQ ID N0 4 Cys-Gly-Ser-
Ala-Gly-Phe-Asp-Phe-Ser-Phe-Leu-Pro-Gln was synthesized
in an analogous manner.
21~74~6
-- lg --
Example 2
Activation of peptide~
The peptide synthesized according to example la) is
activated by acylation with maleinimidohexanoyl-N-
hydroxysuccinimide (MHS). For this 0.1 mmol of the
peptide is dissolved in 20 ml 0.1 mol/l potassium
phosphate buffer pH 7.5, admixed with a solution of
0.1 mmol MHS in 6 ml dioxane and stirred for 20 minutes
at 20C. Subsequently the pH value is adjusted to pH 4
with glacial acetic acid and the reaction mixture is
immediately lyophilized. The lyophilisate is dissolved
in 5 ml water and purified by means of preparative HPLC
on a Waters Delta-Pak~ C18 column (100 A, 15 ~m 50 x
300 mm) using an elution gradient of 100 % A (water 0.1
% trifluoroacetic acid) to 100 % B (99.9 % acetonitrile
0.1 % trifluoroacetic acid).
- 20 - 2 1~74~ ~
Bx~mple 3
Production of immunogens by coupling aotivated peptide~
to carrier proteins
The coupling of activated peptides to keyhole limpet
haemocyanin (KLH), bovine serum albumin (BSA) and A-
galactosidase (AGal) is described. In order to couple
the peptide activated with MHS according to example 2,
it is necessary that the carrier protein has free SH
groups. A-Gal already has these in its natural state and
therefore requires no further pretreatment. In the case
of KLH and BSA the NH2 groups of the ~-amino side chain
of lysine residues are derivatized by treatment with N-
succinimidyl-S-acetylthiopropionate (SATP) and thus
converted into SH groups.
In this way a carrier protein is obtained which has an
increased number of SH groups compared to its native
state. For this 113.51 mg SATP (dissolved in 10 ml
dioxane) is added dropwise to a solution of 1.39 g KLH
in 500 ml 0.1 mol/l potassium phosphate buffer pH 8.5
within 20 minutes. After stirring for 30 minutes at 20C
the pH value of the reaction solution is readjusted with
0.1 mol/l sodium hydroxide solution to pH 8.5 and it is
stirred for a further 24 hours. The solution is
subsequently concentrated with the aid of an Amicon
cell (membrane YM10) to 100 ml, dialysed 3 x 24 hours
against 3 l 0.1 mol/l potassium phosphate buffer pH
8.5/0.05 mol/l sodium chloride each time and
subsequently lyophilized.
In order to cleave the S-acetyl protecting group, 481 mg
of the KLH-SATP lyophilisate is dissolved in 20 ml
21674~
- 21 -
0.1 mol/l potassium phosphate buffer pH 8.5/0.05 mol/l
sodium chloride, admixed with 0.5 ml freshly prepared
1 mol/l hydroxylamine solution and stirred for 90
minutes at 20C.
7.23 mol of the activated peptide obtained according to
example 2 in 4 ml water is added to the derivatized
carrier protein and stirred for 20 hours at 20C.
Subsequently the turbid solution is dialysed twice
against 1 l 0.1 mol/l potassium phosphate buffer pH
8.5/0.05 mol/l sodium chloride. The dialysate is
centrifuged, the clear supernatant is decanted and
lyophilized.
21~7~6
- 22 -
Example 4
Production of polyclonal antiboaies again~t linear
collagen fragments
5 sheep were in each case immunized in a known manner
with the immunogen from example 3. The immunogens
contained the peptide having the sequence stated in
SEQ ID NO 2 which corresponds to the amino acids No. 892
to 907 in the sequence of the a chain of type I
collagen. KLH or ~-galactosidase served as the carrier
protein. The animals were immunized at monthly intervals
with the immunogens in complete Freund's adjuvant. The
dose was 500 ~g per animal and immunization. Blood
samples were taken four months after the initial
immunization and the antibodies obtained were examined
for reaction with collagen fragments.
ELI~A to detect the reaction of the antisera with
collagen fragments
The following material and reagents were used:
Microtitre plates Maxisorp F96, Nunc Company
Coating buffer: 50 mM sodium carbonate pH 9.6
0.1 % NaN3
Incubation buffer: 10 mM sodium phosphate pH 7.4
0.1 % Tween 20
0.9 % NaCl
1 % bovine serum albumin
- 23 - 2 1 67 4 9 ~
ubstrate solution: ABTS~, Boehringer Mannheim GmbH,
catalogue No. 857424.
2 mg/ml vanillin was added to the
solution to increase the signal.
Wash solution: 0.1 % Tween 20
0.9 % NaCl
Each of the wells of the titre plates was filled with
100 ~1 of a solution which contained 10 ~g/ml collagen
fragments in coating buffer. The collagen fragments were
prepared by protease digestion of human collagen from
bones according to the instructions in EP-A-0 505 210.
After incubating for one hour at room temperature while
shaking, it was washed three times with wash solution.
The antisera were diluted 1 : 4000 with incubation
buffer and 100 ~1 of each was incubated in the wells of
the microtitre plate for one hour at room temperature
while shaking. Subsequently the wells were washed three
times with wash solution.
A conjugate of horseradish peroxidase with rabbit
antibodies against the Fc part of sheep-IgG is diluted
in incubation buffer to a concentration of 12.5 mU/ml
and each of the wells of the microtitre plate are filled
with 100 ~1 thereof. After incubating for one hour at
room temperature while shaking, the titre plates are
washed three times with wash solution.
100 ~1 substrate solution is added and incubated until
there is a clear colour development (10 - 60 minutes).
The absorbance is determined as the difference between
the measurements at 405 and 492 nm.
21S7~fi
- 24 -
The sera of most animals showed a strong reaction with
the collagen fragments on the solid phase. The serum of
an animal which had not been immunized only exhibited a
weak measurement signal under the same conditions. The
results are given in table 1.
Table
B-galactosidase KLH
animal No. absorbance animal No. absorbance
1 1.05 1 1.16
2 1.18 2 2.62
3 1.49 3 1.28
4 0.48 4 1.42
> 2.70 5 1.81
Mice were immunized in an analogous manner with
immunogens according to example 3 and the antisera were
obtained. The three antisera 223/20, 241/13 and 242/2
were particularly suitable. The antiserum 223/20 was
derived from a mouse which had been immunized with a
decapeptide corresponding to SEQ ID NO 4 coupled to KLH.
The antiserum 241/13 was derived from a mouse which had
been immunized with a peptide corresponding to SEQ ID
NO 2 of 16 amino acids in length coupled to KLH.
The antiserum 242/2 was derived from a mouse which had
been immunized with a peptide corresponding to SEQ ID
NO 2 of 16 amino acids in length coupled to BGal.
- 25 - 2167~g 8
Bxample 5
Determination of collagen and its degradation products
in body fluids by means of a competitive test
The wells of a 96-well microtitre plate are coated
overnight at 4C with streptavidin according to
EP-A-0 344 578 (100 ~l of a solution of 1 ~g/ml in PBS)
and unspecific binding sites that are still free are
blocked for 2 hours at room temperature by incubation
with 300 ~l BSA (bovine serum albumin, 10 mg/ml).
The decapeptide having the sequence shown in SEQ ID NO 3
which had been prepared according to example lb) is
biotinylated at its amino terminus with D-biotinyl-~-
amidocaproic acid-N-succinimide ester (Boehringer
Mannheim, catalogue No. 1008960) according to the
manufacturer's instructions. The biotinylated peptide is
dissolved at a concentration of 10 ng/ml in PBS, 0.05 %
Tween 20, 1 % BSA and bound to the streptavidin-coated
microtitre plate by incubating 100 ~l per well for 1
hour. Subsequently unbound peptide is removed by washing
three times with PBS, 0.05 % Tween 20.
150 ~l of the sample to be examined (serum, plasma or a
standard) is in each case incubated with 150 ~l of the
antibody according to the invention according to example
4 for 2 hours at 37C (or overnight at 4C). 100 ~l of
this mixture is in each case added to the bound
decapeptide in the wells of a microtitre plate and
incubated for 60 minutes at 37C. In this process only
the antibody excess of the antiserum which is still
unbound after incubation with the sample can bind to the
immobilized decapeptide.
21~7~96
- 26 -
After washing three times with PBS/0.05 % Tween 20,
bound antibody is detected by subsequent incubation with
a rabbit anti-sheep IgG-POD conjugate (Boehringer
M~nnheim GmbH) and ABTS~ (1 mg/ml).
164 patient sera were measured using the test according
to the invention (MTP competitive test). The results
were related to data determined with a radioimmunoassay
(RIA). This RIA ICTP (telopeptide ICTP [125I] from the
Orion Diagnostica Company, Finnland) is based on cross-
linked collagen fragments which are prepared and
isolated by enzymatic digestion and biochemical methods.
It can be seen from Figure 1 that the method according
to the invention results in measured values that
correlate well with the RIA values which means the
method according to the invention yields clinically
relevant data. A correlation coefficient of 0.959 was
determined.
- 27 - 2~6749~
Example 6
Influence of denaturing reagents on the determination of
a C-terminal peptide from collagen
The biotinylated decapeptide corresponding to SEQ ID
NO 3 is bound to microtitre plates coated with
streptavidin as described in example 5.
As an alternative a fragment of collagen, a C-terminal
telopeptide (CTX) which was prepared according to
Risteli (1993) was adsorbed to the surface of microtitre
plates (Nunc, Maxisorb). For this 100 ~l of a solution
containing 1 ~g/ml CTX is incubated overnight at 4C in
each of the wells of a microtitre plate. Free unspecific
binding sites are blocked by incubation for two hours at
room temperature with 300 ~l bovine serum albumin
(10 mg/ml in PBS).
A CTX solution is used as a sample. This is either pre-
treated with a PBS solution alone (control), a PBS
solution containing 3 M potassium thiocyanate (KSCN) or
a solution containing 1 % tetradecyltriethylammonium
bromide (TTAB). Equal volumes of the CTX solution and
the buffer solutions are mixed and incubated for one
hour. Subsequently the mixture was diluted 1 to 10 with
a PBS solution containing 0.05 % Tween 20~ since
undiluted denaturing agents could influence the immuno-
reactivity of the antibodies.
100 ~l of the samples pretreated in this way are
incubated for one hour at room temperature with 100 ~l
of a solution of a polyclonal mouse antibody in the
microtitre plate which was coated either with the
2~ ~7~9~
- 28 -
decapeptide or CTX as described above. Unbound
antibodies were removed by washing three times with PBS
containing 0.05 ~ Tween 20~. Bound antibodies were
detected with a sheep anti-mouse F(ab)-POD conjugate
(Boehringer Mannheim GmbH, catalogue No. 117 2808) and
ABTS~ (1 mg/ml).
Antisera from mice 223/20, 241/13 and 242/2 described in
example 4 were used as the antisera which were diluted
1:2000 in PBS containing 0.05 ~ Tween 20~ before use.
The results are compiled in Table 2 (measured
absorbances). CTX in PBS without denaturing agents only
showed a significant competition with the mouse serum
242/2. The use of the denaturing reagents KSCN and TTAB
in all cases results in a drastic increase in the
competition even with antisera which did not show
significant competition without denaturing agents.
Table 2
Sample pre-treatment Antiserum Antiserum Antiserum
223/20 241/13 242/2
Control PBS 1.314 0.502 0.563
CTX not denatured 1.125 0.370 0.283
Control KCSN 1.202 0.443 0.562
CTX + KSCN 0.461 0.174 0.259
Control TTAB 0.873 0.217 0.471
CTX + TTAB 0.107 0.090 0.244
2167~96
8EQUENCE PR~.OCOL
(2) Information for SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Phe Asp Phe Ser Phe Leu Pro Xaa
1 5
(2) Information for SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys
1 5 10 15
3~ 2167~9B
(2) Information for SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln
1 5 10
(2) Information for SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Cys Gly Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln
1 5 10
