Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
.~. 2I39592
BOEHRINGER MANNHEIM GMBH 3699/OA/
Immunoassay for the detection of collagen or collagen
fragment-
~
The invention concerns antigens for the production ofantibodies against collagen, a process for the
production of such antigens, antibodies against collagen
which are obtainable by immunization with an antigen
according to the invention as well as the use of such
antibodies for the detection of collagen.
Collagen represents an important structural protein in
the connective tissue of 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 mature collagen of a
particular tissue, such as in particular bone or
cartilage, is the cross-linking of adjacent fibres by
hydroxylysylpyridinoline 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-linkages can be utilized as biological
markers for the specific detection of collagen (Z.
Gunja-Smith et al., Biochem. J. 197 (1981), 759-762).
When extracellular collagen is degraded, hydroxylysyl-
pyridinoline or lysylpyridinoline derivatives which
contain peptide side-chains or free pyridinoline
derivatives with lysyl or hydroxylysyl residues as
" 21395~2
described in W0 91/10141 enter into body fluids such as
blood or urine. The detection of these compounds in body
fluids therefore indicates the degradation of
extracellular collagen as occurs for example in
osteoporosis and as a result of tumours of bone tissue.
Monoclonal antibodies were described in W0 89/12824 for
the detection of hydroxylysyl or lysylpyridinolines with
peptide side-chains which were obtained by immunization
with appropriately cross-linked collagen fragments which
can be isolated from urine. In the method described in
W0 91/08478, collagen is also detected by means of an
antibody against natural i.e. cross-linked degradation
products of collagen produced in vivo.
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 be obtained for example 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
binding to a carrier molecule is necessary in order to
obtain an antigen with an immunogenic effect. The
structure of the epitope should not be changed in this
process by the binding to the carrier molecule. Hence
the 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
2l39592
-- 3
R.H. Burdon and P.H. van Knippenberg, Elsevier,
Amsterdam, New York, Oxford 1988, pages 95-100).
A problem in the chemical synthesis of a defined antigen
that corresponds to a natural degradation product of
cross-linked collagen is that it has not been previously
possible to chemically synthesize the hydroxylysyl or
lysylpyridinoline structure resulting from the cross-
linking.
The object of the invention was therefore to provide a
defined antigen for the production of antibodies against
collagen or collagen fragments for use as the specific
binding partner of the antibody against collagen or
collagen fragments in a competitive immunoassay and as a
standard material to construct a standard or calibration
curve in a competitive immunoassay for the detection of
collagen or collagen fragments.
Hitherto it has always been assumed that for the
detection of collagen or collagen degradation products
in a sample it is necessary to detect the cross-linkage
structures as such or so-called cross-linked peptides
which are formed by the cross-linking of hydroxylysyl or
lysyl residues since this hydroxylsyl pyridinoline or
lysylpyridinoline structure is characteristic for
collagen. Examples of such detection methods are
described in WO 89/12824, WO 91/08478, WO 89/04491 and
WO 91/10141.
It was now surprisingly found that the use of a defined
antigen, a binding partner or a standard material which
contains a synthetic linear peptide that corresponds to
a sequence of the non-helical linear C- or N-terminal
2139592
-- 4
region of collagen is adequate to achieve the above-
mentioned object. The advantages of using synthetic
linear peptides as binding partners in immunoassays, as
a standard material or as an immunogen for 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 exhibits a lower susceptibility 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 which contains a synthetic linear
peptide which corresponds to a sequence of the non-
helical linear C- or N-terminal region of collagen is
incubated with an antibody which is capable of binding
the synthetic linear peptide and with the sample and the
binding of the antibody to the binding partner is
determined in a suitable manner.
The invention furthermore concerns a standard material
for contructing a standard or calibration curve in a
competitive immunoassay for the detection of collagen or
collagen fragments which is characterized in that it
contains an antigen which contains a synthetic peptide
which corresponds to a sequence of the non-helical C- or
N-terminal region of collagen.
The invention in addition also concerns an antigen for
the production of antibodies against collagen or
collagen fragments which contains a synthetic linear
peptide which corresponds to a sequence of the non-
2139592
-- 5 --
helical C- or N-terminal region of collagen and it
concerns antibodies produced using this antigen.
All continuous amino acid sequences of the non-helical
C- or N-terminal region of collagen are suitable as the
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, particularly preferably 8 to 20 amino
acids are preferably used. In this case it is not
necessary that the sequence includes the region of the
cross-linking. It can, however, indeed also overlap this
region. However, in no case is there a hydroxylysyl
pyridinoline or lysylpyridinoline cross-linkage in the
synthetic peptide. The synthetic peptides from the C-
terminal region of collagen have proven to be most
suitable since the non-helical C-terminal region is
larger than the non-helical N-terminal region of
collagen. Thus in this region more potential epitopes
are available than in the N-terminal region. Peptides
with the sequences shown in SEQ ID NO 1, 2 or 3 from the
C-terminal region of the al chain of collagen are
particularly suitable.
The concentration of collagen degradation products is an
important diagnostic marker for the extent of an
osteolysis. with the aid of the synthetic linear
peptides it is possible to perform a competitive
immunoassay 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 against these collagen fragments
2l39s92
-- 6 --
and thus enable a competitive test. Such antibodies
against 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, also be
produced according to the invention by means of 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
may be coupled to a second component. The coupling to
the second component is preferably achieved via the N-
and C-terminal amino acids of the linear peptide. If
necessary a spacer can additionally be inserted between
the peptide and the second component. The second
component can for example serve to indirectly couple the
peptide 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 covalently bound to
biotin. Attachment to the solid phase is then achieved
by means of binding to avidin or streptavidin which has
in turn 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 coupled for example to albumin, immuno-globulin,
~-galactosidase, polymers such as polylysine or dextran
molecules as described in EP-A-O 545 350 or to particles
such as latex. Preferably 30 to 40 peptide molecules are
coupled to each 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
2139592
person skilled in the art.
In the test procedure the antibody can be incubated
simultaneously or sequentially with the sample and the
binding partner which contains the synthetic linear
peptide. Subsequently the amount of bound or unbound
antibodies is determined in the usual manner.
Agglutination tests such as TINIA, or FPIA
(fluorescence-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 techniques (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 antibodies.
The peptides according to the invention have proven to
be particularly suitable for use as defined binding
partners to compete with the sample for binding to the
antibodies. The synthetic linear peptides having the
sequences shown in SEQ ID N0 1, 2 or 3 are particularly
preferred.
After determining the extent of antibody binding to the
binding partner which represents a measure of the amount
of antigen in the sample, the exact amount of antigen in
the sample can be determined in the usual manner by
comparison with a standard treated in the same manner.
Collagen degradation products isolated from natural
material can be used as a standard. However, these are
naturally characterized by a certain variation. An
antigen which contains the synthetic linear peptide
according to the invention has proven to be more
suitable as a standard material. The antigen of this
2139592
.
-- 8
standard can merely be composed of this peptide or be
composed 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 of peptide and carrier, the linear peptide
which corresponds to a sequence of the non-helical C- or
N-terminal region of collagen is synthesized and bound
to the carrier molecule via its N- or C-terminal amino
acid by means of suitable coupling methods. One or
several peptides can be bound per carrier molecule. If
necessary the coupling can be achieved via a spacer. For
certain purposes such as agglutination tests, it may be
advantageous to bind several peptides according to the
invention of different sequences to a carrier molecule
especially if polyclonal antibodies are used in the test
which have not been produced with the aid of the antigen
according to the invention and thus usually recognize
several epitopes.
The already known antibodies against collagen
degradation products can be used as antibodies in the
competitive immunoassay. Antibodies are also suitable
which have been obtained with the aid of an antigen
which contains the linear synthetic peptide according to
the invention.
For the immunization the linear synthetic peptides which
correspond to one or several sequences of the non-
hellcal c- or N-terminal region of collagen have to be
bound to a suitable carrier protein such as for example
keyhole limpet hemocyanin, bovine serum albumin or
edestin.
In order to produce these antigens or immunogens the
X139592
linear peptides are firstly synthesized chemically in
the usual manner. Subsequently the synthetic peptides
are coupled via the N-terminal amino group to the
aforementioned carrier proteins by means of maleinimido-
hexanoic acid-N-hydroxysuccinimide ester. It has
surprisingly turned out that synthetic linear peptides
having the sequences shown in SEQ ID NO 1, 2 or 3 are
particularly suitable for the production of antibodies
which are suitable for a competitive test procedure.
Using the antigens according to the invention which
contain a synthetic linear peptide which corresponds to
a sequence of the non-helical C- or N-terminal region of
collagen, it is possible to obtain antibodies which not
only recognize the peptide according to the invention
but also the degradation products of collagen that occur
in body fluids.
The invention therefore also concerns a process for the
production of antibodies against 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 according to known
methods. The desired antibody is preferably isolated by
immunoadsorption to a peptide having the sequence shown
in SEQ ID NO 1, 2 or 3 coupled to a carrier protein,
preferably Sepharose.
A preferred subject matter of the invention is a process
for the production of monoclonal antibodies against
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
2139592
-
-- 10 --
antibody and isolating the antibody from the cloned
cells or from the culture supernatant of these cells.
The immunization is carried out in animals which are
usually used for this; mice or rabbits are preferably
used.
The spleen cells of the immunized animals are
immortalized by methods familiar to a person skilled in
the art such as e.g. by 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 ~ ined whether an
antibody binds to this antigen.
The invention in addition concerns the polyclonal and
monoclonal antibodies obtainable by the process
according to the invention.
These polyclonal and monoclonal antibodies do 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.
The antibodies according to the invention can therefore
be used in the test procedures described above for the
determination of collagen or collagen fragments.
2139592
-- 11 --
The invention therefore in addition 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
the antibody.
The invention is elucidated in more detail by the
following examples in conjunction with the sequence
protocols.
EQ ID N0 1 shows the sequence of a peptide according
to the invention comprising 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 comprising 16 amino
acids.
EQ ID NO 3 shows the sequence of a peptide according
to the invention comprising 10 amino
acids.
xample
eptide syntheses
Peptides having a partial sequence of the amino acid
sequence of collagen with the sequences shown in the
sequence protocols SEQ ID NO 2 and 3 are prepared by
means of fluorenylmethyloxycarbonyl(Fmoc) solid phase
peptide synthesis on a a) Labortec SP 640 peptide
2139592
.
- 12 -
synthesizer and 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-amide (SEQ ID N0 2)
4.0 equivalents of each of the following Fmoc-amino
acid derivatives are used in the stated order:
Lys with tert. butyloxycarbonyl protecting group
Glu with tert. butyl ester protecting group
Gln withoùt side-chain protecting group
Pro without side-chain protecting group
Pro without side-chain protecting group
Gln without side-chain protecting group
Pro without side-chain protecting group
Leu without side-chain protecting group
Phe without side-chain protecting group
Ser with tert. butyl ether protecting group
Phe without side-chain protecting group
Asp with tert. butyl ester protecting group
Phe without side-chain protecting group
Gly without side-chain protecting group
Ala without side-chain protecting group
Ser with tert. butyl ether protecting group
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) at a loading of 0.87 mmol/g
(JACS 95 (1973), 1328). The coupling reactions are
2139592
~.
- 13 -
carried out for 60 minutes with 4.4 equivalents
dicyclohexylcarbodiimide and 4.8 equivalents
N-hydroxybenzotriazol in dimethylformamide as a
reaction medium in relation to the Fmoc-amino acid
derivative. The synthesis resin washed with isopropanol
is monitored for coupling yield by means of the Kaiser
test (Anal. Biochem. 34 (1970), 595). If this shows
that the conversion is not yet complete, the conversion
is completed by re-coupling under the conditions stated
above. After each step in the synthesis the Fmoc group
is cleaved off within 20 minutes by means of 20 %
piperidine in dimethylformamide. The loading of the
resin is determined after each piperidine treatment by
means of W absorbance of the released fulvene group.
After the synthesis the loading is still 0.68 mmol/g.
Release of the peptide from the synthesis resin and
cleavage of the acid-labile protecting groups is
carried out 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 - 2 hours and then filtered.
The material is then preliminarily 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
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 % trifluoro-acetic acid) to 100 %
buffer B (60 % acetonitrile, 40 % water, 0.1 %
trifluoroacetic acid) within 120 minutes. The identity
of the eluted material is examined by means of fast
2139592
- 14 -
atom bombardment mass spectrometry (FAB-MS).
b) Production of Ala-Gly-Phe-A~p-Phe-Ser-Phe-Leu-Pro-Gln
(SEQ ID NO 3)
The peptide was prepared on 30 mg 4-(2',4'-dimethoxy-
phenyl-Fmoc-aminomethyl)phenoxy resin SA 5030 from the
Advanced Chemtech Company using a loading of
0.47 mmol/g. Each of the following Fmoc amino acid
derivatives were coupled twice to the peptide to be
synthesized which is bound to a solid phase using each
time 140 ~mol of the amino acid derivative together
with 140 ~mol l-hydroxybenzotriazol in dimethyl-
formamide DMF and 10 ~mol N,N-diisopropylcarbodiimide
in DMF:
Glu with trityl protecting group
Ser with tert. butyl protecting group
Asp with tert. butyl protecting group
Pro
Leu
Phe > each without a side-chain protecting group
Gly
Ala
The coupling periods were 30 and 40 minutes. The
cleavage period 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
with DMF. The peptide was released by treating the
resin, from which solvent had been removed by filtration
and which was washed with dichloromethane and methanol,
2139592
- 15 -
with 1 ml of a solution of 90 % trifluoroacetic acid,
3 % thioanisol, 3 % ethanedithiol 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 of a white lyophilisate of a purity of
79 % according to HPLC was obtained. The indentity was
confirmed by mass FAB-spectroscopy.
Example 2
Activation of peptides
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 with
glacial acetic acid to pH 4 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).
2139592
- 16 -
Example 3
Production of immunogens by coupling activated peptides
to carrier proteins
The coupling of activated peptides to keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) and ~-
galactosidase (~Gal) is described. In order to couple
the peptides activated with MHS according to example 2,
it is necessary that the carrier protein carries free SH
groups. ~Gal already has these in the natural form and
therefore requires no further pre-treatment. 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.
Thus 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 re-
adjusted with 0.1 mol/l sodium hydroxide solution to pH
8.5 and stirred for a further 24 hours. The solution is
subsequently concentrated to 100 ml with the aid of an
Amicon cell (membrane YM10), dialyzed for 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
~ 2139592
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 from 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 dialyzed twice against 1 1
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.
Example 4
Production of polyclonal antibodie~ against linear
collagen fragments
5 sheep were immunized in each case 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 amino acids No. 892 to 907
in the sequence of the a-chain of collagen type I. KLH
or B-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
collected four months after the first immunization and
the antibodies obtained were tested for reaction with
collagen fragments.
2139~92
- 18 -
ELISA for testing 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
Substrate solution: ABTS~, Boehringer Mannheim GmbH,
Catalogue No. 857424.
2 mg/ml vanillin was added to the
solution to amplify the signal.
Washing solution: 0.1 % Tween 20
0.9 % NaCl
The wells of the titre plates were each filled with
100 ~l of a solution that contained 10 ~g/ml collagen
fragments in coating buffer. The collagen fragments
were produced from human collagen from bones by protease
digestion according to the instructions in
EP-A-0 505 210. After one hour incubation at room
temperature while shaking it was washed three times with
washing solution.
2139592
.
-- 19 --
The antisera were diluted 1 : 4000 with incubation
buffer and 100 ~1 of each was incubated for 1 hour while
shaking at room temperature in the wells of the
microtitre plate. The wells were subsequently washed
three times with washing solution.
A conjugate of horseradish peroxidase and rabbit
antibodies against the Fc part of sheep IgG is diluted
in incubation buffer to a concentration of 12.5 mU/ml
and the wells of the microtitre plate are each coated
with 100 ~1 thereof. After one hour incubation while
shaking at room temperature, the titre plates are washed
three times with washing solution.
100 ~1 substrate solution is added and incubated until a
colour development becomes visible (10 - 60 minutes).
The absorbance is recorded as a differential measurement
at 405 and 492 nm.
The sera of most of the animals showed a strong reaction
with the collagen fragments on the solid phase. The
serum of a non-immunized animal only showed a weak
measurement signal under the same conditions. The
results are shown in Table 1.
2139592
- 20 -
Table
~-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
Example 5
Determination of collagen and its degradation products
in body fluids by means of a competitive te~t
The wells of a 96-well microtitre plate are coated at
4C overnight with streptavidin (100 ~1 of a solution of
1 ~g/ml in PBS) according to EP-A 0 344 578 and
unspecific binding sites which are still free are
blocked by incubation with 300 ~1 BSA (bovine serum
albumin, 10 mg/ml) for 2 hours at room temperature.
The decapeptide having the sequence shown in SEQ ID NO 3
which was synthesized according to example lb) is
biotinylated at the amino terminus using D-biotinyl-~-
amidocaproic acid-N-succinimide ester (Boehringer
Mannheim, Catalogue No. 1008960) according to the
instructions of the manufacturer. The biotinylated
peptide is dissolved in PBS, 0.05 % Tween 20, 1 % BSA at
a concentration of 10 ng/ml and bound to the
2l39s92
- 21 -
streptavidin-coated microtitre plate by a 1 hour
incubation of 100 ~l per well. Subsequently unbound
peptide is removed by washing three times with PBS,
0.0S % Tween 20.
In each case 150 ~l of the sample to be examined (serum,
plasma or a standard) is incubated for 2 hours at 37OC
(or overnight at 4C) according to example 4 with 150 ~l
of the antibody according to the invention. 100 ~l of
this mixture is added in each case to the bound
decapeptide in the wells of the microtitre plate and
incubated for 60 minutes at 37C. In this process only
the excess antibody of the antiserum which is not yet
bound after incubation with the sample can bind to the
immobilized decapeptide.
After washing three times with PBS/0.05 % Tween 20,
bound antibody is detected by subsequent incubation with
an anti-rabbit-IgG-POD conjugate (Boehringer Mannheim
GmbH, Catalogue No. 1238 850) and ABTS~ (1 mg/ml).
164 patient sera were measured using the test according
to the invention (MTP competitive test). The results
were compared to data that were determined using a
radioimmunoassay (RIA). This RIA ICTP (telopeptide ICTP
[125I] from Orion Diagnostica, Finland) is based on
cross-linked collagen fragments which are produced and
isolated by enzymatic digestion and biochemical methods.
It is now apparent from Figure 1 that the method
according to the invention yields measured values which
correlate well with the RIA values, which means that the
method according to the invention generates clinically
relevant data. A correlation coefficient of 0.959 was
determined.
2139592
~1 (a)
-- ,2~S --
SEQUENCE PROTOCOL
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) 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
(D) 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
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) 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
