CA2139592A1 - Immunoassay for the detection of collagen or collagen fragments - Google Patents
Immunoassay for the detection of collagen or collagen fragmentsInfo
- Publication number
- CA2139592A1 CA2139592A1 CA002139592A CA2139592A CA2139592A1 CA 2139592 A1 CA2139592 A1 CA 2139592A1 CA 002139592 A CA002139592 A CA 002139592A CA 2139592 A CA2139592 A CA 2139592A CA 2139592 A1 CA2139592 A1 CA 2139592A1
- Authority
- CA
- Canada
- Prior art keywords
- collagen
- sequence
- peptide
- antigen
- linear peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 84
- 108010035532 Collagen Proteins 0.000 title claims abstract description 84
- 229920001436 collagen Polymers 0.000 title claims abstract description 84
- 238000001514 detection method Methods 0.000 title claims abstract description 14
- 239000012634 fragment Substances 0.000 title claims description 26
- 238000003018 immunoassay Methods 0.000 title description 8
- 239000000427 antigen Substances 0.000 claims abstract description 35
- 102000036639 antigens Human genes 0.000 claims abstract description 35
- 108091007433 antigens Proteins 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 claims abstract description 18
- 230000003053 immunization Effects 0.000 claims abstract description 13
- 238000002649 immunization Methods 0.000 claims abstract description 13
- 230000008569 process Effects 0.000 claims abstract description 10
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- 230000027455 binding Effects 0.000 claims description 22
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- 239000010421 standard material Substances 0.000 claims description 8
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- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 4
- 125000006850 spacer group Chemical group 0.000 claims description 4
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- 125000006239 protecting group Chemical group 0.000 description 13
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- 238000006243 chemical reaction Methods 0.000 description 8
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- 239000011616 biotin Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
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Abstract
The invention concerns antigens for the production of antibodies 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.
Description
.~. 2I39592 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 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 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-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 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 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 .
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 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 -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.
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 .
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 ~.
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 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, 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).
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
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 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 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-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 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 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 .
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 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 -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.
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 .
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 ~.
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 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, 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).
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 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.
.
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.
Table ~-galactosidase KLH
Animal No. Absorbance Animal No. Absorbance 1 1.05 1 1.16 2 1.18 2 2.62
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 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.
.
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.
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 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.
~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 (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 (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
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 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.
~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 (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 (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
Claims (11)
1. Competitive immunoassay for the detection of collagen or collagen fragments in a sample, wherein a binding partner which contains a synthetic linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen is incubated with an antibody that is 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.
2. Method as claimed in claim 1, wherein the synthetic linear peptide corresponds to a sequence of the non-helical C-terminal region of collagen.
3. Method as claimed in one of the claims 1 and 2, wherein the synthetic linear peptide is composed of 5 to 25 amino acids and preferably of 8 to 20 amino acids.
4. Method as claimed in one of the claims 1 to 3, wherein the synthetic peptide corresponds to the sequence shown in SEQ ID NO 1, 2 or 3.
5. Standard material for constructing a standard or calibration curve in a competitive immunoassay for the detection of collagen or collagen fragments, wherein it contains an antigen which contains a synthetic linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen.
6. Process for the production of the standard material as claimed in claim 5, wherein a linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen is synthesized and coupled via a spacer to a suitable carrier molecule via its N- or C-terminal amino acid.
7. Antigen for the production of antibodies against collagen or collagen fragments, wherein it contains a synthetic linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen.
8. Antigen as claimed in claim 7, wherein the synthetic linear peptide is coupled to a suitable carrier protein.
9. Process for the production of the antigen as claimed in claim 7, wherein a linear peptide that corresponds to a sequence of the non-helical C- or N-terminal region of collagen is synthesized and coupled by means of its N- or C-terminal amino acid to a carrier protein if desired via a spacer.
10. Process for the production of antibodies against collagen or collagen fragments, wherein an antigen as claimed in claim 7 or 8 is used for the immunization.
11. Antibodies against collagen or collagen fragments obtainable by immunization with an antigen as claimed in claim 6 or 7 and isolation of the desired antibody from the serum of the immunized animals or by immortalizing the spleen cells of the immunized animals, cloning those immortalized spleen cells which produce the desired antibody and isolating the antibody from the cloned cells or from their culture supernatant.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4225038.2 | 1992-07-29 | ||
| DE4225038A DE4225038C2 (en) | 1992-07-29 | 1992-07-29 | Production and use of antibodies against collagen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2139592A1 true CA2139592A1 (en) | 1994-02-17 |
Family
ID=6464371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002139592A Abandoned CA2139592A1 (en) | 1992-07-29 | 1993-07-28 | Immunoassay for the detection of collagen or collagen fragments |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0659276B2 (en) |
| JP (1) | JP3360826B2 (en) |
| AT (1) | ATE162311T1 (en) |
| CA (1) | CA2139592A1 (en) |
| DE (2) | DE4225038C2 (en) |
| DK (1) | DK0659276T3 (en) |
| ES (1) | ES2114064T5 (en) |
| FI (1) | FI950378A (en) |
| NO (2) | NO950325D0 (en) |
| WO (1) | WO1994003813A1 (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0711415B1 (en) * | 1993-07-28 | 1998-07-22 | Boehringer Mannheim Gmbh | Immunoassay for detecting collagen type i or collagen fragments thereof |
| DK104093D0 (en) * | 1993-09-17 | 1993-09-17 | Osteometer A S | PROCEDURE FOR DETERMINING COLLAGEN FRAGMENTS IN BODY LIQUIDS, TEST KITS AND MEANS FOR EXERCISING THE PROCEDURE AND USING THE PROCEDURE FOR DIAGNOSTICING THE DISEASES OF TABLET METAL |
| GB9506050D0 (en) * | 1995-03-24 | 1995-05-10 | Osteometer A S | Assaying collagen fragments in body fluids |
| DE69520111T2 (en) | 1994-10-17 | 2001-09-06 | Osteometer Biotech As Herlev | Estimation of the fragmentation pattern of collagen in body fluids and diagnosis of disorders related to collagen metabolism |
| DE59505585D1 (en) * | 1994-12-23 | 1999-05-12 | Roche Diagnostics Gmbh | Antigens and antibodies for the detection of collagen I |
| US5750647A (en) * | 1995-05-19 | 1998-05-12 | Washington Research Foundation | Synthetic peptide analogs of NTx |
| US6107047A (en) * | 1996-03-21 | 2000-08-22 | Osteometer Biotech A/S | Assaying protein fragments in body fluids |
| GB9617616D0 (en) | 1996-08-22 | 1996-10-02 | Osteometer Biotech As | Assaying protein fragments in body fluids |
| WO1998026286A2 (en) | 1996-12-09 | 1998-06-18 | Osteometer Biotech A/S | Sandwich assays for collagen type i fragments |
| US6117646A (en) * | 1997-09-22 | 2000-09-12 | Osteometer Biotech A/S | Assaying protein fragments in body fluids |
| US6255056B1 (en) | 1998-06-19 | 2001-07-03 | Washington Research Foundation | Cartilage resorption assays |
| US6916903B2 (en) | 1998-06-19 | 2005-07-12 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
| US6348320B1 (en) | 1998-06-19 | 2002-02-19 | Washington Research Foundation | Cartilage resorption assays measuring type II collagen fragments |
| US6602980B1 (en) | 1998-06-19 | 2003-08-05 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
| CN101854957B (en) * | 2007-09-10 | 2013-05-22 | 通用电气健康护理有限公司 | Radiofluorination methods |
| GB201514658D0 (en) * | 2015-08-18 | 2015-09-30 | Nordic Bioscience As | Immunoassay for collagen type VIII sequences |
| CN113866406B (en) * | 2021-10-18 | 2022-09-27 | 深圳上泰生物工程有限公司 | Kit for specifically detecting sugar-deficient transferrin |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3714634A1 (en) * | 1987-05-02 | 1988-11-17 | Hoechst Ag | METHOD FOR SELECTIVE IMMUNOLOGICAL DETERMINATION OF INTACT PROCOLLAGEN PEPTIDE (TYPE III) AND PROCOLLAGEN (TYPE III) IN BODY LIQUIDS AND MEANS TO IMPLEMENT IT |
| DE58908787D1 (en) * | 1988-04-27 | 1995-02-02 | Hoechst Ag | Monoclonal antibody for the selective immunological determination of intact procollagen peptide (type III) and procollagen (type III) in body fluids. |
| WO1990008195A1 (en) * | 1989-01-13 | 1990-07-26 | President And Fellows Of Harvard College | Monoclonal antibody to human type ix collagen |
| US5081031A (en) * | 1989-12-14 | 1992-01-14 | Regents Of The University Of Minnesota | Synthetic polypeptide with type iv collagen activity |
| GB9014220D0 (en) * | 1990-06-26 | 1990-08-15 | Farmos Yhtymy Oy | Method for the immunlolgical determinition of the carboxyterminal propeptide of type i procollagen |
| GB9105893D0 (en) * | 1991-03-20 | 1991-05-08 | Orion Yhtymae Oy | Bone resorption assay based on a peptide liberated during collagen degradation |
| JPH07504896A (en) * | 1992-01-31 | 1995-06-01 | ベイリンク，デビッド ジェイ． | Aminoprocollagen 1 (1) peptide |
-
1992
- 1992-07-29 DE DE4225038A patent/DE4225038C2/en not_active Expired - Fee Related
-
1993
- 1993-07-28 CA CA002139592A patent/CA2139592A1/en not_active Abandoned
- 1993-07-28 DK DK93917678T patent/DK0659276T3/en active
- 1993-07-28 WO PCT/EP1993/002010 patent/WO1994003813A1/en active IP Right Grant
- 1993-07-28 ES ES93917678T patent/ES2114064T5/en not_active Expired - Lifetime
- 1993-07-28 JP JP50497794A patent/JP3360826B2/en not_active Expired - Fee Related
- 1993-07-28 EP EP93917678A patent/EP0659276B2/en not_active Expired - Lifetime
- 1993-07-28 AT AT93917678T patent/ATE162311T1/en not_active IP Right Cessation
- 1993-07-28 DE DE59308000T patent/DE59308000D1/en not_active Expired - Lifetime
-
1995
- 1995-01-27 NO NO950325A patent/NO950325D0/en not_active Application Discontinuation
- 1995-01-27 FI FI950378A patent/FI950378A/en unknown
-
1996
- 1996-01-26 NO NO960349A patent/NO960349D0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| ATE162311T1 (en) | 1998-01-15 |
| EP0659276A1 (en) | 1995-06-28 |
| JPH07509778A (en) | 1995-10-26 |
| DK0659276T3 (en) | 1998-09-14 |
| DE4225038A1 (en) | 1994-02-03 |
| FI950378A0 (en) | 1995-01-27 |
| JP3360826B2 (en) | 2003-01-07 |
| NO960349L (en) | 1996-01-26 |
| EP0659276B2 (en) | 2008-07-30 |
| NO960349D0 (en) | 1996-01-26 |
| DE59308000D1 (en) | 1998-02-19 |
| DE4225038C2 (en) | 1995-11-30 |
| NO950325L (en) | 1995-01-27 |
| ES2114064T3 (en) | 1998-05-16 |
| WO1994003813A1 (en) | 1994-02-17 |
| ES2114064T5 (en) | 2008-11-16 |
| FI950378A (en) | 1995-01-27 |
| NO950325D0 (en) | 1995-01-27 |
| EP0659276B1 (en) | 1998-01-14 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |