CA2167496A1 - Immunoassay for the detection of collagen or collagen fragments - Google Patents
Immunoassay for the detection of collagen or collagen fragmentsInfo
- Publication number
- CA2167496A1 CA2167496A1 CA002167496A CA2167496A CA2167496A1 CA 2167496 A1 CA2167496 A1 CA 2167496A1 CA 002167496 A CA002167496 A CA 002167496A CA 2167496 A CA2167496 A CA 2167496A CA 2167496 A1 CA2167496 A1 CA 2167496A1
- Authority
- CA
- Canada
- Prior art keywords
- collagen
- peptide
- antibody
- sample
- sequence
- 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
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Classifications
-
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- G01—MEASURING; TESTING
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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Abstract
The invention concerns an immunoassay for the detection of collagen or collagen fragments in a sample using an antibody which recognizes a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen wherein the sample is preferably denatured.
Description
-21~749B
Imm~no~ y for the detection of collagen or collagen fragments The invention concerns an immunoassay for the detection of collagen or collagen fragments in a sample using at least one antibody which recognizes a synthetic linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen in which the sample is preferably denatured.
Collagen is an important structural protein in the connective tissue of the skin, cartilage and bone. 11 types are known which are each composed of three chains.
Each type is composed of 1 - 3 different chains which are denoted al, a2 and a3 (E. Miller et al., in Methods in Enzymology 144, Structural and Contractile Proteins, ed. L. Cunningham, Academic Press Inc. 1987, p. 3 - 41).
A characteristic property of the mature collagen of certain tissues such as bone or cartilage in particular, is the cross-linking of adjacent fibres by hydroxylysyl-pyridinoline or lysylpyridinoline (D. Fujimoto et al., J. Biochem 83 (1978), 863 - 867; D. Eyre et al., Ann.
Rev. Biochem 53 (1984), 717 - 748 and D. Eyre, Methods in Enzymology 144 (1987), 115 - 139). These cross-links can be utilized as chemical markers for the specific detection of collagen (Z. Gunja-Smith et al., Biochem.
J. 197 (1981), 759 - 762). When extracellular collagen is degraded, hydroxylysylpyridinoline or lysyl-pyridinoline derivatives which contain peptide side chains or free pyridinoline derivatives with lysyl or hydroxylysyl residues as described in WO 91/10141 pass into body fluids such as blood or urine. The detection -- 21674gB
of these compounds in body fluids is therefore an indicator for the degradation of extracellular collagen such as that which for example occurs in osteoporosis and as a result of bone tissue tumours. Monoclonal antibodies which were obtained by immunization with appropriately cross-linked collagen fragments which can be isolated from the urine were described in WO 89/12824 for the detection of such hydroxylysylpyridinolines or lysylpyridinolines with peptide side chains. Also in the method described in WO 91/08478 the collagen test is by means of an antibody to natural, i.e. generated in vivo, cross-linked degradation products of collagen.
A disadvantage of these peptides isolated from natural sources is that there is no reliable source for a reproducible production of the antigens or binding partners in the test. A further disadvantage of the peptides isolated from natural sources is the risk of contamination with infectious material.
Defined antigens can for example be obtained by chemical synthesis of a peptide which corresponds to an epitope of the antigen. If small peptides with a molecular weight of about 700 - 1500 D are used for this, then it is necessary to bind them to a carrier molecule in order to obtain an antigen with an immunogenic effect. In this process the structure of the epitope must not be changed by binding to the carrier molecule. Therefore coupling to the carrier molecule has previously been carried out at the ends of the peptide chain at an adequate distance from the presumed epitope region (Laboratory Technics in Biochemistry and Molecular Biology, Synthetic Polypeptides as Antigens, Editors R.H. Burdon and P.H.
van Knippenberg, Elsevier, Amsterdam, New York, Oxford 1988, pages 95 - 100).
2167~g6 A problem in the chemical synthesis of a defined antigen that corresponds to a natural degradation product of cross-linked collagen is the hydroxylysylpyridinoline or lysylpyridinoline structure resulting from the cross-linking the chemical synthesis of which is very complicated.
The object of the invention was therefore to provide a defined antigen for the production of antibodies to collagen or collagen fragments, for use as a specific binding partner of the antibody to collagen or collagen fragments in a competitive immunoassay and as a standard material for establishing a standard or calibration curve in a competitive immunoassay for the detection of collagen or collagen fragments.
Previously it has always been assumed that in order to detect collagen or collagen degradation products in a sample it is necessary to detect the cross-linked structures per se or so-called cross-linked peptides which result from the cross-linking of hydroxylysyl or lysyl residues since this hydroxylysylpyridinoline or lysylpyridinoline structure is characteristic for collagen. Examples of such methods of detection are described in WO 89/12824, WO 91/08478, WO 89/04491 and W0 91/10141.
It has now been surprisingly found that the use of a defined antigen, a binding partner or a standard material containing a synthetic linear peptide which corresponds to a sequence of the non-helical linear C-terminal or N-terminal region of collagen is sufficient for achieving the aforementioned object. The advantages of using synthetic linear peptides as binding 2iS7~9~
partners in immunoassays, as a standard material or as an immunogen in the antibody production are that these peptides, in contrast to peptides from natural sources, can be produced reproducibly with an exactly defined structure. Moreover an immunoassay in which such short synthetic peptides are used is less susceptible to interference.
The invention therefore concerns a competitive immunoassay for the detection of collagen or collagen fragments in a sample which is characterized in that a binding partner containing a synthetic linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen is incubated with an antibody capable of binding the synthetic linear peptide and the sample, and the binding of the antibody to the binding partner is determined in a suitable manner.
It has proven to be particularly advantageous to denature the sample or rather the collagen or collagen fragments present in this sample and thus improve the accessibility of the epitopes to the antibody binding.
The most suitable method is to pretreat the sample before incubation with the antibody with a denaturing agent for proteins which are known to a person skilled in the art. In order to limit or completely avoid interference of the immunological reaction between the sample and the specific antibodies, the denatured sample is preferably additionally diluted before incubation with the antibody. All agents known to a person skilled in the art for this purpose are suitable as denaturing agents. Potassium thiocyanate (KSCN) at a concentration of 2 - 6 M and tetradecyltriethylammonium bromide (TTAB) at a concentration of 0.5 - 2 M have proven to be 21fi74g~
particularly suitable for the denaturation.
The introduction of a denaturation step of the sample enables antibodies which only bind negligibly to non-denatured samples to be used in addition i.e. they bind a denatured sample very well. Therefore as a result of the introduction of this denaturing step more antibodies are available for a diagnostic test for collagen or collagen fragments.
The invention in addition concerns a standard material for establishing a standard or calibration curve in a competitive immunoassay for the detection of collagen or collagen fragments characterized in that it contains an antigen that contains a synthetic peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen.
The invention in addition likewise concerns an antigen for the production of antibodies to collagen or collagen fragments which contains a synthetic linear peptide corresponding to a sequence of the non-helical C-terminal or N-terminal region of collagen and the antibodies produced using this antigen.
All consecutive amino acid sequences of the non-helical C-terminal or N-terminal region of collagen are suitable as synthetic linear peptides. These regions are known from Chu et al., Nature 310, 337-340 (1984), Click et al., Biochemistry 9, 4699-4706 (1970), Morgan et al., J.
Biol. Chem. 245, 5042-5048 (1970) and Bernard et al., Biochemistry 22, 5213-5223 (1983). Peptides comprising 5 to 25 amino acids are preferably used and especially those comprising 8 to 20 amino acids. In this connection ~167~96 it is not necessary that the sequence comprises the cross-linking region. However, it can indeed also overlap this region. However, in no case is a hydroxylysylpyridinoline or lysylpyridinoline cross-link present in the synthetic peptide. Synthetic peptides from the C-terminal region of collagen have proven to be particularly suitable since the non-helical C-terminal region is larger than the non-helical N-terminal region of collagen. Thus more potential epitopes are available in this region than in the N-terminal region. Peptides having the sequence shown in SEQ ID NO. 1, 2, 3 or 4 from the C-terminal region of the al chain of collagen are particularly suitable.
The concentration of degradation products of collagen is an important diagnostic marker for the extent of osteolysis. The synthetic linear peptides enable a competitive immunoassay to be carried out for the detection of collagen or collagen fragments. It has surprisingly turned out that these peptides compete very well with the collagen fragments that occur in natural samples such as plasma, serum or urine for antibodies to these collagen fragments and thus enable a competitive test. Such antibodies to collagen or collagen degradation products are commercially available for example in the telopeptide ICTP [125I] radioimmunoassay kit from the Orion Diagnostica Company Finland. They can, however, be preferably produced according to the invention using the synthetic linear peptide.
For an application in a competitive immunoassay, the synthetic linear peptide can be used directly as a binding partner which is bound to a solid phase or it can be coupled to a second component. Coupling to the second component is preferably achieved via the ~ ~ ~ 7 4 ~ ~
N-terminal and C-terminal amino acids of the linear peptide. Optionally a spacer can in addition be inserted between the peptide and the second component. The second component can for example serve for coupling the peptide indirectly to a solid phase. Examples of this are known to a person skilled in the art. The peptide is preferably coupled to bovine serum albumin and the coupling product is adsorptively bound to a solid phase such as a plastic tube. The peptide can also be bound covalently to biotin. It is then attached to the solid phase by means of binding to avidin or streptavidin which itself has been bound to the solid phase. The second component can also serve as a carrier for several peptides for example in a competitive turbidimetric inhibition immunoassay (TINIA) in which several peptides are for example coupled to albumin, immunoglobulin, B-galactosidase, to polymers such as polylysine or to dextran molecules such as those described in EP-A-O 545 350 or to particles such as latex. Preferably 30 to 40 peptide molecules are coupled per carrier molecule. The peptide can also be coupled to a component which represents a label. Examples of all these test variants are known to a person skilled in the art.
In the test procedure the antibody can be incubated simultaneously or sequentially with the sample and with the binding partner which contains the synthetic linear peptide. Subsequently the amount of bound or non-bound antibody is determined in the usual manner. For example the antibody used can itself be labelled and this label serves directly as a measure of the bound or unbound antibody. It is also possible to use a second labelled antibody which is directed towards the bound or unbound antibody such as towards the Fc part of this antibody.
Agglutination tests such as TINIA or FPIA (fluorescence polarisation immunoassay) (W. Dandliker et al., J. Exp.
Med. 122 (1965), 1029), EMIT (enzyme multiplied immunoassay) (Gunzer et al., Kontakte III (1980), 3 -11) and CEDIA techn;ques (Henderson et al., Clinical Chemistry 32 (1986), 1637 - 1641) can for example serve as competitive test variants for determining the amount of bound or unbound antibody. The peptides according to the invention have proven to be particularly suitable for use as defined binding partners which compete with the sample for binding to the antibodies. The synthetic linear peptides with the sequences shown in SEQ ID N0 1,
Imm~no~ y for the detection of collagen or collagen fragments The invention concerns an immunoassay for the detection of collagen or collagen fragments in a sample using at least one antibody which recognizes a synthetic linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen in which the sample is preferably denatured.
Collagen is an important structural protein in the connective tissue of the skin, cartilage and bone. 11 types are known which are each composed of three chains.
Each type is composed of 1 - 3 different chains which are denoted al, a2 and a3 (E. Miller et al., in Methods in Enzymology 144, Structural and Contractile Proteins, ed. L. Cunningham, Academic Press Inc. 1987, p. 3 - 41).
A characteristic property of the mature collagen of certain tissues such as bone or cartilage in particular, is the cross-linking of adjacent fibres by hydroxylysyl-pyridinoline or lysylpyridinoline (D. Fujimoto et al., J. Biochem 83 (1978), 863 - 867; D. Eyre et al., Ann.
Rev. Biochem 53 (1984), 717 - 748 and D. Eyre, Methods in Enzymology 144 (1987), 115 - 139). These cross-links can be utilized as chemical markers for the specific detection of collagen (Z. Gunja-Smith et al., Biochem.
J. 197 (1981), 759 - 762). When extracellular collagen is degraded, hydroxylysylpyridinoline or lysyl-pyridinoline derivatives which contain peptide side chains or free pyridinoline derivatives with lysyl or hydroxylysyl residues as described in WO 91/10141 pass into body fluids such as blood or urine. The detection -- 21674gB
of these compounds in body fluids is therefore an indicator for the degradation of extracellular collagen such as that which for example occurs in osteoporosis and as a result of bone tissue tumours. Monoclonal antibodies which were obtained by immunization with appropriately cross-linked collagen fragments which can be isolated from the urine were described in WO 89/12824 for the detection of such hydroxylysylpyridinolines or lysylpyridinolines with peptide side chains. Also in the method described in WO 91/08478 the collagen test is by means of an antibody to natural, i.e. generated in vivo, cross-linked degradation products of collagen.
A disadvantage of these peptides isolated from natural sources is that there is no reliable source for a reproducible production of the antigens or binding partners in the test. A further disadvantage of the peptides isolated from natural sources is the risk of contamination with infectious material.
Defined antigens can for example be obtained by chemical synthesis of a peptide which corresponds to an epitope of the antigen. If small peptides with a molecular weight of about 700 - 1500 D are used for this, then it is necessary to bind them to a carrier molecule in order to obtain an antigen with an immunogenic effect. In this process the structure of the epitope must not be changed by binding to the carrier molecule. Therefore coupling to the carrier molecule has previously been carried out at the ends of the peptide chain at an adequate distance from the presumed epitope region (Laboratory Technics in Biochemistry and Molecular Biology, Synthetic Polypeptides as Antigens, Editors R.H. Burdon and P.H.
van Knippenberg, Elsevier, Amsterdam, New York, Oxford 1988, pages 95 - 100).
2167~g6 A problem in the chemical synthesis of a defined antigen that corresponds to a natural degradation product of cross-linked collagen is the hydroxylysylpyridinoline or lysylpyridinoline structure resulting from the cross-linking the chemical synthesis of which is very complicated.
The object of the invention was therefore to provide a defined antigen for the production of antibodies to collagen or collagen fragments, for use as a specific binding partner of the antibody to collagen or collagen fragments in a competitive immunoassay and as a standard material for establishing a standard or calibration curve in a competitive immunoassay for the detection of collagen or collagen fragments.
Previously it has always been assumed that in order to detect collagen or collagen degradation products in a sample it is necessary to detect the cross-linked structures per se or so-called cross-linked peptides which result from the cross-linking of hydroxylysyl or lysyl residues since this hydroxylysylpyridinoline or lysylpyridinoline structure is characteristic for collagen. Examples of such methods of detection are described in WO 89/12824, WO 91/08478, WO 89/04491 and W0 91/10141.
It has now been surprisingly found that the use of a defined antigen, a binding partner or a standard material containing a synthetic linear peptide which corresponds to a sequence of the non-helical linear C-terminal or N-terminal region of collagen is sufficient for achieving the aforementioned object. The advantages of using synthetic linear peptides as binding 2iS7~9~
partners in immunoassays, as a standard material or as an immunogen in the antibody production are that these peptides, in contrast to peptides from natural sources, can be produced reproducibly with an exactly defined structure. Moreover an immunoassay in which such short synthetic peptides are used is less susceptible to interference.
The invention therefore concerns a competitive immunoassay for the detection of collagen or collagen fragments in a sample which is characterized in that a binding partner containing a synthetic linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen is incubated with an antibody capable of binding the synthetic linear peptide and the sample, and the binding of the antibody to the binding partner is determined in a suitable manner.
It has proven to be particularly advantageous to denature the sample or rather the collagen or collagen fragments present in this sample and thus improve the accessibility of the epitopes to the antibody binding.
The most suitable method is to pretreat the sample before incubation with the antibody with a denaturing agent for proteins which are known to a person skilled in the art. In order to limit or completely avoid interference of the immunological reaction between the sample and the specific antibodies, the denatured sample is preferably additionally diluted before incubation with the antibody. All agents known to a person skilled in the art for this purpose are suitable as denaturing agents. Potassium thiocyanate (KSCN) at a concentration of 2 - 6 M and tetradecyltriethylammonium bromide (TTAB) at a concentration of 0.5 - 2 M have proven to be 21fi74g~
particularly suitable for the denaturation.
The introduction of a denaturation step of the sample enables antibodies which only bind negligibly to non-denatured samples to be used in addition i.e. they bind a denatured sample very well. Therefore as a result of the introduction of this denaturing step more antibodies are available for a diagnostic test for collagen or collagen fragments.
The invention in addition concerns a standard material for establishing a standard or calibration curve in a competitive immunoassay for the detection of collagen or collagen fragments characterized in that it contains an antigen that contains a synthetic peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen.
The invention in addition likewise concerns an antigen for the production of antibodies to collagen or collagen fragments which contains a synthetic linear peptide corresponding to a sequence of the non-helical C-terminal or N-terminal region of collagen and the antibodies produced using this antigen.
All consecutive amino acid sequences of the non-helical C-terminal or N-terminal region of collagen are suitable as synthetic linear peptides. These regions are known from Chu et al., Nature 310, 337-340 (1984), Click et al., Biochemistry 9, 4699-4706 (1970), Morgan et al., J.
Biol. Chem. 245, 5042-5048 (1970) and Bernard et al., Biochemistry 22, 5213-5223 (1983). Peptides comprising 5 to 25 amino acids are preferably used and especially those comprising 8 to 20 amino acids. In this connection ~167~96 it is not necessary that the sequence comprises the cross-linking region. However, it can indeed also overlap this region. However, in no case is a hydroxylysylpyridinoline or lysylpyridinoline cross-link present in the synthetic peptide. Synthetic peptides from the C-terminal region of collagen have proven to be particularly suitable since the non-helical C-terminal region is larger than the non-helical N-terminal region of collagen. Thus more potential epitopes are available in this region than in the N-terminal region. Peptides having the sequence shown in SEQ ID NO. 1, 2, 3 or 4 from the C-terminal region of the al chain of collagen are particularly suitable.
The concentration of degradation products of collagen is an important diagnostic marker for the extent of osteolysis. The synthetic linear peptides enable a competitive immunoassay to be carried out for the detection of collagen or collagen fragments. It has surprisingly turned out that these peptides compete very well with the collagen fragments that occur in natural samples such as plasma, serum or urine for antibodies to these collagen fragments and thus enable a competitive test. Such antibodies to collagen or collagen degradation products are commercially available for example in the telopeptide ICTP [125I] radioimmunoassay kit from the Orion Diagnostica Company Finland. They can, however, be preferably produced according to the invention using the synthetic linear peptide.
For an application in a competitive immunoassay, the synthetic linear peptide can be used directly as a binding partner which is bound to a solid phase or it can be coupled to a second component. Coupling to the second component is preferably achieved via the ~ ~ ~ 7 4 ~ ~
N-terminal and C-terminal amino acids of the linear peptide. Optionally a spacer can in addition be inserted between the peptide and the second component. The second component can for example serve for coupling the peptide indirectly to a solid phase. Examples of this are known to a person skilled in the art. The peptide is preferably coupled to bovine serum albumin and the coupling product is adsorptively bound to a solid phase such as a plastic tube. The peptide can also be bound covalently to biotin. It is then attached to the solid phase by means of binding to avidin or streptavidin which itself has been bound to the solid phase. The second component can also serve as a carrier for several peptides for example in a competitive turbidimetric inhibition immunoassay (TINIA) in which several peptides are for example coupled to albumin, immunoglobulin, B-galactosidase, to polymers such as polylysine or to dextran molecules such as those described in EP-A-O 545 350 or to particles such as latex. Preferably 30 to 40 peptide molecules are coupled per carrier molecule. The peptide can also be coupled to a component which represents a label. Examples of all these test variants are known to a person skilled in the art.
In the test procedure the antibody can be incubated simultaneously or sequentially with the sample and with the binding partner which contains the synthetic linear peptide. Subsequently the amount of bound or non-bound antibody is determined in the usual manner. For example the antibody used can itself be labelled and this label serves directly as a measure of the bound or unbound antibody. It is also possible to use a second labelled antibody which is directed towards the bound or unbound antibody such as towards the Fc part of this antibody.
Agglutination tests such as TINIA or FPIA (fluorescence polarisation immunoassay) (W. Dandliker et al., J. Exp.
Med. 122 (1965), 1029), EMIT (enzyme multiplied immunoassay) (Gunzer et al., Kontakte III (1980), 3 -11) and CEDIA techn;ques (Henderson et al., Clinical Chemistry 32 (1986), 1637 - 1641) can for example serve as competitive test variants for determining the amount of bound or unbound antibody. The peptides according to the invention have proven to be particularly suitable for use as defined binding partners which compete with the sample for binding to the antibodies. The synthetic linear peptides with the sequences shown in SEQ ID N0 1,
2, 3 or 4 are particularly preferred.
After determining the extent of antibody binding to the binding partner, which is a measure of the amount of antigen in the sample, it is possible to determine the exact amount of antigen in the sample in the usual manner by comparison with a standard treated in the same manner.
Collagen degradation products isolated from natural materials can be used as a standard. However, these are characterized by a certain intrinsic variability. An antigen containing the synthetic linear peptide according to the invention has proven to be more suitable as a standard material. In this case the antigen of the standard can either be composed solely of this peptide or of this peptide coupled to a suitable carrier which for example serves to improve the water-solubility of the peptide. In order to produce the standard material comprising peptide and carrier, the linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen is synthesized and bound to the carrier molecule via its N-terminal or C-terminal amino acid by suitable coupling ~167496 methods. One or several peptides can be bound per carrier molecule. Optionally the coupling can be achieved via a spacer. For certain purposes, such as for agglutination tests, it may be advantageous to bind several peptides according to the invention with different sequences to a carrier molecule especially if polyclonal antibodies are used in the test that were not produced with the aid of the antigen according to the invention and thus usually recognize several epitopes.
The already known antibodies to collagen degradation products can be used as antibodies in the competitive immunoassay. Antibodies which have been obtained with the aid of an antigen containing the linear synthetic peptide according to the invention are especially suitable.
For the immunization the linear synthetic peptides corresponding to one or several sequences of the non-helical C-terminal or N-terminal region of collagen are preferably bound to a suitable carrier protein such as keyhole limpet haemocyanin, bovine serum albumin or edestin.
In order to produce these antigens or immunogens the linear peptides are firstly chemically synthesized in the usual manner. Subsequently the synthetic peptides are coupled to the aforementioned carrier proteins via the N-terminal amino group using maleinimidohexanoic acid N-hydroxysuccinimide ester. It has surprisingly turned out that synthetic linear peptides having the sequence shown in SEQ ID NO 1, 2, 3 or 4 are particularly suitable for the production of antibodies that are suitable for a competitive test procedure.
The antigens according to the invention containing a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen enable antibodies to be obtained which not only recognize the peptide according to the invention but also the degradation products of collagen occurring in body fluids.
The invention therefore in addition concerns a process for the production of antibodies to collagen or collagen fragments by immunization with an antigen according to the invention and isolation of the desired antibody from the serum of the immunized animals by known methods. The desired antibody is preferably isolated by means of immunosorption to a peptide having the sequence shown in SEQ ID NO 1, 2, 3 or 4 coupled to a carrier protein and preferably to sepharose.
A preferred subject matter of the invention is a process for the production of monoclonal antibodies to collagen or collagen fragments by immunization with an antigen according to the invention, immortalization of the spleen cells of the immunized animals, cloning those immortalized spleen cells which produce the desired antibody and isolation of the antibody from the cloned cells or from the culture supernatant of these cells.
The immunization is carried out in animals that are usually used for this; mice or rabbits are preferably used.
The spleen cells of the immunized animals are 15749~
immortalized by methods familiar to a person skilled in the art such as e.g. the hybridoma technique (Kohler and Milstein, Nature 256 (1975), 495 - 497) or by transformation with the Epstein-Barr virus (EBV
transformation). In order to detect those immortalized cells which produce the desired antibody, a sample of the culture supernatant is incubated in a conventional immunoassay with the antigen according to the invention used for the immunization and it is examined whether an antibody binds to this antigen.
The invention furthermore concerns the polyclonal and monoclonal antibodies that can be obtained by the process according to the invention.
These polyclonal and monoclonal antibodies not only react with the hapten according to the invention used for the immunization but also react well with collagen and with the natural degradation products of collagen found in body fluids. Collagen or fragments thereof present in the sample are preferably denatured which in most cases considerably improves the binding of the antibodies according to the invention.
The antibodies according to the invention can therefore be used in test procedures for the determination of collagen or collagen fragments.
Therefore the invention furthermore concerns an immunoassay for the detection of collagen or collagen fragments in a sample using at least one antibody that recognizes a synthetic linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen which is characterized in - 12 - 21~749~
that the sample is denatured. The competitive immunoassay described above in which the linear peptides according to the invention are additionally used has proven to be most suitable. The use of antibodies according to the invention is not limited at all to a competitive immunoassay. The antibodies can be also used in other test formats such as a sandwich immunoassay.
Antibodies of the state of the art can for example be used as a second antibody in this immunoassay. One only needs to take care that the two antibodies do not compete with one another for the same binding site.
The invention therefore also concerns the use of a polyclonal or monoclonal antibody according to the invention for the determination of osteolysis by incubating the antibody with a tissue sample and determining the collagen degradation product binding to this antibody.
The invention furthermore concerns a test combination for the detection of collagen or collagen fragments in a sample containing a protein denaturing agent in a first reagent and separate from this in a second reagent an antibody which recognizes a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen. The reagents are either present in the form of aqueous, preferably buffered, solutions in which case all common buffers known to a person skilled in the art that do not interfere with the immunological reaction can be used as the buffer or in the form of dry, preferably lyophilized, mixtures that can be reconstituted by the addition of a suitable solvent such as water.
- 13 - ~1~749~
It can also contain other common test additives such as substances that reduce interference, proteins or detergents. The test combination also preferably additionally contains a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen as a binding partner of the antibody. The synthetic linear peptide can either be coupled directly to a carrier or be coupled to a second component which mediates binding to the solid phase. The peptide can also be coupled to a component which represents a label.
Furthermore a standard for establishing a standard or calibration curve may be present in a third reagent which contains an antigen containing a synthetic peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen.
The invention is elucidated in more detail by the following examples in conjunction with the sequence protocols.
EQ ID NO 1 shows the sequence of a peptide according to the invention composed of 9 amino acids in which Xaa denotes an arbitrary amino acid.
EQ ID NO 2 shows the sequence of a peptide according to the invention composed of 16 amino acids.
- 14 - ~1674~
EQ ID NO 3 shows the sequence of a peptide according to the invention composed of 10 amino acids.
EQ ID NO 4 shows the sequence of a peptide according to the invention composed of 13 amino acids.
- 15 _ 216~96 Example Peptide syntheses The peptides having a partial sequence of the amino acid sequence of collagen as shown in the sequence protocols SEQ ID NO 2 and 3 are synthesized by means of fluorenyl-methyloxycarbonyl (Fmoc) solid phase peptide synthesis on a a) Labortec SP 640 peptide synthesizer or b) Zinsser analytic SMPS 350 peptide synthesizer.
a) Production of acetyl-Ser-Ala-Gly-Phe-Asp-Phe-Ser-Phe-Leu-Pro-Gln-Pro-Pro-Gln-Glu-Lys-Amid (SEQ ID N0 2) 4.0 equivalents of each of the following Fmoc amino acid derivatives are used in the stated sequence:
Lys with a tert. butyloxycarbonyl protecting group Glu with a tert. butyl ester protecting group Gln without a side chain protecting group Pro without a side chain protecting group Pro without a side chain protecting group Gln without a side chain protecting group Pro without a side chain protecting group Leu without a side chain protecting group Phe without a side chain protecting group Ser with a tert. butyl ether protecting group Phe without a side chain protecting group Asp with a tert. butyl ester protecting group Phe without a side chain protecting group Gly without a side chain protecting group Ala without a side chain protecting group -~1674g6 acetyl acetic anhydride The amino acids or amino acid derivatives are dissolved in N-methylpyrrolidone.
The peptide is synthesized on 3 g 4-(2',4'-dimethoxy-phenyl-Fmoc-aminomethyl)-phenoxy resin (Tetrahedron Letters 28 (1987), 2107) with a loading of 0.87 mmol/g (JACS 95 (1973), 1328). The coupling reactions are carried out for 60 minutes using 4.4 equivalents in relation to the Fmoc amino acid derivatives of dicyclohexylcarbodiimide and 4.8 equivalents N-hydroxy-benzotriazol in dimethylformamide as the reaction medium. The coupling yield is monitored by means of a Kaiser test (Anal. Biochem. 34 (1970), 595) on the synthesis resin washed with isopropanol. If this does not show a complete conversion, the conversion is completed by recoupling under the conditions stated above. After each step in the synthesis the Fmoc group is cleaved within 20 minutes using 20 % piperidine in dimethylformamide. The resin loading is determined by means of the W absorbance of the released fulvene group after each piperidine treatment. The loading is still 0.68 mmol/g after the synthesis.
The peptide is released from the synthesis resin and the acid-labile protecting groups are cleaved with 80 ml trifluoroacetic acid, 5 ml ethanedithiol, 2.5 g phenol, 2.5 ml m-cresol and 5 ml water within 60 minutes at room temperature.
The reaction solution is subsequently concentrated in a vacuum. The residue is taken up in diisopropyl ether, stirred vigorously for 1 - 2 hours and then filtered.
21~7~9~
The material is then pre-purified by means of gel permeation chromatography on Sephadex G15 using 0.5 %
acetic acid as the eluting agent. The crude material obtained is subsequently filtered and isolated within 120 minutes by means of preparative HPLC on Nucleosil RP18 (column 40 mm x 250 mm 300 A, 5 ~m) using a gradient of 100 % buffer A (water, 0.1 % trifluoroacetic acid) to 100 % buffer B (60 % acetonitrile, 40 % water, 0.1 % trifluoroacetic acid). The identity of the eluted material is determined by means of fast-atom-bombardment-mass spectrometry (FAB-MS).
b) Synthe~i~ of Ala-Gly-Phe-A~p-Phe-8er-Phe-Leu-Pro-Gln ~EQ ID NO 3) The peptide was synthesized on 30 mg 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin SA 5030 from the Advanced Chemtech Company with a loading of 0.47 mmol/g. 140 ~mol of each of the following Fmoc amino acid derivatives was coupled twice together in each case with 140 ~mol l-hydroxybenzotriazol in dimethyl-formamide DMF and 10 ~mol N,N-diisopropyl-carbodiimide in DMF to the solid phase-bound peptide to be constructed:
Glu with a trityl protecting group Ser with a tert. butyl protecting group Asp with a tert. butyl protecting group Pro Leu Phe > each without a side chain protecting group Gly Ala The coupling times were 30 and 40 minutes. The cleavage time was 20 minutes and was carried out with a solution of 50 % piperidine in DMF. The washing steps were carried out eight times after each reaction step using DMF. The peptide was released by treating the resin, that had been filtered free of solvent and washed with dichloromethane and methanol, with 1 ml of a solution of sO % trifluoroacetic acid, 3 % thioanisol, 3 % ethande-dithiol and 3 % thiocresol within 20 minutes and 140 minutes. The product was precipitated by addition of 15 ml cold diisopropyl ether to the pooled filtrate and isolated by filtration. The residue was dissolved in 50 % acetic acid and lyophilized. 8 mg white lyophilisate with a purity of 79 % according to HPLC was obtained. The identity was confirmed by means of FAB
mass spectroscopy.
The peptide with the sequence SEQ ID N0 4 Cys-Gly-Ser-Ala-Gly-Phe-Asp-Phe-Ser-Phe-Leu-Pro-Gln was synthesized in an analogous manner.
21~74~6 -- lg --Example 2 Activation of peptide~
The peptide synthesized according to example la) is activated by acylation with maleinimidohexanoyl-N-hydroxysuccinimide (MHS). For this 0.1 mmol of the peptide is dissolved in 20 ml 0.1 mol/l potassium phosphate buffer pH 7.5, admixed with a solution of 0.1 mmol MHS in 6 ml dioxane and stirred for 20 minutes at 20C. Subsequently the pH value is adjusted to pH 4 with glacial acetic acid and the reaction mixture is immediately lyophilized. The lyophilisate is dissolved in 5 ml water and purified by means of preparative HPLC
on a Waters Delta-Pak~ C18 column (100 A, 15 ~m 50 x 300 mm) using an elution gradient of 100 % A (water 0.1 % trifluoroacetic acid) to 100 % B (99.9 % acetonitrile 0.1 % trifluoroacetic acid).
- 20 - 2 1~74~ ~
Bx~mple 3 Production of immunogens by coupling aotivated peptide~
to carrier proteins The coupling of activated peptides to keyhole limpet haemocyanin (KLH), bovine serum albumin (BSA) and A-galactosidase (AGal) is described. In order to couple the peptide activated with MHS according to example 2, it is necessary that the carrier protein has free SH
groups. A-Gal already has these in its natural state and therefore requires no further pretreatment. In the case of KLH and BSA the NH2 groups of the ~-amino side chain of lysine residues are derivatized by treatment with N-succinimidyl-S-acetylthiopropionate (SATP) and thus converted into SH groups.
In this way a carrier protein is obtained which has an increased number of SH groups compared to its native state. For this 113.51 mg SATP (dissolved in 10 ml dioxane) is added dropwise to a solution of 1.39 g KLH
in 500 ml 0.1 mol/l potassium phosphate buffer pH 8.5 within 20 minutes. After stirring for 30 minutes at 20C
the pH value of the reaction solution is readjusted with 0.1 mol/l sodium hydroxide solution to pH 8.5 and it is stirred for a further 24 hours. The solution is subsequently concentrated with the aid of an Amicon cell (membrane YM10) to 100 ml, dialysed 3 x 24 hours against 3 l 0.1 mol/l potassium phosphate buffer pH
8.5/0.05 mol/l sodium chloride each time and subsequently lyophilized.
In order to cleave the S-acetyl protecting group, 481 mg of the KLH-SATP lyophilisate is dissolved in 20 ml 21674~
0.1 mol/l potassium phosphate buffer pH 8.5/0.05 mol/l sodium chloride, admixed with 0.5 ml freshly prepared 1 mol/l hydroxylamine solution and stirred for 90 minutes at 20C.
7.23 mol of the activated peptide obtained according to example 2 in 4 ml water is added to the derivatized carrier protein and stirred for 20 hours at 20C.
Subsequently the turbid solution is dialysed twice against 1 l 0.1 mol/l potassium phosphate buffer pH
8.5/0.05 mol/l sodium chloride. The dialysate is centrifuged, the clear supernatant is decanted and lyophilized.
21~7~6 Example 4 Production of polyclonal antiboaies again~t linear collagen fragments 5 sheep were in each case immunized in a known manner with the immunogen from example 3. The immunogens contained the peptide having the sequence stated in SEQ ID NO 2 which corresponds to the amino acids No. 892 to 907 in the sequence of the a chain of type I
collagen. KLH or ~-galactosidase served as the carrier protein. The animals were immunized at monthly intervals with the immunogens in complete Freund's adjuvant. The dose was 500 ~g per animal and immunization. Blood samples were taken four months after the initial immunization and the antibodies obtained were examined for reaction with collagen fragments.
ELI~A to detect the reaction of the antisera with collagen fragments The following material and reagents were used:
Microtitre plates Maxisorp F96, Nunc Company Coating buffer: 50 mM sodium carbonate pH 9.6 0.1 % NaN3 Incubation buffer: 10 mM sodium phosphate pH 7.4 0.1 % Tween 20 0.9 % NaCl 1 % bovine serum albumin - 23 - 2 1 67 4 9 ~
ubstrate solution: ABTS~, Boehringer Mannheim GmbH, catalogue No. 857424.
2 mg/ml vanillin was added to the solution to increase the signal.
Wash solution: 0.1 % Tween 20 0.9 % NaCl Each of the wells of the titre plates was filled with 100 ~1 of a solution which contained 10 ~g/ml collagen fragments in coating buffer. The collagen fragments were prepared by protease digestion of human collagen from bones according to the instructions in EP-A-0 505 210.
After incubating for one hour at room temperature while shaking, it was washed three times with wash solution.
The antisera were diluted 1 : 4000 with incubation buffer and 100 ~1 of each was incubated in the wells of the microtitre plate for one hour at room temperature while shaking. Subsequently the wells were washed three times with wash solution.
A conjugate of horseradish peroxidase with rabbit antibodies against the Fc part of sheep-IgG is diluted in incubation buffer to a concentration of 12.5 mU/ml and each of the wells of the microtitre plate are filled with 100 ~1 thereof. After incubating for one hour at room temperature while shaking, the titre plates are washed three times with wash solution.
100 ~1 substrate solution is added and incubated until there is a clear colour development (10 - 60 minutes).
The absorbance is determined as the difference between the measurements at 405 and 492 nm.
21S7~fi The sera of most animals showed a strong reaction with the collagen fragments on the solid phase. The serum of an animal which had not been immunized only exhibited a weak measurement signal under the same conditions. The results are given in table 1.
Table B-galactosidase KLH
animal No. absorbance animal No. absorbance 1 1.05 1 1.16 2 1.18 2 2.62
After determining the extent of antibody binding to the binding partner, which is a measure of the amount of antigen in the sample, it is possible to determine the exact amount of antigen in the sample in the usual manner by comparison with a standard treated in the same manner.
Collagen degradation products isolated from natural materials can be used as a standard. However, these are characterized by a certain intrinsic variability. An antigen containing the synthetic linear peptide according to the invention has proven to be more suitable as a standard material. In this case the antigen of the standard can either be composed solely of this peptide or of this peptide coupled to a suitable carrier which for example serves to improve the water-solubility of the peptide. In order to produce the standard material comprising peptide and carrier, the linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen is synthesized and bound to the carrier molecule via its N-terminal or C-terminal amino acid by suitable coupling ~167496 methods. One or several peptides can be bound per carrier molecule. Optionally the coupling can be achieved via a spacer. For certain purposes, such as for agglutination tests, it may be advantageous to bind several peptides according to the invention with different sequences to a carrier molecule especially if polyclonal antibodies are used in the test that were not produced with the aid of the antigen according to the invention and thus usually recognize several epitopes.
The already known antibodies to collagen degradation products can be used as antibodies in the competitive immunoassay. Antibodies which have been obtained with the aid of an antigen containing the linear synthetic peptide according to the invention are especially suitable.
For the immunization the linear synthetic peptides corresponding to one or several sequences of the non-helical C-terminal or N-terminal region of collagen are preferably bound to a suitable carrier protein such as keyhole limpet haemocyanin, bovine serum albumin or edestin.
In order to produce these antigens or immunogens the linear peptides are firstly chemically synthesized in the usual manner. Subsequently the synthetic peptides are coupled to the aforementioned carrier proteins via the N-terminal amino group using maleinimidohexanoic acid N-hydroxysuccinimide ester. It has surprisingly turned out that synthetic linear peptides having the sequence shown in SEQ ID NO 1, 2, 3 or 4 are particularly suitable for the production of antibodies that are suitable for a competitive test procedure.
The antigens according to the invention containing a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen enable antibodies to be obtained which not only recognize the peptide according to the invention but also the degradation products of collagen occurring in body fluids.
The invention therefore in addition concerns a process for the production of antibodies to collagen or collagen fragments by immunization with an antigen according to the invention and isolation of the desired antibody from the serum of the immunized animals by known methods. The desired antibody is preferably isolated by means of immunosorption to a peptide having the sequence shown in SEQ ID NO 1, 2, 3 or 4 coupled to a carrier protein and preferably to sepharose.
A preferred subject matter of the invention is a process for the production of monoclonal antibodies to collagen or collagen fragments by immunization with an antigen according to the invention, immortalization of the spleen cells of the immunized animals, cloning those immortalized spleen cells which produce the desired antibody and isolation of the antibody from the cloned cells or from the culture supernatant of these cells.
The immunization is carried out in animals that are usually used for this; mice or rabbits are preferably used.
The spleen cells of the immunized animals are 15749~
immortalized by methods familiar to a person skilled in the art such as e.g. the hybridoma technique (Kohler and Milstein, Nature 256 (1975), 495 - 497) or by transformation with the Epstein-Barr virus (EBV
transformation). In order to detect those immortalized cells which produce the desired antibody, a sample of the culture supernatant is incubated in a conventional immunoassay with the antigen according to the invention used for the immunization and it is examined whether an antibody binds to this antigen.
The invention furthermore concerns the polyclonal and monoclonal antibodies that can be obtained by the process according to the invention.
These polyclonal and monoclonal antibodies not only react with the hapten according to the invention used for the immunization but also react well with collagen and with the natural degradation products of collagen found in body fluids. Collagen or fragments thereof present in the sample are preferably denatured which in most cases considerably improves the binding of the antibodies according to the invention.
The antibodies according to the invention can therefore be used in test procedures for the determination of collagen or collagen fragments.
Therefore the invention furthermore concerns an immunoassay for the detection of collagen or collagen fragments in a sample using at least one antibody that recognizes a synthetic linear peptide which corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen which is characterized in - 12 - 21~749~
that the sample is denatured. The competitive immunoassay described above in which the linear peptides according to the invention are additionally used has proven to be most suitable. The use of antibodies according to the invention is not limited at all to a competitive immunoassay. The antibodies can be also used in other test formats such as a sandwich immunoassay.
Antibodies of the state of the art can for example be used as a second antibody in this immunoassay. One only needs to take care that the two antibodies do not compete with one another for the same binding site.
The invention therefore also concerns the use of a polyclonal or monoclonal antibody according to the invention for the determination of osteolysis by incubating the antibody with a tissue sample and determining the collagen degradation product binding to this antibody.
The invention furthermore concerns a test combination for the detection of collagen or collagen fragments in a sample containing a protein denaturing agent in a first reagent and separate from this in a second reagent an antibody which recognizes a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen. The reagents are either present in the form of aqueous, preferably buffered, solutions in which case all common buffers known to a person skilled in the art that do not interfere with the immunological reaction can be used as the buffer or in the form of dry, preferably lyophilized, mixtures that can be reconstituted by the addition of a suitable solvent such as water.
- 13 - ~1~749~
It can also contain other common test additives such as substances that reduce interference, proteins or detergents. The test combination also preferably additionally contains a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen as a binding partner of the antibody. The synthetic linear peptide can either be coupled directly to a carrier or be coupled to a second component which mediates binding to the solid phase. The peptide can also be coupled to a component which represents a label.
Furthermore a standard for establishing a standard or calibration curve may be present in a third reagent which contains an antigen containing a synthetic peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen.
The invention is elucidated in more detail by the following examples in conjunction with the sequence protocols.
EQ ID NO 1 shows the sequence of a peptide according to the invention composed of 9 amino acids in which Xaa denotes an arbitrary amino acid.
EQ ID NO 2 shows the sequence of a peptide according to the invention composed of 16 amino acids.
- 14 - ~1674~
EQ ID NO 3 shows the sequence of a peptide according to the invention composed of 10 amino acids.
EQ ID NO 4 shows the sequence of a peptide according to the invention composed of 13 amino acids.
- 15 _ 216~96 Example Peptide syntheses The peptides having a partial sequence of the amino acid sequence of collagen as shown in the sequence protocols SEQ ID NO 2 and 3 are synthesized by means of fluorenyl-methyloxycarbonyl (Fmoc) solid phase peptide synthesis on a a) Labortec SP 640 peptide synthesizer or b) Zinsser analytic SMPS 350 peptide synthesizer.
a) Production of acetyl-Ser-Ala-Gly-Phe-Asp-Phe-Ser-Phe-Leu-Pro-Gln-Pro-Pro-Gln-Glu-Lys-Amid (SEQ ID N0 2) 4.0 equivalents of each of the following Fmoc amino acid derivatives are used in the stated sequence:
Lys with a tert. butyloxycarbonyl protecting group Glu with a tert. butyl ester protecting group Gln without a side chain protecting group Pro without a side chain protecting group Pro without a side chain protecting group Gln without a side chain protecting group Pro without a side chain protecting group Leu without a side chain protecting group Phe without a side chain protecting group Ser with a tert. butyl ether protecting group Phe without a side chain protecting group Asp with a tert. butyl ester protecting group Phe without a side chain protecting group Gly without a side chain protecting group Ala without a side chain protecting group -~1674g6 acetyl acetic anhydride The amino acids or amino acid derivatives are dissolved in N-methylpyrrolidone.
The peptide is synthesized on 3 g 4-(2',4'-dimethoxy-phenyl-Fmoc-aminomethyl)-phenoxy resin (Tetrahedron Letters 28 (1987), 2107) with a loading of 0.87 mmol/g (JACS 95 (1973), 1328). The coupling reactions are carried out for 60 minutes using 4.4 equivalents in relation to the Fmoc amino acid derivatives of dicyclohexylcarbodiimide and 4.8 equivalents N-hydroxy-benzotriazol in dimethylformamide as the reaction medium. The coupling yield is monitored by means of a Kaiser test (Anal. Biochem. 34 (1970), 595) on the synthesis resin washed with isopropanol. If this does not show a complete conversion, the conversion is completed by recoupling under the conditions stated above. After each step in the synthesis the Fmoc group is cleaved within 20 minutes using 20 % piperidine in dimethylformamide. The resin loading is determined by means of the W absorbance of the released fulvene group after each piperidine treatment. The loading is still 0.68 mmol/g after the synthesis.
The peptide is released from the synthesis resin and the acid-labile protecting groups are cleaved with 80 ml trifluoroacetic acid, 5 ml ethanedithiol, 2.5 g phenol, 2.5 ml m-cresol and 5 ml water within 60 minutes at room temperature.
The reaction solution is subsequently concentrated in a vacuum. The residue is taken up in diisopropyl ether, stirred vigorously for 1 - 2 hours and then filtered.
21~7~9~
The material is then pre-purified by means of gel permeation chromatography on Sephadex G15 using 0.5 %
acetic acid as the eluting agent. The crude material obtained is subsequently filtered and isolated within 120 minutes by means of preparative HPLC on Nucleosil RP18 (column 40 mm x 250 mm 300 A, 5 ~m) using a gradient of 100 % buffer A (water, 0.1 % trifluoroacetic acid) to 100 % buffer B (60 % acetonitrile, 40 % water, 0.1 % trifluoroacetic acid). The identity of the eluted material is determined by means of fast-atom-bombardment-mass spectrometry (FAB-MS).
b) Synthe~i~ of Ala-Gly-Phe-A~p-Phe-8er-Phe-Leu-Pro-Gln ~EQ ID NO 3) The peptide was synthesized on 30 mg 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin SA 5030 from the Advanced Chemtech Company with a loading of 0.47 mmol/g. 140 ~mol of each of the following Fmoc amino acid derivatives was coupled twice together in each case with 140 ~mol l-hydroxybenzotriazol in dimethyl-formamide DMF and 10 ~mol N,N-diisopropyl-carbodiimide in DMF to the solid phase-bound peptide to be constructed:
Glu with a trityl protecting group Ser with a tert. butyl protecting group Asp with a tert. butyl protecting group Pro Leu Phe > each without a side chain protecting group Gly Ala The coupling times were 30 and 40 minutes. The cleavage time was 20 minutes and was carried out with a solution of 50 % piperidine in DMF. The washing steps were carried out eight times after each reaction step using DMF. The peptide was released by treating the resin, that had been filtered free of solvent and washed with dichloromethane and methanol, with 1 ml of a solution of sO % trifluoroacetic acid, 3 % thioanisol, 3 % ethande-dithiol and 3 % thiocresol within 20 minutes and 140 minutes. The product was precipitated by addition of 15 ml cold diisopropyl ether to the pooled filtrate and isolated by filtration. The residue was dissolved in 50 % acetic acid and lyophilized. 8 mg white lyophilisate with a purity of 79 % according to HPLC was obtained. The identity was confirmed by means of FAB
mass spectroscopy.
The peptide with the sequence SEQ ID N0 4 Cys-Gly-Ser-Ala-Gly-Phe-Asp-Phe-Ser-Phe-Leu-Pro-Gln was synthesized in an analogous manner.
21~74~6 -- lg --Example 2 Activation of peptide~
The peptide synthesized according to example la) is activated by acylation with maleinimidohexanoyl-N-hydroxysuccinimide (MHS). For this 0.1 mmol of the peptide is dissolved in 20 ml 0.1 mol/l potassium phosphate buffer pH 7.5, admixed with a solution of 0.1 mmol MHS in 6 ml dioxane and stirred for 20 minutes at 20C. Subsequently the pH value is adjusted to pH 4 with glacial acetic acid and the reaction mixture is immediately lyophilized. The lyophilisate is dissolved in 5 ml water and purified by means of preparative HPLC
on a Waters Delta-Pak~ C18 column (100 A, 15 ~m 50 x 300 mm) using an elution gradient of 100 % A (water 0.1 % trifluoroacetic acid) to 100 % B (99.9 % acetonitrile 0.1 % trifluoroacetic acid).
- 20 - 2 1~74~ ~
Bx~mple 3 Production of immunogens by coupling aotivated peptide~
to carrier proteins The coupling of activated peptides to keyhole limpet haemocyanin (KLH), bovine serum albumin (BSA) and A-galactosidase (AGal) is described. In order to couple the peptide activated with MHS according to example 2, it is necessary that the carrier protein has free SH
groups. A-Gal already has these in its natural state and therefore requires no further pretreatment. In the case of KLH and BSA the NH2 groups of the ~-amino side chain of lysine residues are derivatized by treatment with N-succinimidyl-S-acetylthiopropionate (SATP) and thus converted into SH groups.
In this way a carrier protein is obtained which has an increased number of SH groups compared to its native state. For this 113.51 mg SATP (dissolved in 10 ml dioxane) is added dropwise to a solution of 1.39 g KLH
in 500 ml 0.1 mol/l potassium phosphate buffer pH 8.5 within 20 minutes. After stirring for 30 minutes at 20C
the pH value of the reaction solution is readjusted with 0.1 mol/l sodium hydroxide solution to pH 8.5 and it is stirred for a further 24 hours. The solution is subsequently concentrated with the aid of an Amicon cell (membrane YM10) to 100 ml, dialysed 3 x 24 hours against 3 l 0.1 mol/l potassium phosphate buffer pH
8.5/0.05 mol/l sodium chloride each time and subsequently lyophilized.
In order to cleave the S-acetyl protecting group, 481 mg of the KLH-SATP lyophilisate is dissolved in 20 ml 21674~
0.1 mol/l potassium phosphate buffer pH 8.5/0.05 mol/l sodium chloride, admixed with 0.5 ml freshly prepared 1 mol/l hydroxylamine solution and stirred for 90 minutes at 20C.
7.23 mol of the activated peptide obtained according to example 2 in 4 ml water is added to the derivatized carrier protein and stirred for 20 hours at 20C.
Subsequently the turbid solution is dialysed twice against 1 l 0.1 mol/l potassium phosphate buffer pH
8.5/0.05 mol/l sodium chloride. The dialysate is centrifuged, the clear supernatant is decanted and lyophilized.
21~7~6 Example 4 Production of polyclonal antiboaies again~t linear collagen fragments 5 sheep were in each case immunized in a known manner with the immunogen from example 3. The immunogens contained the peptide having the sequence stated in SEQ ID NO 2 which corresponds to the amino acids No. 892 to 907 in the sequence of the a chain of type I
collagen. KLH or ~-galactosidase served as the carrier protein. The animals were immunized at monthly intervals with the immunogens in complete Freund's adjuvant. The dose was 500 ~g per animal and immunization. Blood samples were taken four months after the initial immunization and the antibodies obtained were examined for reaction with collagen fragments.
ELI~A to detect the reaction of the antisera with collagen fragments The following material and reagents were used:
Microtitre plates Maxisorp F96, Nunc Company Coating buffer: 50 mM sodium carbonate pH 9.6 0.1 % NaN3 Incubation buffer: 10 mM sodium phosphate pH 7.4 0.1 % Tween 20 0.9 % NaCl 1 % bovine serum albumin - 23 - 2 1 67 4 9 ~
ubstrate solution: ABTS~, Boehringer Mannheim GmbH, catalogue No. 857424.
2 mg/ml vanillin was added to the solution to increase the signal.
Wash solution: 0.1 % Tween 20 0.9 % NaCl Each of the wells of the titre plates was filled with 100 ~1 of a solution which contained 10 ~g/ml collagen fragments in coating buffer. The collagen fragments were prepared by protease digestion of human collagen from bones according to the instructions in EP-A-0 505 210.
After incubating for one hour at room temperature while shaking, it was washed three times with wash solution.
The antisera were diluted 1 : 4000 with incubation buffer and 100 ~1 of each was incubated in the wells of the microtitre plate for one hour at room temperature while shaking. Subsequently the wells were washed three times with wash solution.
A conjugate of horseradish peroxidase with rabbit antibodies against the Fc part of sheep-IgG is diluted in incubation buffer to a concentration of 12.5 mU/ml and each of the wells of the microtitre plate are filled with 100 ~1 thereof. After incubating for one hour at room temperature while shaking, the titre plates are washed three times with wash solution.
100 ~1 substrate solution is added and incubated until there is a clear colour development (10 - 60 minutes).
The absorbance is determined as the difference between the measurements at 405 and 492 nm.
21S7~fi The sera of most animals showed a strong reaction with the collagen fragments on the solid phase. The serum of an animal which had not been immunized only exhibited a weak measurement signal under the same conditions. The results are given in table 1.
Table B-galactosidase KLH
animal No. absorbance animal No. absorbance 1 1.05 1 1.16 2 1.18 2 2.62
3 1.49 3 1.28
4 0.48 4 1.42 > 2.70 5 1.81 Mice were immunized in an analogous manner with immunogens according to example 3 and the antisera were obtained. The three antisera 223/20, 241/13 and 242/2 were particularly suitable. The antiserum 223/20 was derived from a mouse which had been immunized with a decapeptide corresponding to SEQ ID NO 4 coupled to KLH.
The antiserum 241/13 was derived from a mouse which had been immunized with a peptide corresponding to SEQ ID
NO 2 of 16 amino acids in length coupled to KLH.
The antiserum 242/2 was derived from a mouse which had been immunized with a peptide corresponding to SEQ ID
NO 2 of 16 amino acids in length coupled to BGal.
- 25 - 2167~g 8 Bxample 5 Determination of collagen and its degradation products in body fluids by means of a competitive test The wells of a 96-well microtitre plate are coated overnight at 4C with streptavidin according to EP-A-0 344 578 (100 ~l of a solution of 1 ~g/ml in PBS) and unspecific binding sites that are still free are blocked for 2 hours at room temperature by incubation with 300 ~l BSA (bovine serum albumin, 10 mg/ml).
The decapeptide having the sequence shown in SEQ ID NO 3 which had been prepared according to example lb) is biotinylated at its amino terminus with D-biotinyl-~-amidocaproic acid-N-succinimide ester (Boehringer Mannheim, catalogue No. 1008960) according to the manufacturer's instructions. The biotinylated peptide is dissolved at a concentration of 10 ng/ml in PBS, 0.05 %
Tween 20, 1 % BSA and bound to the streptavidin-coated microtitre plate by incubating 100 ~l per well for 1 hour. Subsequently unbound peptide is removed by washing three times with PBS, 0.05 % Tween 20.
150 ~l of the sample to be examined (serum, plasma or a standard) is in each case incubated with 150 ~l of the antibody according to the invention according to example 4 for 2 hours at 37C (or overnight at 4C). 100 ~l of this mixture is in each case added to the bound decapeptide in the wells of a microtitre plate and incubated for 60 minutes at 37C. In this process only the antibody excess of the antiserum which is still unbound after incubation with the sample can bind to the immobilized decapeptide.
21~7~96 After washing three times with PBS/0.05 % Tween 20, bound antibody is detected by subsequent incubation with a rabbit anti-sheep IgG-POD conjugate (Boehringer M~nnheim GmbH) and ABTS~ (1 mg/ml).
164 patient sera were measured using the test according to the invention (MTP competitive test). The results were related to data determined with a radioimmunoassay (RIA). This RIA ICTP (telopeptide ICTP [125I] from the Orion Diagnostica Company, Finnland) is based on cross-linked collagen fragments which are prepared and isolated by enzymatic digestion and biochemical methods.
It can be seen from Figure 1 that the method according to the invention results in measured values that correlate well with the RIA values which means the method according to the invention yields clinically relevant data. A correlation coefficient of 0.959 was determined.
- 27 - 2~6749~
Example 6 Influence of denaturing reagents on the determination of a C-terminal peptide from collagen The biotinylated decapeptide corresponding to SEQ ID
NO 3 is bound to microtitre plates coated with streptavidin as described in example 5.
As an alternative a fragment of collagen, a C-terminal telopeptide (CTX) which was prepared according to Risteli (1993) was adsorbed to the surface of microtitre plates (Nunc, Maxisorb). For this 100 ~l of a solution containing 1 ~g/ml CTX is incubated overnight at 4C in each of the wells of a microtitre plate. Free unspecific binding sites are blocked by incubation for two hours at room temperature with 300 ~l bovine serum albumin (10 mg/ml in PBS).
A CTX solution is used as a sample. This is either pre-treated with a PBS solution alone (control), a PBS
solution containing 3 M potassium thiocyanate (KSCN) or a solution containing 1 % tetradecyltriethylammonium bromide (TTAB). Equal volumes of the CTX solution and the buffer solutions are mixed and incubated for one hour. Subsequently the mixture was diluted 1 to 10 with a PBS solution containing 0.05 % Tween 20~ since undiluted denaturing agents could influence the immuno-reactivity of the antibodies.
100 ~l of the samples pretreated in this way are incubated for one hour at room temperature with 100 ~l of a solution of a polyclonal mouse antibody in the microtitre plate which was coated either with the 2~ ~7~9~
decapeptide or CTX as described above. Unbound antibodies were removed by washing three times with PBS
containing 0.05 ~ Tween 20~. Bound antibodies were detected with a sheep anti-mouse F(ab)-POD conjugate (Boehringer Mannheim GmbH, catalogue No. 117 2808) and ABTS~ (1 mg/ml).
Antisera from mice 223/20, 241/13 and 242/2 described in example 4 were used as the antisera which were diluted 1:2000 in PBS containing 0.05 ~ Tween 20~ before use.
The results are compiled in Table 2 (measured absorbances). CTX in PBS without denaturing agents only showed a significant competition with the mouse serum 242/2. The use of the denaturing reagents KSCN and TTAB
in all cases results in a drastic increase in the competition even with antisera which did not show significant competition without denaturing agents.
Table 2 Sample pre-treatment Antiserum Antiserum Antiserum Control PBS 1.314 0.502 0.563 CTX not denatured 1.125 0.370 0.283 Control KCSN 1.202 0.443 0.562 CTX + KSCN 0.461 0.174 0.259 Control TTAB 0.873 0.217 0.471 CTX + TTAB 0.107 0.090 0.244 2167~96 8EQUENCE PR~.OCOL
(2) Information for SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Phe Asp Phe Ser Phe Leu Pro Xaa (2) Information for SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys 3~ 2167~9B
(2) Information for SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln (2) Information for SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Cys Gly Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln
The antiserum 241/13 was derived from a mouse which had been immunized with a peptide corresponding to SEQ ID
NO 2 of 16 amino acids in length coupled to KLH.
The antiserum 242/2 was derived from a mouse which had been immunized with a peptide corresponding to SEQ ID
NO 2 of 16 amino acids in length coupled to BGal.
- 25 - 2167~g 8 Bxample 5 Determination of collagen and its degradation products in body fluids by means of a competitive test The wells of a 96-well microtitre plate are coated overnight at 4C with streptavidin according to EP-A-0 344 578 (100 ~l of a solution of 1 ~g/ml in PBS) and unspecific binding sites that are still free are blocked for 2 hours at room temperature by incubation with 300 ~l BSA (bovine serum albumin, 10 mg/ml).
The decapeptide having the sequence shown in SEQ ID NO 3 which had been prepared according to example lb) is biotinylated at its amino terminus with D-biotinyl-~-amidocaproic acid-N-succinimide ester (Boehringer Mannheim, catalogue No. 1008960) according to the manufacturer's instructions. The biotinylated peptide is dissolved at a concentration of 10 ng/ml in PBS, 0.05 %
Tween 20, 1 % BSA and bound to the streptavidin-coated microtitre plate by incubating 100 ~l per well for 1 hour. Subsequently unbound peptide is removed by washing three times with PBS, 0.05 % Tween 20.
150 ~l of the sample to be examined (serum, plasma or a standard) is in each case incubated with 150 ~l of the antibody according to the invention according to example 4 for 2 hours at 37C (or overnight at 4C). 100 ~l of this mixture is in each case added to the bound decapeptide in the wells of a microtitre plate and incubated for 60 minutes at 37C. In this process only the antibody excess of the antiserum which is still unbound after incubation with the sample can bind to the immobilized decapeptide.
21~7~96 After washing three times with PBS/0.05 % Tween 20, bound antibody is detected by subsequent incubation with a rabbit anti-sheep IgG-POD conjugate (Boehringer M~nnheim GmbH) and ABTS~ (1 mg/ml).
164 patient sera were measured using the test according to the invention (MTP competitive test). The results were related to data determined with a radioimmunoassay (RIA). This RIA ICTP (telopeptide ICTP [125I] from the Orion Diagnostica Company, Finnland) is based on cross-linked collagen fragments which are prepared and isolated by enzymatic digestion and biochemical methods.
It can be seen from Figure 1 that the method according to the invention results in measured values that correlate well with the RIA values which means the method according to the invention yields clinically relevant data. A correlation coefficient of 0.959 was determined.
- 27 - 2~6749~
Example 6 Influence of denaturing reagents on the determination of a C-terminal peptide from collagen The biotinylated decapeptide corresponding to SEQ ID
NO 3 is bound to microtitre plates coated with streptavidin as described in example 5.
As an alternative a fragment of collagen, a C-terminal telopeptide (CTX) which was prepared according to Risteli (1993) was adsorbed to the surface of microtitre plates (Nunc, Maxisorb). For this 100 ~l of a solution containing 1 ~g/ml CTX is incubated overnight at 4C in each of the wells of a microtitre plate. Free unspecific binding sites are blocked by incubation for two hours at room temperature with 300 ~l bovine serum albumin (10 mg/ml in PBS).
A CTX solution is used as a sample. This is either pre-treated with a PBS solution alone (control), a PBS
solution containing 3 M potassium thiocyanate (KSCN) or a solution containing 1 % tetradecyltriethylammonium bromide (TTAB). Equal volumes of the CTX solution and the buffer solutions are mixed and incubated for one hour. Subsequently the mixture was diluted 1 to 10 with a PBS solution containing 0.05 % Tween 20~ since undiluted denaturing agents could influence the immuno-reactivity of the antibodies.
100 ~l of the samples pretreated in this way are incubated for one hour at room temperature with 100 ~l of a solution of a polyclonal mouse antibody in the microtitre plate which was coated either with the 2~ ~7~9~
decapeptide or CTX as described above. Unbound antibodies were removed by washing three times with PBS
containing 0.05 ~ Tween 20~. Bound antibodies were detected with a sheep anti-mouse F(ab)-POD conjugate (Boehringer Mannheim GmbH, catalogue No. 117 2808) and ABTS~ (1 mg/ml).
Antisera from mice 223/20, 241/13 and 242/2 described in example 4 were used as the antisera which were diluted 1:2000 in PBS containing 0.05 ~ Tween 20~ before use.
The results are compiled in Table 2 (measured absorbances). CTX in PBS without denaturing agents only showed a significant competition with the mouse serum 242/2. The use of the denaturing reagents KSCN and TTAB
in all cases results in a drastic increase in the competition even with antisera which did not show significant competition without denaturing agents.
Table 2 Sample pre-treatment Antiserum Antiserum Antiserum Control PBS 1.314 0.502 0.563 CTX not denatured 1.125 0.370 0.283 Control KCSN 1.202 0.443 0.562 CTX + KSCN 0.461 0.174 0.259 Control TTAB 0.873 0.217 0.471 CTX + TTAB 0.107 0.090 0.244 2167~96 8EQUENCE PR~.OCOL
(2) Information for SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Phe Asp Phe Ser Phe Leu Pro Xaa (2) Information for SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys 3~ 2167~9B
(2) Information for SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln (2) Information for SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Cys Gly Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln
Claims (9)
1. Competitive immunoassay for the detection of collagen or collagen fragments in a sample in which one binding partner which contains a synthetic linear peptide that corresponds to a non-helical C-terminal 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, wherein the sample is denatured.
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, 3 or 4.
5. Immunoassay as claimed in claim 1, wherein the sample is denatured before incubation with the antibody.
6. Immunoassay as claimed in claim 1, wherein TTAB or KCSN is used as the denaturing agent.
7. Immunoassay for the detection of collagen or collagen fragments in a sample using at least one antibody that recognizes a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen, wherein the sample is denatured.
8. Test combination for the detection of collagen or collagen fragments in a sample containing a protein denaturing agent in a first reagent and separate therefrom in a second reagent an antibody which recognizes a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen.
9. Test combination as claimed in claim 8, wherein it additionally contains a synthetic linear peptide that corresponds to a sequence of the non-helical C-terminal or N-terminal region of collagen.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| WOPCT/EP93/02010 | 1993-07-28 | ||
| PCT/EP1993/002010 WO1994003813A1 (en) | 1992-07-29 | 1993-07-28 | Immunoassay for detecting collagen or collagen fragments |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2167496A1 true CA2167496A1 (en) | 1995-02-09 |
Family
ID=8165750
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002167496A Abandoned CA2167496A1 (en) | 1993-07-28 | 1994-01-21 | Immunoassay for the detection of collagen or collagen fragments |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0711415B1 (en) |
| JP (1) | JP2703116B2 (en) |
| AT (1) | ATE168779T1 (en) |
| CA (1) | CA2167496A1 (en) |
| DE (1) | DE59406512D1 (en) |
| DK (1) | DK0711415T3 (en) |
| ES (1) | ES2122231T3 (en) |
| FI (1) | FI960392A (en) |
| WO (1) | WO1995004282A1 (en) |
Families Citing this family (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6110689A (en) | 1994-01-21 | 2000-08-29 | Osteometer A/S | Method of assaying collagen fragments in body fluids, a test kit and means for carrying out the method and use of the method to diagnose the presence of disorders associated with the metabolism of collagen |
| GB9506050D0 (en) | 1995-03-24 | 1995-05-10 | Osteometer A S | Assaying collagen fragments in body fluids |
| EP0787301B1 (en) | 1994-10-17 | 2001-02-14 | Osteometer Biotech AS | Estimation of the fragmentation pattern of collagen in body fluids and the diagnosis of disorders associated with the metabolism of collagen |
| 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 |
| DE69705423T2 (en) | 1996-12-09 | 2002-05-16 | Osteometer Biotech As Herlev | SANDWEIGHT TEST FOR DETECTING COLLAGEN FRAGMENTS |
| US6117646A (en) * | 1997-09-22 | 2000-09-12 | Osteometer Biotech A/S | Assaying protein fragments in body fluids |
| US6348320B1 (en) | 1998-06-19 | 2002-02-19 | Washington Research Foundation | Cartilage resorption assays measuring type II collagen fragments |
| US6916903B2 (en) | 1998-06-19 | 2005-07-12 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
| US6255056B1 (en) | 1998-06-19 | 2001-07-03 | Washington Research Foundation | Cartilage resorption assays |
| US6602980B1 (en) | 1998-06-19 | 2003-08-05 | Washington Research Foundation | Collagen type III synthetic peptides for collagen resorption assays |
| DK1149111T3 (en) * | 1999-01-06 | 2009-12-14 | Univ Southern California | Method and composition for inhibiting angiogenesis |
| ES2327812T3 (en) | 1999-07-13 | 2009-11-04 | University Of Southern California | NEW METHODS AND COMPOSITIONS TO INHIBIT THE ANGIOGENESIS USING ANTAGONIST BASES OF MMP-9 AND / OR INTEGRINES BETA-1. |
| US7365167B2 (en) | 2001-11-26 | 2008-04-29 | Cell Matrix, Inc. | Humanized collagen antibodies and related methods |
| US7390885B2 (en) | 2001-11-26 | 2008-06-24 | Cell Matrix, Inc. | Humanized collagen antibodies and related methods |
| US7488792B2 (en) | 2002-08-28 | 2009-02-10 | Burnham Institute For Medical Research | Collagen-binding molecules that selectively home to tumor vasculature and methods of using same |
| JP2004108914A (en) * | 2002-09-18 | 2004-04-08 | Kudo Norio | Method for measuring collagen |
| US7781171B2 (en) | 2003-10-15 | 2010-08-24 | Daiichi Pure Chemicals Co., Ltd. | Mehod of pretreating sample and immunological assay method using the same |
| DE602004020372D1 (en) * | 2003-10-15 | 2009-05-14 | Sekisui Medical Co Ltd | PROCEDURE FOR SAMPLE PREPARATION AND IMMUNOLOGICAL TEST METHOD USING THEREOF |
| CN117561445A (en) | 2021-03-24 | 2024-02-13 | 积水医疗株式会社 | Immunological analysis method of C-terminal peptide of type I collagen |
| WO2024034580A1 (en) * | 2022-08-08 | 2024-02-15 | 株式会社先端生命科学研究所 | Method for detecting ceacam1 |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3479158D1 (en) * | 1983-03-04 | 1989-08-31 | Noctech Ltd | Diagnostic agent, a process for its preparation and its use in diagnostic methods |
| CA1339952C (en) * | 1984-10-29 | 1998-07-14 | William J. Knowles | Immunoassays for denatured protein analytes, particularly hb alc, and monoclonal antibodies thereto |
| 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 |
| EP0339443B1 (en) * | 1988-04-27 | 1994-12-21 | Hoechst Aktiengesellschaft | Monoclonal antibody for the selective immune 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 |
| DE69315989T2 (en) * | 1992-01-31 | 1998-04-23 | David Jeston Baylink | AMINOTERMINAL PROCOLLAGEN 1 (I) PEPTIDE |
| DE4225038C2 (en) * | 1992-07-29 | 1995-11-30 | Boehringer Mannheim Gmbh | Production and use of antibodies against collagen |
-
1994
- 1994-01-21 WO PCT/EP1994/000164 patent/WO1995004282A1/en active IP Right Grant
- 1994-01-21 AT AT94905104T patent/ATE168779T1/en active
- 1994-01-21 DK DK94905104T patent/DK0711415T3/en active
- 1994-01-21 DE DE59406512T patent/DE59406512D1/en not_active Expired - Lifetime
- 1994-01-21 ES ES94905104T patent/ES2122231T3/en not_active Expired - Lifetime
- 1994-01-21 JP JP7505505A patent/JP2703116B2/en not_active Expired - Fee Related
- 1994-01-21 EP EP94905104A patent/EP0711415B1/en not_active Expired - Lifetime
- 1994-01-21 CA CA002167496A patent/CA2167496A1/en not_active Abandoned
-
1996
- 1996-01-29 FI FI960392A patent/FI960392A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPH08509294A (en) | 1996-10-01 |
| WO1995004282A1 (en) | 1995-02-09 |
| JP2703116B2 (en) | 1998-01-26 |
| ATE168779T1 (en) | 1998-08-15 |
| EP0711415A1 (en) | 1996-05-15 |
| FI960392A0 (en) | 1996-01-29 |
| EP0711415B1 (en) | 1998-07-22 |
| ES2122231T3 (en) | 1998-12-16 |
| FI960392A (en) | 1996-01-29 |
| DK0711415T3 (en) | 1999-04-26 |
| DE59406512D1 (en) | 1998-08-27 |
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| EEER | Examination request | ||
| FZDE | Discontinued |