JPH10300749A - Antibody for recognizing collagen fragment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To recognize a collagen fragment specifically and with a high sensitivity by creating an antibody with peptide that is adjusted based on the amino acid sequence of N terminal teropeptide of I-type collagen α 2 chain including amino acid Lys as an immunity source. SOLUTION: An antibody is used to specifically recognize the amino acid residual group 1-7 of the N terminal of I-type collagen α2 chain, namely amino acid sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val (sequence number 1) and is obtained by immunizing an animal with peptide including amino acid sequence that is indicted by at least the sequence number 1 as an immunity source and adjusting serum from the animal. Peptide preferably includes an amino acid sequence being indicted by the sequence number 1 and a plurality of amino acid residual groups are connected before and after it. Further, peptide should normally be as long as 7 amino acids or more and is preferably as long as approximately 15 amino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to type I collagen α2.
The present invention relates to an antibody that recognizes a specific amino acid sequence contained in the N-terminal telopeptide of a chain, an immunoassay for measuring type I collagen or a fragment thereof using the antibody, and a reagent kit used for the immunoassay.

[0002]

2. Description of the Related Art Collagen is a major protein in skin, bone and cartilage and plays an important role in maintaining the structure and function of mature connective tissue. A collagen molecule has a regular helical structure with three polypeptide chains called α chains, forming a rod-like molecule. To date, the existence of two or more types of α-chains is known, and there are five types of major collagens composed of these types, types I, II, III, IV and V. Among them, type I collagen is the most abundant collagen in the body, and is the only collagen detected in calcified bone, and accounts for 90% or more of the bone organic matrix. The telopeptide of type I collagen is a non-helix forming site of collagen molecules, and this region is known to be a portion showing the antigenicity of collagen protein (Chu et al., Na
ture, 310, 337-340 (1984), Click et al., Biochemist.
ry, 9, 4699-4706 (1970), Morganet al., J. Biol. Ch.
em., 245, 5042-5048 (1970), Bernard et al., Bioche.
mistry, 22, 5212-5223 (1983)).

[0003] One characteristic property of mature collagen of certain tissues, especially bone or cartilage, is that adjacent fibers are cross-linked by hydroxylysyl or lysyl pyridinoline (D. Fujimoto et al.). al., J.
Biochem., 83, 863-867 (1978), D. Eyre et al., An
n. Rev. Biochem., 53, 717-748 (1984), D. Eyre, Met.
hods in Enzymology, 144, 115-139 (1987)). These crosslinks are one of the major crosslinks of mature collagen,
A crosslink formed by the condensation of hydroxylysine or lysine in the telopeptide at the N and C terminals of the heavy chain collagen molecule with hydroxylysine or lysine in the triple chain, and is necessary for the completion of collagen fibers. It is something.

[0004] Hydroxylysyl or lysylpyridinoline is excreted as free form or into peptide-bound form in the urine via the blood with the breakdown of collagen due to aging, hormones, mechanical stress, and the like. Since urinary hydroxylysyl or lysylpyridinoline is considered to be almost bone-derived, detection of such compounds in body fluids allows detection of, for example, Page disease, primary hyperparathyroidism, osteoporosis, metastatic bone tumors, etc. Information on the degradation of the resulting mature collagen can be obtained (Fujimoto et al., J. Biochem., 94, 1133-1).
136 (1983), Eyre et al., J. Bone Miner. Res., S210
(1988), Robins et al., In: Christiansen, C., Over.
gaad, K. (eds.) Osteoporosis, Osteopress Aps, Copen
hagen, pp. 465-468 (1990); Paterson, CR, et al.,
Br. J. Cancer, 64, 884-886 (1991), Sano et al., B
r. J. Cancer, 70, 701-703 (1994)).

As for the detection of hydroxylysyl or lysyl pyridinoline, urinary measurement of hydroxylysyl pyridinoline was reported in 1981 (Gunja-Sm).
ithet al., Biochem J., 197, 756-762 (1981)), followed by high performance liquid chromatography (HPLC) (Black et al.
al., Anal. Biochem. J., 169, 197-203 (1988)), enzyme immunoassay (EIA) (Seyedin et al.,
J. Bone Miner. Res., 8, 635-641 (1993), Robins et a
l., J. Bone Miner. Res., 9, 1643-1649 (1994)) have developed a method for measuring total, free and peptide-bound forms of hydroxylysyl or lysyl pyridinoline.

[0006] Hydroxylysyl or lysylpyridinoline is mainly present in the non-helix forming portion (telopeptide portion) at the C-terminus or N-terminus of a collagen molecule, and bone is composed of 90% of type I collagen. A method of measuring the C-terminal or N-terminal portion of type I collagen by utilizing the characteristic described above is called a radioimmunoassay (RIA) (Risteleet).
al., Clin. Chem., 39, 635-640 (1993)), enzyme immunoassay (EIA) (Hanson et al., J. Bone).
Miner., 7, 1251-1258 (1992), Bonde et al., Clin. Ch.
em., 40, 2022-2025 (1994)).

[0007] For the measurement of the N-terminal telopeptide of type I collagen, the cross-linked structure formed by cross-linking of hydroxylysyl or lysyl residues obtained by using the corresponding cross-linked collagen fragment isolated from urine as an immunogen. As such, a monoclonal antibody against a so-called cross-linked peptide is disclosed in WO 89/12824 and WO
No. 91/08478. JP-A-8-
No. 233811 describes a monoclonal antibody that recognizes a specific amino acid sequence (Val-Gly-Leu-Gly) using a synthetic peptide.

[0008] However, recently, it has been reported that the peptide-bound form increases as urinary total hydroxylysyl or lysylpyridinoline increases, or that osteoporosis patients receive the same estrogen preparation or bisphosphonate preparation having the same bone resorption inhibiting action. It has been reported that changes in total hydroxylysyl or lysylpyridinoline differ from changes in free hydroxylysyl or lysylpyridinoline between (Garnero,
P., et al., J. BoneMiner. Res., 10, 641-649 (199
5)), the significance of each measurement is not clear. In addition, the telopeptide moiety may be metabolized and degraded by the liver and kidney, and the significance of the measurement may vary depending on the epitope and sensitivity of the antibody used for the measurement.

[0009]

Accordingly, an object of the present invention is to provide an antibody which specifically recognizes a collagen fragment in a body fluid,
An object of the present invention is to provide an immunoassay having a high specificity or an antigen necessary for producing an antibody.

[0010]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above-mentioned object, and as a result, based on the amino acid sequence of the N-terminal telopeptide of type I collagen α2 containing the amino acid Lys forming a pyridinoline bridge. The present inventors have found that an antibody that specifically and highly sensitively recognizes a collagen fragment can be obtained by preparing an antibody using the peptide prepared as an immunogen as an immunogen, thereby completing the present invention.

That is, the present invention provides an antibody that specifically recognizes the amino acid sequence contained in the N-terminal telopeptide of type I collagen α2 chain shown in SEQ ID NO: 1. According to a preferred embodiment of the present invention, at least SEQ ID NO: 1
The antibody obtained as an immunogen using a peptide containing the amino acid sequence shown in SEQ ID NO: 1; the above antibody having an amino acid sequence shown in SEQ ID NO: 2 is provided.

According to another aspect of the present invention, there is provided an immunoassay for measuring the presence or concentration of type I collagen or a fragment thereof, which comprises using any one of the above-mentioned antibodies; SEQ ID NO.
A competitive immunoassay for measuring the presence or concentration of type I collagen or a fragment thereof, comprising using an antigenic peptide comprising the amino acid sequence shown in 1 above.

According to another aspect of the present invention there is provided a peptide comprising at least the amino acid sequence of SEQ ID NO: 1 for use as an immunogen in combination with a carrier substance. Further, according to another aspect of the present invention, there is provided a reagent kit for use in an immunoassay for measuring the presence or concentration of type I collagen or a fragment thereof, comprising at least one of the above antibodies.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below in more detail. The antibody of the present invention comprises the amino acid residues 1-7 at the N-terminal of the α2 chain of type I collagen, that is, the amino acid sequence Gln-Tyr-As
It specifically recognizes p-Gly-Lys-Gly-Val (SEQ ID NO: 1) and immunizes an animal with at least a peptide containing the amino acid sequence shown in SEQ ID NO: 1 as an immunogen. Is obtained by preparing The peptide used as the immunogen may be any peptide as long as it contains at least the amino acid sequence shown in SEQ ID NO: 1. Examples of the peptide preferably used in the present invention include a peptide containing the amino acid sequence shown in SEQ ID NO: 1 and having a plurality of amino acid residues before and / or after it. Further, the length of the peptide is usually at least 7 amino acids, preferably about 15 amino acids. Most preferably, a peptide having the amino acid sequence shown in SEQ ID NO: 2 is used as the immunogen.

The peptide may be prepared by any method. For example, the N-terminal sequence of the α2 chain of type I collagen Gln-Tyr-Asp-Gly-Lys-Gly-
It is obtained by purifying a collagen fragment having Val (SEQ ID NO: 1), and also by enzymatically or chemically degrading type I collagen (Click et al., Bio).
chemistry, 9, 4699-4706 (1970)). It can also be easily synthesized using a commonly used peptide synthesizer known per se (Merrifield, RB et al., J. A.
m. Chem. Soc., 85, 2149-2154 (1963)). Furthermore, it can also be prepared by preparing a DNA encoding the above peptide, introducing it into an appropriate expression vector, transforming an appropriate host cell, and expressing it.

When immunizing an animal using the peptide thus obtained as an immunogen, an amino acid having a reactive functional group at the amino or carboxyl terminus of the peptide is introduced, and the hapten is bound to a carrier substance. Preferably used for immunogens. Here, examples of the amino acid having a functional group include cysteine, lysine, glutamic acid, and aspartic acid. Carrier substances include bovine serum albumin (BSA), thyroglobulin, keyhole limpet hemocyanin (K
LH) and the like. The coupling between the peptide and the carrier substance can be easily carried out using a suitable binder such as maleimide, glutaraldehyde, carbodiimide and the like.

The animal is immunized with the peptide conjugated to the carrier substance thus obtained. Animal immunization may be performed in the same manner as in the production of normal antibodies. That is, a solution containing the peptide bound to the carrier substance is mixed with an adjuvant, if necessary, and is used for mice, rats, rabbits, guinea pigs,
It is administered subcutaneously or intraperitoneally to an animal such as sheep, which is usually used for producing antibodies. If a booster is performed every two to three weeks after the first immunization, a high-titer antiserum can be obtained. Blood is collected from the immunized animal and serum is prepared. In the present invention, the obtained antiserum can be used as it is without purification, but after inactivating complement by heat treatment of the serum, immunoglobulin is subjected to salting out with ammonium sulfate, ion exchange chromatography or the like. The fraction may be purified. Further, by purifying the antibody using a peptide column on which a specific partial peptide (a peptide containing the sequence shown in SEQ ID NO: 1) is immobilized, the amino acid sequence shown in SEQ ID NO: 1 can be specifically converted. An antibody that recognizes is obtained.

Further, antibody-producing cells are collected from the immunized animal, and fused with cultured cells such as myeloma cells derived from syngeneic animals to produce a hybridoma by a conventional method, and the immunoglobulin fraction is separated from the culture of the hybridoma. By preparing, a monoclonal antibody that recognizes a specific epitope (amino acid sequence shown in SEQ ID NO: 1) can be obtained.

The antibody thus obtained and type I collagen or a fragment thereof contained in a body fluid sample such as serum, plasma or urine (hereinafter, this may be referred to as "antigenic substance")
Can be reacted by a commonly used immunoassay known per se, for example, a sandwich method, a competition method, etc., to detect the degree of antigen-antibody reaction and to determine the presence or concentration of the antigenic substance in the sample.

Either the antigen peptide or the antibody used in the above immunoassay may be labeled with a suitable labeling substance, or may be bound to a solid phase. Here, examples of the labeling substance include enzymes such as peroxidase and alkaline phosphatase; radioactive substances such as [ ¹²⁵ I]; fluorescent substances such as fluorescein and rhodamine; chemiluminescent substances such as acridinium derivatives and isoluminol derivatives; And the solid phase includes, for example, glass such as glass beads and glass test tubes; plastics such as plastic beads, plastic tubes and plastic trays; and latex particles. The extent of the antigen-antibody reaction is determined by a commonly used radioimmunoassay known per se (Radioimmunoassay).
ssay (RIA), latex particle immunoassay (latexpar
ticle immunoassay (LPIA), enzyme immunoassay (enzyme immunoassay; EIA), fluorescence immunoassay (f
luorescence immunoassay (FIA), chemiluminescence immunoassay (CLIA) or chemiluminescence enzyme immunoassay (chemiluminecense enz)
yme immunoassay (CLEIA) or the like.

The antigenic peptide used in the immunoassay may be, for example, a peptide containing at least the amino acid sequence shown in SEQ ID NO: 1, and preferably an N-terminal acetylated peptide is suitable. Specific examples of the antigen peptide include, for example, peptides having the amino acid sequences shown in SEQ ID NOs: 3 and 4. In the present invention, the measurement of an antigenic substance is preferably performed by a sandwich method or a competitive immunoassay, and particularly preferably performed by a competitive immunoassay. The operation procedure is as follows.

That is, a body fluid sample such as serum or urine is mixed with an isotope-labeled antigen and an antibody that reacts with the antigen to form a complex of the antigen and the labeled antigen with the antibody in the sample. Subsequently, the antigen-antibody complex, the free antigen and the antibody are separated by a known method, for example, a polyethylene glycol method, a second antibody method and the like. Next, the antigen concentration in the sample is measured from the amount of isotope in the antigen-antibody complex. In this case, since the binding between the antibody and the isotope-labeled antigen is inhibited depending on the concentration of the antigen in the body fluid sample, by measuring the isotope-labeled antigen in the inhibited antigen-antibody complex, The antigen concentration in the sample can be measured indirectly. Alternatively, a body fluid sample and the antibody are mixed to form an antigen-antibody complex, and then an isotope-labeled antigen is added to further form a labeled antigen-antibody complex. Thereafter, the concentration of the antigen in the sample is measured in the same procedure as in the method described above.

Using the antibody obtained according to the present invention, a reagent kit used for an immunoassay for measuring the presence or concentration of type I collagen or a fragment thereof including the above-mentioned immunoreaction and detection steps, which is known per se, is used. It can be prepared by the method described below. Such a kit is provided with a configuration similar to that of a kit using a normal immune reaction. That is, the reagent kit of the present invention contains at least the antibody of the present invention, and further, as an optional element, a sample diluent,
Wash solution, labeled antibody or labeled antigen peptide, dye, standard peptide, standard peptide for positive control, etc.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Peptide Synthesis Peptide synthesizer (Applied Biosystems Model 430)
A) and the amino acid sequence of the N-terminal telopeptide of the type I collagen α2 chain (SEQ ID NO: 1) according to the standard synthesis protocol of this apparatus based on the 9-fluorenylmethoxycarbonyl (Fmoc) -peptide solid phase synthesis method. ) Having the amino acid sequence shown in SEQ ID NOs: 2 to 4 were synthesized. For synthesis, Cys, Asn, Gl
n is a trityl group; Tyr and Asp are tertiary butyl groups;
As ys, each Fmoc amino acid derivative having a tertiary butyroxycarbonyl group as a side chain protecting group was used, and all other Fmoc amino acid derivatives used did not contain a side chain protecting group.

These Fmoc amino acid derivatives were dissolved in N-methylpyrrolidone, and a peptide was synthesized using 4 equivalents of the Fmoc amino acid derivative with respect to 4-hydroxymethylphenoxymethyl resin (corresponding to 0.25 mmol of amino group). The coupling reaction between the Fmoc amino acid derivative and the resin was carried out in dimethylformamide (DMF) using 1 equivalent of dicyclohexylcarbodiimide (DCC) and 1 equivalent of N-hydroxy-benzotriazole (HOBt) at room temperature for 60 minutes.

After completion of the synthesis, trifluoroacetic acid (TFA)
Using 10 ml, 0.25 ml of ethanedithiol, and 0.25 ml of water, the mixture was reacted at room temperature for 2 hours to cleave the peptide and protecting group from the synthetic resin. Next, the synthetic resin was removed, and the reaction solution was concentrated using an evaporator. The residue was dissolved in water, washed with diisopropyl ether and freeze-dried. The lyophilized crude peptide is
Tskgel 120T (Tosoh Corporation; 6 mm x 250)
eluent A (water, 0.1 μm, 5 μm) column.
% TFA) to eluent B (60% acetonitrile, 40%
% Water, 0.1% TFA) for 60 minutes using preparative high performance liquid chromatography (HPL).
C). The purified peptide is FAB
It was confirmed by mass spectrometry that the peptide had the amino acid sequence shown in SEQ ID NOs: 2 to 4.

As shown in the following examples, the peptide of SEQ ID NO: 2 was used as an immunogen, the peptide of SEQ ID NO: 3 was used as a labeled antigen peptide in the competition method, and the peptide of SEQ ID NO: 4 was used as a standard peptide for preparing a standard curve. .

Example 2 Preparation of immunogen (1) Introduction of maleimide group into porcine thyroglobulin Porcine thyroglobulin (pTG; manufactured by Sigma, 187 m
g) with 10 ml of 0.1 M sodium phosphate buffer (p
H7.2), and 50 mg of sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-
1-Carboxylate (Sulfo-SMCC; Pierce) was added and the mixture was stirred at room temperature for 60 minutes. Then, the reaction mixture was washed with 0.1 M sodium phosphate buffer (pH 7.2).
G-25 column (PD-1) equilibrated with
(0 column; manufactured by Pharmacia) to remove excess reagent.

(2) Preparation of peptide-pTG complex To 10 mg of the maleimidated pTG obtained in (1), 1 ml of sodium phosphate buffer (pH 7.2) was added and stirred, and 0.485 mg of the peptide of SEQ ID NO: 2 was added. Dissolved 0.1 M sodium phosphate buffer (pH 7.2) 0.2
ml was added dropwise and stirred at room temperature for 1 hour. Then 3.3
0.03 ml of a 0.1 M sodium phosphate buffer (pH 7.2) in which 3 mg of cysteine hydrochloride was dissolved was added, and the mixture was further stirred at room temperature for 1 hour. This solution was added to physiological saline 1
L was dialyzed eight times. This dialysate was dispensed and -20
Stored at ° C.

Example 3 Immunization and Blood Collection An emulsion of 1 mg of the peptide-pTG complex obtained in Example 1 and Freund's complete adjuvant was subcutaneously injected into 20 or more backs of rabbits (New Zealand White). Further 0.5 m of peptide-pTG complex every 2 weeks
8 g of Freund's complete adjuvant emulsion
It was injected subcutaneously. Ten days after the eighth immunization, blood was collected from the ear vein to obtain a rabbit antiserum for analysis, and the antibody titer was measured by radioimmunoassay. That is, SEQ ID NO: 3
The antibody titer was measured using the synthetic peptide of [ ¹²⁵ I] -peptide label (labeled antigen peptide), and it was confirmed to be 50,000-fold. Thereafter, whole blood was collected from the carotid artery of the rabbit to obtain serum, and antiserum YMCL02 was obtained.

Example 4 Analysis of Antigen -Resistant Site of Antiserum The reactive site of telopeptide against antiserum YMCL02 was analyzed using the SPOTs system (Cambridge Research Biochemical).
s company). (1) Peptide synthesis According to the SPOTs standard synthesis method based on the Fmoc peptide solid phase synthesis method, 8 consecutive amino acids in the amino acid sequence of SEQ ID NO: 1
8 types of peptides having the amino acid sequence of the residues (SEQ ID NOS: 5 to 5)
12) was synthesized into spots on the SPOTs membrane, respectively. The side chain protecting group of the synthesized peptide is 50% T
Deprotected with FA. The SPOTs membrane with the peptide attached was dried and stored at -20 ° C.

(2) Reactivity test between antiserum and synthetic peptide The reactivity between the antiserum and the peptide synthesized on the membrane was tested according to the SPOTs analysis method. The SPOTs membrane was added to a 200-fold diluted antiserum solution at room temperature.
Shake time. After washing with a Tris buffer (pH 8.0) containing 0.05% Tween-20, a β-galactosidase-labeled anti-rabbit immunoglobulin antibody solution was added.
Shake at room temperature for 2 hours. 0.05% Tween-20
After washing with Tris buffer (pH 8.0),
A -3-indoyl-β-D-galactopyranoside (BCIG) solution was added and shaken at room temperature for 1 hour or more. The reactivity between the antiserum and the synthetic peptide was confirmed by the spot developing a blue color. As shown in Table 1, the antiserum YMCL02 was 2
It was confirmed that the antiserum reacted with Gln-Tyr-Asp-Gly-Lys-Gly-Val (SEQ ID NO: 1), which is a consensus sequence, by reacting with the species peptide.

[0034]

[Table 1] Reactivity of spot number amino acid sequence reactivity with antiserum 1 SEQ ID NO: 5 No 2 SEQ ID NO: 6 No 3 SEQ ID NO: 7 No 4 SEQ ID NO: 8 5 SEQ ID NO: 9 No 6 SEQ ID NO: 10 No 7 SEQ ID NO: 11 Yes 8 SEQ ID NO: 12

Example 5 [ ¹²⁵ I] -peptide label (label
(Antigen peptide) 0.3 mCi of Na ¹²⁵ I was added to 30 μl of 0.5 M phosphate buffer containing 5 μg of the synthetic peptide of SEQ ID NO: 3, and 1.5 μl of chloramine T (1.0 mg / ml) was added. After 5 minutes, 10 μl of ascorbic acid (1.0
mg / ml). Further, 20 μl of a potassium iodide solution (16.6 mg / ml) was added, and the mixture was purified by an HPLC column (TSKgel-ODS120T; manufactured by Tosoh Corporation).

EXAMPLE 6 Competitive Radioimmunoassay 50 μl of urine sample or standard peptide solution of SEQ ID NO: 4
Into a 0.05M borate buffer (pH 8.2; 5 mM ethylenediaminetetraacetic acid, 0.1 M NaCl, 1% BS
A, 250 μl containing 0.05% Tween 20, 0.02% sodium azide) and 100 μl of [ ¹²⁵ I] -peptide label (370 Bq) were added and mixed, and then the rabbit anti-telopeptide antibody obtained in Example 3 was added. (YMCL02) 1
Then, 00 μl was added, mixed, and allowed to stand at 4 ° C. for 20 hours. After adding and mixing 500 μl of goat anti-rabbit γ-globulin antibody, the mixture was allowed to stand at 4 ° C. for 0.5 hour. Centrifugation (3000r
(pm × 20 minutes), the supernatant was removed, and the radioactivity of the precipitate was measured with a gamma-counter (Aloka ARC-600).
The standard curve created by this method is shown in FIG.

Example 7 Urinary N-telopep in healthy subjects and patients
Urine was measured from healthy subjects and four patients with primary hyperparathyroidism and one patient with malignant tumor (HHM) associated with hypercalcemia using the method and calibration curve of Pide Example 6. Subsequently, it was compared with the creatinine value in the urine sample. Creatinine was measured by the Jaffé method, and the amount of telopeptide (the amount of collagen fragments) per 1 μmol of creatinine was calculated. The results are shown in Table 2 below. As is clear from the table, it is considered that in primary hyperparathyroidism and the like, the N-telopeptide level in urine is high, and the amount of dissolved bone is increased.

[0038]

[Table 2] specimen No. Diseases measurement value (ng / [mu] mol creatinine) 1 healthy person 2.01 2 healthy person 3.52 3 healthy subjects 2.89 4 primary hyperparathyroidism 4.98 5 primary hyperparathyroidism 9. 196 Primary hyperparathyroidism 4.24 7 Primary hyperparathyroidism 6.85 8 HHM 24.46

[0039]

The antibody of the present invention has an amino acid sequence Gln-Tyr-As contained in the type I collagen α2 chain N-terminal telopeptide.
It specifically recognizes p-Gly-Lys-Gly-Val (SEQ ID NO: 1), and can specifically measure type I collagen or a fragment thereof using the antibody. In diseases such as primary hyperparathyroidism, crosslinked collagen degradation products are considered to show high levels, and the antibodies and immunoassays of the present invention are useful for measuring the amount of bone lysis in the disease. is there.

[0040]

[Sequence list]

SEQ ID NO: 1 Sequence length: 7 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence

SEQ ID NO: 2 Sequence length: 15 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Cys Gly Gly Asn Phe Ala Ala Gln Tyr Asp Gly Lys Gly Val Gly 1 5 10 15

SEQ ID NO: 3 Sequence length: 17 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Location: 1 Other information: Xaa = Ac-Arg (acetylarginine) sequence Xaa Glu Asp Gly Gly Asn Phe Ala Ala Gln Tyr Asp Gly Lys Gly Val Gly 1 5 10 15

SEQ ID NO: 4 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 5 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 6 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 7 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 8 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 9 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 10 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 11 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 12 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

[0052]

[Brief description of the drawings]

FIG. 1 shows a standard curve of type I collagen α2 chain N-telopeptide by competitive radioimmunoassay (RIA).

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Masato Takada 8-5-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref.

Claims (7)

[Claims] 1. Type I collagen α shown in SEQ ID NO: 1.
An antibody that specifically recognizes an amino acid sequence contained in a two-chain N-terminal telopeptide. 2. The method according to claim 1, wherein a peptide comprising at least the amino acid sequence shown in SEQ ID NO: 1 is obtained as an immunogen.
The antibody according to 1. 3. The antibody according to claim 2, wherein the amino acid sequence is the sequence shown in SEQ ID NO: 2. 4. An immunoassay for measuring the presence or concentration of type I collagen or a fragment thereof, wherein the antibody according to claim 1 is used. 5. The presence or concentration of type I collagen or a fragment thereof, comprising using the antibody according to claim 1 and a peptide comprising at least the amino acid sequence shown in SEQ ID NO: 1. Competitive immunoassay to measure. 6. A peptide comprising at least the amino acid sequence shown in SEQ ID NO: 1 for use as an immunogen in combination with a carrier substance. 7. A reagent kit for use in an immunoassay for measuring the presence or concentration of type I collagen or a fragment thereof, comprising at least the antibody according to claim 1.