JP2559907B2 - Antibodies against human osteocalcin fragments - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antibody which selectively binds to a fragment of human osteocalcin.

[0002]

2. Description of the Related Art Osteocalcin [also known as bone g
la protein (BGP)] is vitamin K in bone.
It is a dependent calcium-binding protein. Its molecular weight is 5,800 and it is composed of 49 amino acid residues. This protein is osteoblast (osteoblast)
Produced from, it accounts for about 20% of the non-collagen protein constituents of bone. This protein contains γ-carb
There are oxyglutamamic acid residues, which have hydroxyapatite and strong affinity, and are therefore presumed to have an important role in bone matrix formation.

This osteocalcin was first found in chicken and bovine bones [Proc. N
atl. Acad. Sci. USA, vol. 7
2, pp3925-3929 (1975), ibid.
73, pp 1447 to 1451 (1976)], and other various human bones including humans [Th.
e Journal of Biological C
chemistry vol. 255, pp86885-8
691 (1980)], the structural similarity is said.

Several groups are challenging the measurement of this osteocalcin. Price et al. [J. Clin. Invest. vol. 66,
pp878-883 (1980)] and Delmas et al. [J. Clin. Invest. vol. 71,
pp1316-1321 (1983)] are cows and humans.
Utilizing the structural similarity of osteocalcin, a human
We are developing a radioimmunoassay for measuring osteocalcin.

Also, Caterwood et al. [Bon
e vol. 6, pp9-13 (1985)], an amino acid sequence common to bovine and human osteocalcin.
A peptide having 37 to 49 residues (C-terminal) counted from the terminal side was synthesized, and a radioimmunoassay was constructed by the same competitive method using the antibody thereof, enabling the measurement of human osteocalcin.

On the other hand, while these methods are measurement methods using a polyclonal antibody, in order to improve the sensitivity and accuracy, Japanese Patent Laid-Open No. 1160493 similarly discloses a monoclonal antibody against bovine osteocalcin ( (Hereinafter referred to as MCA), and the C-terminal ( ⁴⁵ Ph
and MCA recognizes e- ⁴⁹ Val), recognizes the intermediate region ⁽²¹ Gla- ³¹ Glu) sandwich method using two kinds of MCA with MCA is disclosed. Therefore, this method also constitutes a measurement system using the common amino acid sequences of bovine and human osteocalcin to measure the human blood system.

Conventionally, there are various approaches to the measurement of human osteocalcin as described above, but few attempts have been made to selectively measure a specific osteocalcin fragment. This fragment has been shown to appear in bone resorption [J. Cli
n. Invest. vol. 77, pp1762 ~
1769 (1986)]. In an attempt to measure this fragment, we have published its contents in WO 90/09587. However, in this method, it is necessary to measure the total amount of fully human osteocalcin and its fragments, and to calculate the difference between them in combination with the separately measured amount of fully human osteocalcin.

[0008]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an antibody for selectively measuring a human osteocalcin fragment.

[0009]

DISCLOSURE OF THE INVENTION The present invention provides human osteocalcin obtained by immunizing a peptide within the N-terminal 1 to 19 residues of human osteocalcin, the C-terminal of which is the 19th residue. It is an antibody against the fragment. The antibody against the human osteocalcin fragment of the present invention is a peptide within the N-terminal 1 to 19 residues of human osteocalcin, preferably within the N-terminal 10 to 19 residues, and the C-terminal of the peptide is the 19th residue. It is obtained by immunizing. This peptide is also preferably a peptide to which 4 or more amino acids are bound. In addition, it is preferable that this peptide has a functional group having a binding property with a carrier protein, such as cysteine, bonded to its N-terminal side. The antibodies of the present invention are human osteocalcin 1-4.
Human osteocalcin 1-
High reactivity to 19 is preferred. A specific reaction ratio by the solid phase antigen competition method is preferably 2.5 or more, more preferably 10 or more by the solid phase antigen binding method. In the solid-phase antigen competition method, the antigen corresponding to the antibody to be evaluated is solid-phased (solid-phased antigen), and the solid-phased antigen and the separately added antigen compete with each other to select which antibody is selected. The solid-phase antigen-binding method is a method for evaluating whether or not they react with each other, and the antigen corresponding to the antibody to be evaluated and other antigens are both immobilized, and the antibody selectively reacts with which antigen. It is a method of evaluating whether to do.

The antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a polyclonal antibody. Further, the antibody of the present invention includes all antibody molecules, F (ab ') ₂ , Fab' or Fab.
Any one may be used as long as it retains the ability to bind to an antigen, such as a cb fragment. In the case of F (ab ′) ₂ or Fab ′, such a fragment can be obtained by a known method such as a monoclonal antibody or polyclonal obtained as described below, for example, the antibody is cleaved with pepsin to give an F (ab ′) ₂ fragment. Or F (a
b ′) ₂ Fragment is treated to reduce Fa
b'fragment [Niso
noff, A.N. , Et al. : Arch. Bioc
hem. Biophys. 89 230 (196
0), Parham, P .; : J. Immunol.
131 2895 (1983)]. This antibody is
It reacts selectively with the terminal 19-residue human osteocalcin fragment.

The present invention is also an antibody obtained by immunizing a peptide of the N-terminal 45 to 49 residues of human osteocalcin. This peptide is preferably one having a functional group capable of binding to a carrier protein, such as cysteine, attached to its C-terminal side.
This antibody preferably has low reactivity with human osteocalcin 1-49 and high reactivity with N-terminal 45-49, and a specific reaction ratio by the solid phase antigen competition method is 1.5 or more. It is more preferable that the reaction ratio by the solid phase antigen binding method is 5.0 or more.
The antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a polyclonal antibody. In addition, this antibody is a whole antibody molecule, F
As described above, any one may be used as long as it has the ability to bind to an antigen, such as (ab ′) ₂ , Fab ′ or Facb fragment. This antibody selectively reacts with the C-terminal 5-residue human osteocalcin fragment.

Each of the methods for producing the antibody of the present invention will be described below by taking a polyclonal antibody as an example. When the antibody is a monoclonal antibody, the cell fusion method by Keller and Milstein [G. Koehler and Milst
ein, Nature (London), 256 , 49
5-497 (1975)], the hybridoma can be prepared by culturing and secreting the hybridoma and separating from the culture solution. (I) Collection of antibody selectively reacting with N-terminal 19-residue osteocalcin fragment Ia Preparation of immunogen Ia- Preparation of N-terminal ( ¹ Try- ¹⁹ Arg) synthetic peptide N-terminal side of human osteocalcin ^The amino acid sequence described below with H-Cys-Tyr-Leu-Tyr-Gln-Try containing cysteine at the N-terminal of ¹ Try.
-Leu-Gly-Ala-Pro-Val-Pro-
Tyr-Pro-Asp-Pro-Leu-Glu-P
The peptide represented by ro-Arg-OH is synthesized. A peptide synthesizer manufactured by ABI can be used for peptide synthesis. I-a- N-terminal ⁽¹⁵ Pro- ¹⁹ Arg) following amino acid sequence put a cysteine at the N-terminus of the creation peptide ¹⁵ Pro- ¹⁹ Arg synthetic peptide H-Cys-Pro-Leu- Glu-Pro-Arg
The peptide represented by -OH is synthesized.

Binding of Ib Synthetic Peptide to Carrier Protein In immunization, the peptide of the immunogen is converted into a relatively large molecule and administered to the immunized animal. To convert a peptide into a large molecule, there are a method of binding the peptide to a carrier protein and a method of binding to another polymer substance. Examples of the carrier protein include albumin, keyhole limpet hemocyanin (KLH), protein A, etc., but albumin, KLH
Is preferred. In addition, other polymer substances include water-soluble polymers such as polyvinylpyrrolidone. In binding the peptide to the carrier protein,
Use COOH terminal group, NH ₂ terminal group of peptide,
Alternatively, an appropriate functional group capable of binding to the carrier protein is introduced. Carbodiimides are used when utilizing the COOH end group of the peptide, while glutaraldehyde is preferably used when utilizing the NH ₂ end group. Furthermore, when a specific bond is required, a functional group having a property of binding to a carrier protein, such as cysteine, is introduced into the peptide, and a maleimide group or a dithiopyridyl group is introduced into the carrier protein side to bond it. Created by Among them, the method of introducing an appropriate functional group capable of binding to the carrier protein is preferable. Cysteine is preferably used as a suitable functional group. The functional group having the ability to bind to the carrier protein may be introduced either on the N-terminal side or the C-terminal side of the peptide, but among them, the N-terminal side is preferable in the case of an N-terminal ( ¹ Try- ¹⁹ Arg) synthetic peptide.

Preparation of Ic Polyclonal Antibody As a method for immunizing the above-mentioned immunogen, a peptide converted into a high molecular substance is mixed with an adjuvant and administered to an immunized animal to prepare an antibody according to a method known per se. Is adopted. As the adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide or the like is used. Animals to be immunized include goats, rabbits, horses, sheep, dogs, mice, guinea pigs and pigs.

(II) Collection of antibody selectively reacting with C-terminal 5-residue osteocalcin fragment II-a Preparation of immunogen II-a- Preparation of C-terminal ( ⁴⁵ Phe- ⁴⁹ Val) synthetic peptide Peptide ⁴⁵ Phe- Cysteine was added to the C-terminal of ⁴⁹ Val to give the following amino acid sequence: H-Phe-Tyr-Gly-Pro-Val-Cys
The peptide represented by -OH is synthesized. For peptide synthesis, a peptide synthesizer manufactured by ABI can be used. II-b Binding of synthetic peptide to carrier protein Preferably, a conjugate with a carrier protein is prepared in the same manner as in I-b. When forming a conjugate with a carrier protein, it is preferable to introduce a functional group having a binding property with the carrier protein, such as cysteine, into the C-terminal side of the synthetic peptide. II-c Preparation of polyclonal antibody It can be prepared according to I-c.

[0016]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1 (Creation of highly selective antibody at N-terminal 1 to 19 residues) (1) Preparation of immunogen (a) Synthesis of peptide at N-terminal 19 residues Amino acid sequence 19 specific to N-terminal of human osteocalcin 19 Cysteine was added to the N-terminal side of the residue to synthesize the peptide shown in FIG. A peptide synthesizer manufactured by ABI was used for the synthesis. Hereinafter, the name of this peptide is HOst-
It was set to N (19). (A) Synthesis of N-terminal 5 Residue Peptide The peptide shown in FIG. 2 was synthesized by adding cysteine to the N-terminal side of the N-terminal 15 to 19 residue peptide of human osteocalcin. The name of this peptide is HOst-N (1
5-19). (C) Synthetic peptide [HOst-N (19), HOst
-N (15-19)] and carrier protein synthesis Keyhole which is a typical carrier protein
Limpet hemocyanin (KLH) was replaced with N- (m-maleimidobenzoic acid) -N-succinimide ester (MB
It was remaleimidized by S). On the other hand, HOst-N (1
9) or HOst-N (15-19) is freed of SH group with 2-mercaptoethanol to give MBS-ized KL.
HOst-N (19) or HOst-N (15-
19) was added dropwise and the reaction was allowed to react while maintaining the pH at 6.0 to 6.5. After reacting for 3 hours, the mixture was dialyzed, and the obtained product was used as an immunogen. (D) Synthesis of synthetic peptide-carrier protein conjugate for comparison Human osteocalcin N-terminal 1-20
(21st residue cysteine) and human osteocalcin whole molecules 1 to 49 were synthesized by a peptide synthesizer and named as HOst-N (20) and HOst (1-49), respectively. Next, maleimidized KLH and HOst-N (2
0) and a conjugate of HOst (1-49) and KLH was prepared using dicyclohexylcarbodiimide (DCC).

(2) Preparation of antibody (1) Three kinds of synthetic peptides prepared in (c) and (d), HOst-N (15-19) and HOst-N (2)
0), the conjugate of HOst (1-49) and KLH was mixed with complete Freund's adjuvant, and a rabbit was immunized. After confirming the increase in antibody titer, blood was collected and antiserum was collected. On the other hand, HOst-N (19), HOst-N (15-1
9), the KLH conjugate of HOst (1-49) was mixed with complete Freund's adjuvant, and the mouse was immunized. After confirming the increase in antibody titer, blood was collected and antiserum was collected.

(3) Examination of Specificity of N-Terminal 1 to 19 Residues of Antibody (Solid Phase Antigen Competitive Method) N-terminal 1 to 19 [HOst-N (19)] was used as a solid phase of a microplate using its aqueous solution. And after-coating with 1% bovine serum albumin (BSA), the anti-HOst-N (19) antibody prepared in (2) was reacted as a primary antibody. An anti-rabbit IgG / HRP label was reacted as a secondary antibody, and a curve depending on the amount of primary antibody was obtained. Based on this, the optimal antibody amount was determined, and HOst-
The amount of N (19) added again was reacted competitively, and the amount of HOst-N (19) added was plotted along the horizontal axis and the vertical axis corresponds to the amount of anti-HOst-N (19) antibody bound to the solid phase. A calibration curve showing the amount of the obtained was obtained. Using the calibration curve, HOst-
Instead of N (19), native human osteocalcin [HOst (1-49)] was added and a dose-dependent curve was drawn in the same manner. The results are shown in FIG. In the figure,
Black circles show the case where HOst-N (19) was added, and white circles show the case where HOst (1-49) was added.

Similarly, the above operation was carried out using an anti-HOst-N (15-19) antibody to obtain a dose-dependent curve. FIG. 4 shows the results. In the figure, the black triangle is HOst-N.
In the case of adding (19), the white triangle indicates HOst (1-4
The case where 9) is added is shown. M in the dose-dependent curve
ax. The reaction ratio was determined based on the amount of antigen giving 1/2 OD of OD. As a result, the HOst-N (19) selectivity of the antibody was 2.6-fold for the anti-HOst-N (19) antibody and 4.3-fold for the anti-HOst-N (15-19) antibody. .

(4) Antibody HOst-N (19) / HO
Examination of st (1-49) reaction ratio (solid phase antigen binding method) HOst-N (19) and HOst (1-49) were immobilized on a microplate with a buffer solution and after-coated with 1% BSA, Rabbit anti-HOst (1-49) antibody, anti-HOst-N (20) antibody, anti-HOst-N antibody
The reaction was performed using the (15-19) antibody. Then, an anti-rabbit IgG / HRP label was reacted as a secondary antibody, and after washing, a color reaction was performed. A graph was obtained in which the horizontal axis represents the dilution ratio of the antiserum and the vertical axis represents the amount corresponding to the amount of antibody bound to the solid phase (OD _{420 nm} ). This is shown in FIG. The black circle in the figure is HOst
-N (19) bound rabbit anti-HOst (1-49) antibody, filled triangles represent rabbit anti-HOst-N (19) antibody bound, filled squares represent HOst-N.
Rabbit anti-HOst-N (15-19) combined with (19)
Antibodies, open circles are rabbit anti-HOst (1-49) antibodies bound to HOst (1-49), open triangles are HOst (1-49).
49) and the rabbit anti-HOst-N (20) antibody bound to
White squares are rabbit anti-HOs combined with HOst (1-49)
The t-N (15-19) antibody is shown. As a result, rabbit anti-H
Ost (1-49) antibody HOst-N (19) / HO
The st (1-49) reaction ratio is about 1/25 and anti-HOst (1
-49) antibody is highly selective for HOst (1-49), conversely, the same reaction ratio of rabbit anti-HOst-N (15-19) antibody was about 100/1 and HOst-N (19). Highly selective results were obtained.

On the other hand, the mouse anti-HOst-N (20) was similarly prepared.
Antibody, anti-HOst-N (19) antibody and anti-HOs
The reaction was carried out using t-N (15-19) antibody, and the relationship between the dilution ratio of the antiserum and the amount corresponding to the amount of antibody bound to the solid phase was obtained. A graph showing this relationship is shown in FIG. In the figure, black circles are mouse anti-HOst that reacted with HOst-N (19).
-N (20) antibody, black triangles are mouse anti-HOst-N (19) antibodies that reacted with HOst-N (19), and black squares are mouse anti-HOst-N (19) antibodies.
N (15-19) antibody, open circles are HOst (1-49)
Mouse anti-HOst-N (20) antibody that reacted with, and open triangles represent mouse anti-HOst-N (20) antibody that reacted with HOst (1-49).
-N (19) antibody, open squares represent mouse anti-HOst-N (15-19) antibody reacted with HOst (1-49).

As a result, mouse anti-HOst-N (19)
The antibody HOst-N (19) / HOst (1-49) reaction ratio was about 40/1, and mouse anti-HOst-N (15-1).
9) The reaction ratio of the antibody is about 250/1 and HOst-N (1
High selectivity was observed for 9).

Example 2: Preparation of C-terminal 5-residue highly selective antibody (1) Preparation of immunogen (a) Synthesis of C-terminal 5 residue peptide Amino acid sequence 5 specific to C-terminal of human osteocalcin A peptide shown in FIG. 7 was synthesized by adding cysteine to the C-terminal side of the residue, and the name of this peptide was HOst-C.
(5). (A) Synthesis of conjugate of immunogen and carrier protein HOst-C (5) and K were prepared according to Example 1 (1) (c).
A conjugate with LH was synthesized. (2) Preparation of antibody Preparation was carried out according to Example 1 (2).

(3) Examination of specificity of C-terminal 5 residues of antibody (solid-phase antigen competition method) An aqueous solution of C-terminal 5 residues [HOst-C (5)] was immobilized on a microplate and 1%. After-coated with BSA. Then, a primary antibody [anti-HOst-C (5) antibody] is reacted and an anti-rabbit IgG / HRP is used as a secondary antibody.
The label was reacted and a curve dependent on the amount of primary antibody was obtained. The amount of the antibody was fixed at 5 μg / ml, the concentration of HOst-C (5) was changed to several types, and the antibody coexisted with the same antibody to perform a competitive reaction.
Then, after the reaction with a secondary antibody, color development was performed, and HOst was added.
-C (5) amount on the horizontal axis, anti-HOst-C bound to a solid phase
(5) A calibration curve was obtained with the amount of antibody (that is, OD of color development) on the vertical axis. Using the calibration curve, Native human osteocalcin [HOst (1-49)] was added to obtain a dose-dependent curve. The result is shown in FIG. In the figure, the black squares represent the case where HOst-C (5) was added, and the white squares represent HOs.
The case where t (1-49) is added is shown. As a result, the HOst-C (5) selectivity of the antibody was 3.9 times.

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to obtain an antibody having high selectivity for human osteocalcin fragments. Therefore, by using these antibodies, a measurement system with high selectivity for human osteocalcin fragments can be constructed, and the measurement method is expected to reflect bone resorption in bone metabolism.

[Brief description of drawings]

FIG. 1 is the human osteocalcin N synthesized in Example 1.
The peptide of the terminal 19 residues [HOst-N (19)] is shown.

FIG. 2 Human osteocalcin N synthesized in Example 1
Peptides of terminal 15 to 19 residues [HOst-N (15-
19)] is shown.

FIG. 3 shows a dose-dependent curve when an anti-HOst-N (19) antibody was used in Example 1.

FIG. 4 shows the anti-HOst-N (15-1) in Example 1.
9) Shows a dose-dependent curve when an antibody is used.

FIG. 5 shows that in Example 1, rabbit anti-HOst-N (15
-19) shows the selectivity of the antibody for HOst-N (19).

FIG. 6 shows that in Example 1, rabbit anti-HOst-N (15
-19) HO of antibody and anti-HOst-N (19) antibody
The selectivity for st-N (19) is shown.

FIG. 7: Human osteocalcin C synthesized in Example 2
The peptide [HOst-C (5)] of the terminal 5 residues is shown.

FIG. 8 shows a dose-dependent curve when an anti-HOst-C (5) antibody was used in Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C07K 7/08 8517-4H C07K 7/08 (72) Inventor Tadakatsu Nakamoto 4 Asahigaoka, Hino, Tokyo 3-2, Teijin Ltd. Tokyo Research Center (56) Reference JP-A-1-160493 (JP, A) International Publication 90/9587 (WO, A)

Claims (3)

(57) [Claims] 1. The N-terminal side of fully human osteocalcin
Human osteocalcin corresponding to residues 1 to 19 of
An antibody against the fragment, which can be
Thus the reactivity towards the fragment when measured
Is more reactive than fully human osteocalcin
An antibody against human osteocalcin fragment, which is characterized by being 2.5 times or more higher . 2. The human osteocalci according to claim 1.
Antibody to said fragment, said fragment
Has a binding property at 10 to 19 residues as viewed from the N-terminal of
An antibody against a human osteocalcin fragment. 3. The N-terminal of fully human osteocalcin
Human Osteoca corresponding to 45 to 49 residues
An antibody against a lucin fragment,
Reaction to the fragment as determined by law
Responsiveness is the reactivity to fully human osteocalcin
An antibody against a human osteocalcin fragment, which is characterized by being more than 3.9 times higher .