JP2009278908A - Anti-osteocalcin antibody and immunity-measuring method using the same - Google Patents

Abstract

An object of the present invention is to provide a novel anti-osteocalcin antibody capable of highly sensitive immunoassay for osteocalcin and a highly sensitive immunoassay method for osteocalcin using the antibody.
A peptide comprising an amino acid sequence obtained by introducing at least one amino acid mutation into the amino acid sequence of a heavy chain variable region (VH) of an anti-osteocalcin antibody, and detecting sensitivity of osteocalcin in open sandwich immunoassay Is improved compared to the case of using a peptide before introduction of the amino acid mutation.
[Selection figure] None

Description

  The present invention relates to an anti-osteocalcin antibody suitable for open sandwich immunoassay and an osteocalcin immunoassay method using the same.

  The total number of osteoporosis patients in Japan is estimated to be 10 million, about 8 million women and about 2 million men, and this figure is expected to increase as the population ages. Since severe osteoporosis patients are forced to go to bed, early detection by regular diagnosis is important. As a marker molecule for osteoporosis, a vitamin K-dependent calcium-binding protein of 49 amino acids and 5900 molecular weight called osteocalcin (Bone Gla Protein (BGP)) is known. BGP is closely related to bone turnover (especially bone formation), and measuring blood concentration is an indicator of bone metabolism abnormality or therapeutic effect. Until now, immunoassay has mainly been used for BGP detection (Patent Documents 1 to 5), but because of the sandwich method, measurement of BGP degradation products (peptides) was impossible. Since BGP undergoes degradation in blood, quantification with a BGP peptide is desirable in order to evaluate accurate bone turnover (Non-patent Document 1). A competitive immunoassay can be applied to detect peptides. However, since the blood BGP concentration is as low as several nanograms per milliliter, sensitivity and reproducibility are problematic.

In recent years, a non-competitive BGP-C terminal peptide measurement method using a new immunoassay called the open sandwich (OS) method has been developed (Non-patent Document 2). In the open sandwich (OS) method, a VH region polypeptide and a VL region polypeptide of an antibody that specifically recognizes an antigen are prepared, and one polypeptide is labeled with a reporter molecule to form a labeled polypeptide. A method of immobilizing a polypeptide on a solid phase to form an immobilized polypeptide, contacting the antigen-containing sample and the labeled polypeptide with the solid phase, and measuring the amount of the reporter molecule of the labeled polypeptide bound to the immobilized polypeptide It is. The OS method is a non-competitive immunoassay that utilizes a phenomenon in which association constants of V _H and V _L increase in the presence of an antigen, and has an advantage that higher sensitivity can be obtained than the competitive method. Using the KTM219 anti-BGP antibody, BGP measurement by the OS method has already been performed, and detection sensitivity comparable to the blood concentration has been obtained. However, further sensitivity is required for practical use.

Bone 31, 2002, 62-69 Anal. Chem. 79 (16), 6193-6200 (2007) JP 2007-45834 A JP-A-9-329600 JP-A-6-289020 JP-A-6-78788 JP-A-7-55804

  In the conventional BGP quantification, since it was performed by the sandwich method, only the full-length BGP could be detected. However, BGP released into the blood is subject to degradation by proteases and becomes partially undetectable peptides, so if the subject's protease activity is high, the blood concentration may be estimated low. There was a problem. On the other hand, for the quantification of peptides, instrumental analysis such as mass spectrometry and high performance liquid chromatography and immunoassay by competitive methods are used. However, in instrumental analysis, it is difficult to identify BGP peptides having various lengths from various components contained in serum, and there is a problem that expensive equipment is required. In addition, the immunoassay by the competitive method has a problem in sensitivity. The immunoassay based on the OS method enables a non-competitive immunoassay for BGP peptides, is superior to the competitive method in sensitivity, does not require expensive equipment, and allows simple and rapid diagnosis. So far, an OS immunoassay system using an anti-BGP antibody, KTM219, has been developed, and detection sensitivity (about 1 ng / ml) comparable to normal blood BGP concentration has been obtained. However, when practical application was considered, higher sensitivity was required. That is, the present invention has a problem to be solved by providing a novel anti-osteocalcin antibody that enables highly sensitive immunoassay of osteocalcin (BGP), and a highly sensitive immunoassay method for osteocalcin using the antibody. did.

  So far, an open sandwich ELISA using anti-BGP antibody KTM219 using VH and VL has been reported, and non-competitive detection of BGP peptide has become possible (Lim et al. Anal. Chem. 79, 6193 (2007)). . In order to improve the detection sensitivity of open sandwich ELISA for BGP, a VH (BGP) library in which random mutations were introduced into VH gene fragments by error prone PCR was prepared. The VH (BGP) library was expressed as a phage library in which VH was fused to the surface of bacteriophage particles by incorporating it into a phagemid vector and introducing it into E. coli together with a helper phage in order to improve screening efficiency. In the screening, a VH-displayed phage that was expressed as a fusion protein with E. coli-derived maltose-binding protein (MBP) and further mixed with a biotinylated VL and antigen (BGP peptide) to form a strong complex Was recovered using streptavidin immobilized beads. As described above, as a result of introducing random mutations into VH and performing selection, the mutant O2AG2 whose sensitivity in the OS-ELISA measurement system was improved to about 0.1 ng / ml (N31S, V37I, S58T, H82R in VH) 3 mutations), O2AH2 (having 2 mutations of E10V and T69I in VH), single mutant (having V37I mutation in VH), and triple mutant (V37I, S58T, and H82R in VH) ) The present invention has been completed based on these findings.

  That is, according to the present invention, the peptide comprises an amino acid sequence obtained by introducing at least one amino acid mutation into the amino acid sequence of the heavy chain variable region (VH) of an anti-osteocalcin antibody, There is provided the above peptide, wherein the detection sensitivity of osteocalcin is improved as compared with the case where the peptide before introduction of the amino acid mutation is used.

The present invention further provides a peptide comprising an amino acid sequence having at least one or more of the following mutations in the amino acid sequence of the heavy chain variable region (VH) of an anti-osteocalcin antibody.
(1) A mutation in which the 10th amino acid residue of the amino acid sequence of VH is valine in the Kabat numbering system;
(2) a mutation in which the 31st amino acid residue of the amino acid sequence of VH is serine in the Kabat numbering system;
(3) a mutation in which the 37th amino acid residue of the amino acid sequence of VH is isoleucine in the Kabat numbering system;
(4) a mutation in which the 58th amino acid residue of the VH amino acid sequence is threonine in the Kabat numbering system;
(5) a mutation in which the 69th amino acid residue of the VH amino acid sequence is isoleucine in the Kabat numbering system;
(6) a mutation in which the amino acid residue at position 82 of the amino acid sequence of VH is arginine in the Kabat numbering system;

According to the present invention, any of the following peptides is further provided.
(A) A peptide comprising an amino acid sequence having at least one or more of the following mutations in the amino acid sequence of the heavy chain variable region (VH) of the anti-osteocalcin antibody shown in SEQ ID NO: 2.
(1) a mutation in which the 10th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is valine;
(2) a mutation in which the 31st amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is serine;
(3) a mutation in which the 37th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is isoleucine;
(4) a mutation in which the 58th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is threonine;
(5) a mutation in which the 69th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is isoleucine;
(B) a light chain variable region of an anti-osteocalcin antibody, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted in the amino acid sequence of the peptide described in (a) above A peptide capable of specifically recognizing osteocalcin together with a peptide consisting of the amino acid sequence of (VL).

According to the present invention, any of the following peptides is further provided.
(A) A peptide comprising the amino acid sequence of any one of SEQ ID NOs: 5 to 7.
(B) the amino acid sequence of any one of SEQ ID NOs: 5 to 7, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted, and the light chain variable of an anti-osteocalcin antibody A peptide that can specifically recognize osteocalcin together with a peptide consisting of the amino acid sequence of the region (VL).

According to the present invention, any of the following peptides is further provided.
(A) A peptide comprising the amino acid sequence set forth in SEQ ID NO: 8.
(B) the amino acid sequence of SEQ ID NO: 8, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted, and the light chain variable region (VL) of an anti-osteocalcin antibody A peptide capable of specifically recognizing osteocalcin together with a peptide comprising the amino acid sequence of

The present invention further provides a nucleic acid encoding the above-described peptide of the present invention.
The present invention further provides a recombinant vector comprising the above-described nucleic acid of the present invention.
The present invention further provides a transformant transformed with the above-described recombinant vector of the present invention.

The present invention further provides an osteocalcin detection reagent comprising the above-described peptide of the present invention.
The present invention further provides an osteocalcin detection kit comprising at least the peptide of the present invention described above and a peptide consisting of the amino acid sequence of the light chain variable region (VL) of an anti-osteocalcin antibody.
According to the present invention, there is further provided an osteocalcin immunoassay method comprising contacting the above-described peptide of the present invention with a test sample.

  When the antibody of the present invention was used in an open sandwich ELISA, the detection sensitivity was improved about 10 times compared to KTM219, which is a conventional anti-BGP antibody. As a result, sufficient sensitivity can be obtained even with about 0.1 ng / ml antigen below the lower limit of normal human blood BGP concentration, and a highly reproducible and practical BGP peptide diagnostic kit is provided. be able to. Furthermore, blood BGP levels may increase due to osteoarthritis, hyperparathyroidism, hyperthyroidism, bone metastasis of malignant tumors, multiple myeloma, rheumatoid arthritis, etc., but hypoparathyroidism, In contrast, hypothyroidism and Cushing's syndrome have been shown to induce a decrease in blood concentration. In the conventional sandwich method, since the measurable concentration range is about two digits, two measurement systems in which the measurable range is adjusted to the low concentration side and the high concentration side are necessary. However, the high sensitivity of the present invention extends the measurable range of the open sandwich method to 0.1 to 1000 ng / ml, making it possible to diagnose osteoporosis patients and Cushing syndrome patients using the same measurement system. became. In addition, since the open sandwich measurement system of the present invention enables highly sensitive quantification of BGP degradation products, not only total BGP quantification combining both degradation products and undegraded BGP is possible. In addition, it is possible to evaluate the rate of degradation of BGP in the blood by quantifying the decomposed product and the undecomposed product separated by an ultrafiltration membrane or the like, which is also useful in the field of basic medical research.

Hereinafter, the present invention will be described in more detail.
The peptide of the present invention is a peptide corresponding to the heavy chain variable region (VH) of an anti-osteocalcin antibody, by introducing at least one amino acid mutation into the amino acid sequence of the heavy chain variable region (VH) of the anti-osteocalcin antibody. It is a peptide consisting of the obtained amino acid sequence, and is characterized in that the detection sensitivity of osteocalcin in open sandwich immunoassay is improved compared to the case where the peptide before introduction of the amino acid mutation is used.

A specific example of the peptide of the present invention is a peptide comprising an amino acid sequence having at least one or more of the following mutations in the amino acid sequence of the heavy chain variable region (VH) of an anti-osteocalcin antibody.
(1) A mutation in which the 10th amino acid residue of the amino acid sequence of VH is valine in the Kabat numbering system;
(2) a mutation in which the 31st amino acid residue of the amino acid sequence of VH is serine in the Kabat numbering system;
(3) a mutation in which the 37th amino acid residue of the amino acid sequence of VH is isoleucine in the Kabat numbering system;
(4) a mutation in which the 58th amino acid residue of the VH amino acid sequence is threonine in the Kabat numbering system;
(5) a mutation in which the 69th amino acid residue of the VH amino acid sequence is isoleucine in the Kabat numbering system;
(6) a mutation in which the amino acid residue at position 82 of the amino acid sequence of VH is arginine in the Kabat numbering system;

  The Kabat numbering system refers to the numbering system according to the EU index (Kabat et. Al., Sequences of Immunological Interest, NIH, 1991 Fifth edition) by Kabat et al.

  An example of the amino acid sequence of the heavy chain variable region (VH) of an anti-osteocalcin antibody that can be used in the present invention is the amino acid sequence shown in SEQ ID NO: 2. The amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted, and the amino acid of the light chain variable region (VL) of the anti-osteocalcin antibody A peptide capable of specifically recognizing osteocalcin can be used together with a peptide consisting of a sequence.

  In the present invention, “several” in the “amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted” is specifically 1 to 20, preferably 1 to 10. More preferably, the number is 1 to 5, particularly preferably 1 to 3, and most preferably 1 or 2.

  The peptide of the present invention can be obtained by incorporating a nucleic acid encoding the peptide into an expression vector, introducing the vector into an appropriate host cell, and collecting and purifying the cell or cell culture supernatant. As the vector, a phage or a plasmid that can be propagated autonomously in the host cell or can be integrated into the chromosome of the host cell can be used. As the plasmid DNA, a plasmid derived from Escherichia coli, Bacillus subtilis or yeast can be used, and examples of the phage DNA include λ phage. In the present invention, it is preferable to use a phagemid vector. Since the phagemid vector is a plasmid prepared so as to contain a part of the filamentous phage genome, it is necessary to further infect a helper phage after transforming E. coli using the phagemid vector. As a result, a coat protein for particle formation is supplied to obtain a phage in which helper phage particles and phagemid particles are mixed. As a simpler method, it is also possible to use a phage vector containing a necessary DNA sequence. In the case of a phage vector, it is possible to directly obtain a phage by infecting the phage vector with E. coli, and it is not necessary to use a helper phage.

  Examples of the host used for transformation include bacteria (such as E. coli), yeast, animal cells (such as COS cells and CHO cells), and insect cells, and those that can express the peptide of the present invention. There is no particular limitation. The host transformation method is also not particularly limited, and examples thereof include calcium phosphate method, electroporation method, spheroplast method, lithium acetate method, lipofection method, and the like, and are performed using various commercially available transfection reagents. You can also.

  When the peptide of the present invention is produced intracellularly after culturing the transformant, the peptide of the present invention can be recovered by disrupting the cells. When the peptide of the present invention is produced extracellularly, the culture solution is used as it is, or the cells are removed by centrifugation or the like. Then, normal protein isolation and purification methods, ie, solvent extraction, salting-out with ammonium sulfate, desalting, precipitation with organic solvents, anion exchange chromatography using diethylaminoethyl (DEAE) sepharose, etc. Cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using resin such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography The peptide of the present invention can be isolated and purified by using a single method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing or the like alone or in combination.

  In the present invention, osteocalcin can be immunologically measured by bringing the above-described peptide of the present invention (that is, the heavy chain variable region (VH) of the anti-osteocalcin antibody) into contact with the test sample. For example, a VH fragment or VL fragment of an antibody variable region introduced into a host cell by introducing a vector having a gene encoding the heavy chain variable region (VH and light chain variable region (VL) of an anti-osteocalcin antibody into the host cell. A protein containing one of the above and a phage displaying the other of the VH fragment or VL fragment of the antibody variable region, and the recovered protein containing one of the VH fragment or VL fragment of the antibody variable region and the VH fragment of the antibody variable region Alternatively, the interaction between the VH polypeptide and the VL polypeptide can be evaluated by contacting the antigen with a phage displaying the other of the VL fragments and detecting the complex of the VH fragment, the VL fragment and the antigen. The complex detection of VH fragment, VL fragment and antigen can be performed by the open sandwich immunoassay described below.

  Proteinaceous antigens are generally measured by a method using two types of antibodies called a sandwich method. The sandwich method needs to prepare two types of antibodies that can simultaneously bind to an antigen, but has the advantage of high specificity and sensitivity. However, small molecules with a molecular weight of 1000 or less are too small to be sandwiched between two types of antibodies. That is, since a small molecule having a molecular weight of 1000 or less is a unitary antigen having only one antigenic determinant, it is difficult to sandwich between two types of antibodies. For this reason, such small molecules are usually measured by a method called a competitive method. However, the competitive method has the drawbacks that it is difficult to set conditions, the sensitivity is low, and the measurement operation requires considerable attention.

  The present inventors have reported an immunoassay called an open sandwich immunoassay as a method capable of non-competitively measuring even a small molecule without such drawbacks. This method basically uses the principle that “the variable region (antigen-binding site) of an antibody is unstable without an antigen but is stabilized when an antigen binds”. An antibody is composed of two chains, an H chain and an L chain, and each antigen-binding site is called VH and VL, and these constitute a variable region Fv which is the smallest unit capable of recognizing an antigen. Recently, gene fragments encoding VH and VL can be easily cloned using a phage display method or the like, but the binding between VH and VL is non-covalent and unstable in many cases. In most cases, it is used as a single chain antibody (scFv).

  The present inventors have found that this unstable Fv may be stabilized when an antigen binds, and by using this, the antigen concentration can be measured easily and rapidly with higher sensitivity. That is, a VL fragment is immobilized on a plate, and a VH fragment combined with phage or alkaline phosphatase and a sample containing an antigen are mixed and washed once, and then the phage or enzyme immobilized on the plate is immobilized. It has been found that if the amount is measured, it shows a very good correlation with the antigen amount (UEDA, H. et al. Nature Biotechnol. 14, 1714-1718 (1996)).

According to the present invention, for example, the following measurement kit can be produced.
(1) The VL fragment of the anti-osteocalcin antibody is immobilized on a tube or microplate using biotin-avidin interaction or physical adsorption.
(2) A fusion protein of a VH fragment of an anti-osteocalcin antibody and a reporter enzyme (for example, alkaline phosphatase) is prepared, and this is brought into contact with a solid phase on which VL is immobilized together with a sample for a certain period of time.
(3) After washing, the immobilized enzyme activity is measured and used as an index of the antigen concentration in the sample.

It is also possible to produce the following measurement kit.
(1) The VH fragment and VL fragment of the anti-osteocalcin antibody are labeled with two kinds of fluorescent dyes (for example, fluorescein and rhodamine) that overlap each other in absorption and fluorescence spectra.
(2) These are mixed with the sample, and only the fluorescent dye on the short wavelength side is excited with excitation light after about 5 minutes. By measuring the fluorescence intensity derived from two types of fluorescent dyes, the fluorescence energy transfer phenomenon due to the association of VH / VL can be detected. The ratio of the two fluorescence intensities is used as an index of the antigen concentration in the sample. In this method, the antigen concentration can be measured in a short time and without a washing operation, compared to the previous method.

It is also possible to prepare the following measurement kit.
(1) A fusion protein of two kinds of enzyme fragments (for example, LacZΔα and LacZΔω) that are not active alone or increase in activity when they are brought close to each other, each of the VH fragment and VL fragment of an anti-osteocalcin antibody Expressed in E. coli and purified.
(2) Mix two types of fusion protein and sample, wait for a certain period of time, mix with substrate (eg, luminescent substrate Galacton Plus, Tropix, Bedford, MA), and measure the activity of the fusion protein complex in the sample. It is used as an index of antigen concentration. This method can measure the antigen concentration much more sensitively than the previous two methods, and does not include a washing operation.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Abbreviations used in the following examples are as follows.
2YT: Medium containing 1.6% bactotryptone, 1% yeast extract, 0.5% NaCl
2YTAG: 2YT containing 100 μg / ml ampicillin and 1% glucose
2YTAK: 2YT containing 100 μg / ml ampicillin, 50 μg / ml kanamycin and 0.1% glucose
2YTAG plate: 2YT agar containing 100 μg / ml ampicillin and 1% glucose
LB: Medium containing 1% bacto tryptone, 0.5% yeast extract, 0.5% NaCl
LBAC: LB containing 100 μg / ml ampicillin and 34 μl / ml chloramphenicol
LBAC plate: LB agar containing 100 μg / ml ampicillin and 34 μl / ml chloramphenicol
LBAG: LB containing 100 μg / ml ampicillin and 1% glucose
LBACG plate: LB agar containing 100 μg / ml ampicillin and 34 μl / ml chloramphenicol, 1% glucose
LBAG plate: LB agar containing 100 μg / ml ampicillin and 1% glucose
SOC: Medium containing 2% bactotryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 20 mM glucose, 10 mM MgCl ₂
PBS: 10 mM phosphate buffer (pH 7.2) containing 137 mM NaCl and 2.7 mM KCl
2% MPBS: PBS containing 2% (w / v) skim milk
5% IBPBS: PBS containing 5% (v / v) immunoblock (Dainippon Sumitomo Pharma, Osaka)
20% IBPBS: PBS containing 20% (v / v) immunoblock
PBST: PBS containing 0.1% triton-X100
TE: 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA
TAE buffer: 40 mM Tris-acetate (pH 8.3) containing 1 mM EDTA
TALON buffer: 50 mM sodium phosphate (pH 7.0) containing 300 mM NaCl
TALON eluate: TALON buffer (pH 7.0) containing 500 mM imidazole
IPTG: Isopropyl-β-thiogalactopyranoside
PCIA: phenol: chloroform: isoamyl alcohol (25: 24: 1) mixture
PEG / NaCl: 20% Polyethylene glycol 6000, 2.5 M NaCl
DMSO: dimethyl sulfoxide
BGPC8: A synthetic peptide consisting of the C-terminal 8 amino acid residues of BGP (amino acid sequence: YRRFYGPV)

In all experiments, water purified with Milli-Q (Millipore Co., Billerica, MA) was used. Hereinafter, it is expressed as milliQ water. Unless otherwise indicated, ordinary reagents used were those of Sigma (St. Louis, MO), Nacalai Tesque (Kyoto), Wako Pure Chemical (Osaka), Kanto Chemical (Tokyo). Oligo DNA was synthesized at Texas Genomics Japan (Tokyo) or Invitrogen (Tokyo).
T3000 thermocycler (Biometra, Goettingen Germany) was used for Polymerase chain reaction (PCR), and CEQ ^™ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA) was used for DNA sequencing.

The genotypes of E. coli TG-1 and BL21 (DE3) (pLysS) are as follows.
TG-1: supE, hsd Δ5, thi, Δ (lac-proAB) / F '[traD36, proAB ⁺ , lacI ^q , lacZ ΔM15]
BL21 (DE3) (pLysS): F -, ompT, hsdS B (r B - m B -), gal (λcI 857, ind1, Sam7, nin5, lacUV5-T7gene1), dcm (DE3) [pLysS, Cam r]

The primer sequences used for PCR are shown below.
M13RV: 5'-caggaaacagctatgac-3 '(SEQ ID NO: 9)
VH1for2Not: 5'-ctcatgcggccgctgaggagacggtgaccgtggtccc-3 '(SEQ ID NO: 10)
pHENseq: 5'-ctatgcggccccattca-3 '(SEQ ID NO: 11)

Example 1: Preparation of MBP-VL
MBP-VL was expressed by transforming plasmid pMAL-VL (see Lim et al. Anal. Chem. 79, 6193 (2007)) into E. coli BL21 (DE3) (pLysS) by the heat shock method. 1 μl (about 100 ng) of plasmid and 100 μl of BL21 (DE3) (pLysS) competent cells were mixed and allowed to stand on ice for 30 minutes, then heat shocked at 42 ° C. for 45 seconds and immediately left on ice for 2 minutes. Thereafter, 200 μl of SOC medium was added, cured for 30 minutes, applied to an LBAC plate, and cultured at 37 ° C. overnight.

  The resulting colonies were inoculated into 4 ml of LBACG and cultured with shaking at 30 ° C. overnight. A small amount of the cultured medium (4 ml) was added to 1000 ml of LBAC and shaken at 30 ° C. to perform large-scale culture. When O.D. reached 0.5-0.6, 400 μl of 1000 mM IPTG was added, and further cultured with shaking at 16 ° C. overnight. After separating the cell culture broth into supernatant and Escherichia coli pellets using a centrifuge, MBP-VL is recovered from the supernatant by the ammonium sulfate precipitation method and ultrasonic cell disruption from the pellets, respectively. did.

  After adding 430 g of ammonium sulfate to about 1000 ml of the supernatant and stirring overnight at 4 ° C., insoluble matter containing MBP-VL was collected by centrifugation and suspended in 30 ml of TALON buffer. The pellet was suspended in 30 ml of TALON buffer, transferred to a 50 ml centrifuge tube with a lid, disrupted by a sonicator, and the supernatant containing MBP-VL was collected by centrifugation.

  The solution containing MBP-VL obtained above was dialyzed against TALON buffer and then added to 600 μl of TARON affinity resin (Clontech Laboratories, Inc., Mountain View, Calif.). After stirring with a rotator at 4 ° C. for 20 minutes, the resin was recovered by centrifugation at about 2,000 g. After adding TALON buffer and stirring for 10 minutes, the resin was recovered in the same manner. Thereafter, the TALON eluate was added, and the supernatant containing MBP-VL was collected. Then, the buffer was exchanged with PBS and stored at −80 ° C. with 16% glycerol. The purified protein was confirmed by SDS-PAGE.

Example 2: Biotinylation of MBP-VL 17.9 µl of biotinylated reagent (PEO ₄ -Biotin (PIERCE Biotechonolgy, Rockford, IL) against 1 ml of purified MBP-VL solution (PBS buffer, 500 µg / ml) )) Was added and the reaction was carried out at 4 ° C. for 2 hours. After the reaction, unreacted biotin reagent was removed by dialysis.

Example 3: Preparation of a phagemid vector encoding wild type VH (BGP) First, PCR was performed using spFv (BGP) 9A / pKST2 (see Lim et al. Anal. Chem. 79, 6193 (2007)) as a template. Then, a VH fragment of the anti-BGP antibody KTM219 was prepared.
PCR conditions are as follows.

Reaction solution composition
spFv (BGP) 9A / pKST2 (approximately 100 μg / ml) 1 μl
Forward primer VH1for2Not (50 μM) 1 μl
Reverse primer M13RV (50 μM) 1 μl
10x Ex taq buffer (Mg ²⁺ 20 mM) (Takara bio, Otsu) 10 μl
dNTP Mixture (2.5 mM each) (Takara bio) 8μl
5 U / μl Ex Taq DNA polymerase (Takara bio) 1 μl
milliQ water 78 μl

Reaction cycle
1, 94 ℃ 5 min
2, 94 ℃ 30 sec
3, 55 ℃ 30 sec
4, 72 ℃ 30 sec
(2 to 4 25 times)
5, 72 ℃ 10 min
6, 16 ℃ ∞

The PCR product (VH (BGP)) was electrophoresed on a 1.5% agarose gel (TAE buffer), then a band around 400 bp was cut out and the Wizard ^R SV Gel and PCR Clean-Up System (Promega Co., Madison, WI) ). Subsequently, in order to cleave at the Nco I and Not I sites, the obtained 100 μl VH fragment (2 μg) was added to 1 μl Nco I (Roche Applied Science, Basel,
Switzerland, 10 U), 1 μl Not I (Roche Applied Science 10 U), 4 μl 10x BSA solution, 4 μl 10x H buffer (Roche Applied Science), 15 μl milliQ water, and leave at 37 ° C. overnight did. The restriction enzyme-treated VH fragment was subjected to agarose gel electrophoresis and DNA extraction in the same manner as described above, and dissolved in 25 μl of milliQ water. Mix 2 μl each of VH fragment (about 100 μg / ml) and pIT2 vector (about 870 μg / ml, MRC Cambridge), 2.5 μl T4 DNA ligase (Takara bio, 1250 U), 10 μl 10xT4 buffer (Takara bio ), 83 μl milliQ water was added and reacted at 16 ° C. overnight. After mixing with an equal volume of PCIA to completely inactivate the restriction enzyme, recover the supernatant (74 μl) containing DNA, add 7.4 μl 3 M sodium acetate and 148 μl ethanol, and add -80 μl. After standing at 20 ° C. for 20 minutes and centrifuging at 11,600 g at 4 ° C. for 20 minutes, the obtained precipitate was washed with 70% ethanol, dried and suspended in 20 μl of milliQ water. Subsequently, 4 μl of the ligation reaction solution was added to 100 μl of the TG-1 competent cell for electroporation, the ligation product was introduced into the cells by electroporation, and immediately 900 μl of SOC was added. Incubation was performed at 37 ° C. for 30 minutes. After that, colonies obtained by applying to 2YTAG plate and culturing overnight at 37 ° C were cultured overnight in 4 ml of 2YTAG, and collected from E. coli collected by centrifugation using QIAquick miniprep kit (Qiagen, Hilden, Germany) Plasmid DNA was extracted and sequenced according to the protocol of Beckman Coulter, and the gene structure shown in FIG. 1 was confirmed.

Example 4: Library preparation
Error prone PCR was performed using spFv (BGP) 9A / pKST2 as a template to prepare a VH fragment of the anti-BGP antibody KTM219 with a random mutation introduced.
The conditions for error-prone PCR are as follows.

Reaction solution composition
spFv (BGP) 9A / pKST2 (about 100 μg / ml) 2 μl
Forward primer VH1for2Not (50 μM) 2 μl
Reverse primer M13RV (50 μM) 2 μl
10x Ex taq buffer (Mg ²⁺ free) (Takara bio) 20 μl
10 mM MnCl ₂ 10 μl
100 mM dATP 0.7 μl
100 mM dCTP 0.8 μl
100 mM dGTP 0.4 μl
100 mM dTTP 2.7 μl
2% Triton-X100 10 μl
5 U / μl Taq DNA polymerase (Takara bio) 3.2 μl
milliQ water 141.2 μl

Reaction cycle
1, 94 ℃ 5 min
2, 94 ℃ 1 min
3, 55 ℃ 1 min
4, 72 ℃ 1 min
(2 to 4 30 times)
5, 72 ℃ 10 min
6, 16 ℃ ∞

The PCR product (VH (BGP) library) was electrophoresed on a 1.5% agarose gel (TAE buffer), and then a band around 400 bp was cut out and extracted using the Wizard ^R SV Gel and PCR Clean-Up System. Subsequently, in order to cleave at the Nco I and Not I sites, the obtained 100 μl VH (BGP) library (11 μg) was added to 4 μl Nco I (Roche Applied Science 40 U), 4 μl Not I ( Roche Applied Science 40 U), 15 μl 10 × BSA solution, 15 μl 10 × H buffer (Roche Applied Science), 12 μl milliQ water were added, and the mixture was allowed to stand at 37 ° C. overnight. The VH (BGP) library treated with the restriction enzyme was subjected to agarose gel electrophoresis and DNA extraction as described above. Furthermore, 72 ml of the VH (BGP) library solution was dissolved in 15 μl milliQ after PCIA treatment and ethanol precipitation as described above (yield 4.35 μg).

A vector incorporating the VH (BGP) library was prepared as follows.
First, Nco I and Not I treatment of pIT2 (66 μg, 200 μl) was performed. In order to reduce the contribution of self-ligation, we also cut at the Xho I site between Nco I and Not I at the same time. Restriction enzyme treatment: 10 μl Nco I (Roche Applied Science 100 U), 3 μl Not I (Roche Applied Science 120 U), 3 μl Xho I (Roche Applied Science 40 U), 30 μl 10x BSA solution, 30 μl 10x H buffer (Roche Applied Science), 19 μl milliQ water was added, and the mixture was allowed to stand at 37 ° C. for 6 hours. After the treatment, as described above, agarose gel electrophoresis, DNA extraction, PCIA treatment, and ethanol precipitation were performed to obtain 26 μg of a restriction enzyme-treated vector.

Ligation reaction and transformation of VH (BGP) library and pIT2 vector were performed as follows.
VH (BGP) library (290 μg / ml) and pIT2 vector (870 μg / ml) mixed in 2.3, 2.1 μl, 2.3, or 6.9 μl, respectively, 2.5 μl T4 DNA ligase (1250 U), 10 μl 10xT4 buffer 83 μl milliQ water was added and allowed to react at 16 ° C. overnight. Thereafter, in the same manner as described above, PCIA treatment and ethanol precipitation were performed, and the resultant was suspended in 20 μl of milliQ water. Subsequently, 4 μl of the ligation reaction solution was added to 100 μl of TG-1 competent cell for electroporation, and the ligation product was introduced into the cells by electroporation, and immediately 900 μl of SOC was added. The same operation was repeated a total of 5 times, and after all mixing, incubation was performed at 37 ° C. for 30 minutes. Thereafter, a part was sampled, 377 μl of DMSO was added to the remainder, and the mixture was stored at −80 ° C. From the number of colonies obtained by applying the sampled transformant to a 2YTAG plate and culturing at 37 ° C. overnight, it was estimated that a total of 1.3 × 10 ⁸ transformants were obtained. Several colonies were cultured overnight in 4 ml of 2YTAG, and plasmid DNA was extracted from the E. coli collected by centrifugation with the QIAquick miniprep kit, and the sequence was determined. As a result, it was revealed that 3.3 substitutions were introduced per VH gene, and that frameshift mutations occurred in about 20% of clones.

Transformant (VH (BGP) / pIT2 library) stored above is inoculated into 1000 ml of 2YTAG and cultured at 37 ° C. When OD600 reaches about 0.5, The KM13 helper phage (6.8 × 10 ¹¹ cfu) was added. After standing at 30 ° C. for 30 minutes, the cells were centrifuged at 3,300 g, the supernatant was discarded, resuspended in 320 ml 2YTAK, and stirred in a baffled flask at 30 ° C. for 16 hours at 200 rpm. Furthermore, 80 ml of PEG / NaCl was added to 320 ml of the supernatant collected by centrifugation at 3,300 g, and left on ice for 2 hours. Thereafter, the pellet obtained by centrifugation at 3,300 g for 30 minutes was suspended in 10 ml TE, centrifuged at 11,600 g for 10 minutes to remove E. coli, and the supernatant (VH (BGP) phage library) was recovered.

Example 5: Open sandwich-panning
In order to select clones having strong interaction with VL in the presence of antigen from the VH (BGP) phage library, the following open sandwich panning was performed.
First, 12.5 μl of VH (BGP) phage library (10 ¹⁰ cfu) and biotinylated MBP-VL solution (final concentration 0.5 mg / ml), 250 μl of 2% MPBS containing BGPC8 (final concentration 1 ng / ml) After preparing and stirring with a rotator for 30 minutes at room temperature, it was left still for 60 minutes. Meanwhile, 12 μl of a suspension of streptavidin-immobilized magnetic beads (Dynal Biotech, Oslo, Norway) was mixed with 200 μl of 2% MPBS and allowed to stand at room temperature for 60 minutes, and then a magnetic separator (12-Tube Magnet The beads were collected using (Qiagen)). Thereto was added a mixture of the VH (BGP) phage library, biotinylated MBP-VL solution, and BGPC8, and the mixture was stirred with a rotator for 15 minutes at room temperature. Thereafter, the collected beads were washed three times with PBS-T, 200 μl of 1 mg / ml Trypsin solution was added, and the mixture was stirred for 15 minutes with a room temperature rotator. Divide the phage eluate in half, add one to the glycerol stock, add 900 μl of TG-1 culture (OD ₆₀₀ = 0.4) to the other, leave at 37 ° C for 30 minutes, and centrifuge at 11,600 g for 10 minutes did. The pellet (phage-infected TG-1) was suspended in 50 ml 2TYAG and cultured at 37 ° C. until the OD ₆₀₀ was approximately 0.4. After that, antibody-displayed phages were prepared by adding half to the glycerol stock and the remaining helper phages as described above, followed by panning in the second round. TG-1 infected with the second round of phage eluate was seeded on 2YTAG plates, and the obtained colonies were suspended in 4 ml LBAG and sequenced as described above. To obtain monoclonal VH-displayed phage.

Example 6: Monoclonal phage OS-ELISA
OS-ELISA was performed using the monoclonal VH-displayed phage obtained above.
A PBS solution containing 1 μg / ml of MBP-VL was dispensed at 100 μl per well onto a Falcon 3912 microplate and allowed to stand overnight at 4 ° C. After discarding the solution from the microplate, 200 μl of 20% IBPBS was added thereto, and blocking was performed by leaving at room temperature for 2 hours. Next, after washing the microplate with PBS-T, 100 μl of 5% IBPBS containing 10 ¹⁰ cfu / ml V _H- displayed phage obtained above and BGPC8 at various concentrations (0 to 100 ng / ml) was added, and room temperature was added. And left to stand for 60 minutes. In order to detect the immobilized VH-displayed phage by the above operation, the HRP / anti-M13 Monoclonal Conjugate (GE Healthcare UK Ltd.) diluted 1/5000 with 5% IBPBS after washing the microplate with PBS-T. , Amersham Place, England) was added, and the mixture was allowed to stand at room temperature for 1 hour. After washing the microplate three times with PBS-T, the enzyme reaction solution prepared in advance (50 ml 100 mM sodium acetate pH 6.0, 500 μl 10 mg / ml TMBZ (in DMSO), 10 μl H ₂ O ₂ ) Was added to each well in an amount of 100 μl to initiate the reaction. After reacting in the dark for about 5 minutes, 50 μl of 3.2NH ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm was measured with a plate reader (control was 655 nm). As a result, the detection sensitivity of the monoclonal phage OS-ELISA using the wild-type anti-BGP antibody VH-displayed phage is about 1 ng / ml, whereas the two clones (O2AG2, O2AH2) each have about 0.01 ng / ml. It was revealed that the detection sensitivity was significantly improved at 0.1 ng / ml (FIGS. 2 and 3). As a result of the sequence, four amino acid mutations of N31 → S, V37 → I, S58 → T, and H82 → R are found in V2 of O2AG2 (SEQ ID NO: 5), and E10 → in VH of O2AH2 (SEQ ID NO: 8). Two amino acid mutations of V and T69 → I were confirmed.

Of the four mutations observed in O2AG2, a single mutant (SEQ ID NO: 6) in which only the V37 → I mutation was introduced into the wild type, a triple in which three mutations were introduced: V37 → I and S58 → T and H82 → R. A mutant (SEQ ID NO: 7) was prepared as follows.
First, DNA fragments each containing 4 mutation sites were amplified by PCR using pIT2 encoding wild type and O2AG2. The conditions are shown below.

Reaction solution composition (template) (approx. 100 μg / ml) 1 μl
Forward primer pHENseq (100 μM) 0.5 ml
Reverse primer M13RV (100 μM) 0.5 μl
10x Ex taq buffer (Mg ²⁺ 20 mM) (Takara bio) 10 μl
dNTP mixture (2.5 mM each) (Takara bio) 8 μl
5 U / μl Ex Taq DNA polymerase (Takara bio) 1 μl
milliQ water 79 μl

Reaction cycle
1, 96 ℃ 2 min
2, 94 ℃ 30 min
3, 55 ℃ 30 min
4, 72 18 sec
(2 to 4 25 times)
5, 72 ℃ 5 min
6, 16 ℃ ∞

Each PCR product was purified using Wizard ^R SV Gel and PCR Clean-Up System, and then the 37th DNA fragment amplified from O2AG2 and the other 3 DNA fragments amplified from the wild type were mixed in an equivalent amount. Overlap extension PCR was performed under the following conditions. The primers used were M13RV and pHENseq.

DNA fragment mixture 4 μl
10x Ex taq buffer (Mg ²⁺ 20 mM) (Takara bio) 10 μl
dNTP mixture (2.5 mM each) 8 μl
5 U / μl Ex Taq DNA polymerase (Takara bio) 2 μl
milliQ water 76 μl

Reaction cycle
1, 94 ℃ 5 min
2, 94 ℃ 30 sec
3, 56 ℃ 30 sec
4, 72 ℃ 40 sec
(2 to 4 15 times)
5.Add 1 μl of each primer 100 μM and 1 μl Ex taq (Takara bio)
6, 94 ℃ 5 min
7, 58 ℃ 30 sec
8, 72 ℃ 40 sec
(6 to 8 35 times)
9, 72 ℃ 5 min
10, 16 ℃ ∞

  Similarly, the 31st DNA fragment amplified from the wild type and the other 3 DNA fragments amplified from O2AG2 were mixed and overlap extension PCR was performed. The primers used were M13RV and pHENseq.

  As a result, the obtained single mutant and triple mutant VH fragments were inserted into the phagemid vector pIT2 in the same manner as described above, and monoclonal phage OS-ELISA was performed in the same manner (FIG. 2). As a result, it was suggested that amino acid mutations have an additive contribution to the improvement of detection sensitivity.

FIG. 1 shows the structure of a phagemid vector encoding wild type VH (BGP). FIG. 2 shows the results of an open sandwich ELISA using monoclonal VH-displayed phage. FIG. 3 shows the results of an open sandwich ELISA using monoclonal VH-displayed phage. FIG. 4 shows the VH domain (base sequence is shown in SEQ ID NO: 1, amino acid sequence is shown in SEQ ID NO: 2) and VL domain (base sequence is shown in SEQ ID NO: 3, and amino acid sequence is shown in SEQ ID NO: 3), which is an anti-BGP antibody. 4).

Claims (11)

A peptide comprising an amino acid sequence obtained by introducing at least one or more amino acid mutations into the amino acid sequence of the heavy chain variable region (VH) of an anti-osteocalcin antibody, wherein the detection sensitivity of osteocalcin in open sandwich immunoassay is the above-mentioned amino acid The above peptide, which is improved as compared with the case of using a peptide before introducing a mutation. A peptide comprising an amino acid sequence having at least one or more of the following mutations in the amino acid sequence of the heavy chain variable region (VH) of an anti-osteocalcin antibody.
(1) A mutation in which the 10th amino acid residue of the amino acid sequence of VH is valine in the Kabat numbering system;
(2) a mutation in which the 31st amino acid residue of the amino acid sequence of VH is serine in the Kabat numbering system;
(3) a mutation in which the 37th amino acid residue of the amino acid sequence of VH is isoleucine in the Kabat numbering system;
(4) a mutation in which the 58th amino acid residue of the VH amino acid sequence is threonine in the Kabat numbering system;
(5) a mutation in which the 69th amino acid residue of the VH amino acid sequence is isoleucine in the Kabat numbering system;
(6) a mutation in which the amino acid residue at position 82 of the amino acid sequence of VH is arginine in the Kabat numbering system; Any of the following peptides.
(A) A peptide comprising an amino acid sequence having at least one or more of the following mutations in the amino acid sequence of the heavy chain variable region (VH) of the anti-osteocalcin antibody shown in SEQ ID NO: 2.
(1) a mutation in which the 10th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is valine;
(2) a mutation in which the 31st amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is serine;
(3) a mutation in which the 37th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is isoleucine;
(4) a mutation in which the 58th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is threonine;
(5) a mutation in which the 69th amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 is isoleucine;
(B) a light chain variable region of an anti-osteocalcin antibody, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted in the amino acid sequence of the peptide described in (a) above A peptide capable of specifically recognizing osteocalcin together with a peptide consisting of the amino acid sequence of (VL). Any of the following peptides.
(A) A peptide comprising the amino acid sequence of any one of SEQ ID NOs: 5 to 7.
(B) the amino acid sequence of any one of SEQ ID NOs: 5 to 7, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted, and the light chain variable of an anti-osteocalcin antibody A peptide that can specifically recognize osteocalcin together with a peptide consisting of the amino acid sequence of the region (VL). Any of the following peptides.
(A) A peptide comprising the amino acid sequence set forth in SEQ ID NO: 8.
(B) the amino acid sequence of SEQ ID NO: 8, consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, added and / or inserted, and the light chain variable region (VL) of an anti-osteocalcin antibody A peptide capable of specifically recognizing osteocalcin together with a peptide comprising the amino acid sequence of A nucleic acid encoding the peptide according to any one of claims 1 to 5. A recombinant vector comprising the nucleic acid according to claim 6. A transformant transformed with the recombinant vector according to claim 7. The osteocalcin detection reagent containing the peptide in any one of Claim 1 to 5. 6. An osteocalcin detection kit comprising at least the peptide according to claim 1 and a peptide consisting of an amino acid sequence of a light chain variable region (VL) of an anti-osteocalcin antibody. An immunoassay method for osteocalcin, which comprises contacting the test sample with the peptide according to any one of claims 1 to 5.