JP2009050269A - Antibody specifically binding to human hmg-1 and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody binding to human HMG-1, but not binding to human HMG-2 and a reagent for immunologically measuring the human HMG-1, obtaining accurate measured value of HMG-1 not involving error, without measuring HMG-2 therein, and making operation for measurement simple and enabling automation, and to provide a method for immunologically measuring the human HMG-1. <P>SOLUTION: The present invention provides an antibody binding to human HMG-1, but not binding to human HMG-2 and provides a method for producing the antibody. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an antibody that specifically binds to human high mobility group protein-1 that can be a marker for diseases such as sepsis, and an immunoassay reagent and immunity for human high mobility group protein-1 using this antibody. It is related to the method of measuring.

  The present invention is useful in the field of life science such as clinical examination, clinical pathology, immunology and medicine, and in the field of chemistry such as analytical chemistry.

  High Mobility Group Protein (hereinafter sometimes abbreviated as “HMG”) was discovered in 1964 as a large amount of non-histone protein contained in chromatin structure and is universally included in all higher animals and plants. It has a very high degree of conservation of primary structure among tribes. It has also been found that it exists not only in the nucleus but also in the cytoplasm. Although the physiological effect is not clearly understood, HMG relaxes the double helix structure when binding to DNA, so that the transcriptional activity is increased by changing the higher order structure of DNA to the optimal structure during the transcription reaction. It is thought to function as a very wide range of transcriptional promoters and nucleosome relaxing factors.

  There are several types of HMG. For example, high mobility group protein-1 (HMG-1), high mobility group protein-2 (HMG-2), high mobility group protein-3 (HMG-3), high mobility group protein-8 (HMG-8), High Mobility Group Protein-17 (HMG-17), High Mobility Group Protein-I (HMG-I), High Mobility Group Protein-Y (HMG-Y), High Mobility Group Protein-I (Y) (HMG-I ( Y)), high mobility group protein IC (HMG IC) and the like.

  In addition, when the present inventors analyzed the homology of amino acid sequences using genetic information processing software “GENETYX” (Software Development), bovine HMG-1 was compared with human HMG-1. Was 98.6% and porcine HMG-1 homology was 99.1%. Similarly, human HMG-2 has a homology of 81.2%, bovine HMG-2 has a homology of 72.3%, and porcine HMG-. The homology of 2 was 79.4%.

  In 1999, Wang et al. Performed quantitative measurement of HMG-1 in serum (in blood) for the first time by Western blotting using a polyclonal antibody prepared using HMG-1 itself as an immunogen. As a result, Wang et al. Showed that HMG-1 can be a marker for sepsis. Then, it was shown that it is possible to accurately determine HMG-1 in blood in order to discriminate between surviving patients and patients dying among septic patients. That is, the usefulness of not only confirming the presence of HMG-1 in blood but also precise quantification was revealed (H. Wang et al., SCIENCE, Vol. 285, No. 9, 248-251). Page, 1999).

It has been previously shown that antibodies used for the measurement of HMG-1, that is, antibodies that bind to HMG-1, can be prepared by Parkinen et al. And Rep et al. (J. Parkkinen et al., The Journal of Biological Chemistry, 268, 26, 19726-19738, published in 1993; WA Lepp et al., Journal of Immunoassay, 10, 4, 449-465, published in 1989). Using this antibody, Rep et al. State that Solid-phase Enzyme Immunoassay is possible for HMG-1. (In this solid-phase enzyme immunoassay, purified HMG-1 is immobilized on a well of a microplate (microtiter plate), and an antibody that binds to enzyme-labeled HMG-1 is brought into contact therewith to act. It was confirmed that the antibody that binds to HMG-1 binds to purified HMG-1.)
However, since these antibodies are polyclonal antibodies prepared using HMG-1 itself as an immunogen, its amino acid sequence is highly homologous to HMG-1 (81.2%) and binds to HMG-2. It was an end.

  That is, the measuring method of HMG-1 shown by Rep et al. Or Wang et al. Was a measuring method that measured not only HMG-1 but also HMG-2.

  In 2000, Luhiainen et al. Also developed a polyclonal antibody prepared by using genetically engineered rat HMG-1 itself as an immunogen and a part of the amino acid sequence of HMG-1 “Lys Phe Lys Asp Pro Asn”. Using each of the polyclonal antibodies prepared using the peptide consisting of “Ala Pro Lys Arg Pro Pro Ser Ala” as an immunogen, HMG-1 in human blood was measured by an ELISA sandwich method (A. Rouhiainen et al., Thromb). Haemost, 84, 1087-1094, 2000).

  However, the polyclonal antibody prepared by using rat HMG-1 itself as an immunogen used here binds not only to HMG-1 but also to HMG-2 for the reasons described above.

  In addition, a polyclonal antibody prepared by using a peptide consisting of a part of the amino acid sequence of HMG-1 “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” as an immunogen is an amino acid sequence of the peptide used as an immunogen. However, since it contains a part of the amino acid sequence of HMG-2 and is a polyclonal antibody, it also binds not only to HMG-1 but also to HMG-2.

  Therefore, the measurement method disclosed by Ruhiainen et al. Is also a measurement method that measures not only HMG-1 but also HMG-2.

 Thus, until now, there has been no known antibody that binds to HMG-1 but does not bind to HMG-2.

  Therefore, there is no measurement method capable of measuring only HMG-1 without measuring HMG-2 other than the measurement method by the Western blot method of Wang et al. (In this Western blot method, HMG-1, HMG-2, etc. are electrophoresed, the antibody is brought into contact with and bound to this carrier or a carrier to which this carrier has been transferred. Therefore, since the migration positions of HMG-1 and HMG-2 (mobility due to the difference in molecular weight) are different, HMG-1 and HMG-2 can be separated.)

  As described above, an antibody that binds to HMG-1 but does not bind to HMG-2 is not known and did not exist. For this reason, except for the method of Western blotting by Wang et al., An immunological measurement method that can measure only HMG-1 which can be a marker for diseases such as sepsis and does not measure HMG-2. There was no immunoassay reagent. That is, in immunological measurement methods and immunological measurement reagents other than Western blotting, even when trying to measure HMG-1 in a sample, HMG-2 contained in the sample is also measured, and therefore positive. A measurement value of HMG-1 including an error is obtained. This HMG-2 is also measured, and the measured value of HMG-1 including a positive error may cause a diagnosis of a disease such as sepsis to be mistaken.

  The Western blot method is a measurement method that is complicated in operation and requires skill in the technique, and therefore, the number of measurers and measurement facilities that can be implemented is limited. In the Western blot method, it is difficult to automate the measurement.

  Therefore, a measuring method and a measuring reagent that can accurately measure only HMG-1 without measuring HMG-2, and that are simple in operation or can be automated. Was demanded.

  On the other hand, the first object of the present invention is to provide an antibody that binds to human HMG-1 but does not bind to human HMG-2.

  In addition, the second problem of the present invention is that HMG-2 is not measured, an accurate measurement value of HMG-1 that does not include an error can be obtained, and the measurement operation is simple and can be automated. Another object is to provide an immunological measurement reagent for human HMG-1.

  The third problem of the present invention is that HMG-2 is not measured, an accurate measurement value of HMG-1 that does not include an error can be obtained, and the operation is simple and can be automated. It is to provide a method for immunological measurement of human HMG-1.

The present invention includes the following inventions.
(1) An antibody that binds to human high mobility group protein-1 but does not bind to human high mobility group protein-2.
(2) The following formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
The antibody according to (1) above, which is prepared by using a peptide consisting of the amino acid sequence represented by
(3) Features of [1] and [2] below:
[1] The following formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
Consisting of an amino acid sequence obtained by performing deletion, substitution, insertion or addition, or modification of one to several amino acid residues to the amino acid sequence represented by
[2] When an antibody is prepared using the peptide as an immunogen, an antibody that binds to human high mobility group protein-1 but does not bind to human high mobility group protein-2 can be obtained.
The antibody according to (1) above, which is prepared by using a peptide having the above as an immunogen.
(4) Features of [1] and [2] below:
[1] The following formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
Consisting of an amino acid sequence obtained by performing deletion, substitution, insertion or addition, or modification of one to several amino acid residues to the amino acid sequence represented by
[2] It can bind to an antibody prepared using a peptide comprising the amino acid sequence represented by the formula (I) as an immunogen,
The antibody according to (1) above, which is prepared by using a peptide having the above as an immunogen.
(5) The antibody according to any one of (1) to (4), which is a monoclonal antibody.
(6) Use of the antibody according to any one of (1) to (5) above in an immunological measurement method for measuring human high mobility group protein-1 contained in a sample using an antigen-antibody reaction An immunological measurement method comprising:
(7) An immunoassay reagent for measuring human high mobility group protein-1 contained in a sample using an antigen-antibody reaction, wherein the antibody according to any one of (1) to (5) above An immunological measurement reagent comprising:

  The antibody of the present invention is an antibody that binds to human HMG-1 but does not bind to human HMG-2 having a very high homology with human HMG-1.

  Further, the human HMG-1 immunological measurement reagent of the present invention can measure only HMG-1 without measuring HMG-2, and the measurement operation is simple and can be automated. Measurement reagent. Thereby, an accurate measurement value of HMG-1 that does not include an error can be obtained, and erroneous diagnosis can be prevented in diagnosis of diseases such as sepsis.

  And the immunological measurement method of human HMG-1 of the present invention can measure only HMG-1 without measuring HMG-2, and the measurement operation is simple and can be automated. Measurement method. Thereby, an accurate measurement value of HMG-1 that does not include an error can be obtained, and erroneous diagnosis can be prevented in diagnosis of diseases such as sepsis.

Hereinafter, the present invention will be described in detail.
1. Antibodies that bind to human HMG-1 but do not bind to human HMG-2 (1) General Remarks The present invention binds to human high mobility group protein-1 (human HMG-1) but does not An antibody that does not bind to mobility group protein-2 (human HMG-2) (hereinafter sometimes referred to as “the antibody of the present invention”) is an antibody that binds to human HMG-1 and does not bind to human HMG-2. Anything is acceptable.

The antibodies of the present invention include any type of polyclonal antibodies, antisera consisting of polyclonal antibodies, or monoclonal antibodies, and fragments of these antibodies (Fab, F (ab ′) ₂ or Fab ′ etc.) Is also included.
The antibody of the present invention is preferably a monoclonal antibody because the absorption operation can be omitted in the preparation operation.

The method for obtaining the antibody of the present invention is not particularly limited, and examples thereof include the following methods.
[1] By comparing and comparing the amino acid sequence of HMG-1 and the amino acid sequence of HMG-2, the amino acid sequence of HMG-1 having low homology between HMG-1 and HMG-2 is selected. Next, a peptide or protein containing this amino acid sequence is prepared and obtained, and this is used as an immunogen to immunize an animal to obtain the produced antibody. Then, it is confirmed that the antibody binds to human HMG-1 and does not bind to human HMG-2, or such an antibody is selected to obtain the antibody of the present invention.

  The amino acid sequence is preferably a highly hydrophilic portion. This is because the higher the hydrophilicity, the higher the possibility that the amino acid sequence is present on the surface of the HMG-1 molecule, and therefore the higher the possibility that an antibody produced using this as an immunogen can bind to HMG-1. .

[2] A peptide or protein containing all or part of the amino acid sequence of HMG-1 is prepared and obtained, and the animal is immunized using this as an immunogen to obtain the produced antibody. A solution containing the produced antibody and HMG-2 immobilized on a solid phase is brought into contact, and an antibody that binds to HMG-2 is bound to the solid phase via the immobilized HMG-2. Next, by separating the carrier and the solution, an antibody that binds to HMG-2 is removed from the solution, and an antibody that binds to human HMG-1 and does not bind to human HMG-2 is obtained.

[3] The amino acid sequence of HMG-1 is compared with the amino acid sequence of HMG-2, and the amino acid sequence of HMG-1 having low homology between HMG-1 and HMG-2 is selected. Next, a peptide or protein containing this amino acid sequence is prepared and obtained, and this is used as an immunogen to immunize an animal to obtain the produced antibody. A solution containing the produced antibody and HMG-2 immobilized on a solid phase is brought into contact, and an antibody that binds to HMG-2 is bound to the solid phase via the immobilized HMG-2. Next, by separating the carrier and the solution, an antibody that binds to HMG-2 is removed from the solution, and an antibody that binds to human HMG-1 and does not bind to human HMG-2 is obtained.

  In this case as well, the amino acid sequence is preferably a highly hydrophilic portion. This is because the higher the hydrophilicity, the higher the possibility that the amino acid sequence is present on the surface of the HMG-1 molecule, and therefore the higher the possibility that an antibody produced using this as an immunogen can bind to HMG-1. .

  The method [1] is a method suitable for obtaining a monoclonal antibody, and the methods [2] and [3] are suitable methods for obtaining a polyclonal antibody or antiserum. And, the method [2] is more complicated to operate than the method [1], and the operation [3] is more complicated than the method [2]. Since it is complicated, the method [1] is more preferable from the viewpoint of operation.

(2) Immunogen The immunogen for producing the antibody of the present invention will be described below.
As described above, as an immunogen for producing the antibody of the present invention, a peptide or protein containing all or part of the amino acid sequence of HMG-1, or low homology between HMG-1 and HMG-2 A peptide or protein containing the amino acid sequence of HMG-1 can be used.

  The amino acid sequence is preferably a highly hydrophilic portion. This is because the higher the hydrophilicity, the higher the possibility that the amino acid sequence is present on the surface of the HMG-1 molecule, and therefore the higher the possibility that an antibody produced using this as an immunogen can bind to HMG-1. .

  The estimation of the hydrophilicity of each amino acid residue of this amino acid sequence is carried out by the method of Hop et al. (TP Hopp et al., Proc. Natl. Acad. Sci. USA, 78, 3824- 3828, published in 1981) or by Parker et al. (Parker et al., Biochemistry, Vol. 25, pages 5425-5432, published in 1986).

  The inventors of the present invention have described the amino acid sequence of human HMG-1 (L. Wen et al., Nucleic Acids Res., 17, pp. 1197-1214, published in 1989) according to the method of Hop et al. The estimation of the hydrophilicity of the residue was performed. The results are shown in FIG.

  In this figure, the horizontal axis indicates the order from the N-terminal of each amino acid residue of human HMG-1. On the other hand, the vertical axis indicates that the higher the value is on the positive side, the higher the hydrophobicity is. On the contrary, the higher the value is on the negative side, the higher the hydrophilicity is.

Next, the present inventors converted the highly hydrophilic sequence of the amino acid sequence of human HMG-1 into the amino acid sequence of human HMG-2 (M. Yoshida et al., J. Biol. Chem., 267, 6641-1). 6645, published in 1992).
Then, from this highly hydrophilic amino acid sequence, the amino acid sequence of human HMG-1 having low homology between human HMG-1 and human HMG-2 was selected.

  The amino acid sequence selected by the present inventors is “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly from the N-terminal amino acid residue (glycine) to the 11th amino acid residue (lysine) of human HMG-1. Lys ".

  As will be described later, an antibody prepared using a peptide consisting of this amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” as an immunogen binds to human HMG-1, but does not bind to human HMG-2. An antibody that does not bind (an antibody of the present invention).

  The antibody of the present invention can be obtained by preparing as an immunogen a peptide consisting of the amino acid sequence represented by this “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”. As an immunogen for producing glycine, 1 to several amino acids (usually 1 to 8, preferably 1 to 6) in the amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” It may be a peptide consisting of an amino acid sequence obtained by deletion, substitution, insertion or addition, or modification of a residue.

  The amino acid sequence of human HMG-2 corresponding to the amino acid sequence of human HMG-1 is “Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys”, which is the sixth amino acid residue from the N-terminal “ Since “Lys” (HMG-1) is simply replaced by “Asn” (HMG-2), and the only difference in the amino acid sequence between human HMG-1 and human HMG-2 is this point, human HMG Among the immunogens of an antibody that binds to -1 but does not bind to human HMG-2 (the antibody of the present invention), an immunogen derived from the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” It is considered essential to include the sixth amino acid residue “Lys” from the N-terminal of human HMG-1.

  Since there is a report that an antibody can recognize an amino acid sequence consisting of three amino acids (F. Hudecz et al., J. Immunol. Methods, 147, 201-210, published in 1992), Among antibody immunogens, “Pro Lys Lys”, “Asp Pro Lys”, or “Lys Lys Pro” can be defined (considered) as the minimum unit of an immunogen derived from the amino acid sequence. Furthermore, those obtained by adding other amino acids or peptides to these tripeptides can be considered as immunogens of the antibody of the present invention.

Deletion of one to several amino acid residues in the amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” as an immunogen for producing the antibody of the present invention, A peptide comprising an amino acid sequence obtained by substitution, insertion or addition, or modification satisfies one of the following conditions.
(A) a peptide capable of obtaining an antibody that binds to human HMG-1 but does not bind to human HMG-2 when an antibody is prepared using the peptide as an immunogen, or
(B) A peptide capable of binding to an antibody prepared using a peptide having an amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” as an immunogen.

  In addition, when following the method of [2] of said (1), the peptide or protein which contains all or one part of the amino acid sequence of HMG-1 can be prepared and acquired, and this can also be made into an immunogen.

  As the aforementioned immunogen, a peptide consisting of an amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”, an amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” A peptide comprising an amino acid sequence obtained by deletion, substitution, insertion or addition, or modification of one to several amino acid residues, or a peptide or protein comprising all or part of the amino acid sequence of HMG-1 It can be extracted and purified from body fluids, cells, tissues or organs of humans or other animals by a known method.

  The method for obtaining HMG-1 or HMG-2 from human thymus, porcine thymus, bovine thymus, human placenta, neutrophil, HL-60 cell line, etc. is described in the following documents (H Goodwin et al., Biochema Biophysica Acta, 405, 280-291, published in 1975; M. Yoshida et al., J. Biochem., 95, 117-124, published in 1980; 530, 39-46, 1992).

  Since a mixture of bovine HMG-1 and bovine HMG-2 is sold by Wako Pure Chemical Industries, only bovine HMG-1 can be purified and obtained.

  Each of the immunogens can be synthesized by a peptide synthesis method such as a liquid phase method and a solid phase method, and an automatic peptide synthesizer may be used. 1 “Protein Chemistry IV”, Tokyo Kagaku Doujin, 1975, Izumiya et al. “Basics and Experiments of Peptide Synthesis”, Maruzen, 1985, “Sequence Chemistry Laboratory 2 Protein Chemistry” edited by Japanese Biochemical Society , 1987, etc., and it is easy to produce a mutant in which the amino acid sequence is deleted, substituted, inserted or added. In addition, modifications such as introduction of unnatural amino acids, chemical modification of each amino acid residue, or cyclization of the interior of the molecule by introduction of cysteine residues to stabilize the structure may be performed.

  Furthermore, each of the immunogens may be prepared from DNA or RNA having a corresponding nucleobase sequence by using genetic engineering technology, edited by the Japanese Biochemical Society, “Sequential Biochemistry Experiment Course 1 Gene Research Method I”, Tokyo Chemical Doujin, 1986, Japan Biochemical Society, “Sequential Biochemistry Course 1, Genetic Research Method II”, Tokyo Chemical Doujin, 1986, Japan Biochemical Society, “Sequential Biochemistry Experimental Course 1, Genetic Research Method III”, Tokyo Chemical Doujin 1987 and the like.

  By the way, when the immunogen is a low molecular weight substance, it is common to immunize an animal or the like with a carrier bound to the immunogen. However, a specific antibody against the peptide having 5 amino acids is used as an immunogen. Since there is a report that it was produced (Kiyama et al., “Abstract 3 of the 112th Annual Meeting of the Japanese Pharmaceutical Society”, page 122, published in 1992), it is not essential to use a carrier.

  In addition, as a carrier when using a carrier when producing the antibody, mussel hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, poly Known carriers such as alanyl lysine, dipalmityl lysine, tetanus toxoid or polysaccharide can be used.

The immunogen and carrier binding methods include glutaraldehyde method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, maleimidobenzoyl-N-hydroxysuccinimide ester method, bisdiazotized pentidine method or N-succimidyl- A known binding method such as a 3- (2-pyridyldithio) propionic acid method can be used.
Moreover, what made the immunogen adsorb | suck to carriers, such as a nitrocellulose particle | grain, polyvinylpyrrolidone, or a liposome, can also be used as an immunogen.

(3) Antibody acquisition method
[1] Polyclonal antibody and antiserum In the antibody of the present invention, the polyclonal antibody or antiserum can be obtained by the following procedure.

First, a mammal (mouse, rabbit, rat, sheep, goat, horse, etc.) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier.
The amount of immunization of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of immunized animal, the site of immunization, and the like. Each mouse is injected with 0.1 μg to several mg, preferably 50 μg to 1 mg of the immunogen or a conjugate of the immunogen and a carrier at a time. In the case of rabbits, 10 μg to several tens mg of the immunogen or a conjugate of the immunogen and a carrier is immunized once per rabbit.

  The immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection. As the adjuvant, known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant or pertussis adjuvant can be used.

Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the initial immunization, booster injections of the immunogen or the conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks. Also in this case, the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.

  After the initial immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the whole blood is collected and the serum is separated to obtain an antiserum containing the antibody of the present invention .

  The antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.

  The obtained polyclonal antibody is composed of both a polyclonal antibody that binds to HMG-1 but does not bind to HMG-2 and a polyclonal antibody that binds to both HMG-1 and HMG-2. Therefore, this is further passed through an affinity chromatography column immobilized with HMG-2 as a ligand on a solid phase. Polyclonal antibodies that bind to both HMG-1 and HMG-2 bind to the solid phase via the ligand (HMG-2) of this column and are collected. In contrast, a polyclonal antibody that binds to HMG-1 but does not bind to HMG-2 passes through this column without binding to the ligand (HMG-2) of this column. By obtaining the fraction, a polyclonal antibody that binds to human HMG-1 but does not bind to human HMG-2 can be obtained.

  In addition, when an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier exists in the obtained antiserum or polyclonal antibody. It is preferable to carry out. As this removal treatment method, a carrier is added to the obtained polyclonal antibody or antiserum solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography, etc. Can be used.

[2] Monoclonal antibody In the antibody of the present invention, the monoclonal antibody can be obtained by the following operation.
The monoclonal antibody is an antibody-producing cell such as a hybridoma obtained by the cell fusion method of Keller et al. (G. Koehler et al., Nature, 256, 495-497, published in 1975) or a tumorigenic cell by a virus such as Epstan-Barr virus. Can be obtained.

Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.
First, a mammal (mouse, nude mouse, rat, etc., for example, BALB / c of an inbred mouse) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier. . The immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like. Preferably, 0.1 μg to 5 mg of the immunogen or a combination of the immunogen and carrier is immunized per dose.

  The immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing. Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.

Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 1 to 2 weeks. The number of booster injections is generally 2 to 6 times. Also in this case, the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.

  After the first immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the immunogen or the combined immunogen and carrier is added to physiological saline. A solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization. Three to five days after the final immunization, cells having the ability to produce antibodies such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.

  The cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). A cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferred. For example, a P3-X63-Ag8 strain (ATCC) which is a HGPRT-deficient cell line derived from BALB / c mice. TIB9), P3-X63-Ag8-U1 strain (Cancer Research Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8.653 strain (JCRB 0028) Or use SP2 / O-Ag-14 strain (JCRB 0029) etc. Rukoto can.

  Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.

  When myeloma cells are of HGPRT-deficient strain or TK-deficient strain, fusion of cells capable of producing antibodies and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine Only cells (hybridomas) can be selectively cultured and propagated.

  The hybridoma culture supernatant thus obtained is subjected to immunological measurement such as ELISA or Western blot using the immunogen, the combined immunogen and carrier, or human HMG-1. By measuring by this method, a hybridoma that produces an antibody that binds to human HMG-1 or the like can be selected.

  In addition, antibodies that do not bind to human HMG-2 etc. are produced by measuring the culture supernatant of the above hybridoma using an immunoassay such as ELISA or Western blot using human HMG-2 etc. The hybridoma to be selected can be selected.

  By combining these two types of hybridoma selection methods and known cloning methods such as limiting dilution, the antibody of the present invention (monoclonal antibody), ie, human HMG-1, binds to human HMG-2. Can be obtained by isolating a production cell line of an antibody that does not bind (monoclonal antibody).

  The monoclonal antibody-producing cell line is cultured in an appropriate medium, and the antibody (monoclonal antibody) of the present invention can be obtained from the culture supernatant. As the medium, a serum-free medium or a low-concentration serum medium is used. In this case, it is preferable from the viewpoint of easy purification of the antibody, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.

  In addition, a monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and prestimulated with pristane or the like, and after a certain period of time, the antibody (monoclonal antibody) of the present invention is obtained from ascites collected in the abdominal cavity. It can also be obtained.

  The monoclonal antibody thus obtained was purified by a salting-out method using ammonium sulfate, sodium sulfate or the like, a method such as ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods. The antibody (monoclonal antibody) of the present invention can be obtained.

2. Immunological measurement method of human HMG-1 (1) General remarks The immunological measurement method of human HMG-1 of the present invention (hereinafter referred to as “the immunological measurement method of the present invention” or “the measurement method of the present invention”). In the immunological measurement method in which human HMG-1 contained in a sample is measured using an antigen-antibody reaction, “It binds to human HMG-1 but does not bind to human HMG-2”. It is characterized by using an “antibody”.

  That is, in the immunological measurement method of human HMG-1, as an antibody that binds to human HMG-1 that is a measurement target substance, “an antibody that binds to human HMG-1 but does not bind to human HMG-2”. It is the measuring method characterized by using. Thereby, in the measuring method of the present invention, HMG-2 is not measured, and an accurate measured value of HMG-1 that does not include an error can be obtained.

  This “antibody that binds to human HMG-1 but does not bind to human HMG-2” can be used without particular limitation as long as it has such properties, and the above-described effects can be obtained. it can.

  In an immunological assay using two antibodies as antibodies that bind to human HMG-1, at least one of the two antibodies binds to “human HMG-1 but human HMG-1”. Any antibody that does not bind to 2 may be used. The other may be any antibody as long as it is an “antibody that binds to human HMG-1.” Both antibodies may also be “an antibody that binds to human HMG-1 but does not bind to human HMG-2”.

  For example, in an ELISA sandwich method, one or both of an enzyme-labeled antibody and a solid-phased antibody may be “an antibody that binds to human HMG-1 but does not bind to human HMG-2”. That's fine.

The “antibody that binds to human HMG-1 but does not bind to human HMG-2” is not limited to one type, and a plurality of types may be used simultaneously.
The details of the “antibody that binds to human HMG-1 but does not bind to human HMG-2” are described above in “1. It binds to human HMG-1 but does not bind to human HMG-2”. As described in the section “Antibodies that do not bind”.

(2) Immunological measurement method The measurement method of the present invention is an immunological measurement method in which human HMG-1 contained in a sample is measured using an antigen-antibody reaction. As long as “antibodies that bind to human HMG-1 but do not bind to human HMG-2” are used as antibodies that bind to, the measurement principle is not particularly limited. There is an effect.

  Examples of the immunological measurement method include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescence Antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination measurement method, erythrocyte agglutination method, particle agglutination method, JP-A-9-229936, JP-A-10-132919, etc. A specific binding substance for the described measurement target substance (test substance) is immobilized, and a carrier having a surface coated with this, and a particle on which the specific binding substance for the measurement target substance (test substance) is fixed. Measurement method to be used, or ELSA method (Enzyme-linked Ligandsorbent Assay) shown by Dahlbeack et al. (Thro mb. Haemost., 79, 767-772, published in 1998; WO 98/23963).

In the immunological measurement method described above, the measurement method of the present invention can be applied to any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method).
In addition, the measurement in the measurement method of the present invention may be performed by a method or using an apparatus such as an analyzer.

(3) Measurement sample As a sample in the measurement method of the present invention, blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, and other body fluids; stool; organs such as blood vessels or liver; Tissues; cells; or biological samples that may contain HMG-1 such as stool, organs, tissues, or extracts of cells or the like.

(4) Immunological measurement method using labeled antibody Immunological measurement method using labeled antibody such as enzyme immunoassay, fluorescent immunoassay, radioimmunoassay or luminescence immunoassay as the measurement method of the present invention Can be performed by the sandwich method or the competition method, but when the sandwich method is performed, either one of the immobilized antibody and the labeled antibody binds to “human HMG-1. May be any antibody that does not bind to human HMG-2, and both of the immobilized antibody and the labeled antibody are “an antibody that binds to human HMG-1 but does not bind to human HMG-2”. There may be.

  As the solid phase carrier used in the measurement method, polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposome, gelatin, agarose, cellulose, sepharose, glass, metal, A solid support in the form of a microcapsule, a bead, a microplate (microtiter plate), a test tube, a stick, or a test piece made of a material such as ceramics or a magnetic material can be used.

  The solid phase antibody can be prepared by adsorbing and binding an antibody such as the antibody of the present invention and a solid phase carrier by a known method such as a physical adsorption method, a chemical binding method, or a combination thereof.

  In the case of physical adsorption, according to a known method, the antibody and the solid phase carrier are mixed and brought into contact with a solution such as a buffer solution, or the antibody dissolved in the buffer solution is brought into contact with the solid phase carrier, etc. It can be carried out. When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with a known method described in ed. "Shinsei Kagaku Kenkyu Ken 1 Protein IV", published by Tokyo Kagaku Dojin, published in 1991, etc., the antibody and solid phase carrier are divalent crosslinking reagents such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody and the solid phase carrier.

  Further, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, bovine serum albumin (BSA) is applied to the surface or inner wall surface of the solid phase carrier on which the antibody is immobilized. ), Treatment by known methods such as casein, gelatin, protein such as ovalbumin or its salt, surfactant or nonfat dry milk, etc., and blocking treatment (masking treatment) of the solid phase carrier May be.

As the labeling substance, peroxidase (POD), alkaline phosphatase (ALP), β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase, or the like can be used in the case of enzyme immunoassay. In the case of fluorescence immunoassay, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, or the like can be used. In the case of a radioimmunoassay, tritium, iodine 125, iodine 131, or the like can be used. In the luminescence immunoassay method, NADH-FMNH ₂ -luciferase system, luminol-hydrogen peroxide-POD system, acridinium ester system or dioxetane compound system can be used.

  The method for binding an antibody such as the antibody of the present invention to a labeling substance such as an enzyme is described in the Japanese Society of Clinical Pathology “Special Issue 53 Special Issue on Clinical Pathology-Immunoassay for Clinical Examination-Technology and Application”, Clinical Pathology Publications Published in 1983; Japan Biochemical Society edited by “Neurochemistry Experiment Course 1 Protein IV”, Tokyo Kagaku Dojin, published in 1991, etc., in accordance with known methods such as glutaraldehyde, carbodiimide, imide ester or maleimide The bivalent crosslinking reagent can be mixed and brought into contact with each other to react with the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, or the like of the antibody and the labeling substance.

  The operation method of the measurement is a known method or the like (edited by the Japanese Society of Clinical Pathology “Special Issue on Extraordinary Clinical Pathology No. 53: Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, published in 1983; edited by Ishikawa Shinji et al. “Enzyme Immunoassay”, 3rd edition, Medical School, published in 1987; edited by Kitagawa Tsuneki et al., “Protein Nucleic Acid Enzyme Separate Volume No. 31 Enzyme Immunoassay”, Kyoritsu Shuppan, published in 1987).

  For example, “solid phase carrier = solid phase antibody = human HMG-1 = labeled antibody” by reacting the solid phase antibody with the sample and simultaneously reacting the labeled antibody or reacting the labeled antibody after washing. To form a complex. Then, the unbound labeled antibody is washed and separated and included in the sample from the amount of labeled antibody bound to the solid phase carrier via “solid phase antibody = human HMG-1” or the amount of unbound labeled antibody. Only the amount (concentration) of HMG-1 that has been obtained can be measured.

  Specifically, in the case of an enzyme immunoassay, a substrate is reacted with an enzyme labeled with an antibody under the optimum conditions, and the amount of the enzyme reaction product is measured by an optical method or the like. In the case of the fluorescence immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of the radioimmunoassay, the radiation dose by the radioactive substance label is measured. In the case of a luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.

(5) Immunological measurement method by agglutination method The measurement method of the present invention is an immune complex aggregate such as an immunoturbidimetric method, latex turbidimetric method, latex agglutination method, erythrocyte agglutination method, or particle agglutination method In the case where the transmitted light or scattered light is measured by an optical method or measured by visual measurement, a phosphate buffer, glycine buffer, Tris buffer or Good buffer as a solvent. Further, a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor may be included.

  When an antibody such as the antibody of the present invention is used after being sensitized to a solid support, polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride are used as the solid support. -Acrylic acid ester copolymer, vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid heavy Particles made of a material such as coalescence, acrylic acid polymer, latex, gelatin, liposome, microcapsule, erythrocyte, silica, alumina, carbon black, metal compound, metal, ceramics or magnetic material can be used.

  The method for sensitizing an antibody such as the antibody of the present invention to a solid phase carrier can be performed by a known method such as a physical adsorption method, a chemical binding method, or a combination thereof.

  In the case of physical adsorption, according to a known method, the antibody and the solid phase carrier are mixed and brought into contact in a solution such as a buffer solution, or the antibody dissolved in the buffer solution or the like is brought into contact with the solid phase carrier, etc. It can be carried out. When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with a known method described in ed. "Shinsei Kagaku Kenkyu Ken 1 Protein IV", published by Tokyo Kagaku Dojin, published in 1991, etc., the antibody and solid phase carrier are divalent crosslinking reagents such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody and the solid phase carrier.

  Further, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, bovine serum albumin (BSA) is applied to the surface or inner wall surface of the solid phase carrier on which the antibody is immobilized. ), Treatment by known methods such as casein, gelatin, protein such as ovalbumin or its salt, surfactant or nonfat dry milk, etc., and blocking treatment (masking treatment) of the solid phase carrier May be.

  In addition, when the latex turbidimetric method is used as a measurement principle, the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are bonded and aggregated via the measurement target substance (HMG-1). For reasons such as the level of mass formation and the ease of measurement of the generated aggregate mass, the average particle size of the latex particles is preferably 0.04 to 1 μm.

  When the latex turbidimetric method is used as a measurement principle, the concentration of latex particles in which the antibody of the present invention is immobilized is included in the concentration of HMG-1 in the sample, on the latex particle surface of the antibody of the present invention. Since the optimum concentration differs depending on various conditions such as the distribution density in the sample, the particle size of the latex particles, and the mixing ratio of the sample and the measuring reagent, it cannot be generally stated.

  However, in general, a measurement reaction in which an antigen-antibody reaction is performed between “the antibody of the present invention” mixed with a sample and a latex particle and “HMG-1” contained in the sample by mixing a sample and a measurement reagent. Sometimes, the concentration of latex particles on which the antibody of the present invention is immobilized is generally 0.005 to 1% (w / v) in the reaction mixture. The concentration of “latex particles on which the antibody of the present invention is immobilized” in such a concentration as described above in the mixed solution is contained in the measurement reagent.

  In addition, when the indirect agglutination reaction method such as latex agglutination reaction method, erythrocyte agglutination reaction method or particle agglutination reaction method is used as the measurement principle, the particle size of the particles used as the solid phase carrier is not particularly limited, but the average particle The diameter is preferably in the range of 0.01 to 100 μm, and more preferably in the range of 0.5 to 10 μm. And it is preferable that the specific gravity of these particle | grains exists in the range of 1-10, and it is more preferable that it exists in the range of 1-2.

  In addition, as a container used for the measurement when an indirect agglutination reaction method such as a latex agglutination reaction method, an erythrocyte agglutination reaction method or a particle agglutination reaction method is used as a measurement principle, for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc. And a test tube, a microplate (microtiter plate), a tray, and the like. The bottom surface of the solution storage portion (such as a well of a microplate) of these containers preferably has a shape having an inclination from the center to the periphery of the bottom, such as U-type, V-type, or UV-type.

  The measuring operation can be performed by a known method. For example, in the case of measuring by an optical method, the sample is reacted with the antibody or the antibody sensitized to the sample and the solid support, and the endpoint is measured. Transmitted light and scattered light are measured by the method or the rate method. Moreover, when measuring visually, in the said containers, such as a plate and a microplate, the antibody made to sensitize a sample and a solid-phase carrier is made to react, and the state of aggregation is visually determined. In addition, you may measure using apparatuses, such as a microplate reader, instead of measuring visually.

3. Reagent for immunological measurement of human HMG-1 (1) General Remarks The immunological measurement reagent for human HMG-1 of the present invention (hereinafter referred to as “immunological measurement reagent of the present invention” or “measurement reagent of the present invention”). Is characterized in that the above-described antibody of the present invention is used in an immunological measurement reagent for measuring human HMG-1 contained in a sample by utilizing an antigen-antibody reaction. These can be used in the measurement method of the present invention described above. Accordingly, the antibody used in the measurement reagent of the present invention, the measurement principle, and the like are the same as those of the measurement method of the present invention described above.

(2) Other reagent components In the measurement reagent of the present invention, various aqueous solvents can be used as the solvent. Examples of the aqueous solvent include purified water, physiological saline, and various buffer solutions such as Tris buffer, phosphate buffer, or phosphate buffered saline. About the pH of this buffer solution, an appropriate pH may be appropriately selected and used. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3-12.

  The measurement reagent of the present invention includes a solid phase carrier on which an antibody such as the antibody of the present invention is immobilized, a solid phase carrier sensitized with an antibody such as the antibody of the present invention, and / or the above-mentioned Proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or a salt thereof, in addition to reagent components such as a labeled antibody obtained by binding an antibody such as the antibody of the present invention and a labeling substance such as an enzyme; Salts; Various sugars; Nonfat dry milk; Various animal sera such as normal rabbit serum; Various preservatives such as sodium azide or antibiotics; Activating substances; Reaction promoting substances; Sensitivity increasing substances such as polyethylene glycol; Nonspecific reaction suppression Substances, or one or more of various surfactants such as nonionic surfactants, amphoteric surfactants or anionic surfactants may be appropriately contained. And the density | concentration at the time of making these contain in a measuring reagent is although it does not specifically limit, 0.001 to 10% (W / V) is preferable, and 0.01 to 5% (W / V) is especially preferable. .

  Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetic acid Anionic surfactants, such as and the like.

(3) Configuration of measurement reagent The measurement reagent of the present invention can be used alone for measuring HMG-1 in a sample. And it can be sold by itself. Moreover, the measuring reagent of this invention can also be used for the measurement of HMG-1 in a sample in combination with another reagent. And it can also be sold in combination with other reagents. Examples of the other reagents include buffers, sample diluents, reagent diluents, reagents containing labeling substances, reagents containing substances that generate signals such as color development, and signals related to color development. A reagent containing a substance, a reagent containing a substance for performing calibration (calibration), a reagent containing a substance for performing accuracy control, and the like can be given. The other reagent is the first reagent and the measurement reagent of the present invention is the second reagent, or the measurement reagent of the present invention is the first reagent and the other reagent is the second reagent. They can be used and sold in various combinations as appropriate.

EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by these Examples.
[Example 1] (Selection of amino acid sequence of human HMG-1 having high hydrophilicity and low homology with human HMG-2)
An amino acid sequence having high hydrophilicity and low homology with human HMG-2 was selected from the amino acid sequence of human HMG-1.

(1) The amino acid sequence of human HMG-1 is the same as the data of the aforementioned Wen et al. [Wen et al., Nucleic Acids Res. , Vol. 17, pp. 1197-1214, published in 1989]

(2) The estimation of the high hydrophilicity of each amino acid residue in the amino acid sequence of human HMG-1 was carried out by the method of Hop et al. P. Hopp et al., Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828, published in 1981]. The result is as shown in FIG.

(3) Next, a highly hydrophilic sequence among the amino acid sequences of human HMG-1 is converted into the amino acid sequence of human HMG-2 [M. Yoshida et al. Biol. Chem. 267, 6641-6645, published in 1992]. Then, from this highly hydrophilic amino acid sequence, the amino acid sequence of human HMG-1 having low homology between human HMG-1 and human HMG-2 was selected.

(4) Here, the first amino acid sequence selected by the present inventors is “Gly Lys” from the N-terminal amino acid residue (glycine) to the 11th amino acid residue (lysine) of human HMG-1. Gly Asp Pro Lys Lys Pro Arg Gly Lys ”. The amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” of human HMG-1 is equivalent to the amino acid sequence “Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys” of human HMG-2. , The sixth amino acid residue from the N-terminus is different. [In human HMG-1 it is “Lys”, whereas in human HMG-2 it is “Asn”. ]

(5) Next, the second amino acid sequence selected by the present inventors is from the 87th amino acid residue (lysine) to the 100th amino acid residue (alanine) from the N-terminus of human HMG-1. “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala”. The amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” of human HMG-1 corresponds to the amino acid sequence “Lys Lys Lys Asp Pro Asn Ala Pro Lys Arg” of human HMG-2. The 88th amino acid residue from the N-terminal is different from “Pro Pro Ser Ala”. [In human HMG-1 it is “Phe”, whereas in human HMG-2 it is “Lys”. ]

[Example 2] (Synthesis of peptide)
The amino acid sequences “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” and “Cys Lys Phe” in which cysteine is bound to the carrier at the N-terminus of each of the two amino acid sequences selected in Example 1. The peptides “Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” were synthesized respectively.

  First, the amino acid sequence “Cys Gly Lys Gly Asp Pro Lys Lys” was applied by a t-butoxycarbonyl amino acid solid phase method according to the instruction manual using an applied biosystems model 430A peptide automatic synthesizer (Model 430A peptide synthesizer). The peptide “Pro Arg Gly Lys” was synthesized.

In order to suppress side reactions, the peptide synthesized from the resin was eliminated by the hydrogen fluoride method in the presence of dimethylsulfide, p-thiocresol, m-cresol and anisole as a scavenger.
Thereafter, the scavenger was extracted with dimethyl ether, and the synthesized peptide was extracted with 2N acetic acid.

  An anion exchange resin, Dowex 1-X2 (DOWEX 1-X2) was purified by anion exchange column chromatography, and the main peak was analyzed by high performance liquid chromatography (HPLC) on an octadecyl (ODS) column. The pattern was confirmed.

  And it concentrated by lyophilizing | drying with an evaporator, Then, it refine | purified by HPLC and fractionated. In addition, the apparatus and conditions at the time of this HPLC refinement | purification used the reverse phase ODS column YMC-D-ODS-5 (20 mm x 300 mm) of Yamamura Chemical Laboratory, the TWINCLE pump of JASCO Corporation, and JASCO Corporation. A gradient of 0% to 70% of acetonitrile in 0.1% trifluoroacetic acid (TFA) was performed at a flow rate of 7.0 mL / min with a GP-A40 type gradientator, and a UVIDEC-100V type detector (210 nm, manufactured by JASCO Corporation) , 1.28 AUFS).

The synthetic peptide purified and fractionated here was lyophilized with an evaporator and concentrated.
The purity of the obtained synthetic peptide was analyzed by HPLC. The apparatus and conditions were a reverse phase ODS column YMC-R-ODS-5 (4.9 mm × 300 mm) manufactured by Yamamura Chemical Laboratory, Inc., a TWINCLE pump manufactured by JASCO Corporation, and a GP-A40 type gradient manufactured by JASCO Corporation. A gradient of 0% to 70% acetonitrile in 0.1% trifluoroacetic acid (TFA) was performed at a flow rate of 1.0 mL / min for 25 minutes, and a UVIDEC-100V detector (210 nm, 1 nm, manufactured by JASCO Corporation) was used. .28 AUFS). It was found that the synthetic peptide thus obtained had a purity of almost 100%.

  Further, in the same manner as described above, a peptide having an amino acid sequence “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” was synthesized. And when the purity of the obtained synthetic peptide was analyzed by HPLC in the same manner as described above, it was found that the purity was almost 100%.

[Example 3] (Preparation of immunogen)
Ten milligrams of mussel hemocyanin (KLH) (manufactured by Calbiochem) or bovine serum albumin (BSA) (manufactured by Seikagaku Corporation) as a carrier is 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0). 150 μL of a 2.5% maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (Pierce) solution dissolved in N, N-dimethylformamide was added to this and stirred at room temperature for 30 minutes. Reacted.

  A Sephadex G-25 (Sephadex G-25) column which is a gel filtration column equilibrated with 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0) at 4 ° C. Pharmacia-LCB Co., Ltd.] and monitored by absorbance at 280 nm to fractionate MBS-carrier binding components.

  The MBS-carrier binding component was adjusted to pH 7.0 with trisodium phosphate, and the peptide “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” synthesized in Example 2 was added thereto and mixed for reaction for 150 minutes. I let you.

After the reaction, it was dialyzed 3 times against water and then freeze-dried to obtain an immunogen comprising a carrier bound to the peptide.
In addition, the peptide “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” synthesized in Example 2 was also operated as described above to obtain an immunogen comprising a carrier bound to the peptide. .

[Example 4] (Preparation of monoclonal antibody)
Monoclonal antibodies were prepared using the immunogen prepared in Example 3 as follows.
[1] Immunization to animals (1) 100 μg / mL of the immunogen obtained in Example 3 (the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” combined with KLH) 8 weeks old female BALB / c mouse (Nippon Charles River Co., Ltd.) was dissolved in physiological saline (0.9% sodium chloride aqueous solution) and mixed with Freund's complete adjuvant. ) 0.5 mL was injected into the abdomen subcutaneously.

(2) Two weeks after the first immunization, the above immunogen is dissolved in physiological saline to a concentration of 50 μg / mL, and this is mixed with an equal amount of Freund's incomplete adjuvant to give an emulsion. A booster injection was given. This booster injection was performed every 2 weeks.

(3) The antibody titer in the serum of this mouse, which is an immunized animal, was measured every week from the sixth week after the first immunization by enzyme immunoassay (ELISA, EIA). The operation of this ELISA method is shown below.
[1] The immunogen obtained by binding BSA as a carrier to the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” obtained in Example 3 at a physiological saline (5 μg / mL). 0.9% sodium chloride aqueous solution), 100 μL per well was added to a 96-well microplate (manufactured by NUNK), and allowed to stand at 37 ° C. for 2 hours to immobilize the immunogen. It was.
[2] The microplate was washed with a washing solution (phosphate buffered saline containing 0.05% Tween 20 (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2 After washing with .68 mM potassium chloride (pH 7.2)), 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.2) containing 1% BSA was added in an amount of 300 μL per well, and the mixture was incubated at 37 ° C. The mixture was allowed to stand for 2 hours for blocking, and then washed again with a washing solution.
[3] 100 μL of the serum of the mouse to be tested for antibody production was added to each well of the microplate as a sample, and allowed to react at 37 ° C. for 2 hours, and then washed with a washing solution.
[4] Further, as a control, 100 μL of HAT medium was added to each well of the microplate of [2] and allowed to stand at 37 ° C. for 2 hours, and then washed with a washing solution.
[5] Peroxidase (POD) -labeled anti-mouse IgG antibody (Amersham) was diluted 2,000 times with phosphate buffered saline containing 3% BSA, and then microplates according to [3] and [4] The reaction was carried out by adding 100 μL per well and allowing to stand at 37 ° C. for 2 hours.
[6] After washing with a washing solution, a peroxidase reaction solution (3 mM 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) [ABTS] containing 50 mM disodium hydrogen phosphate—24 mM 100 μL per well of 2 μL of 1.7% hydrogen peroxide added to 1 mL of citrate buffer immediately before use) was allowed to react at room temperature. After 15 minutes, 50 μL of 6N sulfuric acid was added per well to stop the reaction.
[7] The absorbance at 415 nm was measured with an EIA plate reader (manufactured by Bio-Rad).

(4) Since it was recognized that the antibody titer reached a plateau after 18 weeks from the first immunization, it was obtained in Example 3 in which 800 μg / mL with physiological saline was subcutaneously subcutaneously in the abdomen of this immunized mouse. 0.5 mL of the immunogen (the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” combined with KLH) was injected. On the third day, the spleen was obtained from the mouse of this immunized animal.

  The above operations (1) to (4) are carried out by applying KLH to the peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” obtained in the other Example 3. The spleen was obtained from the mouse of the immunized animal.

[2] Proliferation of myeloma cells The P3-X63-Ag8-U1 strain (cancer research research source bank 9085), which is a myeloma cell line deficient in hypoxanthine, guanine, phosphoribosyl transferase from BALB / c mice, Growth was performed in RPMI 1640 tissue culture medium (Biocell) containing 10% serum and supplemented with glutamine, penicillin and streptomycin.

  In this method, the myeloma cells were grown in a medium-sized bottle for cell culture (manufactured by NUNK, 200 mL) until about 80% of the bottom surface of the bottle occupied by the cells. The number of cells was counted by trypan blue dye exclusion method and hemocytometer.

[3] Cell fusion (1) The spleen obtained from the mouse of the immunized animal in [1] above was thoroughly loosened using a stainless steel mesh # 200, and filtered while washing with RPMI1640 medium without serum. Thereafter, centrifugation was performed at 200 g to separate spleen cells. Furthermore, the spleen cells were washed again three times with RPMI 1640 medium solution without serum.

(2) The spleen cells and the proliferated P3-X63-Ag8-U1 strain myeloma cells were mixed at a ratio of 5 to 1, followed by centrifugation. The mixed cells were slowly suspended in a RPMI 1640 medium containing 50% polyethylene glycol 1500 (PEG 1500, manufactured by Roche Diagnostics). Then, this was gradually diluted with RPMI1640 medium solution so that the polyethylene glycol concentration finally became 5%.

(3) From this, the cells were separated by centrifugation and gradually dispersed in a growth medium consisting of S-clone medium (Sanko Junyaku Co., Ltd.) containing 5% hybridoma cloning factor (Origen). Then, cells having a cell number of 10 ⁶ cells / 100 μL per well were planted in the wells of a flat-bottomed 96-well microplate (manufactured by Nunk) and cultured at 37 ° C. in 5% carbon dioxide.

(4) On the first day after cell fusion, each well was supplemented with 100 μL of HAT medium (the above growth medium supplemented with 0.01 mM hypoxanthine, 1.6 μM thymidine and 0.04 μM aminopterin, All were made by Tokyo Chemical Industry Co., Ltd.). For the next 3 days, about half of the HAT medium was replaced with fresh HAT medium every day, and thereafter, the same replacement was performed every 2 to 3 days.

(5) The cells were observed with a microscope. Hybridoma (fusion cell) clones appear from day 10 onwards, and after 14 days, in order to examine the production of antibodies that recognize the amino acid sequence indicated by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”, The supernatant was screened by ELISA. The operation of this ELISA method was carried out in the same manner as in the above [1] (3).

(6) In the screening of (5) above, a hybridoma of a well that has been found to produce an antibody that recognizes the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” has 24 wells. The cells were spread on a plate and cultured. As the cell density increased, the small and medium-sized bottles were scaled up and cultured.

(7) The hybridoma was cultured and maintained in an HT medium (HAT medium not containing aminopterin and hybridoma cloning factor).

(8) When the production of an antibody recognizing the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” was examined in the same manner as in the above (5) by ELISA, the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys” was determined. It binds to the immunogen (in which the carrier is BSA) obtained in Example 3 consisting of a peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” including “Pro Arg Gly Lys”, and Identified three hybridomas producing antibodies that did not bind.

  The above operations (1) to (8) are carried out by applying KLH to the peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” obtained in the other Example 3. The above-mentioned mouse spleen obtained by immunization with the above-mentioned mouse was similarly performed, and “Cys Lys Phe Lys Asp Pro Asn Ala” containing the amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala”. Six hybridomas producing an antibody that binds to the immunogen obtained in Example 3 consisting of a peptide represented by “Pro Lys Arg Pro Pro Ser Ala” (carrier is BSA) and does not bind to BSA were confirmed. .

[4] Hybridoma subcloning (A) When the immunogen is a conjugate of a peptide and a carrier represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” (1) obtained in [3] above, Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys "which produces an antibody that binds to the immunogen obtained in Example 3 (the carrier is BSA) and does not bind to BSA. Each of the hybridomas was subcloned by the limiting dilution method. The number of cells of these hybridomas was counted by trypan blue dye exclusion method and hemocytometer.
These hybridomas are suspended at a ratio of 0.5 growth cells and 1 growth cell per 100 μL of HT medium. One well of a 96-well flat-bottom microplate is suspended. 100 μL was dispensed per unit. The medium was changed every 2 to 3 days to grow the hybridoma.

(2) Two weeks later, the number of colonies in each well was examined under a microscope, and the immunogen obtained in Example 3 consisting of a peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” ( The hybridoma producing an antibody that binds to the carrier with BSA and does not bind to BSA was examined by ELISA in the same manner as described above. One colony was present in one well, and two hybridomas (wells) producing such an antibody could be obtained.

(3) This was transferred to a 24-well plate and cultured for 2 weeks until cell growth was good.

(4) Next, the reactivity of the antibodies produced by these hybridomas with human HMG-1 prepared in Reference Example 2 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate is replaced with the human HMG-1 prepared in Reference Example 2, and the sample is replaced with the culture supernatant of each hybridoma (each well). Except for this, the procedure was the same as in (3) of [1] above.
As a result, it was found that one of the hybridomas is a cell line that produces an antibody that binds to the human HMG-1.

(5) This hybridoma was cloned again in the same manner as in the above (1) and (2), and antibody production was examined for each well. One colony hybridoma was present in each well, and The immunogen obtained in Example 3 consisting of a peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” containing the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” One hybridoma producing an antibody that binds to BSA) and does not bind to BSA was obtained.

(6) The reactivity of the antibody produced by this hybridoma clone with the human HMG-1 was examined again by ELISA as in (4) above. As a result of this study, it was confirmed that the antibody (monoclonal antibody) produced by this hybridoma clone was an antibody (monoclonal antibody) that binds to human HMG-1.

(7) Next, the reactivity of the antibody produced by this hybridoma with each of bovine HMG-1 and bovine HMG-2 prepared in Reference Example 3 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate was changed to bovine HMG-1 or bovine HMG-2 prepared in Reference Example 3, and the sample was mixed with this hybridoma (this well). The procedure was the same as in the above [1] (3) except that the culture supernatant was replaced.

As a result of this examination, it was confirmed that the antibody produced by this hybridoma binds to bovine HMG-1, but does not bind to bovine HMG-2.
The amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” adopted as the amino acid sequence of the immunogen peptide and the amino acid sequence “Gly Lys Gly Asp Pro” of HMG-2 corresponding to this sequence Asn Lys Pro Arg Gly Lys is the same sequence in both human and bovine.

  Therefore, from the above, it was revealed that the antibody (monoclonal antibody) produced by this hybridoma is an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2. .

  This hybridoma was named MD77 and was registered as FERM P-18404 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1-6 East 1st, Tsukuba, Ibaraki, Japan) July 4, 2001 Deposited at the end.

(B) When the immunogen is a conjugate of a peptide and a carrier represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” (1) “Cys Lys obtained in [3] above” Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala "which produces an antibody that binds to the immunogen obtained in Example 3 (carrier is BSA) and does not bind to BSA Each of the hybridomas was subcloned by the limiting dilution method. The number of cells of these hybridomas was counted by trypan blue dye exclusion method and hemocytometer.

  These hybridomas are suspended at a ratio of 0.5 growth cells and 1 growth cell per 100 μL of HT medium. One well of a 96-well flat-bottom microplate is suspended. 100 μL was dispensed per unit. The medium was changed every 2 to 3 days to grow the hybridoma.

(2) Two weeks later, the number of colonies in each well was examined under a microscope, and obtained in Example 3 consisting of a peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” Hybridomas that produce antibodies that bind to the immunogen (carrier BSA) but not to BSA were examined by ELISA as described above. One colony was present in one well, and four hybridomas (wells) producing such an antibody could be obtained.

(3) This was transferred to a 24-well plate and cultured for 2 weeks until cell growth was good.

(4) Next, the reactivity of the antibodies produced by these hybridomas with human HMG-1 prepared in Reference Example 2 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate is replaced with the human HMG-1 prepared in Reference Example 2, and the sample is replaced with the culture supernatant of each hybridoma (each well). Except for this, the procedure was the same as in (3) of [1] above.
As a result, it was found that one of the hybridomas is a cell line that produces an antibody that binds to the human HMG-1.

(5) This hybridoma was cloned again in the same manner as in the above (1) and (2), and antibody production was examined for each well. One colony hybridoma was present in each well, and Obtained in Example 3 consisting of a peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” containing the amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” Two hybridomas were produced that produced antibodies that bound to the immunogen (carrier BSA) and did not bind to BSA.

(6) The reactivity of each antibody produced by these two hybridoma clones with human HMG-1 was examined again by ELISA as in (4). As a result of this examination, it was confirmed that each antibody (monoclonal antibody) produced by these two hybridoma clones was an antibody (monoclonal antibody) that binds to the human HMG-1.

(7) Next, the reactivity of each antibody produced by these two hybridomas with each of bovine HMG-1 and bovine HMG-2 prepared in Reference Example 3 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate was changed to bovine HMG-1 or bovine HMG-2 prepared in Reference Example 3, and the samples were hybridomas (this well). The procedure was the same as in (3) of [1] except that the culture supernatant was replaced.
As a result of this examination, it was confirmed that each antibody produced by these two hybridomas binds to both bovine HMG-1 and bovine HMG-2.

  The amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” adopted as the amino acid sequence of the immunogen peptide, and the amino acid sequence “Lys Lys” of HMG-2 corresponding to this sequence “Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” is the same sequence in both human and bovine.

  Therefore, from the above, it is clear that each antibody (monoclonal antibody) produced by these two hybridomas is an antibody (monoclonal antibody) that binds to both human HMG-1 and human HMG-2. became.

  These hybridomas are named MD78 and are registered as FERM P-18405 at the Patent Organism Depository Center (National Institute of Advanced Industrial Science and Technology) (1st, 1st East, 1st Street, Tsukuba, Ibaraki, Japan). Deposited as of April 4th.

[5] Production of monoclonal antibody (1) Each monoclonal antibody-producing cell line (hybridoma) obtained in [4] above is put in a medium-sized bottle (manufactured by NUNK) one by one. Was cultured in HT medium until cells occupied.
(2) Thereafter, these hybridomas were scraped off and collected by centrifugation at 200 g for 5 minutes. Next, this was washed 3 times with RPMI 1640 medium containing no serum and then suspended in 2 mL of RPMI 1640 medium.
(3) Into the abdominal cavity of a male BALB / c mouse (Charles River Japan) previously treated with 2,6,10,14-tetramethylpentadecane, 1 mL of the hybridoma suspension obtained in (2) above Was injected. This was repeated again if no abdominal swelling was observed within 2 weeks of injection.
(4) Ascites was collected when swelling of the abdomen of this mouse was observed. This was centrifuged at 200 g for 5 minutes, and a supernatant containing the monoclonal antibody produced from the hybridoma was separated from the hybridoma and obtained.

[6] Purification of monoclonal antibody (1) To 10 mL of each supernatant liquid containing the monoclonal antibody produced from hybridoma obtained in [5] above, 1.8 g of sodium sulfate was added at 22 ° C. with stirring. After sodium was completely dissolved, stirring was continued for 1 hour for salting out.
(2) This was centrifuged at 7000C (7000 g, 15 minutes), and the precipitate obtained by separating from the supernatant was dissolved in 2 mL of 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride. .
(3) Next, this was sufficiently dialyzed against 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride, and then centrifuged at 1000 g for 20 minutes to remove insoluble matters.
(4) A flow rate of 0.4 mL / min was applied to a DEAE-cellulose ion exchange column (manufactured by Selva) [1 × 10 cm] which had been equilibrated with a 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride. And 2 mL of the eluate was collected.
(5) It was confirmed from the absorbance at 280 nm that immunoglobulin G (IgG) was contained in the flow-through fraction of the eluate, and this was collected and concentrated to 2 mL.
(6) Furthermore, this was subjected to protein A-Sepharose CL-4B affinity chromatography (Pharmacia-LCB Co., Ltd.) for purification to obtain a purified monoclonal antibody.

  In the case where the immunogen is a conjugate of the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” and a carrier, it binds to human HMG-1, but binds to human HMG-2. The amount of antibody not obtained (monoclonal antibody) was 0.5 mg as the amount of protein.

  In addition, it binds to both human HMG-1 and human HMG-2 when the immunogen is a conjugate of a peptide and a carrier represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” The obtained amount of antibody (monoclonal antibody) was 0.4 mg as the amount of protein.

[Example 5] (Preparation of peroxidase-labeled antibody)
An antibody that binds to human HMG-1 obtained in Example 4 but does not bind to human HMG-2 was labeled with peroxidase to prepare a peroxidase-labeled antibody.
(1) Introduction of maleimide group into peroxidase After dissolving 4 mg of peroxidase (from horseradish) in 0.3 mL of 0.1 M phosphate buffer (pH 7.0), N-succinimidyl-4- (N-maleimide) A solution prepared by dissolving 1.0 mg of methyl) cyclohexane-1-carboxylic acid in 60 μL of N, N′-dimethylformamide was added and reacted at 30 ° C. for 60 minutes. Thereafter, dialysis was performed overnight with 0.1 M phosphate buffer (pH 6.0). Through the above operation, a maleimide group was introduced into the peroxidase.

(2) Introduction of thiol group into antibody Human HMG in the case where the immunogen obtained in Example 4 is a conjugate of a peptide and a carrier represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” -1 but not human HMG-2 (monoclonal antibody) in 0.5 mL of 0.1 M phosphate buffer solution (pH 6.5) containing 10 mg / mL concentration, A solution prepared by dissolving 0.6 mg of S-acetylmercaptosuccinic anhydride in 10 μL of N, N′-dimethylformamide was added and reacted at room temperature for 30 minutes.

  Thereafter, 20 μL of 0.1 M EDTA, 0.1 mL of 0.1 M Tris-HCl buffer (pH 7.0), and 0.1 mL of 1 M hydroxylamine hydrochloride (pH 7.0) were added thereto. And left at 30 ° C. for 5 minutes.

This was then passed through a Sephadex G-25 column that had been equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA and subjected to simple gel filtration chromatography. The acetylmercaptosuccinic anhydride was removed and the antibody fraction was collected.
Through the above operation, a thiol group was introduced into an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2.

(3) Preparation of labeled antibody The peroxidase introduced with the maleimide group prepared in the above (1) and the antibody introduced with the thiol group prepared in the above (2) were mixed one-on-one and reacted at 30 ° C. for 20 hours. Peroxidase was introduced (labeled) into the antibody. This was then passed through a column of Ultragel AcA34 that had been equilibrated with 0.1 M phosphate buffer (pH 6.5) and subjected to gel filtration chromatography. Each fraction of the gel filtration chromatography was confirmed by 10% polyacrylamide electrophoresis, and only the fraction of the antibody bound with peroxidase was collected so that unbound peroxidase was not mixed. The antibody fraction bound to peroxidase was concentrated to obtain a peroxidase-bound antibody, that is, a peroxidase-labeled antibody. And the protein concentration of the solution containing this peroxidase labeled antibody was measured.

[Example 6] (Microplate immobilized antibody)
The antibody that binds to both human HMG-1 and human HMG-2 obtained in Example 4 was immobilized on a microplate to prepare a microplate-immobilized antibody.
(1) Human HMG-1 and human when the immunogen obtained in Example 4 is a conjugate of a peptide and a carrier represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” Antibodies that bind to both HMG-2 (monoclonal antibodies) were added to phosphate buffered saline (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.68 mM potassium chloride ( After adjusting the pH to 15 μg / mL according to pH 7.2)), add 100 μL per well to a 96-well microplate (manufactured by NUNK), and allow to stand at 37 ° C. for 2 hours. And adsorbed to solid phase.
(2) This antibody-immobilized microplate was washed with a washing solution (phosphate buffered saline (pH 7.2) containing 0.05% Tween 20) and then 10 mM containing 1% BSA. Potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.2) was added in an amount of 300 μL per well, allowed to stand at 37 ° C. for 2 hours for blocking, and then washed again with a washing solution.

  Through the above operation, a microplate-immobilized antibody was prepared by immobilizing an antibody (monoclonal antibody) that binds to both human HMG-1 and human HMG-2 on a microplate.

[Example 7] (Immunoassay reagent and immunoassay method for human HMG-1 using an antibody that binds to human HMG-1 but does not bind to human HMG-2)
The peroxidase-labeled antibody prepared in Example 5 and the microplate immobilized antibody prepared in Example 6 were used as immunological measurement reagents, and were prepared in human HMG-1 prepared in Reference Example 2 and Reference Example 3. Measurement was performed by enzyme immunoassay (sandwich method) of bovine HMG-1. Then, a calibration curve in this immunological measurement method was prepared.

1. Reagent
[1] Peroxidase-labeled antibody A peroxidase-labeled antibody prepared by linking peroxidase to an antibody that binds to human HMG-1 but does not bind to human HMG-2, prepared in Example 5, was determined by enzyme immunoassay. Used as an enzyme-labeled antibody in the sandwich method.
[2] Microplate-immobilized antibody A microplate-immobilized antibody prepared by immobilizing an antibody that binds to both human HMG-1 and human HMG-2 in each well of the microplate, prepared in Example 6, It was used as an immobilized antibody in the sandwich method of enzyme immunoassay.
[3] Washing solution Phosphate buffered saline (pH 7.2) containing 0.05% Tween 20 was prepared and used as a washing solution.
[4] Peroxidase substrate solution 2 μL of 1.7% per 1 mL of 50 mM disodium hydrogen phosphate-24 mM citrate buffer containing 3 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) What added hydrogen peroxide just before use was prepared, and it was set as the label | marker peroxidase substrate, ie, peroxidase substrate liquid.
[5] Reaction Stop Solution A 6N sulfuric acid aqueous solution was prepared and used as a reaction stop solution.

2. sample
[1] Sample containing human HMG-1 A solution containing human HMG-1 prepared in Reference Example 2 was mixed with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7) containing 0.01% sodium azide. 4) and was sufficiently dialyzed. The protein concentration of the solution containing the human HMG-1 after dialysis was determined by a protein assay (manufactured by Bio-Rad). Then, the solution containing the human HMG-1 is diluted with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide, and the human HMG- Samples having a concentration of 360 ng / mL, 720 ng / mL, or 1,080 ng / mL were prepared, respectively.
[2] Sample containing bovine HMG-1 A solution containing bovine HMG-1 prepared in Reference Example 3 was mixed with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7) containing 0.01% sodium azide. 4) and was sufficiently dialyzed. The protein concentration of the solution containing bovine HMG-1 after dialysis was determined by a protein assay (Bio-Rad). Then, the solution containing bovine HMG-1 was diluted with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide to obtain bovine HMG-1 concentration. Prepared samples of 10 ng / mL, 50 ng / mL, 100 ng / mL or 500 ng / mL, respectively.
[3] Sample of 0 ng / mL 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide was added to human HMG-1 concentration and bovine HMG-1 A sample having a concentration of 0 ng / mL was used.

3. Measurement by enzyme immunoassay (sandwich method)
[1] Samples containing 3 types of human HMG-1 prepared in 2 above, samples containing 4 types of bovine HMG-1 and samples of 0 ng / mL were each diluted twice with physiological saline. did.
[2] 100 μL of each sample diluted in [1] above is added to the well of the above-mentioned microplate-immobilized antibody and allowed to stand at 37 ° C. for 2 hours to immobilize the antibody on the microplate And antigen-antibody reaction with HMG-1 contained in the sample.
[3] Next, each well of the microplate-immobilized antibody was washed with the washing solution 1 described above.
[4] The 1 peroxidase-labeled antibody was diluted 1,000 times with phosphate buffered saline containing 3% BSA. Next, 100 μL of this was added to each well of the microplate-immobilized antibody subjected to the washing operation of [3] above, and then allowed to stand at 37 ° C. for 2 hours. As a result, a reaction for binding the peroxidase-labeled antibody to HMG-1 bound to the antibody immobilized on the microplate was performed.
[5] Thereafter, each well of the microplate-immobilized antibody was washed with the washing solution of 1.
[6] Next, 100 μL of the above-mentioned peroxidase substrate solution was added to each well of the microplate-immobilized antibody. And it was made to react at room temperature.
[7] 15 minutes after the addition of the peroxidase substrate solution, 100 μL of the reaction stop solution of 1 was added to each well of the microplate-immobilized antibody to stop the reaction of the labeled peroxidase. .
[8] Next, the absorbance (450 nm) of the solution in each well of the microplate-immobilized antibody was measured with a microplate reader (Bio-Rad).
[9] The measured values of the samples, that is, calibration curves, obtained by the above operation are shown in the figure. The measured values of the sample containing human HMG-1 and the sample of 0 ng / mL, that is, a calibration curve are shown in FIG. Moreover, the measured value of the sample containing the bovine HMG-1 and the sample of 0 ng / mL, that is, a calibration curve is shown in FIG.

  In these figures, the horizontal axis represents the concentration of human HMG-1 or bovine HMG-1 contained in the sample, and the vertical axis represents the measured absorbance value at 450 nm. However, the measured value of absorbance was obtained by subtracting the absorbance of phosphate buffered saline (pH 7.2) containing 3% BSA as a blinded value.

4). Summary From FIG. 2, in the sample containing human HMG-1, the absorbance obtained in proportion to the concentration of human HMG-1 contained in the sample increased, and was proportional to the concentration of human HMG-1 contained in the sample. It was found that measurements could be obtained.
Therefore, it was confirmed that the human HMG-1 contained in the sample can be accurately measured by the measurement reagent and the measurement method of the present invention.

  From FIG. 3, in the sample containing the bovine HMG-1, the absorbance obtained in proportion to the contained bovine HMG-1 concentration increased, and the measurement was proportional to the bovine HMG-1 concentration contained in the sample. It turns out that the value can be obtained.

[Example 8] (Measurement of serum sample by immunological measurement reagent and immunological measurement method of human HMG-1 of the present invention)
Regarding the immunological measurement reagent and immunological measurement method of human HMG-1 of the present invention, HMG-1 contained in a serum sample was measured to confirm the accuracy at the time of measurement of the serum sample.

1. Measurement Reagent “[1] Peroxidase-labeled antibody”, “[2] Microplate immobilized antibody”, “[3] Washing solution”, “[4] Peroxidase substrate solution” in “1. And “[5] Reaction stop solution” were used.

2. sample
[1] The solution containing bovine HMG-1 prepared in Reference Example 3 was sufficiently dialyzed with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide. did. The protein concentration of the solution containing bovine HMG-1 after dialysis was determined by a protein assay (Bio-Rad).

  Then, the solution containing bovine HMG-1 was diluted with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide to obtain bovine HMG-1 concentration. Prepared solutions of 40 ng / mL, 200 ng / mL or 400 ng / mL, respectively.

[2] The three types of solutions prepared in [1] were each diluted 2-fold with physiological saline. And the sample which does not contain the serum whose bovine HMG-1 density | concentration is 20 ng / mL, 100 ng / mL, or 200 ng / mL, respectively was prepared.
[3] In addition, each of the three solutions prepared in [1] was diluted 2-fold with human serum. And the serum sample whose bovine HMG-1 density | concentration is 20 ng / mL, 100 ng / mL, or 200 ng / mL, respectively was prepared.

3. Measurement by enzyme immunoassay (sandwich method)
[1] 100 μL of each sample diluted in 2 above is added to the well of the microplate-immobilized antibody in 1, and left at 37 ° C. for 2 hours to immobilize the antibody and sample immobilized on the microplate The antigen-antibody reaction with HMG-1 contained in was carried out.
[2] Next, each well of the microplate-immobilized antibody was washed with the washing solution 1 described above.
[3] The 1 peroxidase-labeled antibody was diluted 1,000 times with phosphate buffered saline containing 3% BSA. Next, 100 μL of this was added to each well of the microplate-immobilized antibody subjected to the washing operation of [2], and then allowed to stand at 37 ° C. for 2 hours. As a result, a reaction for binding the peroxidase-labeled antibody to HMG-1 bound to the antibody immobilized on the microplate was performed.
[4] Thereafter, each well of the microplate-immobilized antibody was washed with the washing solution of 1.
[5] Next, 100 μL of the above-mentioned peroxidase substrate solution was added to each well of the microplate-immobilized antibody. And it was made to react at room temperature.
[6] 15 minutes after the addition of the peroxidase substrate solution, 100 μL of the reaction stop solution of 1 was added to each well of the microplate-immobilized antibody to stop the reaction of the labeled peroxidase.
[7] Next, the absorbance (450 nm) of the solution in each well of the microplate-immobilized antibody was measured with a microplate reader (Bio-Rad).
[8] The measured values of each sample obtained by the above operation are shown in the table. The measured value of absorbance was obtained by subtracting the absorbance of phosphate buffered saline (pH 7.2) containing 3% BSA as a blinded value.

4). Summary From the above table, at each bovine HMG-1 concentration, the measured value (absorbance) of the sample without serum diluted with physiological saline and the measured value (absorbance) of the serum sample diluted with human serum are almost the same. It turns out that it is the same.

  Serum has various components including various components, but the immunoassay reagent and immunoassay method of the present invention can control the concentration of HMG-1 even in a sample containing such serum or serum. It was confirmed that it can be measured accurately.

  As described above, the antibodies used in the immunological measurement reagents and immunological measurement methods of the present invention that were measured as described above were immunized at the same amino acid sequence between human and bovine. The immunological measurement reagent and immunological measurement method of the present invention in Example 7 can accurately measure both the human HMG-1 and the bovine HMG-1. As it has been confirmed, it is clear that the same result can be obtained even if a sample obtained by diluting human HMG-1 with serum instead of bovine HMG-1 is used as a sample.

[Example 9] (Confirmation of reactivity of antibody with human HMG-1 and human HMG-2)
An antibody (monoclonal antibody) that binds to human HMG-1 prepared in Example 4 but does not bind to human HMG-2, binds to both human HMG-1 and human HMG-2 prepared in Example 4 Reaction of each of the antibody (monoclonal antibody) and the antibody (polyclonal antibody) that binds to both human HMG-1 and human HMG-2 prepared in Reference Example 4 with human HMG-1 and human HMG-2 Sex was confirmed by Western blot.

1. Western blotting (1) Antibody that binds to human HMG-1 but does not bind to human HMG-2 (monoclonal antibody prepared in Example 4)
[1] Human serum was subjected to electrophoresis using a titan gel lipoprotein electrophoresis kit (manufactured by Helena Laboratories). The support was an agarose gel, which was contacted with 2 μL of the human serum.
[2] Then, barbital buffer (pH 8.8) was used as an electrophoresis buffer, and electrophoresed by applying electricity at a voltage of 90 V for 75 minutes. The electrophoresis was performed by preparing two sets of the same ones, and the following operations were performed in the same manner.
[3] The transfer after the electrophoresis of [2] was performed by a dry method using a Nova Blot Electrophoretic Transfer Kit (Pharmacia-LCB) according to the instruction manual.

First, the agarose gel subjected to the electrophoresis in [2] was placed on a transfer device. Next, on this agarose gel, a 9 cm × 9 cm nitrocellulose membrane (manufactured by Bio-Rad) was overlaid, and 48 mM tris (hydroxymethyl) aminomethane, 39 mM glycine, 0.0375% (W / V) sodium dodecyl sulfate. Transfer was performed at a current of 65 mA for 2 hours using a transfer buffer solution (SDS) and 20% (V / V) methanol.
[4] The nitrocellulose membrane subjected to this transfer was subjected to phosphate buffered saline containing 1% BSA (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride and 2.68 mM. Blocking was performed by immersing in 20 mL of an aqueous solution containing potassium chloride (pH 7.2) at 4 ° C. overnight.
[5] Next, this was washed by shaking for 10 minutes in 20 mL of a cleaning solution (phosphate buffered saline containing 0.05% Tween 20). This operation was performed three times.
[6] 80 μg of an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 prepared in Example 4 was dissolved in 20 mL of phosphate buffered saline, and dissolved in this solution. The nitrocellulose membrane subjected to the above operation [5] was reacted by being immersed for 2 hours at room temperature.
[7] The nitrocellulose membrane subjected to the operation of [6] was washed by shaking in 20 mL of a cleaning solution for 10 minutes. This was done three times.
[8] Next, a peroxidase-labeled anti-mouse IgG antibody (manufactured by Dako) was diluted 500-fold with a phosphate buffered saline containing 3% BSA to prepare a 20 mL solution. The nitrocellulose membrane of [7] was allowed to react by being immersed for 2 hours at room temperature.
[9] The nitrocellulose membrane was washed with shaking in 20 mL of a washing solution for 10 minutes. This operation was performed three times.
[10] The nitrocellulose membrane of [9] above was added to 20 mL of phosphate buffered saline containing 0.025% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide at room temperature. Color was developed by dipping for a minute.

  By the above operation, the results of Western blotting for the antibody (monoclonal antibody) prepared in Example 4 that binds to human HMG-1 but does not bind to human HMG-2 were obtained.

(2) Antibody that binds to both human HMG-1 and human HMG-2 (monoclonal antibody prepared in Example 4)
“An antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 prepared in Example 4” in [6] of (1) above was prepared in “Example 4”. , Except that it is changed to “Antibody that binds to both human HMG-1 and human HMG-2 (monoclonal antibody)”, the procedure is performed as in [1] to [10] of the above (1). The results of Western blotting were performed on the prepared antibody (monoclonal antibody) that binds to both human HMG-1 and human HMG-2.

(3) Antibody that binds to both human HMG-1 and human HMG-2 (polyclonal antibody prepared in Reference Example 4)
“An antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2, prepared in Example 4,” prepared in [6] of (1) above was prepared in “Reference Example 4. , Except that it is changed to “Antibody that binds to both human HMG-1 and human HMG-2 (polyclonal antibody)”, the procedure is performed as in [1] to [10] of the above (1). The results of Western blotting were performed on the prepared antibody (polyclonal antibody) that binds to both human HMG-1 and human HMG-2.

2. Experimental Results (1) Results of Western Blotting The results of Western blotting in (1), (2) and (3) above are shown in FIG.
In this figure, “MA1” is the result for an antibody that binds to human HMG-1 but does not bind to human HMG-2 (the monoclonal antibody prepared in Example 4), and “MA2” is human HMG. 1 is a result of an antibody that binds to both human-1 and human HMG-2 (monoclonal antibody prepared in Example 4), and “PA” is an antibody that binds to both human HMG-1 and human HMG-2 (Reference Example) 4 is a result of the polyclonal antibody prepared in 4.).

In this figure, the band “1” is a band due to human HMG-1 contained in human serum, and the band “2” is a band due to human HMG-2 contained in human serum.
[Separately, instead of human serum, purified bovine HMG-1 and bovine HMG-2 were each electrophoresed and subjected to Western blotting, and confirmed from the position of the obtained band. . (The homology of the amino acid sequences of human HMG-1 and bovine HMG-1, and human HMG-2 and bovine HMG-2 are so high that they migrate at approximately the same position in electrophoresis.)]

(2) Control
The operation of [6] in (1) above is performed for each of the other nitrocellulose membranes that have been subjected to the operation of [1] in (1), (2), and (3) above. However, the operations from [7] onward in the above (1) were performed in the same manner, and this was used as a control.

In this control (control) in which neither an antibody that binds to human HMG-1 but does not bind to human HMG-2 nor an antibody that binds to both human HMG-1 and human HMG-2 is allowed to act. No color development was observed at any of the positions where the human HMG-1 band appeared and the positions where the human HMG-2 band appeared.
From this, it was confirmed that non-specific color development did not occur in each Western blotting method.

3. 4. Summary FIG. 4 shows that antibodies (monoclonal antibodies) that bind to both human HMG-1 and human HMG-2 prepared in Example 4 and both human HMG-1 and human HMG-2 prepared in Reference Example 4 It can be seen that the antibody that binds (polyclonal antibody) develops color at both the position where human HMG-1 migrates and the position where human HMG-2 migrates.
From this, it was confirmed that both human HMG-1 and human HMG-2 exist in human serum.

  The antibody that binds to both human HMG-1 and human HMG-2 (monoclonal antibody) and the antibody that binds to both human HMG-1 and human HMG-2 (polyclonal antibody) are certainly human HMG- It was confirmed from the results of this Western blotting that it binds to both 1 and human HMG-2.

  In addition, as shown in FIG. 4, the antibody that binds to human HMG-1 prepared in Example 4 but does not bind to human HMG-2 (monoclonal antibody) develops color at the position where human HMG-1 migrates. Although it can be seen, it can be seen that no color is seen at the position where human HMG-2 migrates.

  From this, the antibody that binds to human HMG-1 but does not bind to human HMG-2 does not bind to human HMG-2, although it does bind to human HMG-1. This was confirmed from the result of Western blotting.

[Reference Example 1] (Preparation of human HMG-1 gene)
Human HMG-1 was cloned from human brain cDNA by DNA amplification. The PCR product was modified with BamHI and HindIII, subcloned into the BamHI-HindIII site of the sequence vector pCAL-n vector (Stratagene, California, USA), and the DNA sequence was confirmed.

[Reference Example 2] (Preparation of human HMG-1 by DNA recombination method)
In order to express the PCR product modified with BamHI-HindIII as a fusion protein with glutathione-S-transferase, it was subcloned into the BamHI-HindIII site of the pEX expression vector.

Next, this recombinant plasmid was transformed into E. coli. E. coli / JM1 was transformed.
After the transformed cells were cultured at about 1 L, IPTG was induced, and glutathione-S-transferase / human HMG-1 fusion protein was transformed into E. coli. It was expressed in E. coli / JM1.

  This expressed E. coli. After collecting E. coli / JM1, about 30 mL of phosphate buffered saline (PBS) [including 137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate and 5.59 mM disodium hydrogen phosphate Aqueous solution (pH 7.2)].

And this was crushed by ultrasonication and the supernatant liquid was collect | recovered after centrifugation.
The glutathione-S-transferase / human HMG-1 fusion protein contained in the supernatant was purified with a glutathione column (Pharmacia).

Next, from this purified glutathione-S-transferase / human HMG-1 fusion protein, factor Xa was allowed to act to excise human HMG-1.
Human HMG-1 was obtained and prepared by the above DNA recombination operation.

[Reference Example 3] (Preparation of bovine HMG-1 and bovine HMG-2)
Bovine HMG-1 and bovine HMG-2 were obtained from the bovine thymus by the method of Sanders et al. Sanders et al. B. R. C. 78, 1034-1042, published in 1977].
[1] First, 500 g of bovine thymus was crushed in 600 mL of a buffer containing 140 mM sodium chloride and 0.5 mM PMSF.
[2] Next, the crushed material was centrifuged with a centrifuge, and the supernatant was removed.
[3] To this, a buffer solution containing 140 mM sodium chloride and 0.5 mM PMSF was added and stirred, and then centrifuged with a centrifuge to remove the supernatant. This washing operation was repeated twice.
[4] Next, 300 mL of 0.75 M perchloric acid was added to the resulting precipitate. And after centrifuging with a centrifuge, the supernatant liquid was fractionated. 400 mL of 0.75 M perchloric acid was added to the remaining precipitate. Also for this, after centrifuging with a centrifuge, the supernatant was collected. This supernatant was combined with the previously collected supernatant. The precipitate was discarded.
[5] 0.75 M perchloric acid was added to the combined supernatant to a total volume of 1,000 mL. Next, after centrifuging with a centrifuge, the supernatant was filtered through a glass filter (grade 4).
[6] A mixture of 3,500 mL of acetone and 21 mL of concentrated hydrochloric acid was added to the filtrate of the filtration. Since turbidity occurred, the mixture was centrifuged with a centrifuge and the supernatant was collected. To this supernatant, 2,500 mL of acetone was added. Then, since turbidity occurs again, this was centrifuged with a centrifuge to separate the supernatant, and the remaining precipitate was collected.
[7] The collected precipitate was naturally dried at room temperature.

Through the above operation, approximately 200 mg of a protein fraction containing HMG-1 and HMG-2 was obtained.
[8] The protein fraction containing HMG-1 and HMG-2 is dissolved in 10 mL of 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and then this 200 mM sodium chloride is contained. Dialysis was sufficiently performed with 7.5 mM sodium borate buffer (pH 9.0).
[9] After this dialysis, it was added to a CM-Sephadex C25 column that had been equilibrated with 7.5 mM sodium borate buffer (pH 9.0).

After that, elution was performed with 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and cation exchange chromatography was performed.
This elution pattern is shown in FIG. In this figure, the vertical axis indicates the absorbance at 280 nm, and the horizontal axis indicates the number of the eluted fraction.

[10] As a result of 20% SDS-polyacrylamide electrophoresis, the elution fraction indicated by “A” and the elution fraction indicated by “B” in FIG. It was confirmed that the elution fraction indicated by “C” and the elution fraction indicated by “D” were fractions containing bovine HMG-2.

  Accordingly, the elution fraction indicated by “A” and the elution fraction indicated by “B” in FIG. 5 are mixed and collected, and further the elution fraction indicated by “C” and the elution fraction indicated by “D”. Were collected and mixed.

[Reference Example 4] (Preparation of polyclonal antibody)
A polyclonal antibody was prepared using bovine HMG-1 prepared in Reference Example 3 as follows.
[1] Immunization to animals (1) The bovine HMG-1 obtained in Reference Example 3 above was dissolved in physiological saline (0.9% sodium chloride aqueous solution) to 400 μg / mL, and this was completely Freund's. An equal volume of adjuvant was mixed with each other as an emulsion, and 0.5 mL of the rabbit (Kitayama Labes Co., Ltd.) was subcutaneously injected into the abdomen.

(2) Two weeks after the first immunization, the above immunogen is dissolved in physiological saline so as to be 300 μg / mL, and this is mixed with an equal amount of Freund's incomplete adjuvant to give an emulsion. A booster injection was given. This booster injection was performed every 2 weeks.

(3) The antibody titer in the serum of this rabbit as an immunized animal was measured every week from the 6th week after the first immunization by enzyme immunoassay (ELISA, EIA). The operation of this ELISA method is shown below.
[1] Bovine HMG-1 obtained in Reference Example 3 was dissolved in physiological saline so as to have a concentration of 5 μg / mL, and this was added to a 96-well microplate (manufactured by NUNK) at 100 μL per well at 37 ° C. The bovine HMG-1 was immobilized on the plate at rest for 2 hours.
[2] The microplate was washed with a washing solution (phosphate buffered saline containing 0.05% Tween 20 (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride and 2 After washing with an aqueous solution containing 68 mM potassium chloride (pH 7.2))), 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution (pH 7.2) containing 1% BSA was added in an amount of 300 μL per well. Then, the mixture was allowed to stand at 37 ° C. for 2 hours for blocking and then washed again with a washing solution.
[3] The rabbit serum to be tested for antibody production is diluted 1,000-fold, 10,000-fold, and 100,000-fold with physiological saline, and 100 μL of these is added to each well of the microplate. The reaction was allowed to stand for 2 hours at 37 ° C., and then washed with a washing solution.
[4] Also, as a control, 100 μL of 0.1 M phosphate buffered saline containing 1% BSA was added to each well of the microplate of [2] and allowed to stand at 37 ° C. for 2 hours. Washed.
[5] Peroxidase (POD) -labeled anti-rabbit IgG antibody (Amersham) was diluted 5,000 times with phosphate buffered saline containing 3% BSA, and then microplates according to [3] and [4] The reaction was carried out by adding 100 μL per well and allowing to stand at 37 ° C. for 2 hours.
[6] After washing with a washing solution, a peroxidase reaction solution (3 mM 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) [ABTS] containing 50 mM disodium hydrogen phosphate—24 mM 100 μL per well of 2 μL of 1.7% hydrogen peroxide added to 1 mL of citrate buffer immediately before use) was allowed to react at room temperature. After 15 minutes, 50 μL of 6N sulfuric acid was added per well to stop the reaction.
[7] The absorbance at 415 nm was measured with an EIA plate reader (manufactured by Bio-Rad).

(4) Since it was recognized that the antibody titer reached a plateau after 12 weeks from the first immunization, a whole blood was collected from the immunized rabbit. And antiserum was obtained from the blood of this rabbit.
(5) With respect to the antiserum obtained in the above (4), sodium sulfate was added at a ratio of 0.18 g with respect to 1 mL of this antiserum at 22 to 25 ° C. with stirring, and further after the sodium sulfate was completely dissolved. Salting was performed by continuing stirring for 30 minutes.
(6) This was centrifuged (7000 g, 15 minutes) at 22 to 25 ° C., and the precipitate obtained by separating from the supernatant was dissolved in 1 mL of phosphate buffered saline.
(7) Next, this was sufficiently dialyzed against phosphate buffered saline, and then centrifuged at 1000 g for 20 minutes to remove insolubles.
(8) This was passed through a DEAE-cellulose ion exchange column (manufactured by Selva) [1 × 2.5 cm] equilibrated with phosphate buffered saline, and the eluate was collected.
(9) It was confirmed from the absorbance at 280 nm that immunoglobulin G (IgG) was contained in the flow-through fraction of the eluate, and this was collected and concentrated.

(10) Next, this was passed through a column on which human HMG-1 prepared in Reference Example 2 was immobilized, and affinity chromatography was performed. This operation is shown below.
[1] Affinity chromatography in which 1 g of CNBr-Sepharose (Pharmacia Biotech) was reacted with 2 mg of human HMG-1 prepared in Reference Example 2 according to the instruction manual, and HMG-1 was immobilized. A chromatography column was prepared.
[2] The column was equilibrated with phosphate buffered saline, and then passed through the component (polyclonal antibody) concentrated in (9) above.
[3] This was thoroughly washed with phosphate buffered saline and then passed through 0.1 M acetate buffer (pH 3.0).
[4] The eluted fractions were collected, dialyzed with phosphate buffered saline, and then concentrated.

  By the above affinity chromatography operation, a polyclonal antibody that binds to human HMG-1 was collected.

(11) The rabbit polyclonal antibody obtained by the above operation is capable of binding to human HMG-1. It can also bind to human HMG-2 having high homology with human HMG-1.

[Example 10] (Confirmation of antibody reactivity with human HMG-1 and human HMG-2)
The antibody (monoclonal antibody) that binds to human HMG-1 prepared in Example 4 but does not bind to human HMG-2 was tested for reactivity with human HMG-1 and human HMG-2 by Western blotting. Confirmed.

  In Example 9, the reactivity of human HMG-1 and human HMG-2 contained in human serum with each antibody was confirmed. In this example, human HMG prepared from human cells was used. -1 and human HMG-2 were confirmed for reactivity with the antibody.

1. Western blotting (1) Reaction of antibodies (monoclonal antibodies) that bind to human HMG-1 but do not bind to human HMG-2 to human HMG-1 and human HMG-2 electrophoresed and transcribed Western blotting
[1] Electrophoresis of human HMG-1 and human HMG-2 prepared from human cells (HL60 cells) in Reference Example 5 described later using a Titan Gel Lipoprotein Electrophoresis Kit (manufactured by Helena Laboratories) went. The support was an agarose gel, and 7.5 mM borate buffer (pH 9) containing 200 mM sodium chloride in which human HMG-1 and human HMG-2 were dissolved to 0.5 mg / mL, respectively. 0.0) was contacted.
[2] Then, barbital buffer (pH 8.8) was used as an electrophoresis buffer, and electrophoresed by applying electricity at a voltage of 90 V for 75 minutes. The electrophoresis was performed by preparing two sets of the same ones, and the following operations were performed in the same manner.
[3] The transfer after the electrophoresis of [2] was performed by a dry method using a Nova Blot Electrophoretic Transfer Kit (Pharmacia-LCB) according to the instruction manual.

  First, the agarose gel subjected to the electrophoresis in [2] was placed on a transfer device. Next, on this agarose gel, a 9 cm × 9 cm nitrocellulose membrane (manufactured by Bio-Rad) was overlaid, and 48 mM tris (hydroxymethyl) aminomethane, 39 mM glycine, 0.0375% (W / V) sodium dodecyl sulfate. Transfer was performed at a current of 65 mA for 2 hours using a transfer buffer solution (SDS) and 20% (V / V) methanol.

[4] The nitrocellulose membrane subjected to this transfer was subjected to phosphate buffered saline containing 1% BSA (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride and 2.68 mM. Blocking was performed by immersing in 20 mL of an aqueous solution containing potassium chloride (pH 7.2) at 4 ° C. overnight.
[5] Next, this was washed by shaking for 10 minutes in 20 mL of a cleaning solution (phosphate buffered saline containing 0.05% Tween 20). This operation was performed three times.
[6] 80 μg of an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 prepared in Example 4 was dissolved in 20 mL of phosphate buffered saline, and dissolved in this solution. The nitrocellulose membrane subjected to the above operation [5] was reacted by being immersed for 2 hours at room temperature.
[7] The nitrocellulose membrane subjected to the operation of [6] was washed by shaking in 20 mL of a cleaning solution for 10 minutes. This was done three times.
[8] Next, a peroxidase-labeled anti-mouse IgG antibody (manufactured by Dako) was diluted 500-fold with a phosphate buffered saline containing 3% BSA to prepare a 20 mL solution. The nitrocellulose membrane of [7] was allowed to react by being immersed for 2 hours at room temperature.
[9] The nitrocellulose membrane was washed with shaking in 20 mL of a washing solution for 10 minutes. This operation was performed three times.
[10] The nitrocellulose membrane of [9] above was added to 20 mL of phosphate buffered saline containing 0.025% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide at room temperature. Color was developed by dipping for a minute.

  By the above operation, the reactivity of the antibody (monoclonal antibody) prepared in Example 4 that binds to human HMG-1 but does not bind to human HMG-2 with human HMG-1 and human HMG-2 The result of Western blotting was obtained.

(2) Western blotting in which an antibody (polyclonal antibody) that binds to human HMG-2 was reacted with a human HMG-1 and human HMG-2 electrophoresed and transcribed For reference, the above human HMG-1 An antibody that binds to human HMG-2 but does not bind to human HMG-2 (monoclonal antibody) (polyclonal antibody) that binds to commercially available human HMG-2 [Product name: Anti HMG-2 (C-19), [1] to [10] of (1) above except that Human, product number: SC-8758, manufacturer: Santa Cruz (USA), sales: Cosmo Bio Inc.) is used in [6] of (1) above. Then, a band was obtained and used to confirm the position of the human HMG-2 band in this Western blot method.

(3) Western in which bovine HMG-1 and bovine HMG-2 were electrophoresed and transcribed with an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 Blot method As a reference, in place of the above-mentioned human HMG-1 and human HMG-2, except that an equal volume mixture of bovine HMG-1 and bovine HMG-2 prepared from bovine thymus in Reference Example 3 was used. The operation was performed as described in [1] to [10] in (1) to obtain a band, which was used for confirmation of the position of the bovine HMG-1 band in this Western blotting method.

2. Experimental Results (1) Results of Western blotting The results of Western blotting in 1 above are shown in FIG.
In this figure, the lane “H [1]” is the lane of the Western blotting method of (1) above, and the lane of “H [2]” is the lane of the Western blotting method of (1) (2). The lane of “B [1]” is the lane of the Western blotting method of (1) above (3).

From FIG. 6, the following can be understood.
• In the “H [1]” lane, only one band is recognized.
In the lane “H [1]”, no band is recognized at the band position in the lane “H [2]”.
・ Bands are recognized at the same position in the lanes “H [1]” and “B [1]”.

That is,
[1] It can be seen that no band is observed in the lane of “H [1]” at the position of the lane of “H [2]” reacted with an antibody (polyclonal antibody) that binds to human HMG-2. .
[2] In the “H [1]” lane where human HMG-1 and human HMG-2 prepared from human cells were electrophoresed, only one band was observed, and the position of the band was human HMG- It is the same as the position of the only band in the lane of “B [1]” in which bovine HMG-1 having a high homology with 1 and bovine HMG-2 having a high homology with human HMG-2 were migrated. I understand that.

  From the above, it was confirmed that the antibody (monoclonal antibody) prepared in Example 4 binds to human HMG-1 prepared from human cells and does not bind to human HMG-2 prepared from human cells. It was.

(2) Control
For the other nitrocellulose membrane that has been operated up to the operation of [1] [1] [5], the operation of [6] is not performed, but the operations of [7] and after are performed in the same manner. Was used as a control.
In (2) and (3) above, a control was prepared in the same manner.

In the control (control) in which none of these antibodies was allowed to act, no color development was observed.
From this, it was confirmed that non-specific color development did not occur in each Western blotting method.

[Reference Example 5] (Preparation of human HMG-1 and human HMG-2 from human cells)
Human HMG-1 and human HMG-2 were prepared from human cells (HL60 cells).
[1] First, 3 L of the supernatant of a culture solution of human cells (HL60 cells) cultured in RPMI 1640 was concentrated to about 250 mL.
[2] Next, sodium chloride was added so that the final concentration was 200 mM.
[3] This was centrifuged with a centrifuge (10,000 rpm, 30 minutes), and the supernatant was collected and filtered with a filter having a pore size of 0.45 μm.
[4] The filtrate was passed through a high trap heparin column (manufactured by Amersham Pharmacia).
[5] The column was washed with a 10 mM sodium phosphate buffer solution (pH 7.0) containing 200 mM sodium chloride.
[6] Next, a sodium phosphate buffer solution (pH 7.0) was eluted from the column with a sodium chloride concentration gradient of 200 mM to 2,000 mM.
[7] Each elution fraction was subjected to SDS-polyacrylamide electrophoresis, and the fraction containing human HMG-1 and human HMG-2 was identified based on its mobility. This fraction was a fraction eluted when the sodium chloride concentration was 500 mM to 1,000 mM.
[8] The fraction containing human HMG-1 and human HMG-2 of [7] above is applied to a CM-Sephadex C25 column that has been equilibrated with 7.5 mM sodium borate buffer (pH 9.0). I passed.
After that, elution was performed with 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and cation exchange chromatography was performed.
[9] Each fraction eluted here was subjected to SDS-polyacrylamide electrophoresis, and the fraction containing human HMG-1 and the fraction containing human HMG-2 were identified from their mobility.
By the above operation, human HMG-1 was prepared from human cells (HL60 cells), and human HMG-2 was also prepared.

SEQ ID NO: 1: Peptide synthesized based on the amino acid sequence of human HMG-1 SEQ ID NO: 2: Peptide synthesized based on the amino acid sequence of human HMG-1

It is the figure which showed the result of having estimated the high hydrophilicity of each amino acid residue by the method of Hop et al. About all the amino acid sequences of human HMG-1. It is the figure which showed the analytical curve created by measuring the sample containing human HMG-1 with the immunological measuring reagent of this invention, and the immunological measuring method. It is the figure which showed the analytical curve created by measuring the sample containing bovine HMG-1 with the immunological measurement reagent of this invention, and the immunological measurement method. It is the figure which showed the result of the western blotting method which confirmed the reactivity with human HMG-1 and human HMG-2 of each antibody. It is the figure which showed the elution pattern of the cation exchange chromatography in preparation of bovine HMG-1 and bovine HMG-2. It is the figure which showed the result of the western blotting method which confirmed the reactivity with human HMG-1 and human HMG-2 of each antibody.

Claims (10)

  An antibody that binds to human high mobility group protein-1 but does not bind to human high mobility group protein-2. Formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
The antibody according to claim 1, which is prepared by using a peptide consisting of the amino acid sequence represented by   The antibody according to claim 1 or 2, which is a monoclonal antibody.   The antibody according to claim 3, which is a monoclonal antibody produced by the hybridoma MD77 (FERM P-18404).   The amino acid sequence of human high mobility group protein-1 is compared with the amino acid sequence of human high mobility group protein-2, and the amino acid sequence of human high mobility group protein-1 having low homology is selected. Immunizing an animal with a peptide containing an amino acid sequence as an immunogen, obtaining the produced antibody, binding to human high mobility group protein-1 and not binding to human high mobility group protein-2 The antibody according to any one of claims 1 to 3, which is an antibody obtained by confirming or selecting   An animal is immunized with the whole amino acid sequence of human high mobility group protein-1 or a peptide or protein containing the amino acid sequence represented by the above formula (I) as an immunogen, and binds to human high mobility group protein-1 A polyclonal antibody that does not bind to human high mobility group protein-2 and a polyclonal antibody that binds to both human high mobility group protein-1 and human high mobility group protein-2 Then, the obtained polyclonal antibody is passed through a solid phase carrier immobilized on a solid phase with human high mobility group protein-2 as a ligand, and contacted with human high mobility group protein-1 and human high mobility. A polyclonal antibody that binds to any of the two group protein-2s is bound to the solid phase via the human high mobility group protein-2 immobilized on the solid phase as a ligand to the solid phase carrier and bound to the ligand. The antibody according to claim 1 or 2, which is a polyclonal antibody obtained as a fraction passed through a solid phase carrier without passing through.   An animal is immunized with the whole amino acid sequence of human high mobility group protein-1 or a peptide or protein containing the amino acid sequence represented by the above formula (I) as an immunogen, and binds to human high mobility group protein-1 A polyclonal antibody that does not bind to human high mobility group protein-2 and a polyclonal antibody that binds to both human high mobility group protein-1 and human high mobility group protein-2 Then, the obtained polyclonal antibody is passed through an affinity chromatography column in which human high mobility group protein-2 is immobilized on a solid phase using human high mobility group protein-2 as a ligand. A polyclonal antibody that binds to both TEIN-1 and human high mobility group protein-2 is bound to the solid phase via human high mobility group protein-2 immobilized on the solid phase as a ligand to the column, The antibody according to claim 1 or 2, which is a polyclonal antibody obtained as a fraction passed through a column without binding to the ligand.   The amino acid sequence of human high mobility group protein-1 is compared with the amino acid sequence of human high mobility group protein-2, and the amino acid sequence of human high mobility group protein-1 having low homology is selected. Immunizing an animal with a peptide containing an amino acid sequence as an immunogen, obtaining the produced antibody, binding to human high mobility group protein-1 and not binding to human high mobility group protein-2 The method for producing an antibody according to any one of claims 1 to 3, wherein the antibody is obtained by confirming or selecting.   An animal is immunized with the whole amino acid sequence of human high mobility group protein-1 or a peptide or protein containing the amino acid sequence represented by the above formula (I) as an immunogen, and binds to human high mobility group protein-1 A polyclonal antibody that does not bind to human high mobility group protein-2 and a polyclonal antibody that binds to both human high mobility group protein-1 and human high mobility group protein-2 Then, the obtained polyclonal antibody is passed through a solid phase carrier immobilized on a solid phase with human high mobility group protein-2 as a ligand, and contacted with human high mobility group protein-1 and human high mobility. A polyclonal antibody that binds to any of the two group protein-2s is bound to the solid phase via the human high mobility group protein-2 immobilized on the solid phase as a ligand to the solid phase carrier and bound to the ligand. The method for producing an antibody according to claim 1 or 2, wherein a fraction obtained by passing through a solid phase carrier is obtained.   An animal is immunized with the whole amino acid sequence of human high mobility group protein-1 or a peptide or protein containing the amino acid sequence represented by the above formula (I) as an immunogen, and binds to human high mobility group protein-1 A polyclonal antibody that does not bind to human high mobility group protein-2 and a polyclonal antibody that binds to both human high mobility group protein-1 and human high mobility group protein-2 Then, the obtained polyclonal antibody is passed through an affinity chromatography column in which human high mobility group protein-2 is immobilized on a solid phase using human high mobility group protein-2 as a ligand. A polyclonal antibody that binds to both TEIN-1 and human high mobility group protein-2 is bound to the solid phase via human high mobility group protein-2 immobilized on the solid phase as a ligand to the column, The method for producing an antibody according to claim 1 or 2, wherein a fraction passing through the column is obtained without binding to the ligand.

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