JP2013096783A - Data detection method, diagnostic drug and diagnostic kit for determining pulmonary adenocarcinoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a data detection method for determining existence of crisis of pulmonary adenocarcinoma in a subject, a degree of advance or a prognosis condition, and to provide a kit to be used for the method.SOLUTION: The data detection method measures human calnexin contained in a subject's specimen by using an antibody to be specifically coupled with the human calnexin and determines the existence of crisis of pulmonary adenocarcinoma in the subject, a degree of progress or a prognosis condition on the basis of the measurement result. As an antibody to be specifically coupled with the human calnexin, a monoclonal antibody is used.

Description

  The present invention relates to a method for determining lung adenocarcinoma using an antibody that specifically binds to human calnexin (hereinafter abbreviated as CANX if necessary). The present invention also relates to a diagnostic agent and diagnostic kit for lung adenocarcinoma containing an antibody that specifically binds to human calnexin.

Lung cancer has a high incidence and is representative of refractory cancer along with pancreatic cancer. When a patient notices a medical condition, it often progresses to an untreatable state. Traditionally, lung cancer has been diagnosed using a combination of X-rays and cytology of sputum, but it is often difficult to detect while the stage is in an early stage and is ready for treatment. It was.
Lung cancer is classified into small cell lung cancer with high drug sensitivity and non-small cell lung cancer with low drug sensitivity. Small cell lung cancer accounts for 20% of all lung cancer and non-small cell lung cancer accounts for 80% of all lung cancer. Non-small cell lung cancer is further classified into lung adenocarcinoma, squamous cell carcinoma, and large cell cancer. Lung adenocarcinoma is a very important cancer that accounts for 80% of non-small cell lung cancer.

Until now, a specific antigen (HCAVIII) for human non-small cell lung cancer has been identified and isolated as a lung cancer marker, and a method for using an antibody against the lung cancer marker for diagnosis and treatment is known (Patent Literature). 1). In addition, a specific monoclonal antibody against an epitope found in the amino acid of LCGA related to human non-small cell lung cancer protein is known (Patent Document 2).
Human calnexin (NCBI Refseq, No. NP001737) belongs to the calreticulin family, is expressed as a protein of about 67 kDa, and is known as a calcium binding protein that interacts with a newly synthesized glycoprotein in the endoplasmic reticulum. Human calnexin plays a role in quality control of proteins synthesized in the endoplasmic reticulum, and is also involved in protein folding and secretion, and antigen presentation. It is considered that the cytoplasmic domain of human calnexin is cleaved by caspase and that the fragment suppresses apoptosis (Non-patent Document 1). Compared to the primary tissue of melanoma, the expression of human calnexin is significantly reduced in metastatic lesions of melanoma, and it is thought that the decreased expression of human calnexin contributes to the metastatic nature of melanoma cells (non-patented) Reference 2).
It has not been known to detect lung adenocarcinoma of non-small cell lung cancer separately from other carcinomas or other types of lung cancer using non-small cell lung cancer markers and monoclonal antibodies.

Japanese National Patent Publication No. 10-503087 Special Table 2003-501444 Japanese Unexamined Patent Publication No. 01-052800 Journal of Biochemistry, 136, 3, 399-405, 2004 Cancer Letters 203, 2, 225-231, 2004

The present invention measures human calnexin in a subject's specimen using an antibody that specifically binds to human calnexin, and determines whether or not the subject has developed lung adenocarcinoma based on the measurement results. Or it aims at providing the data detection method which determines the condition of a prognosis. Another object of the present invention is to provide a diagnostic agent for lung adenocarcinoma containing an antibody that specifically binds to human calnexin and a kit for diagnosing lung adenocarcinoma.

  The inventors of the present invention produced a monoclonal antibody using lung adenocarcinoma cells or a disrupted product thereof and identified a lung adenocarcinoma-specific marker by proteomic technology using the acquired antibody. Found that lung adenocarcinoma could be distinguished from other carcinomas and other types of lung cancer, and completed the present invention. That is, the present invention relates to the following (1) to (12).

(1) Measure human calnexin in a subject's sample using an antibody that specifically binds to human calnexin, and determine whether or not the subject has developed lung adenocarcinoma, A method to determine the prognosis status.
(2) The method according to (1), wherein the antibody that specifically binds to human calnexin is a monoclonal antibody.
(3) The method according to (2), wherein the monoclonal antibody that specifically binds to human calnexin is a monoclonal antibody produced by hybridoma FERM BP-11318.
(4) The method according to any one of (1) to (3), wherein the specimen is blood.
(5) A diagnostic agent for lung adenocarcinoma comprising an antibody that specifically binds to human calnexin.
(6) The diagnostic agent according to (5), wherein the antibody that specifically binds to human calnexin is a monoclonal antibody.
(7) The diagnostic agent according to (6), wherein the monoclonal antibody that specifically binds to human calnexin is a monoclonal antibody produced by hybridoma FERM BP-11318.
(8) A lung adenocarcinoma diagnostic kit comprising an antibody that specifically binds to human calnexin.
(9) The kit according to (8), wherein the antibody that specifically binds to human calnexin is a monoclonal antibody.
(10) The kit according to (9), wherein the monoclonal antibody that specifically binds to human calnexin is a monoclonal antibody produced by hybridoma FERM BP-11318.
(11) A monoclonal antibody that specifically binds to human calnexin produced by hybridoma FERM BP-11318.
(12) Hybridoma FERM BP-11318.

  The present invention measures human calnexin in a subject's specimen using an antibody that specifically binds to human calnexin, and determines whether or not the subject has developed lung adenocarcinoma based on the measurement results. Alternatively, a data detection method for determining a prognostic situation is provided. The present invention also provides a diagnostic agent and kit for lung adenocarcinoma containing an antibody that specifically binds to human calnexin.

The reactivity of the antibody contained in the culture supernatant of hybridoma KU-Lad-001 and A549 cells fixed with AMeX is shown. It is a figure explaining the purification method of the antigen protein which is overexpressed in a human lung adenocarcinoma cell and couple | bonds with the said monoclonal antibody. It is the flowchart explaining the identification and confirmation method of the refined antigen protein.

(A) The antigen-antibody mixture concentrated by the procedure shown in FIG. 2 is developed by SDS-PAGE and the result of zinc staining is shown. The second lane from the left shows the SDS-PAGE of the recovered product recovered from the Sepharose beads after the antigen-antibody reaction of the ascites antibody of KU-Lad-001 and the A549 soluble protein mixture. In the fourth lane from the left, the culture supernatant antibody of KU-Lad-001 is used instead of the ascites antibody of KU-Lad-001, and the subsequent operation is the same as in the second lane. The other lanes show the results of the control experiment. Only the second and fourth lanes from the left indicate that the target antigen protein is concentrated. (B) The result of Western blotting using KU-Lad-001 antibody on another gel obtained by SDS-PAGE by the same operation as in FIG. 4A is shown. 2nd from left (KU-Lad-001 ascites antibody and A549 soluble protein mixture), 4th lane (KU-Lad-001 culture supernatant antibody and A549 soluble protein mixture) and rightmost lane (before concentration) The target antigenic protein is detected only in the A549 soluble protein mixture).

The results of dot blotting of patient and healthy human serum and subsequent reaction with KU-Lad-001 are shown. The antibody is highly reactive with lung cancer patient sera including lung adenocarcinoma, squamous cell carcinoma, and small cell carcinoma, but has a strong reaction with healthy human serum. Indicates not to be shown. It is the figure which showed the reactivity of the formalin fixed and paraffin embedded A549 cell and KU-Lad-001 antibody. The KU-Lad-001 antibody has been shown to be suitable for staining of formalin-fixed and paraffin-embedded specimens.

The result of having examined whether the KU-Lad-001 antibody reacts with human normal bronchial epithelium and human normal lung tissue of human normal alveolar epithelium is shown. The result of having examined whether the KU-Lad-001 antibody reacts with the lung adenocarcinoma tissue extract | collected from four lung adenocarcinoma patients (FIG. 8A-D) is shown. The result of having examined whether the KU-Lad-001 antibody reacts with the lung squamous cell carcinoma tissue extract | collected from four lung squamous cell carcinoma patients (FIG. 9A-D) is shown.

Whether KU-Lad-001 antibody reacts with lung squamous cell carcinoma tissue collected from patients with small cell lung cancer (SCLC, FIG. 10A to C) and large cell neuroendocrine carcinoma (LCNEC, FIG. 10D) The result of examination is shown. The result of the positive rate in the tumor cell according to each histological type of lung cancer is represented. The results of staining intensity for each histological type of lung cancer are shown. The result of the staining score for each histological type of lung cancer is shown. The results of quantifying the amount of the antigen protein human calnexin recognized by the KU-Lad-001 antibody contained in 33 cases of lung adenocarcinoma patient serum and 56 cases of healthy subject serum are shown.

The antibody that specifically binds to human calnexin used in the data detection method of the present invention for determining the presence or absence of lung adenocarcinoma, the degree of progression, or the status of prognosis is specific to human calnexin. The antibody is not particularly limited as long as it can bind, and may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable. An antibody that specifically binds to human calnexin can be obtained by immunizing human calnexin or a fragment thereof. Examples of human calnexin include a polypeptide having the amino acid sequence represented by SEQ ID NO: 1.
A monoclonal antibody that specifically binds to human calnexin is obtained by, for example, culturing a hybridoma prepared from an antibody-producing cell taken from a mouse immunized with human calnexin having the amino acid sequence represented by SEQ ID NO: 1 or a fragment thereof. Can be manufactured.
Human calnexin or a fragment thereof can be used as an immunogen as it is, but a desired immunogen can also be prepared using the protein or a fragment thereof.

In order to efficiently perform immunity induction caused by the immunogen, it is also one preferred embodiment that the antigen protein or a fragment thereof is bound to a carrier. It is particularly preferred when the antigen protein or fragment thereof is 100 amino acids or less.
The antigen protein or fragment thereof and the carrier can be bound by a reaction that causes a covalent bond between the respective functional groups, with or without a linker. Examples of the functional group include a carboxyl group, an amino group, a glycidyl group, a thiol group, a hydroxyl group, an amide group, an imino group, a hydroxysuccinyl ester group, a maleimide group, and an isothiocyanate group. By performing the condensation reaction, the protein or a fragment thereof and the carrier can be bound.
Examples of the bonding method without using a linker include a method using a carbodiimide compound such as EDC. It is also possible to use NHS or a derivative thereof as a substance that enhances this reaction. The condensation reaction between the isothiocyanate group and the amino group does not require any other reagent, and can be combined only by mixing under neutral to weakly alkaline conditions.

The linker may be any molecule as long as it is a molecule that binds the functional groups of the carrier protein and the side chain of the peptide to each other. A preferred embodiment is, for example, a molecule having a first reactive group capable of reacting with an amino acid residue of a carrier protein and a second reactive group capable of reacting with a functional group on the side chain of the peptide. It is preferable that the first reactive group and the second reactive group are different groups. Examples of the reactive group include allyl azide, carbodiimide, hydrazide, hydroxymethylphosphine, imide ester, isocyanate, maleimide, NHS-ester, PFP-ester, psoralen, pyridyl disulfide, vinyl sulfone, and isothiocyanate.
A desired antibody or antibody fragment can be produced using the immunogen. It is also preferable to administer the immunogen together with an adjuvant in order to efficiently induce immunity with the immunogen. Examples of adjuvants include Freund's adjuvant and alum.

  Examples of antibodies that can be produced include polyclonal antibodies and monoclonal antibodies. Examples of the polyclonal antibody include an antiserum of an immunized animal or a polyclonal antibody purified from the antiserum. The polyclonal antibody of the present invention includes any polyclonal antibody that specifically binds to the above human calnexin. Specifically, a warm-blooded animal is immunized with human calnexin, Examples include polyclonal antibodies prepared by collecting serum from animals and removing fractions reactive to human calnexin by known affinity chromatography. Examples of the warm-blooded animals include mice, rats, hamsters, guinea pigs, rabbits, goats, horses, cows, sheep, goats, pigs, chickens and the like.

Examples of monoclonal antibodies include antibodies or antibody fragments produced by hybridomas, or recombinant antibodies or antibody fragments produced by a transformant transformed with an expression vector containing an antibody gene.
The method for producing a hybridoma that produces the monoclonal antibody comprises administering the above-mentioned immunogen to a warm-blooded animal usually once every 2 to 6 weeks, about 2 to 10 times in total, and antibody titer from the immunized warm-blooded animal. It is possible to perform the final immunization by selecting an individual in which spleen is observed, and the spleen or lymph node is taken out 2 to 5 days later and fused with mouse myeloma cells. The methods described above can be applied to the production of hybridomas and the preparation of monoclonal antibodies.
Examples of the gene recombinant antibody include antibodies produced by gene recombination, such as humanized antibodies, human antibodies or antibody fragments. Examples of the recombinant antibody of the present invention include antibodies that retain the characteristics of a monoclonal antibody.

The humanized antibody includes an antibody having an Fc region of a human antibody such as a human chimeric antibody or a human type complementarity determining region (hereinafter referred to as CDR) transplanted antibody.
The human chimeric antibody consists of a warm-blooded antibody antibody heavy chain (hereinafter referred to as H chain) V region (hereinafter referred to as antibody heavy chain variable region as VH), and antibody light chain (referred to as L chain). Yes) V region (hereinafter referred to as antibody variable region light chain VL) and human antibody heavy chain constant region (hereinafter referred to as CH) or human antibody light chain constant region (hereinafter referred to as CL) ).
The human chimeric antibody of the present invention is used for animal cells having cDNA encoding VH and VL obtained from a hybridoma producing a human calnexin-specific mouse monoclonal antibody and having genes encoding human antibody CH and human antibody CL. A human chimeric antibody expression vector inserted into an expression vector can be constructed and introduced into animal cells for expression and production.
The structure of the human chimeric antibody of the present invention may belong to any immunoglobulin (Ig) class, but it is IgG type, and further IgG C, IgG2, IgG3, IgG4 and other immunoglobulin C regions belonging to IgG type. Is preferred.

The human CDR-grafted antibody is a human antibody that constructs a gene encoding the V region in which the VH and VL CDR sequences of a human calnexin-specific mouse monoclonal antibody are replaced with the CDR sequences of any human antibody VH and VL, respectively. It can be produced by introducing the gene into an expression vector for animal cells having a gene encoding CH of human and CL of human antibody, respectively, and expressing it.
The structure of the human CDR-grafted antibody C region of the present invention may belong to any immunoglobulin (Ig) class, but may be an IgG type, or an immunoglobulin such as IgG1, IgG2, IgG3, or IgG4 belonging to an IgG type. The C region is preferred.
A human antibody originally means an antibody that naturally exists in the human body, but a human antibody phage library created by recent advances in genetic engineering, cell engineering, and developmental technology, and human antibodies Also included are antibodies obtained from production transgenic animals.

The antibody present in the human body is, for example, a human antibody-producing cell that produces a human antibody is isolated, immortalized by infecting an EB virus or the like, and single-cloned to produce a lymphocyte that produces the antibody. It can be cultured as a regenerative cell, and the antibody can be purified from the culture.
A human antibody phage library is a library of antibody fragments such as Fab and scFv expressed on the phage surface by inserting antibody cDNA prepared from human antibody-producing cells such as B cells or lymphocytes into a phage vector. That means. From the library, phages expressing antibody fragments having a desired antigen-binding activity can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can also be converted into a human antibody molecule consisting of two complete heavy chains and two complete light chains using genetic engineering techniques.

Human calnexin antibody-producing cells can be isolated using an antigen or a fragment thereof. As a preferred embodiment, the antibody-producing cells are labeled via an antigen or a fragment thereof and isolated from other cells, and the antibody-producing cells are bound to an insoluble carrier using an antigen or a fragment thereof bound to an insoluble carrier. However, it is not limited to these methods.
As a human antibody-producing transgenic animal, an animal in which a human antibody gene is integrated into the genome is used. Specifically, a human antibody-producing transgenic animal can be created by introducing a human antibody gene into mouse ES cells, transplanting the ES cells into other mouse early embryos, and generating them. The method for producing a human antibody from a human antibody-producing transgenic animal is to purify the antibody from the culture by obtaining and culturing the human antibody-producing hybridoma by a conventional hybridoma production method in warm-blooded animals. be able to.

  Examples of the antibody fragment of the present invention include an antibody fragment obtained by digesting an antibody with an appropriate peptidase or an antibody fragment produced by gene recombination. The antibody fragments are Fab (Fragment of antigen binding), Fab ′, F (ab ′) 2, single chain antibody (Single chain Fv; hereinafter referred to as scFv) that specifically react with human calnexin. ) (Science, 242, 423, 1988), dimerization variable region (also called Diabody) (Nature Biotechnol., 15, 629, 1997), and disulfide stabilized V region fragment (hereinafter referred to as dsFv) (Molecular Immunol., 32, 249, 1995). The antibody fragment also includes a peptide containing the above-described VH and VL CDR amino acid sequences (hereinafter referred to as a CDR-containing peptide) (J. Biol. Chem., 271, 2966, 1996). Alternatively, VH and VL are not integrated by disulfide bonds, but are expressed as separate molecules (represented as VH and VL molecules, respectively) (Nature Biotechnology, 14, 1714, 1996) as antibody fragments. .

  Fab is composed of the N-terminal half of the H chain obtained by cleaving the upper peptide part of the two disulfide bonds that crosslink the two H chains at the hinge region of IgG with the enzyme papain, and the entire L chain. It is an antibody fragment having an antigen binding activity of about 30,000 molecular weight.

The Fab of the present invention can be obtained by treating an anti-human calnexin antibody with the enzyme papain. Alternatively, a Fab can be produced by inserting a DNA encoding the Fab fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. .
Fab ′ is an antibody fragment having an antigen binding activity of about 50,000 molecular weight obtained by cleaving the disulfide bond between the hinges of F (ab ′) 2 below.
The Fab ′ of the present invention can be obtained by treating the anti-human calnexin antibody F (ab ′) 2 with a reducing agent dithiothreitol. Also, Fab ′ is produced by inserting a DNA encoding the Fab ′ fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. You can also.
F (ab ') 2 is obtained by treating the lower part of two pairs of disulfide bonds in the hinge region of IgG with the enzyme trypsin, and has an antigen-binding activity of about 100,000 molecules with two Fab regions bound at the hinge region. It is an antibody fragment having
The F (ab ′) 2 of the present invention can be obtained by treating an anti-human calnexin antibody with a reducing agent dithiothreitol. Alternatively, the DNA encoding the F (ab ′) 2 fragment of the antibody is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the vector is introduced into a prokaryotic or eukaryotic expression, and expressed. ab ′) 2 can be produced.

scFv is represented by a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P). VH and VL contained in the scFv used in the present invention can be any of the anti-human calnexin antibodies of the present invention.
The scFV of the present invention synthesizes VH and VL cDNA from mRNA prepared from an anti-human calnexin antibody-producing hybridoma, and inserts the DNA encoding the scFv fragment of the antibody into a prokaryotic expression vector or eukaryotic expression vector. The scFv can be produced by expressing the vector by introducing it into a prokaryotic or eukaryotic organism.

Diabody is an antibody fragment in which scFv having the same or different antigen binding specificity forms a dimer, and is an antibody fragment having a bivalent antigen-binding activity for the same antigen or a bispecific antigen-binding activity for different antigens. .
The diabody of the present invention synthesizes VH and VL cDNAs from mRNA prepared from an anti-human calnexin antibody-producing hybridoma, and inserts the DNA encoding the diabody fragment of the antibody into a prokaryotic expression vector or eukaryotic expression vector. Diabodies can be produced by expressing the vectors by introducing them into prokaryotes or eukaryotes.

dsFv is a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are linked by a disulfide bond. The amino acid residue to be substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. [Protein Engineering, 7, 697 (1994)]. VH and VL contained in the dsFv of the present invention can be any anti-human calnexin antibody.
The dsFv of the present invention synthesizes VH and VL cDNAs from mRNA prepared from an anti-human calnexin antibody-producing hybridoma, and inserts the DNA encoding the dsFv fragment of the antibody into a prokaryotic or eukaryotic expression vector. Then, the vector can be expressed by introducing it into a prokaryote or eukaryote to produce dsFv.

The peptide containing CDR is composed of at least one region of CDR of VH or VL. A peptide containing a plurality of CDRs can be produced by binding directly or via an appropriate peptide linker.
VH and VL molecules can be produced by synthesizing VH and VL cDNAs from mRNA prepared from anti-human calnexin antibody-producing hybridomas and expressing them in a suitable combination of expression vector and host. In order to obtain a molecule having a good affinity with a human calnexin molecule, it is also a preferred embodiment that the expression vector is a phage expression vector and the host is E. coli. A VH molecule and a VL molecule can also be expressed and produced as a fusion protein with a desired protein.

(Production of antibody derivatives)
The anti-human calnexin antibody or antibody fragment used in the present invention includes a derivative of an antibody in which a radioisotope, protein, drug or the like is chemically or genetically introduced or bound to the antibody or antibody fragment. To do.
The derivative of the antibody used in the present invention is an N-terminal side or C-terminal side of the H chain or L chain of the anti-human calnexin antibody or antibody fragment used in the present invention, and further appropriate substitution in the antibody or antibody fragment. It can be produced by chemically linking a radioisotope, protein, or drug to a group or a side chain, and further to a sugar chain in an antibody or antibody fragment (Introduction to Antibody Engineering, Osamu Kinmitsu, Co., Ltd.) Jinjinshokan, 1994).

Alternatively, DNA obtained by ligating DNA encoding an anti-human calnexin antibody or antibody fragment and DNA encoding the protein to be bound is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the vector is inserted into a prokaryotic or eukaryotic expression vector. It can be expressed and genetically engineered by introducing it into a eukaryote.
The drug may be any substance such as a radioisotope, protein, or small molecule.
Examples of the drug used in the detection method, quantification method, detection reagent, quantification reagent or diagnostic agent of the derivative of the antibody of the present invention include a label used in a usual immunoassay. Examples of the label include radioactive isotopes, enzymes such as alkaline phosphatase, peroxidase, luciferase, luminescent substances such as acridinium ester and lophine, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC) and the like. Examples include fluorescent substances. As the drug, a substance such as biotin can also be used as a label. The substance is bound to the substance, such as avidin, to which a radioactive isotope, an enzyme such as alkaline phosphatase, peroxidase or luciferase, a luminescent substance such as acridinium ester or lophine, or a fluorescent substance such as FITC or RITC is bound. It is a substance that makes it possible to use a secondary labeling substance that can bind specifically and with high affinity.

  The antibody that binds to human calnexin or a fragment thereof used in the present invention is an antibody production B of a mouse immunized with a cell that produces the human calnexin or a fragment thereof having the amino acid sequence represented by SEQ ID NO: 1 or a fragment thereof, etc. It can be produced by preparing a hybridoma from a cell, screening a hybridoma that reacts with human calnexin or a fragment thereof from a variety of hybridomas obtained, and culturing the obtained hybridoma. Hereinafter, a method for producing a hybridoma that produces a monoclonal antibody according to the present invention using cells or a disrupted product thereof will be described.

(Preparation of antibody-producing B cells)
Preparation of antibody-producing cells according to the present invention is carried out by administering human calnexin or a fragment thereof, or a fragment thereof, to a warm-blooded animal, taking out the spleen or lymph node, and cell fusion with mouse myeloma cells. To do.
Cells that produce human calnexin or a fragment thereof may be either lung cancer cells that have been surgically excised from a patient or subcultured cell lines. Examples of the subculture cell lines include human LCNEC cell lines LCN1, LCN2, and lung adenocarcinoma cell A549. These cell lines can also be used for screening of monoclonal antibodies by immunostaining.
When the lung cancer cell is used for sensitization of a warm-blooded animal, it is preferably used in a form such as a homomodulate of the cell or a cell as it is.

The warm-blooded animal is not particularly limited, and examples thereof include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, chickens, and the like, and particularly small animals such as mice, rats, and guinea pigs. Is preferred.
The cells and the like are administered to a site where antibody production is possible, for example, intraperitoneally, intravenously, or subcutaneously. When using a mouse, it is preferably administered from the abdominal cavity or the tail vein. In addition, the administration of the cells or the like may be administered alone or together with complete Freund's adjuvant or incomplete Freund's adjuvant in order to enhance antibody production ability.

The cells and the like are administered at about 10 ³ to 10 ⁸ or equivalent thereof per kg body weight of the warm-blooded animal. For example, when mice are used, 10 ³ to 10 ⁷ mice / mouse are administered.
The cells are administered to warm-blooded animals usually once every 2 to 6 weeks, about 2 to 10 times in total. In addition, an individual having an antibody titer is selected from the immunized warm-blooded animals, the final immunization is performed, and spleen or lymph nodes are collected 2 to 5 days later, and the antibody-producing cells contained therein are myeloma. Fuse with cells.

(Production of hybridoma)
Hybridomas are prepared by fusing the obtained antibody-producing cells and myeloma cells. When using a mouse, examples of the myeloma cell include cell lines such as SP2 / 0, NS-1, P3U1, and AP-1. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. PEG, particularly PEG 1000 to PEG 6000 is preferable. The PEG is preferably added at a concentration of about 10 to 80%.
The preferred number ratio between the antibody-producing cells and the myeloma cells is usually about 1: 1 to 20: 1. These cells can be efficiently fused by incubating usually at 20 to 40 ° C., preferably 30 to 37 ° C., usually for 1 to 10 minutes.
Next, the cell-fused cell group is cultured for 5 to 25 days using an animal cell culture medium (for example, RPMI1640) containing fetal bovine serum 10 to 20% to form a cell colony.

  The cell colony is subjected to a limiting dilution method in which cells are seeded at a rate of 1 cell / well twice, and binding to human calnexin or a fragment thereof is performed by screening using a culture supernatant of a well in which a hybridoma is confirmed. A hybridoma in which antibody production has been confirmed is defined as a single clone.

  The first limiting dilution method uses a 96-well plate and 200 μl of RPMI-1640 medium (final concentration of 15% fetal bovine serum and 10% BM-Condimed H1 (Roche Diagnostics) to contain two hybridomas per well. Stick, Tokyo)). After one to two weeks, one or two hybridoma colonies are visible in each well. At this point, it is further examined whether the hybridoma maintains the antibody production ability using the same screening method as described above. Hybridomas are collected from positive wells and added to 200 μl RPMI-1640 medium (final concentration 15% fetal calf serum and 10% BM-Condimed H1) using a 96-well plate to contain 0.5 cells per well. In this case, one hybridoma colony is usually observed by well after about 1 week to 10 days. Using this supernatant, antibody production is confirmed using the same screening method. This hybridoma has become a single clone at this point, and this culture supernatant is used as a monoclonal antibody.

The screening of the hybridoma can also be performed by detecting an antibody specific for a cell or the like used as an antigen and / or an IgG antibody of the hybridoma regardless of specificity. The screening can employ a method for detecting the antibody without any particular limitation.
As an example of the former, for example, the cells used as the antigen are brought into contact with the culture supernatant, and an anti-immunoglobulin antibody or protein G labeled with a radioactive substance, an enzyme, a fluorescent dye, or the like is added, and the cells or the like are added. And a method for detecting a monoclonal antibody bound to. Specific examples include immunostaining. Here, examples of the anti-immunoglobulin antibody include an anti-mouse immunoglobulin antibody when the cell used for cell fusion is a mouse.

As an example of the latter, the antibody contained in the culture supernatant is immobilized on a membrane or the like, and is contacted with an anti-immunoglobulin antibody or protein G labeled with a radioactive substance, an enzyme, a fluorescent dye or the like to form a complex. Thus, there is a method for detecting the antibody. Specific examples include immunoblotting.
In the screening of the cell colonies, even if the culture supernatant is positive in any part of the cells, etc., and negative in the method using the cells, the culture supernatant is against human calnexin or a fragment thereof. Colonies that have been confirmed to be producing antibodies can be selected to produce single clone hybridomas.

The hybridoma strain thus produced includes a hybridoma strain produced using the lung adenocarcinoma cell line A549 cells as an immunogen and producing a monoclonal antibody that specifically binds to human calnexin. Hybridoma strain KU-Lad-001, which produces a monoclonal antibody that specifically binds to human calnexin, was registered as FERM BP-11318 at the Biotechnology Research Institute (IPOD), Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology. It has been deposited.
(Preparation of monoclonal antibody)
First, the obtained single clone hybridoma is cultured, and the monoclonal antibody that reacts with the lung cancer cells is recovered from the culture supernatant, and the antibody is purified.

  The hybridoma is usually cultured in an animal cell culture medium (for example, RPMI1640) containing 10 to 20% fetal bovine serum by adding hypoxanthine, aminopterin, thymidine (HAT), and the like by a conventional method. Can do. After obtaining the necessary amount of the culture supernatant, the monoclonal antibody is separated and purified. The separation and purification can be performed by an ordinary immunoglobulin separation and purification method. For example, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, adsorption / desorption method by ion exchanger (eg DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase, protein G, etc. Only an antibody can be collected with an active adsorbent, and the antibody can be obtained by dissociating the bond.

Examples of the method for confirming the binding state between the antigen or fragment thereof and the antibody or antibody fragment used in the present invention include enzyme immunoassay, Western blotting, dot blotting, and immunoprecipitation.
Enzyme immunoassay refers to the reaction of human calnexin or a human calnexin-expressing microorganism, animal cell or insect cell extract with an antibody or antibody fragment against human calnexin, and further, alkaline phosphatase or peroxidase, etc. After reacting an enzyme-labeled anti-mouse Ig antibody or IgG antibody or antibody fragment, if the enzyme substrate is a substance that produces a chromogen, illuminant or phosphor as a result of the enzyme reaction, the enzyme In this method, the reaction is measured with an absorptiometer, a luminescence photometer, or a fluorometer. The method can also use a derivative labeled with an antibody or antibody fragment that binds directly to human calnexin with an enzyme, and reacts with a derivative labeled with the antibody or antibody fragment with biotin or the like, and then or At the same time, after reacting the secondary labeling substance labeled with an enzyme, the enzyme reaction may be measured with an appropriate measuring instrument or the like.

  Western blotting refers to human calnexin or human calnexin-expressing microorganisms, cell extracts of animal cells or insect cells by SDS-polyacrylamide gel electrophoresis [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988]. After fractionation, the fractionated protein group is blotted from gel to a PVDF membrane or nitrocellulose membrane, and this membrane is reacted with an antibody or antibody fragment that binds to human calnexin, and further, a fluorescent substance such as FITC, peroxidase, biotin This is a method of confirming after reacting an anti-mouse Ig antibody or IgG antibody or antibody fragment with an enzyme label such as

With the dot blotting method, human calnexin or microorganisms expressing human calnexin, cell extracts of animal cells or insect cells are blotted onto a nitrocellulose membrane, and the membrane is reacted with an antibody that binds to human calnexin, Furthermore, this is a method of confirming after reacting a fluorescent substance such as FITC, an anti-mouse Ig antibody or IgG antibody or antibody fragment to which an enzyme label such as peroxidase or biotin is applied.
Immunoprecipitation refers to the reaction of human calnexin or human calnexin-expressing microorganisms, animal cells or insect cell extracts with antibodies or antibody fragments that bind to human calnexin, followed by protein G-sepharose, etc. In this method, a carrier having a specific binding ability to immunoglobulin is added to precipitate the antigen-antibody complex.

(Method for measuring human calnexin in specimen)
The present invention also relates to a method for immunologically detecting or quantifying human calnexin in a specimen using human calnexin or a fragment thereof. Examples of specimens include human-derived biological samples, microorganisms that are considered to express human calnexin, cells or tissues derived from animals or insects, and human-derived biological samples are preferred.
Examples of the biological sample in the present invention include tissue or cells at a disease site, tissue or cells in a body fluid, and the like. Examples of the body fluid sample include blood, lymph, peritoneal fluid, sputum, alveolar lavage fluid, urine, sweat and the like. In the present invention, a sample obtained by processing the tissue, cell, body fluid or the like can also be used as a biological sample. Examples of the processing include dilution, concentration, extraction, and pathological slice preparation. Examples of the treatment for preparing a pathological section include a series of treatments such as formalin fixation, excision, embedding, thin slice, and extension.

By the immunological detection or quantification method of the present invention, it is possible to determine the presence or absence, the degree of progression, or the prognosis of a disease involving human calnexin. Examples of diseases involving human calnexin include lung adenocarcinoma. The disease site may be a primary lesion or a metastatic lesion. An antibody or antibody fragment that binds to human calnexin used in the present invention is used to immunologically detect human calnexin in a specimen. Alternatively, methods for quantification include immunoassay methods such as immunological measurement methods, immune cell staining methods, immunohistochemical staining methods, Western blotting methods, dot blotting methods, immunoprecipitation methods [Monoclonal Antibody Experiment Manual (Kodansha Scientific Fick, 1987), follow-up biochemistry experiment course 5 immunobiochemical research method (Tokyo Kagaku Doujin, 1986)].

Immunological assay methods include enzyme immunoassay (EIA or ELISA), luminescent immunoassay, fluorescent immunoassay (FIA), and radioimmunoassay (RIA) depending on the labeling method. An enzyme immunoassay is preferable.
As the label used in the enzyme immunoassay, any known enzyme (edited by Koji Ishikawa et al., Enzyme immunoassay, Medical School) can be used. For example, alkaline phosphatase, peroxidase, luciferase and the like can be used. When the enzyme substrate is a substance that produces a chromogen as a result of the enzyme reaction, the dye color reaction is measured with an absorptiometer or the like. When the enzyme substrate is a substance that generates a luminescent substance as a result of the enzyme reaction, the luminescent substance-forming reaction is measured with a luminescence detector or the like.

As the label used in the luminescence immunoassay, any known luminescent substance [Imai Kazuhiro, Bioluminescence and Chemiluminescence, Yodogawa Shoten; Clinical Laboratory 42 (1998)] can be used. For example, acridinium and its derivatives, ruthenium complex compounds, lophine and the like can be used. As the ruthenium complex compound, for example, those described in Clin. Chem. 37, 9, 1534-1539 (1991) are preferable. The compound emits electrochemically with an electron donor.
As a label used in the fluorescence immunoassay, any known fluorescent substance (Akira Kawao, fluorescent antibody method, Soft Science) can be used. For example, FITC, RITC, etc. Examples of other fluorescent substances include quantum dot (Science, 281, 2016-2018, 1998). Alternatively, it may be a phycobiliprotein such as phycoerythrin, a fluorescent protein such as GFP (Green fluorescent Protein) or a related protein thereof.

  Radioimmunoassay (RIA) is an anti-mouse Ig obtained by reacting a sample considered to contain human calnexin with an antibody or antibody fragment that binds to human calnexin used in the present invention, and further applying a radioactive substance label. This is a method in which an antibody, an IgG antibody or an antibody fragment is reacted and then measured with a scintillation counter or the like. In this method, a derivative obtained by directly labeling the human calnexin monoclonal antibody or antibody fragment used in the present invention with a radioactive substance can also be used, and the derivative labeled with the antibody or antibody fragment can be reacted with biotin or the like. Then, after or simultaneously with the secondary labeling substance labeled with the radioactive substance, a method of measuring with a scintillation counter or the like may be used.

  The immunological measurement method is a method for measuring the amount of antibody or the amount of antigen using the antigen or antibody to which the above-described various labels are applied. The immunological assay of the present invention may be any method as long as it is a method for detecting or measuring an antigen. For example, the competitive method and the sandwich method [Immunology Illustrated 5th Edition (Nankodo)] can be mentioned, and the sandwich method is preferable.

  The sandwich method is a method in which a first antibody is bound to a solid phase, an antigen to be measured is trapped, and a labeled second antibody is reacted. The antibody used in the sandwich method may be either a polyclonal antibody or a monoclonal antibody, and antibody fragments such as Fab, Fab ′, and F (ab) 2 described above may be used. The combination of two types of antibodies used in the sandwich method may be a combination of antibodies or antibody fragments that recognize different epitopes, or a combination of a polyclonal antibody and a monoclonal antibody or antibody fragment. Specific examples include human calnexin, which is the monoclonal antibody of the present invention, and an antibody or antibody fragment combination that recognizes an epitope different from the epitope of human calnexin.

  The immunocytostaining method is an anti-mouse immunoglobulin labeled with a fluorescent substance such as FITC by reacting a sample considered to contain human calnexin with an antibody or antibody fragment that binds to human calnexin used in the present invention. (Ig) A method in which a fluorescent dye is measured by flow cytometry or a fluorescence microscope after reacting an antibody, an immunoglobulin G (IgG) antibody or an antibody fragment. In this method, a derivative labeled with an antibody or antibody fragment that binds directly to the human calnexin used in the present invention with a fluorescent substance can also be used, and a derivative labeled with the antibody or antibody fragment with biotin or the like can be used. It may be a method of reacting and then reacting with a secondary labeling substance labeled with a fluorescent substance, or measuring a fluorescent dye by flow cytometry or a fluorescence microscope after the reaction.

It is also possible to implement the above embodiment using a light emitting material such as acridinium ester or lophine instead of the fluorescent material. In this case, a luminescence measuring device can be used as the measuring device.
In immunohistochemical staining, a tissue piece collected from a human or animal living tissue is fixed with a fixative such as formalin or ethanol, or is not fixed, and the tissue piece is frozen or embedded in paraffin. , Preparing a tissue section from the tissue piece, fixing the tissue section to a glass or other slide glass, removing unnecessary substances such as deparaffin, and further improving the reactivity between the antigen and the antibody as necessary. This is a method of observing or quantifying the expression of human calnexin contained in a tissue section using a microscope or a camera after reacting with the antibody or antibody fragment of the present invention.
In the above immunostaining method, an immunostaining method using a fluorescent substance, a luminescent substance, an enzyme, and a radioactive substance-labeled antibody is possible, and it is detected or measured by the same procedure as the above-described immunological measurement method suitable for each label It can be performed.
The methods described above can be applied to the Western blotting method, dot blotting method and immunoprecipitation method.

(Manufacture of reagents and kits)
Reagents or kits can be produced using antibodies or antibody fragments that bind to human calnexin. Examples of reagents or kits based on the sandwich ELISA, immune cell staining method, and immunohistochemical staining method are exemplified below, but the present invention is not limited thereto.
A human calnexin detection reagent or quantification reagent containing the anti-human calnexin antibody of the present invention is used in the detection method or quantification method using the above-described immunological assay, and includes human carnenes containing components capable of performing the method. If the substance is substantially the same as each component of the detection reagent or quantitative reagent for xin, or a substance that is substantially the same as a part thereof, it is included in the reagent of the present invention even if the structure or form is different. Is done.

(1) Sandwich ELISA
Constituent elements include anti-human calnexin antibody, carrier for immobilizing anti-human calnexin antibody, solid phase on which anti-human calnexin antibody is immobilized, labeled secondary antibody used for detection or antibody fragment thereof In addition, it may be in the form of a kit containing a biological sample processing reagent, a biological sample diluent, a reaction buffer, a washing solution, a labeled detection reagent or a human calnexin standard as necessary. May be.
The anti-human calnexin antibody is not particularly limited as long as it is an antibody that specifically binds to human calnexin. For example, human calnexin produced by the hybridoma human calnexin of the present invention is preferable. The anti-human calnexin antibody used in the present invention can be a fragmented antibody fragment as necessary.
Examples of the antibody fragment include a peptide containing Fab, Fab ′, F (ab ′) 2, scFv, dsFv, diabody and CDR, VH or a fusion protein thereof, VL or a fusion protein thereof.
The above antibody or antibody fragment can be detected using a secondary antibody, or can be directly detected by labeling the antibody.
When detecting using a secondary antibody, as the secondary antibody, any antibody can be used as long as it can bind to the antibody. The antibody may be a polyclonal antibody or a monoclonal antibody, or Fab, Peptides containing Fab ′, F (ab ′) 2, scFv, dsFv, diabody and CDR can be used. The antibody or antibody fragment used for the secondary antibody is used after being labeled for detection.

As the substance for labeling the anti-human calnexin antibody or antibody fragment or the secondary antibody or antibody fragment for detecting the antibody, the labeling method described in detail in the immunological assay can be used.
As a method of labeling an antibody or antibody fragment with the above-mentioned label, there are a method of genetically binding and a method of chemically binding. The genetic engineering method is performed according to the method described in the literature [Proc. Natl. Acad. Sci. USA, 93, 974 (1996); Proc. Natl. Acad. Sci. USA, 93, 7826 (1996)]. be able to. The chemical bonding can be performed according to the method described in the literature [Science, 261, 212 (1993)]. Moreover, the method of chemically combining radioisotopes can be performed according to the method described in the literature [Antibody Immunoconjugates and Radiopharmaceuticals, 3, 60 (1990); Anticancer Research, 11, 2003 (1991)].

Examples of methods for measuring the amount of label include an absorbance method (colorimetric method), a fluorescence method, a luminescence method, and a radioactivity method. When the label is an enzyme, the amount of the label can be measured by reacting the enzyme substrate with the enzyme and measuring the produced substance.
When the enzyme is peroxidase, the amount of peroxidase can be measured by, for example, an absorbance method or a fluorescence method. Examples of the method for measuring the amount of peroxidase by the absorbance method include a method in which peroxidase is reacted with a combination of hydrogen peroxide and an oxidative coloring chromogen as a substrate, and the absorbance of the reaction solution is measured with a spectrophotometer, etc. There is. Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen.

  A leuco-type chromogen is a substance that is converted into a pigment by itself in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Specifically, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis (dimethylamino) ) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamino) diphenylamine sodium salt (DA-64), 4,4'-bis (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA).

  The oxidative coupling chromogen is a substance that forms a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and an aniline (Trinder reagent), a combination of a coupler and a phenol. Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine. Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-ethyl-N- (2-hydroxy -3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-di (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (3-sulfopropyl) aniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3 , 5-Di Methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- ( 3-methylphenyl) -N'-succinylethylenediamine (EMSE), N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -4-fluoro-3,5-dimethoxyaniline (F-DAOS). Examples of the phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).

As a method for measuring the amount of peroxidase by a fluorescence method, for example, there is a method of measuring the intensity of generated fluorescence by reacting peroxidase with a combination of hydrogen peroxide and a fluorescent substance as its substrate. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.
Examples of the compound exhibiting chemiluminescence as a substrate for peroxidase include a luminol compound and a lucigenin compound.

  When the enzyme is alkaline phosphatase, the amount of alkaline phosphatase can be measured, for example, by a luminescence method. As a method for measuring the amount of alkaline phosphatase by the luminescence method, for example, there is a method in which alkaline phosphatase and its substrate are reacted and the luminescence intensity of the generated luminescence is measured with a luminescence intensity meter. Examples of alkaline phosphatase substrates include 3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), 2-chloro- 5- {4-methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.13,7] decan] -4-yl} phenyl phosphate disodium salt (CDP-StarTM ), 3- {4-methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.13,7] decan] -4-yl} phenyl phosphate disodium salt (CSPDTM) ), [10-methyl-9 (10H) -acridinylidene] phenoxymethyl phosphate disodium salt (LumigenTM APS-5) and its analogs (H. Akhavan-Tafti, Z. Arghavani et al John Wiley and Sons, Chichester, 497-500 (1997)).

When the enzyme is β-D-galactosidase, the amount of β-D-galactosidase can be measured by, for example, an absorbance method (colorimetric method). Examples of the method for measuring the amount of β-D-galactosidase by the absorbance method (colorimetric method) include a method of reacting β-D-galactosidase and its substrate and measuring the absorbance of the reaction solution with a spectrophotometer. is there. Examples of the substrate for β-D-galactosidase include 2-chloro-4-nitrophenyl β-D-galactoside.
When the enzyme is luciferase, the amount of luciferase can be measured by, for example, a luminescence method. As a method for measuring the amount of luciferase by the luminescence method, for example, there is a method of reacting luciferase with its substrate and measuring the luminescence intensity of the generated luminescence with a luminescence intensity meter or the like. Examples of luciferase substrates include luciferin and coelenterazine.

When the label is a radioisotope, the amount of radioisotope can be determined by measuring the radioactivity with a scintillation counter or the like.
As the carrier for immobilizing the anti-human calnexin antibody, any carrier can be used as long as it can bind and hold the antibody, but materials obtained by molding various polymer materials to suit the application are used. .
The shape of the carrier for immobilizing peptides etc. is tubes, beads, plates, fine particles such as latex, sticks, etc., and the materials are polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin , Polymer materials such as agarose, cellulose, polyethylene terephthalate, glass, ceramics, magnetic particles and metals.

As a method for immobilizing an antibody, a known method such as a physical method and a chemical method or a combination thereof can be used. Examples of physical bonds include physical adsorption. Examples of chemical bonds include covalent bonds and non-covalent bonds. Examples of non-covalent bonds include electrostatic bonds, hydrogen bonds, hydrophobic bonds, and coordinate bonds. For example, a 96-well immunoassay microterplate made of polystyrene is prepared by subjecting an antibody to a hydrophobic solid phase.
The antibody to be immobilized may be directly immobilized on the solid phase, or may be immobilized on the solid phase after the antibody is passed through biotin-avidin, glutathione-glutathione transferase or the like.
The solid phase on which the anti-human calnexin antibody is immobilized protects the functional group remaining on the carrier by blocking. As a substance used for blocking in an immunological measurement method, a protein, a surfactant, a commercially available blocking reagent and the like are usually used. As a normal protein used for the blocking of the present invention, BSA, KLH, casein and the like are used. Is used.

  Examples of the diluted solution of the biological sample include an aqueous solution containing a stabilizer in a surfactant or a buffer. When whole blood is used as a sample, an aqueous solution is prepared in an isotonic solution with salts, saccharides, buffers, etc. for the purpose of preventing changes in serum component concentration due to expansion and contraction of blood cells such as red blood cells. It is preferred that The salts are not particularly limited, and examples thereof include alkali metal halide salts such as sodium chloride and potassium chloride. The saccharide is not particularly limited, and examples thereof include sugar alcohols such as mannitol and sorbitol.

The reaction buffer may be any solution as long as the antibody on the antibody-immobilized solid phase and the antigen in the biological sample can undergo a binding reaction. Moreover, you may add surfactant, antiseptic | preservative, a stabilizer, a reaction accelerator, an enzyme activity regulator, etc. as needed.
Any washing solution can be used as long as it can usually remove and wash unreacted substances and does not affect the antigen-antibody reaction. If necessary, a buffer, a surfactant, a protein such as BSA or casein, an antiseptic or a stabilizer may be added. For example, PBS-Tween Tablets (2130-10, BioVision) is used.

  The buffer used for the sample dilution, reaction buffer, or washing solution is not particularly limited as long as the buffer used in the buffer has a buffering capacity, but has a pH of 1 to 11, for example, lactic acid buffer or citrate buffer. Agent, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer (except when the label is alkaline phosphatase), triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur Buffers, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer, glycine buffer, Good buffer, etc. It is done. Good buffering agents include, for example, MES (2-morpholinoethanesulfonic acid) buffer, bis-tris [bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane] buffer, ADA [N- (2-acetamido) imino Diacetic acid] buffer, PIPES [piperazine-N, N′-bis (2-ethanesulfonic acid)] buffer, ACES {2- [N- (2-acetamido) amino] ethanesulfonic acid} buffer, MOPSO ( 3-morpholino-2-hydroxypropanesulfonic acid) buffer, BES {2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid} buffer, MOPS (3-morpholinopropanesulfonic acid) buffer , TES <2- {N- [Tris (hydroxymethyl) methyl] amino} ethanesulfonic acid> buffer, HEPES [N- (2-hydroxyethyl) -N '-(2-sulfoethyl) piperazi ], DIPSO {3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid} buffer, TAPSO <2-hydroxy-3-{[N-tris (hydroxymethyl) ) Methyl] amino} propanesulfonic acid> buffer, POPSO [piperazine-N, N'-bis (2-hydroxypropane-3-sulfonic acid)] buffer, HEPPSO [N- (2-hydroxyethyl) -N ' -(2-hydroxy-3-sulfopropyl) piperazine] buffer, EPPS [N- (2-hydroxyethyl) -N '-(3-sulfopropyl) piperazine] buffer, tricine [N-tris (hydroxymethyl)] Methylglycine] buffer, bicine [N, N-bis (2-hydroxyethyl) glycine] buffer, TAPS {3- [N-tris (hydroxymethyl) methyl] aminopropanesulfonic acid} buffer, CHES [2- (N-cyclohe Shiruamino) ethanesulfonic acid] buffer, CAPSO [3- (N- cyclohexylamino) -2-hydroxy propanesulfonic acid] buffer, CAPS [3- (N- cyclohexylamino) propanesulfonic acid] and the like buffers.

Examples of enzyme activity regulators and enzyme stabilizers include metal ions such as magnesium ions, manganese ions, and zinc ions. The content of these metal ions in the reagent is not particularly limited as long as the enzyme is stabilized in the measurement.
Examples of the preservative include sodium azide and antibiotics. The content of these preservatives in the reagent is not particularly limited as long as it is such a content that the substance to be measured in the sample is appropriately measured in the measurement.
Examples of human calnexin standard substances include human calnexin obtained by gene recombination techniques or obtained from biological samples, cells expressing human calnexin, and partial peptides of human calnexin. .

(2) Immune cell staining method The component includes a combination of a labeled anti-human calnexin antibody or an antibody fragment thereof, or an anti-human calnexin antibody and a labeled secondary antibody or an antibody fragment thereof used for detection, If necessary, it may be in the form of a kit containing a biological sample treatment reagent, a biological sample diluent, a reaction buffer, a washing solution, blocking, a hemolytic agent, a fixative, or a human calnexin standard. The embodiment of the antibody fragment and the secondary antibody is the same as the substance exemplified in (1) Sandwich ELISA.

The label used for the labeled anti-human calnexin antibody and the labeled secondary antibody is one of the labeling substances exemplified in the above immunoassay (enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, radioimmunoassay). A fluorescent label is a desirable embodiment.
As the biological sample processing reagent, biological sample diluent, reaction buffer solution, washing solution, blocking, or human calnexin standard substance, the substances exemplified in the above section (1) Sandwich ELISA can be used. As hemolytic agents, those containing ammonium chloride are generally used, but are not limited thereto. A fixative containing ethanol or formaldehyde is used, but is not limited thereto. BD FACS Lysing Solution and BD PharmLyse (both Becton Dickinson) can be used as commercially available products.

(3) Immunohistochemical staining method As a component, the monoclonal antibody or antibody fragment that binds to human calnexin used in the present invention may be a monoclonal antibody or antibody fragment labeled with a labeling substance. Known reagents can be used as necessary reagents. Examples of other reagents necessary for detection include a labeled secondary antibody used for detection or an antibody fragment thereof, a known substance used for high-sensitivity staining described below, and the like. In the form of a kit, including a solution, blocking reagent or solution, endogenous substance blocking reagent or solution, antibody dilution, reaction buffer, washing solution, labeled detection reagent or human calnexin standard May be. The embodiment of the antibody fragment and the secondary antibody is the same as the substance exemplified in (1) Sandwich ELISA.

Labels used for labeled anti-human calnexin antibodies or antibody fragments, labeled secondary antibodies or antibody fragments are exemplified in the above immunological measurements (enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, radioimmunoassay) However, it is more preferable to use the labeling substances exemplified in the enzyme immunoassay method and the fluorescence immunoassay method.
Regarding the reaction conditions of the monoclonal antibody or antibody fragment, labeled secondary antibody or antibody fragment that binds to human calnexin used in the present invention, the antibody concentration is 0.01 to 100 μg / mL, preferably 0.1 to 30 μg / mL. mL. The antibody reaction temperature is usually in the range of 4 ° C. to 37 ° C., and the antibody reaction time can be appropriately set according to the reaction temperature, but at 4 ° C., it is 1 hour to 24 hours, and at room temperature, it is 10 It is preferably within 8 minutes.

  Reagents used for blocking the activity of endogenous substances include 0.2 to 2% periodic acid aqueous solution, 1 to 5% hydrogen peroxide, 0.1 to 0.5 for the removal of endogenous peroxidase. % Hydrogen peroxide-added methanol, 0.1-0.5% hydrogen peroxide, 0.05-0.2% sodium azide aqueous solution, etc. are used. A dilute solution of levamisole is used to inhibit endogenous alkaline phosphatase activity. In order to block the activity of endogenous biotin, it is possible to react 0.01-0.1% avidin aqueous solution and then react 0.001-0.01% biotin aqueous solution. In order to inhibit the activity of the endogenous substance, the above liquid can be reacted at room temperature for 5 minutes to 1 hour, but is not limited to this reaction temperature and reaction time.

The operation of blocking the activation of endogenous substances is performed after reacting an enzyme-labeled or enzyme-unlabeled monoclonal antibody or antibody fragment that binds to human calnexin used in the present invention with a diluent. May be.
As the washing solution, PBS, phosphate buffer, Tris-HCl buffer or the like can be used. This pH is 5.5 to 9.0, preferably 6.0 to 8.5. The concentration is 0.2 to 0.005 mol / L, preferably 0.1 to 0.01 mol / L.
As the blocking solution, normal animal serum, bovine serum albumin, skim milk, casein solution and the like are preferable, but not limited thereto. As animals used for normal animal serum, all animal species other than humans or mice can be used, but goats, sheep, rabbits, pigs and the like are preferable. When using enzyme-labeled anti-mouse Ig or IgG, it is preferable to use normal serum of the same type as the animal species from which the anti-mouse Ig or IgG antibody was obtained. The serum concentration can be arbitrarily selected within the range of 0.1 to 20%, and preferably 1 to 5%. The blocking temperature can be appropriately set within the range of 4 ° C to 37 ° C. Although the blocking time can be appropriately set according to the reaction temperature, it is preferably 10 minutes or more and 1 hour or less at room temperature.

  As the antibody dilution solution, PBS containing bovine serum albumin is preferably used, but is not limited thereto. The concentration of bovine serum albumin can be arbitrarily selected within the range of 0.1 to 5%. Furthermore, it is also possible to add a surfactant. As the type of surfactant, Triton X-100 (X-100, Sigma-Aldrich), Tween 20 (40350-02, Kanto Chemical Co., Inc.) and the like can be used. The concentration of the surfactant is 0.005 to 0.5%, preferably 0.01 to 0.2%.

  As the coloring reaction solution, a 3,3′-diaminobenzidine (DAB) solution can be used when peroxidase is used as a labeling enzyme. It is desirable to add hydrogen peroxide water during use. As a sensitizer for DAB color development, imidazole can be added. Further, a 3-amino-9-ethylcarbazole solution and a 4-chloro-1-naphthol solution can be used. When alkaline phosphatase is used as a labeling enzyme, each salt of fast red, fast red violet or fast blue can be used as a color former. Neufuchsin solution (105226, Merck) or BCIP / NBT solution (B6404, Sigma-Aldrich) can also be used. As the color development solution of glucose oxidase, for example, a mixed solution of glucose, PMS (phenazine methosulfate) and chromogen is used. The chromogen may be any chromogen as long as it is reduced by PMS to produce an insoluble pigment. Examples of the chromogen include tetrazolium salts such as NBT, TNBT, BSPT, and INT (349-01503 Wako Pure Chemical Industries, Ltd.). The color development reaction time can be appropriately selected from 30 seconds to 20 minutes, but more preferably 1 to 10 minutes.

  In tissues embedded in formalin-fixed paraffin, the antigen is often hidden by aldehyde fixation, and contact between the antigen and the antibody may be inhibited. In this case, the staining property can be improved by the antigen activation treatment. As the activation treatment, for example, proteolytic enzyme treatment or heat treatment can be used. Any proteolytic enzyme can be used as long as it has no cleavage activity on the antibody recognition site on the antigen, but a limited degrading enzyme is particularly preferable. For example, trypsin, pronase, pepsin, actinase, ficin, proteinase K, Examples include subtilisin and papain.

  Examples of the heat treatment include, but are not limited to, heating operations using a microwave irradiator, a microwave oven, an autoclave, a pressure cooker, a water bath, a steamer, a hot plate, or the like in water or an arbitrary buffer solution. Examples of the buffer used for the heat treatment include citrate buffer, EDTA solution, sodium hydroxide citrate buffer, Tris hydrochloride buffer, borate buffer, glycine hydrochloride buffer, carbonate buffer, acetate buffer. Although there are liquids, it is not particularly limited to these. The salt concentration of the buffer is 0.0005 to 0.2 mol / L, preferably 0.001 to 0.1 mol / L. The pH of the buffer solution is 5.0 to 10.0, preferably pH 5.5 to 9.5. Furthermore, an aqueous urea solution, an aqueous aluminum chloride solution, an aqueous periodic acid solution, or the like can also be used. The concentration of these aqueous solutions is 0.1 to 7%, preferably 1 to 5%.

Moreover, alkali treatment and acid treatment are also possible. As the alkali treatment, NaOH, KOH or the like is used, and as the acid treatment, hydrochloric acid, formic acid, acetic acid or the like is used. Furthermore, a protein denaturant can also be used. Urea, guanidine, etc. can be used as the protein denaturant.
The following high-sensitivity staining methods (“Revised 4th edition, Watanabe / Nakane Enzyme Antibody Method”, Hiroshi Nakura, Yoshiyuki Nagamura, Hiroshi Tsutsumi, edited by Interdisciplinary Planning Co., Ltd.) can be performed for the purpose of improving staining intensity. These high-sensitivity staining methods exhibit excellent effects when used after the reaction of the monoclonal antibodies and antibody fragments of the present invention.

  As a high-sensitivity staining method, a PAP (peroxidase anti-peroxidase) method, a four-step PAP method, a double bridge PAP method, a PAP method using a Fab fragment, and a hapten sandwich method can be used. In addition, ABC (avidin biotinylated-peroxidase complex) method, LAB (labeled avidin-biotin) method, BRAB (bridge avidin-biotin) method, LSAB (labelled strept avidin) using streptavidin, which applies the principle of avidin-biotin complex formation biotin) method, SABC (streptavidin biotin complex) method, CSA (catalyzed signal amplification) method using biotinylated tyramid, a method using anti-avidin antibody as a modification of ABC method, a method using biotinylated protein A, PAP and A method of combining ABC and an ABC method using a colloidal gold-labeled anti-peroxidase antibody can be used. Furthermore, a method using enzyme-labeled protein A can also be used.

As another embodiment of the high sensitivity staining method, there is an enzyme labeled polymer method. In this method, a chromogenic enzyme and a primary antibody or a chromogenic enzyme and a secondary antibody bound to a high molecular polymer such as dextran or amino acid is used. The monoclonal antibody of the present invention is used as a primary antibody to form a high molecular polymer. Either the mode of binding or the mode of binding an anti-mouse Ig antibody or anti-mouse IgG antibody as a secondary antibody to a macromolecular polymer is possible.
An immunocolloid method using a metal-labeled antibody can also be used. Furthermore, it is also possible to use a chromogenic enzyme such as alkaline phosphatase or glucose oxidase instead of peroxidase. A combination of an avidin / biotin complex and these chromogenic enzymes or an APAPP (alkaline phosphatase-antialkaline phosphatase) method can also be used. Alternatively, an indirect method using a peroxidase-labeled anti-FITC antibody can also be performed.

  Commercially available kits using the above principles can also be used. Examples of commercially available kits include Vector Stain Kit (Vector), Strept AB Complex (DAKO), AB Complex (DAKO), SAB-PO kit (Nichirei), and SAB-AP kit (Nichirei). ), Super Sensitive Ready-to-Use Detection Kit (BioGenex), Polyvalant staining kit (ScyTek), EPOS system (DAKO), EnVision system (DAKO), Super Sensitive Non-Biotin HRP Ready- There are to-Use Detection Kit (manufactured by BioGenex) and Simple stain system (manufactured by Nichirei).

  Examples of the LAB method and the LSAB method include the following conditions. After the monoclonal antibody or antibody fragment binding to human calnexin used in the present invention or after the end of the endogenous substance activity blocking operation, it is washed with a washing solution and reacted with biotin-labeled anti-mouse Ig antibody or biotin-labeled anti-mouse IgG antibody. Then, an embodiment in which avidin-labeled peroxidase is reacted is possible. Regarding the reaction conditions for biotin-labeled anti-mouse Ig antibody or biotin-labeled anti-mouse IgG antibody, the antibody concentration is 0.1 to 100 μg / mL, preferably 5 to 75 μg / mL, more preferably 20 to 50 μg / mL. be able to. Regarding the antibody reaction temperature and time, conditions similar to those for the reaction of the monoclonal antibody or antibody fragment that binds to human calnexin used in the present invention can be selected. Regarding the reaction conditions for avidin-labeled peroxidase, the concentration can be 0.1 to 100 μg / mL, preferably 5 to 75 μg / mL, more preferably 20 to 50 μg / mL. Regarding the antibody reaction temperature and time, conditions similar to those for the reaction of the monoclonal antibody or antibody fragment that binds to human calnexin used in the present invention can be selected.

  Examples of human tissue collection include a method of collecting a tissue containing a target tissue such as a lesion site obtained by surgery or biopsy. The collected tissue can be used as a specimen as it is. Typical techniques for fixation include freezing unfixed, preparing frozen sections and then fixing with cold acetone, etc., freezing after fixation, preparing frozen sections, and embedding in paraffin after fixation The method etc. are mentioned. The immobilization is broadly divided into immobilization that causes cross-linking formation in protein / peptide chains centering on aldehydes, and immobilization by an organic solvent system based on protein coagulation precipitation. Examples of the aldehyde-based fixative include 10-20% unbuffered formalin, 10% buffered formalin, buffered 4% paraformaldehyde, PLP (periodate-lysine-paraformaldehyde), Zamboni solution, Buan solution (saturated picric acid: formalin stock solution: Glacial acetic acid = 15: 5: 1), glutaraldehyde. Examples of the organic solvent-based fixing solution include ethanol, acetone, Carnoy's solution (ethanol: chloroform: glacial acetic acid = 6: 3: 1), and metacan solution (methanol: chloroform: glacial acetic acid = 6: 3: 1). In addition, special fixatives (Revised 4th edition, Watanabe / Nakane Enzyme Antibody Method, Hiroshi Nakura, Yoshiyuki Nagamura, Hiroshi Tsutsumi) Formalin fixative, formalin / ethanol mixture (formalin stock solution: 100% ethanol: water = 1: 8: 1), ethanol acetate / formalin mixture (formalin stock solution: glacial acetic acid: ethanol = 2: 1: 17) Used.

  In the case of unfixed freezing, chop the tissue, place it in a frozen embedding agent (OCT compound, etc.), quickly freeze it with dry ice / acetone, dry the acetone, seal it, and store it in a deep freezer. The method is used. As a freezing method after fixation, the tissue is cut into small pieces, fixed using any of the above-described fixing solutions, washed with, for example, PBS or PBS containing 10 to 20% sucrose, and freeze-embedded (OCT compound). Etc.) and rapidly frozen with dry ice / acetone, dried with acetone, sealed and stored in a deep freezer. As a method of embedding in paraffin after fixation, use a method in which the tissue is minced, fixed with any of the above-mentioned fixing solutions, dehydrated and permeable with ethanol, xylene, etc., embedded in paraffin, and stored as a paraffin block. It is done. As the fixing method, immersion fixing, injection fixing, deaeration fixing, freeze replacement fixing, freeze drying fixing, and the like are used. In the case of immersion fixation, the fixing time is preferably from half a day to one week, but generally 1 to 2 days is more preferable. It is also possible to reduce the fixed time to several hours by using a microwave or a home microwave oven.

For blood cells and microorganisms collected from human or animal tissues, cultured cells such as humans or animals or insects, if they are floating cells, a method of attaching them to a glass slide using cytospin, centrifugation After pelleting the cells by the method, a method of forming a clot and freezing or embedding it in paraffin is mentioned. Examples of the method for forming a clot include a method of encapsulating in agarose or a method of adding fibrinogen and then thrombin to form an artificial fibrin clot.
A preferred method for preparing the section is a method using a cryostat in the case of a frozen section, and a method using a microtome is preferable in the case of a paraffin section.

  Examples of the staining flow of the enzyme-labeled antibody method in the case of a frozen section include the following embodiments. After the frozen section is washed with an appropriate washing solution, the activity of the endogenous substance is blocked and then blocked with a blocking solution. A solution obtained by diluting the enzyme-labeled monoclonal antibody or antibody fragment of the present invention with a diluent is reacted, washed with a washing solution, and then subjected to a color reaction. Alternatively, an enzyme-unlabeled monoclonal antibody or antibody fragment of the present invention diluted with a diluent is reacted, washed with a washing solution, and then an enzyme-labeled anti-mouse Ig or IgG antibody diluted with a diluent is reacted. It is also possible to carry out a color reaction after washing again with a washing solution. As appropriate, nuclear staining can be performed using methyl green or the like. Alternatively, staining with hematoxylin or the like can be performed to clarify the cell image.

In the case of a paraffin section, a deparaffinization operation is performed with xylene, toluene, benzene, etc., followed by washing with 100% ethanol and 70% ethanol and then washing with water. The operation after blocking the activity of the endogenous substance is the same as described above.
The use of an automatic immunostaining device is also a method that can be used to complete the present invention. HIS-20 (Sakura Seiki), i6000 (Kyowa Medex), ST5050 (Leica), HX benchmark (Roche Diagnostics), Autostainer (DAKO), Horizon (DAKO) But not limited to this).

  As a method for qualitatively or quantitatively determining an immunostained image, a method suitable for the labeling substance of the antibody can be used. That is, when an enzyme is selected as a labeling substance, an optical microscope, a confocal laser scanning microscope, etc., and when a fluorescent substance is selected as a labeling substance, a fluorescence microscope, a confocal laser scanning microscope, a fluorescence image analyzer, etc. In the case of a radioactive substance, it is possible to use an autoradiography, a radiographic image analyzer or the like, and in the case of a luminescent substance, a luminescent image analyzer or the like.

With respect to an image obtained with a microscope, the form and shading of a staining positive site can be detected with the naked eye, thereby enabling qualitative or quantitative determination.
Alternatively, image data can be input to the image analysis device as a digital signal using a digital image input system such as a digital camera in order to perform image processing that suits the purpose of the image obtained with the microscope and express it clearly. can do. The image analyzer can measure the area, shortest diameter, longest diameter, circumference, etc. of positive staining sites, and measure the transmittance, optical concentration, absorbance, etc. of positive staining sites. Quantitative analysis according to the purpose such as product positive rate and distribution becomes possible.

(Example 1) AMeX fixation First, AMeX (acetone / methanol / xylene) fixation of A549 cell line (Human Science Research Resource Bank) derived from human lung adenocarcinoma was performed according to the following procedure.
Centrifuge the cultured A549 cell line at 1,000 rpm for 10 minutes, remove the supernatant, add 10 ml of phosphate buffered saline (PBS-) that does not contain divalent ions, and after stirring, further 1,000 rpm for 10 minutes And centrifuged. After removing the supernatant, 30 ml of acetone at 4 ° C. was added and fixed at 4 ° C. for 16-18 hours. After fixation, the supernatant was discarded, acetone at 4 ° C. was newly added, and a dehydration reaction was performed while returning to room temperature using a permeator for 15 minutes at room temperature.
Furthermore, the supernatant was discarded, room temperature acetone was added, and the mixture was stirred on a permeator for 15 minutes to remove as much water as possible. Thereafter, methyl benzoate was added and a substitution reaction was performed twice for 15 minutes in the same manner. Further, a penetration reaction is carried out in the same manner using xylene, and finally, it is placed in paraffin having a melting temperature of 58 to 60 ° C., and paraffin is permeated into the cell specimen over 4 hours while reducing the pressure with a vacuum pump. The block was stored at 4 ° C. The block was cut into approximately 3 μm sections with a microtome, picked up on a silane-coated slide glass, and dried at 40 ° C. for 1 day. Then, it put into the plastic bag with a zipper and preserve | saved at 4 degreeC.

(Example 2) Production of hybridoma ^Seven 1 × 10 ⁷ A549 cell lines, a lung adenocarcinoma of human lung cancer, were immunized three times every other week into the peritoneal cavity of a 5-week-old female Balb / c mouse. Three days after the third immunization, 1 μl of blood was collected from the tail vein of the immunized mouse and diluted 100-fold with phosphate buffered saline (0.0075 M, pH 7.4; hereinafter abbreviated as PBS). Using the solution, AMeX-embedded cell specimens of the A549 cell line used as an immunogen were immunostained.
Next, the antibody titer of the immunized mouse by the immunostaining method was confirmed as follows.
The A549 cell line sliced to approximately 3 μm was deparaffinized with xylene for 15 minutes. After that, the sections were passed through a descending ethanol series (100% ethanol 3 tanks → 95% ethanol 1 tank → 90% ethanol 1 tank → 80% ethanol 1 tank → 70% ethanol 1 tank) for 10 seconds, gradually increasing the ethanol concentration. The dexylene operation was performed while lowering. Thereafter, the section was washed with running water, and 3% hydrogen peroxide solution was placed on the section and reacted for 10 minutes to inhibit endogenous peroxidase activity.

  After further washing with running water, the sections are soaked in 0.075 M TBS (pH 7.5) for 5 minutes and then treated with 2% normal pig serum / TBS for 10 minutes to prevent non-specific protein reactions. Then, after removing the excess solution on the section, 100 μl of mouse serum diluted 100-fold as described above was placed on the section and allowed to react at room temperature for 2 hours in a wet chamber. After the reaction, it was washed 3 times for 5 minutes each with TBS, and a high-sensitivity polymer reagent labeled with HRP (ChemMate ENVISIN, DAKO Japan, Kyoto) was added dropwise to the sections as a secondary antibody and reacted at room temperature for 30 minutes. After the reaction, the plate was washed with TBS three times for 5 minutes each, and then 100 μl of Stable DAB solution (Invitrogen, Tokyo) as a substrate reaction solution of HRP enzyme was dropped and reacted for 2 to 3 minutes to visualize the antigen-antibody product. After that, after staining with Meyer's hematoxylin for 30 seconds, ascending ethanol series (70% ethanol 1 tank → 80% ethanol 1 tank → 90% ethanol 1 tank → 95% ethanol 1 tank → 100% ethanol 3 tanks) Then, after passing several tanks of xylene, a chemical encapsulant and Enteran (Mutou, Tokyo) were dropped, and a cover glass was placed to make a permanent specimen. After the encapsulation, the localization of the antigen recognized by the antibody contained in the serum of the mouse immunized with a microscope was confirmed.

By the above confirmation method, a mouse in which the A549 cell line was stained and an increase in antibody titer in the blood was confirmed was selected, and one week after the third immunization, final immunization was performed in the same manner. Three days later, spleen cells were removed from Balb / c mice, and the spleen cells and myeloma cells SP2 / 0 were subjected to cell fusion using polyethylene glycol. The ratio of the myeloma cells to the spleen cells was 1: 5, and the spleen cells were prepared to be 1.8 × 10 ⁵ per well of a 96-well plate.
8 to 23 days after the cell fusion, 100 μl of culture supernatant was recovered from the well in which a hybridoma colony appeared, and the supernatant was used as a primary antibody.

(Example 3) Screening of hybridomas by immunostaining method Using the supernatant as a primary antibody, a specimen of the A549 cell line is immunostained, and a colony in which the cell line is positive contains a target monoclonal antibody-producing hybridoma It was judged and selected. The immunostaining method is the same as the immunostaining method using immunized mouse serum as a primary antibody. The reactivity between the antibody contained in the hybridoma culture supernatant and A549 cells fixed with AMeX is shown in FIG. The A549 cells fixed with Amex were strongly stained with the antibody, indicating that the antigen protein recognized by the antibody was overexpressed in the A549 cells.

(Example 4) Cloning of hybridoma Using the supernatant of the well in which the presence of the target hybridoma was confirmed, the A549 cell line was immunostained and immunoblotted according to the procedure described above, and monoclonal antibody production was confirmed. The confirmed one was used as a single clone hybridoma.
By such a procedure, a hybridoma cell line KU-Lad-001 producing a monoclonal antibody that specifically binds to the A549 cell line was obtained.
The method of ascites of hybridoma was performed as follows. 500 μl of pristane was injected into the abdominal cavity of BALB / c mice (about 5 weeks after birth). One week later, hybridomas were planted and cultured in 10 cm dishes so as to be subconfluent. After 1 week, all the cells in the dish were collected by centrifugation at 1200 rpm for 10 minutes, the supernatant after centrifugation was removed, suspended in 500 μl RPMI1640 · 10% FCS, and 4 cells per mouse. The number of cells corresponding to 5 dishes was injected intraperitoneally into mice. After about 10 days, cervical dislocation was performed after ether anesthesia of a mouse in which ascites sufficiently accumulated in the abdominal cavity. The abdominal cavity was slightly opened, and ascites was collected using a sterile cotton plug pasteur so as not to damage the internal organs. Ascites was centrifuged at 1200 rpm for 10 minutes to remove blood cells, etc., and then dispensed in an amount of about 500 μl each and stored at −80 ° C. until use.

Example 5 Identification and Confirmation of Antigen (Human Calnexin) against the Monoclonal Antibody FIG. 2 shows a method for purifying an antigen that is overexpressed in human lung adenocarcinoma cells and binds to the monoclonal antibody.
Separation and purification of the antigen was performed using Sepharose beads (Protein G sepharose 4B FF, manufactured by GE Healthcare) to which protein G that recognizes and binds to the Fc region of the antibody was immobilized. The culture supernatant of the monoclonal antibody-producing cells was reacted with the Sepharose beads, and the antibody in the culture supernatant was immobilized on Sepharose beads (FIG. 2 (1)). After that, about 20 mg (one 10 cm dish) of human lung adenocarcinoma cultured cells (A549) was added to RIPA buffer (0.1% SDS, 1% Triton-X100, 0.2% sodium deoxycholate, 0.05M Tris-HCl, 0.15M NaCl pH7 .6, 2 mM CaCl2, 2 mM MgCl2, Complete mini (Roche Applied Science, 1 tablet / 10 ml), pH 7.6) Add 800 μl and solubilize on ice for 30 minutes to obtain the appropriate amount of the soluble protein mixture. The above monoclonal antibody was reacted with immobilized Sepharose beads. Thus, only the antigen was trapped on the sepharose beads by an antigen-antibody reaction between the antibody immobilized on the sepharose beads and the antigen in the soluble protein mixture (FIG. 2 (2)). Then add about 20 μl of denaturant (SDS) solution (2% SDS, 62.5 mM Tris-Cl (pH 8.5), 5% -mercaptoethanol) to denature protein G on Sepharose beads. Separation from the sepharose beads and the antibody and antigen were recovered (FIG. 2 (3)).

Next, the obtained antigen protein was identified by the proteome technique. FIG. 3 is a flowchart illustrating a method for identifying and confirming a purified antigen protein. The identification was performed by combining two analysis methods. That is, the purified antigen is divided into two and each is developed by SDS-PAGE, and one gel (Gel 1) is stained with zinc to identify a band that seems to be an antigen (FIG. 3 (1)). This was digested in a gel and the antigen was identified using a mass spectrometer. On the other gel (Gel 2), it was confirmed by Western blotting that the protein in the band was an antigen (FIG. 3 (2)).
The antigen was identified by the procedure shown in FIG. That is, the concentrated antigen and antibody mixture was developed by SDS-PAGE and stained with zinc.

Zinc staining was performed as follows. A zinc staining kit (2 97-57701, manufactured by Wako Pure Chemical Industries, Ltd.) was used. First, the electrophoresis buffer adhering to the gel after electrophoresis was washed off with MilliQ water (water purified with MilliQ manufactured by Millipore). The gel was transferred to a container containing 40 ml of the zinc staining kit solution A and shaken for 5 minutes. Next, the solution was transferred to a container containing 40 ml of the B solution of the zinc staining kit and shaken until a band was clearly formed. The solution B was discarded and washed 5 times with MilliQ water for 5 minutes. The band was read with a scanner (Epson, GT-9800F) and the image was saved.
After zinc staining, the antigen band on the gel was cut out and digested in gel with a digestive enzyme (trypsin) to form a peptide fragment group, and the molecular weight and amino acid sequence information of each peptide fragment were measured with a mass spectrometer. This result was searched using a database to identify a known protein corresponding to the antigen.

  In the SDS-PAGE, polyacrylamide gel was used, Tris, glycine, and SDS solution were used as developing solutions, and the developing was performed at a current of 20 mA for about 1.5 hours. The antigen band was detected by zinc staining. The result is shown in FIG. 4A. The second lane from the left shows the SDS-PAGE of the recovered product recovered from the Sepharose beads after the antigen-antibody reaction of the ascites antibody of KU-Lad-001 and the A549 soluble protein mixture. In the fourth lane from the left, the culture supernatant antibody of KU-Lad-001 is used instead of the ascites antibody of KU-Lad-001, and the subsequent operation is the same as that of the second lane. The other lanes show the results of the control experiment. Only the second and fourth lanes from the left indicate that the target antigen protein is concentrated.

For antigen purification, protein G-bound Sepharose beads were used as described above. For this reason, the antigen is trapped on the Sepharose beads, and the antibody is present together with the antigen in the solution recovered with the denaturing agent. Thus, the antibody light and heavy chains and antigen are detected when stained with zinc. In FIG. 4A, bands other than the light chain and heavy chain of the antibody are present in addition to the band indicated by the dotted line □ (denoted as antigen) in the figure. In the immunoprecipitation method performed this time, a protein adsorbed non-specifically to Sepharose beads is often judged as an antigen. However, when a sample obtained by reacting only the antibody with Sepharose beads and a sample obtained by reacting only the protein mixture with Sepharose beads were electrophoresed simultaneously as control samples, no band indicated by a dotted line □ was observed in these control samples. Therefore, the band was found to be an antigen protein band.
FIG. 4B shows the result of Western blotting using the antibody on another gel obtained by SDS-PAGE in the same manner as in FIG. 4A. This detection was performed by chemiluminescence.

  Specifically, Western blotting was performed by the following method. The protein in the gel prepared for Western blotting is transferred to a PVDF membrane (Millipore), and the transferred PVDF membrane is in 2% normal pig serum (NSS) / TBS (0.01 MTris-HCl pH 7.5, 150 mM NaCl) Blocking was performed at room temperature for 60 minutes. After blocking, the PVDF membrane was reacted with KU-Lad-001 antibody for 60 minutes at room temperature. PVDF membrane was washed 3 times for 5 minutes in TBS containing 0.1% Tween20 (TBS-T), and then reacted with horseradish peroxidase-labeled rabbit anti-mouse IgG antibody diluted 1000-fold with 2% NSS for 30 minutes at room temperature I let you. The PVDF membrane was washed 3 times for 5 minutes in TBS-T, transferred to TBS, and then lightly washed 3 times with TBS. Thereafter, the PVDF membrane was incubated with Immobilon Western (Millipore) for 1 minute at room temperature, and then chemiluminescence was detected.

In FIG. 4B, the second from the left (KU-Lad-001 ascites antibody and A549 soluble protein mixture), the fourth lane (KU-Lad-001 culture supernatant antibody and A549 soluble protein mixture) and the rightmost lane Only the (anti-concentration A549 soluble protein mixture) shows that the target antigen protein is detected.
Since Western blotting uses an antibody that recognizes a mouse IgG antibody as a secondary antibody, the light chain and heavy chain of the antibody are also detected. The part where luminescence is detected in the other part is the antigen. This antigen site was consistent with the zinc-stained antigen site in FIG. 4A, and it was confirmed that the protein concentrated by the immunoprecipitation method in FIG. 2 was the antigen.

In-gel digestion for antigen identification was performed as follows.
The gel piece from which the antigen band of FIG. 4A was cut was decolorized with a decoloring solution of a zinc staining kit (manufactured by Wako Pure Chemical Industries, Ltd.), and then completely decolorized with 50% acetonitrile and 50 mM ammonium bicarbonate. The gel pieces were washed with distilled water, dehydrated in 100% acetonitrile for 15 minutes, and dried for 45 minutes with a centrifugal evaporator. About 20 μl of 20 ng / μl trypsin (Promega, Madison, Wis.) Dissolved in 50 mM acetic acid was added to the dried gel piece and absorbed in ice for 45 minutes. Thereafter, the excess trypsin solution was removed, 25 mM ammonium bicarbonate was added to such an extent that the gel was immersed, and digestion in the gel was performed at 37 ° C. for 24 hours. After completion of the enzymatic digestion, the solution around the gel piece was completely recovered and stored temporarily in ice. Furthermore, in order to collect the peptide fragments still remaining in the gel, a 50% acetonitrile / 5% trifluoroacetic acid solution was added to the extent that the gel was immersed, and the mixture was stirred at room temperature for 20 minutes. After stirring, the supernatant was added to the stock solution. The enzyme digested peptide mixture collected in this manner was analyzed with a mass spectrometer to identify the protein.

The identification of the protein by a mass spectrometer is as follows.
First, the peptide mixture obtained by in-gel digestion is measured with a mass spectrometer (autoflex III TOF / TOF, Bruker Daltonik GmbH, Bremen, Germany), and MS spectrum and tandem MS spectrum (MS / MS spectrum) are measured. It was. The database search was performed using the identification program MASCOT search (http://www.matrixscience.com/). As a result, proteins that best matched the mass spectrometry data were selected from the database. The outline of protein identification by this program is as follows.

(1) The program trypsin digests all the proteins on the database on a computer to create an enzyme digestion peptide list.
(2) Molecular weight of one peptide (referred to as P1) obtained by measurement A peptide having a molecular weight range of MW ± 1 is selected from the list of (1).
(3) The MS / MS spectra of all peptides selected in (2) are simulated on the computer from the amino acid sequences.
(4) By comparing the MS / MS spectrum of P1 obtained by measurement with the MS / MS spectrum simulated in (3), P1 may be a peptide fragment of which part of which protein on the database based on the degree of coincidence A peptide having high or high possibility is given a score according to the degree of matching.
(5) The above operations (2) to (4) are carried out for all peptides measured with a mass spectrometer.
(6) Based on the results of (5), select the protein that most closely matches the mass spectrometry data from the proteins on the database. At this time, from the most likely protein to the 15th most likely protein, a score is assigned according to each possibility.

The criteria for determining whether the protein with the highest score is a truly analyzed protein are mainly the following two points.
1. The first protein has a significantly higher score than others. 2. When the protein contains 3 or more types of peptides judged to be most likely in the above (4).
The identification and confirmation confirmed that the antigen protein of the monoclonal antibody obtained from the A549 cell line of human lung adenocarcinoma was human calnexin.

(Example 6) Serological cancer screening with monoclonal antibodies
Immobilon-P membrane (Millipore) was soaked in methanol for 20 seconds to make it hydrophilic. After the reaction, the membrane was transferred to TBS. Albumin and IgG in 60 μl of patient and healthy subject serum were removed using ProteoExtract Albumin / IgG Removal kit (Merck), and desalted and concentrated using VIVASPIN (Sartorius). The final volume of each serum was adjusted to 60 μl with PBS (−). Each pre-treated serum was diluted 20-fold with PBS (-), and the treated membrane and diluted serum were set in a dot blot apparatus (manufactured by Kaken Genex, Inc., Chiba). Dots were made on the membrane a total of 5 times per second (about 0.5 μl). Two membranes were prepared for each experiment. The membrane was removed from the device, placed in a small container containing TBS, and washed for 10 minutes. Thereafter, the mixture was placed in a small container containing 50 ml of a blocking agent (0.5% casein, Sigma-Aldrich) and blocked for 1 hour while permeating. Thereafter, the membrane was washed with TBS-T (TBS added with Tween 20 at a final concentration of 0.1%) for 5 minutes.

The first membrane is about 1 ml of the hybridoma culture supernatant as the primary antibody, and the other membrane is about 1 ml of antibody dilution (0.5% casein diluted 20-fold with TBS-T) on a 5 x 10 cm membrane. Was dropped on the membrane and reacted at room temperature for 30 minutes. After the reaction, the membrane washed 5 times for 3 minutes each with TBS-T is a 5 × 10 cm HRP labeled rabbit anti-mouse IgG (Fab ') ² (Dako Japan) diluted as a secondary antibody 1,000-fold with an antibody diluent. About 1 ml of the membrane was used and reacted at room temperature for 30 minutes. After the reaction, it was washed with TBS-T for 5 minutes each for 3 minutes, then reacted with HRP chemiluminescence reagent Immobilon Western (MILLIPORE) for 1 minute, and the chemiluminescence was detected with a luminescence detector (Kaken Genex). When there are many target antigens in the serum used, strong luminescence can be detected. When it is not present or extremely low, almost no light is emitted. The staining results were processed and stored with DotBlotChipSystem ver.4.0 (manufactured by Dynacom) linked to this apparatus. The reactivity of the antibody with lung cancer patient serum is shown in FIG.
The antibody showed strong reactivity with lung cancer patient sera including lung adenocarcinoma, squamous cell carcinoma, and small cell carcinoma, but it is strong against healthy human serum. There was no reaction. This result indicates that the antibody is an antibody that distinguishes lung cancer patients from healthy individuals.

(Example 7) Reactivity of the antibody with formalin-fixed and paraffin-embedded cell specimen
A549 cells were centrifuged at 1,000 rpm for 10 minutes, and the supernatant was discarded. 10 ml of phosphate buffered saline (PBS-) containing no divalent ions was added, and after stirring, the mixture was centrifuged at 1,000 rpm for 10 minutes. After discarding the supernatant, 30 ml of 10% formalin was added and fixed at 4 ° C. for 16-18 hours. After fixation, the supernatant was discarded, 70% ethanol, 80% ethanol, and 90% ethanol were added, and each was dehydrated while stirring on a permeator for 2 hours. Further, 100% ethanol was added, and the mixture was permeated at room temperature for 2 hours. Then, it was changed to fresh 100% ethanol and left at room temperature all day and night to complete dehydration. On the next day, it was changed to chloroform, and a penetration operation was performed while permeating twice for 2 hours at room temperature. Finally, it was placed in paraffin having a melting temperature of 58-60 ° C., and paraffin was permeated into the cell specimen while reducing the pressure with a vacuum pump for 4 hours, then embedded in paraffin, and the block was stored at 4 ° C. The sections were sliced into about 3 μm using a microtome, picked up on a silane-coated slide glass, and dried at 40 ° C. for 1 day. Then, it put into the plastic bag with a zipper and preserve | saved at 4 degreeC. The immunostaining method using formalin-fixed and paraffin sections is shown below.

  Tissue sections sliced into 3 μm of various lung cancer tissues fixed in 10% formalin for diagnosis in the cell specimen and pathology laboratory and embedded in paraffin were deparaffinized with xylene for 15 minutes. After that, the sections were passed through a descending ethanol series (100% ethanol 3 tanks → 95% ethanol 1 tank → 90% ethanol 1 tank → 80% ethanol 1 tank → 70% ethanol 1 tank) for 10 seconds, gradually increasing the ethanol concentration. The dexylene operation was performed while lowering. Thereafter, the sections were washed with running water, and 3% hydrogen peroxide solution was placed on the sections and reacted for 10 minutes to inhibit endogenous peroxidase activity. After further washing with running water, the sections were placed in a plastic container containing 200 ml of 0.01M citrate buffer (pH 6.0) with a final concentration of 0.1% and Tween 20 added. Antigenic activation was performed for a minute. After the treatment, the temperature of the entire container was lowered to room temperature with a fan.

  When the solution returned to room temperature, the sections were soaked in 0.075 M TBS (pH 7.5) for 5 minutes and then treated with 2% normal porcine serum / TBS for 10 minutes to prevent nonspecific protein reactions. Thereafter, the excess solution on the section was removed with tissue paper or the like, and then 100 μl of the hybridoma culture supernatant was placed on the section and reacted in a wet box at room temperature for 2 hours. After the reaction, it was washed 3 times for 5 minutes each with TBS, and a high-sensitivity polymer reagent labeled with HRP (ChemMate ENVISIN, DAKO Japan) as a secondary antibody was dropped onto the sections and reacted at room temperature for 30 minutes. After the reaction, the plate was washed with TBS three times for 5 minutes, and then 100 μl of Stable DAB solution (Invitrogen, Tokyo) as a substrate reaction solution of HRP enzyme was dropped and reacted for 2 to 3 minutes to visualize the antigen-antibody product. After that, after staining with Meyer's hematoxylin for 30 seconds, ascending ethanol series (70% ethanol 1 tank → 80% ethanol 1 tank → 90% ethanol 1 tank → 95% ethanol 1 tank → 100% ethanol 3 tanks) Then, after passing several tanks of xylene, a chemical encapsulant and Enteran (Mutou, Tokyo) were dropped, and a cover glass was placed to make a permanent specimen.

After encapsulation, the localization and extent of the antigen recognized by the antibody were confirmed with a microscope. As a result, reactivity was also observed with formalin-fixed A549 cells (FIG. 6). That is, the antibody was shown to be suitable for staining of formalin-fixed and paraffin-embedded specimens.
Next, the reactivity of various human lung cancer tissues embedded with formalin and embedded in paraffin was examined.
FIG. 7 shows the results of examining whether the antibody reacts with normal human lung tissue. As shown in FIG. 7, no staining with the antibody was observed in human normal bronchial epithelium and human normal alveolar epithelium. That is, it was shown that the antigen protein recognized by the antibody was hardly expressed in these human normal tissues.
FIG. 8 shows the results of examining whether or not the antibody reacts with lung adenocarcinoma tissues collected from four lung adenocarcinoma patients. As shown in FIG. 8, strong staining was observed in all four tissues. From this result, it was found that the antigen protein recognized by the antibody was strongly expressed in human lung adenocarcinoma.

FIG. 9 shows the results of examining whether or not the antibody reacts with lung squamous cell carcinoma tissue collected from four lung squamous cell carcinoma patients. As shown in FIG. 9, weak staining was observed in all four tissues. From this result, it was found that the antigen protein recognized by the antibody was only weakly expressed in human lung squamous cell carcinoma.
FIG. 10 shows whether or not the antibody reacts with lung squamous cell carcinoma tissues collected from patients with small cell lung cancer (SCLC, FIGS. 10A to C) and large cell neuroendocrine carcinoma (LCNEC, FIG. 10D). The result of examination was shown. As shown in FIG. 10, only a weaker staining was observed in small cell lung cancer (SCLC) than in squamous cell lung cancer. Therefore, it was found that in small cell lung cancer, the antigen protein recognized by the antibody was expressed only very weakly. In lung large cell neuroendocrine cancer (LCNEC), although it is moderately staining, almost all cancer cells in the tissue were stained. Therefore, it was found that the antigen protein recognized by the antibody is moderately expressed in pulmonary neuroendocrine cancer.
Table 1 summarizes the results of examining the reactivity with 87 cases of formalin-fixed and paraffin-embedded human lung cancer tissues.

As shown in Table 1, in lung adenocarcinoma (AD), 54 out of 59 cases were positive, and the positive rate was 91.5%. In squamous cell carcinoma (SCC), 17 out of 24 cases were positive, and the positive rate was 70.8%. In small cell lung cancer (SCLC), 2 out of 3 cases were positive, and the positive rate was 66.7%. In pulmonary large cell neuroendocrine cancer (LCNEC), one of the cases was positive and the positive rate was 100%.
The stainability of immunostaining of formalin-fixed and paraffin-embedded human lung cancer tissues was evaluated as follows. The positive rate in tumor cells was classified into 5 levels from 0 to 4 as shown in Table 2. As shown in Table 2, the staining intensity was classified into three levels: intensity, medium, and weakness. The staining score was calculated by multiplying the positive rate score in the tumor cells by the staining intensity score.

Table 1 summarizes the staining scores (Score) of immunostaining of formalin-fixed and paraffin-embedded human lung cancer tissues. The scores for lung adenocarcinoma (AD), squamous cell carcinoma (SCC), small cell lung cancer (SCLC), and large cell neuroendocrine carcinoma (LCNEC) are 6.9, 3.0, 1.3, and 8. 0.
Furthermore, the positive rate (FIG. 11), staining intensity (FIG. 12), and staining score (FIG. 13) in tumor cells for each tissue type were determined. There were few cases of small cell lung cancer and pulmonary neuroendocrine cancer, but in comparison between lung adenocarcinoma and squamous cell carcinoma, lung adenocarcinoma clearly showed positive rate and staining intensity in tumor cells compared to squamous cell carcinoma And the staining score was found to be significantly higher. In the two groups of lung adenocarcinoma and squamous cell carcinoma, the risk rate by the Mann-Whitney U test of tumor cell positive rate, staining intensity, and staining score was p <0.0005, p = 0.012, and p <0.0005, respectively. This result indicates that human calnexin is expressed more highly in lung adenocarcinoma than in squamous cell carcinoma. As a result, it was found that examining human calnexin expression in human lung cancer tissue in which formalin is fixed and embedded in paraffin is useful for determination of lung adenocarcinoma.

(Example 8) Expression of recombinant protein in E. coli and purification of expressed protein Fusion proteins of various antigen proteins were added with a histidine tag (His) added to the N-terminus and a FLAG tag added to the C-terminus. After expression of fusion protein expression vectors of various antigens into DH5α or BL21, expression of recombinant proteins was induced. As a preculture, Escherichia coli was cultured with shaking at 37 ° C. overnight in an LB liquid medium containing 50 μg / mL ampcillin. 10 mL of the preculture was added to 1 L of the main culture (LB liquid medium containing 50 μg / mL ampcillin), and cultured with shaking at 37 ° C. until OD600 reached 0.5. IPTG was added to 0.5 mM to induce recombinant protein expression for 16 hours. After E. coli was collected, it was suspended in a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4), and E. coli was disrupted with an ultrasonic crusher (OHTAKE WORKS). The disrupted Escherichia coli crushed material was centrifuged (18,000 g for 30 minutes), and the supernatant was used as the soluble fraction. This soluble fraction was passed through a column packed with Nickel Sepharose (Ni Sepharose 6, GE Healthcare), and the column was washed with a binding buffer of about 20 times the bed volume. The desired purified recombinant protein was eluted with an elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4). The expression of the target protein was confirmed by SDS-PAGE (the gel was ATTO e-pagel) followed by CBB staining (CBB Stain One, Nacalai Tesque).

Example 9 Production and Purification of Polyclonal Antibody As an antigen, a recombinant protein expressed and purified in 100 μg of E. coli by one immunization was used. Guinea pigs were used as immunized animals. After a total of 4 immunizations, whole blood collection and polyclonal antibodies were obtained.
The prepared polyclonal antibody was purified using a carrier to which a His protein having a FLAG tag added at the C-terminal was bound. The method followed the protocol (CNBr-activated Sepharose 4B, GE-Healthcare). First, 5 mg of His protein purified on a Ni Sepharose column was bound to 0.3 mg of a previously swollen and washed CNBr-activated Sepharose 4B (GE-Healthcare) carrier at 4 ° C. overnight with stirring. It was. This carrier was packed into a column, washed with various buffers, and reacted with 0.5 mg of polyclonal antibody at 4 ° C. overnight. The flow-through solution was collected and used as a purified antibody.

(Example 10) Synthesis of antigen protein by cell-free system For protein expression by cell-free system of wheat, WEPRO1240H Expression Kit (manufactured by Cell Free Science) was used. The method followed the protocol of this kit. The plasmid was extracted using Plasmid Midi-Prep Kit (Qiagen). The target gene was transcribed using SP6 RNA polymerase using a plasmid in which the target gene was incorporated downstream of the SP6 promoter as a template. Furthermore, the reaction solution was reacted with wheat extract to express the recombinant protein. After confirming the expression of the recombinant protein by SDS-PAGE, the supernatant (soluble fraction) was collected by centrifugation and purified using an anti-FLAG affinity gel.

(Example 11) Construction of sandwich ELISA system 50 μL each of the monoclonal antibody diluted 1000-fold with PBS was added to wells of an ELISA microplate (F8 MAXISORP, NUNC) and stirred at 4 ° C. The plate was allowed to stand overnight to immobilize the plate. As a control, a solid phase plate was prepared by adding 50 μL of PBS alone. The reaction solution in the well was removed, and a PBS solution (1% BSA-PBS) containing 300 μL of 1% Bovine Serum Albumin (BSA, Intergen) was added and blocked at room temperature for 3 hours. This plate was washed three times with PBST (0.05% Tween 20-PBS) using a plate washer (Bio-Rad), and 50 μL of standard antigen (diluted with 1% BSA-PBS) was added. And left for 1 hour. Protein expressed and purified using a wheat cell-free system was used as a standard antigen. In addition, 50 μL of a sample obtained by diluting a serum of a healthy subject or a lung adenocarcinoma patient 10-fold with 1% BSA-PBS was reacted. Again, the plate was washed three times with PBST, 50 μL of a polyclonal antibody solution (diluted 100-fold with 1% BSA-PBS) was added, and the mixture was allowed to stand at room temperature for 1 hour. After washing with PBST three times, 50 μL of a solution obtained by diluting HRP-Rabbit Anti-Guinea Pig IgG [H + L] (manufactured by Invitrogen) 6,000-fold with 1% BSA-PBS was added, and left at room temperature for 1 hour. After washing three times with PBST, 50 μL of TM blue (SeraCare Life Sciences) was added for color development, and 50 μL of 0.5 M sulfuric acid was added to stop the reaction. Absorbance (A450) was measured using a microplate reader. FIG. 14 shows a summary of the measurement results of human calnexin contained in the serum of 56 normal subjects (Normal) and 33 cases of lung adenocarcinoma (AD).

The value of lung adenocarcinoma patient serum was found to be statistically significant and higher than that of healthy subject (Mann-Whitney U test had a risk factor of 0.002). As a result, it was found that the amount of human calnexin in lung adenocarcinoma patient serum was significantly higher than the amount of human calnexin in healthy subject serum.
Based on the above, the antigen protein human calnexin recognized by the monoclonal antibody is present in the serum of lung adenocarcinoma patients at a higher concentration than that in healthy subject serum, and human calnexin is measured using serum as a specimen. Revealed that it was possible to determine whether or not the patient had lung adenocarcinoma.

  According to the present invention, there are provided a method for determining the presence or absence, the degree of progression or the prognosis of a subject's lung adenocarcinoma, a diagnostic agent for lung adenocarcinoma, and a diagnostic kit for lung adenocarcinoma. The present invention is useful for detecting data used for diagnosis of lung adenocarcinoma.

  (SEQ ID NO: 1) SEQ ID NO: 1 shows the amino acid of human calnexin (calnexin; NCBI Refseq, No. NP001737). The human calnexin of SEQ ID NO: 1 includes a mutant due to amino acid substitution.

Claims (12)

Measure human calnexin in a subject's specimen using an antibody that specifically binds to human calnexin, and determine whether the patient has lung adenocarcinoma, its degree of progression, or prognosis Data detection method for determining The method according to claim 1, wherein the antibody that specifically binds to human calnexin is a monoclonal antibody. The method according to claim 2, wherein the monoclonal antibody that specifically binds to human calnexin is a monoclonal antibody produced by hybridoma FERM BP-11318. The method according to claim 1, wherein the specimen is blood. A diagnostic agent for lung adenocarcinoma comprising an antibody that specifically binds to human calnexin. The diagnostic agent according to claim 5, wherein the antibody that specifically binds to human calnexin is a monoclonal antibody. The diagnostic agent according to claim 6, wherein the monoclonal antibody that specifically binds to human calnexin is a monoclonal antibody produced by hybridoma FERM BP-11318. A lung adenocarcinoma diagnostic kit comprising an antibody that specifically binds to human calnexin. The kit according to claim 8, wherein the antibody that specifically binds to human calnexin is a monoclonal antibody. The kit according to claim 9, wherein the monoclonal antibody that specifically binds to human calnexin is a monoclonal antibody produced by hybridoma FERM BP-11318. A monoclonal antibody that specifically binds to human calnexin produced by the hybridoma FERM BP-11318. Hybridoma FERM BP-11318.