WO2019009346A1 - 抗ヒトIgG4モノクローナル抗体、およびその抗体を利用したヒトIgG4測定試薬 - Google Patents
抗ヒトIgG4モノクローナル抗体、およびその抗体を利用したヒトIgG4測定試薬 Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to an anti-human IgG4 monoclonal antibody and a human IgG4 measurement reagent using the antibody.
- the use of the anti-human IgG4 antibody of the present invention makes it possible to specifically detect and measure human IgG4 in a sample, and is extremely effective in the field of diagnosis and clinical examination such as IgG4 related diseases.
- Non-Patent Document 1 A comprehensive diagnostic criteria for IgG4 related diseases 2011 was proposed, and a diagnostic criteria for hyperpigmentation 4 with an IgG4 level in blood of 135 mg / dL or higher was formulated.
- a diagnostic criteria for hyperpigmentation 4 with an IgG4 level in blood of 135 mg / dL or higher was formulated.
- patients to be examined cases exceeding 5000 mg / dL are also reported, and at the field of clinical examination, examination results showing pseudo low value are regarded as a problem (Non-patent Document 2).
- the blood IgG4 measurement reagent is required to be applied to a general-purpose general biochemistry automatic analyzer introduced widely in hospitals and inspection centers.
- IgG4 is a heterotetrameric molecule with a molecular weight of about 146000 and it is suitable to use an antibody to detect such molecules. Therefore, the first problem is the specificity of the antibody which is an important component of the measurement reagent.
- IgG4 is one of the IgG subclasses, and is known to have very high homology with the other subclasses IgG1, IgG2 or IgG3 (Non-patent Document 3, Non-patent Document 4).
- IgG1 65%, IgG2: 23%, IgG3: 8%, IgG4: 4% in normal healthy serum, and the standard value in healthy subjects is IgG1: 351 to 962; IgG2: 239 to 838 LgG3: 8.5-140; lgG4: 4.5-117 (mg / dL), and preparation of an antibody with excellent specificity which can react only with IgG4 is essential because of the small amount ratio of IgG4 .
- Indirect antibody method such as indirect enzyme antibody method (ELISA method), indirect fluorescent antibody method, CLEIA method (chemiluminescent enzyme immunoassay method) as a measurement method using an antibody; single radial immunodiffusion method (SRID) ) And double immunodiffusion (DID), and other methods such as immunodiffusion, immunoturbidimetry (TIA), and latex (particle) aggregation method classified into immunonephelometry (NIA).
- the latex (particle) based on the detection of an aggregate based on the binding of an antigen and an antibody, which can be detected even at low concentrations of target molecules, is currently used frequently in the field of clinical examination.
- IgG4 immunoturbidimetry
- NIA immunonephelometry
- zone phenomenon aggregation suppression due to antigen excess
- prozone phenomenon aggregation suppression due to antigen excess
- the measured value may be pseudo low, which may lead to an erroneous diagnosis.
- IgG4 as described above, there is a difference of nearly 1000 times in concentration between healthy subjects and hyper-IgG4 patients, and there have been reported cases where prozone phenomenon actually occurred in the reagent for measuring IgG4 (Non-patent literature 2). Therefore, as a second problem, it is required to measure a high concentration of IgG4 as a blood IgG4 measurement reagent.
- a monoclonal antibody against human IgG4 is known (Patent Document 1).
- the IgG subclass has high specificity to IgG4, it may not be suitable for antibody: antigen aggregate formation since generally one recognition site (epitope) of a monoclonal antibody is present.
- the problem 2 is also solved by pseudo-multivalenting a monoclonal antibody.
- Patent Document 2 is a method of biotinylation of a monoclonal antibody to form a multimer with streptavidin
- Patent Document 3 is a method of forming a multimer using Protein A, an anti-immunoglobulin antibody, etc.
- Document 4 describes a method for immobilizing a monoclonal antibody on an insoluble carrier.
- the steps for multivalent formation of monoclonal antibodies are complicated, and in particular, it is difficult to always control the antibody valence constant, and it corresponds to a general-purpose automatic analyzer based on the immunoturbidimetric assay principle. It has the disadvantage of requiring cost and time to maintain its performance as a reagent.
- Patent Document 5 There is also known a method for obtaining a polyclonal antibody with high specificity for any of the IgG subclasses (Patent Document 5). According to this method, it is possible to obtain an antigen-specific polyclonal antibody by immune tolerance without requiring the above-mentioned purification step, but there is individual difference of animals to be immunized to the extent of induction of immune tolerance. Also, because it takes time to cause immune tolerance, it is necessary to obtain a large number of immunized animals to obtain a specific antibody against the desired IgG4 constantly, and in an appropriate manner in terms of cost and animal protection. It is hard to say that it exists.
- the present invention provides a monoclonal antibody having high specificity and affinity to IgG4, a hybridoma producing the same, a method of detecting IgG4 using the monoclonal antibody, and a kit used therefor With the goal.
- antibodies are immobilized on an insoluble carrier, to which a sample containing the antigen to be measured is mixed to cause an immune agglutination reaction based on the antigen-antibody reaction.
- This is a method of measuring an antigen to be measured.
- it is desirable to use a plurality of types of antibodies that recognize various epitopes such as polyclonal antibodies. This is because aggregation is caused by binding of a plurality of antibodies (insoluble carriers) to one antigen molecule. Instead, each antibody molecule may have low affinity.
- the immunological particle aggregation inhibition method (latex (particle) aggregation inhibition method) is an antigen immobilized on an insoluble carrier by mixing an insoluble carrier on which the antigen is immobilized, a free antibody against the antigen, and a sample containing the antigen. And the antigen contained in the sample compete with each other, and as a result, aggregation formation is suppressed, whereby the amount of the antigen to be measured is measured.
- prozone phenomenon is not observed even when there is an antigen concentration far exceeding the measurement range. It is an ideal way to avoid risks.
- the use of polyclonal antibodies is not suitable for such methods.
- the inventors of the present invention have succeeded in producing a monoclonal antibody suitable for the immunological particle aggregation inhibition method (latex (particle) aggregation inhibition method) for human IgG4 detection as a result of intensive studies.
- the constitution of the present invention according to the present invention is as the following [1] to [21].
- An antibody to human IgG4 having a heavy chain variable region and a light chain variable region, wherein the complementarity determining region (hereinafter referred to as CDR) 1, 2 and 3 of the heavy chain variable region are respectively sequenced
- CDRs 1, 2 and 3 of the light chain variable region are the amino acid sequences represented by SEQ ID NOs: 12, 13 and 14 respectively Or the monoclonal antibody according to [2]; [4] The monoclonal antibody according to [3], wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 5 and the light chain variable region has the amino acid sequence of SEQ ID NO: 6; [5] The monoclonal antibody according to [4], which is produced by hybridoma MaI 4-08.
- a monoclonal antibody preferably a monoclonal antibody according to [1] or [2], wherein CDRs 1, 2 and 3 of the light chain variable region of the antibody are the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20 respectively ;
- the monoclonal antibody according to [6] wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 7 and the light chain variable region has the amino acid sequence of SEQ ID NO: 8;
- the monoclonal antibody according to [7] which is produced by hybridoma MaI 4-09.
- a bivalent antibody or bivalent antibody fragment [10] The antibody fragment of [9], which is F (ab ') 2 .
- [11] A method for detecting and detecting human IgG4 in a sample using the monoclonal antibody according to any of [1] to [8] or the antibody fragment according to [9] or [10]; [12] The method according to [11], which is an immunoturbidimetric assay or an immunological paradox. [13] The method according to [12], which is an immunological particle agglutination method; [14] The method according to [12], which is an immunological particle aggregation preventing method; [15] The method according to [11], which is immunohistochemistry (IHC) staining; [16] The method according to [11] by flow cytometry.
- IHC immunohistochemistry
- a kit for measuring human IgG4 comprising the monoclonal antibody according to any of [1] to [8] or the antibody fragment according to [9] or [10].
- kits for the method according to [14] or [17] (1) The monoclonal antibody according to any one of [1] to [8] or the antibody fragment according to [9] or [10]; (2) a kit comprising the isolated human IgG4 or a peptide fragment thereof; and (3) an insoluble carrier; [20] The kit according to [19], wherein (2) the isolated human IgG4 or a peptide fragment thereof is adsorbed to (3) the insoluble carrier. [21] (3) The kit according to [19] or [20], wherein the insoluble carrier is latex particles.
- Embodiments of the present invention make it possible to efficiently detect and quantify IgG4 in a biological sample, and can be useful for the diagnosis of various diseases.
- IgG4-related diseases systemic, chronic inflammatory diseases characterized by high levels of IgG4 and marked IgG4-positive plasma cell infiltration into affected organs
- diseases as Mikulicz disease (Mikulicz disease), Kuttner's tumor, lacrimal glanditis, IgG4-related eye disease, IgG4-related respiratory disease, inflammatory pseudotumor, mediastinal fibrosis, enteritis, sclerosing cholangitis, IgG4-related liver Disorders, autoimmune pancreatitis (AIP), IgG4-related kidney disease, retroperitoneal fibrosis, prostatitis, autoimmune pituitary inflammation, thyroiditis, hypertrophic dura, IgG4-related lymphadenopathy, arthritis, inflammatory abdomen
- IgG4-related diseases systemic, chronic inflammatory diseases
- FIG. 2 shows the reaction absorbance of ELISA for each human globulin in the MaI 4-08 producing antibody.
- Evaluation of Specificity of MaI 4-09 Producing Antibody by ELISA FIG. 3 shows the reaction absorbance of ELISA for each human globulin in the MaI 4-09 producing antibody. Measurement of antibody absorbance change using MaI 4-08 producing antibody
- Fig. 4 shows a standard sample of human IgG4, buffer containing MaI 4-08 producing antibody (first reagent), human IgG4 sensitized latex containing buffer (second reagent Shows the change in absorbance upon reaction and reaction.
- FIG. 6 shows a standard sample of human IgG4, buffer containing MaI 4-05 producing antibody (first reagent), buffer containing human IgG 4 sensitized latex (A second reagent) is mixed and it shows the absorbance change at the time of making it react.
- the other conditions are the same as in FIG. Measurement of the amount of change in antibody absorbance using HP6025 (comparative example)
- FIG. 6 shows a standard sample of human IgG4, buffer containing MaI 4-05 producing antibody (first reagent), buffer containing human IgG 4 sensitized latex (A second reagent) is mixed and it shows the absorbance change at the time of making it react.
- FIG. 6 shows a standard sample of human IgG4, buffer containing MaI 4-05 producing antibody (first reagent), buffer containing human IgG 4 sensitized latex (A second reagent) is mixed and it shows the absorbance change at the time of making it react.
- the other conditions are the same as in FIG. Measurement of the amount of change
- FIG. 7 shows a standard sample of human IgG4, a buffer containing HP6025 (first reagent), and a buffer containing human IgG4-sensitized latex (second reagent) , Shows the change in absorbance upon reaction.
- the other conditions are the same as in FIG.
- FIG. 8 shows 0 to 1000 mg / dL human IgG4 serial dilution sample, buffer containing MaI 4-08 producing antibody (first reagent), human IgG 4 sensitized latex containing The buffer solution (second reagent) is mixed, and the measured value upon reaction is shown.
- FIG. 9 shows 0 to 1000 mg / dL human IgG4 serial dilution sample, buffer containing MaI 4-09 producing antibody (first reagent), human IgG 4 sensitized latex containing The buffer solution (second reagent) is mixed, and the measured value upon reaction is shown.
- Evaluation of resistance to prozone phenomenon using MaI 4-08 producing antibody Fig. 10 shows 0 to 8000 mg / dL human IgG4 serial dilution series sample, buffer containing MaI 4-08 producing antibody (first reagent), human IgG4 sensitivity The measured value at the time of mixing and reacting the production latex containing buffer solution (2nd reagent) is shown. Evaluation of resistance to prozone phenomenon using MaI 4-09 producing antibody FIG.
- FIG. 11 shows human IgG 4 serial dilution series sample of 0 to 8000 mg / dL, buffer containing MaI 4-09 producing antibody (first reagent), human IgG 4 sensitivity The measured value at the time of mixing and reacting the production latex containing buffer solution (2nd reagent) is shown.
- Evaluation of specificity of MaI 4-08 producing antibody to human IgG4 FIG. 12 shows each sample of human IgG1, human IgG2, human IgG3 and human IgG4 of 3000 mg / dL, buffer containing MaI 4-08 producing antibody (first reagent), The measurement value at the time of mixing and reacting human IgG4 sensitized latex containing buffer solution (2nd reagent) is shown.
- FIG. 13 shows each sample of human IgG1, human IgG2, human IgG3 and human IgG4 of 3000 mg / dL, buffer containing MaI 4-09 producing antibody (first reagent), The measurement value at the time of mixing and reacting human IgG4 sensitized latex containing buffer solution (2nd reagent) is shown.
- the alignment of the amino acid sequence H1 (P01861, SEQ ID NO: 4) of the human IgG4 heavy chain constant region obtained from Uniprot and the amino acid sequence of each peptide H2 to 12 in which the sequence is partially deleted is shown. 99th to 110th show a hinge region.
- the alignment of the substituted amino acid sequence (H-31 to H-39) is shown in (bold).
- Western blot analysis of MaI 4-08 antibody against peptide H1-12 Western blot analysis of peptides H1-12 for MaI 4-09 producing antibody Western blot analysis of MaI 4-08 producing antibodies for peptides H10 and H31-39 Western blot analysis for peptides H10 and H31-39 of MaI 4-09 Western blot analysis of peptide H1-12 of MaI 4-05
- the monoclonal antibody according to the embodiment of the present invention is produced, for example, by a hybridoma obtained by immunizing an animal using purified human IgG4 as an immunogen and fusing anti-human IgG4 antibody-producing cells produced by the animal with bone marrow tumor cells. Be done.
- the animals used are not limited, but non-human animals such as mice, rats, guinea pigs, hamsters, rabbits and the like. In particular, mice in which the IgG subtype is IgG1, IgG2a, IgG2b, and IgG3 are preferred.
- the above hybridoma can be obtained by the following method. That is, human IgG4 is mixed together with already known ones such as Freund's complete and incomplete adjuvants, aluminum hydroxide adjuvant and pertussis adjuvant, etc. to make a sensitive adjuvant solution, and divided into several times to divide mice and rats. And other animals are immunized by intraperitoneal or subcutaneous injection every 1 to 3 weeks.
- the amount of sensitizing antigen is between 1 ⁇ g and 100 mg, but generally about 50 ⁇ g is preferable.
- the number of times of immunization is generally 2 to 7 but various methods are known.
- antibody-producing cells derived from spleen or the like are fused with cells having proliferation ability in a test tube such as bone marrow tumor cells (myeloma cells).
- Antibody-producing cells can be obtained from spleens of mice, nude mice, rats and the like.
- fusion can be performed by using polyethylene glycol (PEG) according to the Köhler and Milstein method (Nature. 256, 495. 1975) which is already known per se.
- the fusion can also be performed by Sendai virus, an electrofusion method.
- hybridomas are selected from colonies formed by cells surviving in HAT medium and HT medium from the fused cells by the limiting dilution method.
- the colony culture supernatant prepared from the fused cells in a 96-well well or the like contains an antibody against human IgG4, place the supernatant on an assay plate on which human IgG4 is immobilized, After the reaction, monoclonal antibody-producing clones against human IgG4 can be selected by ELISA in which a secondary labeled antibody such as an anti-mouse immunoglobulin-HRP labeled antibody is reacted. Besides HRP, enzymes such as alkaline phosphatase, fluorescent substances, radioactive substances and the like can be used as the labeling substance of the labeled antibody.
- screening of human IgG4 specific antibodies can be performed by simultaneously carrying out an ELISA using an assay plate bound with only the blocking agent BSA. That is, clones positive in human IgG4 plates and negative in ELISA with BSA can be selected.
- hybridoma is particularly capable of binding to human IgG4, and binding to other human immunoglobulins (other IgG (IgG1, IgG2, IgG3), IgA) Preferred are hybridomas that produce antibodies that do not bind (IgA1, IgA2), IgD, IgE and IgM).
- “binds to human IgG4 and does not bind to other human immunoglobulins” includes human IgG4 as a solid phase antigen, for example, when the measurement conditions other than the following are simply set in a common ELISA.
- the ratio of absorbance when using one without any human immunoglobulin as a control shows a ratio of 40 or more, preferably 50 or more, more preferably 100 or more, and human as a solid phase antigen It is meant that the ratio of the absorbance when showing those without any human immunoglobulin as a control (blank) when containing other than IgG4 is 5 or less, preferably 2 or less. It also means that it exhibits higher binding affinity to human IgG4 than other human immunoglobulins, and can be evaluated using binding rate constant and dissociation constant.
- the specific epitope of the heavy chain constant region of human IgG4 is 4.0 ⁇ 10 5 or more, usually 4.0 ⁇ 10 5 to 6.0 ⁇ 10 5 And a dissociation constant of 5.0 ⁇ 10 ⁇ 10 or less, usually 5.0 ⁇ 10 ⁇ 10 to 3.0 ⁇ 10 ⁇ 10 .
- the above-mentioned hybridoma can be cultured in a medium usually used for cell culture, such as ⁇ -MEM, RPMI 1640, ASF, S-clone, etc., and the monoclonal antibody can be recovered from the culture supernatant.
- a medium usually used for cell culture such as ⁇ -MEM, RPMI 1640, ASF, S-clone, etc.
- the monoclonal antibody can be recovered from the culture supernatant.
- an animal from which a hybridoma is derived and a nude mouse may be subjected to pristane treatment in advance, and ascites fluid may be stored by intraperitoneally injecting cells into the animal, and a monoclonal antibody may be recovered from the ascites fluid.
- a usual method can be used as a method of recovering a monoclonal antibody from the above supernatant and ascites fluid. For example, salting out with ammonium sulfate, sodium sulfate and the
- Human IgG4 in a sample can be detected with high sensitivity and specificity by an immunoassay using a monoclonal antibody according to an embodiment of the present invention.
- the target sample includes blood, serum, plasma and the like collected and isolated from the sample. It may be a biopsy sample from the whole body, for example, from the pituitary, lacrimal gland, salivary gland, thyroid, prostate, bronchial epithelium, alveolar septum, pancreatic duct, retroperitoneum, biliary wall, or rheumatoid arthritis synovium.
- immunoassay a biochemical test assay that measures the level of material contained in a biological sample using the reaction of antigen and antibody.
- a sandwich ELISA method As a detection method by the "immunoassay" using the monoclonal antibody according to the embodiment of the present invention, a sandwich ELISA method, a chemiluminescent enzyme immunoassay (CLEIA method), a fluorescence immunoassay (FIA), latex (particles)
- CLIA method chemiluminescent enzyme immunoassay
- FIA fluorescence immunoassay
- latex (particles) latex (particles)
- the aggregation method may, for example, be mentioned, but the latex (particle) aggregation prevention method is more preferable.
- ELISA method refers to ELISA (Enzyme-Linked Immunosorbent Assay) / EIA (Enzyme Immuno Assay)) and refers to a method of detecting and quantifying the concentration of a substance contained in a sample using an enzyme reaction. The target is detected and measured by using chromogenic luminescence based on the enzyme reaction as a signal.
- competitive ELISA refers to an ELISA that measures the proportion of "labeled antigen” and "antigen present in a sample” binding to an antibody under competitive conditions.
- an antigen is reacted with an antibody to form a precipitate of the immune complex, the aggregate is irradiated with light, and the attenuation (absorbance) of the irradiated light due to scattering is measured by an automatic analyzer. And the amount of antigen contained in the sample.
- an antibody is reacted with an antigen to form a precipitate of an immune complex, light is applied to the complex, and scattered light is measured to detect the antigen contained in the sample.
- the “latex (particle) aggregation method” is a method of solidifying an antibody on latex particles, aggregating latex particles in the presence of an antigen, and observing the aggregation to detect an antigen.
- an antigen is immobilized on latex particles, and "the antigen bound to the latex particle” and “the antigen present in the sample” bind to the antibody at any ratio under competitive conditions. This is a method of observing the aggregation of latex particles and indirectly detecting “the antigen present in the sample”.
- the attenuation (absorbance) of the irradiation light due to scattering may be measured (immunoturbidimetric method), or the scattered light may be measured (immune narcotic method) to observe the aggregation.
- the antigen bound to the latex particle does not necessarily have to be the same molecule as “the antigen present in the sample”, and in the binding to the antibody according to the present invention or an antigen binding fragment thereof It may be any molecule that competes with the “antigen present therein.
- the antibody according to the present invention is used in the “latex (particle) aggregation inhibition method,” “the antigen bound to latex particles” and “the antigen present in the sample” contain the epitope of the antibody. Is preferred.
- Epitope refers to the portion of the antigen that the antibody recognizes.
- Human IgG4 has a molecular weight of about 146000 consisting of two light chain molecules consisting of variable region VL and constant region CL; two heavy chain molecules consisting of variable region VH and constant region CH1, hinge region, constant region CH2 and constant region CH3
- antibodies do not recognize the whole but recognize and bind only a relatively small portion of the antigen.
- In order to function as an epitope at least 10 amino acid residues, more preferably 5 amino acid residues in length are required. This antibody binding moiety is also referred to as "epitope" or "antigenic determinant".
- the antibody according to the embodiment of the present invention is required to recognize IgG4, and not to recognize IgG1, IgG2 and IgG3. Since the light chain is common to IgG1-IgG4, it is desirable for the epitope to be in the heavy chain, especially the heavy chain constant region.
- the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4 are shown by SEQ ID NOS: 1, 2, 3 and 4, respectively, and it is desirable to use low homology portions of SEQ ID NOS: 1-3 and SEQ ID NO: 4 as epitopes.
- the epitope of the antibody according to the preferred embodiment of the present invention consists of all or part of the amino acid sequence from the 221st to the 327th amino acid of the heavy chain constant region of human IgG4 shown in SEQ ID NO: Including.
- the peptide comprising the epitope, more specifically, all or one of the amino acid sequence from position 221 to position 327 of the heavy chain constant region of human IgG4 shown in SEQ ID NO: 4
- a peptide consisting of a portion (usually 5 to 50, preferably 10 to 30) and containing the 299th glutamic acid.
- latex (particle) aggregation method although aggregation does not occur unless a plurality of monoclonal antibodies (polyclonal) which usually have different epitopes are used, since IgG4 itself is a dimer of a light chain and a heavy chain, it is a monoclonal antibody However, there are some that can be applied to the agglutination reaction. Furthermore, if a plurality of antibody molecules are adsorbed to latex particles, aggregation may occur even if the antibody molecule is a monovalent antibody molecule (one antigen (recognition) binding site per molecule).
- the “monovalent antibody” refers to a Fab fragment that can be treated with papain with a natural antibody, single-chain variable fragment (scFV), etc., in which a light chain variable region and a heavy chain variable region are linked genetically. Is this.
- the reaction within the measurement system may vary, so Polyclonal antibodies are not suitable.
- the antibody used is a monovalent antibody molecule, the antibody-mediated agglutination between latex particles (with antigen adsorbed) does not occur, which is also unsuitable.
- immunoglobulin types Two or more antigen (recognition) binding sites are required per molecule, and naturally occurring immunoglobulin types (IgG (divalent), IgA (tetravalent), IgD (divalent), IgE (divalent) and IgM (divalent). It is necessary to be a multimer antibody of F (ab ') 2 (divalent) which can be obtained by pepsin treatment) or the above-mentioned "monovalent antibody”.
- Antigen (recognition) binding site indicates the minimum site of an antibody capable of binding to an antigen. While not limited thereto, the sites containing the heavy chain and light chain complementarity determining regions (CDRs) are referred to. It may include the V L region that is the variable region of the light chain and the V H region that is the variable region of the heavy chain. These may be linked by genetic engineering or via SS bond or the like.
- Latex originally refers to a colloidal water dispersion of a polymer resin, but in the present application, "latex particles" refer to a polymer resin having a uniform particle size.
- the latex particle is not particularly limited as long as it is a latex particle that can be used in a conventional immunoassay, but one having polystyrene as a main material is desirable.
- the particle size of the latex particles used in the present application may be 50 nm to 500 nm, preferably 75 nm to 306 nm, more preferably 100 nm to 111 nm.
- kits for immunoassaying human IgG4 which comprises (1) an antibody of the present invention, (2) isolated human IgG4 or a peptide fragment thereof and (3) It can be carried out using a kit comprising a solid support.
- a solid support and an isolated human IgG4 or peptide fragment solution thereof are separately prepared, and when measuring IgG4, the antibody may be adsorbed to the solid support, May be provided in a state of being adsorbed to a solid support.
- the washing solution for example, Tris buffer containing a surfactant can be used.
- the kit of the present invention can also contain a sample dilution solution as needed.
- a buffer such as Tris can be used as the sample dilution liquid.
- a chelating agent such as EDTA ⁇ 2Na, or an inorganic salt such as sodium chloride may be added to the buffer solution.
- isolated human IgG4 or a peptide fragment thereof is used as the "antigen bound to latex particles" as described above, and therefore needs to contain the epitope of the antibody of the present invention.
- a naturally-occurring IgG4 molecule may be isolated and purified, but a genetically engineered “peptide fragment” thereof, for example, a part or all of the constant region of the light chain or heavy chain of human IgG4 may be cloned It does not matter.
- Part or all of the constant region of the heavy chain of human IgG4 may contain part or all of the amino acid sequence of SEQ ID NO: 4.
- IgG4 can also be measured by a sandwich assay ELISA method using the monoclonal antibody of the present invention.
- the monoclonal antibody in addition to the antibody of the present invention, another antibody against IgG4 can be used.
- the specific example of the method of measuring IgG4 by a sandwich assay is as follows.
- the antibody of the present invention as a primary antibody is adsorbed on a solid support, for example, a plate, reacted with an analyte, for example, IgG4 in serum, the solid support is washed, and then adsorbed IgG4 and biotin IgG4 is detected by reacting with a secondary antibody such as a monoclonal antibody or polyclonal antibody against another biotinylated IgG4, reacting with peroxidase labeled streptavidin, then performing a peroxidase enzyme reaction and then a color reaction. can do.
- a secondary antibody that is directly enzyme-labeled with peroxidase, alkaline phosphatase or the like.
- the substance to be bound to the secondary labeled antibody is not limited to the enzyme depending on the measurement method, and may be a radioisotope, a fluorescent substance, a magnetic substance, a colloid or the like.
- kits for sandwich assay ELISA when performing sandwich assay ELISA using the antibody of the present invention, it can be carried out using a kit for sandwich assay ELISA.
- a kit for immunoassaying IgG4 comprising i) a solid support, ii) an antibody of the present invention, iii) labeled
- IgG4 can also be measured by using a kit comprising an antibody against IgG4, and iv) a component for detecting the label.
- the component for detecting the label is a component for measuring that the antibody is labeled, and when the label is biotin, a reagent containing peroxidase labeled streptavidin, a peroxidase enzyme substrate of tetramethylbenzidine, and hydrogen peroxide
- the label is alkaline phosphatase
- it is a reagent containing p-nitrophenyl phosphate.
- the kit may optionally contain a washing solution. In the present invention, when this kit is used, it is preferable to include a washing solution for binding IgG4 in the sample to the antibody and then removing components that are not adsorbed to the solid phase support.
- the washing solution for example, Tris buffer containing a surfactant can be used.
- the kit of the present invention can also contain a sample dilution solution as needed.
- a buffer such as Tris can be used as the sample dilution liquid.
- a chelating agent such as EDTA ⁇ 2Na, or an inorganic salt such as sodium chloride may be added to the buffer solution.
- Such detection by tissue immunostaining can be carried out using a kit comprising i) the monoclonal antibody of the present invention, ii) a labeled secondary antibody and iii) a coloring reagent as components.
- the labeled secondary antibody include animal-derived anti-IgG antisera and anti-IgG polyclonal antibodies labeled with an enzyme such as peroxidase or alkaline phosphatase, etc.
- an enzyme used for labeling is colored
- reagents such as commonly used chromogenic substrates are used.
- chemiluminescence enzyme immunoassay (CLEIA method)
- fluorescence immunoassay (FIA) or latex agglutination method using the antibody of the present invention
- FIA method for known chemiluminescence enzyme immunoassay
- a kit, a kit for fluorescence immunoassay (FIA) or a kit for latex agglutination can be used.
- Example 1 Preparation and Evaluation of Monoclonal Antibody (1) Selection and Preparation of Antigen for Preparation of Monoclonal Antibody A known method of producing a hybridoma that recognizes a human IgG4 (for example, Kohler and Milstein, Nature (1975) 256, Prepared according to p.495-497). IgG4 used as an antigen was prepared from human serum. Specifically, human serum was fractionated and dialyzed by ammonium sulfate precipitation, and then the IgG4 antigen was purified according to the product insert of CaptureSelect IgG4 Affinity Matrix (Thermo Fisher Scientific Co., Ltd.).
- the emulsified suspension was prepared by thoroughly mixing the same volume of Freund's complete adjuvant (Sigma Aldrich) with 0.5 mg / mL human IgG4 purified by the above method from immune human serum until it was emulsified.
- Four-week-old Balb / c mice were anesthetized with diethyl ether, and the above emulsified suspension was intraperitoneally administered at 50 ⁇ g of human IgG4 per mouse.
- the mice were each injected with the emulsion suspension prepared in Freund's incomplete adjuvant (Sigma Aldrich) as described above, six times at intervals of about two weeks.
- the emulsified suspension was similarly injected about 3 weeks after the above sixth injection as a final immunization.
- antibody dilution buffer (10 mM sodium dihydrogen phosphate; 150 mM sodium chloride; 0.05% polyoxyethylene (20) sorbitan monolaurate; 0.05% bovine serum albumin; pH 7.4 100 ⁇ L each of anti-mouse IgG (H + L) HRP-labeled antibody (Life Technologies) diluted 10000 folds in 1) was shaken at the same conditions as above.
- 100 ⁇ L of SureBlue (KPL) was added. After standing at room temperature for 15 minutes, 100 ⁇ L of 0.5 mol / L sulfuric acid (Wako Pure Chemical Industries, Ltd.) was added.
- the wavelength of 450 nm was measured by a microplate reader (BIO-RAD). Among a total of 4481 wells, 192 wells in which the ability to bind to IgG4 was confirmed were selected. The selected hybridomas were cloned and culture was continued.
- the precipitate was added with 24 mL of a coating buffer containing 3% bovine serum albumin, suspended, subjected to ultrasonication for complete dispersion, and then stirred at room temperature for 1 hour.
- 12 mL of HEPES buffer solution 2 is added to the precipitate obtained by centrifugation again, suspended, subjected to ultrasonication for complete dispersion, and adjusted to a latex concentration of 0.12% with HEPES buffer solution 2
- the obtained human IgG4-sensitized latex particle suspension was obtained and used as a second reagent common to each first reagent.
- the composition of each reagent is as follows.
- HEPES buffer solution 1 25 mM 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (Saikyo Chemical Industries); 100 mM 3- (cyclohexylamino) -1-propanesulfonic acid (Saikyo Chemical Industries); 150 mM sodium chloride (sum Kojun Pharmaceutical Co., Ltd .; 1 mM EDTA ⁇ 2Na (Wako Pure Chemical Industries) 3.3% dextran 70 (Tokyo Chemical Industry); 0.1% Block Ace (Dainippon Sumitomo Pharmaceutical Co., Ltd.); 0.09% sodium azide Photo pure drug industry); pH 7.40.
- Coat buffer 25 mM 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (Saikyo Chemical Industries); 150 mM sodium chloride (Wako Pure Chemical Industries); 1 mM EDTA ⁇ 2 Na (Wako Pure Chemical Industries); 3% bovine serum albumin (Merck Millipore); 0.09% sodium azide (Wako Pure Chemical Industries, Ltd.); pH 7.50.
- HEPES buffer solution 2 500 mM 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (Saikyo Chemical Industries); 25 mM 2-morpholinoethanesulfonic acid (Wako Pure Chemical Industries); 1 mM EDTA ⁇ 2Na (Wako Pure Chemical Industries) 150 mM L-arginine hydrochloride (Wako Pure Chemical Industries); 0.075% Procrine 950 (Sigma Aldrich); pH 6.00.
- the antibody produced from 5 out of 8 clones showed the largest absorbance change at 0.0 mg / dL, and the absorbance change decreased as the human IgG4 concentration increased. Therefore, 5 clones (MaI 4-09, MaI 4-08, MaI 4-01, MaI 4-06, MaI 4-03) showed adaptability to the immunological aggregation inhibition method. On the other hand, regarding the other 3 clones (MaI4-02, MaI4-04, MaI4-05), the largest change in absorbance at 0.0 mg / dL and no decrease in the change in absorbance due to the increase in human IgG4 concentration were observed. It did not apply to the immunological aggregation prevention method (FIG. 1). Among the 5 clones producing antibodies showing adaptability to the immunological aggregation inhibition method, the top 2 clones producing antibodies with high absorbance change at 0.0 mg / dL are selected and used for the subsequent analysis It was.
- NITE P-02112 Hybridoma MaI 4-08
- NITE P-02113 Hybridoma MaI 4-09
- NITE ABP-02112 MaI 4-08, June 8, 2018
- NITE ABP-02113 MaI 4-09
- the adjusted sample was added to a 96-well plate (Thermo Fisher) in 100 ⁇ L portions. It shaken at 700 rpm and 25.0 degreeC conditions for 1 hour with the shaker. After draining and washing three times with 300 ⁇ L of PBST, 100 ⁇ L of blocking buffer was added and stored overnight at 4 ° C. Similarly, after washing with PBST, 100 ⁇ L each of MaI 4-08 and MaI 4-09 producing antibodies adjusted with an antibody dilution buffer to 1 ⁇ g / mL were added and shaken under the same conditions as described above.
- Example 2 Preparation of measurement reagent and measurement using MaI 4-08, MaI 4-09 producing antibody (1)
- Preparation of reagent 1 Preparation of first reagent (R1) 2- [4- (2-hydroxyethyl)- The MaI 4-08 or MaI 4-09 producing antibody solution was added to 1-piperazinyl] ethanesulfonic acid (HEPES) buffer solution 1 to a concentration of 15 ⁇ g / mL, and used as a first reagent. Further, as a control reagent, a solution was similarly prepared in which the MaI 4-05 producing antibody and the commercial antibody HP6025 (Merck Millipore) were similarly added to the HEPES buffer solution 1.
- HEPES 1-piperazinyl] ethanesulfonic acid
- the precipitate was added with 24 mL of a coating buffer containing 3% bovine serum albumin, suspended, subjected to ultrasonication for complete dispersion, and then stirred at room temperature for 1 hour.
- 12 mL of HEPES buffer solution 2 is added to the precipitate obtained by centrifugation again, suspended, subjected to ultrasonication for complete dispersion, and adjusted to a latex concentration of 0.12% with HEPES buffer solution 2
- the obtained human IgG4-sensitized latex particle suspension was obtained and used as a second reagent common to each first reagent.
- composition of each reagent is as follows.
- Table 8 and FIGS. 4, 5, 6, and 7 show the change in absorbance when the concentration of human IgG4 was measured using the above-described reagent.
- reagents prepared with MaI 4-08 and MaI 4-09 producing antibodies can be applied to the latex (particle) agglutination prevention method, and it is revealed that they are useful for measuring human IgG4 concentration using an automatic analyzer It became.
- the dilution linearity was evaluated in the case where a MaI 4-08 producing antibody or a MaI 4-09 producing antibody was used as a mouse anti-human IgG 4 monoclonal antibody used for the first reagent.
- a sample prepared by serially diluting a human IgG4 concentration adjusted to 1000 mg / dL with saline containing 0.85% sodium chloride was used as a sample.
- Physiological saline was used as it is for the sample of human IgG4 concentration 0.0 mg / dL.
- the resistance to the prozone phenomenon was evaluated in the case where the MaI 4-08 producing antibody or the MaI 4-09 producing antibody was used as the mouse anti-human IgG 4 monoclonal antibody used as the first reagent. .
- a standard human serum adjusted to a human IgG4 concentration of 8000 mg / dL was serially diluted with saline containing 0.85% sodium chloride and used as a sample.
- the measurement of human IgG4 concentration is made by reacting 120 ⁇ L of the first reagent and 120 uL of the second reagent with 3 uL of sample using Hitachi 7180 automatic analyzer, 18-18 photometric points between the main wavelength 570 nm and the sub wavelength 800 nm (R2 addition).
- a calibration curve was prepared from the results in Table 6, and the human IgG4 concentration in the sample was calculated.
- Non-patent Document 6 reported as an anti-IgG4 monoclonal antibody, there are antibodies not suitable for the immunological particle aggregation inhibition method.
- analysis of antibody interaction and epitope analysis were performed.
- Example 3 Analysis of Interaction
- the MaI 4-08 and MaI 4-09-producing antibodies and the MaI 4-05-producing antibody and HP 6025 as a control were prepared to be 1 mg / mL.
- 1 mg / mL of IgG4 purified from human serum was prepared.
- IgG4 was immobilized on a chip by amine coupling method, and sensorgram acquisition using a Biacore model was performed.
- the resulting association rate constant (k a), dissociation rate constant (k d) and dissociation constant (KD) became as shown in Table 11.
- the binding rate constant k a of the MaI 4-05 producing antibody is less than one tenth of that of the MaI 4-08 and Ma I 4-09 producing antibodies, and the binding rate constant k a of the HP 6025 antibody is MaI 4-08 and It was 100-fold or less of the MaI 4-09-producing antibody.
- Example 4 Analysis of Epitopes
- the amino acid sequence of human IgG4 heavy chain constant region was obtained from Uniprot (P01861, SEQ ID NO: 4).
- a peptide was designed in which a GST tag was linked to the N terminus of the amino acid sequences H1-12 (FIG. 14) shown in SEQ ID NOs: 4 and 25-35, respectively, in which the obtained amino acid sequence was partially deleted.
- H1 (SEQ ID NO: 4) consists of the amino acid sequence of the full length human IgG4 heavy chain constant region.
- H2 consists of the amino acid sequence from 1 to 274 of human IgG4 heavy chain constant region (SEQ ID NO: 25).
- H3 consists of the amino acid sequence from 1 to 220 of human IgG4 heavy chain constant region (SEQ ID NO: 26).
- H4 consists of the amino acid sequence from 1 to 165 of human IgG4 heavy chain constant region (SEQ ID NO: 27).
- H5 consists of the amino acid sequence from 1 to 110 of human IgG4 heavy chain constant region (SEQ ID NO: 28).
- H6 consists of the amino acid sequence from 1 to 55 of human IgG4 heavy chain constant region (SEQ ID NO: 29).
- H7 consists of the amino acid sequence from 56 to 327 of human IgG4 heavy chain constant region (SEQ ID NO: 30).
- H8 consists of the amino acid sequence from 111 to 327 of human IgG4 heavy chain constant region (SEQ ID NO: 31).
- H9 consists of the amino acid sequence from the 166th position to the 327th position of human IgG4 heavy chain constant region (SEQ ID NO: 32).
- H10 consists of the amino acid sequence from 221 to 327 of the human IgG4 heavy chain constant region (SEQ ID NO: 33).
- H11 consists of the amino acid sequence from 275 to 327 of the human IgG4 heavy chain constant region (SEQ ID NO: 34).
- H12 consists of the 99th to 220th amino acid sequence of human IgG4 heavy chain constant region (SEQ ID NO: 35).
- H10 SEQ ID NO: 33
- the human IgG4 specific sequence is partially substituted with the corresponding amino acid of human IgG1 heavy chain constant region, respectively
- a peptide was designed in which a GST tag was linked to the N-terminus of the amino acid sequence H31-39 (FIG. 15) represented by 36-44.
- H31 is one obtained by replacing the glutamine residue at position 235 of the human IgG4 heavy chain constant region with an arginine residue based on the amino acid sequence of H10 (SEQ ID NO: 36).
- H32 is based on the amino acid sequence of H10, and the 289th arginine residue of human IgG4 heavy chain constant region is substituted with a lysine residue (SEQ ID NO: 37).
- H33 is the amino acid sequence of H10 in which the glutamic acid residue at position 299 of human IgG4 heavy chain constant region is substituted with a glutamine residue (SEQ ID NO: 38).
- H34 is one obtained by replacing the leucine residue at position 325 of the human IgG4 heavy chain constant region with a proline residue based on the amino acid sequence of H10 (SEQ ID NO: 39).
- H35 based on the amino acid sequence of H10, the glutamine residue at position 235 of human IgG4 heavy chain constant region to arginine residue, arginine residue at position 289 to lysine residue, and glutamate residue at position 299 In the group, a leucine residue at position 325 is substituted for a proline residue (SEQ ID NO: 40).
- H36 is the one in which all of the IgG4 specific sequences in the amino acid sequence from 221 to 327 of the human IgG4 heavy chain constant region are substituted with the corresponding amino acid sequence of IgG1 (SEQ ID NO: 41).
- H37 is the amino acid sequence of H10 in which the 289th arginine residue of human IgG4 heavy chain constant region is substituted with a lysine residue and the 325th leucine residue with a proline residue (SEQ ID NO: 42) .
- H38 based on the amino acid sequence of H10, the 289th arginine residue of human IgG4 heavy chain constant region to a lysine residue, the 299th glutamic acid residue to a glutamine, and the 325th leucine residue to a proline residue It is substituted (SEQ ID NO: 43).
- H39 is one obtained by replacing the glutamine residue at position 299 of human IgG4 heavy chain constant region with glutamic acid residue based on the amino acid sequence of H36 (SEQ ID NO: 44).
- a nucleotide sequence suitable for protein expression in E. coli is designed according to GenScript's protocol, and a vector pGEX-6P-1 (GE Healthcare Biosciences) in which each nucleotide sequence is inserted is prepared. did.
- the prepared expression plasmid was introduced into E. coli BL21 strain to obtain a transformant.
- the transformants were cultured in a conventional manner to induce protein expression by IPTG. After collecting the cells by centrifugation, the cells were dissolved in a normal solution and subjected to ultrasonic treatment. 6 ⁇ L of SDS sample buffer was added to 6 ⁇ L of the obtained lysate. Each prepared sample was subjected to heat treatment at 95 ° C. for 5 minutes.
- the resulting labeled antibodies were each diluted 1000-fold with PBST. After washing the PVDF membrane with PBST, the prepared antibody solutions were added to the PVDF membrane, and reacted at room temperature for 1 hour. After washing the PVDF membrane with PBST, the signal was detected with Image Quant Las 4000 using ECL Prime Western Blotting Detection Reagents (GE Healthcare).
- the epitope of the MaI 4-08 and MaI 4-09 producing antibodies is present in the amino acid sequence from the 221st position to the 327th position of the human IgG4 heavy chain constant region which is essential to contain the 299th glutamic acid residue of the human IgG4 heavy chain constant region. It became clear to do (FIG. 18 and FIG. 19).
- the MaI 4-05-producing antibody has an amino acid sequence from the 99th to the 110th, that is, a sequence containing the hinge region (H-1, H-2, H-3, H-4, H-5, H-7, H- The signal was confirmed only in 12). Therefore, it was revealed that the epitope of the MaI4-05-producing antibody is present in the 99th to 110th amino acid sequence of human IgG4 heavy chain constant region (FIG. 20).
- composition of each reagent is as follows.
- the competitor (IgG1) in the reaction system is used by using an antibody having high reactivity and selectivity to the target substance to be measured (human IgG4).
- the target substance can be specifically and precisely measured excluding the influence of (3) and the like.
- the anti-human IgG4 antibody of the present invention it is possible to specifically detect and measure human IgG4 in a sample, and is extremely effective in the field of diagnosis and clinical examination such as IgG4 related diseases.
Abstract
Description
本発明の抗ヒトIgG4抗体を用いれば、試料中のヒトIgG4を特異的に検出測定することが可能であり、IgG4関連疾患などの診断や臨床検査の分野において極めて有効である。
IgG4は約146000の分子量を持つヘテロテトラマー分子であり、かかる分子を検出するのには抗体を用いるのが適している。
従って第1の課題として、測定試薬の重要な構成要素となる抗体の特異性が挙げられる。IgG4はIgGサブクラスの1つであり、その他のサブクラスであるIgG1、IgG2またはIgG3との相同性が非常に高いことが知られている(非特許文献3、非特許文献4)。通常健常者血清においておよそIgG1:65%、IgG2:23%、IgG3:8%、IgG4:4%の割合で存在しており、健常者における基準値はIgG1:351~962;IgG2:239~838;IgG3:8.5~140;IgG4:4.5~117(mg/dL)であり、IgG4の量比の少なさからIgG4にのみ反応できる優れた特異性を有する抗体の作製が必須となる。
しかしながら血液や尿といった試料中の抗原濃度が測定範囲を大きく上回っている場合、抗原過剰による凝集抑制が誘導される可能性がある(地帯現象またはプロゾーン現象と呼ばれる)。その結果、測定値が疑似的に低値となり、誤った診断につながる恐れがある。IgG4の場合、上記のように健常者と高IgG4血症患者との間で1000倍近い濃度差があり、実際にIgG4測定試薬においてプロゾーン現象が発生した事例が報告されている(非特許文献2)。
従って第2の課題として、血中IgG4測定試薬とし高濃度のIgG4も測定することが求められている。
この問題を解決する方法としては、モノクローナル抗体を疑似的に多価にすることで、前記課題2をも解決される方法が挙げられる。例えば、特許文献2にはモノクローナル抗体をビオチン化しストレプトアビジンにて多量体を形成する方法が、特許文献3にはプロテインAや抗イムノグロブリン抗体等を用いて多量体を形成する方法が、さらに特許文献4には不溶性担体へモノクローナル抗体を固定化する方法が記載されている。しかしながら、いずれの方法もモノクローナル抗体の多価形成するための工程が煩雑であり、特に抗体の価数を常に一定に制御することが難しく、免疫比濁法を測定原理とした汎用自動分析装置対応の試薬としての性能を維持するのにコストと時間を要するという欠点がある。
ポリクローナル抗体を得るための方法である、一般的な動物実験的手法に基づいたヤギ、ヒツジ、またはウサギ等へのヒトIgG4の免疫による該抗血清の取得は、産生される抗体のそのほとんどが各IgGサブクラスに共通した部分に対するものとなり、IgG4以外の他のサブクラスにも反応しうる抗体の吸収工程が必要になり、IgG4に対する特異的な抗体を入手するまでに、非常に煩雑な精製工程を必要とする。該抗血清に含まれるIgG4に対する特異的な抗体の相対的または絶対的な存在量と、前記の精製工程により、所望のIgG4に対する特異的な抗体の収量は非常に少なくなるという問題が懸念される。
またIgGサブクラスのいずれかに特異性の高いポリクローナル抗体を取得する方法が知られている(特許文献5)。この方法によると、上記のような精製工程を必要とせず、免疫寛容により抗原特異的なポリクローナル抗体の取得が可能であるが、免疫寛容の惹起の程度には免疫される動物個体差が存在し、また免疫寛容を引き起こすまでに時間を要することから、定常的に所望のIgG4に対する特異的な抗体を取得するには多数の被免疫動物を必要とし、コスト並びに動物愛護の観点から適当な方法であるとは言い難い。
[1]ヒトIgG4に対して特異的に結合するモノクローナル抗体であって、前記抗体のエピトープが、配列番号4に示すヒトIgG4の重鎖定常領域の221番目から327番目のアミノ酸配列に存在し、299番目のグルタミン酸を含む、モノクローナル抗体;
[2]前記ヒトIgG4に、解離定数5.0×10-10以下で結合する[1]に記載のモノクロナール抗体;
[3]重鎖可変領域及び軽鎖可変領域を有するヒトIgG4に対する抗体であって、重鎖可変領域の相補性決定領域(Complementarity Determining Region;以下、CDRと記す)1、2および3がそれぞれ配列番号9、10および11で示されるアミノ酸配列であり、軽鎖可変領域のCDR1、2および3が、それぞれ配列番号12、13および14で示されるアミノ酸配列である、モノクローナル抗体、好ましくは[1]又は[2]に記載のモノクローナル抗体;
[4]重鎖可変領域が配列番号5のアミノ酸配列を有し、軽鎖可変領域が配列番号6のアミノ酸配列を有する[3]に記載のモノクローナル抗体;
[5]ハイブリドーマMaI4-08が産生する[4]に記載のモノクローナル抗体。
[7]重鎖可変領域が配列番号7のアミノ酸配列を有し、軽鎖可変領域が配列番号8のアミノ酸配列を有する[6]に記載のモノクローナル抗体;
[8]ハイブリドーマMaI4-09が産生する[7]に記載のモノクローナル抗体。
[10]F(ab’)2である、[9]に記載の抗体断片。
[12]免疫比濁法又は免疫比ろう法である[11]に記載の方法;
[13]免疫学的粒子凝集法である[12]に記載の方法;
[14]免疫学的粒子凝集阻止法である[12]に記載の方法;
[15]免疫組織化学(IHC)染色である[11]に記載の方法;
[16]フローサイトメトリーによる[11]に記載の方法。
(1)[1]~[8]のいずれかに記載のモノクローナル抗体或いは[9]又は[10]に記載の抗体断片;
(2)単離ヒトIgG4又はそのペプチド断片;及び
(3)不溶性担体
を含むキット;
[20](2)単離ヒトIgG4又はそのペプチド断片が、(3)不溶性担体に吸着している、[19]に記載のキット。
[21](3)不溶性担体がラテックス粒子である、[19]又は[20]に記載のキット。
[23]IgG4関連疾患診断に用いるための、[18]~[21]のいずれかに記載のキット。
[24]配列番号4に示すヒトIgG4の重鎖定常領域の221番目から327番目のアミノ酸配列の全部又は一部からなり、299番目のグルタミン酸を含む、単離されたペプチド。
上記融合法としては、既にそれ自体公知であるケーラーとミルスタインの定法(Nature.256,495.1975)によってポリエチレングリコール(PEG)を用いることで融合できる。センダイウィルス、電気融合法によっても融合を行うことができる。
ここで「ヒトIgG4に結合し、他のヒト免疫グロブリンに結合しない」とは、たとえば、簡便には通常のELISAにおいて、下記以外の測定条件を同一とした際、固相抗原としてヒトIgG4を含む場合にいずれのヒト免疫グロブリンを含まないものを対照(ブランク)としたときの吸光度の比が40以上、好ましくは50以上、より好ましくは100以上を示すものであり、かつ、固相抗原としてヒトIgG4以外を含む場合にいずれのヒト免疫グロブリンを含まないものを対照(ブランク)としたときの吸光度の比が5以下、好ましくは2以下、を示すものであることをいう。
また、他のヒト免疫グロブリンよりもヒトIgG4に対して高い結合親和性を示すものであることをいい、結合速度定数と解離定数を用いて評価することもできる。具体的には、本発明の好ましい実施形態による抗体では、ヒトIgG4の重鎖定常領域の特定のエピトープに、4.0×105以上、通常4.0×105~6.0×105の結合速度定数、並びに5.0×10-10以下、通常5.0×10-10~3.0×10-10の解離定数で結合する。
上記の上清、腹水よりモノクローナル抗体を回収する方法としては、通常の方法を用いることができる。たとえば硫酸アンモニウム、硫酸ナトリウムなどによる塩析法やクロマトグラフィー、イオン交換クロマトグラフィー、プロテインA、プロテインGなどによるアフィニティクロマトグラフィーなどが挙げられる。
「競合ELISA法」とは「標識した抗原」と「試料中の存在する抗原」が、競合条件下でどの割合で抗体に結合するかを測定するELISA法を意味する。
「ラテックス(粒子)凝集阻止法」とは、抗原をラテックス粒子に固相化し、「ラテックス粒子に結合した抗原」と「試料中の存在する抗原」が、競合条件下でどの割合で抗体に結合するかを、ラテックス粒子の凝集を観察し、間接的に「試料中の存在する抗原」を検出する方法である。
いずれの場合も、その凝集の観察に、散乱による照射光の減衰(吸光度)を計測(免疫比濁法)してもよいし、散乱した光を測定(免疫比ろう法)してもよい。
本発明の実施形態に係る抗体は、IgG4を認識し、IgG1、IgG2及びIgG3を認識しないことが求められる。軽鎖はIgG1~IgG4で共通するため、エピトープは重鎖、とりわけ、重鎖定常領域にあることが望ましい。IgG1、IgG2、IgG3及びIgG4の重鎖定常領域は各々配列番号1、2、3及び4で示され、配列番号1~3と配列番号4の相同性の低い部分をエピトープとすることが望ましく、後述する実施例で実証する通り、特に、ヒトIgG4のCH3領域、より具体的には、配列番号4に示すヒトIgG4の重鎖定常領域の221番目から327番目のアミノ酸配列の範囲内に存在し、299番目のグルタミン酸を含むエピトープが他のIgGサブクラスへの交差反応が無くIgG4への特異的な結合を達成する上で望ましい。換言すると、本発明の好ましい実施形態に係る抗体のエピトープは、配列番号4に示すヒトIgG4の重鎖定常領域の221番目から327番目のアミノ酸配列の全部又は一部からなり、299番目のグルタミン酸を含む。従って、本発明の好ましい他の実施形態によれば、エピトープを含むペプチド、より具体的には、配列番号4に示すヒトIgG4の重鎖定常領域の221番目から327番目のアミノ酸配列の全部又は一部(通常、5個から50個、好ましくは10個から30個)からなり、299番目のグルタミン酸を含むペプチドも提供される。
「1価抗体」とは、天然の抗体をパパインで処理して出来るFab断片や、遺伝子工学的に、軽鎖可変領域と重鎖可変領域を結合させた、scFV(single-chain variable fragment)などがこれにあたる。
さらに、用いる抗体が一価抗体分子だと、抗体を介した(抗原の吸着した)ラテックス粒子間の凝集反応が起こらないため、同じく不適である。抗原(認識)結合部位が、1分子あたり2つ以上必要であり、天然のイムノグロブリンタイプ(IgG(2価)、IgA(4価)、IgD(2価)、IgE(2価)及びIgM(10価))や、ペプシン処理でできるF(ab’)2(2価)や上記「1価抗体」のマルチマー抗体である必要がある。
本願に用いるラテックス粒子の粒子径は、50nm~500nm、好ましくは75nm~306nm、より好ましくは100nm~111nmであってよい。
このキットにおいては、固相支持体と単離ヒトIgG4又はそのペプチド断片溶液とを別々に作製しておき、IgG4を測定する際、抗体を固相支持体に吸着させてもよく、あらかじめ、抗体を固相支持体に吸着させた状態で提供してもよい。このキットにおいては、検体中の単離ヒトIgG4又はそのペプチド断片を抗体に結合させた後、固相支持体に吸着しなかった成分を除去するために、洗浄液を含むことが好ましい。洗浄液としては、例えば、界面活性剤を含むトリス緩衝液を使用することができる。
さらに、本発明のキットには、必要に応じ、検体希釈液を加えて含むこともできる。検体希釈液としては、例えば、トリス等の緩衝液を使用できる。その緩衝液には、必要に応じて、EDTA・2Na等のキレート剤、食塩等の無機塩を加えてもよい。
サンドイッチアッセイELISA法で本発明の測定方法を実施するときは、例えば、IgG4を免疫測定するためのキットであって、i)固相支持体、ii)本発明抗体、iii)標識された、別なIgG4に対する抗体、及びiv)標識を検出するための成分を含むキットを用いることによっても、IgG4を測定することができる。
標識を検出するための成分とは、抗体が標識されたものを測定するための成分で、標識がビオチンの場合、ペルオキシダーゼ標識ストレプトアビジン、テトラメチルベンジジンのペルオキシダーゼ酵素基質、及び過酸化水素を含む試薬であり、標識がアルカリホスファターゼの場合、p-ニトロフェニルリン酸を含む試薬である。このキットには、必要に応じ、洗浄液を含んでいてもよい。
本発明において、このキットを使用するときは、検体中のIgG4を抗体に結合させた後、固相支持体に吸着しなかった成分を除去するために、洗浄液を含むことが好ましい。洗浄液としては、例えば、界面活性剤を含むトリス緩衝液を使用することができる。さらに、本発明のキットには、必要に応じ、検体希釈液を加えて含むこともできる。検体希釈液としては、例えば、トリス等の緩衝液を使用できる。その緩衝液には、必要に応じて、EDTA・2Na等のキレート剤、食塩等の無機塩を加えてもよい。
(1)モノクローナル抗体作製用抗原の選択と準備
ヒトIgG4を認識するモノクローナル抗体を産生するハイブリドーマを公知の方法(例えばKohler and Milstein,Nature (1975)256,p.495-497)に準じて作製した。抗原として用いるIgG4はヒト血清より調製した。具体的にはヒト血清を硫安沈殿により分画・透析後、CaptureSelectIgG4 Affinity Matrix(サーモフィッシャーサイエンティフィック株式会社)の製品添付文書に従い、IgG4抗原を精製した。
ヒト血清より上記の方法で精製した0.5mg/mLヒトIgG4と同体積量のフロイント完全アジュバント(シグマアルドリッチ)を乳化するまでよく混和し、乳化懸濁液を作製した。4週齢Balb/cマウスをジエチルエーテル麻酔し、上記の乳化懸濁液を1匹あたり50μgのヒトIgG4にて腹腔内投与を実施した。約2週間間隔で6回、上記と同様にフロイント不完全アジュバント(シグマアルドリッチ)にて作製した乳化懸濁液をそれぞれマウスに注射した。最終免疫として上記6回目の注射から約3週間後、同様に乳化懸濁液を注射した。
最終免疫より3日後にジエチルエーテル麻酔下にて外科的摘出された脾臓を無菌的に分散し脾臓細胞を調製した。ポリエチレングリコールを用いて、脾臓細胞数と骨髄腫細胞P3-X63-Ag8-U1(P3U1)数の融合比率を5:1にて融合を実施した。融合細胞を10% FBS(HyClone社)ASF(コスモ・バイオ株式会社)HAT(コスモ・バイオ株式会社)培地に分散し、48ウェルプレート(住友ベークライト)に分注して37℃、5% CO2条件にて培養した。以下のスクリーニングに用いたハイブリドーマコロニー数は4481であった。
得られたハイブリドーマのスクリーニングを1)IgG4への結合能確認、2)特異性確認、3)免疫学的凝集阻止法への適応性確認、という3段階にて実施した。
上記抗原として用いた精製したヒトIgG4を0.1μg/ウェルになるように96ウェルプレート(Nunc)に分注した。シェイカーにて700rpm、25.0℃の条件で1時間振盪した。廃液し300μLのPBST(10mMリン酸二水素ナトリウム;150mM塩化ナトリウム;0.05%ポリオキシエチレン(20) ソルビタンモノラウレート;pH7.4)にて3回洗浄後、100μLのブロッキング緩衝液(10mMリン酸二水素ナトリウム;150mM塩化ナトリウム;1%ブロックエース粉末(大日本住友製薬);pH7.4)を添加し、1晩以上4℃にて保存した。同様にPBSTにて洗浄後、ハイブリドーマコロニーの培養上清を100μLずつ添加し、上記と同条件にて振盪させた。同様にPBSTにて洗浄後、抗体希釈緩衝液(10mMリン酸二水素ナトリウム;150mM塩化ナトリウム;0.05%ポリオキシエチレン(20) ソルビタンモノラウレート;0.05%ウシ血清アルブミン;pH7.4)にて10000倍希釈した抗マウスIgG(H+L) HRP標識抗体(Life Technologies)を100μLずつ添加し、上記と同条件にて振盪させた。同様にPBSTにて洗浄後、SureBlue(KPL)を100μL添加した。室温にて15分間静置した後、0.5mol/L硫酸(和光純薬工業)を100μL添加した。波長450nmをマイクロプレートリーダー(BIO-RAD)にて測定した。合計4481ウェル中、IgG4への結合能が確認された192ウェルを選定した。選定されたハイブリドーマを単クローン化し、培養を続けた。
1)にて選定されたクローンから産生される抗体の特異性を評価した。Myeloma ヒトIgG1(EMD Millipore)、Myeloma ヒトIgG2(EMD Millipore)、Myeloma ヒトIgG3(シグマアルドリッチ)、Myeloma ヒトIgG4(EMD Millipore)を1μg/mLになるようにPBS(-)にて調整した。調整した試料を96ウェルプレート(Thermo Fisher)に100μLずつ添加した。シェイカーによる振盪以降の操作を上記1)と同様に行った。192クローン中、Myeloma ヒトIgG1、2、3と比べて、Myeloma ヒトIgG4に強い反応を示す8クローンを選定した。
2)にて選定されたクローンから産生される抗体において、免疫学的凝集阻止法への適用性を評価した。
1)第一試薬(R1)の調製
2-[4-(2-ヒドロキシエチル)-1-ピペラジニル]エタンスルホン酸(HEPES)緩衝溶液1に、上記の通り選定された8クローン由来の産生抗体をそれぞれ含む溶液を15μg/mLの濃度になるように加え、第一試薬とした。
2)第二試薬(R2)の調製
2%濃度の粒径107nmのポリスチレン製のラテックス粒子溶液18mLに、ヒト血清から通常の方法で粗精製したヒトIgG4 0.35mg/mL溶液18mLを加え、室温にて1時間撹拌してラテックス粒子にヒトIgG4を物理吸着させた。その後20,000rpmで90分間遠心分離を行い、上清を廃棄して沈殿物を回収した。この沈殿物に3%濃度のウシ血清アルブミンを含むコート緩衝液を24mL加えて懸濁し、超音波処理を行って完全に分散させた後、室温で1時間撹拌した。次いで、再び遠心分離を行って得られた沈殿物にHEPES緩衝溶液2を12mL加え懸濁し超音波処理を行って完全に分散させた後、HEPES緩衝溶液2にてラテックス濃度0.12%に調整したヒトIgG4感作ラテックス粒子懸濁液を得て、各第一試薬に対し共通する第二試薬として用いた。
なお、各試薬の組成は以下の通りである。
HEPES緩衝溶液1
25mM 2-[4-(2-ヒドロキシエチル)-1-ピペラジニル]エタンスルホン酸(埼京化成工業);100mM 3-(シクロヘキシルアミノ)-1-プロパン スルホン酸(埼京化成工業);150mM 塩化ナトリウム(和光純薬工業);1mM EDTA・2Na(和光純薬工業);3.3%デキストラン70(東京化成工業);0.1% ブロックエース(大日本住友製薬);0.09% アジ化ナトリウム(和光純薬工業);pH7.40。
コート緩衝液
25mM 2-[4-(2-ヒドロキシエチル)-1-ピペラジニル]エタンスルホン酸(埼京化成工業);150mM 塩化ナトリウム(和光純薬工業); 1mM EDTA・2Na(和光純薬工業);3% ウシ血清アルブミン(メルクミリポア);0.09% アジ化ナトリウム(和光純薬工業);pH7.50。
HEPES緩衝溶液2
500mM 2-[4-(2-ヒドロキシエチル)-1-ピペラジニル]エタンスルホン酸(埼京化成工業); 25mM 2-モルホリノエタンスルホン酸(和光純薬工業);1mM EDTA・2Na(和光純薬工業);150mM L-アルギニン塩酸塩(和光純薬工業);0.075% プロクリン950(シグマアルドリッチ);pH6.00。
標準試料として、ヒトIgG4濃度をそれぞれ0.0、1.6、6.3、12.5、25.0、50.0mg/dLに調整した標準ヒト血清を用いた。なお0.0mg/dLには生理食塩水を用いた。日立7180形自動分析装置を用い、試料3μLに対し第一試薬120μL、第二試薬120μLを反応させ、主波長570nm、副波長800nmにて18~28測光ポイント間(R2添加後約1分後から4分後に相当)において、2ポイントエンド法による吸光度変化量を測定した(n=2)。8クローン中5クローンより産生される抗体は、0.0mg/dL時に最も大きい吸光度変化量を示し、ヒトIgG4濃度が上昇するに従って吸光度変化量が減少した。よって、5クローン(MaI4-09、MaI4-08、MaI4-01、MaI4-06、MaI4-03)は免疫学的凝集阻止法への適応性を示した。一方で他3クローン(MaI4-02、MaI4-04、MaI4-05)に関しては0.0mg/dL時における最も大きい吸光度変化量、及びヒトIgG4濃度上昇に伴う吸光度変化量の減少が認められず、免疫学的凝集阻止法へ適応しなかった(図1)。免疫学的凝集阻止法への適応性を示した抗体を産生する5クローンのうち、0.0mg/dL時の吸光度変化量が高い抗体を産生する上位2クローンを選定し、以降の解析に用いた。
上記で選定されたハイブリドーマMaI4-08及びMaI4-09は、2015年9月1日付で独立行政法人製品評価技術基盤機構内特許微生物寄託センターに対して2015年9月1日に受領番号NITE AP-02112(MaI4-08)及びNITE AP-02113(MaI4-09)で受領され、2015年9月14日生存確認後、受託番号NITE P-02112(MaI4-08)及びNITE P-02113(MaI4-09)が付与された(受託証2015年9月30日発行)。
以下に、寄託を特定する内容を記載する。
[1]寄託機関の名称・あて名
名称:独立行政法人 製品評価技術基盤機構産業技術総合研究所 特許微生物寄託センター
あて名:日本国千葉県木更津市かずさ鎌足2-5-8(郵便番号292-0818)
[2]寄託日:2015年9月1日
[3]受託番号 NITE P-02112(ハイブリドーマMaI4-08)
NITE P-02113(ハイブリドーマMaI4-09)
その後、同じハイブリドーマを国際寄託するために、独立行政法人製品評価技術基盤機構内特許微生物寄託センターに対して微生物移管請求を行い、2018年6月8日に受領番号NITE ABP-02112(MaI4-08)及びNITE ABP-02113(MaI4-09)で受領された。
上記ハイブリドーマより抗体を産生させる方法として、公知の方法(例えば モノクローナル抗体:生化学実験法 東京化学同人)に準じて行った。具体的にはモノクローナル抗体を産生するためにはin Virtoでの培養及びIn Vivoでの培養がある。In Virtoでの培養としては培養液中におけるハイブリドーマの培養等があり、一方でIn Vivoでの培養方法としてマウス腹水を作製する方法等があるが、In Virtoでの培養を選択し、モノクローナル抗体を含む培養液上清を取得した。
上記よりモノクローナル抗体を精製する方法として、公知の方法(例えばモノクローナル抗体:生化学実験法 東京化学同人)に準じて行った。モノクローナル抗体の精製としてアフィニティークロマトグラフィー精製が一般的であり、プロテインGやプロテインA等を担持したセファロースカラムを用いるが、プロテインGを担持したプロテインGセファロースカラム(GEヘルスケア)を選択し、精製を行った。得られたモノクローナル抗体における280nmの吸光度を測定し、抗体濃度を確認した。濃度を調整後、0.45μm以下フィルターで濾過滅菌した。
MaI4-08、MaI4-09産生抗体をそれぞれIso-Gold Rapid Mouse-Monoclonal Isotyping Kit(with κ and λ)(BioAssay Works)の製品添付文書に従い、アイソタイプの同定を実施した。アイソタイプは表1の通りであった。
Myeloma ヒトIgA1(Abcam)、Myeloma ヒトIgA2(Abcam)、Myeloma ヒトIgD(Abcam)、Myeloma ヒトIgE(Abcam)、Myeloma ヒトIgM(Abcam)、Myeloma ヒトIgG1(EMD Millipore)、Myeloma ヒトIgG2(EMD Millipore)、Myeloma ヒトIgG3(シグマアルドリッチ)、Myeloma ヒトIgG4(EMD Millipore)を1μg/mLになるようにPBS(-)にて調整した。調整した試料を96ウェルプレート(Thermo Fisher)に100μLずつ添加した。シェイカーにて700rpm、25.0℃の条件で1時間振盪した。廃液し300μLのPBSTにて3回洗浄後、100μLのブロッキング緩衝液を添加し、1晩以上4℃にて保存した。同様にPBSTにて洗浄後、1μg/mLになるように抗体希釈緩衝液にて調整したMaI4-08、MaI4-09産生抗体をそれぞれ100μLずつ添加し、上記と同条件にて振盪させた。同様にPBSTにて洗浄後、抗体希釈緩衝液にて4000倍希釈した抗マウスIgG HRP標識抗体(Abcam)を100μLずつ添加し、上記と同条件にて振盪させた。同様にPBSTにて洗浄後、SureBlue(KPL)を100μL添加した。MaI4-08、MaI4-09それぞれ5、3分後に0.5mol/L硫酸(和光純薬)を100μL添加した。波長450nmをマイクロプレートリーダー(BIO-RAD)にて測定した。その結果、他調整試料と比べMyeloma ヒトIgG4に強いシグナルが確認された(表2及び3、図2及び3)。
MaI4-08、MaI4-09はそれぞれFusion Antibodies社のプロトコルによって、mRNAの抽出、クローニング及びシークエンシグを行った。解析した結果、MaI4-08の可変領域は配列番号5(重鎖可変領域)及び配列番号6(軽鎖可変領域)であり、MaI4-09の可変領域は配列番号7(重鎖可変領域)及び配列番号8(軽鎖可変領域)であり、CDRのアミノ酸配列は表4の通りになった。
(1)試薬の調製
1)第一試薬(R1)の調製
2-[4-(2-ヒドロキシエチル)-1-ピペラジニル]エタンスルホン酸(HEPES)緩衝溶液1に、MaI4-08またはMaI4-09産生抗体溶液を15μg/mLの濃度になるように加え、第一試薬とした。また対照試薬として、MaI4-05産生抗体及び市販抗体HP6025(メルクミリポア)を同様にHEPES緩衝溶液1に添加した溶液を調製した。
2)第二試薬(R2)の調製
2%濃度の粒径107nmのポリスチレン製のラテックス粒子溶液18mLに、ヒト血清から通常の方法で粗精製したヒトIgG4 0.35mg/mL溶液18mLを加え、室温にて1時間撹拌してラテックス粒子にヒトIgG4を物理吸着させた。その後20,000rpmで90分間遠心分離を行い、上清を廃棄して沈殿物を回収した。この沈殿物に3%濃度のウシ血清アルブミンを含むコート緩衝液を24mL加えて懸濁し、超音波処理を行って完全に分散させた後、室温で1時間撹拌した。次いで、再び遠心分離を行って得られた沈殿物にHEPES緩衝溶液2を12mL加え懸濁し超音波処理を行って完全に分散させた後、HEPES緩衝溶液2にてラテックス濃度0.12%に調整したヒトIgG4感作ラテックス粒子懸濁液を得て、各第一試薬に対し共通する第二試薬として用いた。
標準試料として、ヒトIgG4濃度をそれぞれ1.6、6.3、12.5、25、50mg/dLに調整した標準ヒト血清を用いた。本測定では検体試料の測定を20倍希釈にして測定することを前提とするため、最終的な標準試料濃度は20倍となり、31.3、125、250、500、1000mg/dLとなる。以降に記載する試料濃度は全て20倍した最終的な試料濃度とする。ヒトIgG4濃度0.0mg/dLの試料には塩化ナトリウムを0.85%含む生理食塩水を用いた。ヒトIgG4濃度の測定は日立7180形自動分析装置を用い、試料3μLに対し第一試薬120μL、第二試薬120μLを反応させ、主波長570nm、副波長800nmにて18~28測光ポイント間(R2添加後約1分後から4分後に相当)において、2ポイントエンド法による吸光度変化量を測定した(n=2)。
第一試薬に用いるマウス抗ヒトIgG4モノクローナル抗体としてMaI4-08産生抗体またはMaI4-09産生抗体を用いた場合について、希釈直線性の評価を行った。試料としてヒトIgG4濃度を1000mg/dLに調整した標準ヒト血清を、塩化ナトリウムを0.85%含む生理食塩水により段階希釈した試料を検体として用いた。ヒトIgG4濃度0.0mg/dLの試料には生理食塩水をそのまま用いた。ヒトIgG4濃度の測定は日立7180形自動分析装置を用い、試料3μLに対し第一試薬120μL、第二試薬120μLを反応させ、主波長570nm、副波長800nmにて18~28測光ポイント間(R2添加後約1分後から4分後に相当)において、2ポイントエンド法による吸光度変化量を測定した(n=2)。表6の結果から検量線を作成し、試料中のヒトIgG4濃度を算出した。
第一試薬に用いるマウス抗ヒトIgG4モノクローナル抗体としてMaI4-08産生抗体またはMaI4-09産生抗体を用いた場合について、プロゾーン現象への耐性評価を行った。試料としてヒトIgG4濃度を8000mg/dLに調整した標準ヒト血清を、塩化ナトリウムを0.85%含む生理食塩水により段階希釈し、検体として用いた。ヒトIgG4濃度の測定は日立7180形自動分析装置を用い、試料3uLに対し第一試薬120μL、第二試薬120uLを反応させ、主波長570nm、副波長800nmにて18~28測光ポイント間(R2添加後約1分後から4分後に相当)において、2ポイントエンド法による吸光度変化量を測定した(n=2)。表6の結果から検量線を作成し、試料中のヒトIgG4濃度を算出した。
第一試薬に用いるマウス抗ヒトIgG4モノクローナル抗体としてMaI4-08産生抗体またはMaI4-09産生抗体を用いた場合について、ヒトIgG4に対する特異性の評価を行った。試料として3000mg/dLに調整したMyeloma ヒトIgG4溶液(EMD Millipore)を用いた。また対照試料として、同様に調整したMyeloma ヒトIgG1溶液(EMD Millipore)、Myeloma ヒトIgG2溶液(EMD Millipore)、Myeloma ヒトIgG3溶液(シグマアルドリッチ)を用いた。ブランク試料には生理食塩水を用いた。ヒトIgG4濃度の測定は日立7180形自動分析装置を用い、試料3μLに対し第一試薬120μL、第二試薬120μLを反応させ、主波長570nm、副波長800nmにて18~28測光ポイント間(R2添加後約1分後から4分後に相当)において、2ポイントエンド法による吸光度変化量を測定した(n=2)。表6の結果から検量線を作成し、試料中のヒトIgG4濃度を算出した。
結果を図8~13、表9及び10にそれぞれ示した。
本発明のハイブリドーマが産生したマウス抗ヒトIgG4モノクローナル抗体を第一試薬に用いた場合、0~1000mg/dLの検量範囲で十分な希釈直線性を有することが判明した(図8及び9)。また、プロゾーン現象については8000mg/dLまで測定値の低下は見られなかった(図10及び11)。さらに、IgG1、IgG2、IgG3への反応性はほとんど認められなかった(図12及び13、表9及び10)。
MaI4-08及びMaI4-09の産生抗体と、対照としてMaI4-05産生抗体及びHP6025とを1mg/mLになるように調製した。またヒト血清より精製した1 mg/mLのIgG4を調製した。株式会社住化分析センターのプロトコルに沿って、IgG4をアミンカップリング法にてチップに固定化し、Biacore機種を用いたセンサーグラム取得を行った。得られた結合速度定数(ka)、解離速度定数(kd)及び解離定数(KD)は表11の通りとなった。
3種類の抗体のうち、MaI4-05産生抗体の結合速度定数kaはMaI4-08およびMaI4-09産生抗体の10分の1以下であり、HP6025抗体の結合速度定数kaはMaI4-08およびMaI4-09産生抗体の100分の1以下であった。
UniprotよりヒトIgG4重鎖定常領域のアミノ酸配列を取得した(P01861、配列番号4)。得られたアミノ酸配列を部分的に欠損させたそれぞれ配列番号4及び25~35で示されるアミノ酸配列H1~12(図14)のN末端にGSTタグを連結したペプチドを設計した。H1(配列番号4)はヒトIgG4重鎖定常領域全長のアミノ酸配列からなるものである。H2はヒトIgG4重鎖定常領域の1から274番目までのアミノ酸配列からなるものである(配列番号25)。H3はヒトIgG4重鎖定常領域の1から220番目までのアミノ酸配列からなるものである(配列番号26)。H4はヒトIgG4重鎖定常領域の1から165番目までのアミノ酸配列からなるものである(配列番号27)。H5はヒトIgG4重鎖定常領域の1から110番目までのアミノ酸配列からなるものである(配列番号28)。H6はヒトIgG4重鎖定常領域の1から55番目までのアミノ酸配列からなるものである(配列番号29)。H7はヒトIgG4重鎖定常領域の56から327番目までのアミノ酸配列からなるものである(配列番号30)。H8はヒトIgG4重鎖定常領域の111から327番目までのアミノ酸配列からなるものである(配列番号31)。H9はヒトIgG4重鎖定常領域の166から327番目までのアミノ酸配列からなるたものである(配列番号32)。H10はヒトIgG4重鎖定常領域の221から327番目までのアミノ酸配列からなるものである(配列番号33)。H11はヒトIgG4重鎖定常領域の275から327番目までのアミノ酸配列からなるものである(配列番号34)。H12はヒトIgG4重鎖定常領域の99から220番目までのアミノ酸配列からなるものである(配列番号35)。
さらに、MaI4-08、MaI4-09産生抗体に関しては299番目のグルタミン酸が保存された条件(H-10、H-31、H-32、H-34、H-37)においてシグナルが確認されたが、299番目のグルタミン酸をグルタミンに置換した条件(H-33、H-35、H-36、H-38)ではシグナルは確認されなかった。したがってMaI4-08,MaI4-09産生抗体のエピトープはヒトIgG4重鎖定常領域の299番目のグルタミン酸残基を含むことを必須とするヒトIgG4重鎖定常領域の221番目から327番目のアミノ酸配列に存在することが明らかとなった(図18及び図19)。
一方、MaI4-05産生抗体は、99から110番目のアミノ酸配列、すなわちヒンジ領域を含む配列(H-1、H-2、H-3、H-4、H-5、H-7、H-12)にのみシグナルが確認された。したがって、MaI4-05産生抗体のエピトープはヒトIgG4重鎖定常領域の99から110番目のアミノ酸配列に存在することが明らかとなった(図20)。
Claims (20)
- ヒトIgG4に対して特異的に結合するモノクローナル抗体であって、前記抗体のエピトープが、配列番号4に示すヒトIgG4の重鎖定常領域の221番目から327番目のアミノ酸配列に存在し、299番目のグルタミン酸を含む、モノクローナル抗体。
- 前記ヒトIgG4に、解離定数5.0×10-10以下で結合する請求項1に記載のモノクロナール抗体;
- 重鎖可変領域及び軽鎖可変領域を有するヒトIgG4に対する抗体であって、重鎖可変領域の相補性決定領域(Complementarity Determining Region;以下、CDRと記す)1、2および3がそれぞれ配列番号9、10、および11で示されるアミノ酸配列であり、軽鎖可変領域のCDR1、2および3が、それぞれ配列番号12、13および14で示されるアミノ酸配列である、モノクローナル抗体。
- 重鎖可変領域が配列番号5のアミノ酸配列を有し、軽鎖可変領域が配列番号6のアミノ酸配列を有する請求項3に記載のモノクローナル抗体。
- ハイブリドーマMaI4-08が産生する請求項4に記載のモノクローナル抗体。
- 重鎖可変領域及び軽鎖可変領域を有するヒトIgG4に対する抗体であって、重鎖可変領域の相補性決定領域CDR1、2および3がそれぞれ配列番号15、16、および17で示されるアミノ酸配列であり、抗体の軽鎖可変領域のCDR1、2および3が、それぞれ配列番号18、19および20で示されるアミノ酸配列であるモノクローナル抗体。
- 重鎖可変領域が配列番号7のアミノ酸配列を有し、軽鎖可変領域が配列番号8のアミノ酸配列を有する請求項6に記載のモノクローナル抗体。
- ハイブリドーマMaI4-09が産生する請求項7に記載のモノクローナル抗体。
- 請求項1~8のいずれか一項に記載のモノクローナル抗体の抗原結合部位を2つもつ、二価抗体分子又は二価抗体断片。
- F(ab’)2である、請求項9に記載の抗体断片。
- 請求項1~8のいずれか一項に記載のモノクローナル抗体或いは請求項9又は10に記載の二価抗体分子又は二価抗体断片を用いて、試料中のヒトIgG4を測定検出する方法。
- 免疫学的粒子凝集阻止法である請求項11に記載の方法。
- 請求項12に記載の方法であって;
(1)試料中のヒトIgG4と請求項1~8のいずれか一項に記載のモノクローナル抗体或いは請求項9又は10に記載の二価抗体分子又は二価抗体断片を結合させ;次いで
(2)ヒトIgG4又はそのペプチド断片が固定された不溶性担体を加えて、試料中のヒトIgG4と結合しなかった上記モノクローナル抗体或いは二価抗体分子又は二価抗体断片との間で凝集反応を起こし、そして
(3)凝集された不溶性担体を検出することにより、試料中のヒトIgG4を検出測定する方法。 - 請求項1~8のいずれか一項に記載のモノクローナル抗体或いは請求項9又は10に記載の二価抗体分子又は二価抗体断片を含む、ヒトIgG4の測定キット。
- 請求項13に記載の方法のためのキットであって;
(1)請求項1~8のいずれか一項に記載のモノクローナル抗体或いは請求項9又は10に記載の二価抗体分子又は二価抗体断片;
(2)単離ヒトIgG4又はそのペプチド断片;及び
(3)不溶性担体
を含むキット。 - (2)単離ヒトIgG4又はそのペプチド断片が、(3)不溶性担体に吸着している、請求項15に記載のキット。
- (3)不溶性担体がラテックス粒子である、請求項15又は16に記載のキット。
- IgG4関連疾患の診断を補助するための、請求項11~13のいずれか一項に記載の方法。
- IgG4関連疾患診断に用いるための、請求項14~17のいずれか一項に記載のキット。
- 配列番号4に示すヒトIgG4の重鎖定常領域の221番目から327番目のアミノ酸配列の全部又は一部からなり、299番目のグルタミン酸を含む、単離されたペプチド。
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CN115327137B (zh) * | 2022-10-11 | 2023-01-17 | 苏州惠中生物科技有限公司 | 一种人免疫球蛋白g4亚型化学发光免疫检测试剂盒 |
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KR102577474B1 (ko) | 2017-11-21 | 2023-09-12 | 인튜어티브 서지컬 오퍼레이션즈 인코포레이티드 | 직관적인 움직임을 위한 마스터/도구 정합과 제어를 위한 시스템 및 방법 |
US11877816B2 (en) | 2017-11-21 | 2024-01-23 | Intuitive Surgical Operations, Inc. | Systems and methods for master/tool registration and control for intuitive motion |
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EP3650547A4 (en) | 2021-03-10 |
CN111566214B (zh) | 2024-03-08 |
US20210140973A1 (en) | 2021-05-13 |
JPWO2019009346A1 (ja) | 2020-05-07 |
CN111566214A (zh) | 2020-08-21 |
US11372001B2 (en) | 2022-06-28 |
JP6982226B2 (ja) | 2021-12-17 |
TW201920274A (zh) | 2019-06-01 |
EP3650547A1 (en) | 2020-05-13 |
TWI791551B (zh) | 2023-02-11 |
KR20200024304A (ko) | 2020-03-06 |
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