JP2015086208A - MONOCLONAL ANTIBODIES THAT RECOGNIZE SUGAR CHAIN DEFICIENT HUMAN IgA1 HINGE REGION AND USES THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monoclonal antibody highly specific for sugar chain-deficient IgA1 characteristic of IgA nephropathy, which is useful in examination of IgA nephropathy etc.SOLUTION: Provided is a monoclonal antibody against sugar chain deficient human IgA1, that specifically recognizes human IgA1 hinge region to which a sugar chain not containing sialic acid and galactose is bonded.

Description

  The present invention relates to an antibody useful for testing for IgA nephropathy. Specifically, the present invention relates to a monoclonal antibody that recognizes a sugar chain-deficient human IgA1 hinge part and a test method for IgA nephropathy and the like using the antibody.

  IgA nephropathy accounts for about 40% of chronic nephritis in Japan, and about 40% of those suffering from end stage renal failure without treatment. The number of chronic maintenance dialysis patients in Japan continued to increase, reaching about 270,000 in 2008. Among them, the estimated number of dialysis patients with this disease as the primary disease is estimated to be tens of thousands, and the development of treatment and prevention methods for this disease is considered to be one of the important issues from the medical economic viewpoint of reducing dialysis medical expenses. .

  At present, the only test for IgA nephropathy is biopsy, and the establishment of a simple and highly accurate test method is desired. In fact, several research groups are actively researching with the goal of establishing a test for IgA nephropathy. As one of them, Moldoveanu et al. (Non-patent Document 1) and our research group (Non-patent Document 2) have reported that a lectin (helix) that recognizes IgA1 and N-acetylgalactosamine (hereinafter also referred to as “GalNAc”). aspersa: HAA) and reported that a significant increase was observed in patients with IgA nephropathy. However, the results of the ROC curve analysis denied the possibility of clinical application of “degree of binding to HAA” as a marker for diagnosis of this disease. In addition, there was no association between the clinicopathological severity or the ablation pulse treatment and the "degree of binding with HAA", suggesting that it is not effective as an indicator of the degree of disease progression or therapeutic effect.

  The research group of the inventors of the present application has advanced research under the above background, and has reported an antibody useful for testing IgA nephropathy and a test method using the antibody (Patent Document 1).

  In addition, the prior patent document regarding the test | inspection method (diagnosis method) of IgA nephropathy is disclosed below (patent documents 2-5). In Patent Document 2, a method for diagnosing IgA nephropathy using the difference in binding ability between IgA1 molecules, in Patent Document 3, a method for diagnosing IgA nephropathy using an antibody against the IgA1 hinge region, and in Patent Document 4, IgA1 Methods for diagnosing IgA nephropathy using the number of sugar chains bound to the hinge as an index have been proposed. Patent Document 5 discloses a monoclonal antibody that recognizes an IgA1 hinge portion containing an O-linked sugar chain to which galactose is not bound.

JP 2010-285419 A JP-A-9-311132 Japanese Patent Laid-Open No. 10-111290 Japanese Patent Laid-Open No. 10-111291 International Publication No. 2011/081189 Pamphlet

Moldoveanu A, Wyatt RJ, Lee JY, et al: Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels.Kidney Int 11: 1148-1154, 2007 Shimozato S, Hiki Y, Odani H, et al: Serum under-galactosylated IgA1 is increased in Japanese patients with IgA nephropathy. Nephrol Dial Transplant 6: 1931-1939, 2008

  Based on the above background, the present invention provides a monoclonal antibody that has high specificity for sugar chain-deficient IgA1, which is characteristic of IgA nephropathy, and is useful for testing diseases associated with sugar chain-deficient IgA1 (particularly IgA nephropathy). The issue is to provide. It is also an object to provide a test method or test kit using the antibody. Although the above-mentioned Patent Document 5 shows an antibody that recognizes a sugar chain-deficient IgA1 hinge, its reactivity with clinical specimens has not been studied, and whether it is an antibody that can be clinically applied. It is unknown. In addition, the rat-derived antibody class is IgG2a or IgG2b, and the antibody class is different from the monoclonal antibody disclosed in the present application.

In order to solve the above-mentioned problems, the present inventors diligently studied with the aim of obtaining a monoclonal antibody highly specific for sugar chain-deficient IgA1. As a result, it was confirmed that the monoclonal antibody showing a specific binding property to the sugar chain-deficient IgA1 was successfully produced, and that the produced monoclonal antibody was extremely effective for examination (diagnosis) of IgA nephropathy. The present invention described below is mainly based on the above results and knowledge.
[1] A monoclonal antibody that specifically recognizes a human IgA1 hinge portion to which a sugar chain containing sialic acid and galactose is bound, which is an antibody against sugar chain-deficient human IgA1.
[2] The following sequence: VPST (GalNAc) PPT (GalNAc) PS (GalNAc) PS (GalNAc) TPPT (GalNAc) PSPS (SEQ ID NO: 1) (where V is valine, P is proline, S is serine) , T represents threonine, and GalNac in parentheses represents O-linked N-acetylgalactosamine bound to the immediately preceding amino acid residue), and was prepared as an antibody against a synthetic hinge glycopeptide [1 ].
[3] The monoclonal antibody according to [1] or [2], which does not recognize a human IgA1 hinge part to which no sugar chain is bound.
[4] The monoclonal antibody according to any one of [1] to [3], which is a mouse-derived antibody.
[5] The monoclonal antibody according to [4], wherein the antibody class is an antibody of IgG3, IgG1, or IgG2b.
[6] The monoclonal antibody according to any one of [1] to [5], which is an antibody produced by the hybridoma 35A12 strain or an antibody that competitively reacts with the antibody.
[7] A method for testing a disease associated with sugar chain-deficient IgA1, which comprises detecting the sugar chain-deficient human IgA1 with the monoclonal antibody according to any one of [1] to [6].
[8] The test method according to [7], wherein the disease is an autoimmune disease.
[9] The test method according to [7], wherein the disease is IgA nephropathy.
[10] The inspection method according to [9], including the following steps (1) to (3):
(1) A step of bringing the monoclonal antibody according to any one of [1] to [6] into contact with a human specimen;
(2) detecting the immune complex produced by step (1);
(3) A step of determining, based on the detection result, the presence or absence or possibility of IgA nephropathy, or the possibility of developing or developing IgA nephropathy.
[11] The inspection method according to [9], comprising the following steps (i) to (iv):
(i) contacting the human specimen with the immobilized monoclonal antibody according to any one of [1] to [6];
(ii) contacting the anti-human IgA antibody after removing non-specific binding components;
(iii) detecting the anti-human IgA antibody specifically bound after removing the non-specific binding component;
(iv) A step of determining, based on the detection result, whether or not IgA nephropathy is suffering or developing, or the possibility of developing or causing IgA nephropathy.
[12] The test method according to [10] or [11], wherein the human sample is a human sample after sialidase treatment.
[13] The test method according to any one of [10] to [12], wherein the human sample is a serum sample.
[14] A reagent for testing a disease associated with sugar chain-deficient IgA1, comprising the monoclonal antibody according to any one of [1] to [6].
[15] The test reagent according to [14], wherein the disease is an autoimmune disease.
[16] The test reagent according to [14], wherein the disease is IgA nephropathy.
[17] A test kit comprising the test reagent according to any one of [14] to [16].
[18] The test kit according to [17], further comprising a labeled antibody against the test reagent.

A dose-dependent curve using sialidase-treated serum as a specimen. X-axis is serum dilution factor, Y-axis is absorbance (490 nm). Results of ELISA method. The binding properties of the serum specimen and the monoclonal anti-hinge glycopeptide (HGP) antibody were compared among the IgA nephropathy patient group, the healthy group, and other renal disease groups. Sialidase-treated serum was used as a specimen. The Y axis is absorbance (490 nm). Receiver operating characteristic curves (ROC curve) drawn between IgA nephropathy patient group and other renal disease group (upper left) and between IgA nephropathy patient group and healthy group (lower right) .

  The first aspect of the present invention relates to a monoclonal antibody that specifically recognizes a sugar chain-deficient human IgA1 hinge region. Unless stated otherwise, the antibodies herein are in an isolated state. An “isolated antibody” includes an antibody that is natural and has not been subjected to any external manipulation (artificial manipulation), that is, an antibody that has been produced in a human body and remains there. Not included. The isolated antibody may contain components other than the antibody (for example, purified water, buffer solution, preservation solution, protective agent).

  The antibody of the present invention specifically recognizes a sugar chain-deficient human IgA1 hinge region as a feature. Preferably, the monoclonal antibody of the present invention does not recognize a human IgA1 hinge region to which no sugar chain is bound. There are two subtypes of human IgA, IgA1 and IgA2. The IgA1 subtype is predominant in glomerular IgA in IgA nephropathy. IgA1 has a characteristic structure in which a sugar chain is added to the hinge part. There are Ser (serine) and Thr (threonine) capable of binding O-linked sugar chains at the IgA1 hinge. O-linked sugar chains are generally composed of N-acetylgalactosamine (GalNAc) and galactose (Gal) from the inside, and sialic acid (NeuAc) can be bound to the outside. Decreased galactose content is observed in the O-linked sugar chain bound to the sugar chain-deficient human IgA1 hinge. In the present specification, “decreased galactose content” means that the Gal content of the sugar chain bound to the hinge part is lower than that of a healthy person. In order to distinguish from the hinge part of a healthy person's IgA1, the hinge part of such a feature is expressed as a “sugar chain-deficient human IgA1 hinge part” for convenience in this specification. From the results of the mass spectrometric analysis, the Gal content of the “glycan-deficient human IgA1 hinge part” is compared with the number of Gals in the sugar residues constituting the sugar chain of the entire hinge part. Sugar chain) is estimated to be about 80-90%. The number of sugar chains that bind to the sugar chain-deficient human IgA1 hinge region recognized by the antibody of the present invention is not particularly limited, but is 4 to 5, for example.

  The antibody of the present invention specifically recognizes a human IgA1 hinge portion to which a sugar chain not containing sialic acid and galactose is bound. The “sugar chain not containing sialic acid and galactose” refers to a sugar chain composed of GalNAc residues.

  The antibody of the present invention is a monoclonal antibody and exhibits high specificity for sugar chain-deficient human IgA1. A “monoclonal antibody” is an antibody obtained from a clone derived from a single antibody-producing cell, and exhibits a much higher specificity than a polyclonal antibody that is a mixture of a plurality of types.

The method for preparing the monoclonal antibody of the present invention is not particularly limited, but typically, the monoclonal antibody of the present invention can be prepared by an immunological technique. For example, it can be prepared by the following procedure. First, an antigen is prepared and used to immunize animals such as rabbits, goats, sheep, mice, rats, and guinea pigs. Preferably, a mouse is used. As an antigen, a glycopeptide having the following sequence may be used. V represents valine, P represents proline, S represents serine, T represents threonine, and GalNac in parentheses represents O-linked N-acetylgalactosamine bound to the immediately preceding amino acid residue.
VPST (GalNAc) PPT (GalNAc) PS (GalNAc) PS (GalNAc) TPPT (GalNAc) PSPS (SEQ ID NO: 1)

  An example of the above-mentioned antigen is a glycopeptide corresponding to the hinge part of IgA1 antibody, and GalNAc is added at five positions. In the antigen, there are a total of nine sites to which O-linked sugar chains can bind, as in the human IgA11 hinge region. It is also possible to employ an antigen to which GalNAc has been added at one to nine (for example, one, two, three, four) of these nine places. When the number of GalNAc addition sites is 5 or less, it is preferable to select an addition site from among the 5 sites to which GalNAc has been added in the above example of antigen. The five sites correspond to sites where sugar chain addition is frequently observed in human IgA1. The antigen can be prepared by using a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method).

  In order to enhance the immunity-inducing action, it is preferable to use an antigen to which a carrier protein is bound. As the carrier protein, KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

  Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone producing an antibody having high specificity for the antigen is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in the abdominal cavity and purifying it from ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

As shown in Examples described later, the present inventors succeeded in obtaining a plurality of monoclonal antibodies that specifically recognize a sugar chain-deficient human IgA1 hinge region by a method using a mouse as an immunized animal. For identification, these antibodies were assigned clone numbers of No. 35A12, No. 35H10, No. 38A7, No. 43C5, No. 44H8 and No. 54B9. The subclasses of these antibodies were determined as No.35A12 was IgG3 (κ chain), No.35H10 was IgG1 (κ chain), No.43C5 was IgG2b (κ chain), and No.44H8 was IgG1 (κ chain). . The hybridoma producing the clone No. 35A12 (35A12 strain) is deposited with a predetermined depository as follows.
Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818, Japan) Date of deposit (date of receipt): November 1, 2013 Number: NITE ABP-01744

Based on the above facts, in a preferred embodiment, the monoclonal antibody of the present invention is derived from a mouse. The antibody class is IgG3, IgG1, or IgG2b. On the other hand, the monoclonal antibody of the present invention can be identified by the hybridoma 35A12 strain produced. In one aspect, a monoclonal antibody that competitively reacts with an antibody produced by the hybridoma is provided. In general, antibody competition occurs when the epitopes are identical, the epitopes partially match, or the epitope positions are close.
What is a competitive reaction?

  The second aspect of the present invention relates to a test method which is one of the uses of the monoclonal antibody of the present invention. It is known that sugar chain deficiency IgA1 is involved in the progression of IgA nephropathy. In addition, glycan deficiency IgA1 is also involved in the pathogenesis of diseases other than IgA nephropathy, specifically autoimmune diseases such as systemic lupus erythematosus and Henoch-Schonlein purpura. It has been reported (Seminars in Nephrology 28 (1), 78 (2008)). Therefore, in the test method of the present invention, sugar chain-deficient human IgA1 is detected using the monoclonal antibody of the present invention in order to test for diseases involving sugar chain-deficient IgA1. The “disease associated with sugar chain insufficiency IgA1” is not particularly limited, and includes IgA nephropathy, systemic lupus erythematosus, Henoch-Schenlein purpura chronic glomerulonephritis and the like.

  As described above, the monoclonal antibody of the present invention specifically recognizes the sugar chain-deficient IgA1 hinge region. Therefore, according to the test method using the monoclonal antibody of the present invention, sugar chain-deficient IgA1 in a sample can be specifically detected. The detection results indicate whether or not a disease associated with sugar chain deficiency IgA1 (especially IgA nephropathy) is present or present, or a disease associated with sugar chain deficiency IgA1 (particularly IgA nephropathy). It is useful information to know the possibility. For example, when targeting a patient with IgA nephropathy, it is possible to obtain information useful for evaluation or grasping of the patient's disease state, evaluation of therapeutic effect, and the like. Thus, the test method of the present invention can be used for monitoring the therapeutic effect. On the other hand, when the subject is a person other than the patient, for example, a person who has not been certified with IgA nephropathy, useful information for early diagnosis is obtained, and prevention or early treatment intervention is possible. Furthermore, the cause of this disease may be multiple, and IgA nephropathy that develops due to this sugar chain-deficient IgA1 may be identified as a subtype by this technique.

  Examples of detection methods include ELISA (Enzyme-Linked ImmunoSorbent Assay), radioimmunoassay, FACS (fluorescence activated cell sorting), immunoprecipitation, immunoblotting, immunoturbidimetry, electrochemiluminescence, chemiluminescence, fluorescence A qualitative or quantitative method such as a luminescence method may be mentioned. An immunohistochemical staining method can also be employed. Among them, it is preferable to detect sugar chain-deficient IgA1 using ELISA methods (sandwich ELISA, competitive ELISA, etc.). The ELISA method has many advantages such as high detection sensitivity, high specificity, excellent quantitativeness, simple operation, and suitability for simultaneous processing of multiple samples.

In one aspect of the inspection method of the present invention, the following steps (1) to (3) are performed.
(1) A step of bringing the antibody of the present invention into contact with a human specimen
(2) detecting the immune complex produced in step (1)
(3) A step of determining whether or not IgA nephropathy is affected or present or based on the detection result, or whether or not IgA nephropathy is affected

  In step (i), a human specimen is first prepared and contacted with the antibody of the present invention (monoclonal antibody that specifically recognizes a sugar chain-deficient human IgA1 hinge). This contact operation is typically performed in a well, but is not limited thereto. The human specimen may be prepared from a biological fluid such as serum, plasma, urine, spinal fluid, ascites, pleural effusion of the subject (IgA nephropathy patient or healthy subject). Serum is preferably used. If serum is used, simple measurement is possible. Further, preferably, a specimen after sialidase treatment is used. That is, a sample obtained by removing sialic acid from the sugar chain of the hinge part in advance is used. The specificity of the antibody becomes clear by sialidase treatment. A commercially available sialidase (for example, Boehringer Mannheim, Roche Diagnostics) may be used.

  When the sample contains sugar chain-deficient human IgA1, as a result of step (1), specific binding by the antibody of the present invention occurs, resulting in an immune complex (complex of antibody of the present invention and sugar chain-deficient IgA1). Body) is formed. In step (2), the immune complex is detected.

  In step (3), based on the detection result of step (2), the presence or absence or possibility of IgA nephropathy, or the possibility of developing or developing IgA nephropathy is determined. Determining “possibility to develop or develop IgA nephropathy” is “prediction of the occurrence or development of IgA nephropathy”. Thus, the present invention can also be used for predicting the morbidity or development of IgA nephropathy. Furthermore, the cause of this disease may be multiple, and IgA nephropathy that develops due to this sugar chain-deficient IgA1 may be identified as a subtype by this technique.

The determination here may be either qualitative or quantitative. Examples of qualitative judgment and quantitative judgment are shown below. It should be noted that the determination here can be automatically / mechanically performed without depending on the determination of a person having specialized knowledge such as a doctor or a laboratory technician, as is apparent from the determination criteria.
(Example 1 of qualitative judgment)
When the detected value is higher than the reference value, “I am suffering from IgA nephropathy”, “I am likely to be suffering from IgA nephropathy”, or “I am likely to be suffering from IgA nephropathy in the future” When the detected value is lower than the reference value, "I do not suffer from IgA nephropathy", "It is highly possible that I do not have IgA nephropathy", or " It is determined that the possibility of being affected is low.
(Example 2 of qualitative judgment)
When detected (ie, positive) “I am suffering from IgA nephropathy”, “I am likely to be suffering from IgA nephropathy”, or “I am likely to suffer from IgA nephropathy in the future When it is judged as “high” and is not detected (ie, when it is negative), it is “not likely to have IgA nephropathy”, “highly likely not to have IgA nephropathy”, or “in the future, IgA nephropathy” Is less likely to be affected ”.

(Example 1 of quantitative determination)
As shown below, “Possibility of suffering from IgA nephropathy (%)” is preset for each detection value range, and “Possibility of suffering from IgA nephropathy (%) Is determined.
Detection value ab: The possibility of having IgA nephropathy is 10% or less Detection value bc: The possibility of having IgA nephropathy is 10% to 30%
Detection values cd: 30% to 50% likely to have IgA nephropathy
Detection values d to e: Possibility of suffering from IgA nephropathy 50% to 70%
Detection values ef: 70% to 90% likely to have IgA nephropathy

(Example 2 of quantitative determination)
As shown below, “Possibility of suffering from IgA nephropathy in the future (%)” is preset for each range of detection values, and from the detection value “Possibility of suffering from IgA nephropathy in the future (%) Is predicted.
Detected values a to b: 10% or less likely to have IgA nephropathy in the future Detected values bc to 10 to 30% likely to have IgA nephropathy in the future
Detection values cd: 30% to 50% likely to have IgA nephropathy in the future
Detection value de: 50% to 70% possibility of suffering from IgA nephropathy in the future
Detected values ef: 70% to 90% likely to have IgA nephropathy in the future

In another aspect of the present invention, the following steps (i) to (iv) are performed.
(i) a step of bringing the immobilized antibody of the present invention into contact with a human specimen
(ii) removing non-specific binding components and then contacting with an anti-human IgA antibody
(iii) detecting the anti-human IgA antibody specifically bound after removing the non-specific binding component
(iv) A step of determining whether or not IgA nephropathy is present or present, or the possibility of developing or developing IgA nephropathy based on the detection result

  In step (i), first, the antibody of the present invention (monoclonal antibody that specifically recognizes the sugar chain-deficient human IgA1 hinge region), that is, the antibody of the present invention immobilized on an insoluble support (hereinafter, “solid phase”) is used. (Referred to as “antibody”). As the insoluble support, for example, a resin such as polystyrene resin, polycarbonate resin, silicon resin, nylon resin, or a water-insoluble substance such as glass can be used. For example, a commercially available anti-human IgA antibody (for example, Jackson Immuno Reseach Labs) may be used. The form of the insoluble support is not particularly limited. Examples of forms of insoluble supports are wells, plates.

  Next, the human specimen is brought into contact with the immobilized antibody. By this operation, if sugar chain-deficient IgA1 is present in the human sample, it is trapped in the immobilized antibody. As the human specimen, biological fluids such as serum, plasma, urine, spinal fluid, ascites, pleural effusion of the subject (IgA nephropathy patient or healthy person) are used. Serum is preferably used. If serum is used, simple measurement is possible. Preferably, a human sample that has been treated with sialidase in advance is used.

  In the subsequent step (ii), the non-specific binding component is washed away and then contacted with an anti-human IgA antibody. As a result, when the sugar chain-deficient IgA1 is trapped in the immobilized antibody, the anti-human IgA antibody binds to the sugar chain-deficient IgA1 and an immune complex is formed. On the other hand, when the sugar chain-deficient IgA1 is not trapped in the immobilized antibody, specific binding by the anti-human IgA antibody does not occur. Thus, the amount of immune complex formed varies depending on the state of capture of the sugar chain-deficient IgA1 in the solid-phased antibody (reflecting the sugar chain state of the IgA1 hinge part in the human specimen). In the present invention, this difference is used to determine IgA nephropathy. An anti-IgA1 antibody is preferably used as the anti-human IgA antibody. The anti-IgA1 antibody may be polyclonal or monoclonal.

In step (iii), the non-specific binding component is washed away, and then the specifically bound antibody (in other words, immune complex) is detected. For example, if the anti-human IgA antibody used in step (ii) is labeled in advance, the labeled amount may be detected directly after washing away non-specific binding components. On the other hand, it is also possible to detect using a so-called secondary antibody. In this case, after removing the non-specific binding component, contacting the labeled antibody to the antibody used in step (ii) (step (iii-1)), removing the non-specific binding component, A step (step (iii-2)) of detecting the labeled antibody bound thereto is carried out. For labeling of the antibody or secondary antibody used in step (ii), for example, fluorescent dyes such as fluorescein, rhodamine, Texas red, oregon green, horseradish peroxidase, microperoxidase, alkaline phosphatase, β-D-galactosidase, etc. Enzymes, chemical or bioluminescent compounds such as luminol and acridine dyes, radioisotopes such as ³² P, ¹³¹ I, and ¹²⁵ I, biotin, and the like can be used. Many labeled antibodies (for example, labeled anti-rabbit IgG antibodies) that can be used as secondary antibodies are commercially available, and the labeled antibodies can also be used.

  In step (iv), based on the detection result, the presence or absence or possibility of IgA nephropathy, or the possibility of developing or developing IgA nephropathy is determined. Since this step is the same as step (3) in the above embodiment, its description is omitted.

  The third aspect of the present invention relates to a test reagent for diseases involving sugar chain deficiency IgA1. Although the reagent of the present invention can be suitably used in the test method of the present invention, it can also be used as an experimental tool for studying the onset mechanism and symptom progression mechanism of diseases (particularly IgA nephropathy) involving sugar chain deficiency IgA1. Can be used. Furthermore, the cause of this disease may be multiple, and IgA nephropathy that develops due to this sugar chain-deficient IgA1 may be identified as a subtype such as “sugar chain-deficient IgA nephropathy” by this method. On the other hand, in view of the property of recognizing a GalNAc residue on an O-linked sugar chain, it can be used as a research tool for mucous membranes, mucins and the like. The test reagent of the present invention contains the antibody of the present invention. The test reagent of the present invention may be provided in a state of being immobilized on an insoluble support (solid phase reagent). Alternatively, it may be provided in a labeled state (fluorescent label, enzyme label, etc.).

  The fourth aspect of the present invention provides a kit (test kit) that can be used in the test method of the present invention. The kit of the present invention comprises the test reagent of the present invention. Reagents, diluents, reaction containers and the like necessary for carrying out the inspection method of the present invention can be included in the kit. The kit of the present invention usually includes instructions for use. By using the kit, the test method of the present invention can be carried out more easily and in a shorter time.

  In a preferred embodiment, a secondary antibody (labeled antibody against the test reagent of the present invention) is included in addition to the test reagent of the present invention. According to the kit containing the secondary antibody, a series of operations such as formation of an immune complex by contact between the test reagent of the present invention and a sample, binding of a labeled antibody to the immune complex, and measurement of the amount of bound label are performed. Can be implemented.

  A standard sample may be included in the kit of the present invention. The standard sample here serves as a reference for evaluating the detection result using the kit of the present invention. For example, a sugar chain-deficient IgA1 or sugar chain-deficient IgA1 hinge peptide or a part thereof is used as a standard sample. Standard samples can be prepared by synthesis or by separation and purification from the serum of patients with IgA nephropathy.

1. Preparation of mouse monoclonal antibody against human IgA1 hinge peptide (1) Human IgA1 hinge glycopeptide synthesis Protected peptide resin is a sugar corresponding to human IgA1 hinge described later by tert-butoxycarbonyl method (Boc method) using an automatic synthesizer. A peptide (synthetic human IgA1 hinge glycopeptide, hereinafter abbreviated as sHGP) was synthesized. The obtained protected glycopeptide resin was treated with anhydrous hydrogen fluoride, and the crude glycopeptide was purified by reverse phase HPLC to obtain the desired sHGP with high purity. sHGP has the following structure in which GalNAc is added to five sites that are said to have a high frequency of O-glycan side chains in the 19-mer amino acid sequence of the human IgA1 hinge.
VPST (GalNAc) PPT (GalNAc) PS (GalNAc) PS (GalNAc) TPPT (GalNAc) PSPS-NH ₂ (SEQ ID NO: 1)

(2) Immunization to mice Two types of synthetic peptides (human IgA1 hinge glycopeptide (sHGP) and human IgA1 hinge peptide), which are haptens, were subjected to KLH (manufactured by 77600 Thermo) and glutaraldehyde method. OVA (77120 Thermo) was bound to obtain antigens for immunization (KLM binding) and antibody screening (OVA binding).

  Equal amounts of the prepared antigen (KLM-conjugated sHGP) and adjuvant (TiterMaxGold: G3 Funakoshi) were mixed, and 100 µg of antigen was subcutaneously administered to BALB / cA mouse 6 old female (manufactured by CLEA Japan) at intervals of 2 weeks. The administration was divided into two portions, and 40 μg of the antigen solution alone was subcutaneously administered 1 to 2 weeks later. Three days later, whole blood was collected under anesthesia, and the spleen and various lymph nodes were collected.

(3) Cell fusion
Mouse myeloma cells (P3UI) were subcultured by adding 10% FBS (S156 BWT) to RPMI1640 medium (118-119 GIBCO) supplemented with pyruvic acid, glutamic acid, penicillin and streptomycin (RPMI medium). What was cultured was used. Cell fusion was performed as follows. 1 ml of PEG (783641 Roche) was reacted with the sensitized cell pellet, RPMI1640 medium was added, and the mixture was allowed to stand in a 37 ° C. incubator for 10 minutes, and then cells were collected. Subsequently, using a HAT medium (21060-017 GIBCO), hybridomas were selectively cultured as usual.

(4) Antibody screening A 96-well microplate for ELISA was used for the primary screening of specific antibodies. After binding an OVA-linked glycopeptide to each well, the cells in the selective culture book were reacted. Subsequently, enzyme-labeled anti-mouse IgG (1030-04 Cosmo Bio) was reacted to select a monoclonal antibody positive well.

Next, in order to select a monoclonal antibody that reacts with an OVA-bound glycopeptide antigen but does not react with an OVA-bound peptide antigen without a sugar as a secondary screening, the same assay is performed and the target antibody candidate is selected. Sorted out. The selected cells were transferred to a culture plate and expanded. Clinical evaluation was performed using the culture supernatant as a sample. Specifically, using IgA nephropathy patient serum and healthy subject serum, the reactivity of each antibody was evaluated by ELISA, and clones that responded not only to synthetic peptides but also to IgA in vivo were selected. Selected clones were established as single clones by limiting dilution. By detailed examination using the culture supernatant of established clones, it was finally narrowed down to 6 clones (35A12, 35H10, 38A7, 43C5, 44H8, 54B9). The 35A12 strain that produces an antibody capable of clearly distinguishing IgA nephropathy patients from healthy individuals was selected as a superior strain. The 35A12 strain was deposited as follows.
Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818, Japan) Date of deposit (date of receipt): November 1, 2013 Number: NITE ABP-01744

(5) Determination of subclass The subclass was determined by a commercially available immunochromatography method (monoclonal antibody isotyping kit 1493027 Roche) using the culture supernatant of each sufficiently cloned hybridoma strain. The operation followed the attached manual. As a result of the examination, the subclass of the antibody produced by the 35A12 strain is IgG3 (κ chain), the subclass of the antibody produced by the 35H10 strain is IgG1 (κ chain), the subclass of the antibody produced by the 43C5 strain is IgG2b (κ chain), The subclass of the antibody produced by the 44H8 strain was determined to be IgG1 (κ chain).

2. Evaluation using clinical specimens
The binding of the monoclonal anti-HGP antibody produced by the 35A12 strain and serum IgA1 was compared among the IgA nephropathy patient group, other renal disease groups, and healthy subjects.

2-1. Materials and Methods (1) Serum Serum collected at the time of renal biopsy of 29 IgA nephropathy patients diagnosed by renal biopsy was used. As controls, 27 patients with other kidney diseases (ANCA-related nephritis 11 cases, SLE nephropathy 6 cases, membranous nephropathy 4 cases, diabetic nephropathy 1 case, minimal change group 1 case, nephrosclerosis / renal Serum collected from 23 patients with retronephritis (1 patient), cholesterol embolism (1 patient), obese nephropathy (1 patient) and healthy subjects (sex and age combined with IgA nephropathy patient group) were used.

(2) ELISA method (2-1) Preparation of ELISA plate
Add 50 μl of a monoclonal antibody adjusted to 10 μl / ml (0.05 M bicarbonate buffer (pH 9.6) containing (IgG fraction)) to each well of a polystyrene microtiter plate (Corning, NY, USA). (O / N) left at 4 ° C., and the monoclonal antibody was coated on the plate.After washing 3 times with 0.01M PBS-0.05% Tween-20 (pH 7.4, hereinafter abbreviated as PBST), 1% bovine serum albumin ( PBST solution containing BSA (Sigma Chemical Company, St Louis, MO, USA) was added at 100 μl / well, and the mixture was allowed to stand at room temperature for 1 hour for blocking.

(2-2) Serial sialidase treatment
To remove the neuraminic acid residue bound to the GalNAc residue in the IgA1 hinge O-linked sugar side chain, 5 volumes of 1.5 mU / ml neuraminidase (Vibrio cholerae, Roche Diagnostic Corp., Indianapolis, IN, USA) 0.01M acetate buffer (pH 5) was added and left at 37 ° C. for 2 hours.

(2-3) Dose-dependent curve Dose-dependent curve using samples obtained by serial dilution (1/25, 1/100, 1/400, 1/1600, 1/6400) of serum treated with sialidase by the ELISA method described below (Fig. 1). The experiment was performed with the optimum dilution concentration (100-400) determined.

(2-4) Operation of ELISA The serum after sialidase treatment was diluted to an optimal magnification with PBST containing 1% BSA (hereinafter, dilution buffer). Each specimen thus obtained was added at 50 μl / well and incubated at 4 ° C. overnight. After washing three times with PBST, 50 μl / well of 5000-fold diluted HRP-labeled goat anti-human IgA antibody (IgG Fab fraction, MBL Co. LTD, Japan Nagoya) was added. After standing at room temperature for 2 hours, the plate was washed 5 times, and 0.1 M citrate buffer containing 50 μl of O-phenylenediamine (0.5 mg / ml) -H ₂ O ₂ (0.1%, Pierce) was added for color development. The color reaction was stopped with 50 μl of 1 M sulfuric acid, and the absorbance (492 nm) of each well was measured.

(2-5) Comparison between three groups and statistical analysis Bonferroni corrected Mann-Whitney U test was used for comparison between the three groups. A P value of less than 0.05 was considered statistically significant. In order to examine the possibility of clinical application of this antibody, draw receiver operating characteristic curves (ROC curve) between the IgA nephropathy patient group and other renal disease group and healthy group, respectively. The area of under the curve (AUC) was calculated. When AUC exceeded 0.9, it was judged to have sensitivity and specificity that can be applied clinically.

2-2. Results As shown in FIG. 2, the IgA nephropathy patient group binds to this antibody compared to the other kidney disease group (P = 2.8 × 10 ⁻⁶ ) and the healthy group (P = 1.2 × 10 ⁻⁶ ). Serum IgA was significantly increased. The AUC calculated from the ROC curves drawn between the IgA nephropathy patient group and the other kidney disease group (upper left of FIG. 3) and between the IgA nephropathy patient group and the healthy subject group (lower right of FIG. 3), respectively. The sensitivity / specificity of this antibody was considered to be clinically applicable.

3. Summary As described above, it was confirmed that the monoclonal anti-HGP antibody that was successfully obtained showed IgA nephropathy patient-specific binding properties and was useful for detection or testing of IgA nephropathy. In addition, as a result of measuring also when the sialidase treatment of serum was omitted, the specificity of the antibody became clear by performing the sialidase treatment, and a significant difference was recognized.

  The antibody of the present invention is used in a test method for diseases (particularly IgA nephropathy) involving sugar chain-deficient IgA1. For example, if the antibody of the present invention is used, IgA nephropathy can be examined with high sensitivity and specificity.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

As shown in Examples described later, the present inventors succeeded in obtaining a plurality of monoclonal antibodies that specifically recognize a sugar chain-deficient human IgA1 hinge region by a method using a mouse as an immunized animal. For identification, these antibodies were assigned clone numbers of No. 35A12, No. 35H10, No. 38A7, No. 43C5, No. 44H8 and No. 54B9. The subclasses of these antibodies were determined as No.35A12 was IgG3 (κ chain), No.35H10 was IgG1 (κ chain), No.43C5 was IgG2b (κ chain), and No.44H8 was IgG1 (κ chain). . The hybridoma producing the clone No. 35A12 (35A12 strain) is deposited with a predetermined depository as follows.
Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818 Japan) Date of deposit (Reception date): November 1, 2013
Accession Number: NITE BP-01744

Next, in order to select a monoclonal antibody that reacts with an OVA-bound glycopeptide antigen but does not react with an OVA-bound peptide antigen without a sugar as a secondary screening, the same assay is performed and the target antibody candidate is selected. Sorted out. The selected cells were transferred to a culture plate and expanded. Clinical evaluation was performed using the culture supernatant as a sample. Specifically, using IgA nephropathy patient serum and healthy subject serum, the reactivity of each antibody was evaluated by ELISA, and clones that responded not only to synthetic peptides but also to IgA in vivo were selected. Selected clones were established as single clones by limiting dilution. By detailed examination using the culture supernatant of established clones, it was finally narrowed down to 6 clones (35A12, 35H10, 38A7, 43C5, 44H8, 54B9). The 35A12 strain that produces an antibody capable of clearly distinguishing IgA nephropathy patients from healthy individuals was selected as a superior strain. The 35A12 strain was deposited as follows.
Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818 Japan) Date of deposit (Reception date): November 1, 2013
Accession Number: NITE BP-01744

Claims (18)

  A monoclonal antibody that specifically recognizes a human IgA1 hinge region to which a sugar chain containing no sialic acid and galactose is bound, which is an antibody against sugar chain-deficient human IgA1.   The following sequence: VPST (GalNAc) PPT (GalNAc) PS (GalNAc) PS (GalNAc) TPPT (GalNAc) PSPS (SEQ ID NO: 1) (where V is valine, P is proline, S is serine, T is 2. An antibody prepared as an antibody against a synthetic hinge glycopeptide consisting of threonine, and GalNac in parentheses represents O-linked N-acetylgalactosamine bound to the immediately preceding amino acid residue, respectively. Antibodies.   The monoclonal antibody according to claim 1 or 2, which does not recognize a human IgA1 hinge part to which no sugar chain is bound.   The monoclonal antibody according to any one of claims 1 to 3, which is a mouse-derived antibody.   The monoclonal antibody according to claim 4, wherein the antibody class is an antibody of IgG3, IgG1, or IgG2b.   The monoclonal antibody according to any one of claims 1 to 5, which is an antibody produced by the hybridoma 35A12 strain or an antibody that competitively reacts with the antibody.   A test method for a disease involving sugar chain deficiency IgA1, which comprises detecting the sugar chain deficiency human IgA1 by the monoclonal antibody according to any one of claims 1 to 6.   The test | inspection method of Claim 7 whose said disease is an autoimmune disease.   The test | inspection method of Claim 7 whose said disease is IgA nephropathy. The inspection method according to claim 9, comprising the following steps (1) to (3):
(1) A step of contacting the monoclonal antibody according to any one of claims 1 to 6 with a human specimen;
(2) detecting the immune complex produced by step (1);
(3) A step of determining, based on the detection result, the presence or absence or possibility of IgA nephropathy, or the possibility of developing or developing IgA nephropathy. The inspection method according to claim 9, comprising the following steps (i) to (iv):
(i) contacting the human specimen with the immobilized monoclonal antibody according to any one of claims 1 to 6;
(ii) contacting the anti-human IgA antibody after removing non-specific binding components;
(iii) detecting the anti-human IgA antibody specifically bound after removing the non-specific binding component;
(iv) A step of determining, based on the detection result, whether or not IgA nephropathy is suffering or developing, or the possibility of developing or causing IgA nephropathy.   The test method according to claim 10 or 11, wherein the human sample is a human sample after sialidase treatment.   The test | inspection method as described in any one of Claims 10-12 whose human sample is a serum sample.   A reagent for testing a disease involving a sugar chain-deficient IgA1, comprising the monoclonal antibody according to any one of claims 1 to 6.   The test reagent according to claim 14, wherein the disease is an autoimmune disease.   The test reagent according to claim 14, wherein the disease is IgA nephropathy.   The test kit containing the test reagent as described in any one of Claims 14-16.   The test kit according to claim 17, further comprising a labeled antibody against the test reagent.

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