JP5696273B2 - Diagnosis of idiopathic normal pressure hydrocephalus with sugar chain biomarkers - Google Patents

Description

  The present invention relates to a glycoprotein marker that can be used for diagnosis of idiopathic normal pressure hydrocephalus, and more specifically to a diagnostic method, a diagnostic kit, and the like for iNPH using this glycoprotein.

  Idiopathic normal pressure hydrocephalus (iNPH) is a senile cerebrospinal fluid metabolism disorder characterized by dementia and ventricular enlargement. iNPH is considered to be a cure for dementia because it can be completely cured by simple surgery. However, because the main symptoms (dementia and ventricular enlargement) are similar to Alzheimer's disease, most of the disease has been misdiagnosed as Alzheimer's disease.

  Conventionally, the definitive diagnosis of iNPH was based on whether a large amount (30 mL) of cerebrospinal fluid was removed by lumbar puncture, reducing pressure symptoms due to excessive cerebrospinal fluid, and whether the symptoms were alleviated (tap test). However, this method places a heavy burden on the patient and results are qualitative. For this reason, there are many false negatives (cases in which the tap test does not become positive despite iNPH). Also, because iNPH is an elderly disease, only a few milliliters of cerebrospinal fluid can be removed due to lumbar deformity. In this case, determination is impossible and surgery is not performed. The small amount of cerebrospinal fluid obtained at this time is discarded because there is no appropriate diagnostic marker.

Patent Document 1 describes a method of diagnosing Alzheimer's disease using a glycoprotein sugar chain in serum as a biomarker.
However, a glycoprotein that can be used as a biomarker that can be used for diagnosis of idiopathic normal pressure hydrocephalus has not been known so far.
JP 2006-317210 A

  The object of the present invention is to find a biomarker capable of accurately diagnosing idiopathic normal pressure hydrocephalus, which was often mistakenly diagnosed as Alzheimer's disease, and to reduce the burden on the patient compared with conventional diagnostic methods. It is to provide a new diagnostic method with a small amount.

  Since cerebrospinal fluid metabolism abnormalities are suspected in iNPH, the present inventors have found that the cerebrospinal fluid of iNPH patients has glycoproteins with sugar chains having a structure different from that of healthy individuals, or are characterized by cerebrospinal fluid. The amount of glycoprotein with a typical sugar chain structure might change. Based on this idea, as a result of diligent research to solve the above problems, there is a glycoprotein that is significantly reduced in cerebrospinal fluid samples derived from iNPH patients compared to cerebrospinal fluid samples derived from healthy subjects. For the first time. The present inventors have discovered that this glycoprotein is transferrin having N-acetylglucosamine (GlcNAc) as a terminal sugar, and named this transferrin isoform as transferrin-1. Transferrin that is usually present in serum has already been reported to have a sugar chain structure of sialic acid (Sia) -galactose (Gal) -N-acetylglucosamine (GlcNAc) at the non-reducing end (N-Linked sugar chains of glycoproteins in human plasma in Comprehensive Glycoscience, Vol. 1 (Chapter 1.02.2.2), pp44-47, Ed. Kamerling, JP). Therefore, the present inventors have shown for the first time that there is an abundance of transferrin having a glycoform with a short sugar chain in cerebrospinal fluid compared to serum transferrin. The present inventors have succeeded in establishing a diagnostic method using this cerebrospinal fluid characteristic transferrin-1 as a diagnostic marker for iNPH, and completed the present invention.

That is, the present invention
[1] A test method for diagnosing idiopathic normal pressure hydrocephalus in a subject, comprising measuring the amount of transferrin-1 in a body fluid collected from the subject.
[2] Measuring the amount of transferrin-2 in the body fluid, and calculating the value of transferrin-2 / transferrin-1.
The method according to [1] above, further comprising:
[3] A substance that specifically binds to the protein part of transferrin, and a substance that specifically binds to the sugar chain part of transferrin-1.
The method according to [1] above, wherein one or more substances selected from the group consisting of:
[4] The method according to [3] above, further using a substance that specifically binds to the sugar chain portion of transferrin-2;
[5] a substance that specifically binds to the protein portion of transferrin,
A substance that specifically binds to the sugar chain part of transferrin-1, and a substance that specifically binds to the sugar chain part of transferrin-2;
The method according to [3] above, wherein
[6] The method according to any one of [3] to [5] above, wherein the substance that specifically binds to the protein portion of transferrin is an anti-human transferrin antibody;
[7] The method according to any one of [3] to [5] above, wherein the substance that specifically binds to the sugar chain portion of transferrin-1 is lectin;
[8] The method according to [4] or [5] above, wherein the substance that specifically binds to the sugar chain portion of transferrin-2 is lectin;
[9] The method according to [7] above, wherein the substance that specifically binds to the sugar chain moiety of transferrin-1 is PVL lectin;
[10] The method described in [8] above, wherein the substance that specifically binds to the sugar chain portion of transferrin-2 is SSA lectin;
[11] The method according to [1] or [2] above, wherein the body fluid is cerebrospinal fluid;
[12] The method according to [1] or [2] above, wherein the measurement is performed using a sandwich ELISA;
[13] A substance that specifically binds to the protein portion of transferrin, and a substance that specifically binds to the sugar chain portion of transferrin-1.
A diagnostic kit for idiopathic normal pressure hydrocephalus comprising one or more substances selected from the group consisting of:
[14] The kit according to [13], further comprising a substance that specifically binds to a sugar chain part of transferrin-2;
[15] A substance that specifically binds to the protein portion of transferrin,
A substance that specifically binds to the sugar chain part of transferrin-1, and a substance that specifically binds to the sugar chain part of transferrin-2;
A kit according to [13] above, comprising:
[16] The kit according to any one of [13] to [15] above, wherein the substance that specifically binds to the protein portion of transferrin is an anti-human transferrin antibody;
[17] The kit according to any one of [13] to [15] above, wherein the substance that specifically binds to the sugar chain portion of transferrin-1 is a lectin;
[18] The kit according to any one of [13] to [15] above, wherein the substance that specifically binds to the sugar chain portion of transferrin-2 is lectin;
[19] The kit according to [17] above, wherein the substance that specifically binds to the sugar chain portion of transferrin-1 is PVL lectin;
[20] The kit according to [18] above, wherein the substance that specifically binds to the sugar chain moiety of transferrin-2 is SSA lectin;
[21] The kit according to [13] above, wherein a substance that specifically binds to the protein portion of transferrin is immobilized on a support.
Etc.

  By using the novel iNPH diagnostic marker of the present invention, iNPH can be accurately diagnosed even from a small amount of body fluid (eg, cerebrospinal fluid) sample. In addition, the burden on the subject at the time of the examination is less than that of the conventional method.

  The present invention provides a method for diagnosing iNPH using the amount of transferrin-1 in a body fluid (preferably cerebrospinal fluid) sample or the ratio of transferrin-1 and transferrin-2 in a sample as a diagnostic marker.

  The amino acid sequence of human transferrin is Accession No. : NM — 001063 (SEQ ID NO: 2), and the nucleic acid sequence is represented by SEQ ID NO: 1. Transferrin is a glycoprotein that binds and carries diatomic iron. Serum transferrin has a molecular weight of approximately 80 KDa (698 amino acid residues), and it has been reported that biantennary N-glycans are added to the 432rd and 630th asparagines (N-Linked sugar chains of glycoproteins in human). plasma in Comprehensive Glycoscience, Vol. 1 (Chapter 1.02.2.2), pp44-47, Ed. Kamerling, JP). Serum transferrin has a Sia-Gal-GlcNAc structure at the end of the sugar chain, just like normal serum proteins.

  In addition to transferrin having the same sugar chain structure as previously known serum transferrin (transferrin-2), the present inventors have added a transferrin isoform having a sugar chain structure different from that (transferrin- Newly found that 1) exists. The protein portion (core protein) of transferrin in cerebrospinal fluid is the same as serum transferrin.

  In this specification, the transferrin isoform in cerebrospinal fluid which is considered to have a sugar chain structure different from serum transferrin is referred to as transferrin-1. Transferrin-1 was hardly sialic acid-modified by lectin array analysis and stained with PVL lectin specific for GlcNAc, so its sugar chain has a sialo and a galacto structure. It is thought to have a GlcNAc terminus.

  In this specification, the transferrin isoform which is considered to have the same sugar chain structure as serum transferrin present in the spinal fluid is referred to as transferrin-2. The sugar chain of transferrin-2 is thought to have a Sia-Gal-GlcNAc terminus.

  In the present specification, serum transferrin, transferrin-1 and transferrin-2 are sometimes collectively referred to as transferrin.

  Glycoproteins can be detected / measured / quantified (as used herein, these terms are interchangeable) using substances that specifically bind to the glycoprotein. Such substances include substances that bind to the protein part of glycoprotein or a part thereof (eg, antibody), substances that bind to the sugar chain part of glycoprotein or part thereof (eg, lectin, sugar chain antibody), etc. Is mentioned. In the present specification, these substances are also referred to as “protein binding substances” and “sugar chain binding substances”, respectively. In the present specification, the protein part of a glycoprotein or a part thereof (peptide) may be simply referred to as a protein part, and the sugar chain part or part thereof may be simply referred to as a sugar chain part.

The present invention provides a test method for diagnosing iNPH, comprising measuring the amount of transferrin-1 in a body fluid derived from a subject. When the amount of transferrin-1 is significantly reduced as compared with a body fluid sample derived from a healthy person, the subject can be diagnosed as an iNPH patient.
Although there are individual differences in the amount of transferrin-1 present in body fluids, it is possible to evaluate the effect of the treatment by monitoring changes in the amount of transferrin-1 in patients treated with iNPH .

  For the measurement of transferrin-1, a substance that specifically binds to the protein portion of transferrin and a substance that specifically binds to the sugar chain portion of transferrin-1 are used alone or in combination. When a substance that specifically binds to the protein part of transferrin or a substance that specifically binds to the sugar chain part of transferrin-1 is used alone, the amount of transferrin-1 is measured by Western blot etc. using these substances for detection To do. If the band positions of transferrin-1 and -2 are known, it is possible to measure the amount of transferrin-1 even with a substance that specifically binds to the protein portion of transferrin alone. When these binding substances are used in combination, a sandwich method or the like can be used.

  In addition to measuring the amount of transferrin-1 in body fluids, the present invention also measures the amount of transferrin-2 and the ratio of transferrin-2 to transferrin-1 (the value of transferrin-2 / transferrin-1 In other words, the present invention provides a test method for iNPH diagnosis, which comprises calculating a sugar chain index. Examples of the method for measuring transferrin-2 include the same methods as described above for the measurement of transferrin-1. When this sugar chain index is significantly reduced as compared with a body fluid sample derived from a healthy person, the subject can be diagnosed as an iNPH patient. The glycan index is particularly useful for definitive diagnosis of iNPH.

  When measuring both transferrin-1 and 2 by Western blotting, if the position of each band on electrophoresis is known, a substance that specifically binds to the protein portion of transferrin alone can measure both amounts simultaneously. Is possible. For example, by performing electrophoresis of purified transferrin-1 and transferrin-2 simultaneously with a sample derived from a subject, it is easy to distinguish both isoforms in the sample. In this case, each band may be analyzed with a densitometer or the like to calculate the ratio of transferrin-1 and transferrin-2.

  When both transferrin-1 and 2 are measured by the sandwich method, etc., a substance that specifically binds to the protein part of transferrin, a substance that specifically binds to the sugar chain part of transferrin-1, and transferrin-2 A substance that specifically binds to the sugar chain moiety is used.

  A sugar chain-binding substance that specifically binds to transferrin-1 is a substance that binds to the sugar chain part of transferrin-1 but does not substantially bind to the sugar chain part of transferrin-2, that is, transferrin-1 This refers to a substance that does not substantially bind to a structure common to the sugar chain structure and the sugar chain structure of transferrin-2. The same applies to a sugar chain binding substance that specifically binds to transferrin-2. As used herein, “substantially does not bind” refers to a substance that does not bind to the substance at all or exhibits a significantly low bond. It is preferable not to bind at all. As long as these conditions are satisfied, any combination of sugar chain binding substances can be used.

  In particular, when there are many unrelated glycoproteins having a sugar chain to which a sugar chain-binding substance specific for transferrin-1 or 2 is present in a body fluid sample derived from a subject, the protein-binding substance and the sugar It is preferable to measure the amount of each isoform in combination with a chain binding substance.

  When using a combination of a glycan-binding substance and a protein-binding substance, the binding specificity of the glycan-binding substance to a glycan structure that does not exist in either transferrin-1 or 2 does not need to be considered. .

When the sandwich method is used for measurement of transferrin-1 or 2, such measurement includes ELISA, immunochromatography, radioimmunoassay (RIA), fluorescence immunoassay (FIA method), chemiluminescence immunoassay, evanescent wave analysis, etc. Can be used. These methods are known to those skilled in the art, and any method may be selected. Moreover, these methods should just be implemented according to a normal procedure, and the setting of actual reaction conditions etc. is in the normal technical range of those skilled in the art.
Of these, it is particularly preferable to use a lectin / antibody sandwich ELISA using an antibody and a lectin as a protein binding substance and a sugar chain binding substance, respectively.

  In the sandwich method, either a protein binding substance or a sugar chain binding substance is bound to a solid phase. Hereinafter, the immobilized binding substance is referred to as a “capturing agent” and the other is referred to as a “detecting agent”. Since transferrin-1 and 2 have a common protein portion, it is preferable from the viewpoint of simplicity of operation to use a protein binding substance (preferably an antibody) as a capture agent.

  Supports (solid phase) for immobilizing the capture agent include plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose (trademark)). , Membranes (eg, nitrocellulose membrane, PVDF membrane), gels (eg, polyacrylamide gel) and the like. Among them, plates, beads and membranes are preferably used, and plates are most preferably used from the viewpoint of easy handling.

  The capture agent may be immobilized by any method as long as sufficient binding strength can be obtained. For example, the capture agent is immobilized by covalent bond, ionic bond, physical adsorption or the like. Or you may use the support body which solidified the capture agent beforehand.

The detection agent may be indirectly or directly labeled with a labeling substance. Examples of labeling substances include fluorescent substances (eg, FITC, rhodamine), radioactive substances (eg, ¹³ C, ³ H), enzymes (eg, alkaline phosphatase, peroxidase (horseradish peroxidase, etc.), glucose oxidase, β-galactosidase ), Etc. Alternatively, the detection agent may be labeled with biotin, (strept) avidin may be labeled with the labeling substance, and the binding between biotin and (strept) avidin may be used.

  When an enzyme is used as a labeling substance, detection is performed using an appropriate substrate corresponding to the enzyme used. For example, when peroxidase is used as an enzyme, o-phenylenediamine (OPD), tetramethylbenzidine (TMB) or the like is used as a substrate. When alkaline phosphatase is used, p-nitrophenyl phosphate (PNPP) or the like is used. Is used. As the enzyme reaction stop solution and the substrate solution, conventionally known ones can be appropriately selected and used according to the selected enzyme.

  The capture agent forms a complex with transferrin-1 or 2 in the body fluid sample. Transferrin-1 and 2 in the body fluid are detected and quantified by measuring a signal generated by applying a detection agent to the complex. The measurement of the signal may be performed using an appropriate measuring device depending on the labeling substance used.

Since the binding between the sugar chain and the lectin is weaker than the binding with the antibody (generally, the binding constant of the antigen-antibody reaction is 10 ⁶ to 10 ⁹ M ^-1 , but the binding constant between the lectin and the sugar chain is 10 ⁴ to 10 ⁷ M ⁻¹ ), when a lectin is used as a sugar chain binding substance, it is preferable to detect a signal using an evanescent wave excitation type fluorescence detection method. The evanescent wave excitation type fluorescence detection method is different in refractive index of glass (solid phase) and water (liquid phase) 2 when light is incident on the end surface (side surface) of the slide glass under conditions where total reflection occurs. In the case of interphase, this method utilizes the fact that light with a very short range called “evanescent wave” (called “near field light”) oozes out from the interface only in the near field of several hundred nm. By this method, excitation light of the fluorescent material is incident from the end face, and only the fluorescent material existing in the near field is excited to perform fluorescence observation. The evanescent wave excitation type fluorescence detection method is described in Kuno et al., Mol Cell Proteomics, 2008 Aug 11, etc. For this detection, GlycoStation ^™ Reader 1200 (Mortex) or the like can be used.

  It is preferable to use a lectin as the sugar chain binding substance. “Lectin” is a general term for proteins that recognize and bind to a specific sugar chain structure. A sugar chain is generally composed of a plurality of types of sugars, and each sugar has a variety of binding modes, and thus has a diverse and complicated structure. As lectins, those derived from many animals and plants are known. For example, galectin which is an animal lectin family having affinity for galactose; C-type lectin which is a calcium ion-dependent animal lectin family; glycosaminoglycan Annexin, legume lectin, lysine, and the like that exhibit a certain affinity.

  The difference between transferrin-1 and 2 is that transferrin-1 has GlcNAc at the non-reducing end and transferrin-2 has Siaα2,6Gal at the non-reducing end. Therefore, it is preferable to use a lectin that specifically recognizes the GlcNAc terminus and a lectin that specifically recognizes the Siaα2,6Gal terminus as the substance that specifically binds to each sugar chain moiety.

  As the lectin that specifically binds to the sugar chain portion of transferrin-1, any lectin may be used as long as it can specifically recognize a sugar chain having GlcNAc at the non-reducing end. Among such lectins, PVL (Psathyrella Velutina) lectin is most preferable. PVL lectin is a lectin derived from Musinatake that belongs to basidiomycete, a kind of fungus, and GlcNAc is known to recognize a sugar chain structure present at the non-reducing end.

  As a lectin that specifically binds to the sugar chain portion of transferrin-2, any lectin may be used as long as it can specifically recognize a sugar chain having Siaα2,6Gal at the non-reducing end. Examples include, but are not limited to, SSA (Sambucus sieboldian agglutinin) lectin, SNA (Sambucus nigraagglutinin) lectin, and TJA-I (Trichosanthes japonica agglutinin-I) lectin. SSA lectin is preferable.

The lectin can be appropriately selected according to the sugar chain structure of the glycoprotein to be detected. Examples of techniques for analyzing the sugar chain structure of glycoprotein include frontal affinity chromatography (FAC), lectin microarray, sugar chain profiling, MS or MS ⁿ (mass spectrometry and tandem mass spectrometry). If the sugar chain structure of a glycoprotein is determined, an appropriate lectin can be selected based on the information. Information on lectins can be obtained from the lectin frontier database (LfDB) or the homepage of the National Institute of Advanced Industrial Science and Technology, Glycomedical Engineering Research Center.

  A sugar chain antibody can also be used as the sugar chain binding substance. When using a sugar chain antibody, it is preferable to use an antibody that specifically binds to a sugar chain having GlcNAc at the non-reducing end and an antibody that specifically binds to a sugar chain having Siaα2,6Gal at the non-reducing end.

  As a substance that specifically binds to the protein portion of transferrin, an antibody is preferably used. A commercially available antibody may be used as such an antibody, but an antibody specific for the protein portion of transferrin may be prepared by a method known per se based on the sequence information of transferrin.

  Examples of commercially available antibodies include, but are not limited to, Bethyl Laboratories, Inc. anti-human transferrin antibodies (eg, A200-107A, A80-128, A80-128A, A80-128P, etc.).

  Based on the sequence information of human transferrin, a partial peptide thereof is prepared, and an anti-human transferrin antibody can be prepared according to a method known per se as described below. As these peptides, any peptide may be used as long as the prepared anti-transferrin antibody does not cross-react with an irrelevant antigen that may be contained in the sample. Deletions and the like may be included. For example, the peptide may be a peptide close to or far from a transfer chain sugar chain-binding amino acid residue. Alternatively, in the detection of transferrin-1 or 2, it is also conceivable to use an antibody that recognizes the protein portion and the sugar chain portion together.

  The antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known antibody or antiserum production methods. For the production of an antibody, the protein part of transferrin or the sugar chain part of transferrin-1 or 2 is used as an antigen (antigen of the present invention).

<Preparation of monoclonal antibody>
Monoclonal antibody-producing cell The antigen of the present invention is administered to a mammal at a site where antibody production is possible by administration, together with a carrier or a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.

  When producing monoclonal antibody-producing cells, select a mammal with an antigen titer from an antigen-immunized mammal, such as a mouse, and collect spleen or lymph nodes 2-5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells to be fused with myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled antigen of the present invention with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.

  Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to be used and the number of myeloma cells is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.

  Various methods can be used for screening monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which the antigen of the present invention is adsorbed directly or together with a carrier, and then radioactive. A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A; A method of detecting a monoclonal antibody bound to a solid phase by adding a hybridoma culture supernatant to a solid phase adsorbed with an anti-immunoglobulin antibody or protein A, adding the antigen of the present invention labeled with a radioactive substance or enzyme, etc. Can be mentioned.

  Selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto, but can usually be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1-10% fetal calf serum, or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.

Phage display antibody library Another approach to making monoclonal antibodies is to use phage display. In this method, PCR mutations may be introduced in addition to CDRs, so there are few reports of HAHA production in the clinical stage, but there is no risk of infection with heterologous viruses derived from the host animal. And the specificity of the antibody is infinite (antibodies against prohibited clones, sugar chains, etc. can be easily produced).

Examples of the method for preparing a phage display antibody library include, but are not limited to, the following.
The phage to be used is not particularly limited, but usually filamentous phage (Ff bacteriophage) is preferably used. As a method for displaying a foreign protein on the phage surface, there is a method of expressing and displaying on a coat protein as a fusion protein with any of the coat proteins of g3p and g6p to g9p, but g3p or g8p is often used. This is a method of fusing to the N-terminal side. The phage display vector includes 1) a foreign protein fused to the coat protein gene of the phage genome, and all the coat proteins displayed on the phage surface are presented as fusion proteins with the foreign protein. ) A gene that encodes the fusion protein is inserted separately from the wild-type coat protein gene, and the fusion protein and the wild-type coat protein are expressed simultaneously, or 3) E. coli having a phagemid vector having the gene encoding the fusion protein Infecting a helper phage having a wild type coat protein gene to produce a phage particle that simultaneously expresses the fusion protein and the wild type coat protein. Infectious ability Because dividing, for the production of antibody libraries 2) or 3) of types used.

Specific examples of the vector include those described in Holt et al. (Curr. Opin. Biotechnol., 11: 445-449, 2000). For example, pCES1 (see J. Biol. Chem., 274: 18218-18230, 1999) is a DNA encoding a κ light chain constant region downstream of a g3p signal peptide under the control of one lactose promoter and a g3p signal peptide. This is a Fab expression type phagemid vector in which DNA encoding CH3, His-tag, c-myc tag, and g3p coding sequence are arranged via an amber stop codon (TAG) downstream. When introduced into Escherichia coli having an amber mutation, Fab is displayed on the g3p coat protein, but when expressed in an HB2151 strain without an amber mutation, a soluble Fab antibody is produced. Moreover, as the scFv expression type phagemid vector, for example, pHEN1 (J. Mol. Biol., 222: 581-597, 1991) is used.
On the other hand, examples of the helper phage include M13-KO7 and VCSM13.
In addition, as another phage display vector, a sequence containing a codon encoding cysteine is linked to the 3 ′ end of the antibody gene and the 5 ′ end of the coat protein gene, and both genes are separated separately (not as a fusion protein). Examples include those designed to be expressed and capable of displaying an antibody on a coat protein on the surface of a phage via SS bonds between the introduced cysteine residues (CysDisplay ^™ technology of Morphosys).

  Examples of antibody libraries include naive / non-immunized libraries, synthetic libraries, immune libraries, and the like.

  A naive / non-immunized library is obtained by RT-PCR of VH and VL genes possessed by normal animals (such as humans) and randomly cloning them into the above phage display vectors. It is a library. Usually, mRNA from lymphocytes such as peripheral blood, bone marrow, and tonsils of normal individuals is used as a template. In order to eliminate V gene bias such as disease history, an amplification of only IgM-derived mRNA that has not undergone class switching due to antigen sensitization is called a naive library. Representative examples include CAT libraries (see J. Mol. Biol., 222: 581-597, 1991; Nat. Biotechnol., 14: 309-314, 1996), MRC libraries (Annu Rev. Immunol., 12: 433-455, 1994), Dyax library (J. Biol. Chem., 1999 (supra); Proc. Natl. Acad. Sci. USA, 14: 7969-7974, 2000).

  A synthetic library selects functional specific antibody genes in B cells, and replaces a portion of an antigen binding region such as CDR3 of a V gene fragment with DNA encoding a random amino acid sequence of an appropriate length. It is a library. Since a library can be constructed from a combination of VH and VL genes that produce functional scFv and Fab from the beginning, it is said to be excellent in antibody expression efficiency and stability. Representative examples include Morphosys' HuCAL library (see J. Mol. Biol., 296: 57-86, 2000), BioInvent's library (see Nat. Biotechnol., 18: 852, 2000), And a library of Crucell (see Proc. Natl. Acad. Sci. USA, 92: 3938, 1995; J. Immunol. Methods, 272: 219-233, 2003).

  The immunized library can be used for the above naive / non-immunized live from lymphocytes collected from individuals whose blood antibody titer against the target antigen has increased or human lymphocytes that have been artificially immunized with the target antigen by the in vitro immunization method. In the same manner as in the case of the rally, mRNA was prepared, and the VH and VL genes were amplified by RT-PCR method and made into a library. Since the target antibody gene is contained in the library from the beginning, the target antibody can be obtained even from a relatively small size library.

The greater the diversity of the library, the better. However, in reality, the number of phages that can be handled by the following panning operations (10 ¹¹ to 10 ¹³ phages) and the number of phages necessary for isolation and amplification of clones by normal panning (100 In consideration of (˜1,000 phage / clone), about 10 ⁸ to 10 ¹¹ clones are appropriate, and an antibody having a Kd value of usually about 10 ⁻⁹ order can be screened with a library of about 10 ⁸ clones.

The process of selecting antibodies against the target antigen by the phage display method is called panning. Specifically, for example, a carrier on which an antigen is immobilized is contacted with a phage library, and after washing away unbound phage, the bound phage is eluted from the carrier, and the phage is propagated by infecting E. coli. The phages displaying antigen-specific antibodies are concentrated by repeating a series of operations 3 to 5 times. As a carrier for immobilizing an antigen, various carriers used in normal antigen-antibody reaction and affinity chromatography, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass, Examples include microplates, tubes, membranes, columns, beads, and the like made of metal, and surface plasmon resonance (SPR) sensor chips. For the immobilization of the antigen, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. For example, a biotin- (strept) avidin system is preferably used. When the endogenous ligand that is the target antigen is a small molecule such as a peptide, special care must be taken so that the moiety used as the antigenic determinant is not covered by binding to the carrier. For washing of unbound phage, blocking solution such as BSA solution (1-2 times), PBS containing a surfactant such as Tween (3-5 times) and the like can be used sequentially. There are also reports that the use of citrate buffer (pH 5) and the like is preferable. For elution of specific phages, acids (eg, 0.1M hydrochloric acid) are usually used, but cleavage by specific proteases (eg, gene sequences encoding trypsin cleavage sites at the junctions of antibody genes and coat protein genes) In this case, since the wild-type coat protein is displayed on the surface of the eluted phage, even if all of the coat protein is expressed as a fusion protein, infection and growth of E. coli are possible) Competitive elution with soluble antigen, or reduction of SS binding (for example, with CysDisplay ^™ , after antigen panning, the antigen-specific phage is recovered by dissociating the antibody and coat protein using an appropriate reducing agent. Elution) is also possible. In the case of elution with acid, after neutralization with Tris or the like, the eluted phage is infected with E. coli, and after culturing, the phage is recovered by a conventional method.

  When the phages displaying antigen-specific antibodies are concentrated by panning, they are infected with E. coli and then seeded on a plate for cloning. The phages are collected again, and the antigen binding activity is confirmed by measurement using the above-described antibody titer measurement method (eg, ELISA, RIA, FIA, etc.) or FACS or SPR.

  Isolation and purification of antibodies from phage clones displaying selected antigen-specific antibodies can be achieved, for example, when using a vector in which an amber stop codon is introduced at the junction between the antibody gene and the coat protein gene as a phage display vector. When the phage is infected with E. coli without an amber mutation (eg HB2151 strain), soluble antibody molecules are produced and secreted into the periplasm or medium. Can be recovered and purified using the same purification technique as described above. If His-tag or c-myc tag is introduced, it can be easily purified using IMAC or anti-c-myc antibody column. In addition, when utilizing cleavage by a specific protease during panning, the antibody molecule is separated from the phage surface when the protease is acted on, so the target antibody is purified by carrying out the same purification operation. be able to.

  Separation and purification of monoclonal antibodies can be carried out in the same way as in the separation and purification of ordinary polyclonal antibodies (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchanger (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method for obtaining an antibody by dissociating the binding using an active adsorbent such as protein A or protein G) Can be done according to

<Preparation of polyclonal antibody>
The polyclonal antibody can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (antigen of the present invention) and a carrier protein is prepared, a mammal is immunized in the same manner as the above-described monoclonal antibody production method, and an antibody-containing substance against the antigen of the present invention is immunized from the immunized animal. It can be produced by collecting and purifying the antibody.

  Regarding the complex of immune antigen and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of carrier and hapten should be such that antibodies can be efficiently produced against haptens that are immunized by cross-linking to carriers. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin and the like are about 0.1 to 20 in weight ratio with respect to hapten 1. Preferably, a method of coupling at a ratio of about 1 to 5 is used.

  Various coupling agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.

  The condensation product is administered to a mammal at a site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration can usually be carried out about once every 2 to 6 weeks, about 3 to 10 times in total. Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably from blood.

  The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

The antibody of the present invention is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen. In addition to a complete antibody molecule, for example, Fab, Fab ′, F (ab ') ₂ such as fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having protein stabilizing action such as polyethylene glycol (PEG) They may be modified derivatives thereof.

  In the method of the present invention, when the amount of transferrin-1 is significantly reduced in a body fluid sample derived from a subject as compared to the amount in a body fluid sample (eg, cerebrospinal fluid) derived from a healthy person, the subject Determined to be iNPH patient. Since the amount of transferrin-1 in cerebrospinal fluid varies greatly among individuals, the ratio of transferrin-1 and transferrin-2, not the absolute amount of transferrin-1, should be used as a diagnostic marker for the definitive diagnosis of iNPH. More preferred. In the method of the present invention, the value of transferrin-2 / transferrin-1 (referred to herein as “sugar chain index”) is used as a diagnostic index. The relative decrease in transferrin-1 is expressed as an increase in glycan index.

  For example, in the case of Example 2, the sugar chain index value in the cerebrospinal fluid sample of a healthy person was 0.96 ± 0.24 (mean ± S.D.), Whereas in iNPH patients, it increased to about 2.96 ± 1.65. From this result, when the glycan index value derived from the subject is 1.44 or more (average value of normal values + 2 × S.D.), The subject can be determined as an iNPH patient.

  Specific numerical values of the sugar chain index or any other parameter that can be set based on the amount of transferrin-1 and / or transferrin-2 in the body fluid sample used as a criterion are false negative and It is desirable to set to minimize false positive results. Such numerical values can be appropriately set by those skilled in the art. In order to reduce false negative / false positive results as much as possible, the method of the present invention may be combined with other test methods as necessary. Examples of such examination methods include examination of clinical symptoms (dementia, gait disturbance, urinary incontinence, etc.), head CT, head MRI, RI cisternography, MRI cisternography, intracranial pressure measurement, cerebral blood flow Measurement and the like. For example, it is assumed that the test according to the present invention is performed in a patient who is strongly suspected of iNPH by the above test method.

  For calculation of the glycan index, it is preferable to use the results of quantification using PVL lectin and SSA lectin as substances that specifically bind to the sugar chain portions of transferrin-1 and 2, respectively. Quantification may be performed using the lectin.

  Examples of the method for measuring the amount of transferrin-1 and 2 include, but are not limited to, a method using a lectin as described in Example 2 and a method of performing Western blotting using an anti-transferrin antibody. The value of the sugar chain index serving as a diagnostic criterion for iNPH can be appropriately set according to the type of binding substance used for measurement of transferrin-1 and 2, the measurement method, and the like.

  Examples of the body fluid used in the method of the present invention include, but are not limited to, cerebrospinal fluid, blood, saliva, tears, nasal discharge, and preferably cerebrospinal fluid. Even when a body fluid other than cerebrospinal fluid is used, transferrin can be detected and quantified by the same method as in the above-described cerebrospinal fluid. These body fluid samples may be collected by a commonly used method. Moreover, since saliva, tears, nasal discharge and the like can be collected by a non-invasive method, it is preferable to use them in order to reduce the burden on the patient.

  When cerebrospinal fluid is used as the body fluid, the cerebrospinal fluid can be collected from the subject by a normal method such as lumbar puncture. The amount of the cerebrospinal fluid sample to be collected is preferably selected within a range that does not increase the burden on the subject, and is preferably 2 ml or less, more preferably 1 ml or less. Also, an amount of at least 200 μl, preferably at least 60 μl is sufficient for use in the diagnosis of the present invention.

  As the cerebrospinal fluid or other samples, those collected from the subject may be used as they are, or operations such as dilution may be performed as necessary. When diluting, for example, it is diluted 20 times with a buffer solution or the like.

  The present invention also includes an idiopathic normal pressure head comprising one or more substances selected from the group consisting of a substance that specifically binds to the protein portion of transferrin and a substance that specifically binds to the sugar chain portion of transferrin-1 A diagnostic kit for the disease is provided. The kit may further include a substance that specifically binds to the sugar chain portion of transferrin-2. The kit also includes a substance that specifically binds to the protein part of transferrin, a substance that specifically binds to the sugar chain part of transferrin-1, and a substance that specifically binds to the sugar chain part of transferrin-2. Can be a thing. Each binding substance is as described above.

  The kit may further include a container (the above-mentioned support and the like), various reagents and buffers necessary for each reaction, instructions including a test protocol, and the like.

  In this kit, a substance that specifically binds to the protein portion of transferrin may be preliminarily immobilized on a support.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

Example 1
The textbook states that the glycoprotein in cerebrospinal fluid is a leaked serum protein. However, based on the assumption that glycoproteins with sugar chains unique to the central nervous system exist, the present inventors have different sugar chains in (and presumed to be) sugar chains in cerebrospinal fluid and serum. Protein screening was performed. Several dozen or more kinds of specific antibodies against glycoproteins in serum are commercially available. Most of these antibodies are antibodies against the protein portion of the glycoprotein. Since the mobility on SDS-PAGE often differs when the sugar chain structure is different, Western blotting was performed using these antibodies. Glycoproteins in cerebrospinal fluid showing a different mobility band from the corresponding glycoproteins in serum were used as candidates for cerebrospinal fluid markers. Western blotting conditions are very common.
Even when the mobility of candidate molecules differs between serum and cerebrospinal fluid, this difference in mobility is not necessarily based on the difference in sugar chain structure. Therefore, the N-glycanase was used to remove N-linked sugar chains from the glycoprotein and the Rf value was examined before and after digestion to examine changes in the Rf value due to N-glycan digestion.

  Among antibodies against various glycoproteins, Western blotting was performed using an anti-transferrin antibody (Cat. No. A80-128A; BETHYL Laboratories, Inc.). As shown in FIG. Different band patterns were obtained. Of the two bands observed in the cerebrospinal fluid sample, the one showing the same Rf value as the transferrin in serum was designated as transferrin-2, and the one showing the lower Rf value was designated as transferrin-1. This suggested that transferrin-1 may have a characteristic sugar chain structure different from serum transferrin and transferrin-2. When transferrin-1, transferrin-2, and serum transferrin as a control were treated with N-glycanase, the mobility of all bands increased and showed the same mobility (not shown). From this, it was shown that the difference in mobility between transferrin-1, transferrin-2 and serum transferrin is based on the difference in the structure of their N-glycans.

  In order to determine the sugar chain structure of each transferrin and search for lectins that can distinguish them, transferrin-1 and transferrin-2 were completely purified from cerebrospinal fluid (FIG. 1B) and analyzed on a lectin microarray together with serum transferrin ( Figure 2).

Purification of transferrin-1 and 2 Cerebrospinal fluid samples are desalted by dialysis. After that, albumin is removed with HiTrap Blue. Next, purification is performed by anion exchange chromatography (Q Sepharose). For adsorption, 20 mM Tris-HCl (pH 8.0) buffer is used. After washing the column with the same buffer, elution with a linear concentration gradient of sodium chloride is performed using a buffer obtained by adding 1 M sodium chloride to the same buffer as the final buffer. As will be described later, since transferrin-1 does not have sialic acid (negative charge), it is eluted at the beginning of the sodium chloride concentration gradient. Transferrin-2 elutes as a major peak immediately thereafter. In some cases, each peak is purified by re-chromatography with Q Sepharose.
Lectin microarray transferrin-1 and -2 were purified to near homogeneity on SDS-PAGE (FIG. 1B). Simultaneously with both samples, commercially available purified transferrin (serum-derived; apo-Transferrin human (Sigma: T4382)) was used as a control. Purified transferrin is added onto a lectin microarray where 43 lectins are spotted on a glass slide. Thereafter, an anti-human transferrin antibody is added, and a biotinylated secondary antibody is added for detection. Fluorescently labeled streptavidin is added to detect the signal. Since the evanescent wave is detected in this experimental system, the signal can be detected in real time without washing away excess reagents.
As a result, as shown in FIG. 2, serum transferrin and transferrin-2 gave very similar signal patterns, but the signal pattern of transferrin-1 was completely different from them. For example, serum transferrin and transferrin-2 gave very strong signals to those showing affinity for Siaα2,6Gal such as SSA, SNA, TJA-I lectin. On the other hand, transferrin-1 hardly reacted with these lectins. From these results, it was suggested that transferrin-1 has a very unique sugar chain structure different from serum transferrin and transferrin-2. Detailed analysis of the lectin signal suggested that transferrin-1 has GlcNAc as a terminal sugar.

Example 2
Since cerebrospinal fluid metabolism abnormalities are suspected in iNPH, in order to investigate whether or not glycoprotein transferrin-1 unique to cerebrospinal fluid can be used as a diagnostic marker for iNPH, changes in transferrin-1 are We examined the cerebrospinal fluid samples of healthy subjects. Based on the results of the lectin microarray, PVL lectin was selected as a probe for specifically detecting the sugar chain of transferrin-1.
Since biotinylated PVL is not commercially available, unlabeled PVL lectin (Wako Pure Chemicals, Cat. No.165-17591) was purchased and biotinylated in the presence of 10 mM GlcNAc. 10 mM GlcNAc is used so that the sugar chain binding site of PVL lectin is biotinylated and is not inactivated. PVL lectin is biotinylated agent Ez-Link NHS-Biotin (Pierce, Cat. No. 21336) and 10 mM sodium phosphate buffer, pH 7.0, 130 mM NaCl, 10% glycerol for 4 hours at room temperature. Reacted. The reaction is stopped with 100 mM Tris, dialyzed against 10% glycerol, and stored frozen.
Sample buffer for SDS-PAGE is added to the cerebrospinal fluid sample, and electrophoresis is performed using a polyacrylamide gel having a concentration gradient of 5 to 20%. The glycoprotein is transferred to a nitrocellulose membrane according to a conventional method. After blocking with bovine serum albumin / PBS (phosphate buffered saline), biotinylated PVL (3.3 μg / ml) was reacted at room temperature for 1 hour. The membrane was washed with PBS, 0.1% tween, 5 min x 3 times. Detection is performed with streptavidin conjugated with HRP (Horseradish Peroxidase) (Thermo SCIENTIFIC, # 21126, 1 mg / ml is diluted 1: 2000 in 1% BSA / PBS). Pierce Super Signal is used as the chemiluminescent substrate. As a detector, Ato's Cool Saver AE-6955 is used. In Western blotting of transferrin, for example, BETHYL Laboratories, Inc. Cat. No. A80-128A Lot No. A80-128A-5 is used as an anti-human transferrin antibody.
Using cerebrospinal fluid samples from healthy subjects and iNPH patients, the amount of transferrin-1 in the cerebrospinal fluid was measured (FIG. 3B). As a result, it was shown that the amount of transferrin-1 in the cerebrospinal fluid sample of iNPH patients tended to decrease compared with that in healthy subjects, but on the other hand, the total protein amount and transferrin protein amount in human cerebrospinal fluid There are many individual differences, and it was shown that it is not optimal to use the absolute amount of transferrin-1 as a diagnostic index. Therefore, we examined whether the ratio of transferrin-1 and 2 can be used as a diagnostic index. In the same manner as described above for transferrin-1, the transferrin-2 signal was detected using biotinylated SSA lectin (Seikagaku Biobusiness Co., Ltd., # 300442) (FIG. 3A). The conditions are the same as those for detection of transferrin-1, except for the exposure time to the detector. The SSA signal / PVL signal (transferrin-2 / transferrin-1) was divided and defined as the sugar chain index. Since the PVL signal is the denominator, the value of the sugar chain index increases when the value of the PVL signal decreases. Transferrin-1 and 2 were quantified by lectin blot, and the glycan index was determined to be 0.96 ± 0.24 (mean ± SD) for disease control, while it increased to about 2. 3-fold ± 2.96 ± 1.65 for patients with iNPH. It became clear that (Figure 4).
From the above, it was shown that the ratio of transferrin-1 and 2 can be a diagnostic index for iNPH.

Example 3
FIG. 4 shows the results of the lectin blot. The lectin / antibody sandwich ELISA method is optimal for screening a large number of specimens. The sandwich ELISA method can be performed using a Human Transferrin ELISA Quantitation Kit (BETHYL). An anti-human transferrin antibody (BETHYL Laboratories, Inc. Cat. No. A80-128A) is adsorbed to the plate in the presence of 0.05M carbonate buffer (pH 9.6). After washing with TBS (50 mM Tris, (pH 8.0) 140 mM NaCl), overnight blocking is performed with a blocking buffer (TBS + 10% Block Ace (No. UK-B80, Snow Brand Milk Products)). Cerebrospinal fluid samples are treated with 6M urea and then added to the plate in 0.05% NP40 / TBS. Wash well with TBST (TBS + 0.05% Tween 20) and add biotinylated PVL. After washing with TBST, TMB (Catalog No. 50-7600, KPL) is used as a chromogenic substrate and quantified with a plate reader.
Using this method, a calibration curve was generated for each transferrin. Transferrin (corresponding to transferrin-1) having a GlcNAc-terminal sugar chain used for preparing a calibration curve was prepared by converting transferrin containing Sia α2,6 galactose purified from serum to sialidase (Catalog No. 24229-74, Nacalai Tesque) and It was prepared by treatment with galactosidase (Catalog No. 100570, Seikagaku Corporation).
The principle of the method used in this example is shown in FIG. The figure on the left shows how to detect sugar chains using an antibody (BETHYL Laboratories, Inc. Cat. No. A80-128A) (capture antibody) as a capture substance and PVL lectin as a detection substance. The figure shows a method for detecting transferrin-1 core protein using antibodies as both capture and detection substances. The antibody (detection antibody) used as the detection substance was the one contained in the Human Transferrin ELISA Quantitation Kit (BETHYL). The lower graph of FIG. 5 shows the results of a calibration curve prepared using transferrin having the GlcNAc terminal sugar chain prepared in this way. Transferrin-2 is also detected in the same manner using SSA lectin.
From these results, it was shown that transferrin can be quantitatively measured by using a lectin and an antibody as detection substances.

  Since transferrin-1 is a diagnostic marker that can be measured even in tens of microliters of cerebrospinal fluid, the diagnostic method of the present invention has traditionally benefited from surgical treatment despite being potentially curable. It becomes possible to diagnose iNPH as a false negative patient or an indeterminate case that has been left unattended. Therefore, the present invention is socially important not only from the patient's healing but also from the viewpoint of reducing the burden on the caregiver while the aging society is progressing remarkably.

FIG. 1A shows that, unlike serum, there are two isoforms (transferrin-1 and 2) in cerebrospinal fluid. FIG. 1B shows the purification results of each isoform in cerebrospinal fluid. FIG. 2 shows lectin microarray results for transferrin-1 and -2 and serum transferrin in cerebrospinal fluid. FIG. 3A is a diagram showing the results of detection of transferrin using an antibody and a lectin. FIG. 3B shows the results of staining transferrin-1 in a cerebrospinal fluid sample (Control) derived from healthy subjects and cerebrospinal fluid samples derived from iNPH patients with PVL lectin. FIG. 4 is a diagram showing sugar chain indexes in a cerebrospinal fluid sample (Control) derived from a healthy person and a cerebrospinal fluid sample derived from an iNPH patient. FIG. 5 shows a schematic diagram of the transferrin-1 detection method (upper) and a calibration curve (lower) obtained by each method.

Claims (21)

Has a-sialylated or a-galacto structure as a sugar chain in a body fluid collected from a subject, you see contains measuring the amount of transferrin having GlcNAc terminus, body fluid is cerebrospinal fluid, blood, saliva, tears and nasal A test method for diagnosing idiopathic normal pressure hydrocephalus in the subject, wherein the test method is selected from the group consisting of : Measuring the amount of transferrin-2 in the body fluid, and calculating the value of transferrin having a sialo or a galacto structure as transferrin-2 / sugar chain and having a GlcNAc terminus,
The method of claim 1, further comprising: A substance that specifically binds to the protein part of transferrin, and a substance that specifically binds to the sugar chain part of transferrin having an a-sialo or a-galacto structure as a sugar chain and having a GlcNAc terminus,
The method according to claim 1, wherein one or more substances selected from the group consisting of:   The method according to claim 3, further comprising using a substance that specifically binds to the sugar chain portion of transferrin-2. A substance that specifically binds to the protein portion of transferrin,
A substance that has an a-sialo or a-galacto structure as a sugar chain and specifically binds to the sugar chain part of transferrin having a GlcNAc terminus, and a substance that specifically binds to the sugar chain part of transferrin-2;
The method according to claim 3, wherein   The method according to any one of claims 3 to 5, wherein the substance that specifically binds to the protein portion of transferrin is an anti-human transferrin antibody.   The method according to any one of claims 3 to 5, wherein the substance that specifically binds to a sugar chain part of transferrin having an a-sialo or a-galacto structure as a sugar chain and having a GlcNAc terminus is lectin.   The method according to claim 4 or 5, wherein the substance that specifically binds to the sugar chain portion of transferrin-2 is lectin.   The method according to claim 7, wherein the substance that specifically binds to a sugar chain part of transferrin having an a-sialo or a-galacto structure as a sugar chain and having a GlcNAc terminus is PVL lectin.   The method according to claim 8, wherein the substance that specifically binds to the sugar chain portion of transferrin-2 is SSA lectin.   The method according to claim 1 or 2, wherein the body fluid is cerebrospinal fluid.   The method according to claim 1 or 2, wherein the measurement is performed using a sandwich ELISA. A substance that specifically binds to the protein part of transferrin, and a substance that specifically binds to the sugar chain part of transferrin having an a-sialo or a-galacto structure as a sugar chain and having a GlcNAc terminus,
A kit for use in the method of claim 3, comprising one or more substances selected from the group consisting of:   The kit according to claim 13, further comprising a substance that specifically binds to a sugar chain portion of transferrin-2. A substance that specifically binds to the protein portion of transferrin,
A substance that has an a-sialo or a-galacto structure as a sugar chain and specifically binds to the sugar chain part of transferrin having a GlcNAc terminus, and a substance that specifically binds to the sugar chain part of transferrin-2;
The kit according to claim 13, comprising:   The kit according to any one of claims 13 to 15, wherein the substance that specifically binds to the protein portion of transferrin is an anti-human transferrin antibody.   The kit according to any one of claims 13 to 15, wherein the substance that specifically binds to a sugar chain part of transferrin having an a-sialo or a-galacto structure as a sugar chain and having a GlcNAc terminus is a lectin.   The kit according to any one of claims 13 to 15, wherein the substance that specifically binds to the sugar chain portion of transferrin-2 is a lectin.   The kit according to claim 17, wherein the substance that specifically binds to a sugar chain part of transferrin having an a-sialo or a-galacto structure as a sugar chain and having a GlcNAc terminus is PVL lectin.   The kit according to claim 18, wherein the substance that specifically binds to the sugar chain part of transferrin-2 is SSA lectin.   The kit according to claim 13, wherein a substance that specifically binds to the protein portion of transferrin is immobilized on a support.