WO2013024867A1 - Antibody and use thereof - Google Patents

Antibody and use thereof Download PDF

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WO2013024867A1
WO2013024867A1 PCT/JP2012/070747 JP2012070747W WO2013024867A1 WO 2013024867 A1 WO2013024867 A1 WO 2013024867A1 JP 2012070747 W JP2012070747 W JP 2012070747W WO 2013024867 A1 WO2013024867 A1 WO 2013024867A1
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reelin
antibody
amino acid
active
seq
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PCT/JP2012/070747
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French (fr)
Japanese (ja)
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光治 服部
孝夫 河野
真利 鯉江
有紗 久永
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公立大学法人名古屋市立大学
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Publication of WO2013024867A1 publication Critical patent/WO2013024867A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

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  • the present invention relates to antibodies and their use. More specifically, the present invention relates to a novel anti-active Reelin antibody that specifically binds to active Reelin and does not bind to inactive Reelin, and variable regions of the H chain and L chain of this antibody.
  • the present invention relates to an encoding nucleic acid or a nucleic acid encoding a CDR sequence portion in these regions, a hybridoma cell producing this antibody, an active Reelin measurement method using this antibody, and an active Reelin measurement kit used therefor.
  • Reelin is a large secreted protein consisting of over 3400 amino acids that is important for brain function. It has been reported that Reelin is essential for the formation of a characteristic layered structure of the brain, and that even humans fail to form brain structures when Reelin is deficient. On the other hand, Reelin is also expressed in the adult brain, and it has become clear that a decrease in expression level and an increase in degradation contribute to diseases such as schizophrenia, Alzheimer's disease, and mood disorders.
  • Patent Document 1 discloses a Reelin epitope region polypeptide and a polynucleotide encoding the Reelin, thereby further studying Reelin's function and resulting from Reelin gene abnormality and neuronal arrangement abnormality. It is said that it can provide diagnostic and therapeutic means for brain disorders.
  • Patent Document 2 proposes a method for measuring Reelin as a biomarker for nondestructively evaluating or predicting DIIA levels in other important components of the central nervous system.
  • Reelin the extracellular matrix protein deficient in reeler mutantmice, is processed by a metalloproteinase.” Lambert de Rouvroit C, de Bergeyck V, Cortvrindt C, Bar I, Eeckhout Y, 1999 : 214-7. “Mechanism and significance of specific proteolytic cleavage ofReelin.” Kohno S, Kohno T, NakanoY, Suzuki K, Ishii M, Tagami H, Baba A, Hattori M. Biochem Biophys Res Commun27 1 .
  • Nt site an unknown site close to the N-terminal side of the amino acid sequence of Reelin protein
  • Ct site an unknown site close to the side
  • test samples prepared for research or measurement of Reelin include a mixture of full-length Reelin that has not undergone intramolecular degradation and degraded Reelin that has undergone intramolecular degradation.
  • full-length Reelin is active, but degraded Reelin that has undergone intramolecular degradation at Nt ⁇ ⁇ site is inactive (see Non-Patent Document 2), while Ct site Degraded Reelin that has undergone intramolecular degradation has been found to maintain somewhat reduced activity.
  • electrophoresis was performed using the difference in molecular weight between full-length Reelin and decomposed Reelin.
  • electrophoresis of full-length Reelin and large-scale Reelin, which are large proteins is very time-consuming and lacks practicality in terms of efficiency.
  • the present invention has a technical problem to be solved to provide a means for selectively measuring active Reelin easily and quickly even with a large amount of sample.
  • the inventor of the present application has specifically identified the degradation site of N-t site, which has not been elucidated in the amino acid sequence of Reelin protein. Furthermore, the present inventors succeeded in producing a hybridoma cell that produces a monoclonal antibody using a peptide having this amino acid sequence as an antigen. As a result, Reelin that has not undergone degradation at N-t site, that is, an anti-reactive Reelin antibody that specifically binds only to active Reelin, was obtained, thereby completing the present invention.
  • the configuration of the first invention for solving the above-described problems is an anti-active Reelin antibody that is an antibody that specifically binds to active Reelin but does not bind to inactive Reelin.
  • the category of “antibody” is not limited, but a monoclonal antibody produced by a hybridoma cell having the necessary conditions is particularly preferable.
  • Active Reelin refers to Reelin that has not undergone degradation at N-t site
  • active Reelin refers to Reelin that has undergone degradation at N-t site.
  • the configuration of the second invention for solving the above problem is an anti-active Reelin antibody, which is an antibody that specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
  • the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is an eight amino acid sequence centering on the unresolved Nt site degradation site of Reelin protein. Occurs in this amino acid sequence, particularly between proline (Pro) and alanine (Ala) in this amino acid sequence. As will be described later in the sequence listing, these eight amino acid sequences and their degradation sites were actually determined from mouse Reelin, but the above amino acid sequences and their degradation sites also exist in human Reelin. Is exactly the same.
  • “having (having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing)” means consisting of the amino acid sequence or partially including this amino acid sequence. Also in the following description of the present specification, when “having” a specific amino acid sequence or base sequence has the same meaning as described above.
  • the configuration of the third invention for solving the above-mentioned problem is that an H chain variable region having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing and an L chain variable region having the amino acid sequence shown in SEQ ID NO: 3 of the sequence listing It is an anti-active Reelin antibody which is an antibody comprising a polypeptide having the same.
  • the constitution of the fourth invention for solving the above-mentioned problem is that the heavy chain variable region having an amino acid sequence in which 1 to several amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And an L chain variable region having an amino acid sequence in which 1 to several amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 3 in the Sequence Listing, and has the following (1 ), An anti-active Reelin antibody, which is an antibody corresponding to at least one of (2).
  • an amino acid sequence in which one to several amino acids are substituted for the amino acid sequence shown in SEQ ID NO: 2 or 3 in the sequence listing includes conservative substitution of amino acids.
  • a “conservative substitution” is the replacement of a given amino acid residue with another chemically or functionally similar amino acid residue so that the overall function of the polypeptide remains substantially unchanged.
  • Examples of chemically or functionally similar amino acids include hydrophobic amino acids (Ala, Ile, Leu, Phe, Pro, Trp, Val, Met), polar but uncharged amino acids (Asn, Cys, Gln, Gly, Ser, Thr, Tyr), basic amino acids (Arg, His, Lys), acidic amino acids (Asp, Glu), etc. (translated by EECornn et al., Nobuo Tamiya et al., “Corn Stamp Biochemistry” 5th edition "Tokyo Chemical Doujinshi, p56-58, 1988).
  • the structure of the fifth invention for solving the above-mentioned problems is shown in the heavy chain variable region including all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6 in the sequence listing, and SEQ ID NOs: 7 to 9 in the sequence listing.
  • It is an anti-active Reelin antibody, which is an antibody consisting of a polypeptide having an L chain variable region containing all of the CDR sequence consisting of an amino acid sequence.
  • “(including all of the CDR sequences)” means including all of the CDR sequences in part. Also in the following description of the present specification, when “including” a specific amino acid sequence or base sequence, it has the same meaning as described above.
  • the structure of the sixth invention for solving the above-described problems is a nucleic acid corresponding to any of (3) to (8) below.
  • nucleic acids encoding the amino acid sequences shown in SEQ ID NOs: 4 to 9 are nucleic acids encoding the six CDR sequences defined in the fifth invention.
  • Nucleic acid is a concept including sense / antisense DNA or RNA, hybridization probe, PCR primer, template for producing a fusion gene, and the like. Including things.
  • the structure of the seventh invention for solving the above-mentioned problems includes all of the base sequences (3) to (8) described in the sixth invention, and the antiactivity according to any one of the first to fifth inventions A nucleic acid encoding a type Reelin antibody.
  • the “nucleic acid” is a concept including sense / antisense DNA or RNA.
  • the hybridoma cell according to the eighth invention described above is a patent microorganism deposit center (NPMD: NITE Patent) of the National Institute of Technology and Evaluation (NITE) in Japan. Microorganisms Depositary), and the receipt number “NITE ABP-1403” has been issued on August 9, 2012.
  • NPMD NITE Patent
  • NITE ABP-1403 the receipt number “NITE ABP-1403” has been issued on August 9, 2012.
  • this hybridoma cell can be prepared based on common general technical knowledge. Therefore, at least in the Japanese patent system, this does not apply when patent deposits are required.
  • the structure of the ninth invention for solving the above-mentioned problem is that active Reelin in a test sample is measured by an antigen-antibody reaction using the anti-active Reelin antibody according to any one of the first to fifth inventions. This is an active Reelin measuring method.
  • a tenth aspect of the present invention for solving the above-described problems comprises the anti-active Reelin antibody according to any one of the first to fifth aspects of the invention, and is used for measuring active Reelin in a test sample.
  • the anti-active Reelin antibody of the first invention specifically binds to active Reelin and does not bind to inactive Reelin, this antibody is used to detect inactive Reelin in a test sample. Without this, only active Reelin can be measured selectively. Therefore, even for a large amount of samples, it has become possible to selectively measure active Reelin simply and quickly using, for example, a multi-well plate.
  • the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is an amino acid sequence of 4 amino acids on both sides of the Nt ⁇ site degradation site of Reelin protein, which has not been elucidated so far.
  • the active Reelin antibody does not bind to inactive Reelin that has undergone degradation at Nt site, but specifically binds to Nt site non-degradable Reelin that has not undergone degradation at this site.
  • N-t site non-degradable Reelin is an active form, and this includes full-length Reelin and degraded Reelin that has undergone only intramolecular degradation at C-t site.
  • most of the intramolecular degradation of Reelin occurs at Nt site, and relatively little degradation occurs at Ct site. Therefore, the presence of degraded Reelin that has undergone only intramolecular decomposition at Ct site is measured. It can also be virtually ignored as within the error range.
  • the heavy chain variable region and the light chain variable region of the anti-active Reelin antibody according to the present invention are defined. Therefore, the anti-active Reelin antibody of the third invention can be expected to have the same action and effect as the anti-active Reelin antibody of the second invention.
  • anti-reactivity with a slight “sequence change” that has the same function as the anti-active Reelin antibody of the third invention and does not affect the antibody function in the amino acid sequences of the H chain variable region and L chain variable region A type Reelin antibody is provided.
  • the anti-active Reelin antibody of the fifth invention can be expected to have the same actions and effects as the anti-active Reelin antibody of the second invention.
  • nucleic acids according to the sixth and seventh inventions and the hybridoma cells according to the eighth invention are both extremely useful as means for producing or researching the anti-active Reelin antibody according to the present invention.
  • an active Reelin measurement method capable of selectively measuring only active Reelin without detecting inactive Reelin easily and quickly even for a large amount of samples.
  • the tenth invention provides an active Reelin measurement kit for effectively carrying out the active Reelin measurement method of the ninth invention.
  • the full-length Reelin and the decomposed Reelin are shown in a diagram. It is a figure of western blotting which shows the evaluation result of an anti-active type Reelin antibody.
  • the anti-active Reelin antibody according to the present invention is an antibody corresponding to any one of the following 1) to 5).
  • Reelin protein itself is well-known, description of the amino acid sequence etc. is abbreviate
  • An antibody that specifically binds to active Reelin and does not bind to inactive Reelin 2) Specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing It is an antibody.
  • An antibody comprising a polypeptide having an H chain variable region having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and an L chain variable region having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
  • an H chain variable region having an amino acid sequence in which one to several amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; and the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing
  • H chain variable region including all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6 in the sequence listing, and L chain including all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 7 to 9 of the sequence listing
  • An antibody comprising a polypeptide having a variable region.
  • the above-mentioned various antibodies include polyclonal antibodies and monoclonal antibodies, and monoclonal antibodies are particularly preferable. Further, these antibodies include peroxidase, ⁇ -D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase and other enzymes, delphinium for detection and measurement purposes. In addition, a labeled anti-active Reelin antibody bound with a fluorescent label such as, a radioisotope label or isotope label, biotin and the like is also included.
  • nucleic acid encodes the CDR sequence consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6 and the CDR sequence consisting of the amino acid sequences shown in SEQ ID NOs: 7 to 9, respectively (3) to (8) Or a nucleic acid corresponding to any of the above.
  • examples of the nucleic acid according to the present invention include nucleic acids that include all the base sequences (3) to (8) described above and encode the anti-active Reelin antibody described above.
  • nucleic acids include, in addition to those directly defined above, nucleic acids that hybridize with these nucleic acids under stringent conditions and exhibit functions equivalent to these nucleic acids.
  • hybridizes under stringent conditions means that in a suitable hybridization method such as colony hybridization method, plaque hybridization method or Southern blot hybridization method, one polynucleotide ( DNA) or the other polynucleotide (DNA) can be hybridized to a fragment of the polynucleotide.
  • a suitable hybridization method such as colony hybridization method, plaque hybridization method or Southern blot hybridization method, one polynucleotide ( DNA) or the other polynucleotide (DNA) can be hybridized to a fragment of the polynucleotide.
  • one polynucleotide or a fragment of the polynucleotide immobilized on the filter is hybridized with the other polynucleotide at a predetermined temperature (X ° C) in the presence of 0.7 to 1 M NaCl.
  • X ° C a predetermined temperature
  • the filter is washed under an X ° C condition using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate).
  • the “X ° C.” is at least 50 ° C. or more, more preferably 60 ° C. or more, and further preferably 65 ° C. or more.
  • Mammalian cells other than humans can be used as myeloma cells that should constitute hybridoma cells by cell fusion and spleen cells immunized with a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
  • mammals such as monkeys, rats and mice that are commonly used for this purpose are preferred. Mice are particularly preferred because they have established a myeloma cell system and are easy to handle.
  • Reelin is present throughout the body, including in the blood, even if normal (normal) mice are immunized with Reelin-derived peptides, they may be recognized as “autoantibodies” and eliminated. Very expensive. Thus, for example, the use of “Reelin-deficient mice” provided by Jackson Lab.
  • a method for obtaining spleen cells immunized with a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing a method for obtaining hybridoma cells by cell fusion, a method for culturing hybridoma cells, an anti-active Reelin antibody ( As a method for obtaining a monoclonal antibody), various known methods can be arbitrarily employed.
  • the method for measuring active Reelin is a method for measuring active Reelin in a test sample by an antigen-antibody reaction using any one of the above-mentioned anti-active Reelin antibodies.
  • antigen-antibody reaction using any one of the above-mentioned anti-active Reelin antibodies.
  • various known methods utilizing this type of antigen-antibody reaction can be arbitrarily employed, and are not particularly limited.
  • Test samples include blood and other body fluids collected from humans, mammals, model animals showing phenotypes suitable for Reelin deficiency, etc., and compositions prepared from these body fluids (for example, dilution with pH buffer solution) Etc.), or these freeze-dried products can be used arbitrarily.
  • the kit for measuring active Reelin according to the present invention comprises any one of the above anti-reactive Reelin antibodies, and is used for measuring active Reelin in a test sample.
  • This measurement kit may include any other necessary or beneficial components that this type of kit may have, such as dilution buffers or other components.
  • Immunity 200 ⁇ L of an antigen peptide solution (0.8 mg / mL), which is a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, is sonicated for 1 minute in an Eppendorf tube using SONIFIER250 on the same volume of Freund's adjuvant and ice. (Output 15%, Duty Cycle 20%) to obtain an emulsion.
  • myeloma cells (PAI cells) cultured in a 10 cm dish in the presence of 5% CO 2 at 37 ° C. were suspended and collected in 50 mL tubes. Thereafter, the mixture was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, 10 mL of DMEM was added, and the number of cells was counted.
  • PAI cells myeloma cells
  • HAT medium 100 ⁇ L / well of HAT medium was added in advance, and seeded at 100 ⁇ L / well on a 96-well plate that had been reacted in the presence of CO 2 at 37 ° C., 5%.
  • Table 1 the table described as “5.0 ⁇ 10 ⁇ 5 cells / well” indicates the group designated as 5.0 ⁇ 10 5 cells / well, and the table expressed as “2.5 ⁇ 10 ⁇ 5 cells / well” corresponds to the above 2.5.
  • a group of ⁇ 10 5 cells / well is shown.
  • “A” to “H” in the vertical direction indicate “A” to “H” in the 96-well plate, and “1” to “12” in the horizontal direction indicate 96 well. “1” to “12” of the plate are shown. Among these, H12 used mouse serum after completion of immunization as a positive control.
  • the medium of 70-80% confluent HEK293T cells (cultured in a 10cm dish) was replaced with Opti-MEM (Invitrogen), and 15 ⁇ g and 30 ⁇ L of Lipofectamine 2000 (Invitrogen) pCrl (a vector in which the full length of Reelin cDNA was inserted into pcDNA3) was used for transfection. After 5 hours, 50 Units / mL The medium was replaced with Opti-MEM containing Penicillin + 50 mg / mL Streptomycin, and the cells were cultured at 37 ° C. in the presence of 5% CO 2 . After 48 hours, the supernatant was collected using a 0.45 ⁇ m sterilizing filter and a 5 mL syringe, and the supernatant was mixed with 4 ⁇ SDS sumpling buffer 3: 1 to prepare a sample.
  • Opti-MEM Invitrogen
  • pCrl a vector in which the full length of Reelin cDNA
  • MRNA was extracted from 4.2 ⁇ 10 6 cells of the hybridoma cell line 2F3 cultured using RNeasy Protect Mini Kit (QIAGEN) for mRNA purification.
  • the cells were dissolved in 350 ⁇ L of RLT buffer and homogenized in a syringe. After adding an equal amount of 70% ethanol, the cells were transferred to an RNeasy spin column and centrifuged at 10400 rpm for 15 seconds to remove the lower layer solution. 700 ⁇ L of RW1 buffer was added to the RNeasy spin column, and centrifuged at 10400 rpm for 15 seconds to remove the lower layer solution.
  • cDNA was synthesized using PrimeScript Reverse Transcriptae (Takara Bio Inc.) for cDNA synthesis. After mixing Oligo-dT primer 1 ⁇ L, dNTP (2.5mM) 4 ⁇ L, mRNA of 2F3 strain 4.3 ⁇ g, 65 °C After incubating for 5 minutes, it was rapidly cooled on ice.
  • reaction solution 5 ⁇ buffer 0.5 ⁇ L of RNase inhibitor, and 1 ⁇ L of reverse transcriptase (5 U / ⁇ L) derived from AMV (Avian Myeloblastosis Virus) were added to make 20 ⁇ L with RNase-free sterilized water.
  • PCR primers were designed based on this homology.
  • the nucleotide sequence of the PCR primer is as follows: the H chain N-terminus is SEQ ID NO: 10 in the sequence listing, the H chain C-terminus is SEQ ID NO: 11 in the sequence listing, the L chain N-terminus is SEQ ID NO: 12 in the sequence listing, and the L chain C-terminus is the sequence listing. SEQ ID NO: 13 were as shown respectively.
  • H chain and L chain genes were amplified by the PCR method using a PCR device manufactured by Nippon Bio-Radola Corporation.
  • the reaction solution is 1 ⁇ L using the reaction solution obtained by synthesizing cDNA as a template for each of the H chain and L chain, 20 pmol each of the H chain or L chain N-terminal and C-terminal primers, and 10 ⁇ PCR buffer.
  • the conditions of the PCR method were a dissociation reaction at 94 ° C. for 1 minute, an annealing reaction at 45 ° C. for 1 minute, and an extension reaction at 72 ° C. for 2 minutes, and these were performed for 30 cycles.
  • the cloned strains were selected by colony PCR using the target gene amplified after PCR as an index, and then plasmids were extracted from these strains and their nucleotide sequences were determined.
  • the H chain variable region is composed of 504 bases (168 amino acids) shown in SEQ ID NO: 2 of the sequence listing
  • the L chain variable region is composed of 390 bases (130 amino acids) shown in SEQ ID NO: 3 of the sequence listing. It was done. From the sequence and molecular weight of the homologous region, it was determined that these base sequences of cDNA encoded the H chain and L chain of the IgG monoclonal antibody.
  • Each region of 94 to 144 (L1 region), base numbers 190 to 210 (L2 region), and base numbers 307 to 333 (L3 region) is a portion corresponding to the hypervariable region of the Kabat database, Indicates the area that determines the bond.
  • Samples were poured at 30 ⁇ L per lane, separated by SDS-polyacrylamide electrophoresis (SDS-PAGE), and transferred to a PVDF membrane (Millipore) using a semi-dry transfer machine (Trans-blot SD cell, Bio-Red).
  • the PVDF membrane after the transfer was blocked with 5% skim milk (Morinaga Milk Industry) in TBST for 30 minutes. After blocking, a PVDF membrane and 5% skim milk in TBST containing the primary antibody were incubated at room temperature for 2 hours. Thereafter, the PVDF membrane was washed with TBST for 5 minutes ⁇ 4 times, and 5% skim milk in TBST containing a secondary antibody labeled with HRP was incubated at room temperature for 1 hour.
  • SDS-PAGE SDS-polyacrylamide electrophoresis
  • a commercially available mouse anti-Reelin N-terminal recognition monoclonal antibody G10 (1: 3000 Millipore) and an antibody obtained from the 2F3 strain (2F3 antibody) were used.
  • Anti-mouse IgG HRP-linked antibody (1: 4000, GE Healthcare) was used as a secondary antibody.
  • FIG. 1 In the upper part of FIG. 1, the full-length Reelin designed, and its Nt site and Ct site are shown.
  • “NR6” shown in the middle of FIG. 1 is a fragment on the amino terminal side when full-length Reelin is cleaved at Ct site
  • “NR2” shown in the lower part of FIG. 1 is a full-length Reelin cut at Nt site. It is a fragment on the amino terminal side when Therefore, an antibody that recognizes all of full-length Reelin, NR2 fragment, and NR6 fragment is an antibody that recognizes active Reelin and inactive Reelin without discrimination. In contrast, an antibody that recognizes full-length Reelin and an NR6 fragment but not an NR2 fragment is an antibody that recognizes only active Reelin.
  • the G10 antibody could detect the NR2 band, whereas the 2F3 antibody did not detect the NR2 band. From this, it is considered that the 2F3 antibody recognizes only the Reelin that is not degraded at Nt site, that is, the active Reelin.
  • the present invention provides a means for selectively measuring active Reelin easily and quickly even with a large amount of sample.

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Abstract

Provided is a means whereby active reelin can be selectively measured in a simple and rapid fashion, even in high-volume samples. An anti-active reelin antibody that binds specifically to active reelin but does not bind to inactive reelin; alternatively, an anti-active reelin antibody that binds specifically to a peptide having the amino acid sequence shown by SEQ ID NO: 1 in the sequence listing.

Description

抗体とその利用Antibodies and their use
　本発明は抗体とその利用に関する。更に詳しくは本発明は、活性型リーリンに対して特異的に結合し、不活性型リーリンに対しては結合しない新規な抗活性型リーリン抗体と、この抗体のＨ鎖、Ｌ鎖の可変領域をコードする核酸又はこれらの領域におけるＣＤＲ配列部分をコードする核酸と、この抗体を生産するハイブリドーマ細胞と、この抗体を利用する活性型リーリン測定方法、及びそのために用いる活性型リーリン測定用キットに関する。 The present invention relates to antibodies and their use. More specifically, the present invention relates to a novel anti-active Reelin antibody that specifically binds to active Reelin and does not bind to inactive Reelin, and variable regions of the H chain and L chain of this antibody. The present invention relates to an encoding nucleic acid or a nucleic acid encoding a CDR sequence portion in these regions, a hybridoma cell producing this antibody, an active Reelin measurement method using this antibody, and an active Reelin measurement kit used therefor.
　リーリンは、脳の機能に重要な、３４００を超えるアミノ酸からなる巨大な分泌タンパク質である。リーリンが脳の特徴的な層構造の形成に必須であること、ヒトでもリーリンが欠損すると脳構造形成不全が起こることも報告されている。一方、リーリンは成体の脳でも発現しており、その発現量の低下や分解亢進が統合失調症、アルツハイマー病、気分障害等の疾患の一因となることが明らかになってきている。
　リーリンに関して、例えば下記の特許文献１ではリーリンのエピトープ領域ポリペプチド及びこれをコードするポリヌクレオチドを開示し、これによってリーリンの機能を更に研究すると共に、リーリン遺伝子の異常並びにニューロンの配置異常に起因する脳障害に対する診断・治療手段を提供できるとしている。又、下記の特許文献２では、中枢神経系の他の重要成分におけるＤＩＩＡレベルを非破壊的に評価又は予想するためのバイオマーカーとしてのリーリンを測定する方法を提案している。 Reelin is a large secreted protein consisting of over 3400 amino acids that is important for brain function. It has been reported that Reelin is essential for the formation of a characteristic layered structure of the brain, and that even humans fail to form brain structures when Reelin is deficient. On the other hand, Reelin is also expressed in the adult brain, and it has become clear that a decrease in expression level and an increase in degradation contribute to diseases such as schizophrenia, Alzheimer's disease, and mood disorders.
Regarding Reelin, for example, the following Patent Document 1 discloses a Reelin epitope region polypeptide and a polynucleotide encoding the Reelin, thereby further studying Reelin's function and resulting from Reelin gene abnormality and neuronal arrangement abnormality. It is said that it can provide diagnostic and therapeutic means for brain disorders. Patent Document 2 below proposes a method for measuring Reelin as a biomarker for nondestructively evaluating or predicting DIIA levels in other important components of the central nervous system.
特開２００２－１７３６１号公報JP 2002-17361 A 特表２００７－５２４６７４号公報Special table 2007-524673
　リーリンについては、更に、公知の各種文献により、あるいは本願発明者の研究により、以下の点が分かっている。 Regarding Reelin, the following points are known from various known literatures or from the research of the present inventors.
　即ち、リーリンは分子内で特異的な分解を受けるが、その分解部位として、リーリンタンパク質のアミノ酸配列におけるＮ末端側に近い未知のサイト（以下、このサイトを「N-t site」と呼ぶ）とＣ末端側に近い未知のサイト（以下、このサイトを「C-t site」と呼ぶ）とがある（非特許文献１参照）。実際には、リーリンの分子内分解の大部分はN-t siteで起こり、C-t siteで起こる分解は比較的少ない。 That is, Reelin undergoes specific degradation within the molecule, but as its degradation site, an unknown site close to the N-terminal side of the amino acid sequence of Reelin protein (hereinafter, this site is referred to as “Nt site”) and the C-terminal There is an unknown site close to the side (hereinafter, this site is referred to as “Ct site”) (see Non-Patent Document 1). In fact, most of the intramolecular degradation of Reelin occurs at N-t site, and relatively little degradation occurs at C-t site.
　従って、リーリンの研究又は測定用に調製された被験試料には、一般的に、分子内分解を受けていない全長リーリンと分子内分解を受けた分解型リーリンとが混在している。そして前記したリーリンの生理的機能について、全長リーリンは活性であるが、N-t siteでの分子内分解を受けた分解型リーリンは不活性であり（非特許文献２参照）、一方、C-t siteでの分子内分解を受けた分解型リーリンは、幾分低下した活性を維持することが分かっている。 Therefore, in general, test samples prepared for research or measurement of Reelin include a mixture of full-length Reelin that has not undergone intramolecular degradation and degraded Reelin that has undergone intramolecular degradation. With regard to the physiological functions of Reelin, full-length Reelin is active, but degraded Reelin that has undergone intramolecular degradation at Nt あ り site is inactive (see Non-Patent Document 2), while Ct site Degraded Reelin that has undergone intramolecular degradation has been found to maintain somewhat reduced activity.
　以上の点を踏まえれば、リーリンの機能の研究のため、あるいはリーリンの発現量低下等に基づく疾患の診断・治療手段の開発のため、被験試料中のリーリンの測定を行う場合には、活性型リーリンのみを選択的に測定する必要がある。 Based on the above points, when measuring Reelin in a test sample for the study of Reelin's function, or for the development of a disease diagnosis / treatment method based on a decrease in Reelin's expression level, etc. Only Reelin needs to be measured selectively.
　このような要求に応えるために、従来は、例えば全長リーリンと分解型リーリンとの分子量の差を利用して電気泳動を行っていた。しかし、巨大タンパク質である全長リーリンや分解型リーリンの電気泳動は非常に時間がかかり、効率面で実用性に欠けていた。しかも大量のサンプルについて同時に測定を行うことは、手間の面で事実上は不可能であった。 In order to meet such demands, conventionally, for example, electrophoresis was performed using the difference in molecular weight between full-length Reelin and decomposed Reelin. However, electrophoresis of full-length Reelin and large-scale Reelin, which are large proteins, is very time-consuming and lacks practicality in terms of efficiency. Moreover, it is practically impossible to measure a large number of samples at the same time.
　又、被験試料中の不活性型リーリンを検出することなく活性型リーリンのみを選択的に測定するためには、その目的に適う抗体、特にモノクローナル抗体の利用が特に有効であると考えられるが、このような抗原特異性を備える抗体は、未だ提案も提供もされていない。 In addition, in order to selectively measure only active Reelin without detecting inactive Reelin in a test sample, it is considered that the use of an antibody suitable for that purpose, particularly a monoclonal antibody, is particularly effective. Antibodies with such antigen specificity have not yet been proposed or provided.
　そこで本発明は、大量のサンプルであっても簡便、迅速に活性型リーリンを選択的に測定できる手段を提供することを、解決すべき技術的課題とする。 Therefore, the present invention has a technical problem to be solved to provide a means for selectively measuring active Reelin easily and quickly even with a large amount of sample.
　本願発明者は、リーリンタンパク質のアミノ酸配列において未解明であったN-t siteの分解部位を具体的に突き止めた。更に、このアミノ酸配列を有するペプチドを抗原とするモノクローナル抗体を生産するハイブリドーマ細胞を作製することに成功した。その結果、N-t siteでの分解を受けていないリーリン、即ち活性型リーリンのみに特異的に結合する抗活性型リーリン抗体を得るに至り、本発明を完成した。 The inventor of the present application has specifically identified the degradation site of N-t site, which has not been elucidated in the amino acid sequence of Reelin protein. Furthermore, the present inventors succeeded in producing a hybridoma cell that produces a monoclonal antibody using a peptide having this amino acid sequence as an antigen. As a result, Reelin that has not undergone degradation at N-t site, that is, an anti-reactive Reelin antibody that specifically binds only to active Reelin, was obtained, thereby completing the present invention.
　（第１発明の構成）
　上記課題を解決するための第１発明の構成は、活性型リーリンに対して特異的に結合し、不活性型リーリンに対しては結合しない抗体である、抗活性型リーリン抗体である。 (Configuration of the first invention)
The configuration of the first invention for solving the above-described problems is an anti-active Reelin antibody that is an antibody that specifically binds to active Reelin but does not bind to inactive Reelin.
　上記の第１発明において「抗体」のカテゴリーは限定されないが、必要な条件を備えたハイブリドーマ細胞によって生産されるモノクローナル抗体であることが特に好ましい。「活性型リーリン」とは、N-t siteでの分解を受けていないリーリンを言い、「不活性型リーリン」とは、N-t siteでの分解を受けたリーリンを言う。 In the first invention, the category of “antibody” is not limited, but a monoclonal antibody produced by a hybridoma cell having the necessary conditions is particularly preferable. “Activated Reelin” refers to Reelin that has not undergone degradation at N-t site, and “inactive Reelin” refers to Reelin that has undergone degradation at N-t site.
　（第２発明の構成）
　上記課題を解決するための第２発明の構成は、配列表の配列番号１に示すアミノ酸配列を有するペプチドに対して特異的に結合する抗体である、抗活性型リーリン抗体である。 (Configuration of the second invention)
The configuration of the second invention for solving the above problem is an anti-active Reelin antibody, which is an antibody that specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
　上記の第２発明において、配列表の配列番号１に示すアミノ酸配列は、未解明であったリーリンタンパク質のN-t siteの分解部位を中心とする８個のアミノ酸配列であり、N-t siteでの分解は、このアミノ酸配列中で、特にこのアミノ酸配列中のプロリン（Pro）とアラニン（Ala）との間で起こる。なお、配列表において後述するように、この８個のアミノ酸配列及びその分解部位は、実際にはマウスのリーリンから割り出したものであるが、ヒトのリーリンにおいても上記のアミノ酸配列及びその分解部位は全く同一である。 In the second invention, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is an eight amino acid sequence centering on the unresolved Nt site degradation site of Reelin protein. Occurs in this amino acid sequence, particularly between proline (Pro) and alanine (Ala) in this amino acid sequence. As will be described later in the sequence listing, these eight amino acid sequences and their degradation sites were actually determined from mouse Reelin, but the above amino acid sequences and their degradation sites also exist in human Reelin. Is exactly the same.
　又、「（配列表の配列番号１に示すアミノ酸配列を）有する」とは、当該アミノ酸配列からなり、又は、このアミノ酸配列を一部に含むことを意味する。本願明細書の以下の記載においても、特定のアミノ酸配列又は塩基配列について「有する」と言うときは、同上の意味である。 In addition, “having (having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing)” means consisting of the amino acid sequence or partially including this amino acid sequence. Also in the following description of the present specification, when “having” a specific amino acid sequence or base sequence has the same meaning as described above.
　（第３発明の構成）
　上記課題を解決するための第３発明の構成は、配列表の配列番号２に示すアミノ酸配列を有するＨ鎖可変領域と、配列表の配列番号３に示すアミノ酸配列を有するＬ鎖可変領域とを有するポリペプチドからなる抗体である、抗活性型リーリン抗体である。 (Configuration of the third invention)
The configuration of the third invention for solving the above-mentioned problem is that an H chain variable region having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing and an L chain variable region having the amino acid sequence shown in SEQ ID NO: 3 of the sequence listing It is an anti-active Reelin antibody which is an antibody comprising a polypeptide having the same.
　（第４発明の構成）
　上記課題を解決するための第４発明の構成は、配列表の配列番号２に示すアミノ酸配列に対して１～数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を有するＨ鎖可変領域と、配列表の配列番号３に示すアミノ酸配列に対して１～数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を有するＬ鎖可変領域とを有するポリペプチドからなり、以下の（１）、（２）の少なくとも一方に該当する抗体である、抗活性型リーリン抗体である。 (Configuration of the fourth invention)
The constitution of the fourth invention for solving the above-mentioned problem is that the heavy chain variable region having an amino acid sequence in which 1 to several amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing And an L chain variable region having an amino acid sequence in which 1 to several amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 3 in the Sequence Listing, and has the following (1 ), An anti-active Reelin antibody, which is an antibody corresponding to at least one of (2).
　（１）活性型リーリンに対して特異的に結合し、不活性型リーリンに対しては結合しない。 (1) It specifically binds to active Reelin and does not bind to inactive Reelin.
　（２）配列表の配列番号１に示すアミノ酸配列を有するペプチドに対して特異的に結合する。 (2) It specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
　上記の第４発明において、「配列表の配列番号２又は３に示すアミノ酸配列に対して１～数個のアミノ酸が置換されたアミノ酸配列」の好ましい例として、アミノ酸の同類置換を挙げることができる。「同類置換」とは、ポリペプチドの機能が全体的に実質的に不変のまま維持されるように、所定のアミノ酸残基を化学的又は機能的に類似した別のアミノ酸残基で置換することを言う。化学的又は機能的に類似したアミノ酸の例示として、疎水性アミノ酸（Ala、Ile、Leu、Phe、Pro、Trp、Val、Met）同士、極性だが電荷のないアミノ酸（Asn、Cys、Gln、Gly、Ser、Thr、Tyr）同士、塩基性アミノ酸（Arg、His、Lys）同士、酸性アミノ酸（Asp、Glu）同士、等が挙げられる（E.E.Cornnら著、田宮信雄ら訳、「コーン・スタンプ生化学  第５版」東京化学同人刊、p56-58、1988）。 In the fourth invention, a preferred example of “an amino acid sequence in which one to several amino acids are substituted for the amino acid sequence shown in SEQ ID NO: 2 or 3 in the sequence listing” includes conservative substitution of amino acids. . A “conservative substitution” is the replacement of a given amino acid residue with another chemically or functionally similar amino acid residue so that the overall function of the polypeptide remains substantially unchanged. Say. Examples of chemically or functionally similar amino acids include hydrophobic amino acids (Ala, Ile, Leu, Phe, Pro, Trp, Val, Met), polar but uncharged amino acids (Asn, Cys, Gln, Gly, Ser, Thr, Tyr), basic amino acids (Arg, His, Lys), acidic amino acids (Asp, Glu), etc. (translated by EECornn et al., Nobuo Tamiya et al., “Corn Stamp Biochemistry” 5th edition "Tokyo Chemical Doujinshi, p56-58, 1988).
　（第５発明の構成）
　上記課題を解決するための第５発明の構成は、配列表の配列番号４～６に示すアミノ酸配列からなるＣＤＲ配列の全てを含むＨ鎖可変領域と、配列表の配列番号７～９に示すアミノ酸配列からなるＣＤＲ配列の全てを含むＬ鎖可変領域とを有するポリペプチドからなる抗体である、抗活性型リーリン抗体である。 (Structure of the fifth invention)
The structure of the fifth invention for solving the above-mentioned problems is shown in the heavy chain variable region including all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6 in the sequence listing, and SEQ ID NOs: 7 to 9 in the sequence listing. It is an anti-active Reelin antibody, which is an antibody consisting of a polypeptide having an L chain variable region containing all of the CDR sequence consisting of an amino acid sequence.
　上記の第５発明において、「（～ＣＤＲ配列の全てを）含む」とは、当該ＣＤＲ配列の全てを一部に含むことを意味する。本願明細書の以下の記載においても、特定のアミノ酸配列又は塩基配列について「含む」と言うときは、同上の意味である。 In the fifth aspect of the present invention, “(including all of the CDR sequences)” means including all of the CDR sequences in part. Also in the following description of the present specification, when “including” a specific amino acid sequence or base sequence, it has the same meaning as described above.
　（第６発明の構成）
　上記課題を解決するための第６発明の構成は、以下（３）～（８）のいずれかに該当する、核酸である。 (Structure of the sixth invention)
The structure of the sixth invention for solving the above-described problems is a nucleic acid corresponding to any of (3) to (8) below.
　（３）配列表の配列番号４に示すアミノ酸配列をコードする核酸。 (3) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
　（４）配列表の配列番号５に示すアミノ酸配列をコードする核酸。 (4) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing.
　（５）配列表の配列番号６に示すアミノ酸配列をコードする核酸。 (5) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing.
　（６）配列表の配列番号７に示すアミノ酸配列をコードする核酸。 (6) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing.
　（７）配列表の配列番号８に示すアミノ酸配列をコードする核酸。 (7) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing.
　（８）配列表の配列番号９に示すアミノ酸配列をコードする核酸。 (8) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing.
　上記の第６発明において、配列番号４～９に示すアミノ酸配列をそれぞれコードする核酸は、第５発明に規定した６種のＣＤＲ配列をコードする核酸である。又、「核酸」とはセンス／アンチセンスＤＮＡ又はＲＮＡや、ハイブリダイゼーション用プローブ、ＰＣＲ用プライマー、融合遺伝子作製時における鋳型等を含む概念であり、１本鎖、２本鎖又は３本鎖のものを包含する。 In the sixth invention, the nucleic acids encoding the amino acid sequences shown in SEQ ID NOs: 4 to 9 are nucleic acids encoding the six CDR sequences defined in the fifth invention. “Nucleic acid” is a concept including sense / antisense DNA or RNA, hybridization probe, PCR primer, template for producing a fusion gene, and the like. Including things.
　（第７発明の構成）
　上記課題を解決するための第７発明の構成は、第６発明に記載した（３）～（８）の全ての塩基配列を含み、第１発明～第５発明のいずれかに記載の抗活性型リーリン抗体をコードする、核酸である。 (Structure of the seventh invention)
The structure of the seventh invention for solving the above-mentioned problems includes all of the base sequences (3) to (8) described in the sixth invention, and the antiactivity according to any one of the first to fifth inventions A nucleic acid encoding a type Reelin antibody.
　上記の第７発明において、「核酸」とは、センス／アンチセンスＤＮＡ又はＲＮＡを含む概念である。 In the seventh aspect of the present invention, the “nucleic acid” is a concept including sense / antisense DNA or RNA.
　（第８発明の構成）
　上記課題を解決するための８発明の構成は、哺乳動物のミエローマ細胞と、配列表の配列番号１に示すアミノ酸配列を有するペプチドで免疫された哺乳動物の脾細胞との融合によって作製され、第１発明～第５発明のいずれかに記載の抗活性型リーリン抗体を生産する、ハイブリドーマ細胞である。 (Configuration of the eighth invention)
The configuration of 8 inventions for solving the above-mentioned problems is produced by fusion of a mammalian myeloma cell and a spleen cell of a mammal immunized with a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. A hybridoma cell that produces the anti-active Reelin antibody according to any one of the first to fifth inventions.
　上記の第８発明に係るハイブリドーマ細胞は、日本国の独立行政法人製品評価技術基盤機構（NITE : National Institute of Technology and Evaluation）における特許微生物寄託センター（NPMD : NITE Patent
Microorganisms Depositary）に既に寄託しており、２０１２年８月９日付けで受領番号「NITE ABP-1403」を交付されている。しかし、このハイブリドーマ細胞は、免疫すべきペプチドのアミノ酸配列が開示された状況下においては、専門家であれば技術常識に基づいて作製することが可能である。従って、少なくとも日本国の特許制度においては、特許寄託が必要とされる場合には該当しない。 The hybridoma cell according to the eighth invention described above is a patent microorganism deposit center (NPMD: NITE Patent) of the National Institute of Technology and Evaluation (NITE) in Japan.
Microorganisms Depositary), and the receipt number “NITE ABP-1403” has been issued on August 9, 2012. However, in the situation where the amino acid sequence of the peptide to be immunized is disclosed, this hybridoma cell can be prepared based on common general technical knowledge. Therefore, at least in the Japanese patent system, this does not apply when patent deposits are required.
　（第９発明の構成）
　上記課題を解決するための第９発明の構成は、第１発明～第５発明のいずれかに記載の抗活性型リーリン抗体を用いた抗原－抗体反応により、被験試料中の活性型リーリンを測定する、活性型リーリン測定方法である。 (Structure of the ninth invention)
The structure of the ninth invention for solving the above-mentioned problem is that active Reelin in a test sample is measured by an antigen-antibody reaction using the anti-active Reelin antibody according to any one of the first to fifth inventions. This is an active Reelin measuring method.
　（第１０発明の構成）
　上記課題を解決するための第１０発明の構成は、第１発明～第５発明のいずれかに記載の抗活性型リーリン抗体を含んで構成され、被験試料中の活性型リーリンを測定するために用いるものである、活性型リーリン測定用キットである。 (Configuration of the tenth invention)
A tenth aspect of the present invention for solving the above-described problems comprises the anti-active Reelin antibody according to any one of the first to fifth aspects of the invention, and is used for measuring active Reelin in a test sample. An active Reelin measurement kit to be used.
　第１発明の抗活性型リーリン抗体は、活性型リーリンに対して特異的に結合し不活性型リーリンに対しては結合しないため、この抗体を用いて、被験試料中の不活性型リーリンを検出することなく活性型リーリンのみを選択的に測定することができる。そのため、大量のサンプルについても、例えばマルチウエルプレートを用いて、簡便、迅速に活性型リーリンを選択的に測定することが可能になった。 Since the anti-active Reelin antibody of the first invention specifically binds to active Reelin and does not bind to inactive Reelin, this antibody is used to detect inactive Reelin in a test sample. Without this, only active Reelin can be measured selectively. Therefore, even for a large amount of samples, it has become possible to selectively measure active Reelin simply and quickly using, for example, a multi-well plate.
　第２発明に関して、配列表の配列番号１に示すアミノ酸配列は、従来は未解明であったリーリンタンパク質のN-t siteの分解部位の両側の各４個のアミノ酸配列であるため、第２発明の抗活性型リーリン抗体は、N-t siteでの分解を受けた不活性型リーリンに対しては結合せず、このサイトでの分解を受けていないN-t site非分解型リーリンに対して特異的に結合する。 Regarding the second invention, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is an amino acid sequence of 4 amino acids on both sides of the Nt の site degradation site of Reelin protein, which has not been elucidated so far. The active Reelin antibody does not bind to inactive Reelin that has undergone degradation at Nt site, but specifically binds to Nt site non-degradable Reelin that has not undergone degradation at this site.
　N-t site非分解型リーリンは活性型であり、これには全長リーリンとC-t siteでの分子内分解のみを受けた分解型リーリンとが包含される。なお、前記のようにリーリンの分子内分解の大部分はN-t siteで起こり、C-t siteで起こる分解は比較的少ないため、C-t siteでの分子内分解のみを受けた分解型リーリンの存在は、測定誤差の範囲内として事実上無視することも可能である。 N-t site non-degradable Reelin is an active form, and this includes full-length Reelin and degraded Reelin that has undergone only intramolecular degradation at C-t site. As mentioned above, most of the intramolecular degradation of Reelin occurs at Nt site, and relatively little degradation occurs at Ct site. Therefore, the presence of degraded Reelin that has undergone only intramolecular decomposition at Ct site is measured. It can also be virtually ignored as within the error range.
　第３発明には、本発明に係る抗活性型リーリン抗体のＨ鎖可変領域とＬ鎖可変領域が規定されている。従って第３発明の抗活性型リーリン抗体も、第２発明の抗活性型リーリン抗体と同様の作用・効果を期待できる。 In the third invention, the heavy chain variable region and the light chain variable region of the anti-active Reelin antibody according to the present invention are defined. Therefore, the anti-active Reelin antibody of the third invention can be expected to have the same action and effect as the anti-active Reelin antibody of the second invention.
　第４発明によって、第３発明の抗活性型リーリン抗体と機能が同等で、そのＨ鎖可変領域及びＬ鎖可変領域のアミノ酸配列において抗体機能に影響しない僅かな「配列の変更」を伴う抗活性型リーリン抗体が提供される。 According to the fourth invention, anti-reactivity with a slight “sequence change” that has the same function as the anti-active Reelin antibody of the third invention and does not affect the antibody function in the amino acid sequences of the H chain variable region and L chain variable region A type Reelin antibody is provided.
　第５発明には、本発明に係る抗活性型リーリン抗体のＨ鎖可変領域におけるＣＤＲ配列の全てとＬ鎖可変領域におけるＣＤＲ配列の全てが規定されている。従って第５発明の抗活性型リーリン抗体も、第２発明の抗活性型リーリン抗体と同様の作用・効果を期待できる。 In the fifth invention, all of the CDR sequences in the heavy chain variable region and all of the CDR sequences in the light chain variable region of the anti-active Reelin antibody according to the present invention are defined. Therefore, the anti-active Reelin antibody of the fifth invention can be expected to have the same actions and effects as the anti-active Reelin antibody of the second invention.
　第６発明及び第７発明に係るそれぞれの核酸、第８発明に係るハイブリドーマ細胞は、いずれも、本発明に係る抗活性型リーリン抗体の生産手段あるいは研究手段として極めて有用である。 The nucleic acids according to the sixth and seventh inventions and the hybridoma cells according to the eighth invention are both extremely useful as means for producing or researching the anti-active Reelin antibody according to the present invention.
　第９発明によって、大量のサンプルについても、簡便、迅速に、不活性型リーリンを検出することなく活性型リーリンのみを選択的に測定することができる活性型リーリン測定方法が提供される。 According to the ninth invention, there is provided an active Reelin measurement method capable of selectively measuring only active Reelin without detecting inactive Reelin easily and quickly even for a large amount of samples.
　第１０発明によって、第９発明の活性型リーリン測定方法を有効に実施するための活性型リーリン測定用キットが提供される。 The tenth invention provides an active Reelin measurement kit for effectively carrying out the active Reelin measurement method of the ninth invention.
全長リーリンと分解型リーリンを図案化して示す。The full-length Reelin and the decomposed Reelin are shown in a diagram. 抗活性型リーリン抗体の評価結果を示すwestern blottingの図である。It is a figure of western blotting which shows the evaluation result of an anti-active type Reelin antibody.
　次に、本発明を実施するための形態を、その最良の形態を含めて説明する。 Next, modes for carrying out the present invention will be described including the best mode.
　〔抗活性型リーリン抗体〕
　本発明に係る抗活性型リーリン抗体は以下の１）～５）のいずれかに該当する抗体である。なお、リーリンタンパク質自体は周知であるため、本明細書では、そのアミノ酸配列等の説明を省略する。 [Anti-active Reelin antibody]
The anti-active Reelin antibody according to the present invention is an antibody corresponding to any one of the following 1) to 5). In addition, since Reelin protein itself is well-known, description of the amino acid sequence etc. is abbreviate | omitted in this specification.
　１）活性型リーリンに対して特異的に結合し、不活性型リーリンに対しては結合しない抗体である
　２）配列表の配列番号１に示すアミノ酸配列を有するペプチドに対して特異的に結合する抗体である。 1) An antibody that specifically binds to active Reelin and does not bind to inactive Reelin 2) Specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing It is an antibody.
　３）配列表の配列番号２に示すアミノ酸配列を有するＨ鎖可変領域と、配列表の配列番号３に示すアミノ酸配列を有するＬ鎖可変領域とを有するポリペプチドからなる抗体である。 3) An antibody comprising a polypeptide having an H chain variable region having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and an L chain variable region having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
　４）配列表の配列番号２に示すアミノ酸配列に対して１～数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を有するＨ鎖可変領域と、配列表の配列番号３に示すアミノ酸配列に対して１～数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を有するＬ鎖可変領域とを有するポリペプチドからなり、（１）活性型リーリンに対して特異的に結合し、不活性型リーリンに対しては結合しない、（２）配列表の配列番号１に示すアミノ酸配列を有するペプチドに対して特異的に結合する、の少なくとも一方に該当する抗体である。 4) an H chain variable region having an amino acid sequence in which one to several amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; and the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing A polypeptide having an L chain variable region having an amino acid sequence in which one to several amino acids are substituted, deleted or added, and (1) specifically binds to active Reelin and It is an antibody that does not bind to active Reelin and (2) specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
　５）配列表の配列番号４～６に示すアミノ酸配列からなるＣＤＲ配列の全てを含むＨ鎖可変領域と、配列表の配列番号７～９に示すアミノ酸配列からなるＣＤＲ配列の全てを含むＬ鎖可変領域とを有するポリペプチドからなる抗体である。 5) H chain variable region including all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6 in the sequence listing, and L chain including all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 7 to 9 of the sequence listing An antibody comprising a polypeptide having a variable region.
　上記の各種抗体には、ポリクロ－ナル抗体、モノクロ－ナル抗体が包含されるが、特にモノクロ－ナル抗体が好ましい。更に、これらの抗体には、検出・測定の目的のためにペルオキシダ－ゼ、β－Ｄ－ガラクトシダ－ゼ、アルカリフォスファタ－ゼ、グルコ－ス－６－リン酸脱水素酵素等の酵素、デルフィニウム等の蛍光標識、放射性同位元素標識または同位元素標識、ビオチン等を結合させた標識化抗活性型リーリン抗体も包含される。 The above-mentioned various antibodies include polyclonal antibodies and monoclonal antibodies, and monoclonal antibodies are particularly preferable. Further, these antibodies include peroxidase, β-D-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase and other enzymes, delphinium for detection and measurement purposes. In addition, a labeled anti-active Reelin antibody bound with a fluorescent label such as, a radioisotope label or isotope label, biotin and the like is also included.
　〔核酸〕
　本発明に係る核酸としては、上記した配列番号４～６に示すアミノ酸配列からなるＣＤＲ配列、配列番号７～９に示すアミノ酸配列からなるＣＤＲ配列をそれぞれコードする、以下（３）～（８）のいずれかに該当する核酸が挙げられる。 [Nucleic acid]
The nucleic acid according to the present invention encodes the CDR sequence consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6 and the CDR sequence consisting of the amino acid sequences shown in SEQ ID NOs: 7 to 9, respectively (3) to (8) Or a nucleic acid corresponding to any of the above.
　（３）配列表の配列番号４に示すアミノ酸配列をコードする核酸。 (3) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
　（４）配列表の配列番号５に示すアミノ酸配列をコードする核酸。 (4) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing.
　（５）配列表の配列番号６に示すアミノ酸配列をコードする核酸。 (5) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing.
　（６）配列表の配列番号７に示すアミノ酸配列をコードする核酸。 (6) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing.
　（７）配列表の配列番号８に示すアミノ酸配列をコードする核酸。 (7) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing.
　（８）配列表の配列番号９に示すアミノ酸配列をコードする核酸。 (8) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing.
　更に本発明に係る核酸としては、上記した（３）～（８）の全ての塩基配列を含み、前記のいずれかに記載した抗活性型リーリン抗体をコードする核酸も挙げられる。 Furthermore, examples of the nucleic acid according to the present invention include nucleic acids that include all the base sequences (3) to (8) described above and encode the anti-active Reelin antibody described above.
　これらの核酸には、上記に直接に規定するものの他、これらの核酸とストリンジェントな条件下でハイブリダイズし、かつ、これらの核酸と同等の機能を示す核酸が含まれる。 These nucleic acids include, in addition to those directly defined above, nucleic acids that hybridize with these nucleic acids under stringent conditions and exhibit functions equivalent to these nucleic acids.
　ここに「ストリンジェントな条件下でハイブリダイズする」とは、コロニーハイブリダイゼーション法，プラークハイブリダイゼーション法又はサザンブロットハイブリダイゼーション法等の適宜なハイブリダイゼーション法において、以下の条件下で一方のポリヌクレオチド（ＤＮＡ）又は該ポリヌクレオチドの断片に対し他方のポリヌクレオチド（ＤＮＡ）がハイブリダイズできることを言う。 Here, “hybridizes under stringent conditions” means that in a suitable hybridization method such as colony hybridization method, plaque hybridization method or Southern blot hybridization method, one polynucleotide ( DNA) or the other polynucleotide (DNA) can be hybridized to a fragment of the polynucleotide.
　即ち、フィルターに固定化された一方のポリヌクレオチド又は該ポリヌクレオチドの断片に対し、０．７～１ＭのＮａＣｌの存在下、所定温度（Ｘ°Ｃ）下で他方のポリヌクレオチドのハイブリダイゼーションを行った後、０．１～２倍程度のＳＳＣ溶液（１倍濃度のＳＳＣ溶液の組成は、１５０ｍＭ塩化ナトリウム，１５ｍＭクエン酸ナトリウムよりなる）を用いてＸ°Ｃの条件下でフィルターを洗浄した場合に、他方のポリヌクレオチドを同定できることを言う。そして上記の「Ｘ°Ｃ」とは、少なくとも５０°Ｃ以上であり、より好ましくは６０°Ｃ以上であり、更に好ましくは６５°Ｃ以上である。 That is, one polynucleotide or a fragment of the polynucleotide immobilized on the filter is hybridized with the other polynucleotide at a predetermined temperature (X ° C) in the presence of 0.7 to 1 M NaCl. After that, when the filter is washed under an X ° C condition using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). In addition, it means that the other polynucleotide can be identified. The “X ° C.” is at least 50 ° C. or more, more preferably 60 ° C. or more, and further preferably 65 ° C. or more.
　〔ハイブリドーマ細胞〕
　細胞融合によりハイブリドーマ細胞を構成すべきミエローマ細胞と、配列表の配列番号１に示すアミノ酸配列を有するペプチドで免疫された脾細胞としては、ヒトを除く哺乳動物の細胞を用いることができる。哺乳動物としてはこの種の目的に常用されるサル，ラット，マウス等の哺乳動物が好ましい。ミエローマ細胞の系が確立され、扱い易いと言う理由から特にマウスが好ましい。 [Hybridoma cells]
Mammalian cells other than humans can be used as myeloma cells that should constitute hybridoma cells by cell fusion and spleen cells immunized with a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. As mammals, mammals such as monkeys, rats and mice that are commonly used for this purpose are preferred. Mice are particularly preferred because they have established a myeloma cell system and are easy to handle.
　又、リーリンは血中をはじめ体内のいたるところに存在するため、普通の（正常な）マウスにリーリン由来のペプチドを免疫しても、「自己抗体」と認識されて排除されてしまう可能性が非常に高い。そこで、例えば、米国のJackson Lab.社が提供している「リーリン欠損マウス」の使用がとりわけ好ましい。 In addition, since Reelin is present throughout the body, including in the blood, even if normal (normal) mice are immunized with Reelin-derived peptides, they may be recognized as “autoantibodies” and eliminated. Very expensive. Thus, for example, the use of “Reelin-deficient mice” provided by Jackson Lab.
　配列表の配列番号１に示すアミノ酸配列を有するペプチドで免疫された脾細胞を取得する方法、細胞融合によりハイブリドーマ細胞を取得する方法、ハイブリドーマ細胞を培養する方法、その培地から抗活性型リーリン抗体（モノクローナル抗体）を取得する方法等については、公知の各種の方法を任意に採用することができる。 A method for obtaining spleen cells immunized with a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, a method for obtaining hybridoma cells by cell fusion, a method for culturing hybridoma cells, an anti-active Reelin antibody ( As a method for obtaining a monoclonal antibody), various known methods can be arbitrarily employed.
　〔活性型リーリン測定方法〕
　本発明の活性型リーリン測定方法は、上記いずれかの抗活性型リーリン抗体を用いた抗原－抗体反応により、被験試料中の活性型リーリンを測定する方法である。その具体的な実施形態としては、この種の抗原－抗体反応を利用する公知の各種の方法を任意に採用することができ、特段に限定されない。 [Method for measuring active Reelin]
The method for measuring active Reelin according to the present invention is a method for measuring active Reelin in a test sample by an antigen-antibody reaction using any one of the above-mentioned anti-active Reelin antibodies. As specific embodiments thereof, various known methods utilizing this type of antigen-antibody reaction can be arbitrarily employed, and are not particularly limited.
　被験試料としては、ヒト、哺乳動物、又はリーリン欠損症等に好適な表現型を示すモデル動物等から採取した血液その他の体液、これらの体液から調製した組成物（例えば、ｐＨ緩衝液による希釈物等）、あるいはこれらの凍結乾燥物等を任意に利用することができる。 Test samples include blood and other body fluids collected from humans, mammals, model animals showing phenotypes suitable for Reelin deficiency, etc., and compositions prepared from these body fluids (for example, dilution with pH buffer solution) Etc.), or these freeze-dried products can be used arbitrarily.
　〔活性型リーリン測定用キット〕
　本発明の活性型リーリン測定用キットは、上記いずれかの抗活性型リーリン抗体を含んで構成され、被験試料中の活性型リーリンを測定するために用いるものである。 [Activated Reelin measurement kit]
The kit for measuring active Reelin according to the present invention comprises any one of the above anti-reactive Reelin antibodies, and is used for measuring active Reelin in a test sample.
　この測定用キットは、この種のキットが備えることがある必要又は有益な他の任意の構成要素、例えば希釈用の緩衝液その他の構成要素を備えることができる。 This measurement kit may include any other necessary or beneficial components that this type of kit may have, such as dilution buffers or other components.
　次に本発明の実施例を説明する。本発明の技術的範囲は以下の実施例によって限定されない。 Next, examples of the present invention will be described. The technical scope of the present invention is not limited by the following examples.
　（免疫）
　配列表の配列番号１に示すアミノ酸配列を有するペプチドである抗原ペプチドの溶液（0.8mg/mL）200μLを、等量のフロイントアジュバントと氷上でSONIFIER250を用いてエッペンドルフチューブ内にて１分間超音波処理（output
15％、Duty Cycle 20％）を行い、エマルジョンとした。 (Immunity)
200 μL of an antigen peptide solution (0.8 mg / mL), which is a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, is sonicated for 1 minute in an Eppendorf tube using SONIFIER250 on the same volume of Freund's adjuvant and ice. (Output
15%, Duty Cycle 20%) to obtain an emulsion.
　このエマルジョンを別の注射器に１ｍＬ取り、投与抗原量が40μｇとなるように、雌10週齢リーラーマウスの腹腔内に注射した。この操作を２週間毎に２回行い、ＥＬＩＳＡ法により抗体生産を確認した後、同マウスの腹腔内に抗原注射を行った。 1 mL of this emulsion was taken into another syringe and injected into the abdominal cavity of a female 10-week-old reeler mouse so that the dose of antigen was 40 μg. This operation was performed twice every two weeks, and after confirming antibody production by ELISA, antigen was injected into the abdominal cavity of the mice.
　（細胞の調製）
　免疫終了後のマウス脾細胞を取り出し、1 wellあたり3 mLのDMEMが入ったNet wellで5 mLのピストンを用いて細胞をほぐした。Net wellの下層を15 mL tubeに移した後、各wellを等量のDMEMで洗浄して15 mL
tubeに移した。300ｇで10分間遠心分離した後、上清を捨て、10 mL のDMEMを加えサスペンドした。この操作を3回繰り返した後、細胞数をカウントした。 (Preparation of cells)
The mouse spleen cells after immunization were taken out, and the cells were loosened using a 5 mL piston in a Net well containing 3 mL of DMEM per well. After transferring the lower layer of the Net well to a 15 mL tube, each well was washed with an equal volume of DMEM and 15 mL
Moved to tube. After centrifugation at 300 g for 10 minutes, the supernatant was discarded and 10 mL of DMEM was added and suspended. After repeating this operation three times, the number of cells was counted.
　ミエローマ細胞については、３７℃、５％ CO2存在下、10cm dish内で培養していたミエローマ細胞（PAI細胞）をサスペンドして50 mL tubeに回収した。その後、1000 rpmで５分間遠心分離し上清を捨て、DMEM 10 mLを加え、細胞数をカウントした。 Regarding myeloma cells, myeloma cells (PAI cells) cultured in a 10 cm dish in the presence of 5% CO 2 at 37 ° C. were suspended and collected in 50 mL tubes. Thereafter, the mixture was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, 10 mL of DMEM was added, and the number of cells was counted.
　（ハイブリドーマの作製）
　上記により調製された脾細胞とミエローマ細胞とを、１０：１の細胞数割合で混合した後、1000 rpmで10分間遠心分離した後、上清を捨てPEG（polyethylene glycol 1500）をtubeの壁をつたわらせながら30秒かけて加えた。その後、3 mLのDMEMを30秒かけて加えた。その後FCSを30秒かけて加え1000 rpmで５分間遠心分離した。上清を捨ていわゆるHAT培地を20ml添加し1000
rpmで５分間遠心分離し上清を捨てた。その後、それぞれ5.0×105cells/
wellおよび2.5×105cells/
wellになるように適量のHAT培地を加えサスペンドした。サスペンド後、あらかじめ100μL/wellのHAT培地を入れ、37℃、５％、CO2存在下に反応させておいた96well plate に、100μL/wellで播種した。 (Production of hybridoma)
Spleen cells and myeloma cells prepared as described above were mixed at a cell number ratio of 10: 1, and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded and PEG (polyethylene glycol 1500) was removed from the wall of the tube. It was added over 30 seconds while shaking. Thereafter, 3 mL of DMEM was added over 30 seconds. After that, FCS was added over 30 seconds and centrifuged at 1000 rpm for 5 minutes. Discard the supernatant and add 20 ml of so-called HAT medium.
Centrifugation was performed at rpm for 5 minutes, and the supernatant was discarded. After that, 5.0 × 10 5 cells /
well and 2.5 × 10 5 cells /
An appropriate amount of HAT medium was added to suspend and suspended. After suspending, 100 μL / well of HAT medium was added in advance, and seeded at 100 μL / well on a 96-well plate that had been reacted in the presence of CO 2 at 37 ° C., 5%.
　培養7日後、培養上清を50μl回収し抗原ペプチドを抗原としたＥＬＩＳＡ法により抗体価を測定した。その結果を表1に示す。表１において、「5.0×10＾5cells/well」と表記したテーブルは上記の5.0×105 cells/wellとした群を示し、「2.5×10＾5cells/well」と表記したテーブルは上記の2.5×105 cells/wellとした群を示す。また、表１中の縦方向の「Ａ」～「Ｈ」の区分は96well plateの「Ａ」～「Ｈ」を示し、横方向の「１」～「１２」の区分は96well
plateの「１」～「１２」を示す。これらの内、Ｈ１２ではポジティブコントロールとして免疫終了後のマウスの血清を用いた。 After 7 days of culture, 50 μl of the culture supernatant was collected, and the antibody titer was measured by ELISA using the antigen peptide as an antigen. The results are shown in Table 1. In Table 1, the table described as “5.0 × 10 ^ 5 cells / well” indicates the group designated as 5.0 × 10 5 cells / well, and the table expressed as “2.5 × 10 ^ 5 cells / well” corresponds to the above 2.5. A group of × 10 5 cells / well is shown. In Table 1, “A” to “H” in the vertical direction indicate “A” to “H” in the 96-well plate, and “1” to “12” in the horizontal direction indicate 96 well.
“1” to “12” of the plate are shown. Among these, H12 used mouse serum after completion of immunization as a positive control.
Figure JPOXMLDOC01-appb-T000001
 　70～80％コンフルエント状態のHEK293T細胞（10cm dishで培養）の培地をOpti-MEM（Invitrogen）に置換し、pCrl（pcDNA3にリーリンのcDNA全長を挿入したベクター）15μgと30μLのLipofectamine 2000（Invitrogen）を用いてトランスフェクションを行った。5時間後、50 Units/mL
Penicillin + 50 mg/mL Streptomycinを含むOpti-MEMに培地を交換し、37℃、5％CO2存在下で培養した。48h後に0.45μm滅菌フィルターおよび、5mLシリンジを用いて上清を回収し、上清と4×SDS sumpling bufferを3：1で混合しサンプル化した。
Figure JPOXMLDOC01-appb-T000001
 The medium of 70-80% confluent HEK293T cells (cultured in a 10cm dish) was replaced with Opti-MEM (Invitrogen), and 15μg and 30μL of Lipofectamine 2000 (Invitrogen) pCrl (a vector in which the full length of Reelin cDNA was inserted into pcDNA3) Was used for transfection. After 5 hours, 50 Units / mL
The medium was replaced with Opti-MEM containing Penicillin + 50 mg / mL Streptomycin, and the cells were cultured at 37 ° C. in the presence of 5% CO 2 . After 48 hours, the supernatant was collected using a 0.45 μm sterilizing filter and a 5 mL syringe, and the supernatant was mixed with 4 × SDS sumpling buffer 3: 1 to prepare a sample.
　サンプルを1レーンあたり30μL注ぎ、SDS-ポリアクリルアミド電気泳動（SDS-PAGE）により分離し、セミドライ式転写機（Trans-blot SD cell、Bio-Red）を用いてPVDF膜（Millipore）に転写した。転写後のPVDF膜は5％スキムミルク（森永乳業）in TBSTで30分間ブロッキングを行った。ブロッキング後、PVDF膜と、表１で抗体価が０．４００以上と比較的高かった各クローンから得られた抗体とを室温で2時間インキュベートした。その後、PVDF膜をTBSTで5分間×4回洗浄し、HRP標識された二次抗体を含む5％スキムミルク in TBSTを室温で1時間インキュベートした。そして、PVDF膜をTBSTで5分間×4回洗浄し、Immobilon Western Chemiluminescent HRP
Substrate（Millipore）を用い可視化し、LAS4000（GE Healthcare）にて発光を検出した。その結果、特に活性型リーリンと特異的に結合したのが、表１の「2.5
cells/well」と表記したテーブル中の「F３」のクローンである。このクローンを「2F3株」と名付けた。
（マウスハイブリドーマ細胞からのｍＲＮＡの抽出）
　培養されたハイブリドーマ細胞2F3株の細胞4.2×１０6個から、ｍＲＮＡ精製用の RNeasy Protect Mini Kit（QIAGEN社）を用いて、ｍＲＮＡの抽出を行った。細胞をRLTバッファー350μLに溶解しシリンジ内でホモジナイズした後、等量の70％エタノールを添加したその後、RNeasyスピンカラムに移して10400 rpmで１５秒間遠心分離し、その下層の液を除去した。RW1バッファー700μLをRNeasyスピンカラムに添加し、10400 rpmで１５秒間遠心分離し、その下層の液を除去した。その後、RPEバッファー500μLをRNeasyスピンカラムに添加し、10400 rpmで１５秒間遠心分離し、その下層の液を除去するという操作を2回繰り返した。そして、ＲＮase freeの滅菌水を50μL RNeasyスピンカラムに添加し、10400 rpmで１分間遠心分離しｍＲＮＡを溶出した。その結果、43.4μｇのｍＲＮＡを取得した。 Samples were poured at 30 μL per lane, separated by SDS-polyacrylamide electrophoresis (SDS-PAGE), and transferred to a PVDF membrane (Millipore) using a semi-dry transfer machine (Trans-blot SD cell, Bio-Red). The PVDF membrane after the transfer was blocked with 5% skim milk (Morinaga Milk Industry) in TBST for 30 minutes. After blocking, the PVDF membrane and the antibody obtained from each clone whose antibody titer was relatively high as 0.400 or higher in Table 1 were incubated at room temperature for 2 hours. Thereafter, the PVDF membrane was washed with TBST for 5 minutes × 4 times, and 5% skim milk in TBST containing a secondary antibody labeled with HRP was incubated at room temperature for 1 hour. Then, the PVDF membrane was washed with TBST for 5 minutes x 4 times, and Immobilon Western Chemiluminescent HRP
Visualization was performed using Substrate (Millipore), and luminescence was detected with LAS4000 (GE Healthcare). As a result, it was shown that “2.5” in Table 1 specifically bound specifically to active Reelin.
It is a clone of “F3” in the table labeled “cells / well”. This clone was named “2F3 strain”.
(Extraction of mRNA from mouse hybridoma cells)
MRNA was extracted from 4.2 × 10 6 cells of the hybridoma cell line 2F3 cultured using RNeasy Protect Mini Kit (QIAGEN) for mRNA purification. The cells were dissolved in 350 μL of RLT buffer and homogenized in a syringe. After adding an equal amount of 70% ethanol, the cells were transferred to an RNeasy spin column and centrifuged at 10400 rpm for 15 seconds to remove the lower layer solution. 700 μL of RW1 buffer was added to the RNeasy spin column, and centrifuged at 10400 rpm for 15 seconds to remove the lower layer solution. Thereafter, the operation of adding 500 μL of RPE buffer to the RNeasy spin column, centrifuging at 10400 rpm for 15 seconds, and removing the lower layer solution was repeated twice. Then, RNase-free sterilized water was added to a 50 μL RNeasy spin column, and centrifuged at 10400 rpm for 1 minute to elute mRNA. As a result, 43.4 μg of mRNA was obtained.
　（ｃＤＮＡの合成）
　上記のように取得したハイブリドーマ細胞2F3株のｍＲＮＡから、ｃＤＮＡ合成用のPrimeScript Reverse Transcriptae（タカラバイオ株式会社）を用いてｃＤＮＡの合成を行った。Oligo-ｄＴプライマー１μL,ｄＮＴＰ（2.5ｍＭ）４μL，上記2F3株のｍＲＮＡ4.3μｇを混合後、65℃
５分間保温後氷上で急冷した。その後、反応液５×バッファー４μL，ＲＮase 阻害剤０．５μL，ＡＭＶ（Avian Myeloblastosis Virus）由来の逆転写酵素（５Ｕ／μL）１μLを加え、ＲＮase freeの滅菌水により２０μLとした。 (Synthesis of cDNA)
From the mRNA of the hybridoma cell line 2F3 obtained as described above, cDNA was synthesized using PrimeScript Reverse Transcriptae (Takara Bio Inc.) for cDNA synthesis. After mixing Oligo-dT primer 1μL, dNTP (2.5mM) 4μL, mRNA of 2F3 strain 4.3μg, 65 ℃
After incubating for 5 minutes, it was rapidly cooled on ice. Thereafter, 4 μL of reaction solution 5 × buffer, 0.5 μL of RNase inhibitor, and 1 μL of reverse transcriptase (5 U / μL) derived from AMV (Avian Myeloblastosis Virus) were added to make 20 μL with RNase-free sterilized water.
　上記反応液の作製後、４２℃で６０分間伸長反応を行い、７０℃，１５分間で酵素を失活させた後、氷上で５分間冷した。 After the above reaction solution was prepared, an extension reaction was performed at 42 ° C. for 60 minutes, the enzyme was inactivated at 70 ° C. for 15 minutes, and then cooled on ice for 5 minutes.
　（ＰＣＲ法による抗体遺伝子の増幅）
　抗体遺伝子の可変領域は、Ｈ鎖，Ｌ鎖共にＮ末端、Ｃ末端にそれぞれ相同性を保持していることが知られている。そこで、この相同性を基にＰＣＲプライマーの設計を行った。ＰＣＲプライマーの塩基配列は、Ｈ鎖Ｎ末端が配列表の配列番号１０、Ｈ鎖Ｃ末端が配列表の配列番号１１、Ｌ鎖Ｎ末端が配列表の配列番号１２、Ｌ鎖Ｃ末端が配列表の配列番号１３に、それぞれ示す通りのものとした。これらのＰＣＲプライマーを用いて、日本バイオラッドラポラトリーズ社製のＰＣＲ装置を利用して、ＰＣＲ法によりＨ鎖，Ｌ鎖の遺伝子増幅を行った。 (Amplification of antibody gene by PCR method)
It is known that the variable region of an antibody gene has homology at the N-terminus and C-terminus for both the H and L chains. Therefore, PCR primers were designed based on this homology. The nucleotide sequence of the PCR primer is as follows: the H chain N-terminus is SEQ ID NO: 10 in the sequence listing, the H chain C-terminus is SEQ ID NO: 11 in the sequence listing, the L chain N-terminus is SEQ ID NO: 12 in the sequence listing, and the L chain C-terminus is the sequence listing. SEQ ID NO: 13 were as shown respectively. Using these PCR primers, H chain and L chain genes were amplified by the PCR method using a PCR device manufactured by Nippon Bio-Radola Corporation.
　反応液は、Ｈ鎖，Ｌ鎖のいずれについても、上記でｃＤＮＡを合成した反応液を鋳型として１μL、上記Ｈ鎖又はＬ鎖のＮ末端及びＣ末端プライマーを各20 p mol、１０×ＰＣＲバッファー（タカラバイオ社製）を５μL，2.5ｍＭのｄＮＴＰを３μL，ＤＮＡ合成酵素
Ex Taq（タカラバイオ社製）を0.5μL用い、これらに滅菌水を加えて５０μLとした。 The reaction solution is 1 μL using the reaction solution obtained by synthesizing cDNA as a template for each of the H chain and L chain, 20 pmol each of the H chain or L chain N-terminal and C-terminal primers, and 10 × PCR buffer. (Takara Bio Inc.) 5 μL, 2.5 mM dNTP 3 μL, DNA synthase
Ex Taq (manufactured by Takara Bio Inc.) was used at 0.5 μL, and sterilized water was added to make 50 μL.
　ＰＣＲ法の条件は、解離反応を９４℃で１分、アニーリング反応を４５℃で１分、伸長反応を７２℃で２分とし、これらを３０サイクル行った。 The conditions of the PCR method were a dissociation reaction at 94 ° C. for 1 minute, an annealing reaction at 45 ° C. for 1 minute, and an extension reaction at 72 ° C. for 2 minutes, and these were performed for 30 cycles.
　こうして得られたＰＣＲ反応液の電気泳動を行った処、目的の位置約３５０ bp にバンドを確認することができた。 As a result of electrophoresis of the PCR reaction solution thus obtained, a band could be confirmed at a target position of about 350 bp.
　（抗体遺伝子のクローニング）
　ＰＣＲ法により増幅したＨ鎖，Ｌ鎖の遺伝子をそれぞれpGEM-T Easyベクター（Promega社製）に挿入し、クローニングした。 (Cloning of antibody genes)
The H chain and L chain genes amplified by PCR were inserted into pGEM-T Easy vector (Promega) and cloned.
　（抗体遺伝子の塩基配列決定）
　クローニングした株を、ＰＣＲ後目的の遺伝子が増幅されることを指標としてコロニーＰＣＲにより選択した後、それらの株からプラスミドを抽出し、塩基配列の決定を行った。 (Determining the base sequence of antibody genes)
The cloned strains were selected by colony PCR using the target gene amplified after PCR as an index, and then plasmids were extracted from these strains and their nucleotide sequences were determined.
　その結果、Ｈ鎖可変領域は配列表の配列番号２に示す504塩基（168アミノ酸）、Ｌ鎖可変領域は配列表の配列番号３に示す390塩基（130アミノ酸）で構成されていることが推定された。又、相同領域の配列及び分子量から、ｃＤＮＡのこれらの塩基配列はIgＧモノクローナル抗体のＨ鎖，Ｌ鎖をコードしていると判断された。 As a result, it is estimated that the H chain variable region is composed of 504 bases (168 amino acids) shown in SEQ ID NO: 2 of the sequence listing, and the L chain variable region is composed of 390 bases (130 amino acids) shown in SEQ ID NO: 3 of the sequence listing. It was done. From the sequence and molecular weight of the homologous region, it was determined that these base sequences of cDNA encoded the H chain and L chain of the IgG monoclonal antibody.
　なお、配列番号２の塩基配列における塩基番号163～183（H1領域）、塩基番号226～273（H2領域）、塩基番号370～381（H3領域）、及び、配列番号３の塩基配列における塩基番号94～144（L1領域）、塩基番号190～210（L2領域）、塩基番号307～333（L3領域）の各領域は、前記Ｋａｂａｔのデータベースの超可変領域に相当する部分であり、抗原との結合を決定付ける領域を示す。 In addition, base numbers 163 to 183 (H1 region), base numbers 226 to 273 (H2 region), base numbers 370 to 381 (H3 region) in the base sequence of SEQ ID NO: 2, and base numbers in the base sequence of SEQ ID NO: 3 Each region of 94 to 144 (L1 region), base numbers 190 to 210 (L2 region), and base numbers 307 to 333 (L3 region) is a portion corresponding to the hypervariable region of the Kabat database, Indicates the area that determines the bond.
　（抗体の評価）
　実験方法
　70～80％コンフルエント状態のHEK293T細胞（35 mm dishで培養）の培地をOpti-MEM（Invitrogen）に置換し、pCrl（pcDNA3にリーリンのcDNA全長を挿入したベクター）2.5μgと5μLのLipofectamine 2000（Invitrogen）を用いてトランスフェクションを行った。5時間後、50 Units/mL
Penicillin + 50 mg/mL Streptomycinを含むOpti-MEMに培地を交換し、37℃、5％CO2存在下で培養した。48h後に0.45μm滅菌フィルターおよび、5mLシリンジを用いて上清を回収し、上清と4×SDS sumpling bufferを3：1で混合しサンプル化した。 (Antibody evaluation)
Experimental method The medium of 70-80% confluent HEK293T cells (cultured in a 35 mm dish) was replaced with Opti-MEM (Invitrogen) and 2.5 μg of pCrl (a vector in which the full length of Reelin cDNA was inserted into pcDNA3) and 5 μL of Lipofectamine Transfections were performed using 2000 (Invitrogen). After 5 hours, 50 Units / mL
The medium was replaced with Opti-MEM containing Penicillin + 50 mg / mL Streptomycin, and the cells were cultured at 37 ° C. in the presence of 5% CO 2 . After 48 hours, the supernatant was collected using a 0.45 μm sterilizing filter and a 5 mL syringe, and the supernatant was mixed with 4 × SDS sumpling buffer 3: 1 to prepare a sample.
　サンプルを1レーンあたり30μL注ぎ、SDS-ポリアクリルアミド電気泳動（SDS-PAGE）により分離し、セミドライ式転写機（Trans-blot SD cell、Bio-Red）を用いてPVDF膜（Millipore）に転写した。転写後のPVDF膜は5％スキムミルク（森永乳業）in TBSTで30分間ブロッキングを行った。ブロッキング後、PVDF膜と一次抗体を含む5％スキムミルク in TBSTとを室温で2時間インキュベートした。その後、PVDF膜をTBSTで5分間×4回洗浄し、HRP標識された二次抗体を含む5％スキムミルク in TBSTを室温で1時間インキュベートした。そして、PVDF膜をTBSTで5分間×4回洗浄し、Immobilon Western
Chemiluminescent HRP Substrate（Millipore）を用い可視化し、LAS4000（GE Healthcare）にて発光を検出した。 Samples were poured at 30 μL per lane, separated by SDS-polyacrylamide electrophoresis (SDS-PAGE), and transferred to a PVDF membrane (Millipore) using a semi-dry transfer machine (Trans-blot SD cell, Bio-Red). The PVDF membrane after the transfer was blocked with 5% skim milk (Morinaga Milk Industry) in TBST for 30 minutes. After blocking, a PVDF membrane and 5% skim milk in TBST containing the primary antibody were incubated at room temperature for 2 hours. Thereafter, the PVDF membrane was washed with TBST for 5 minutes × 4 times, and 5% skim milk in TBST containing a secondary antibody labeled with HRP was incubated at room temperature for 1 hour. Then, the PVDF membrane was washed with TBST for 5 minutes x 4 times, Immobilon Western
Visualization was performed using Chemiluminescent HRP Substrate (Millipore), and luminescence was detected with LAS4000 (GE Healthcare).
　抗体
　一次抗体として、市販のマウス抗リーリンN末端認識モノクローナル抗体 G10（1:3000 Millipore）と、2F3株から得られた抗体（2F3抗体）を用いた。又、二次抗体としてAnti-mouse IgG HRP-linked antibody（1:4000、GE Healthcare）を用いた。 As a primary antibody, a commercially available mouse anti-Reelin N-terminal recognition monoclonal antibody G10 (1: 3000 Millipore) and an antibody obtained from the 2F3 strain (2F3 antibody) were used. Anti-mouse IgG HRP-linked antibody (1: 4000, GE Healthcare) was used as a secondary antibody.
　結果
　評価の結果を述べる前に、図１に基づいて簡単な説明を行う。図１の上段には図案化された全長リーリンと、そのN-t site及びC-t siteが示されている。図１の中段に示す「ＮＲ６」とは、全長リーリンがC-t siteで切断された場合におけるアミノ末端側の断片であり、図１の下段に示す「ＮＲ２」とは、全長リーリンがN-t siteで切断された場合におけるアミノ末端側の断片である。従って、全長リーリン、ＮＲ２断片及びＮＲ６断片の全てを認識する抗体は、活性型リーリンと不活性リーリンとを区別なく認識する抗体である。これに対して、全長リーリンとＮＲ６断片を認識するが、ＮＲ２断片を認識しない抗体は、活性型リーリンのみを認識する抗体である。 Before describing the results of the result evaluation, a brief description will be given based on FIG. In the upper part of FIG. 1, the full-length Reelin designed, and its Nt site and Ct site are shown. “NR6” shown in the middle of FIG. 1 is a fragment on the amino terminal side when full-length Reelin is cleaved at Ct site, and “NR2” shown in the lower part of FIG. 1 is a full-length Reelin cut at Nt site. It is a fragment on the amino terminal side when Therefore, an antibody that recognizes all of full-length Reelin, NR2 fragment, and NR6 fragment is an antibody that recognizes active Reelin and inactive Reelin without discrimination. In contrast, an antibody that recognizes full-length Reelin and an NR6 fragment but not an NR2 fragment is an antibody that recognizes only active Reelin.
　上記の評価の結果、図２に示すように、G10抗体ではNR2のバンドが検出できるのに対し、2F3抗体ではNR2バンドは検出されなかった。このことから、2F3抗体はN-t siteで分解がおきていないリーリン、即ち活性型リーリンのみを認識すると考えられる。 As a result of the above evaluation, as shown in FIG. 2, the G10 antibody could detect the NR2 band, whereas the 2F3 antibody did not detect the NR2 band. From this, it is considered that the 2F3 antibody recognizes only the Reelin that is not degraded at Nt site, that is, the active Reelin.
　本発明により、大量のサンプルであっても簡便、迅速に活性型リーリンを選択的に測定できる手段が提供される。 The present invention provides a means for selectively measuring active Reelin easily and quickly even with a large amount of sample.

Claims (10)

  1. 活性型リーリンに対して特異的に結合し、不活性型リーリンに対しては結合しない抗体であることを特徴とする抗活性型リーリン抗体。 An anti-active Reelin antibody, which is an antibody that specifically binds to active Reelin but does not bind to inactive Reelin.
  2. 配列表の配列番号１に示すアミノ酸配列を有するペプチドに対して特異的に結合する抗体であることを特徴とする抗活性型リーリン抗体。 An anti-active Reelin antibody, which is an antibody that specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
  3. 配列表の配列番号２に示すアミノ酸配列を有するＨ鎖可変領域と、配列表の配列番号３に示すアミノ酸配列を有するＬ鎖可変領域とを有するポリペプチドからなる抗体であることを特徴とする抗活性型リーリン抗体。 It is an antibody comprising a polypeptide having an H chain variable region having the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing and an L chain variable region having the amino acid sequence shown in SEQ ID NO: 3 of the sequence listing. Active Reelin antibody.
  4. 配列表の配列番号２に示すアミノ酸配列に対して１～数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を有するＨ鎖可変領域と、配列表の配列番号３に示すアミノ酸配列に対して１～数個のアミノ酸が置換、欠失又は付加されたアミノ酸配列を有するＬ鎖可変領域とを有するポリペプチドからなり、以下の（１）、（２）の少なくとも一方に該当する抗体であることを特徴とする抗活性型リーリン抗体。
    　（１）活性型リーリンに対して特異的に結合し、不活性型リーリンに対しては結合しない。
    　（２）配列表の配列番号１に示すアミノ酸配列を有するペプチドに対して特異的に結合する。     The heavy chain variable region having an amino acid sequence in which one to several amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing An antibody corresponding to at least one of the following (1) and (2), comprising an L chain variable region having an amino acid sequence in which one to several amino acids are substituted, deleted or added An anti-active Reelin antibody characterized by the above.
    (1) It binds specifically to active Reelin and does not bind to inactive Reelin.
    (2) It specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
  5. 配列表の配列番号４～６に示すアミノ酸配列からなるＣＤＲ配列の全てを含むＨ鎖可変領域と、配列表の配列番号７～９に示すアミノ酸配列からなるＣＤＲ配列の全てを含むＬ鎖可変領域とを有するポリペプチドからなる抗体であることを特徴とする抗活性型リーリン抗体。 H chain variable region containing all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6 in the sequence listing, and L chain variable region containing all of the CDR sequences consisting of the amino acid sequences shown in SEQ ID NOs: 7 to 9 of the sequence listing An anti-active Reelin antibody, which is an antibody comprising a polypeptide having
  6. 以下（３）～（８）のいずれかに該当することを特徴とする核酸。
    　（３）配列表の配列番号４に示すアミノ酸配列をコードする核酸。
    　（４）配列表の配列番号５に示すアミノ酸配列をコードする核酸。
    　（５）配列表の配列番号６に示すアミノ酸配列をコードする核酸。
    　（６）配列表の配列番号７に示すアミノ酸配列をコードする核酸。
    　（７）配列表の配列番号８に示すアミノ酸配列をコードする核酸。
    　（８）配列表の配列番号９に示すアミノ酸配列をコードする核酸。     A nucleic acid that falls under any of the following (3) to (8):
    (3) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.
    (4) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing.
    (5) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing.
    (6) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing.
    (7) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing.
    (8) A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing.
  7. 請求項６に記載の（３）～（８）の全ての塩基配列を含み、第１発明～第５発明のいずれかに記載の抗活性型リーリン抗体をコードすることを特徴とする核酸。 A nucleic acid comprising all the base sequences of (3) to (8) according to claim 6 and encoding the anti-active Reelin antibody according to any one of the first to fifth inventions.
  8. 哺乳動物のミエローマ細胞と、配列表の配列番号１に示すアミノ酸配列を有するペプチドで免疫された哺乳動物の脾細胞との融合によって作製され、請求項１～５のいずれかに記載の抗活性型リーリン抗体を生産することを特徴とするハイブリドーマ細胞。 The anti-active type according to any one of claims 1 to 5, which is prepared by fusion of a mammalian myeloma cell and a spleen cell of a mammal immunized with a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. A hybridoma cell characterized by producing a Reelin antibody.
  9. 請求項１～５のいずれかに記載の抗活性型リーリン抗体を用いた抗原－抗体反応により、被験試料中の活性型リーリンを測定することを特徴とする活性型リーリン測定方法。 6. A method for measuring active Reelin, which comprises measuring active Reelin in a test sample by an antigen-antibody reaction using the anti-active Reelin antibody according to any one of claims 1 to 5.
  10. 請求項１～５のいずれかに記載の抗活性型リーリン抗体を含んで構成され、被験試料中の活性型リーリンを測定するために用いるものであることを特徴とする活性型リーリン測定用キット。 An active Reelin measurement kit comprising the anti-active Reelin antibody according to any one of Claims 1 to 5 and used for measuring active Reelin in a test sample.
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