WO2012133898A1 - 未変性Cochlin-tomoprotein(CTP)に反応する抗体及びそれを用いたCTPの測定方法 - Google Patents
未変性Cochlin-tomoprotein(CTP)に反応する抗体及びそれを用いたCTPの測定方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/14—Disorders of ear, nose or throat
Definitions
- the present invention relates to an antibody that reacts with native CTP, a kit containing the same, and an immunoassay method using the same. Furthermore, the present invention relates to a method for screening an antibody that reacts with native CTP.
- Cochlin-tomoprotein (hereinafter referred to as CTP) is an isoform of Cochlin with a molecular weight of 16 kDa that exists in the perilymph, but is found in other body fluids such as cerebrospinal fluid, blood, and saliva. Therefore, it is expected as a diagnostic marker for perilymph fistula (Patent Document 1, Non-Patent Documents 1 to 5).
- the perilymph fistula is a disease in which the inner ear window separating the middle ear from the inner ear has a hole, and the perilymph filling the inner ear leaks into the middle ear, thereby presenting a functional disorder of the inner ear. It is very important to diagnose early because the symptoms improve by closing the inner ear window in the early stage of onset.
- Non-Patent Document 1 The inventor's research group has so far prepared four types of rabbit polyclonal antibodies using synthetic peptides as immunogens in order to measure CTP in biological samples.
- All the antibodies react only with denatured CTP, and no antibody reacts with native CTP. Therefore, in Non-Patent Documents 1 to 5, CTP in a biological sample is measured by Western blotting using an anti-LCCL3 antibody (not reacting with native CTP) that recognizes the modified CTP.
- CTP is present in a wide range of animal species such as mice, pigs and cattle, and since the homology between the animal species is high, the antibody titer is unlikely to rise and only a few clones were obtained. The obtained clone also did not react unless it was in denaturing conditions, and an antibody reacting with native CTP was not obtained.
- CTP Since the amount of CTP present in the living body is extremely small, it is impossible to prepare CTP from a biological sample for use as an immunogen for antibody production. Therefore, it is necessary to prepare an antibody using a synthetic peptide, a recombinant protein or the like as an immunogen. However, as described above, since an antibody against a synthetic peptide and an antibody against an E. coli-expressed recombinant CTP do not react with native CTP, CTP is considered to form a highly tertiary structure in a biological sample. In general, screening for antibody production involves selection of strains that react well with the antigen used for immunization.
- the present invention provides an antibody that recognizes native CTP in order to solve the above problems. Moreover, the measuring method of CTP in the biological sample using the antibody is provided. Furthermore, a method for screening an antibody that recognizes native CTP is provided.
- native CTP can be measured by using an antibody that recognizes an epitope (amino acid numbers 118 to 132 of SEQ ID NO: 1) in the peptide sequence of CTP-C. It was. Further, it has been found that by recognizing an antibody that reacts with native CTP captured by an anti-CTP-C antibody immobilized on a carrier, an antibody that recognizes native CTP can be obtained, The present invention has been completed.
- the present invention is as follows. (1) An anti-CTP antibody which recognizes an antigenic determinant contained in the polypeptide represented by amino acid numbers 118 to 132 of SEQ ID NO: 1 and reacts with native CTP. (2) The anti-CTP antibody according to (1), obtained by using a peptide consisting of amino acid numbers 118 to 132 of SEQ ID NO: 1 as an immunogen. (3) Prepare native CTP captured by a first antibody that reacts with native CTP, and react with native CTP, including a step of selecting a second antibody that recognizes the native CTP Antibody screening method. (4) The method for screening an antibody that reacts with native CTP according to claim 3, wherein the first antibody is the antibody according to (1).
- a method for examining perilymph fistula which comprises the step of immunologically measuring CTP using at least one antibody according to (1) or (2).
- a CTP measurement kit comprising at least one antibody according to (1) or (2).
- Cochlin is a protein encoded by the gene COCH identified as the causative gene of nonsyndromic hereditary deafness DFNA9 (NG Robertson, Nature Genet., 20, 299-303 (1998). )).
- COCH nonsyndromic hereditary deafness DFNA9
- the amino acid sequence of human Cochlin can be found in Nature Genet. , 20, 299-303 (1998).
- CTP is an approximately 16 kDa isoform consisting of an N-terminal fragment of Cochlin. CTP has been confirmed in a wide range of animal species including humans, cows, pigs, guinea pigs, rats, mice and the like, but human CTP is preferred.
- the amino acid sequence of human CTP is shown in SEQ ID NO: 1.
- the portion represented by amino acid numbers 1 to 24 in the amino acid sequence is a signal sequence.
- the antibody of the present invention recognizes an antigenic determinant (hereinafter sometimes referred to as “epitope”) contained in the polypeptide represented by amino acid numbers 118 to 132 of SEQ ID NO: 1. And reacts with native CTP.
- epipe an antigenic determinant contained in the polypeptide represented by amino acid numbers 118 to 132 of SEQ ID NO: 1.
- An antibody that does not react with a protein other than the protein containing the polypeptide represented by amino acid numbers 118 to 132 of SEQ ID NO: 1 is preferable.
- the antibody of the present invention may further react with denatured CTP as long as it reacts with native CTP, and further reacts with a Cochlin isoform containing the polypeptide represented by amino acid numbers 118 to 132 of SEQ ID NO: 1. May be.
- p63 is known in addition to CTP (Ikezono et al., Biochem. Biophys. Acta, 1535, 3,258-265 (2001)) and may react therewith.
- Undenatured CTP refers to CTP that has not been subjected to denaturation treatment that significantly changes the tertiary structure of the protein.
- the denaturing treatment include addition of a protein denaturing agent (surfactant such as SDS, reducing agent such as DTT, urea, acetone, etc.) and heat treatment.
- a sample diluent or the like in an immunoassay may contain a low-concentration surfactant or the like, but such a condition is not a denaturing treatment.
- the anti-CTP antibody can be produced using, for example, a polypeptide consisting of amino acid numbers 118 to 132 of SEQ ID NO: 1 (hereinafter, this may be referred to as “antigen polypeptide”) as an immunogen.
- the antigen polypeptide may be a synthetic polypeptide chemically synthesized according to a known method, or may be produced by genetic recombination or the like.
- the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody.
- carrier proteins such as KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), porcine thyroid globulin, carbodiimide, maleimide, etc.
- the antigen polypeptide is bound using a condensing agent to produce an antigen for immunization (immunogen).
- the antigen polypeptide may be bound to the carrier protein by a commonly used method known per se. For example, in the case of a method of binding an antigen polypeptide by maleimidation using KLH as a carrier protein.
- a bifunctional condensing agent such as Sulfo-SMCC (Sulfosuccimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) to form maleimide and bind to N-terminal or C-terminal.
- Sulfo-SMCC Sulfosuccimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
- cysteine is contained in the amino acid sequence of the selected antigen polypeptide, it can be used for binding.
- carbodiimidized KLH when used, it can be bound by forming a peptide bond by dehydration condensation with an antigen polypeptide.
- a carrier protein is preferably bound to the N-terminal side of the antigen polypeptide.
- the solution containing the immunogen prepared in this manner is mixed with an adjuvant as necessary, and 2 or less are applied subcutaneously or abdominally to animals such as rabbits, mice, rats, guinea pigs, sheep, goats, chickens, etc., which are usually used for antibody production. Immunize every 3 weeks. After immunization, it is preferable to collect blood appropriately on a trial basis and confirm that the titer (antibody titer) is sufficiently increased by immunological methods such as ELISA and Western blotting. Antiserum can be obtained by collecting blood from an animal that has been confirmed to have a sufficient increase in titer and separating the serum. In the case of chickens, a water-soluble fraction is collected from egg yolk collected from chicken eggs to prepare an egg yolk extract, which can also be used in the same manner as antiserum.
- the obtained antiserum or the like can be used as it is without being purified, but it is preferably purified and used by the following method.
- a purification method using Protein A a salting-out method using ammonium sulfate, a method of purifying an immunoglobulin fraction by ion exchange chromatography, or an affinity using a column on which a specific polypeptide is immobilized.
- the purification method include column chromatography.
- the purification method using Protein A and the method using affinity column chromatography are preferably performed either in combination or in combination.
- the polypeptide for purification to be immobilized on the column a polypeptide containing the same sequence or a part of the sequence may be selected and used according to the amino acid sequence of the antigen polypeptide used.
- antibody-producing cells are collected from the spleen of an immunized animal in the same manner as described above, and fused with a cultured cell such as a myeloma cell derived from a syngeneic animal or the like by a conventional method. Fabrication (Milstein et al., Nature, 256, 495 (1975)). After culturing, the antibody titer is appropriately confirmed by ELISA or the like, and a hybridoma that produces a monoclonal antibody that recognizes the target epitope and has a high antibody-producing ability may be selected. The target monoclonal antibody can be obtained from the culture supernatant of the hybridoma thus selected.
- the antibody thus obtained is an antibody that specifically recognizes native CTP. This can be confirmed by collecting a sample known to contain CTP such as perilymph from a suitable animal species and analyzing the reactivity with native CTP in the sample.
- antibody as used herein includes not only full-length antibodies but also antibody fragments.
- the antibody fragment is preferably a functional fragment containing an antigen-binding region of an antibody or a variable region thereof, and examples thereof include F (ab ′) 2 , Fab ′, Fab and the like.
- F (ab ′) 2 and Fab ′ are produced by treating immunoglobulin with a proteolytic enzyme (for example, pepsin or papain), and exist between two H chains in the hinge region. It is an antibody fragment produced by digesting before and after disulfide bonds.
- an L chain consisting of VL (L chain variable region) and CL (L chain constant region) is cleaved upstream of a disulfide bond existing between two H chains in the hinge region
- two homologous antibody fragments in which an H chain fragment consisting of VH (H chain variable region) and CH ⁇ 1 ( ⁇ 1 region in the H chain constant region) are linked by a disulfide bond at the C-terminal region can be produced.
- Each of these two homologous antibody fragments is Fab ′ That's it.
- an antibody fragment that is cleaved downstream of the disulfide bond existing between the two heavy chains in the hinge region to produce a slightly larger antibody fragment than the two Fab's connected in the hinge region is produced. Can do. This antibody fragment is called F (ab ′) 2 .
- the antibody of the present invention can be used as an immobilized antibody supported on an insoluble carrier such as a solid phase carrier, or as a labeled antibody labeled with a labeling substance.
- an insoluble carrier such as a solid phase carrier
- a labeled antibody labeled with a labeling substance Such immobilized antibodies and labeled antibodies are all within the scope of the present invention.
- immobilized antibody refers to an antibody that is supported on an insoluble carrier by physical adsorption or chemical bonding. These immobilized antibodies can be used for detecting or quantifying CTP contained in a sample.
- insoluble carriers that can be used for supporting antibodies include (1) plastics made of polystyrene resin, polycarbonate resin, silicon resin, nylon resin, etc., glass, latex, metal compounds, magnetic materials, and the like.
- the labeled antibody means an antibody labeled with a labeling substance, and these labeled antibodies can be used for detecting or quantifying CTP contained in a sample.
- the labeling substance that can be used in the present invention is not particularly limited as long as it can detect the presence of the labeling substance by binding it to the antibody by physical binding or chemical binding.
- labeling substances include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin or radioisotopes, and more specifically, peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose -Su-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate , dansyl chloride or fluorescent substances such as tetramethylrhodamine isothiocyanate, 3 H, 14 C, radioisotopes such as 125 I or 131 I, biotin, avidin or chemical, Light material, and the like.
- a known method such as glutaraldehyde method,
- radioisotopes and fluorescent materials can provide a detectable signal alone, but enzymes, chemiluminescent materials, biotin and avidin cannot provide a detectable signal by themselves, and therefore one more kind. Reaction with these other substances produces a detectable signal.
- an enzyme at least a substrate is required, and various substrates are used depending on a method for measuring enzyme activity (colorimetric method, fluorescence method, bioluminescence method, chemiluminescence method, etc.).
- biotin at least avidin or enzyme-modified avidin is generally reacted.
- Various color-developing substances depending on the substrate are used as necessary.
- the method for screening an antibody reacting to native CTP of the present invention comprises preparing a native CTP captured by a first antibody that reacts to native CTP. Selecting a second antibody that recognizes denatured CTP.
- the first antibody is preferably an antibody that recognizes an antigenic determinant contained in the polypeptide represented by amino acid numbers 118 to 132 of SEQ ID NO: 1 and reacts with native CTP. That is, by using the antibody of the present invention, a new antibody that reacts with native CTP can be obtained.
- a new antibody that reacts with native CTP is obtained by screening using the above-described antibody of the present invention as the first antibody and using a library of anti-CTP antibodies as the second antibody. Can be sorted.
- the antibody obtained by the screening method is not particularly limited as long as it is an antibody that reacts with native CTP, but is contained in a region other than the higher order structure and the polypeptide represented by amino acid numbers 118 to 132 of SEQ ID NO: 1.
- An antibody that recognizes an antigenic determinant is preferred.
- the measurement method of the present invention uses at least one of the above-described antibodies of the present invention to determine amino acids 118-132 of SEQ ID NO: 1.
- qualitative measurement and quantitative measurement are included.
- the perilymph fistula means that the perilymph present in the inner ear tissue is an inner ear window (either a round window or an oval window or both) or a fissura ante festram (inner ear and middle ear).
- This is a disease that leaks into the tympanic chamber (middle ear) from the bone space between the ears and causes impairment of hearing and balance. The disease can be detected by confirming that perilymph leaks into the middle ear.
- the method for detecting perilymph fistula detects the presence of CTP present only in the perilymph from body fluids that may be present in the middle ear of a patient suspected of suffering from the disease, Is an indicator of the possibility of suffering from perilymph fistula. According to this method, detection can be performed regardless of the cause or mechanism of the onset of perilymph fistula.
- Patients suspected of suffering from the disease also include patients with sudden hearing loss, inner ear hearing loss, Meniere's disease, vestibular neuritis, head position vertigo, inner ear vertigo and the like.
- a sample containing body fluid present in the middle ear of a patient suspected of suffering from perilymph fistula is used.
- body fluids that can exist in the human middle ear include, for example, perilymph, cerebrospinal fluid (hereinafter referred to as “CSF”), blood, saliva, and middle ear mucosa.
- CSF cerebrospinal fluid
- Middle ear mucus and the like it is known that CSF flows into the inner ear through the 8th cranial nerve pathway of the inner ear canal or the cochlear tubule due to surgery or the like, and also flows into the middle ear due to trauma, fracture, inner ear malformation, etc. It has been.
- Blood can be present in the middle ear due to bleeding from trauma, bleeding from the mucosa of the middle ear, and the like. It is known that saliva flows into the middle ear by flowing back from the ear canal in the nasopharynx. There may also be middle ear exudate in patients with exudative otitis media, otorrhea (pus), etc. in patients with chronic otitis media. Although these body fluids cannot be visually discerned, whether or not perilymph is included in the body fluid collected as a sample by collecting and analyzing them and analyzing the presence of CTP in the sample And can be used as an index of the possibility of perilymph fistula. Examples of the sample containing the body fluid include a middle ear washing liquid, a nasal wipe, and an upper throat wipe.
- the body fluid present in the middle ear it can be collected without mixing blood, drugs, etc. as much as possible, and without mixing other proteins, etc. Any method is acceptable.
- the eardrum may be finely incised, a syringe or the like may be inserted and the body fluid may be sucked and collected as it is, or a cotton swab or the like may be inserted to wipe off the existing body fluid.
- a method of injecting an appropriate amount of an appropriate solution such as physiological saline using a syringe or the like and then collecting the solution together with a syringe or the like is preferably used.
- the solution recovered by such a method is referred to as “middle ear cleaning solution”.
- a solution that is physiologically acceptable in composition, pH, temperature and the like and has a small burden on the patient is selected.
- body fluid derived from the middle ear that has reached the upper pharynx and the oropharynx through the ear canal may be collected. Specifically, for example, it can be collected by inserting a cotton swab or the like from the oral cavity or nasal cavity and wiping off body fluid present in the upper pharynx or oropharynx. These can be collected more easily and less invasively than the middle ear washing solution collected by tympanic incision.
- the CTP measuring method of the present invention is also suitably used for diagnosis during head and neck surgery.
- perilymph fistula is caused by bone destruction in cholesteatoma otitis media, outer ear tumors, middle ear tumors, acoustic nerve tumors, etc., and the extent of this lesion can be diagnosed by this test . That is, if the lesion destroys the bone and reaches the inner ear, CTP is detected from the body fluid present in the middle ear, and if it is shallower than that, CTP is not detected.
- surgical treatment may be applied to the round window or oval window, and it can be determined by this measurement whether or not these have been damaged by the surgical operation. Furthermore, it is useful for determining the electrode insertion site of the cochlear implant in cochlear implant surgery. This is particularly useful in cases with inner ear and middle ear malformations. In these examinations, it is possible to directly collect the middle ear washing liquid or the leaked liquid from the lesion site or the surgical operation site and use it for the examination.
- the sample used for the measurement is not particularly limited as long as it contains CTP, and may be a cell culture solution or the like.
- a sample derived from an animal other than a human may be used for grasping the pathological condition of an experimental animal. Although it does not restrict
- the body fluid thus collected is preferably subjected to analysis immediately after collection, but can also be stored under low temperature conditions such as 4 to -80 ° C, preferably -20 to -70 ° C.
- a preservative that suppresses protein denaturation or the like, a preservative for preventing spoilage, or the like may be added as necessary.
- these samples may be subjected to analysis after pretreatment such as removal of blood cells and tissue pieces, concentration, and purification as necessary. These specific methods may be any method known per se, such as concentration and purification of commonly used proteins.
- the method for detecting the presence of CTP is an immunological method using an antibody recognizing the native CTP (hereinafter, sometimes referred to as “anti-native CTP antibody”).
- immunological methods for protein detection include immunity using labeled antibodies such as enzyme immunoassay (ELISA), chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunochromatography, etc. Any method can be used as long as it is a commonly used method known per se, such as a Western blotting method under non-denaturing conditions, a latex agglutination method, and an immunoturbidimetric method. From the viewpoint of measurement accuracy, an immunoassay method using a labeled antibody is preferably used. For the intraoperative diagnosis, since it is desired to obtain a result quickly, enzyme immunoassay (ELISA method), immunochromatography and the like are particularly preferably used.
- the detection method of the present invention is performed by an immunoassay using a labeled antibody such as an enzyme immunoassay (ELISA), a chemiluminescence immunoassay, a fluorescent antibody method, or a radioimmunoassay
- a sandwich method is used. Or it can also carry out by a competition method.
- the sandwich method at least one of the immobilized antibody and the labeled antibody may be an anti-native CTP antibody.
- the amount of leakage to the middle ear of the perilymph can be quantitatively measured by quantifying the luminescent signal. Since it is not necessary to prepare a CTP analog, sandwich type measurement using two types of antibodies that recognize CTP with different epitopes is particularly preferable.
- the solid phase carrier used in the sandwich method may be any insoluble carrier that can be used to support the antibody.
- plastic made of polystyrene resin, polycarbonate resin, silicon resin, nylon resin, etc., glass, latex Plates made of water-insoluble substances such as metal compounds and magnetic materials, those having an internal volume such as test tubes or tubes, beads, balls, filters, membranes, etc.
- cellulose Insoluble carriers used in affinity chromatography such as carriers, agarose carriers, polyacrylamide carriers, dextran carriers, polystyrene carriers, polyvinyl alcohol carriers, polyamino acid carriers, porous silica carriers, etc. it can
- the operation method of measurement is a known method (for example, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983, edited by Yuji Ishikawa et al. "Enzyme immunoassay", 3rd edition, medical school, 1987, edited by Tsuneaki Kitagawa et al., "Protein nucleic acid enzyme separate volume No.31, enzyme immunoassay", Kyoritsu Shuppan, 1987).
- the immobilized antibody and the sample are reacted, and the labeled antibody is reacted at the same time, or the labeled antibody is reacted after washing to form the immobilized antibody-antigen-labeled antibody complex.
- Unbound labeled antibody can be washed and separated, and the amount of antigen in the sample can be measured from the amount of bound labeled antibody.
- ELISA method enzyme immunoassay
- the substrate is reacted with the labeled enzyme under the optimum conditions, and the amount of the reaction product is measured by an optical method or the like.
- the fluorescence immunoassay the fluorescence intensity by the fluorescent substance label is measured, and in the case of the radioimmunoassay, the radiation dose by the radioactive substance label is measured. In the case of chemiluminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.
- the detection method of the present invention is a measurement in which the formation of an immune complex aggregate is measured by an optical method or visually measured, as in the case of latex agglutination or immunoturbidimetry.
- a phosphate buffer, glycine buffer, Tris buffer or Good buffer can be used as a solvent, and further includes a reaction accelerator such as polyethylene glycol and a nonspecific reaction inhibitor. You may not.
- the solid phase carrier includes polystyrene, styrene-butadiene copolymer, (meth) acrylic acid ester polymer, latex, gelatin, liposome, microcapsule, erythrocyte, silica. Particles made of a material such as alumina, carbon black, metal compound, metal, ceramics, or magnetic material can be used.
- the loading method a known method such as a physical adsorption method, a chemical bonding method, or a combination of these methods can be used.
- the measuring operation can be performed by a known method.
- the sample is reacted with an antibody, or the antibody supported on a sample and a solid support is reacted with the end point method or Transmitted light and scattered light are measured by the rate method.
- the sample and the antibody supported on the solid phase carrier are reacted in a container such as a plate or a microtiter plate, and the state of aggregation is visually determined.
- the patient may suffer from perilymph fistula It can be determined that there is.
- the amount of CTP present in the body fluid can also be determined by performing quantification using a commonly used protein quantification method known per se.
- CTP measurement kit The CTP measurement kit of the present invention comprises the antibody of the present invention. If this reagent kit is used, the detection of perilymph fistula of the present invention can be performed simply and quickly when necessary, and the results can be used for differentiation from other diseases, determination of treatment policy, and the like.
- the form of the reagent included in the kit is not particularly limited, and may be solid or liquid (solution, suspension, etc.).
- the reagent can be prepared by dissolving or suspending the antibody in an appropriate solvent (such as a buffer that can stably store the antibody).
- the kit of the present invention may have any configuration as long as it can perform the detection method of the present invention.
- a reagent kit that detects CTP using an immunoassay using a labeled antibody at least a carrier-immobilized antibody and / or a labeled antibody includes an antibody that reacts with native CTP.
- enzyme substrates, buffers such as diluents and washing solutions, positive controls, and the like can be included.
- the reagent kit of the present invention contains at least an antibody that reacts with native CTP in a sample, and can be prepared by combining commonly known reagents and the like.
- Example 1 Production of Anti-CTP (E. coli) Monoclonal Antibody A polyhistidine tag was fused to the N-terminal side of amino acids corresponding to amino acid numbers 32 to 132 of SEQ ID NO: 1 excluding the signal sequence of human CTP to obtain Escherichia coli. And monoclonal antibodies were produced using this as an immunogen.
- E. coli expression vector An E. coli expression vector, which was transformed into E. coli.
- rCTP recombinant CTP
- the bacterial cells induced and expressed were collected by centrifugation and disrupted by ultrasonic treatment.
- the obtained PCR product was incorporated into the expression vector pCR T7 / NT for E. coli according to the attached document of pCR T7 / NT-TOPO TA Expression Kit (Invitrogen), and then transformed into the E. coli BL21 (DE3) pLysS strain attached to the kit. After conversion, recombinant E. coli for rCTP expression was obtained.
- the obtained recombinant Escherichia coli for rCTP expression was inoculated into 1500 ml of ampicillin-added LB medium and cultured with shaking at 37 ° C. When the absorbance at 600 nm of the culture solution reached 0.5, IPTG was added to the culture solution to a final concentration of 0.1 mM, followed by shaking culture at 37 ° C. for 3 hours.
- the washed precipitate was dissolved in 8M urea to obtain an inclusion body solution.
- the resulting inclusion body solution was added to Ni-NTA Agarose equilibrated with a denaturing binding buffer (8 M Urea, 500 mM NaCl, 20 mM Sodium Phosphate pH 7.8), and reacted at room temperature for 1 hour.
- the resin after the reaction was recovered with a funnel equipped with a glass filter, washed with a denaturing binding buffer having a volume 5 times the resin volume, and further denatured washing buffer with a volume 5 times the resin volume (8M Urea, 500 mM NaCl). , 20 mM Sodium Phosphate pH 6.0).
- a denaturation elution buffer (8 M Urea, 500 mM NaCl, 100 mM imidazolol, 20 mM Sodium Phosphate pH 6.0) was added to the washed resin 5 times the resin volume and allowed to react at room temperature for 1 hour.
- the reaction solution was collected with a funnel equipped with a glass filter to obtain Escherichia coli-expressed rCTP.
- PVDF membrane Immobilon-PSQ, Millipore
- the PVDF membrane was reacted with an anti-CTP (E. coli) antibody as a primary antibody, and then reacted with HRP-Rabbit anti-Mouse IgG (H + L) (ZYMED) as a secondary antibody.
- HRP-Rabbit anti-Mouse IgG H + L
- ZYMED ZYMED
- ECL Advance Western Blotting Detection Kit GE Healthcare was used to detect a 16 kDa band derived from CTP, confirming that the anti-CTP (E. coli) antibody recognizes CTP in human biological samples. did.
- Example 2 Preparation of Polyclonal Antibody Anti-LCCL antibody, anti-LCCL1 antibody, anti-LCCL2 antibody and anti-LCCL3 antibody described in JP-A-2004-85552 were used. In addition, three types of antibodies were newly produced using a polypeptide other than these as an antigen. The reaction between the obtained polyclonal antibody and human CTP was confirmed by Western blotting.
- a polypeptide comprising amino acids corresponding to amino acid numbers 34 to 49 of SEQ ID NO: 1 in the sequence listing was selected.
- a polypeptide comprising amino acids corresponding to amino acid numbers 91 to 108 (sometimes referred to as an “anti-CTP-A antibody”) (an antibody prepared using this as an antigen polypeptide may be referred to as an “anti-CTP-B antibody”)
- an antibody prepared using this as an antigen polypeptide may be referred to as an “anti-CTP-B antibody”
- a polypeptide consisting of amino acids corresponding to amino acid numbers 118 to 132 was selected.
- Table 1 shows immunized animals and antigen polypeptides of each antibody, and FIG. 1 shows the positional relationship of these antigen polypeptides on the amino acid sequence described in SEQ ID NO: 1.
- polypeptide consisting of the amino acid sequence selected in (1) above was synthesized.
- the anti-CTP-B antibody and the antigen polypeptide for producing anti-CTP-C antibody since cysteine is not included in the amino acid sequence, a polypeptide in which cysteine is added to the N-terminus of each sequence is used. Synthesized. Bovine thyroglobulin was bound as a carrier protein via cysteine to prepare an immunogen. Immunization was performed by administering 100 ⁇ g of immunogen to one rabbit every 1-2 weeks. After 8 times of immunization, blood was collected, serum was separated and used as antiserum.
- Example 3 Confirmation of reactivity with native CTP (immunoprecipitation) Since the antibodies prepared in Example 1 and Example 2 were able to detect CTP by Western blotting, it was found that denatured CTP or CTP under denaturing conditions was detectable. Therefore, in order to confirm whether these antibodies can also detect native CTP, the following examination was performed. Each antibody produced in Example 1 and Example 2 was bound to a protein G-immobilized carrier and then reacted with porcine perilymph or human perilymph. After the reaction, the precipitate (antigen-antibody complex bound to the protein G-immobilized carrier) and the supernatant (antibody unreacted fraction) were collected by centrifugation, and Western blotting was performed after SDS-PAGE.
- Example 2 Western blotting was performed by the method described in Example 1 (5) except that anti-LCCL3 antibody was used as the primary antibody and Immunoglobulins / HRP [Goat Polyanti-Rabbit] (DAKO) was used as the secondary antibody. It was. The results are shown in Table 2. Those bound to native CTP are indicated by ⁇ , and those not bound are indicated by ⁇ . Only the anti-CTP-C antibody was confirmed to be able to recognize native CTP in the perilymph.
- Example 4 Production of Monoclonal Antibody Recognizing Native CTP
- an attempt was made to produce an anti-CTP (Baculo) monoclonal antibody.
- a FLAG tag was fused to the C-terminal side of the amino acid corresponding to amino acids 1 to 132 of SEQ ID NO: 1 corresponding to the full length of human CTP and expressed in silkworms, and a monoclonal antibody was produced using this as an immunogen.
- a recombinant baculovirus strain was established by incorporating it into a transfer vector with reference to the nucleotide sequence of human Cochlin. By infecting this with silkworm cocoons, rCTP was produced in the silkworm body.
- the silkworm body fluid was affinity purified with an anti-FLAG carrier. Mice were immunized with the obtained rCTP to obtain monoclonal antibodies. Antibody screening was performed by confirming the reaction with rCTP by ELISA and confirming the reaction with native human CTP supplemented with the anti-CTP-C antibody prepared in Example 3.
- a primer for adding a BglII recognition site to the 5 ′ end of the ORF of human CTP and a primer for adding a NheI recognition site to the 3 ′ end are prepared, and a plasmid containing the COCH gene is prepared.
- PCR amplification was performed as a template.
- This PCR product was digested with BglII and NheI and inserted into the BglII and XbaI recognition sites of plasmid pM23 (Katakura Kogyo) to establish a CTP recombinant baculovirus expression system.
- Silkworm silkworms were infected with this CTP recombinant baculovirus.
- the silkworm body fluid after infection was adsorbed on an anti-FLAG antibody column, and then the FLAG tag was cleaved with thrombin to obtain silkworm-expressed rCTP.
- Table 3 shows the results of confirming the reactivity of the 11 types of hybridomas obtained with CTP in human perilymph.
- 3C10, 7C1, and 7G1 a reaction with native CTP in human perilymph was confirmed.
- the difference in the strength of reaction was recognized by the hybridoma between silkworm-expressed rCTP and CTP in human perilymph.
- Example 5 Measurement of native CTP in human perilymph by sandwich ELISA method
- Anti-CTP-C antibody was used as a solid phase antibody, and stepped using PBS by sandwich ELISA with 3C10 antibody prepared in Example 4 CTP in diluted human perilymph was measured. The measurement was performed in the same manner as in Example 4 (4). The result is shown in FIG. It was confirmed that native CTP in human perilymph can be measured in a concentration-dependent manner.
- the antibody of the present invention is useful in fields such as diagnosis, medical care and research.
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Abstract
Description
(1)配列番号1のアミノ酸番号118~132で表されるポリペプチドに含まれる抗原決定基を認識し、未変性CTPに反応することを特徴とする抗CTP抗体。
(2)配列番号1のアミノ酸番号118~132からなるペプチドを免疫原として得られる、(1)に記載の抗CTP抗体。
(3)未変性CTPに反応する第1の抗体で捕捉した未変性のCTPを用意し、該未変性のCTPを認識する第2の抗体を選別する工程を含む、未変性のCTPに反応する抗体のスクリーニング方法。
(4)前記第1の抗体が(1)に記載の抗体である、請求項3に記載の未変性のCTPに反応する抗体のスクリーニング方法。
(5)(1)または(2)に記載の抗体を少なくとも一つ使用する、配列番号1のアミノ酸番号118~132で表されるポリペプチドを含む蛋白質の免疫学的測定方法。
(6)配列番号1のアミノ酸番号118~132で表されるポリペプチドを含む蛋白質がCTPである、(5)に記載の方法。
(7)(1)または(2)に記載の抗体を少なくとも一つ使用してCTPを免疫学的に測定する工程を含む、外リンパ瘻の検査方法。
(8)(1)または(2)に記載の抗体を少なくとも一つ含む、CTP測定キット。
(9)外リンパ瘻の診断のための(8)に記載の測定キット。
なお、本明細書において、蛋白質の精製及び解析、並びに抗体の作製等の手法は、特に明記しない限り、新生化学実験講座(日本生化学会編;東京化学同人)、Antibodies - A Laboratory Manual(E.Harlow, et al., Cold Spring Harbor Laboratory(1988))等の一般的実験書に記載の方法またはそれに準じて行うことができる。
本発明の抗体は、配列番号1のアミノ酸番号118~132で表されるポリペプチドに含まれる抗原決定基(以下、これを「エピトープ」と称することがある)を認識し、未変性CTPに反応することを特徴とする。そして配列番号1のアミノ酸番号118~132で表されるポリペプチドを含む蛋白質以外の蛋白質には反応しない抗体であることが好ましい。なお、本発明の抗体は未変性CTPに反応する限り、さらに変性CTPに反応してもよいし、さらに配列番号1のアミノ酸番号118~132で表されるポリペプチドを含むCochlinのアイソフォームに反応してもよい。ここで、配列番号1のアミノ酸番号118~132で表されるポリペプチドを含むCochlinのアイソフォームとしては、CTP以外にp63が知られており(Ikezono et al., Biochem.Biophys.Acta, 1535,3,258-265(2001))、それに反応してもよい。
未変性のCTPとは、タンパク質の3次構造が著しく変化するような変性処理を施していないCTPを指す。変性処理としては、たとえば、タンパク質変性剤(SDS等の界面活性剤、DTT等の還元剤、尿素、アセトンなど)の添加や加熱処理等が挙げられる。なお、免疫測定法におけるサンプル希釈液等では、低濃度の界面活性剤等が含まれている場合もあるが、このような条件は変性処理とは言わない。
という。またIgGをペプシンで処理すると、ヒンジ領域中の2本のH鎖間に存在するジスルフィド結合の下流で切断されて前記2つのFab'がヒンジ領域でつながったものよりやや大きい抗体フラグメントを製造することができる。この抗体フラグメントをF(ab')2という。
本発明の未変性のCTPに反応する抗体のスクリーニング方法は、未変性CTPに反応する第1の抗体で捕捉した未変性のCTPを用意し、該未変性のCTPを認識する第2の抗体を選別する工程を含む。ここで、第1の抗体としては、上記配列番号1のアミノ酸番号118~132で表されるポリペプチドに含まれる抗原決定基を認識し、未変性CTPに反応する抗体であることが好ましい。
すなわち、上記本発明の抗体を用いることにより、未変性CTPに反応する新たな抗体を得ることができる。例えば、ELISA法において、第1の抗体として上記本発明の抗体を用い、第2の抗体として抗CTP抗体のライブラリーを使用し、スクリーニングを行うことにより、未変性CTPに反応する新たな抗体を選別することができる。なお、スクリーニング方法で得られる抗体は未変性CTPに反応する抗体である限り、特に限定されないが、高次構造や配列番号1のアミノ酸番号118~132で表されるポリペプチド以外の領域に含まれる抗原決定基を認識する抗体であることが好ましい。
本発明の測定方法は、上記本発明の抗体を少なくとも一つ使用して配列番号1のアミノ酸番号118~132で表されるポリペプチドを含む蛋白質、好ましくはCTPを免疫学的に測定する工程を含む。なお、定性的な測定も定量的な測定も含まれる。
本発明において、外リンパ瘻(Perilymph fistula)とは、内耳組織に存在する外リンパが何らかの要因により内耳窓(正円窓、卵円窓のいずれかまたは両者)あるいはfissura ante fenestram(内耳と中耳の間の骨裂隙)から鼓室内(中耳)に漏出して聴覚・平衡感覚の障害を生じる疾患である。該疾患は、外リンパが中耳へ漏出していることを確認することにより検出することができる。本発明の外リンパ瘻の検出方法は、該疾患に罹患していることが疑われる患者の中耳に存在し得る体液のうち、外リンパのみに存在するCTPの存在を検出して、該患者が外リンパ瘻に罹患している可能性の指標とすることを特徴とする方法である。本法によれば、外リンパ瘻発症の要因や機構によらず検出を行うことができる。
該疾患に罹患していることが疑われる患者には、突発性難聴、内耳性難聴、メニエール病、前庭神経炎、頭位めまい症、内耳性めまい等の患者も挙げられる。厳密に言えばこれらは症候診断名であり、原因診断名である外リンパ瘻が、上記の疾患の原因となっていることは以前から指摘されている。したがって、本発明の方法により外リンパ瘻を検査することでこれらの疾患の検査も可能である。
例えば、真珠腫性中耳炎や外耳腫瘍、中耳腫瘍、聴神経腫瘍等では骨破壊により外リンパ瘻をきたすことが知られており、この病変の深さがどの程度であるかを本検査により診断できる。すなわち、病変が骨を破壊し内耳に至っていれば、中耳に存在する体液からCTPが検出され、それよりも浅ければCTPは検出されない。また、鼓室形成術、アブミ骨手術等では、正円窓や卵円窓に外科的処置を加えることがあり、手術操作によりこれらを損傷したか否かを本測定により判断できる。さらには、人工内耳手術において、人工内耳の電極挿入部位を決定するのにも役立つ。特に内耳、中耳奇形を有する症例ではその有用性が高い。これらの検査においては中耳洗浄液、あるいは病変部位や手術操作部位からの漏出液を直接採取して検査に供することができる。
ELISA法による測定では、発光シグナルの定量化により、外リンパの中耳への漏出量を定量的に測定することが可能となる。CTPアナログを用意する必要がないため、異なるエピトープでCTPを認識する2種類の抗体を用いたサンドイッチ型測定が特に好ましい。
また、目視的に測定する場合には、プレートやマイクロタイタープレート等の容器中で、試料と固相担体に担持させた抗体を反応させ、凝集の状態を目視的に判定する。なお、目視的に測定する代わりにマイクロプレートリーダー等の機器を用いて測定を行ってもよい。
本発明のCTP測定キットは、上記本発明の抗体を含む。該試薬キットを用いれば、本発明の外リンパ瘻の検出を必要時に簡便・迅速に行うことができ、その結果を、他の疾患との鑑別や、治療方針の決定等に役立てることができる。
ヒトCTPのシグナル配列を除いた配列番号1のアミノ酸番号32~132に相当するアミノ酸のN末端側にポリヒスチジンタグを融合させて大腸菌内で発現させ、これを免疫原としてモノクローナル抗体を作製した。
まず、ヒトCochlinのヌクレオチド配列を参考にして大腸菌発現用ベクターに組込み、これを大腸菌に形質転換させた。大腸菌にIPTGを加えることによって組込まれたベクターにコードされたrCTP(リコンビナントCTP)を誘導発現させた。誘導発現した菌体を遠心集菌し、超音波処理によって菌体を破砕した。破砕後、遠心分離によって可溶性画分と不溶性画分に分画した。得られた発現タンパク質は不溶性凝集体を形成したので、不溶性画分を尿素にて可溶化し、ニッケルカラムにてアフィニティー精製した。得られたrCTPをマウスに免疫し、モノクローナル抗体を得た。抗体のスクリーニングは、ELISA法によりrCTPとの反応を確認することにより行い、最終的に得られたモノクローナル抗体とヒトCTPとの反応はウェスタンブロット法により確認した。
CTPのシグナル配列を除いたORFの5'末端に翻訳開始コドンを付加するためのプライマー(5'-ATG ATC ACA TGT TTT ACC AG-3':配列番号9)、および3'末端に終止コドンを付加するためのプライマー(5'―TAT TCA TTACTC CTG TGT ACT ACT-3': 配列番号10)を用意し、COCH遺伝子を含むイメージクローン(IMAGE:27789)(クラボウ 大阪)を鋳型としてPCR増幅を行った。この項で示した配列では、開始コドンおよび終止コドンを下線で示した。
得られたPCR産物を、pCR T7/NT-TOPO TA Expression Kit(Invitrogen社)の添付文書に従って大腸菌用発現ベクターpCR T7/NTに組込んだ後、キット添付の大腸菌BL21(DE3)pLysS株に形質転換して、rCTP発現用組換え大腸菌を得た。
得られたrCTP発現用組換え大腸菌をアンピシリン添加LB培地1500mlに接種し、37℃で振とう培養した。培養液の600nmの吸光度が0.5に到達した時点で、培養液にIPTGを終濃度0.1mMとなるように添加し、引き続き37℃で3時間振とう培養した。
該培養液を、4℃、3000rpmで30分間遠心分離し、沈澱した菌体を回収し、10mlの破砕用緩衝液(50mM Tris-HCl pH8.0、50mM NaCl、1mM EDTA)に懸濁した。氷冷しながら菌体懸濁液を超音波破砕機にて処理することにより菌体を破砕し、これを4℃、3000rpmで30分間遠心分離した。その沈殿を封入体洗浄液(0.5% TrironX-100、1mM EDTA)に懸濁し、これを4℃、3000rpmで30分間遠心分離するという洗浄操作を3回繰り返した。洗浄後の沈殿を8M尿素に溶解し、封入体溶液とした。
得られた封入体溶液を変性結合緩衝液(8M Urea、500 mM NaCl、20 mM Sodium Phosphate pH 7.8)で平衡化させたNi-NTA Agaroseに加え、室温で1時間反応させた。反応後の樹脂をグラスフィルター付ロートで回収し、これを樹脂容積の5倍量の変性結合緩衝液で洗浄した後、さらに樹脂容積の5倍量の変性洗浄緩衝液(8M Urea、500 mM NaCl、20 mM Sodium Phosphate pH 6.0)で洗浄した。洗浄後の樹脂に変性溶出緩衝液(8M Urea、500 mM NaCl、100mM imidazol、20 mM Sodium Phosphate pH 6.0)を樹脂容積の5倍量加え、室温で1時間反応させた。該反応液をグラスフィルター付ロートで回収し、大腸菌発現rCTPを得た。
大腸菌発現rCTPをKLHで架橋したものを抗原として、該抗原をC57BL6マウスに50μgずつ2週間おきに3回投与した。最後の免疫感作から1週間後に、大腸菌発現rCTP固定化プレートを用いたELISA法により抗体価が上昇していることを確認した上で、採取した脾臓を細胞融合用ミエローマ(P3U1)とPEG法にて融合した。得られたハイブリドーマをHAT培地にて選択し、抗体産生ハイブリドーマを得た。
96穴プレートに免疫原である大腸菌発現rCTPを固相化し、それとハイブリドーマ培養上清との反応を確認した。検出には、Rabbit anti Mouse IgG/HRP(ZYMED社)を使用した。同時に、ブランクとしてβアクチン固相プレートとの反応も確認し、ブランクとは反応せず、大腸菌発現rCTPに反応する抗体を選別した。最終的に得られた陽性株は1種のみであった(これを「抗CTP(E.coli)抗体」と称することがある)。
試料として用いたヒト外リンパは、患者に対して採取および研究目的の使用について十分な説明を行い、同意を得た上で用いた。
試料を3×Loading buffer(150mM Tris-HCl pH6.8、300mM DTT、6%SDS、0.3% bromophenol blue、30% glycerol)と混合し、100℃で5分間加熱した。該試料を15% polyacrylamide(PAGEL、ATTO社)にアプライし、running buffer(25 mM Tris、192mM Glycine、0.1% SDS)を使用して、20mAで2時間、電気泳動を行った後、セミドライ法によりPVDF膜(Immobilon-PSQ、Millipore社)に転写した。転写後のPVDF膜に、一次抗体として抗CTP(E.coli)抗体を反応させた後、二次抗体としてHRP-Rabbit anti-Mouse IgG(H+L)(ZYMED社)を反応させた。その後、ECL Advance Western Blotting Detection Kit(GEヘルスケア社)を使用してCTPに由来する16kDaのバンドを検出し、抗CTP(E.coli)抗体がヒト生体試料中のCTPを認識することを確認した。
抗LCCL抗体、抗LCCL1抗体、抗LCCL2抗体および抗LCCL3抗体は、特開2004-85552号公報に記載のものを使用した。また、これらとは別のポリペプチドを抗原として、新たに3種類の抗体を作製した。得られたポリクローナル抗体とヒトCTPとの反応はウェスタンブロット法により確認した。
新たな抗原ポリペプチドとしては、配列表の配列番号1のアミノ酸番号34~49に相当するアミノ酸よりなるポリペプチド(これを抗原ポリペプチドとして作製した抗体を「抗CTP-A抗体」と称することがある)、同じくアミノ酸番号91~108に相当するアミノ酸よりなるポリペプチド(これを抗原ポリペプチドとして作製した抗体を「抗CTP-B抗体」と称することがある)、更にアミノ酸番号118~132に相当するアミノ酸よりなるポリペプチド(これを抗原ポリペプチドとして作製した抗体を「抗CTP-C抗体」と称することがある)をそれぞれ選択した。
上記(1)において選択されたアミノ酸配列からなるポリペプチドを合成した。ここで、抗CTP-B抗体、抗CTP-C抗体作製用抗原ポリペプチドについては、該アミノ酸配列中にシステインが含まれていないので、それぞれの配列のN末端にシステインが付加されたポリペプチドを合成した。システインを介し、キャリア蛋白質としてウシサイログロブリンを結合させて免疫原とした。
免疫は、ウサギ1羽に対して1~2週間おきに免疫原を100μg投与することにより行った。8回免疫後、採血を行い、血清を分離しこれを抗血清とした。これらの抗血清は、実施例1(4)と同様なELISA法により、それぞれ、各免疫原である抗原ポリペプチドおよび大腸菌発現rCTPとの反応を確認した後、別途作成した抗原ポリペプチド結合カラムを用いて精製した。
一次抗体に抗CTP-A抗体、抗CTP-B抗体、または抗CTP-C抗体を使用し、二次抗体としてImmunoglobulins/HRP[Goat Polyclonal Anti-Rabbit](DAKO社)を使用した以外は、実施例1(5)に記載の方法で行い、各ポリクローナル抗体は、いずれもヒト生体試料中のCTPを認識することを確認した。
実施例1および実施例2で作製した抗体が、ウェスタンブロット法によりCTPを検出できたことから、変性されたCTPもしくは変性条件下にあるCTPは検出可能であることがわかった。そこで、これらの抗体が未変性のCTPも検出可能であるかを確認するために、以下の検討を行った。
実施例1および実施例2で作製した各抗体をプロテインG固定化担体に結合させた後、ブタ外リンパ又はヒト外リンパと反応させた。反応後、遠心分離により沈殿(プロテインG固定化担体に結合した抗原抗体複合体)および上清(抗体未反応画分)を回収し、それぞれSDS-PAGE後にウェスタンブロッティングを行った。どちらの画分にCTPが検出されるかを調べることで、各抗体が未変性CTPを認識するか否かを確認した。
具体的には、PBSで平衡化した20μgのProteinG on Sepharose 4B(GEヘルスケア)に各抗体30μgを反応させた後、未結合抗体を除去した。この抗体結合体に、PBSで10倍に希釈したブタ外リンパまたはヒト外リンパの20μlを添加し、4℃で一晩振盪反応させた。反応後、4℃、3000rpmで2分間遠心分離し、上清と沈殿をそれぞれ回収した。
ウェスタンブロッティングは、一次抗体に抗LCCL3抗体を使用し、二次抗体としてImmunoglobulins/HRP[Goat Polyclonal Anti-Rabbit](DAKO社)を使用した以外は、実施例1(5)に記載の方法で行った。その結果を表2に示す。未変性CTPと結合したものを○、結合しなかったものを×で示した。抗CTP-C抗体のみが、外リンパ中の未変性CTPを認識できることが確認された。
未変性CTPを認識するモノクローナル抗体の作製のため、抗CTP(Baculo)モノクローナル抗体の作製を試みた。
ヒトCTPの全長にあたる配列番号1のアミノ酸番号1~132に相当するアミノ酸のC末端側にFLAGタグを融合させてカイコ体内で発現させ、これを免疫原としてモノクローナル抗体を作製した。
まず、ヒトCochlinのヌクレオチド配列を参考にしてトランスファーベクターに組込み、組換えバキュロウイルス株を樹立した。これをカイコ蛹に感染させることによって、カイコ体内にrCTPを生産させた。このカイコ体液から、抗FLAG担体にてアフィニティー精製した。得られたrCTPをマウスに免疫し、モノクローナル抗体を得た。抗体のスクリーニングは、ELISA法によりrCTPとの反応を確認すると共に、実施例3で作製した抗CTP-C抗体に補足させた未変性のヒトCTPとの反応を確認することにより行った。
ヒトCTPのORFの5'末端にBglII認識部位を付加するためのプライマーおよび3'末端にNheI認識部位を付加するためのプライマーを用意し、COCH遺伝子を含むプラスミドを鋳型としてPCR増幅を行った。このPCR産物をBglIIとNheIで消化し、プラスミドpM23(片倉工業)のBglIIとXbaI認識部位に挿入し、CTP組換えバキュロウィルス発現系を樹立させた。このCTP組換えバキュロウィルスをカイコ蛹に感染させた。感染後のカイコ体液を抗FLAG抗体カラムに吸着させた後、トロンビンによりFLAGタグを切断することで、カイコ発現rCTPを得た。
カイコ発現rCTPを抗原として、該抗原をBalb/cマウスに50μgずつ隔日で4回フットパッド免疫した。その後、採取したリンパ細胞をミエローマ細胞(P3U1)とPEG法にて融合した。得られたハイブリドーマをHAT培地にて選択し、抗体産生ハイブリドーマを得た。
実施例1(4)と同様なELISA法により、免疫原であるカイコ発現rCTPとハイブリドーマ培養上清との反応を確認した。検出には、Goat anti-mouse IgG-POD F(ab')2(MBL社)を使用した。
その結果を表3に示す。11種のハイブリドーマにおいて、カイコ発現rCTPとの反応が確認された。
EIAプレート(MAXISORP、Nunc社)に5μg/ml抗CTP-C抗体を100μl/well添加し、4℃で一晩静置した。翌日、25%ブロックエース(大日本住友製薬)を300μl/well添加し、37℃で2時間ブロッキングした。各ウェルを洗浄用緩衝液(0.05%Tween-20、20mM PBS pH7.4)で洗浄後、PBSで40倍希釈したヒト外リンパを100μ/well添加し、室温で1時間振盪反応させた。各ウェルを洗浄した後、一次抗体としてハイブリドーマ培養上清を100μ/well添加し、室温で2時間振盪反応させた。各ウェルを洗浄した後、二次抗体として、10%ブロックエースで2000倍希釈したRabbit anti-mouse Immunoglobulins/HRP(Dako)を添加し、室温で1時間振盪反応させた。各ウェルを洗浄した後、SureBlue Reserve TMB microwell substrate(KPL社)を100μ/well添加し、室温で15分間反応させた後、stop solutionを添加して反応を停止させた。マイクロプレートリーダーで波長450nmの吸光度を測定した。
得られた11種のハイブリドーマとヒト外リンパ中のCTPとの反応性を確認した結果を表3に示す。3C10、7C1、および7G1において、ヒト外リンパ中の未変性CTPとの反応が確認された。また、ハイブリドーマにより、カイコ発現rCTPとヒト外リンパ中のCTPとで、反応の強弱に違いが認められた。
固相抗体として抗CTP-C抗体を用い、実施例4で作製した3C10抗体とのサンドイッチELISAにより、PBSを用いて段階希釈したヒト外リンパ中のCTPを測定した。測定は実施例4(4)と同様に行った。その結果を図2に示す。ヒト外リンパ中の未変性CTPを濃度依存的に測定できることを確認した。
Claims (9)
- 配列番号1のアミノ酸番号118~132で表されるポリペプチドに含まれる抗原決定基を認識し、未変性Cochlin-tomoprotein(CTP)に反応することを特徴とする抗体。
- 配列番号1のアミノ酸番号118~132からなるペプチドを免疫原として得られる、請求項1に記載の抗CTP抗体。
- 未変性CTPに反応する第1の抗体で捕捉した未変性のCTPを用意し、該未変性のCTPを認識する第2の抗体を選別する工程を含む、未変性のCTPに反応する抗体のスクリーニング方法。
- 前記第1の抗体が請求項1記載の抗体である、請求項3に記載の未変性のCTPに反応する抗体のスクリーニング方法。
- 請求項1または2に記載の抗体を少なくとも一つ使用する、配列番号1のアミノ酸番号118~132で表されるポリペプチドを含む蛋白質の免疫学的測定方法。
- 配列番号1のアミノ酸番号118~132で表されるポリペプチドを含む蛋白質がCTPである、請求項5に記載の方法。
- 請求項1または2に記載の抗体を少なくとも一つ使用してCTPを免疫学的に測定する工程を含む、外リンパ瘻の検査方法。
- 請求項1または2に記載の抗体を少なくとも一つ含む、CTP測定キット。
- 外リンパ瘻の診断のための請求項8に記載の測定キット。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004085552A (ja) | 2002-06-27 | 2004-03-18 | Nippon Medical School | 外リンパ瘻の検出方法 |
US20090075306A1 (en) * | 2006-09-07 | 2009-03-19 | The Cleveland Clinic Foundation | Diagnostic assay for detecting and monitoring age related and noise induced hearing loss |
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US20020086988A1 (en) * | 2000-03-03 | 2002-07-04 | Conklin Darrell C. | Full length expressed polynucleotides and the polypeptides they encode |
US7863005B2 (en) | 2002-06-27 | 2011-01-04 | Nippon Medical School Foundation | Method for detecting perilymphatic fistula |
US8003765B2 (en) * | 2003-04-09 | 2011-08-23 | Stony Brook Anaesthesiology, University Faculty Practice Corporation | Methods, antibodies and kits for detecting cerebrospinal fluid in a sample |
-
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004085552A (ja) | 2002-06-27 | 2004-03-18 | Nippon Medical School | 外リンパ瘻の検出方法 |
US20090075306A1 (en) * | 2006-09-07 | 2009-03-19 | The Cleveland Clinic Foundation | Diagnostic assay for detecting and monitoring age related and noise induced hearing loss |
Non-Patent Citations (19)
Title |
---|
"Enzyme-linked immunoassay", 1987, IGAKUSYOIN |
"Immunoassay for laboratory test, technology and application", vol. 53, 1983, RINSYO BYORI |
"Proteins, nucleic acids and Enzymes", 1987, KYORITSU SYUPPAN, article "Enzyme-linked immunoassay" |
"Shin Kagakujikken Kouza", TOKYO KAGAKU DOJIN CO., LTD. |
E. HARLOW ET AL.: "Antibodies - A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY |
IKEZONO ET AL., ACTA OTO-LARYNGOLOGICA, vol. 130, 2010, pages 881 - 887 |
IKEZONO ET AL., AUDIOL NEUROTOL, vol. 14, 2009, pages 338 - 344 |
IKEZONO ET AL., AUDIOL NEUROTOL, vol. 15, 2010, pages 168 - 174 |
IKEZONO ET AL., BIOCHEM BIOPHYS RES COMMUN, vol. 314, 2004, pages 440 - 446 |
IKEZONO ET AL., BIOCHEM. BIOPHYS. ACTA, vol. 1535, no. 3, 2001, pages 258 - 265 |
IKEZONO T. ET AL.: "Cochlin-tomoprotein: a novel perilymph-specific protein and a potential marker for the diagnosis of perilymphatic fistula", AUDIOL. NEUROTOL., vol. 14, no. 5, 15 April 2009 (2009-04-15), pages 338 - 344, XP009179610 * |
IKEZONO T. ET AL.: "CTP (Cochlin-tomoprotein) detection in the profuse fluid leakage (gusher) from cochleostomy", ACTA OTO-LARYNGOLOGICA, vol. 130, 2010, pages 881 - 887, XP009179613 * |
IKEZONO T. ET AL.: "The performance of cochlin- tomoprotein detection test in the diagnosis of perilymphatic fistula", AUDIOL. NEUROTOL., vol. 15, no. 3, 24 September 2009 (2009-09-24), pages 168 - 174, XP009179611 * |
LI L. ET AL.: "Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament", ACTA OTO-LARYNGOLOGICA, vol. 130, 2010, pages 868 - 880, XP008171419 * |
MILSTEIN ET AL., NATURE, vol. 256, 1975, pages 495 |
NATURE GENET., vol. 20, 1998, pages 299 - 303 |
ROBERTSON, NATURE GENET., vol. 20, 1998, pages 299 - 303 |
See also references of EP2692735A4 |
TETSURO IKEZONO, RINSHO KENSA, vol. 49, no. 11, December 2005 (2005-12-01), pages 1259 - 1263 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2022043219A (ja) * | 2016-10-24 | 2022-03-15 | シスメックス株式会社 | 糖ペプチドと反応するモノクローナル抗体およびその用途 |
CN107347179A (zh) * | 2017-03-28 | 2017-11-14 | 吉林市东杰科技开发有限公司 | 一种基于ReactNative实现LBS的方法 |
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