WO2011136332A1 - 軽度に酸化された酸化低密度リポタンパク質に対するモノクローナル抗体およびこれを産生するハイブリドーマ - Google Patents
軽度に酸化された酸化低密度リポタンパク質に対するモノクローナル抗体およびこれを産生するハイブリドーマ Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- C07—ORGANIC CHEMISTRY
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- 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
Definitions
- the present invention includes a monoclonal antibody against lightly oxidized oxidized low density lipoprotein, a hybridoma producing the monoclonal antibody, a kit for detecting lightly oxidized oxidized low density lipoprotein and a biological sample collected from a subject. Relates to a method for detecting lightly oxidized oxidized low density lipoproteins.
- LDL Low Density Lipoprotein
- Oxidation of low-density lipoprotein occurs when the active oxygen becomes excessive, that is, an oxidative stress state is caused by disruption of the balance between generation and elimination of active oxygen in the living body.
- LDL is a huge molecule in which apolipoprotein B is bound to lipids such as cholesterol, phospholipids, and neutral fats.
- unsaturated fatty acids in the constituent lipids are oxidized by active oxygen. .
- chain oxidation reactions occur one after another, and lipid peroxides and aldehydes are produced via conjugated dienes.
- Apolipoprotein B is also oxidized and cleaved by this chain oxidation reaction or direct oxidation reaction with active oxygen.
- LDL loses a spherical structure, the negative charge increases, and the affinity for the receptor also changes, resulting in oxidized LDL having properties different from those of normal LDL (native-LDL).
- Oxidative stress is involved because oxidized LDL is present in arteriosclerotic lesions and is detected at higher concentrations in the serum of patients with hyperlipidemia, diabetes, liver disease, etc. than healthy individuals. It is considered to be an important substance related to various diseases. Thus, antibodies that specifically recognize oxidized LDL have been developed for the purpose of elucidating, treating, diagnosing, and evaluating these diseases.
- an oxidized phospholipid antibody that recognizes as an antigen a phospholipid having a fatty acid cleaved by oxidation obtained from oxidized LDL purified from an arteriosclerotic lesion, or native- Anti-MDA-LDL antibody (Non-patent document 2 and Non-patent document 3) obtained by using malondialdehyde-modified LDL (MDA-LDL) prepared from LDL as an immunogen, acetylated LDL, MDA- from native-LDL in serum
- An antibody obtained by preparing LDL and metal oxide LDL and using solutions containing equal amounts of these as immunogens can be mentioned.
- LDL is a huge particle having many sites that can be oxidized, the degree of oxidation varies.
- antioxidants such as vitamin C in the blood
- highly oxidized oxidized LDL highly oxidized LDL
- lightly oxidized LDL lightly oxidized LDL
- highly oxidized LDL present as oxidized LDL present in blood vessel walls and atherosclerotic lesions. It is considered.
- the antibody described in Non-Patent Document 1 is an antibody against highly oxidized LDL and not an antibody against lightly oxidized LDL.
- Non-Patent Document 2 since a site recognized as an antigen exists inside LDL, it is necessary to pre-process the sample to expose the antigen site.
- Non-Patent Document 1 since the antibodies described in Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3 and Patent Document 1 are not antibodies obtained using oxidized LDL present in serum as an immunogen, serum samples When it is used on plasma samples, it produces a false positive reaction, has low sensitivity, and it is difficult to detect the effects of drug administration or lifestyle improvement.
- the present invention provides a monoclonal antibody against lightly oxidized oxidized LDL, which can serve as an important tool in research and development related to oxidized LDL, and also provides oxidized LDL lightly oxidized using the monoclonal antibody. It is an object of the present invention to provide a kit for simply detecting oxidase and a method for easily detecting lightly oxidized oxidized LDL contained in a biological sample collected from a subject.
- the present inventors prepared and selected a hybridoma using an immunogen as a triglyceride-rich low-density lipoprotein (TG-rich LDL), which is a mildly oxidized LDL that appears in the serum of patients with liver disease.
- TG-rich LDL triglyceride-rich low-density lipoprotein
- the inventors have found that the monoclonal antibody produced by the hybridoma has the property that the degree of reaction with respect to lightly oxidized LDL is large and the degree of reaction with respect to highly oxidized LDL is small, and the following inventions have been completed.
- NASH non-alcoholic steatohepatitis
- a kit for detecting oxidized low density lipoprotein comprising the monoclonal antibody according to any one of (1) to (11).
- a method for detecting oxidized low density lipoprotein contained in a biological sample collected from a subject, wherein the monoclonal antibody according to any one of (1) to (11) is collected from a subject comprising: a step of specifically reacting with oxidized low density lipoprotein contained in a biological sample to form a complex; and a step of detecting the complex.
- a monoclonal antibody a hybridoma producing a monoclonal antibody
- a kit for detecting lightly oxidized oxidized LDL a kit for detecting lightly oxidized oxidized LDL
- a method for detecting lightly oxidized oxidized LDL contained in a biological sample collected from a subject To elucidate, treat, diagnose and evaluate various diseases related to oxidized LDL such as TG-rich LDL, and to develop pharmaceutical compositions with excellent medicinal properties. Can contribute and provide.
- the diagrams on the upper and lower right sides are diagrams showing the results of measuring the concentration of each lipid in each eluate obtained by performing the gel filtration chromatography.
- TC represents total cholesterol
- PL represents phospholipid
- FC represents free cholesterol
- TG neutral fat.
- the horizontal axis indicates the fraction, that is, the size, and the size is smaller as the position is in the positive direction (right direction) of the horizontal axis.
- the results of a homology search with the known amino acid sequence of the amino acid sequences of CDR1 and CDR2 It is a figure which shows the highest thing with high homology. It is a figure which shows the highest thing with high homology as a result of homology search with respect to the known amino acid sequence about the amino acid sequence of CDR3 of G11-6-VH and CDR1 of G11-6-VL. It is a figure which shows the highest thing with high homology as a result of homology search with the known amino acid sequence about the amino acid sequence of CDR2 and CDR3 of G11-6-VL.
- FIG. 4B is a diagram (B) showing the result of dividing the absorbance measurement value of each sample by the serum LDL-C concentration value of each sample.
- FIG. 3B is a diagram (B) showing the results of ELISA performed with an MDA-LDL antibody adhered thereto.
- FIG. 6 is a view showing the results of performing ELISA with G11-6 antibody immobilized on metal oxide LDL of ⁇ a->, ⁇ b->, ⁇ c->, and ⁇ d->.
- the vertical axis represents the absorbance of the ELISA, and the horizontal axis represents the oxidation time of the metal oxide LDL used as the ELISA sample.
- 5A is a graph showing the results of lipid peroxide concentration measurement by the TBARS method and the results of ELISA with G11-6 antibody immobilized (A; vertical axis indicates the absorbance of the ELISA and the excess of the metal oxide LDL of ⁇ a->). Lipid oxide concentration, the horizontal axis indicates the oxidation time of metal oxide LDL as a sample of ELISA and TBARS method), and the result of measurement of lipid peroxide concentration by TBARS method for ⁇ c-> metal oxide LDL (B; vertical axis shows ELISA absorbance and lipid peroxide concentration, horizontal axis shows ELISA and TBARS method sample oxidation time of metal-oxidized LDL) Respectively).
- FIG. 1 shows the result of having performed ELISA which fixed G11-6 antibody about the metal oxide LDL of ⁇ a->, ⁇ a-1>, and ⁇ a + 1>
- A the vertical axis
- a horizontal axis is the horizontal axis
- the oxidation time of the metal oxide LDL used as the ELISA sample is shown), and the results of the lipid peroxide concentration measurement by the TBARS method for the metal oxide LDL of ⁇ a->, ⁇ a-1>, and ⁇ a + 1> are shown.
- FIG. 2 is a diagram (B; the vertical axis represents the lipid peroxide concentration, and the horizontal axis represents the oxidation time of the metal oxide LDL as a sample of the TBARS method).
- FIG. 6 is a view showing the results of ELISA with G11-6 antibody immobilized on metal oxide LDL of ⁇ a->, ⁇ a-1>, ⁇ a + 1>, ⁇ a-1w> and ⁇ a + 1w>.
- the vertical axis represents the absorbance of the ELISA
- the horizontal axis represents the oxidation time of the metal oxide LDL used as the ELISA sample.
- ELISA with G11-6 antibody immobilized For metal oxidized LDL, ELISA with G11-6 antibody immobilized, ELISA with antioxidant phospholipid antibody immobilized, ELISA with anti-MDA-LDL antibody immobilized, ELISA with anti-apolipoprotein B antibody immobilized, TBARS method It is a figure which shows the result of having performed the lipid peroxide density
- the vertical axis represents the absorbance and lipid peroxide concentration measured by ELISA and conjugated diene, and the horizontal axis represents the oxidation time of the metal oxide LDL as a sample.
- the monoclonal antibody against lightly oxidized oxidized LDL according to the present invention has a property that the degree of reaction to lightly oxidized oxidized LDL is large and the degree of reaction to highly oxidized oxidized LDL is small. It is a monoclonal antibody.
- lightly oxidized LDL means a relatively small amount of oxides such as lipid peroxides and aldehydes generated by oxidation reaction, and cleaved apolipoprotein B, and is negative in comparison with native-LDL.
- Highly charged oxidized LDL that is, lightly oxidized LDL
- highly oxidized LDL refers to relatively high oxides such as lipid peroxides, aldehydes, and cleaved apolipoprotein B produced by oxidation reaction. It refers to oxidized LDL having a large number and having a negative charge significantly larger than that of native-LDL, that is, highly oxidized oxidized LDL.
- both lightly oxidized LDL and highly oxidized LDL can be prepared according to a conventional method, for example, by oxidizing native-LDL in serum using a metal.
- the degree of oxidation of the obtained oxidized LDL increases in proportion to the concentration of the metal that acts on native-LDL or the time that it acts. For example, by reacting native-LDL having a final concentration of about 0.493 g / L with copper sulfate having a final concentration of about 3.29 ⁇ mol / L at 37 ° C.
- the final concentration is about
- the final concentration is about
- the final concentration is about 0.476 g / L.
- the native-LDL is reacted with copper sulfate having a final concentration of about 23.81 ⁇ mol / L at 37 ° C. for H3 hours (0 ⁇ H3 ⁇ 8), or the final concentration is about 0.476 g / L of native-LDL.
- Lightly oxidized LDL can be obtained by reacting copper sulfate having a concentration of about 47.62 ⁇ mol / L at 37 ° C. for H4 hours (0 ⁇ H4 ⁇ 8), with a final concentration of 0.493 g.
- the native concentration of about 0.493 g / L of native-LDL is about 6.579 ⁇ mol / L.
- the final concentration of native-LDL was about 0.476 g / L, and the final concentration of about 23.81 ⁇ mol / L copper sulfate was added at 37 ° C.
- H6 hours H6 ⁇ 8
- native sulfate LLD native sulfate LLD having a final concentration of about 0.476 g / L to copper sulfate having a final concentration of about 47.62 ⁇ mol / L at 37 ° C. for H7 hours (H7 ⁇ 8).
- highly oxidized LDL can be obtained.
- a monoclonal antibody has the property that the degree of reaction to lightly oxidized LDL is large and the degree of reaction to highly oxidized LDL is small is, for example, using a monoclonal antibody as a solid-phased antibody, And it can confirm by ELISA which uses an anti- apolipoprotein B antibody for a detection antibody. That is, in this ELISA, if the degree of reaction when using highly oxidized LDL as a sample is small compared to the degree of reaction when using lightly oxidized LDL as a sample, the monoclonal antibody used for the immobilized antibody Will have the property that the degree of reaction to lightly oxidized LDL is large and the degree of reaction to highly oxidized LDL is small.
- the lightly oxidized LDL can include lightly oxidized LDL of mammals and birds such as humans, mice, rats, monkeys (primates excluding humans), goats, dogs, pigs, guinea pigs, rabbits, sheep, chickens and the like. Is preferably human lightly oxidized LDL. Further, it is preferable that the lightly oxidized LDL is TG-rich ⁇ LDL.
- TG-rich LDL is LDL that appears at least in the serum of patients with liver disease.
- TG-rich LDL is characterized by a high content of triacylglycerol (triglyceride, tri-O-acylglycerol; TG, TAG), which is a neutral fat.
- triacylglycerol triglyceride, tri-O-acylglycerol
- TG TAG
- normal LDL with a high cholesterol content It is a lipoprotein different from (native-LDL).
- VLDL very low density lipoprotein
- HDL high density lipoprotein
- TG-rich LDL foams cultured macrophages are directly proportional (Nagasaka H. et al., J. Pediatr, Vol. 146). Pp. 329-335, 2005), while TG peroxidation is hardly detected in the plasma of healthy volunteers, whereas a significant increase in TG peroxidation is observed in the plasma of patients with liver disease (Hui SP. Et al., Lipids, 38, pp.
- TG-rich LDL is considered to be a kind of oxidized LDL. Furthermore, highly oxidized LDL cannot be present in a large amount in serum, but TG-rich® LDL is present in a large amount in the serum of patients with liver disease. Therefore, TG-rich® LDL is considered to be a kind of mildly oxidized LDL. ing.
- TG-rich LDL is an LDL with a large weight ratio of TG compared to native-LDL.
- TG-rich LDL is LDL that foams macrophages.
- Such TG-rich LDL has an oxidized form of intermediate density lipoprotein ⁇ Intermediate en Density Lipoprotein; IDL (also referred to as mid-band, which includes a remnant lipoprotein corresponding to IDL as a fraction) ⁇ (ie, oxidized form)
- IDL also referred to as mid-band, which includes a remnant lipoprotein corresponding to IDL as a fraction
- remnant lipoproteins which is referred to as small dense LDL (sd-LDL, modified LDL), which has a particle size of 25.5 nm or less and a fraction of 1.040 to 1.063 when fractionated by specific gravity.
- An oxidized form of lipoprotein corresponding to LDL is included.
- FIG. 1 and Table 1 show typical examples of the measurement results of the weight ratio of TG in TG-rich LDL.
- the upper and lower left figures in FIG. 1 show gel filtration chromatography while measuring the absorbance at 280 nm for the total lipoprotein fraction separated from the serum of healthy subjects (upper figure) and liver disease patients (lower figure).
- FIG. 1 is a diagram showing the results of the graph, and the upper and lower right diagrams in FIG. 1 show the total lipoprotein fraction separated from the serum of healthy subjects (upper figure) and liver disease patients (lower figure) by ultracentrifugation, respectively. It is a figure which shows the result of having performed the gel filtration chromatography and measuring the density
- Table 1 shows the results of measuring the concentration of each lipid by the above method for 4 patients with liver disease and 7 healthy subjects, and calculating the average value and standard deviation.
- VLDL and HDL disappear in patients with liver disease, and TG-rich LDL having a particle size similar to that of native-LDL is present at a high concentration.
- the weight ratio of TG in TG-rich LDL present at a high concentration is clearly larger than the weight ratio of TG in native-LDL.
- Table 1 since the standard deviation value of each lipid weight ratio in TG-rich LDL is large, it can be understood that the TG weight ratio in TG-rich LDL has some variation.
- TG-rich LDL can be measured by polyacrylamide gel electrophoresis, high performance liquid chromatography (HPLC), or the like to obtain results that are close to native-LDL. May vary to some extent due to differences or severity of disease.
- a result approximated to native-LDL or a result showing that it is slightly negative compared to native-LDL can be obtained by measuring by agarose gel electrophoresis or the like. It may vary to some extent depending on the individual difference of the examiner and the severity of the disease.
- TG-rich LDL is collected from the serum of patients with liver disease, but is considered to be present not only in liver diseases but also in the sera of patients with various diseases, particularly those with oxidative stress-related diseases and those suspected of suffering from such diseases. It has been.
- diseases include acute hepatitis, chronic hepatitis, cirrhosis, liver fibrosis, benign recurrent intrahepatic cholestasis, biliary atresia, steatohepatitis, non-alcoholic steatohepatitis (Non-alcoholic steatohepatitis;
- hyperlipidemia such as hypercholesterolemia, high LDL cholesterolemia, low HDL cholesterolemia, hypertriglyceridemia, cerebral infarction, ischemic heart disease, aortic aneurysm, nephrosclerosis And arteriosclerotic diseases such as obstructive arteriosclerosis, diabetes, hypertension and the like.
- an antibody has a Y chain in which two polypeptides each of a light chain (L chain; molecular weight of about 25,000) and a heavy chain (H chain; molecular weight of about 50,000 to 77,000) are bound to each other by a disulfide bond.
- the basic structure is a letter-shaped heterotetramer.
- the Y-shaped tip portion is called a variable region, and the other portion is called a stationary region.
- the variable region of the light chain is referred to as the VL region
- the variable region of the heavy chain is referred to as the VH region.
- the variable region is an antigen recognition site, and its amino acid sequence varies depending on the type of antibody. On the other hand, the amino acid sequence of the constant region is relatively little changed between antibodies.
- variable regions a region that directly contacts the antigen and plays a central role in binding to the antigen has a particularly large change in amino acid sequence between antibodies, and this region is a complementarity determining region (CDR) or hypervariable. It is called an area.
- An area other than the CDR in the variable area is referred to as a framework area (FR).
- FR framework area
- variable regions (VH region and VL region) in the present invention are healthy as long as the monoclonal antibody according to the present invention has the property that the degree of reaction to lightly oxidized LDL is large and the degree of reaction to highly oxidized LDL is small. As long as it has the property of specifically reacting with oxidized small dense LDL in the elderly, it has the property of specifically reacting with oxidized LDL having the same particle size as native-LDL in NASH patients, or in patients with dyslipidemia Any amino acid sequence may be used as long as it has a property of specifically reacting with oxidized remnant lipoprotein. Examples of the amino acid sequence of the variable region (VH region and VL region) in the present invention include the following (i) to (v).
- the amino acid sequence of the variable region can be confirmed according to a conventional method.
- a conventional method for example, first, RNA is extracted from a hybridoma that produces the monoclonal antibody according to the present invention described later, and a reverse transcription reaction is performed to obtain cDNA.
- the variable region cDNA sequence is amplified by PCR using primers of a known sequence suitable for variable region amplification, and if necessary, the sequence is cloned using a sequencer. The DNA sequence is determined by doing. Finally, it can be confirmed by converting the determined DNA sequence of the variable region into an amino acid sequence according to a triplet codon.
- “react” is used interchangeably with “interact”, “couple”, and “recognize”. Further, in the present invention, it is sufficient if it is clear that an antibody “reacts specifically” with a specific antigen (immunogen) is reactive with the specific antigen.
- the phrase “specifically reacts” with respect to the specific antigen includes not only reacting with other antigens at all, but also includes reacting with other antigens and significantly reacting with the specific antigen.
- the hybridoma according to the present invention produces the monoclonal antibody according to the present invention.
- the hybridoma according to the present invention can be prepared using any method that can be appropriately selected by those skilled in the art. Such methods include, for example, the hybridoma method (Nature, 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (ImmunologyoToday, Vol. 4, page 72, 1983). And EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985), etc., and methods having the following steps (i) to (iv): Can be mentioned.
- an immunogen is prepared.
- the immunogen may be prepared by any method.
- the TG-rich LDL fraction is separated from the serum, concentrated by ultrafiltration, and then dialyzed. Can do.
- a method for confirming that TG-rich LDL is contained in the serum a method that can be appropriately selected by those skilled in the art can be mentioned, and as such a method, there is an agarose gel electrophoresis method. it can.
- the ⁇ -position band was not detected in the subject serum, and moved to the anode side compared to the healthy subject serum. When a ⁇ -position band is detected at the position, it can be determined that the subject serum contains TG-rich LDL.
- a method for separating the TG-rich LDL fraction from serum a method that can be appropriately selected by those skilled in the art can be mentioned, and as such a method, for example, a previously reported method (Ciba H. et al., J. Biol. Lipid Res., 38, 1204-1216, 1997 / Hirano T. et al., J. Aeroclerosis and h Thrombosis, 12, 67-72, 2005) using density gradient centrifugation.
- a method for separating the TG-rich LDL fraction by subjecting the total lipoprotein fraction separated from the serum to gel filtration chromatography can be mentioned.
- the animal is immunized using the isolated TG-rich LDL fraction as an immunogen. Immunization can be performed using a conventional method as appropriate.
- the animal to be immunized is not particularly limited, and examples thereof include mice, rats, monkeys (primates excluding humans), goats, dogs, pigs, guinea pigs, rabbits, sheep, chickens and the like.
- antibody-producing cells are collected from the immunized animal.
- antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells.
- the collected antibody-producing cells are fused with other cells. Examples of methods for collecting antibody-producing cells and cell fusion methods include methods that can be appropriately selected by those skilled in the art.
- a cell to be fused with the antibody-producing cell a cell having a strong proliferation ability is preferably used. For example, a generally available myeloma cell line can be used.
- the cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having properties are preferred.
- a HAT selection medium including hypoxanthine, aminopterin, and thymidine
- the hybridoma produced is screened for a hybridoma producing a monoclonal antibody having a low degree of reaction with respect to native-LDL and a high degree of reaction with respect to metal-oxidized LDL.
- the screening can be performed by a method that can be appropriately selected by those skilled in the art using a conventional method such as an ELISA method or an immunoblot method.
- ELISA with a native-LDL immobilized is performed on the culture supernatant of the hybridoma, the ELISA reaction is detected with an anti- (immunized animal) antibody labeled appropriately, and native- A hybridoma can be identified by identifying a culture supernatant that has a low degree of reaction for LDL and a high degree of reaction for metal-oxidized LDL.
- the hybridoma obtained by the method having the above steps (i) to (iv) can be used as the hybridoma according to the present invention.
- the cell is purified by further cloning or screening, and the hybridoma according to the present invention can do.
- cloning or screening methods include methods such as a limiting dilution method, a trypsin filter paper method, and a penicillin cap method.
- the monoclonal antibody produced by the hybridoma thus obtained can be used as the monoclonal antibody according to the present invention, and the monoclonal antibody produced by the antibody-producing cells collected in step (iii) of the method can be used as the monoclonal antibody. It can be a monoclonal antibody according to the invention.
- Examples of a method for extracting a monoclonal antibody from a hybridoma or antibody-producing cell include conventional methods such as a cell culture method and ascites formation method.
- the extracted monoclonal antibody can be purified by, for example, appropriately selecting a known method such as ammonium sulfate salting-out method, HPLC, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or a combination thereof.
- a known method such as ammonium sulfate salting-out method, HPLC, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or a combination thereof.
- the determination of the class of the obtained monoclonal antibody can be performed according to a conventional method as appropriate.
- the immunized animal is a mouse
- it can be performed using an IsoStripe mouse monoclonal antibody isotyping kit (Roche) or the like.
- the class of the monoclonal antibody according to the present invention any of IgG, IgA, IgM, IgD and IgE can be used.
- the monoclonal antibody according to the present invention can be used after being labeled with biotin, a radioisotope, a fluorescent substance, an enzyme or the like, if necessary. Further, if necessary, it can be used by being fixed to a carrier such as a polystyrene plate, a polypropylene plate, a silicon plate, a polystyrene microbead, a magnetic bead or a latex particle.
- a carrier such as a polystyrene plate, a polypropylene plate, a silicon plate, a polystyrene microbead, a magnetic bead or a latex particle.
- the monoclonal antibody according to the present invention can be obtained as a human antibody by immunizing an animal having human immunoglobulin producing ability, if necessary, and can be derived from an immunized animal using genetic engineering techniques. It can also be obtained as a chimeric antibody composed of a variable region and a human-derived constant region, and has a hypervariable region derived from an immunized animal, and the other antibody regions can also be obtained as humanized antibodies derived from humans.
- a monoclonal antibody according to the present invention a monoclonal antibody produced by a hybridoma with a deposit number of NITE BP-916, which was commissioned on March 17, 2010, to the Patent Microorganism Depositary, National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation. Although an antibody can be mentioned, it is not limited to this.
- the hybridoma according to the present invention can include, but is not limited to, a hybridoma in which the accession number is NITE-BP-916.
- a monoclonal antibody that reacts specifically with oxidized small ⁇ dense LDL in healthy subjects and specifically reacts with oxidized LDL having the same particle size as native-LDL in NASH patients.
- Monoclonal antibodies and monoclonal antibodies that specifically react with oxidized remnant lipoproteins in patients with dyslipidemia are also encompassed by the monoclonal antibodies according to the present invention.
- “healthy person” means a person who is healthy without injury (Iwanami Shoten, Kojien, 6th edition) and who is not suffering from a disease involving at least oxidized LDL.
- the present invention provides a kit for detecting lightly oxidized oxidized LDL according to the present invention.
- the kit according to the present invention comprises the monoclonal antibody according to the present invention, but does not impair the characteristics of secondary antibodies, labeling substances and other substances useful for the implementation of immunological detection means, buffers, instruments, etc. As long as it is included as a component.
- the present invention provides a method for detecting lightly oxidized oxidized LDL contained in a biological sample collected from a subject. It should be noted that the method for detecting lightly oxidized oxidized LDL according to the present invention may include an incubation step, a washing step, etc. as long as the method for detecting lightly oxidized oxidized LDL according to the present invention is not impaired. Good.
- a method for detecting lightly oxidized oxidized LDL according to the present invention is a method for detecting oxidized LDL contained in a biological sample collected from a subject, (I) A step of forming a complex by specifically reacting the monoclonal antibody according to the present invention with oxidized LDL contained in a biological sample collected from a subject (complex formation step) (Ii) Step of detecting the complex (detection step) The above steps (i) and (ii) are included.
- any method that can be appropriately selected by those skilled in the art can be exemplified.
- a solution containing the monoclonal antibody according to the present invention and a biological sample are mixed to form a complex, and the biological sample is fixed to a carrier to form the monoclonal antibody according to the present invention.
- Examples thereof include a method of reacting with a solution containing the same, a method of allowing the monoclonal antibody according to the present invention to adhere to a carrier and reacting with a biological sample.
- a different aspect of the method for detecting lightly oxidized oxidized LDL according to the present invention is a method for detecting oxidized LDL contained in a biological sample collected from a subject, (I) Step of fixing the monoclonal antibody according to the present invention to a carrier (fixing step) (Ii) a step of specifically reacting the monoclonal antibody fixed to a carrier with oxidized LDL contained in a biological sample collected from a subject to form a complex (fixed complex forming step) (Iii) Step of detecting the complex (fixed complex detection step)
- the above steps (i) to (iii) are included.
- any carrier can be used for fixing the monoclonal antibody according to the present invention.
- a carrier include the same carriers as those described above.
- the method for fixing is not particularly limited, and can be appropriately set depending on the carrier used.
- the kit for detecting lightly oxidized oxidized LDL and the method for detecting lightly oxidized oxidized LDL according to the present invention include elucidation, diagnosis and evaluation of severity of various diseases involving not only liver disease but also oxidized LDL. In addition to evaluation of therapeutic effects, it can be used for evaluation of the degree of oxidation of lipoproteins.
- a monoclonal antibody against lightly oxidized oxidized LDL according to the present invention a hybridoma producing the monoclonal antibody, a lightly oxidized oxidized LDL detection kit, and a lightly oxidized material contained in a biological sample collected from a subject
- a method for detecting the oxidized LDL will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.
- Example 1 Production of monoclonal antibody using TG-rich LDL as an immunogen (1) Confirmation of TG-rich LDL by agarose gel electrophoresis Blood collected from one end-stage patient and one healthy subject was collected at room temperature. After standing for 1 hour, centrifugation was performed at 3500 rpm and 4 ° C. for 10 minutes to obtain the serum of end-stage patients with liver disease and the serum of healthy subjects. 1.5 ⁇ L of the obtained serum was applied to an agarose gel (Universal Gel / 8; Helena Laboratories) and electrophoresed at 100 V and 150 W for 45 minutes in a barbital buffer solution with a pH of 8.6 and an ionic strength of 0.06. Then, the gel was dried using a dryer.
- agarose gel Universal Gel / 8; Helena Laboratories
- Triton X-100 2 drops are added to 20 mL of methanol containing 0.03% (w / v) Fat Red 7B (Helena Laboratories), and 4 mL of 0.1 mol / L sodium hydroxide aqueous solution is further added. After dyeing the dried gel for 10 minutes in the staining solution prepared by the above, decolorization was performed by immersing in a 75% (v / v) aqueous methanol solution for 10 seconds.
- potassium bromide Kanto Chemical Co., Inc.
- a sample solution (12 mL) was placed in a centrifuge tube (40PA; Hitachi Koki Co., Ltd.) and filled with a specific gravity solution.
- Centrifugation was performed for 18 hours under conditions of 40000 rpm and 15 ° C., and the upper layer (d ⁇ 1.225 kg / L) was recovered as a total lipoprotein fraction.
- About 8 mL of the collected total lipoprotein fraction was added in an amount of 2 to 3 mL under a nitrogen gas atmosphere using an Amicon stirring cell Model 8050 (Millipore) and an ultrafiltration membrane Amicon XM50 (Millipore) according to the attached instruction manual.
- the eluate was collected 9 mL (3 mL ⁇ 3 fractions) before and after the absorbance peak to obtain 18 mL (3 mL ⁇ 6 fraction) of the TG-rich LDL fraction.
- aqueous solution containing sodium dodecyl sulfate (SDS) was prepared, and a solution prepared by mixing this aqueous solution with a 4% (w / v) aqueous copper sulfate solution at a volume ratio of 100: 1 was prepared as a reaction solution. Further, an equal amount of a phenol reagent (foreign thiocult reagent; Wako Pure Chemical Industries, Ltd.) was mixed with deionized water to prepare a foreign thiocult reagent solution.
- SDS sodium dodecyl sulfate
- bovine serum albumin (BSA) aqueous solution was prepared as a standard solution. 3 mL of the prepared reaction solution was added to 1 mL each of the TG-rich LDL solution and standard solution of Example (2) [2-3] and allowed to react at room temperature for 30 minutes. Subsequently, 300 ⁇ L of the prepared foreign thiocult reagent solution was added with vigorous stirring, followed by reaction at room temperature for 45 minutes. Thereafter, the absorbance at 660 nm was measured. From the comparison with the measured value of the standard solution, the protein concentration of the TG-rich LDL solution of Example (2) [2-3] was calculated.
- BSA bovine serum albumin
- the hybridoma was cultured for about 10 days using a RPMI1640 medium containing penicillin / streptomycin, 10% (w / v) fetal calf serum (FCS) and a HAT solution according to a conventional method until colonies were confirmed.
- a RPMI1640 medium containing penicillin / streptomycin, 10% (w / v) fetal calf serum (FCS) and a HAT solution according to a conventional method until colonies were confirmed.
- Example (4) [4-3] ELISA in which native-LDL and metal oxide LDL are fixed After diluting the native-LDL solution of Example (4) [4-1] and the metal-oxidized LDL solution of Example (4) [4-2] to 20 ⁇ g / mL with PBS, respectively, A plate (Nunc MaxiSorp; Nalge Nunc International) was placed at 50 ⁇ L / well and reacted at 4 ° C. overnight to fix native-LDL and metal oxide LDL on the plate. After removing the liquid and blocking by adding 150 ⁇ L / well of PBS containing 1% (w / v) BSA and incubating at 37 ° C.
- Tween 20 is contained Washed 4 times with PBS (0.05% Tween-PBS). Subsequently, 50 ⁇ L / well of the culture supernatant of each colony of this Example (3) was put into a well to which native-LDL was fixed and a well to which metal oxide LDL was fixed, and reacted at room temperature for 1 hour. Washed 4 times with 0.05% Tween-PBS.
- Disodium p-nitrophenyl phosphate hydrate (Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL using 10 mmol / L Diethanolamine solution containing 0.5 mmol / L MgCl 2 was added at room temperature to 30 ⁇ L / well. Reacted for 1 minute. Subsequently, the absorbance was measured at a main wavelength of 405 nm and a sub wavelength of 600 nm using a microplate reader (Bio-Rad Laboratories). Colonies were selected by identifying a culture supernatant having a low absorbance for the well to which native-LDL was fixed, and a culture supernatant having a high absorbance for the well to which the metal-oxidized LDL was fixed.
- G11-6 of Example (4) was intraperitoneally injected into a mouse injected with 2,6,10,14-tetramethyl-2-pentadecenoic acid (pristen). Ascites containing a monoclonal antibody (G11-6 antibody) produced by G11-6 was obtained.
- the obtained ascites was subjected to a saturated ammonium sulfate (saturated ammonium sulfate) precipitation method according to a conventional method to obtain a crude monoclonal antibody solution. Specifically, while the obtained ascites was cooled with ice, an equal amount of saturated ammonium sulfate was slowly added dropwise and then centrifuged to remove the supernatant. Subsequently, a 50% saturated ammonium sulfate solution was added to the precipitate and centrifuged again to remove the supernatant, followed by washing. The precipitate was dissolved in PBS to obtain a crude G11-6 antibody solution.
- saturated ammonium sulfate saturated ammonium sulfate
- Example 2 Class determination of G11-6 antibody Class of G11-6 antibody of Example 1 (5) by immunochromatography using IsoStrip mouse monoclonal antibody isotyping kit (Roche) according to the attached instructions. Judgment was made. As a result, it was revealed that the class of the G11-6 antibody is IgM.
- RNA extraction Hybridoma G11-6 of Example 1 (4) was placed in a 25 cm 3 flask containing 10 mL of 10% FCS-containing RPMI1640 medium. Inoculated and cultured for 72 hours under 5% CO 2 atmosphere and 37 ° C. Subsequently, centrifugation was performed for 5 minutes at room temperature and 8500 rpm, the supernatant was removed, and the cell pellet was recovered. Using an absolute RNA Miniprep kit (STRATAGENE), RNA was extracted from the collected cell pellets according to the attached instructions.
- STRATAGENE absolute RNA Miniprep kit
- ⁇ -mercaptoethanol was added to 600 ⁇ L of Lysis buffer, and this was added to the cell pellet. After pipetting with a syringe of 18G (outer diameter 1.2 mm, inner diameter 0.94 mm), the entire amount is stored in Prefilter Spin Cup (Cup1), and centrifuged at room temperature and 14000 rpm for 5 minutes, About 600 ⁇ L of filtrate was collected. 600 ⁇ L of 70% (v / v) ethanol was added thereto and mixed by inversion to obtain about 1200 mL of ethanol mixed solution.
- RNA Binding Binding Spin (Cup2), centrifuged at room temperature and 14000 rpm for 1 minute, the filtrate was removed, and the remaining ethanol mixture was added at about 500 ⁇ L to normal temperature and 14000 rpm. Centrifugation was performed again for 1 minute under the conditions described above. Thereafter, the filtrate was removed, 600 ⁇ L of Low Salt Wash Buffer was added, and the filtrate was further removed by centrifugation at room temperature and 14000 rpm for 1 minute. Subsequently, the mixture was further centrifuged for 2 minutes at room temperature and 14000 rpm, and the filtrate was removed.
- Cup2 RNA Binding Binding Spin
- DNase Digestion Buffer 50 ⁇ L and reconstituted RNase-Free DNaseI 5 ⁇ L were mixed and added to Cup2, and incubated at 37 ° C. for 15 minutes. Thereafter, 600 ⁇ L of High-Salt Wash Buffer was added to Cup 2 and centrifuged at room temperature and 14000 rpm for 1 minute, and then the filtrate was removed. After adding 600 ⁇ L of Low Salt Wash Buffer to Cup2, the mixture was centrifuged at room temperature and 14000 rpm for 1 minute, and then the filtrate was removed. Further, 300 ⁇ L of Low Salt Wash Buffer was added to Cup2, and centrifuged at room temperature and 14000 rpm for 2 minutes, and then Cup2 was transferred to a new 1.5 mL Eppendorf tube.
- RNA concentration of the RNA solution was measured using NanoDrop1000 (Thermo Scientific), it was 128 ng / ⁇ L. Therefore, the RNA concentration was adjusted to about 100 ng / ⁇ L by adding an appropriate amount of DEPC water to the RNA solution.
- Reaction solution A about 100 ng / ⁇ L RNA solution 8 ⁇ L, 10 mmol / L dNTP mix 1 ⁇ L, 50 ng / ⁇ L Random Hexamers 1 ⁇ L
- Reaction solution B 10 ⁇ RT Buffer 2 ⁇ L, 25 mmol / L MgCl 2 4 ⁇ L, 0.1 mol / L DTT 2 ⁇ L, 40 U / mL RNaseOUT TM 1 ⁇ L
- reaction solution A was mixed and incubated at 65 ° C. for 5 minutes, and then left on ice for 1 minute.
- reaction solution B is added to reaction solution A and incubated at room temperature for 2 minutes, and then 1 ⁇ L of Superscript TM II RT is added, and then at room temperature for 10 minutes, at 42 ° C. for 50 minutes, and at 70 ° C. for 15 minutes.
- the reverse transcription reaction was performed by incubating in the order of, and cDNA was prepared.
- the obtained cDNA solution was stored at 4 ° C.
- the solution containing the amplified G11-6-VH DNA sequence (VH-PCR product solution) and the solution containing the amplified G11-6-VL DNA sequence (VL-PCR product solution) were stored at 4 ° C.
- VH ligation reaction solution consisting of 4 ⁇ L of G11-6-VH-DNA solution, 1 ⁇ L of Salt Solution and 1 ⁇ L of TOPO vector, and 4 ⁇ L of G11-6- A VL ligation reaction solution comprising a VL-DNA solution, 1 ⁇ L of Salt Solution and 1 ⁇ L of TOPO vector was prepared, and the mixture was allowed to stand at room temperature for 5 minutes to insert the DNA fragment into the vector.
- a solution containing E. coli transformed with the VH ligation reaction solution was used as a VH E. coli solution, and a solution containing E. coli transformed with the VL ligation reaction solution was used as a VL E. coli solution.
- VH E. coli solution was applied to each plate of the VH plate group
- VL E. coli solution was applied to each plate of the VL plate group by adding 10 ⁇ L, 50 ⁇ L, and 100 ⁇ L, respectively, and then all the plates were incubated at 37 ° C. for 15 hours. . Subsequently, a total of 8 colonies appearing on the plate were picked up using a sterilized toothpick from a total of 8 from the VH plate group and 7 from the VL plate group.
- VL E. coli pellet was used as the VL plasmid solution
- VL plasmid solution was used as the VL plasmid solution.
- VH plasmid solution and VL plasmid solution were stored at 4 ° C.
- Sequence reaction solution composition Ready Reaction Mix 2 ⁇ L, Sequencing Buffer 1 ⁇ L, T3 primer 1 ⁇ L, DEPC water 5 ⁇ L, template DNA 1 ⁇ L
- Sequence reaction conditions After a reaction at 96 ° C. for 10 seconds, each reaction was carried out for 25 cycles of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 3 minutes, and then allowed to stand at 4 ° C.
- SAM TM Solution (Applied Biosystems) 45 ⁇ L was added to a sequencing reaction solution (VH sequencing reaction solution) using the VH plasmid solution as a template DNA and a sequencing reaction solution (VL sequencing reaction solution) using the VL plasmid solution as a template DNA.
- VH sequencing reaction solution a sequencing reaction solution
- VL sequencing reaction solution a sequencing reaction solution using the VL plasmid solution as a template DNA.
- TATEC MicroMixer E-36
- the mixture was centrifuged at room temperature and 14000 rpm for 10 seconds to collect the supernatant, and set on a sequencer (3730xl DNA Analyzer; Applied Biosystems) to analyze the DNA sequence. Subsequently, the amino acid sequence and the CDR1, CDR2 and CDR3 of the complementarity determining region (CDR) were estimated from the obtained DNA sequence using software (MacVector; MacVector). The results are shown below.
- VQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGNSSDSYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVAYCTRVYGRAMDYWGQGTSVTVSS (Sequence No. 7) CDR1 of VH region; SYWMH (SEQ ID NO: 8) CDR2 of VH region; AIYPGNSDTSNYQKFKG (SEQ ID NO: 9) CDR3 of VH region; VYGRAMDY (SEQ ID NO: 10)
- Example 3 Reactivity of ELISA in which G11-6 antibody was fixed to serum of liver disease patients (1) Preparation of serum samples Serum was collected from 1 liver disease patient and 1 healthy subject, respectively.
- anti-apolipoprotein B antibody bound to the column was eluted by flowing 0.1 mol / L Glycine-HCl (pH 2.7) at a flow rate of about 0.2 mL / min.
- the eluate was fractionated 0.5 mL each.
- Example 2 [2-1] 27 ⁇ L of the prepared biotin-labeled solution was added to 1 mL of the anti-apolipoprotein B antibody solution of ⁇ 2-1-1>, and reacted at room temperature for 4 hours with shaking.
- the ELISA reaction was measured using an oxidized LDL ELISA kit (oxidized LDL measuring reagent “MX”; Kyowa Medex) according to the attached instruction manual. Specifically, 100 ⁇ L / well of a reaction buffer solution is placed on a plate to which a mouse antioxidant phospholipid monoclonal antibody is fixed, and the serum sample of this example (1) is diluted 250 times using the attached specimen diluent. Then, each 20 ⁇ L / well was added and incubated at 37 ° C. for 2 hours, followed by washing 4 times using the attached washing solution.
- MX oxidized LDL measuring reagent
- peroxidase-labeled goat anti-human apolipoprotein B polyclonal antibody was added at 100 ⁇ L / well, incubated at 37 ° C. for 1 hour, and then washed 4 times with the washing solution.
- the 3,3 ′, 5,5 ′,-tetramethylbenzidine solution was added at 100 ⁇ L / well, incubated at 37 ° C. for 30 minutes, and 0.5 mol / L sulfuric acid was added at 50 ⁇ L / well to stop the reaction. Thereafter, the absorbance was measured at a main wavelength of 450 nm and a sub-wavelength of 620 nm using a microplate reader (Multiskan FC; Thermo Scientific Fisher).
- the ELISA reaction was measured using an oxidized LDL ELISA kit (Oxidized LDL Eliser “Daiichi”; Sekisui Medical) according to the attached instruction manual. Specifically, the plate to which the mouse anti-MDA-LDL monoclonal antibody was fixed was washed three times using the attached washing solution. Subsequently, the serum sample of this Example (1) was diluted 2000 times using a sample diluent (HEPES buffer), each was added at 100 ⁇ L / well, reacted at room temperature for 2 hours, and then the attached washing solution was used. And washed 3 times.
- HEPES buffer sample diluent
- ⁇ -galactosidase-labeled mouse anti-apolipoprotein B monoclonal antibody was added at 100 ⁇ L / well, reacted at room temperature for 1 hour, and then washed 3 times with the attached washing solution.
- 100 ⁇ L / well of the substrate o-nitrophenyl- ⁇ -D-galactopyranoside solution was added and allowed to react at room temperature for 2 hours, and then the reaction was stopped by adding 100 ⁇ L / well of sodium carbonate solution.
- Absorbance was measured at a dominant wavelength of 415 nm and a minor wavelength of 655 nm using a microplate reader (NOVAPATH; Bio-Rad Laboratories).
- FIG. 8 shows the results of Examples (2), (3), and (4).
- the G11-6 antibody in the ELISA to which the G11-6 antibody was fixed, an absorbance of 0.212 was confirmed in patients with liver disease, but almost no absorbance was confirmed in healthy subjects.
- the absorbance of 0.602 was confirmed in the liver disease patients, and the absorbance of 0.094 was confirmed in the healthy subjects.
- the ELISA to which the anti-MDA-LDL antibody was fixed an absorbance of 0.146 was confirmed in patients with liver disease, and an absorbance of 0.179 was confirmed in healthy subjects.
- the antioxidant phospholipid antibody and the anti-MDA-LDL antibody react not only to the liver disease patient serum but also to the healthy subject serum, whereas the G11-6 antibody However, it has been shown to be highly specific.
- Example 4 Correlation between severity of liver disease and reactivity in ELISA with G11-6 antibody fixed (1) Determination of severity of liver disease by agarose gel electrophoresis From 9 patients with liver disease and 14 healthy subjects Each serum was collected and subjected to agarose gel electrophoresis according to the method described in Example 1 (1).
- FIG. 9 shows typical migration results of healthy subject serum and liver disease patient serum.
- liver disease patients in which the ⁇ -position band (corresponding to HDL) is completely deleted as compared with the migration pattern of healthy subjects are determined as a severe group, and the ⁇ -position band (corresponding to HDL).
- the liver disease patients who did not have a deficiency were determined to be in the mild moderate group, 6 of the 9 liver disease patients were in the mild moderate group and 3 were in the severe group.
- Example 3 (2) ELISA to which G11-6 antibody is fixed
- ELISA with G11-6 antibody immobilized was performed.
- a total of 23 samples of 14 healthy subjects, 6 mild to moderate groups, and 3 to severe groups were used in this Example (1).
- the measured values of absorbance were tabulated for each group of healthy subjects, mild to moderate groups, and severe groups, and average values were calculated. The result is shown in FIG. Comparison between each group was statistically significant when P ⁇ 0.05 by performing one-way analysis of variance and multiple comparison test by Scheffe's method.
- the absorbance of the healthy group was 0.052 ⁇ 0.024
- the absorbance of the mild group was 0.105 ⁇ 0.074
- the absorbance of the severe group was 0.699. It was ⁇ 0.942.
- a significant difference was recognized between the severe group and the healthy group at P ⁇ 0.05.
- a significant difference was recognized between P and 0.05 between the severe group and the mild group.
- Total cholesterol (TC); Cholestest CHO (Sekisui Medical) Neutral fat (TG); Excellator TG (Sekisui Medical) Phospholipid (PL); Pure Auto S PL (Sekisui Medical) Cholesterol in high density lipoprotein (HDL-C); Cores test N HDL (Sekisui Medical) Cholesterol in low density lipoprotein (LDL-C); Cholestest LDL (Sekisui Medical)
- Example 3 (2) ELISA to which G11-6 antibody is fixed ELISA with G11-6 antibody fixed was performed by the method described in Example 3 (2) [2-2]. However, instead of the serum sample of Example 3 (1), a total of 20 samples of 1 healthy subject, 7 patients with dyslipidemia, and 12 patients with liver disease were used in this example (1). The result is shown in FIG. Further, the absorbance value of each sample was divided by the serum concentration of LDL-C of each sample measured in this Example (1) using the following formula. The result is shown in FIG.
- the measured value of the absorbance of the healthy person is almost zero, while the measured value of the absorbance of the dyslipidemic patient 1 to the dyslipidemic patient 7 is approximately the measured value of the absorbance of the healthy person. Big in comparison.
- the measured value of the absorbance of dyslipidemic patient 1 is larger than the measured value of the absorbance of other dyslipidemic patients, and the measured value of the absorbance of liver disease patients 1 to 10 and liver disease patients 12 is large. It can be seen that the same or larger than that.
- the absorbances of liver disease patients 1 to 12 are larger than those of healthy subjects and are generally greater than those of dyslipidemic patients. . From these results, it was confirmed that the ELISA to which the G11-6 antibody was fixed had a small response in healthy subjects, but a relatively large response in dyslipidemic patients and liver disease patients.
- Example 6 Reactivity of ELISA to which G11-6 antibody is fixed to gel filtration eluate of serum from patients with various diseases (1) Reactivity of various ELISA to gel filtration eluate of total lipoprotein fraction Serum from healthy subjects Were collected and subjected to density gradient centrifugation according to a previous report (Hirano T. et al., J. Atheroclerosis and Thrombosis, Vol. 12, pp. 67-72, 2005) to separate lipoproteins.
- each elution solution of elution numbers 5, 7, 9, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, and 28 was used as a sample in Example 3 (2) [2-2. ],
- G11-6 antibody is immobilized by the method described in the above, and elution numbers 5, 7, 9, 11, 12, 13, 14, 15, 16, 18, 20 using the attached sample diluent , 22, 25 and 28 were diluted 10-fold, and these samples were used as samples, and an ELISA to which an antioxidant phospholipid antibody was fixed by the method described in Example 3 (3) was further added to the attached sample dilution.
- the eluates of elution numbers 5, 7, 9, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, and 28 were diluted 500 times using the solutions, and these were used as samples.
- FIG. 12A shows the results of TC concentration measurement and various ELISAs for the liver disease patient serum
- FIG. 12B shows the results for the healthy subject serum.
- the TC concentration reached the maximum at elution number 13 in patients with liver disease.
- the ELISA to which the G11-6 antibody was fixed the ELISA to which the antioxidant phospholipid antibody was fixed, and the ELISA to which the anti-MDA-LDL antibody was fixed had the highest absorbance.
- the TC concentration was highest in elution number 13 in young healthy individuals, whereas ELISA and antioxidant phospholipid antibody in which G11-6 antibody was fixed in elution number 14 The absorbance was maximized in both the ELISA to which the antibody was fixed and the ELISA to which the anti-MDA-LDL antibody was fixed.
- Example 3 (2) [2 -2] is used to fix the G11-6 antibody to the elution number 1, 3, 5, 8, 10, 11, 12, 13, 14, 15,
- Each eluate of 16, 18, and 24 was diluted 500 times, and these were used as samples, respectively, and ELISA with an anti-MDA-LDL antibody fixed thereto was performed by the method described in Example 3 (4).
- dyslipidemia patient (n 2).
- the elution number 13 shows the TC concentration, TG concentration, PL concentration, the absorbance of the ELISA to which the G11-6 antibody is immobilized, and the absorbance of the ELISA to which the anti-MDA-LDL antibody is immobilized. Both became the maximum.
- the TC concentration, the TG concentration, and the PL concentration in the elution number 13, and the absorbance of the ELISA to which the G11-6 antibody was fixed in the elution number 11, were the elution number 14 or In the elution number 15, the absorbance of the ELISA to which the anti-MDA-LDL antibody was fixed was maximized.
- the G11-6 antibody has a small response to the oxidized small dense LDL.
- the oxidized small dense LDL is highly oxidized in patients with dyslipidemia. The present inventors believe that this is the case.
- the TC concentration and PL concentration at elution number 13, the TG concentration at elution number 13 or elution number 5, and the ELISA with G11-6 antibody immobilized at elution number 16 were fixed.
- the absorbance of the ELISA to which the anti-MDA-LDL antibody was fixed at the elution number 15 was maximized.
- the G11-6 antibody was also applied to the eluate obtained by subjecting the serum directly to gel filtration chromatography without separating the total lipoprotein fraction by density gradient centrifugation.
- the immobilized ELISA was confirmed to have sufficient sensitivity to specifically detect oxidized LDL.
- Example 7 Reactivity of ELISA to which G11-6 antibody is immobilized against metal-oxidized LDL (1) Preparation of native-LDL fraction [1-1] Separation of lipoproteins by density gradient centrifugation It was collected and subjected to density gradient centrifugation according to a previous report (Hirano T. et al., J. Atherocleosis and Thrombosis, Vol. 12, pp. 67-72, 2005) to separate lipoproteins.
- the B fraction is different from the A and C fractions and does not contain HDL and contains LDL. Therefore, the B fraction was used as a native-LDL solution.
- Example (1) [1-1] The B fraction of Example (1) [1-1], ie, the native-LDL solution, was dialyzed overnight at 4 ° C. using PBS as a dialysis solution. Thereafter, the protein concentration was measured by the method described in Example 1 (2) [2-4] and diluted to 0.5 mg / mL with PBS.
- ⁇ A->, ⁇ b->, ⁇ c-> and ⁇ d-> were used immediately after incubation for each set time.
- ⁇ A-1> was used after 24 hours of storage at 4 ° C. following incubation for each set time.
- ⁇ A-1w> was used after incubation at each set time for 1 week at 4 ° C.
- ⁇ A + 1> was used after dialysis overnight at 4 ° C. using PBS as a dialysis solution following incubation for each set time.
- ⁇ A + 1w> was used after incubation at each set time, followed by dialysis overnight at 4 ° C. using PBS as a dialysis solution, and then storing at 4 ° C. for 6 days.
- the absorbance reaches a maximum at an oxidation time of 0.5 hours, the absorbance decreases at an oxidation time of 1 hour or longer, and reaches an absorbance similar to that of native-LDL at an oxidation time of 8 hours or longer. It was.
- the ELISA to which the G11-6 antibody is immobilized has a large reaction with respect to the lightly oxidized oxidized LDL (lightly oxidized LDL), and the unoxidized native-LDL and the highly oxidized oxidized It was confirmed that the reaction was small for LDL (highly oxidized LDL).
- lipid peroxide concentration of metal oxide LDL by TBARS method The metal oxide LDL of ⁇ a-> and ⁇ c-> in Example (2) was attached using TBARS Assay Kit (Cayman Chemical Co.). The lipid peroxide concentration was measured according to the instructions for use. Specifically, a color-developing reagent was prepared by mixing equal amounts of acetic acid and sodium hydroxide, and adding and dissolving Thiobarbituric acid to 36.8 mmol / L. After adding 1 mL of the coloring reagent and 25 ⁇ L of the SDS solution to 25 ⁇ L each of the metal oxide LDL solutions of ⁇ a-> and ⁇ c-> in Example (2) and incubating at 100 ° C.
- the lipid peroxide concentration showed similar changes between ⁇ a-> and ⁇ c->. That is, in any case, the lipid peroxide concentration increased until the oxidation time passed 2 hours, and the lipid peroxide concentration gradually decreased after the oxidation time passed 4 hours.
- the ELISA in which the G11-6 antibody was fixed a change in absorbance different between ⁇ a-> and ⁇ c-> was detected. That is, in the case of ⁇ a->, the absorbance increases until the oxidation time passes 2 hours, the absorbance decreases rapidly after the oxidation time passes 4 hours, and the oxidation time is 24 hours, the same as native-LDL.
- the absorbance reached a maximum at an oxidation time of 0.5 hours, and the absorbance decreased sharply after an oxidation time of 1 hour, and the oxidation time was 8 hours.
- the absorbance was about the same as that of native-LDL.
- the absorbance increases until the oxidation time passes 1 hour, and the oxidation time reaches 2 hours. As time passed, the absorbance decreased, and the absorbance was about the same as that of native-LDL after an oxidation time of about 16 hours. In the case of ⁇ a + 1>, the absorbance increases until the oxidation time passes 2 hours, the absorbance decreases after the oxidation time passes 4 hours, and the oxidation time is about 16 hours, which is about the same as native-LDL. The absorbance was.
- the same change in absorbance was detected between ⁇ a-1w> and ⁇ a + 1w>.
- the absorbance reached a maximum at an oxidation time of 0.5 hours, and the oxidation time was 1 hour.
- the absorbance decreased.
- the absorbance of ⁇ a-1w> and ⁇ a + 1w> decreased after 1 hour of oxidation time.
- Example 8 Reactivity of ELISA in which G11-6 antibody is immobilized against metal oxide LDL (comparison with reactivity of ELISA in which various antibodies are immobilized) (1)
- Preparation of metal oxide LDL Copper sulfate was added to 120 ⁇ L of native-LDL solution (0.5 mg / mL) of Example 7 (1) [1-3] so that the concentration was 3.33 ⁇ mol / L, and the temperature was 37 ° C.
- Metal-oxidized LDL solutions of various degrees of oxidation were prepared by incubating for 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours and 24 hours. The prepared metal oxide LDL solution was immediately used as an ELISA sample.
- sandwich ELISA was carried out using anti-apolipoprotein B antibody as a solid phase antibody and anti-apolipoprotein B antibody as a detection antibody. Specifically, ELISA was performed by the method described in Example 3 (2) [2-2]. However, in place of 5 ⁇ g / mL of the G11-6 antibody of Example 1 (5), 10 ⁇ g / mL of the goat anti-apolipoprotein B polyclonal antibody of Example 3 (2) ⁇ 2-1-2> was used.
- Example 3 replaced with the serum sample of Example 3 (1) diluted 20 times, and the metal oxidation LDL solution prepared in this Example (1) diluted to 0.1 microgram / mL was used.
- ALP-SA Zymed Laboratories
- ALP-SA Zymed Laboratories
- 250 times was used instead of ALP-SA (Zymed Laboratories) diluted 250 times.
- FIG. 23 shows the results of Examples (2), (3), and (4).
- the absorbance increased until the oxidation time passed 3 hours, the absorbance decreased after the oxidation time passed 4 hours, and the oxidation time 8 Absorbance was comparable to that of native-LDL over time, and the absorbance was lower than before oxidation when the oxidation time was 24 hours.
- the absorbance increased until the oxidation time passed 8 hours, and the absorbance slightly decreased after the oxidation time of 24 hours.
- the absorbance increases until the oxidation time passes 3 hours, the absorbance decreases after the oxidation time passes 4 hours, and the native-LDL is reached after an oxidation time of 24 hours. Absorbance was comparable. In an ELISA using an anti-apolipoprotein B antibody as a solid phase antibody and a detection antibody, the absorbance was high until the oxidation time passed 8 hours, and the absorbance slightly decreased after 24 hours. In the TBARS method, the lipid peroxide concentration increased until the oxidation time passed 4 hours, and decreased slightly when the oxidation time passed 8 hours. In the measurement of the conjugated diene, the absorbance rapidly increased until the oxidation time passed 3 hours, and after 4 hours, the absorbance gradually increased even during the oxidation time.
- the ELISA to which the G11-6 antibody was immobilized had a large reaction with lightly oxidized LDL and a small reaction with unoxidized native-LDL and highly oxidized LDL.
- the change in absorbance of the ELISA to which the G11-6 antibody is immobilized is different from any change in absorbance of the ELISA to which the anti-MDA-LDL antibody is immobilized and the ELISA to which the antioxidant phospholipid antibody is immobilized.
- the G11-6 antibody recognizes a site different from the anti-MDA-LDL antibody and the antioxidant phospholipid antibody as an antigen in oxidized LDL.
- the site of the G11-6 antibody as an antigen does not correlate with the lipid peroxide concentration responsible for reactivity to thiobarbituric acid and conjugated dienes and apolipoprotein B.
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Abstract
Description
(ii)N末端から順に、配列番号8のアミノ酸配列、配列番号9のアミノ酸配列および配列番号10のアミノ酸配列を含むアミノ酸配列、
(iii)N末端から順に、配列番号13のアミノ酸配列、配列番号14のアミノ酸配列および配列番号15のアミノ酸配列を含むアミノ酸配列、
(iv)配列番号7のアミノ酸配列、
(v)配列番号12のアミノ酸配列。
(i)本発明に係るモノクローナル抗体を、被検者から採取した生体試料に含まれる酸化LDLに特異的に反応させて複合体を形成させる工程(複合体形成工程)
(ii)前記複合体を検出する工程(検出工程)
以上(i)および(ii)の工程を有する。
(i)本発明に係るモノクローナル抗体を担体に固着する工程(固着工程)
(ii)担体に固着した前記モノクローナル抗体を、被検者から採取した生体試料に含まれる酸化LDLに特異的に反応させて複合体を形成させる工程(固着複合体形成工程)
(iii)前記複合体を検出する工程(固着複合体検出工程)
以上(i)~(iii)の工程を有する。
(1)アガロースゲル電気泳動によるTG-rich LDLの確認
肝疾患末期患者1名および健常者1名から採取した血液を室温で1時間静置した後、3500rpm、4℃の条件下で10分間遠心分離を行うことにより、肝疾患末期患者血清および健常者血清を得た。得られた血清を1.5μLずつアガロースゲル(ユニバーサルゲル/8;ヘレナ研究所社)に塗布し、pH8.6、イオン強度0.06のバルビタール緩衝液中において、100V、150Wで45分間電気泳動を行った後、ドライヤーを用いてゲルを乾燥させた。続いて、0.03%(w/v)のFat Red 7B(ヘレナ研究所社)を含むメタノール20mLにTritonX-100を2滴加え、さらに0.1mol/Lの水酸化ナトリウム水溶液を4mL加えることにより調製した染色液に、乾燥させたゲルを10分間浸漬して染色を行った後、75%(v/v)メタノール水溶液に10秒間浸漬することにより脱色を行った。
[2-1]密度勾配遠心法による総リポタンパク質画分の分離
本実施例(1)でTG-rich LDLが含まれることが確認された肝疾患末期患者血清について、既報(Chiba H.ら、J.Lipid Res.、第38巻、第1204-1216頁、1997年/Hirano T.ら、J.Atherosclerosis and Thrombosis、第12巻、第67~72頁、2005年)に従って密度勾配遠心法を行い、総リポタンパク質画分を得た。具体的には、本実施例(1)の肝疾患末期患者血清に5,5’-ジチオビス(2-ニトロ安息香酸)(DTNB;和光純薬工業社)およびEDTA-2Na(同仁化学研究所社)をそれぞれ0.7mmol/Lおよび2.7mmol/L(pH7.4)となるよう加え、さらに適量の臭化カリウム(関東化学社)を加えることにより比重dがd=1.225kg/Lとなるように調整し、サンプル溶液とした。続いて、0.20mol/Lの塩化ナトリウム、0.27mmol/LのEDTA-2Na(pH7.4)および1mmol/Lの水酸化ナトリウムを含む水溶液(d=1.006kg/L)に、適量の臭化カリウム(関東化学社)を加えることにより比重dがd=1.225kg/Lとなるように調整し、比重液とした。サンプル溶液12mLを遠心管(40PA;日立工機社)に入れ、比重液を満たし、超遠心機himac CP60E ultracentrifuge(日立工機社)およびローターRPV-50T rotor(日立工機社)を用いて、40000rpm、15℃の条件下で18時間遠心分離を行って、上層(d<1.225kg/L)を総リポタンパク質画分として回収した。回収した総リポタンパク画分約8mLを、アミコン攪拌式セルModel 8050(ミリポア社)および限外濾過膜アミコンXM50(ミリポア社)を用いて、付属の使用書に従い、窒素ガス雰囲気下で2~3mLに濃縮した。
本実施例(2)[2-1]の総リポタンパク質画分について、既報(Chiba Hら、J.Lipid Res.、第38巻、第1204~1216頁、1997年/Hirano T.ら、J.Atherosclerosis and Thrombosis、第12巻、第67~72頁、2005年)に従ってゲル濾過クロマトグラフィーを行い、TG-rich LDL画分を得た。具体的には、以下の器具、試薬および条件を用いて、280nmの吸光度を計測しながらゲル濾過クロマトグラフィーを行い、溶出液を3mLずつ分取した。
カラム ;Sepharose CL-4B(GEヘルスケア社)
緩衝液 ;0.15mol/LのNaCl、0.27mmol/LのEDTA-2Na および3mmol/LのNaN3を含む5mmol/LのTris-HCl 緩衝液(pH7.4)200mL
条件 ;クロマトチャンバー 4℃
流速 0.15mL/分
本実施例(2)[2-2]のTG-rich LDL画分18mLを、アミコン攪拌式セルModel 8050(ミリポア社)および限外濾過膜アミコンXM50(ミリポア社)を用いて、付属の使用書に従い、窒素ガス雰囲気下で2~3mLに濃縮した。その後、リン酸緩衝液(PBS)を透析液として、透析膜(セルロースチューブ20/32;三光純薬社)を用いて、4℃で一晩透析し、TG-rich LDL溶液2~3mLを得た。透析中、透析液の交換を3回行った。
本実施例(2)[2-3]のTG-rich LDL溶液について、既報に従い、Lowry変法を用いてタンパク質濃度を測定した(Markwell MA.ら、Anal.Biochem.、第87巻、第206~210頁、1978年)。具体的には、2%(w/v)の炭酸ナトリウム、0.4%(w/v)の水酸化ナトリウム、0.16%(w/v)の酒石酸塩および1%(w/v)のドデシル硫酸ナトリウム(SDS)を含む水溶液を調製し、この水溶液と4%(w/v)の硫酸銅水溶液とを体積比100:1に混合した溶液を反応溶液として調製した。また、脱イオン水に等量のフェノール試薬(フォーリン・チオカルト試薬;和光純薬社)を混合してフォーリン・チオカルト試薬液を調製した。また、500μg/mLの牛血清アルブミン(BSA)水溶液を標準溶液として調製した。本実施例(2)[2-3]のTG-rich LDL溶液および標準溶液それぞれ1mLに、調製した反応溶液を3mLずつ加えて室温で30分間反応させた。続いて、調製したフォーリン・チオカルト試薬液300μLを強く撹拌しながら加えた後、室温で45分間反応させた。その後、660nmの吸光度をそれぞれ測定した。標準溶液の測定値との比較から、本実施例(2)[2-3]のTG-rich LDL溶液のタンパク質濃度を算出した。
本実施例(2)[2-4]で算出した結果に基づき、本実施例(2)[2-3]のTG-rich LDL溶液のタンパク質濃度を、PBSを用いて1mg/mLに調整した後、4℃で保存した。
本実施例(2)[2-5]の0.5~1mg/mLのTG-rich LDL溶液0.1mLを孔径0.45mmのフィルター(DISMIC-25CS;ADVANTEC社)に通した後、常法に従って、百日咳菌アジュバントを注射したBALB/cマウスに3回腹腔内注射することにより免疫を行った。その後、常法に従って、免疫したマウスから脾臓細胞を採取し、50%ポリエチレングリコール1500(Roche社)を用いてマウスミエローマ細胞株P3U1と融合させてハイブリドーマを作製した。ハイブリドーマは、常法に従って、ペニシリン/ストレプトマイシン、10%(w/v)のウシ胎児血清(FCS)およびHAT溶液を含むRPMI1640培地を用いて、コロニーが確認できるまで約10日間培養した。
[4-1]native-LDL画分の調製
本実施例(2)[2-1]、[2-2]、[2-3]、[2-4]および[2-5]に記載の方法に従って、健常者血清からnative-LDL溶液を調製した。
本実施例(4)[4-1]のnative-LDL溶液1mg/mLに、25μmol/Lとなるよう硫酸銅を添加し、37℃で24時間インキュベートした後、PBSを透析液として4℃で一晩透析を行うことにより、金属酸化LDL溶液を調製した。
本実施例(4)[4-1]のnative-LDL溶液および本実施例(4)[4-2]の金属酸化LDL溶液を、PBSを用いてそれぞれ20μg/mLに希釈した後、96穴プレート(Nunc MaxiSorp;Nalge Nunc International社)に50μL/ウェル入れて4℃で一晩反応させることにより、native-LDLおよび金属酸化LDLをそれぞれプレート上に固着させた。液体を除去して1%(w/v)BSAを含むPBSを150μL/ウェル入れ、37℃で2時間インキュベートすることによりブロッキングを行った後、0.05%(v/v)のTween20を含むPBS(0.05%Tween-PBS)を用いて4回洗浄した。続いて、native-LDLを固着させたウェルおよび金属酸化LDLを固着させたウェルに本実施例(3)の各コロニーの培養上清を50μL/ウェル入れて、室温で1時間反応させた後、0.05%Tween-PBSを用いて4回洗浄した。続いて、PBSで500倍に希釈したビオチン標識ラット抗マウスk鎖(Zymed Laboratories社)を50μL/ウェルずつ入れ、室温で1時間反応させた後、0.05%Tween-PBSを用いて4回洗浄した。その後、0.05%Tween-PBSで500倍に希釈したアルカリフォスファターゼ標識ストレプトアビジン(ALP-SA;Zymed Laboratories社)を50μL/ウェル入れて室温で30分間反応させた後、0.05%Tween-PBSを用いて4回洗浄した。次に、0.5mmol/LのMgCl2を含む10mmol/LのDiethanolamine溶液を用いて1mg/mLに調整したDisodium p-nitrophenyl phosphate hexahydrate(和光純薬社)を100μL/ウェル入れて、室温で30分間反応させた。続いて、マイクロプレートリーダー(Bio-Rad Laboratories社)を用いて、主波長405nmおよび副波長600nmで吸光度を測定した。native-LDLを固着させたウェルについては吸光度が小さい培養上清を、金属酸化LDLを固着させたウェルについては吸光度が大きい培養上清をそれぞれ特定することにより、コロニーの選択を行った。
本実施例(4)[4-3]で選択したコロニーの細胞を、96穴プレートに1個/ウェルとなるように播き、ペニシリン-ストレプトマイシン、10%(v/v)のFCS、Hypoxanthine、ThymidineおよびHybridoma Fusion Cloning Supplement(Roche社)を含むRPMI1640培地(10%FCS-HT-HFCS-RPMI1640)を用いて培養を行った。これらの培養上清について、本実施例(4)[4-3]に記載の方法に従い、再度ELISAを行い、native-LDLを固着させたウェルについては吸光度が小さい培養上清を、かつ金属酸化LDLを固着させたウェルについては吸光度が大きい培養上清を特定することにより、クローンの選択を行った。その結果、得られたハイブリドーマをG11-6と命名した。
常法に従って、2,6,10,14‐テトラメチル‐2‐ペンタデセン酸(プリステン)を注射したマウスの腹腔内に、本実施例(4)のG11-6を接種して培養し、G11-6が産生するモノクローナル抗体(G11-6抗体)を含む腹水を得た。
溶離液;50mmol/L NaPB溶液(pH7.2)
システムコントローラ;CBM-20A(島津製作所社)
送液ポンプ:LC-20AD(島津製作所社)
オートサンプラー;SIL-20A(島津製作所社)
カラムオーブン;CTO-20AC(島津製作所社)
検出器;SPD-20A(島津製作所社)
条件;流速 0.5mL/分
検出波長 280nm
IsoStripマウスモノクローナル抗体アイソタイピングキット(Roche社)を用いて、付属の使用書に従い、イムノクロマトグラフィー法により実施例1(5)のG11-6抗体のクラス判定を行った。その結果、G11-6抗体のクラスはIgMであることが明らかになった。
(1)RNAの抽出
実施例1(4)のハイブリドーマG11-6を、10mLの10%FCS含有RPMI1640培地を入れた25cm3のフラスコに播種し、5%CO2雰囲気および37℃の条件下で72時間培養した。続いて、常温、8500rpmの条件下で5分間遠心分離を行って、上清を除去し、細胞ペレットを回収した。Absolutely RNA Miniprep kit(STRATAGENE社)を用いて、付属の使用書に従い、回収した細胞のペレットからRNAの抽出を行った。
本実施例(1)のRNA溶液を鋳型として、SuperScriptTM First-Strand Synthesis System for RT-PCR(インビトロジェン社)を用いて、付属の使用書に従い逆転写反応を行い、cDNAを調製した。具体的には、まず、下記の組成の反応液Aおよび反応液Bを調製した。
反応液B;10×RT Buffer 2μL、25mmol/L MgCl2 4μL、0.1mol/L DTT 2μL、40U/mL RNaseOUTTM 1μL
本実施例(2)のcDNA溶液を鋳型として、サーマルサイクラー(GeneAmp PCR System 2400;Applied Biosystems社)を用いてPCRを行い、G11-6抗体の重鎖可変領域(G11-6-VH)およびG11-6抗体の軽鎖可変領域(G11-6-VL)のDNA配列をそれぞれ増幅した。PCRに用いたプライマー、PCR反応溶液組成およびPCR反応条件は下記のとおりである。
G11-6-VH(5’プライマー;MuIgVH5’-B);5’-GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT-3’(RはAまたはG、SはCまたはG、YはCまたはT、WはAまたはTを表す。;配列番号1)
G11-6-VH(3’プライマー;MuIgMVH3’);5’-CCCAAGCTTACGAGGGGGAAGACATTTGGGAA-3’(配列番号2)
G11-6-VL(5’プライマー;MuIgkVL5’-B);5’- GGGAATTCATGGAGACAGACACACTCCTGCTAT-3’(配列番号3)
G11-6-VL(3’プライマー;MuIgkVL3’);5’-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3’(配列番号4)
PCR反応溶液組成;10×PCR Buffer(Mg2+フリー) 2.5μL、50mmol/L MgCl2 0.75μL、2.5mmol/L dNTP mix 2.0μL、250pmol/L 5’プライマー 1.0μL、250pmol/L 3’プライマー 1.0μL、cDNA溶液 1.0μL、Platinum Taq DNA polymerase 0.25μL、DEPC水 16.5μL
PCR反応条件;94℃で2分の反応の後、94℃で30秒、50℃で30秒、72℃で2分の各反応を1サイクルとして40サイクル行い、その後72℃で6分の反応を行った。
本実施例(3)のVH-PCR産物溶液およびVH-PCR産物溶液から5μLずつ分取して、それぞれ2μLのLoading buffer(6×Orange DNA Loading Dye;Fermentas社)を添加し、VH泳動用溶液およびVL泳動用溶液とした。VH泳動用溶液、VL泳動用溶液およびDNAラダー(O’GeneRulerTM 100bp DNA Ladder Plus;Fermentas社)を、0.007%(v/v)エチジウムブロマイド(ニッポンジーン社)を含む3%(w/v)アガロースゲル(NuSieve GTG Agarose;CAMBREX社)にアプライした後、陽極側の泳動用バッファー(1×TAE バッファー;ニッポンジーン社)に2μLのエチジウムブロマイド(ニッポンジーン社)を添加して混和し、100Vで、約40分間電気泳動を行った。その後、UV検出器(Dolphin-View;KURABO社)を用いて、泳動像の観察を行った。その結果を図3に示す。
本実施例(3)のVH-PCR産物溶液およびVL-PCR産物溶液から15μLずつ分取して、実施例(4)に記載の方法により電気泳動を行った。続いて、VH-PCR産物溶液を泳動したアガロースゲルからは約450bpのバンドを、VL-PCR産物溶液を泳動したアガロースゲルからは約400bpのバンドをそれぞれ切り出した後、QIAquick gel extraction kit(Qiagen社)を用いて、付属の使用書に従い、DNA断片を含む溶液を回収し、G11-6-VH-DNA溶液およびG11-6-VL-DNA溶液とした。その後、TOPO TA Cloning Kit for Sequencing(Invitrogen社)を用いて、4μLのG11-6-VH-DNA溶液、1μLのSalt Solutionおよび1μLのTOPOベクターからなるVHライゲーション反応液と、4μLのG11-6-VL-DNA溶液、1μLのSalt Solutionおよび1μLのTOPOベクターからなるVLライゲーション反応液とを調製し、室温で5分間静置することにより、DNA断片のベクターへの挿入を行った。
本実施例(5)のVHライゲーション反応液2μLおよびVLライゲーション反応液2μLに、それぞれ、TOP10 Chemically competent E.coli(One Shot TOP10 Chemically Competent E.coli;Invitrogen社)を添加して混和した後、氷中で30分間静置した。続いて、42℃で30秒間静置した後、直ちに氷中で冷却し、E.coli増殖用培地であるS.O.C.medium(Invitrogen社)を250μLずつ加えて37℃で1時間静置することにより、大腸菌の形質転換を行った。VHライゲーション反応液により形質転換した大腸菌を含む溶液をVH大腸菌液とし、VLライゲーション反応液により形質転換した大腸菌を含む溶液をVL大腸菌液とした。
本実施例(6)のVH大腸菌培養液1サンプルおよびVL大腸菌培養液1サンプルについて、常温および3000rpmの条件下で10分間遠心分離を行った後、上清を除去してVH大腸菌ペレットおよびVL大腸菌ペレットを回収した。その後、QIAprep Spin Miniprep Kit(Fermentas社)を用いてプラスミドの精製を行い、VHプラスミド溶液およびVLプラスミド溶液を得た。具体的には、VH大腸菌ペレットおよびVL大腸菌ペレットのそれぞれにP1溶液を250μLずつ加えて懸濁し、1.5mLエッペンドルフチューブに移した後、これにP2溶液を250μLずつ加えて転倒混和し、数分静置して大腸菌を溶解させた。続いて、N3溶液を350μLずつ加えて転倒混和して中和し、常温および14000rpmの条件下で1分間遠心分離を行った後、それぞれ上清を回収して、カラムに添加した。続いて、このカラムを常温および14000rpmの条件下で1分間遠心分離を行って濾液を除去した後、カラムにPE溶液を750μLずつ加えて、常温および14000rpmの条件下で1分間遠心分離を行って濾液を除去することによりカラムを洗浄した。カラムをそれぞれ新しい1.5mLエッペンドルフチューブに移した後、EB溶液を50μLずつ加えて1分間静置した。その後、常温および14000rpmの条件下で2分間遠心分離を行って濾液を回収し、これをプラスミド溶液とした。VL大腸菌ペレットから得られたプラスミド溶液をVLプラスミド溶液とし、VL大腸菌ペレットから得られたプラスミド溶液をVLプラスミド溶液とした。VHプラスミド溶液およびVLプラスミド溶液は4℃で保存した。
本実施例(7)のVHプラスミド溶液およびVLプラスミド溶液をそれぞれ鋳型DNAとして、BigDye Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems社)およびT3プライマー(ATTAACCCTCACTAAAGGGA;配列番号5)を用いてシークエンス反応を行った。シークエンス反応溶液組成およびシークエンス反応条件は以下のとおりである。
シークエンス反応条件;96℃で10秒間の反応の後、96℃で10秒、50℃で5秒、60℃で3分の各反応を1サイクルとして25サイクル行い、その後4℃で静置した。
GTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCCACCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGAGTCTACGGTAGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(配列番号6)
VQLQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTRVYGRAMDYWGQGTSVTVSS(配列番号7)
VH領域のCDR1;SYWMH(配列番号8)
VH領域のCDR2;AIYPGNSDTSYNQKFKG(配列番号9)
VH領域のCDR3;VYGRAMDY(配列番号10)
GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA(配列番号11)
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK(配列番号12)
VL領域のCDR1;RASKSVSTSGYSYMH(配列番号13)
VL領域のCDR2;LVSNLES(配列番号14)
VL領域のCDR3;QHIRELT(配列番号15)
本実施例(8)のG11-6-VH、G11-6-VL、およびそれらのCDR1、CDR2およびCDR3の各アミノ酸配列について、ソフトウェア(MacVector;MacVector社)を用いて、ClustalW解析およびBLAST解析を行い、既知のアミノ酸配列との相同性を検索した。相同性検索の結果、相同性が高かった最上位のものを図4~7に示す。
(1)血清サンプルの調製
肝疾患患者1名および健常者1名からそれぞれ血清を採取し、サンプルとした。
[2-1]ビオチン標識抗アポリポタンパク質B抗体(検出用抗体)の調製
〈2-1-1〉抗アポリポタンパク質B抗体の精製
抗アポリポタンパク質Bポリクローナル抗体を含むヤギ抗血清(WatPa;Enterprises社)について、実施例1(5)に記載の方法により、常法に従い飽和硫安沈殿法を行って、粗抗アポリポタンパク質B抗体溶液を得た。続いて、粗抗アポリポタンパク質B抗体溶液について、常法に従いアフィニティカラムクロマトグラフィー法を行って、精製抗アポリポタンパク質B抗体を得た。具体的には、まず、粗抗アポリポタンパク質B抗体溶液をPBSで10倍希釈した後、以下の器具・機器および条件を用いてアフィニティカラムに循環して通過させた。
送液ポンプ;ペリスタルティックポンプ(SJ-1215;ATTO社)
条件;4℃、流速約0.2mL/分
Dimethylsulfoxide(和光純薬社)に、10mmol/LとなるようN-hydroxysuccinimide ester of biotin(EZ-Link NHS-Biotin Reagents;サーモフィッシャーサイエンティフィック社)を溶解することによりビオチン標識液を調製した。本実施例(2)[2-1]〈2-1-1〉の抗アポリポタンパク質B抗体溶液1mLに、調製したビオチン標識液を27μL添加して、室温で4時間、振盪しながら反応させた後、PBSを透析液として透析を行い、未反応のビオチンを除去することにより、ビオチン標識抗アポリポタンパク質B抗体を調製した。その後、PBSを用いてタンパク質濃度0.01mg/mLに希釈した。
実施例1(5)のG11-6抗体について、PBSを用いてタンパク質濃度5μg/mLに希釈した。これを、96穴プレート(Nunc MaxiSorp;Nalge Nunc International社)に50μL/ウェル入れて、37℃で2時間インキュベートすることによりG11-6抗体をプレート上に固着させた。液体を除去して1%(w/v)BSAを含むPBSを150μL/ウェル入れ、37℃で2時間インキュベートすることによりブロッキングを行った後、0.05%Tween-PBSを用いて4回洗浄した。続いて、PBSを用いてそれぞれ20倍に希釈した本実施例(1)の血清サンプルを50μL/ウェル入れて、4℃で一晩反応させた後、0.05%Tween-PBSを用いて4回洗浄した。次に、本実施例(2)[2-1]〈2-1-2〉のビオチン標識ヤギ抗アポリポタンパク質B抗体を50μL/ウェル入れ、室温で1時間反応させた後、0.05%Tween-PBSを用いて4回洗浄した。続いて、0.05%Tween-PBSで250倍に希釈したALP-SA(Zymed Laboratories社)を50μL/ウェル入れて室温で30分間反応させた後、0.05%Tween-PBSを用いて4回洗浄した。さらに、0.5mmol/LのMgCl2を含む10mmol/LのDiethanolamine溶液を用いて1mg/mLに調整したDisodium p-nitrophenyl phosphate hexahydrate(和光純薬社)を100μL/ウェル入れ、室温で60分間発色反応を行った後、マイクロプレートリーダー(Multiskan FC;サーモサイエンティフィックフィッシャー社)を用いて、主波長405nmおよび副波長620nmで吸光度を測定した。
本実施例(1)の血清サンプルについて、酸化LDLのELISAキット(酸化LDL測定試薬「MX」;協和メデックス社)を用いて、付属の使用書に従いELISAの反応を測定した。具体的には、マウス抗酸化リン脂質モノクローナル抗体が固着されたプレートに反応用緩衝液を100μL/ウェル入れ、付属の検体希釈液を用いて本実施例(1)の血清サンプルを250倍に希釈し、それぞれ20μL/ウェル入れて37℃で2時間インキュベートした後、付属の洗浄液を用いて4回洗浄した。続いて、ペルオキシダーゼ標識ヤギ抗ヒトアポリポタンパク質Bポリクローナル抗体を100μL/ウェル入れ、37℃で1時間インキュベートした後、前記洗浄液を用いて4回洗浄した。次に、3,3’,5,5’,-テトラメチルベンジジン溶液を100μL/ウェル入れて37℃で30分間インキュベートし、0.5mol/Lの硫酸を50μL/ウェル入れることにより反応を停止させた後、マイクロプレートリーダー(Multiskan FC;サーモサイエンティフィックフィッシャー社)を用いて、主波長450nmおよび副波長620nmで吸光度を測定した。
本実施例(1)の血清サンプルについて、酸化LDLのELISAキット(酸化LDLエライザ「第一」;積水メディカル社)を用いて、付属の使用書に従いELISAの反応を測定した。具体的には、付属の洗浄液を用いてマウス抗MDA-LDLモノクローナル抗体が固着されたプレートを3回洗浄した。続いて、検体希釈液(HEPES緩衝液)を用いて本実施例(1)の血清サンプルを2000倍に希釈し、それぞれ100μL/ウェル入れて室温で2時間反応させた後、付属の洗浄液を用いて3回洗浄した。続いて、β-ガラクトシダーゼ標識マウス抗アポリポタンパク質Bモノクローナル抗体を100μL/ウェル入れ、室温で1時間反応させた後、付属の洗浄液を用いて3回洗浄した。次に、基質であるo-ニトロフェニル-β-D-ガラクトピラノシド溶液を100μL/ウェル入れて、室温で2時間反応させた後、炭酸ナトリウム液を100μL/ウェル入れて反応を停止させ、マイクロプレートリーダー(NOVAPATH;Bio-Rad Laboratories社)を用いて、主波長415nmおよび副波長655nmで吸光度を測定した。
(1)アガロースゲル電気泳動による肝疾患重症度の判定
肝疾患患者9名、健常者14名からそれぞれ血清を採取し、実施例1(1)に記載の方法に従ってアガロースゲル電気泳動を行った。健常者血清および肝疾患患者血清の典型的な泳動結果を図9に示す。
実施例3(2)[2-2]に記載の方法に従い、G11-6抗体を固着させたELISAを行った。ただし、サンプルは実施例3(1)の血清サンプルに代えて、本実施例(1)の健常者群14名、軽中度群6名および重度群3名の血清合計23サンプルをそれぞれ用いた。吸光度の測定値を健常者群、軽中度群および重度群の群別に集計し、平均値を算出した。その結果を図10に示す。各群間の比較は、一元配置分散分析およびScheffeの方法による多重比較検定を行い、 P<0.05である場合に、統計学的に有意であるとした。
(1)血清脂質項目の測定
健常者1名、脂質異常症患者7名(脂質異常症1、脂質異常症2、・・・脂質異常症7とする)および肝疾患患者12名(肝疾患1、肝疾患2、・・・肝疾患12とする)から血清合計20サンプルを採取し、以下の試薬および日立自動分析装置7170(日立ハイテクノロジーズ社)を用いて、付属の使用書に従い、血清中脂質濃度を測定した。続いて、測定値を健常者、脂質異常症患者および肝疾患患者の別に集計して、平均値を算出した。その結果を表2に示す。
中性脂肪(TG);エクセライザ TG(積水メディカル社)
リン脂質(PL);ピュアオートS PL(積水メディカル社)
高密度リポタンパク質中のコレステロール(HDL-C);コレステストN HDL(積水メディカル社)
低密度リポタンパク質中のコレステロール(LDL-C);コレステスト LDL(積水メディカル社)
実施例3(2)[2-2]に記載の方法により、G11-6抗体を固着させたELISAを行った。ただし、サンプルは実施例3(1)の血清サンプルに代えて、本実施例(1)の健常者1名、脂質異常症患者7名および肝疾患患者12名の血清合計20サンプルを用いた。その結果を図11のAに示す。また、各サンプルの吸光度の測定値について、以下の式を用いて、本実施例(1)で測定した各サンプルのLDL-Cの血清中濃度で除した。その結果を図11のBに示す。
(1)総リポタンパク質画分のゲル濾過溶出液に対する各種ELISAの反応性
健常者から血清を採取し、既報(Hirano T.ら、J.Atherosclerosis and Thrombosis、第12巻、第67~72頁、2005年)に従って密度勾配遠心法を行い、リポタンパク質を分離した。具体的には、肝疾患患者1名および若年健常者1名から採取したそれぞれの血清2mLを比重dがd=1.225kg/Lとなるように調整し、超遠心機OptimaMAX ultracentrifuge(ベックマン・コールター社)およびローターMLN-80(ベックマン・コールター社)を用いて、50000rpm、15℃の条件下で20時間遠心分離を行った後、上層(d<1.225kg/L)を総リポタンパク質画分として回収した。続いて、実施例1(5)に記載の方法によりゲル濾過クロマトグラフィーを行って、溶出液を0.5mLずつ分取し、分取した順に溶出番号1、溶出番号2、・・・・溶出番号28とした。その後、溶出番号5、7、9、11、12、13、14、15、16、18、20、22、25および28の各溶出液についてコレステストCHO(積水メディカル社)および日立自動分析装置7170(日立ハイテクノロジーズ社)を用いて、付属の使用書に従い、TC濃度を測定した。
肝疾患患者1名、非アルコール性脂肪肝炎(NASH)患者1名、脂質異常症患者2名(脂質異常症1、脂質異常症2とする)および健常者2名(健常者1、健常者2とする)から血清合計6サンプルを採取し、実施例1(5)に記載の方法によりゲル濾過クロマトグラフィーを行って、溶出液を0.5mLずつ分取し、分取した順に溶出番号1、溶出番号2、・・・・溶出番号28とした。その後、溶出番号1~28の各溶出液について実施例5(1)に記載の方法によりTC濃度、TG濃度およびPL濃度を測定した。
(1)native-LDL画分の調製
[1-1]密度勾配遠心法によるリポタンパク質の分離
健常者から血清を採取し、既報(Hirano T.ら、J.Atherosclerosis and Thrombosis、第12巻、第67~72頁、2005年)に従って密度勾配遠心法を行い、リポタンパク質を分離した。具体的には、健常者血清2mLを比重dがd=1.019kg/Lとなるように調製し、超遠心機OptimaMAX ultracentrifuge(ベックマン・コールター社)およびローターMLN-80(ベックマン・コールター社)を用いて、40000rpm、15℃の条件下で20時間遠心分離を行った後、上層(d<1.019kg/L)をA画分として回収した。続いて、下層を比重dがd=1.063kg/Lとなるように調製し、上記の超遠心機およびローターを用いて50000rpm、15℃の条件下で18時間遠心分離を行った。ここで得られた上層(1.019kg/L<d<1.063kg/L)をB画分とし、下層をC画分としてそれぞれ回収した。
本実施例(1)[1-1]の健常者血清、A画分、B画分およびC画分について、市販リポタンパク質分析キット(リポフォー;常光社)を用いて、付属の使用書に従い、ポリアクリルアミドゲル電気泳動法を行った。その結果を図17に示す。
本実施例(1)[1-1]のB画分すなわちnative-LDL溶液について、PBSを透析液として4℃で一晩透析を行った。その後、実施例1(2)[2-4]に記載の方法によりタンパク質濃度測定を行い、PBSを用いて0.5mg/mLに希釈した。
本実施例(1)[1-3]のnative-LDL溶液(0.5mg/mL)120μLに、下記の濃度および量の硫酸銅を添加し、37℃で0.5時間、1時間、2時間、4時間、8時間および24時間インキュベートすることにより、下記の通り様々な酸化度の金属酸化LDL溶液を調製した。
〈a-1〉 250μmol/L硫酸銅1.6μL 透析なし 4℃で24時間保管
〈a-1w〉250μmol/L硫酸銅1.6μL 透析なし 4℃で1週間保管
〈a+1〉 250μmol/L硫酸銅1.6μL 透析あり 4℃で一晩透析
〈a+1w〉250μmol/L硫酸銅1.6μL 透析あり 4℃で一晩透析の後、4℃で6日間保管
〈b-〉 500μmol/L硫酸銅1.6μL 透析なし 酸化処理終了直後に使用
〈c-〉 500μmol/L硫酸銅6.0μL 透析なし 酸化処理終了直後に使用
〈d-〉 1000μmol/L硫酸銅6.0μL 透析なし 酸化処理終了直後に使用
本実施例(2)の〈a+1〉の金属酸化LDL溶液について、実施例1(1)に記載の方法により、アガロースゲル電気泳動を行った。その結果を図18に示す。
実施例3(2)[2-2]に記載の方法により、G11-6抗体を固着させたELISAを行った。ただし、サンプルは実施例3(1)の血清サンプルに代えて、本実施例(2)の〈a-〉、〈b-〉、〈c-〉および〈d-〉の金属酸化LDLをそれぞれ用いた。その結果を図19に示す。
本実施例(2)の〈a-〉および〈c-〉の金属酸化LDLについて、TBARS Assay Kit(ケイマンケミカル社)を用いて、付属の使用書に従い過酸化脂質濃度測定を行った。具体的には、等量の酢酸と水酸化ナトリウムを混合し、36.8mmol/LとなるようThiobarbituric acidを加えて溶解することにより発色試薬を調製した。本実施例(2)の〈a-〉および〈c-〉の金属酸化LDL溶液25μLずつに対し、1mLの発色試薬および25μLのSDS溶液をそれぞれ加えて混合し、100℃で1時間インキュベートした後、1分間氷中に置いて反応を停止させた。続いて室温、12000rpmの条件下で10分間遠心分離を行った後、上清を回収して96穴マイクロプレート(住友ベークライト社)に150μL/ウェル入れ、波長550nmの吸光度をマイクロプレートリーダー(Model680;Bio-Rad Laboratories社)を用いて測定した。〈a-〉についての結果を、本実施例(4)の〈a-〉についての結果と合わせて図20のAに、〈c-〉についての結果を、本実施例(4)の〈c-〉についての結果と合わせて図20のBに、それぞれ示す。
実施例3(2)[2-2]に記載の方法により、G11-6抗体を固着させたELISAを行った。ただし、サンプルは実施例3(1)の血清サンプルに代えて、本実施例(2)の〈a-1〉および〈a+1〉の金属酸化LDLをそれぞれ用いた。その結果を、本実施例(4)の〈a-〉についての結果と合わせて図21のAに示す。
実施例3(2)[2-2]に記載の方法により、G11-6抗体を固着させたELISAを行った。ただし、サンプルは実施例3(1)の血清サンプルに代えて、本実施例(2)の〈a-1w〉および〈a+1w〉の金属酸化LDLのうち、硫酸銅添加後のインキュベート時間が0.5時間、1時間、2時間および4時間のものをそれぞれ用いた。その結果を、本実施例(6)のG11-6抗体を固着させたELISAの結果と合わせて図22に示す。
(1)金属酸化LDLの調製
実施例7(1)[1-3]のnative-LDL溶液(0.5mg/mL)120μLに、3.33μmol/Lとなるよう硫酸銅を添加し、37℃で0.5時間、1時間、2時間、3時間、4時間、6時間、8時間および24時間インキュベートすることにより、様々な酸化度の金属酸化LDL溶液を調製した。調製した金属酸化LDL溶液は速やかにELISAのサンプルとして使用した。
本実施例(1)で調製した金属酸化LDLをサンプルとして、実施例3(2)[2-2]に記載の方法によりG11-6抗体を固着させたELISAを、付属の検体希釈液を用いて、本実施例(1)で調製した金属酸化LDLを2500倍に希釈し、これらをサンプルとして実施例3(3)に記載の方法により抗酸化リン脂質抗体を固着させたELISAを、付属の検体希釈液を用いて、本実施例(1)で調製した金属酸化LDLを1000倍に希釈し、これらをサンプルとして実施例3(4)に記載の方法により抗MDA-LDL抗体を固着させたELISAを、それぞれ行った。
実施例7(5)に記載の方法によりTBARS法を行い、本実施例(1)で調製した金属酸化LDLの過酸化脂質濃度を測定した。
脂質の酸化の指標とするため、各酸化時間の金属酸化LDLにおける共役ジエンを測定した。具体的には、PBSを用いて本実施例(1)で調製した金属酸化LDLを0.04mg/mLに希釈し、分光光度計(V-530;日本分光社)を用いて波長234nmの吸光度を測定した。
Claims (16)
- モノクローナル抗体を固相化抗体とし、かつ抗アポリポタンパク質B抗体を検出用抗体とするELISA(Enzyme-linked immunosorbent assay)において、下記(a)で示される抗原と前記モノクローナル抗体との反応の度合いと比較して、下記(b)で示される抗原と前記モノクローナル抗体との反応の度合いが小となる、酸化低密度リポタンパク質に対し特異的に反応するモノクローナル抗体;
(a)最終濃度0.493g/Lの正常な低密度リポタンパク質(native-LDL)に最終濃度3.29μmol/Lの硫酸銅を37℃で0.5時間反応させて得られる金属酸化低密度リポタンパク質、
(b)最終濃度0.493g/Lの正常な低密度リポタンパク質(native-LDL)に最終濃度3.29μmol/Lの硫酸銅を37℃で24時間反応させて得られる金属酸化低密度リポタンパク質。 - 酸化低密度リポタンパク質がトリグリセリド高含有低密度リポタンパク質(TG-rich LDL)である、請求項1に記載のモノクローナル抗体。
- 酸化低密度リポタンパク質がヒトの酸化低密度リポタンパク質である、請求項1または請求項2に記載のモノクローナル抗体。
- 健常者において酸化型のsmall dense LDLと特異的に反応するモノクローナル抗体。
- 非アルコール性脂肪性肝炎(Non-alcoholic steatohepatitis;NASH)患者においてnative-LDLと同様の粒子サイズの酸化LDLと特異的に反応するモノクローナル抗体。
- 脂質異常症患者において酸化型のレムナントリポタンパク質と特異的に反応するモノクローナル抗体。
- 配列番号10のアミノ酸配列を含む可変領域を含んでなる、請求項1から請求項6のいずれかに記載のモノクローナル抗体。
- 下記(a)の可変領域および/または(b)の可変領域を含んでなる、(1)から(7)のいずれかに記載のモノクローナル抗体;(a)N末端から順に、配列番号8のアミノ酸配列、配列番号9のアミノ酸配列および配列番号10のアミノ酸配列を含む可変領域、(b)N末端から順に、配列番号13のアミノ酸配列、配列番号14のアミノ酸配列および配列番号15のアミノ酸配列を含む可変領域。
- 配列番号7のアミノ酸配列からなる可変領域を含んでなる、請求項1から請求項8のいずれかに記載のモノクローナル抗体。
- 配列番号12のアミノ酸配列からなる可変領域を含んでなる、請求項1から請求項9のいずれかに記載のモノクローナル抗体。
- 受託番号がNITE BP-916であるハイブリドーマにより産生されるモノクローナル抗体。
- 請求項1から請求項11のいずれかに記載のモノクローナル抗体を産生するハイブリドーマ。
- 受託番号がNITE BP-916である、請求項12に記載のハイブリドーマ。
- 請求項1から請求項11のいずれかに記載のモノクローナル抗体を含んでなる、酸化低密度リポタンパク質検出用キット。
- 被検者から採取した生体試料に含まれる酸化低密度リポタンパク質を検出する方法であって、
請求項1から請求項11のいずれかに記載のモノクローナル抗体を、被検者から採取した生体試料に含まれる酸化低密度リポタンパク質に特異的に反応させて複合体を形成させる工程と、
前記複合体を検出する工程と
を有する、前記方法。 - 被検者から採取した生体試料に含まれる酸化低密度リポタンパク質を検出する方法であって、
請求項1から請求項11のいずれかに記載のモノクローナル抗体を担体に固着する工程と、
担体に固着した前記モノクローナル抗体を、被検者から採取した生体試料に含まれる酸化低密度リポタンパク質に特異的に反応させて複合体を形成させる工程と、
前記複合体を検出する工程と
を有する、前記方法。
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CN104914255A (zh) * | 2015-05-21 | 2015-09-16 | 北京协和洛克生物技术有限责任公司 | 一种检测样本中氧化低密度脂蛋白浓度的试剂盒及其制备方法 |
CN111855988A (zh) * | 2019-04-25 | 2020-10-30 | 常州博闻迪医药股份有限公司 | 一种氧化低密度脂蛋白荧光检测试剂盒及制备方法 |
WO2022049858A1 (ja) * | 2020-09-07 | 2022-03-10 | 国立大学法人北海道大学 | 非アルコール性脂肪性肝炎の診断方法 |
EP4080221A4 (en) * | 2020-02-04 | 2023-06-28 | Denka Company Limited | Method for assisting detection of non-alcoholic steatohepatitis |
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US11725002B2 (en) * | 2017-04-28 | 2023-08-15 | Ken-ichi YAMADA | Detection reagents and kit for identifying oxidized state and glycated state of low-density lipoproteins |
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CN116836940B (zh) * | 2023-07-27 | 2024-01-09 | 广州市进德生物科技有限公司 | 一种氧化低密度脂蛋白的单克隆抗体及分泌其单克隆抗体的杂交瘤细胞株与应用 |
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CN104914255A (zh) * | 2015-05-21 | 2015-09-16 | 北京协和洛克生物技术有限责任公司 | 一种检测样本中氧化低密度脂蛋白浓度的试剂盒及其制备方法 |
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EP4080221A4 (en) * | 2020-02-04 | 2023-06-28 | Denka Company Limited | Method for assisting detection of non-alcoholic steatohepatitis |
WO2022049858A1 (ja) * | 2020-09-07 | 2022-03-10 | 国立大学法人北海道大学 | 非アルコール性脂肪性肝炎の診断方法 |
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