JP2007106696A - Anti-quercetin monoclonal antibody, cell producing the same, method for detection of quercetin and detection reagent thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means that can exactly and simply carry out the detection of quercetin metabolic product. <P>SOLUTION: Monoclonal antibody-producing cell strain confronting to the quercetin metabolic product; the monoclonal anti-body that is produced by the cell strains and specifically reacting with the quercetin metabolic product; the detection agent including the monoclonal antibody; and a method for immunological detection of the quercetin in samples by using the detection reagent. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel monoclonal antibody against glucuronide conjugate which is a main in vivo metabolite of quercetin, a cell line capable of producing the antibody, a method for immunological detection of quercetin glycoside using the antibody, and The present invention relates to a detection reagent.

  Quercetin is a type of flavonoid that is widely contained in foods (mainly apples, onions, green tea, etc.) consumed in daily life, most of which is metabolized in vivo such as conjugates It exists in plasma as a metabolite. This is known to have an antioxidant action, an anti-inflammatory action, a lipid absorption inhibitory action, a brain cell transmission substance enhancing action, and the like. Therefore, the intake of quercetin can be expected to prevent lifestyle-related diseases (cancer, diabetes, arteriosclerosis, hyperlipidemia, etc.) and aging. In particular, as one of the main onset factors of these lifestyle-related diseases, the influence of excessive active oxygen (harmful reactive oxygen) in the living body can be considered. Quercetin (metabolite) can exert an action of removing such active oxygen by an antioxidant action.

  By measuring quercetin present in the form of a metabolite in blood or urine, the degree of antioxidant in the living body, for example, the ability to remove (eliminate) the active oxygen possessed by the living body can be known.

  Until now, high-performance liquid chromatography (HPLC) or gas chromatography connected with an electrochemical detector, a mass spectrometer, or the like has been used for detection (measurement) of quercetin in vivo.

  However, the above conventional methods require time, labor, skill, etc. for sample processing before analysis, and detection is difficult or detection results are not accurate due to interference of chromatography with coexisting substances. There was a case. Moreover, since these methods require relatively expensive equipment and columns, there is a disadvantage that a large amount of cost is required for their renewal.

  Accordingly, development of a technique capable of detecting quercetin, particularly quercetin present in vivo as a metabolite in vivo, in a more rapid, simple, accurate and accurate manner is desired in the art.

In order to develop the above technique, it is first considered to establish an antibody specific for a quercetin metabolite. If such an antibody is obtained, the quercetin metabolite in the biological sample can be easily identified and quantified by an immunodetection technique using the antibody. However, there is currently no known antibody that can specifically recognize quercetin metabolites.
Moon et al., “Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin.” Free Radic Biol Med., 2001, 30 (11), 1274-85.

  An object of this invention is to provide the detection technique of the quercetin metabolite requested | required in this industry. Another object of the present invention is to provide a monoclonal antibody against a quercetin metabolite that can be used in the detection technique and a cell line that produces the antibody.

  As a result of repeated research, the present inventor has established a hybridoma having the ability to produce a new monoclonal antibody capable of achieving the above object, and succeeded in obtaining a desired monoclonal antibody from the hybridoma. The present invention has been completed as a result of further research based on this success.

  The present invention provides the inventions described in Items 1 to 7 below.

  Item 1. An anti-quercetin monoclonal antibody that recognizes a glycoside including a glucuronide conjugate of quercetin and does not substantially cross-react with quercetin and its methyl conjugate and sulfate conjugate.

  Item 2. An anti-quercetin monoclonal antibody produced from a fusion cell of a mouse spleen cell immunized with an antigen having quercetin-3-glucuronide as a hapten and a syngeneic mouse myeloma cell.

  Item 3. A fusion cell line of a mouse spleen cell immunized with an antigen having quercetin-3-glucuronide as a hapten and a syngeneic mouse myeloma cell, which has an ability to produce an anti-quercetin monoclonal antibody.

  Item 4. An anti-quercetin monoclonal antibody that cultures the cell line of Item 3, recognizes glucuronide conjugates and glycosides of quercetin, and does not substantially cross-react with quercetin and its methyl conjugate and sulfate conjugate. A method for producing an anti-quercetin monoclonal antibody.

  Item 5. A detection reagent for immunological detection of a quercetin glycoside, comprising the anti-quercetin monoclonal antibody according to Item 1 or 2.

  Item 6. A quercetin glycoside in the sample, comprising a step of contacting the sample containing the quercetin glycoside with the monoclonal antibody according to Item 1 or 2 to generate an immune complex. A method of immunological detection.

  Item 7. The detection method according to Item 6, wherein the immunological detection of quercetin glycoside is performed by sandwich ELISA.

  In this specification, “substantially does not cross-react” with respect to a monoclonal antibody means, for example, that B / B0 at a concentration of 0.1-10 μM (measurement sensitivity range) is 0.8 or more in the competitive ELISA described in the Examples. Say.

  Further, in this specification, the term “detection” described as “immunological detection” or the like not only confirms the presence or absence of quercetin metabolites but also the presence of quercetin metabolites. It shall be used in a broad sense, including measuring (quantifying) the degree (amount) of.

  The present invention provides an anti-quercetin monoclonal antibody having a high binding activity to a quercetin metabolite and a high specificity thereto, a cell line capable of producing the antibody, and a method for producing them.

  By using the monoclonal antibody of the present invention, a glucuronide conjugate of quercetin, which is a main quercetin metabolite present in a sample, can be easily detected with high sensitivity and high accuracy. Moreover, this detection result is specific to a quercetin glucuronide conjugate that is free from errors due to contamination with other quercetin analogs such as methyl conjugates and sulfate conjugates. In addition, this specificity can clearly distinguish substances having a similar structure to quercetin such as isoflavones and catechins that can be ingested on a daily basis, and other antioxidant substances that normally exist in the living body. It can be sufficiently distinguished from vitamins and glutathione. Therefore, according to the use of the antibody of the present invention, quercetin in vivo can be easily and accurately detected. The present invention also provides a reagent and a detection method for immunological detection of quercetin using the monoclonal antibody of the present invention.

  In particular, the immunological detection method of the present invention has an advantage that enables the evaluation of quercetin metabolite (glucuronic acid conjugate) as an antioxidant in the body. That is, the glucuronide conjugate is one of the major quercetin metabolites in rat and human blood, and is a metabolite retaining the B-ring catechol structure that exerts strong antioxidant properties (Moon et al , Free Radic. Boil. Med, 30, 1274-1285, 2001), it is considered to be a metabolite that contributes most to blood antioxidant properties after quercetin intake. On the other hand, sulfate conjugates and methylates found in the blood also include those in which the B ring 3 'position is conjugated (Day et al., Free Radic. Res. 35, 941-952, 2001). Therefore, the contribution to these antioxidant properties is not so high. From this, it is considered that measuring quercetin glucuronide conjugate is extremely effective for evaluating the antioxidant property brought about by quercetin intake.

  The anti-quercetin monoclonal antibody of the present invention is also considered useful for basic research such as pharmacokinetic analysis of quercetin and analysis of antioxidant expression mechanism.

  Hereinafter, the anti-quercetin monoclonal antibody of the present invention will be described in accordance with its production procedure, and then an immunological detection method (measurement method) using the antibody of the present invention will be described in detail.

(1) The hapten antigen quercetin and its metabolite are low molecular compounds that are not themselves immunogenic. In the present invention, quercetin glucuronide conjugate, which is one of the main in vivo metabolites of quercetin, more specifically quercetin-3-glucuronide (hereinafter referred to as “Q3GA”) as a hapten, protein etc. A conjugate obtained by binding to an appropriate polymer substance (carrier) is used as an immunogen (hapten antigen).

  Examples of easily available and inexpensive carriers include carrier proteins such as bovine serum albumin, ovalbumin, and hemocyanin. Of these, keyhole limpet hemocyanin (KLH) is preferred. Such a carrier protein and quercetin are bound by using a general crosslinking agent such as maleimidobenzoyloxysuccinimide, 1-ethyl-3- (3-methylaminopropyl) carbodiimide, N-hydroxysuccinimide, glutaraldehyde and the like. And can be carried out according to a conventional method. More specifically, a desired hapten antigen can be obtained by first binding the cross-linking agent to a hapten in an appropriate buffer, and then binding a carrier protein to the resulting cross-linked product.

  In particular, since Q3GA has a free carboxyl group in its glucuronic acid moiety, the bond between this and the carrier protein can also directly peptide bond the amino group of the protein using the carboxyl group, Furthermore, an amino group can be bound to the carboxyl group using an appropriate binding reagent such as a carbodiimide reagent, and the carrier protein can be peptide-bonded via the amino group. In the present invention, a hapten antigen in which a carrier protein is bound using such a carbodiimide reagent is particularly suitable.

(2) Immunization Pretreatment The hapten antigen can be immunized by administering it to an appropriate mammal as an immunogen. Prior to this immunization, the hapten antigen can be mixed with a suitable adjuvant to enhance its immune response. Examples of adjuvants may be any form such as a water-in-oil emulsion (for example, incomplete Freund's adjuvant), a water-in-oil-in-water emulsion, or an oil-in-water emulsion. More specifically, the adjuvant includes aluminum hydroxide gel, silica adjuvant, powdered bentonite, tapioca adjuvant and the like. Furthermore, in addition to these, cells and cell walls such as BCG and Propionibacterium acnes, cell components such as trehalose dicholate (TDM); lipopolysaccharide (LPS) which is an endotoxin of Gram-negative bacteria, and lipid A fraction; β-glucan (polysaccharide); muramyl dipeptide (MDP); bestatin; synthetic compound such as levamisole; protein or peptide substance derived from biological components such as thymic hormone, thymic hormone humoral factor, and tuftsin; Complete Freund's adjuvant) can also be used as an adjuvant. These adjuvants can be easily obtained as commercial products. The adjuvant exhibits an effect of enhancing or suppressing the immune response depending on the administration route, dose, and administration timing of the immunogen. Further, depending on the type of adjuvant to be used, the antibodies obtained vary in blood antibody production against the immunogen, induction of cellular immunity, immunoglobulin class, and the like. Therefore, it is preferable to appropriately select an adjuvant according to the target immune response. The handling of the selected adjuvant, for example, the method of mixing with the hapten antigen, etc., can follow the methods known in the art for each adjuvant.

(3) Immunization A mammal can be immunized according to a conventional monoclonal antibody production technique.

  Here, the mammal is not particularly limited, but generally, a mouse, rat, cow, rabbit, goat, sheep, guinea pig and the like can be used. Preferred mammals are mice and rats, more preferably mice. These mammals can be selected in consideration of compatibility with plasmacytoma cells that are subsequently cell-fused for the production of the antibody of the present invention. Examples of mice include A / J strain, Balb / c strain, DBA / 2 strain, C57BL / 6 strain, C3H / He strain, SJL strain, NZB strain, and CBA / JNCrj strain. Among these, Balb / c strain mice show high antibody titers in serum after immunization, and according to their use, it is possible to obtain monoclonal antibodies with extremely high affinity with quercetin. It is known that blood antibody titer is related to the ease of producing specific hybridomas. In addition, Balb / c mice are commonly used for mass production of antibodies using ascites after the establishment of cell lines. Therefore, when large-scale production of such antibodies is desired, the use of Balb / c strain mice is preferred. The age of the experimental animal varies depending on the animal species to be used and is not particularly limited, but in the case of a mouse or rat, it is typically about 4 weeks to about 12 weeks, preferably about 6 to about 10 weeks, more preferably about 7 weeks old.

  Mammal immunization is performed according to methods known in the art. For example, a hapten antigen is dissolved (suspended) in physiological saline or a buffer solution according to a conventional method, and a solution mixed with an appropriate adjuvant as necessary is injected subcutaneously, intradermally, intravenously or intraperitoneally into a mammal. By administration. Since the immune response varies depending on the type and strain of mammal being immunized, the immunization schedule can be altered appropriately for the animal used. The administration of the immunogen is generally repeated several times after the first immunization. The booster immunization can be performed, for example, 2 weeks, 4 weeks, 6 weeks and 8 weeks after the first immunization. More specifically, for example, the immunogen is diluted with physiological saline-containing phosphate buffer (PBS), physiological saline or the like to an appropriate concentration, and optionally used in combination with the above-mentioned adjuvant to test animals every 2-14 days. For example, in the case of mice, the total dose of immunogen is preferably about 100-500 μg / mouse. More preferably, mice are used as test animals, and the immunogen is administered 4-5 times or more every 2 weeks, so that the total dose of the immunogen is about 100 μg / mouse or more.

  Thus, desired antibody-producing cells can be prepared in the mammalian body. As immune cells (plasma cells), splenocytes isolated about 3 days after the final administration are preferred.

(4) Confirmation of antibody production After immunization, blood is collected from mammals. Whether the obtained blood has binding activity to quercetin metabolites (hereinafter, this activity may be simply referred to as “quercetin binding activity”). It is confirmed whether the antibody with respect to quercetin (metabolite) is produced in the mammal body by assaying whether it is. Examples of suitable assays include enzyme immunoassay (ELISA, Immunochemistry, 8 , 871-874 (1971); Engvall, E., Meth. Enzymol., 70 , 419-439 (1980)), radioimmunoimmunization Examples include assay method (RIA) and fluorescent antibody method. In order to obtain an antibody having a particularly high quercetin-binding activity, it is desirable to select an antiserum exhibiting a high antibody titer by the above assay method or the like.

(5) A mammal in which production of an antibody having boost quercetin-binding activity has been confirmed can be further boosted (additional injection of immunogen) to enlarge the spleen. The amount of immunogen administered as a boost is preferably about 4 to 5 times the amount of immunogen initially administered, but can be appropriately increased or decreased based on this. Boosting is typically performed using an emulsion of immunogen and incomplete Freund's adjuvant. However, the immunogen administered in the final immunization (additional injection of immunogen several days before cell fusion) is preferably a pure product to which no adjuvant is added. The administration route may be any of subcutaneous, intradermal, intravenous and intraperitoneal.

(6) Cell fusion After the final immunization, spleen cells as plasma cells (immune cells) are removed from the immunized mammal and fused with cells derived from myeloma (plasmacytoma cells, myeloma cells).

  Since the proliferation ability of the fusion cell (hybridoma) depends on the type of myeloma cell to be used, it is preferable to use a myeloma cell having an excellent proliferation ability for cell fusion. The myeloma cells are preferably derived from a mammal of the same strain that is compatible with the mammal from which the immune cells to be fused are derived. Myeloma cells may be prepared fresh or commercially available. Examples of mouse myeloma cell lines include P3U1 (P3-X63-Ag8-U1), Sp2 / O Ag14, FO · 1, S194 / 5.XX0 BU.l, P3 / NS1 / 1 Ag4 1, and the like. Of these, P3U1 is preferred. Examples of rat-derived myeloma cell lines include 210.RCY3.Ag.1.2.3 and YB2 / 0.

  Cell fusion is performed according to methods known in the art (eg, the method of Koehler and Milstein (Koehler and Milstein, Nature, 256: 495 (1975)), the method of Kosbor et al. (Kosbor et al., (1983), Immunol. Today, 4: 72), Kotte et al. (Cote et al., (1983), Proc. Natl. Acad. Sci. USA., 80: 2026), Kore et al. (Cole et al., Monoclonal antibodies and cancer therapy, Alan, R. Liss Inc., New York, NY, pp. 77-96 (1985), etc.) As an example of a cell fusion method, for example, polyethylene glycol (PEG) is used as a fusion promoter. Examples thereof include a method, a method using Sendai virus (HVJ), a method using electric current, etc. Among these, a method using polyethylene glycol is preferable.

  The usage ratio of immune cells to myeloma cells is not different from that in the usual production method of this kind of antibody. For example, it is common to use immune cells about 1-10 times as much as myeloma cells. Examples of the medium for the fusion reaction include various media usually used for the growth of the above myeloma cells, such as RPMI-1640 media, DMEM media, MEM media, and other media commonly used for this kind of cell culture. Usually, it is better to remove serum supplements such as fetal calf serum (FCS) from these media. For fusion, a predetermined amount of the above immune cells and myeloma cells are mixed well in the above medium, and a PEG solution that has been pre-warmed to about 37 ° C., for example, having an average molecular weight of about 1000-6000 is usually added to about 30- It is done by adding and mixing at a concentration of 60W / V%. Thereafter, a desired hybridoma can be prepared by repeating the operation of adding an appropriate medium successively, centrifuging, and removing the supernatant.

(7) Screening and cloning of hybridomas Hybridomas are separated by culturing in a normal selection medium such as HAT medium (medium containing hypoxanthine, aminopterin and thymidine). The culture in the HAT medium may be performed for a time sufficient for the cells other than the target hybridoma (unhybridoma etc.) to die, usually several days to several weeks. The resulting hybridoma is subjected to screening and cloning using the desired antibody-producing ability as an index.

  The ability to produce a desired antibody as an index for screening is determined by assaying the specific binding activity of the antibody for the quercetin metabolite based on a method known in the art. More specifically, this assay can be performed according to the same method as described above for confirmation of antibody production, that is, the ELISA method, RIA method, fluorescent antibody method and the like. Among these, the ELISA method is preferable because the antibody can be easily detected with high sensitivity. Specific examples of this method will be described in detail in Examples below. In addition, the immune antigen can be used for this screening.

  Methods known in the art can be used for hybridoma cloning. Examples of cloning methods include the well-known limiting dilution method and soft agar method. Of these, the limiting dilution method is preferred because it is easy to operate, has a proven track record, and is highly reproducible.

  In order to efficiently select a desired cell from many hybridomas obtained by cell fusion, cell selection is preferably performed from the initial stage of cloning.

  The cloned hybridoma can be cultured in large quantities by in vivo and in vitro culture methods. The in vitro culture method can be carried out by culturing the hybridoma in an appropriate serum medium or serum-free medium, and the desired hybridoma is produced in the medium. According to this culture, a relatively high purity desired antibody can be obtained as a culture supernatant. In the in vivo culture method, the hybridoma is injected and inoculated into the abdominal cavity of a mammal that is compatible with the hybridoma, such as a mouse, and the desired antibody is recovered in large amounts as mouse ascites.

  Hybridomas producing the desired monoclonal antibodies can be subcultured in normal media and can be stored semi-permanently in liquid nitrogen.

  As a specific example of the hybridoma, a hybridoma named “14A2” obtained according to the method described in Examples described later can be exemplified.

(8) Purification of antibody The culture supernatant of antibody-producing hybridoma and ascites fluid from mice can be used as a crude antibody solution as they are. These can be purified by conventional methods, for example, by appropriately combining DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method and the like to obtain purified antibodies. . The purified antibody usually has a purity of about 90% or more, preferably about 95% or more, more preferably about 98% or more.

  The antibody of the present invention reacts with a quercetin metabolite, i.e., a glucuronide conjugate of quercetin, such as Q3GA, but does not substantially react with the aglycon quercetin and the methyl and sulfate conjugates of quercetin. It is characterized in that it has a physical property (specificity).

  Therefore, the antibody of the present invention is particularly useful as a detection reagent for immunological detection of quercetin present in the form of a metabolite in a living body. The present invention also provides a detection reagent using such an antibody of the present invention and a method for immunologically detecting quercetin (metabolite) in a sample using the reagent.

(9) Immunological detection method using the antibody of the present invention The immunological detection (measurement) method targeting quercetin (metabolite) includes, for example, enzyme immunoassay (EIA), enzyme immunometric assay (ELISA) ), Fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay, immunoblot, Western blot, and the like.

  In Western blotting, for example, a sample solution is subjected to acrylamide gel electrophoresis, transferred to a membrane, reacted with the antibody of the present invention, and a reaction product (immunocomplex) produced is detected using a labeled second antibody ( And measuring the amount of labeled second antibody bound to the complex).

  One preferred specific method for immunological detection using the antibody of the present invention is ELISA. This ELISA method can be performed according to a general competitive method, a sandwich method, or the like, and can also be performed in a liquid phase system or a solid phase system.

  An example of a preferred ELISA method is performed as follows. That is, first, a standard antigen (quercetin-Q3GA conjugate) is immobilized on an appropriate carrier and blocked. Next, the sample containing the quercetin metabolite desired to be detected and the antibody of the present invention are contacted with the above-mentioned immobilized labeled antigen, and the antibody-quercetin metabolite immune complex of the present invention and the antibody-standard antigen immune complex of the present invention are obtained. Generate competitively. The amount of the produced antibody-quercetin metabolite immune complex of the present invention can be measured, and the amount of quercetin metabolite in the sample can be determined from a calibration curve prepared in advance.

  When the reactivity between the antibody of the present invention and the standard antigen is known in advance by a calibration curve or the like, a sample containing a quercetin metabolite can be immobilized and used instead of the standard antigen immobilized above.

  In the above preferred ELISA method, the antibody of the present invention can also be used as the first antibody, and the second antibody against the first antibody can be labeled and used. In this case, the amount of the antibody-quercetin metabolite immune complex of the present invention can be easily determined by measuring the labeled amount of the labeled second antibody bound thereto. As a modification of the above method, the first antibody can be labeled with an enzyme, for example, without using the labeled second antibody. Furthermore, a method in which the first antibody is labeled with biotin and an enzyme is bound to avidin or streptavicin instead of the second antibody can be employed as a modification of the above method.

  According to the immunological detection method of the present invention, a quercetin metabolite can be specifically detected with high accuracy and high sensitivity. This detection and measurement result is useful for, for example, evaluation of the body's antioxidant level due to dietary quercetin, which can be used to know the ability of the living body to remove (eliminate) active oxygen, and in turn the life due to ingestion of quercetin. It is possible to obtain an effective index for predicting the preventive effect of habitual diseases (cancer, diabetes, arteriosclerosis, hyperlipidemia, etc.) and aging.

  In addition, since the antibody of the present invention can also recognize quercetin glycosides contained in plants, it can be applied to the measurement of the amount of quercetin contained in food ingredients such as vegetables. This makes it possible to evaluate the antioxidant titer per unit food.

  In carrying out the immunological detection method of the present invention, it is easy to use a kit (detection kit) containing a detection reagent containing the antibody of the present invention. In addition to the antibody of the present invention (detection reagent), such a kit may further include any other reagent components necessary for carrying out the detection (measurement). Examples thereof include standard antigens, labeled antibodies, assay buffers, chromogenic reagents, substrates, stabilizers and the like.

  Samples that can detect (including quantitative) quercetin by applying the immunodetection method of the present invention include blood (serum, plasma, etc.), biological samples such as urine, vegetables and other plants (edible), Of the extract.

  Hereinafter, a preferred embodiment of the method for immunologically detecting quercetin using the antibody of the present invention will be described in detail. In this embodiment, the antibody is included in the first antibody bound to the solid phase and the mobile phase. The kit of the present invention containing the second antibody used in the above is utilized. Here, as the first antibody bound to the solid phase, the antibody of the present invention, in particular, the monoclonal antibody produced by the cell line 14A2 shown in the Examples described later can be advantageously used. Further, the second antibody is not particularly limited to the above monoclonal antibody, and may be a polyclonal antibody or a monoclonal antibody as long as it has binding ability to a quercetin metabolite. These can be produced according to conventional methods. The second antibody is preferably a monoclonal antibody, particularly the same monoclonal antibody as the first antibody.

  These polyclonal and monoclonal antibodies having quercetin-binding activity can be produced, for example, according to the method described in detail in Example 1 below, and antiserum obtained by the method can be used as the polyclonal antibody.

  The second antibody can be arbitrarily labeled by a method known in the art. Examples of the label include enzyme labels, dye labels, magnetic labels, radioactive labels, labels with colored particles (gold colloid, latex, etc.), and the like.

  The kit of the present invention may also contain a specification describing the use of the antibody in a sandwich assay of quercetin metabolites, together with one or more containers containing the first antibody and the second antibody. In addition, the kit may contain suitable reagents, washing solutions, dilution buffers, etc. for detection of the label or for detection of positive and negative controls.

  According to one preferred embodiment, the second antibody is first reacted with a sample sample containing quercetin metabolite in a liquid phase to form a label-antibody-quercetin metabolite complex. Next, the reaction solution containing the complex is used as a mobile phase to react with the immobilized first antibody. As a result, the first antibody and the second antibody bind in a sandwich manner via the quercetin metabolite. Therefore, the label is immobilized on the solid phase via the quercetin metabolite only when the quercetin metabolite is present in the specimen sample.

  Furthermore, by using the antibody of the present invention, the localization of the quercetin metabolite in the human body can be observed, whereby the onset of diseases such as arteriosclerosis and the action of the quercetin metabolite on the arteriosclerosis action can be observed. Involvement can be examined. To observe the localization, an immunohistochemical staining method according to a conventional method (see, for example, Kawai et al., J Biol Chem. (2003) 278, 21040-9) should be performed on any tissue section such as a lesion. Can do this.

EXAMPLES Examples are given below to describe the present invention in more detail, but the present invention is not limited to the following examples.

  In addition, the enzyme immunoassay (ELISA method) used for evaluation of the antiserum, culture supernatant and monoclonal antibody obtained in the following examples was performed as follows.

Enzyme immunoassay (ELISA )
(A) Immunogen coating
An antigen solution was prepared by suspending Q3GA-HSA (human serum albumin) (manufactured by Sigma-Aldrich) in phosphate buffered saline (PBS) to a concentration of 0.001 mg / mL. 50 μL / well of the antigen solution was injected into each well of a microplate (polystyrene high-binding flat bottom # 442404, manufactured by NUNK), and stored overnight in saturated water vapor at room temperature. Immediately before the experiment, the excess antigen solution was removed with an aspirator.

(B) Blocking Into each well of the microplate prepared in (A) above, 200 μL / well of a 4% “Block Ace” (blocking agent, manufactured by Dainippon Pharmaceutical Co., Ltd.) aqueous solution was injected and left at 37 ° C. for 60 minutes. . Thereafter, the block ace solution was removed. When the subsequent experiment was not performed on the same day, it was stored in saturated steam at 4 ° C. in this state. Similar results can be obtained by using 1% BSA-PBS solution instead of Block Ace solution.

(C) Antibody reaction 100 μL / well of antibody solutions (antiserum, culture supernatant, purified antibody, etc.) diluted to various concentrations with 1% HSA-PBS were injected into the wells prepared in (B) above. After leaving at 37 ° C. for 1.5 hours, the remaining TPBS was removed by washing 3 times with PBS containing 0.05% Tween-20 (manufactured by Bio-Rad) (hereinafter abbreviated as “TPBS”).

(D) Reaction of the second antibody As the second antibody, a horseradish peroxidase-labeled anti-mouse IgG antibody (Chemicon international) diluted 5000 times was dissolved in a TPBS solution. 100 μL / well of the second antibody solution was injected into each well prepared in (C) and allowed to stand at 37 ° C. for 1 hour. TPBS was removed by washing 3 times with TPBS.

(E) Substrate reaction and termination Tetramethylbenzidine (TMB) coloring reagent (BD Bioscience) was injected at 100 μL / well. The color reaction was stopped by injecting 50 μL / well of 2N sulfuric acid.

(F) Measurement Using a microplate reader (Bio-Rad model 450, manufactured by Bio-Rad), the absorbance at 450 nm of the well prepared in (E) was measured.

  In this example, enzyme immunoassay was used as the immunoassay, but RIA method, fluorescent antibody method, etc. may also be used.

Preparation of immunogens Quercetin-3-glucuronide (Q3GA) was obtained by the method of Moon et al. (Moon, JH., Et al., "Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral Administration of quercetin ", Free Radical Biology and Medicine, 2001, June 1; 30 (1), 1271-1285). That is, in accordance with the Koenings-Knorr method (Knoenings and Knorr, Ber., 34, 957, 1901, Flowers, Carbohydr. Res. 18, 211-218, 1971), glucuronide obtained by introducing glucuronic acid into the hydroxyl group of quercetin is obtained. Among them, Q3GA having glucuronic acid bonded at the 3-position was purified using HPLC.

  The obtained Q3GA was used as a hapten, 11 μmol of which was dissolved in 200 μL of dimethylformamide (DMF), and 11 μmol of 1-ethyl-3- (3-methylaminopropyl) carbodiimide (EDC) (Pierce) and N -Hydroxysuccinamide (NHS) (Pierce) was added and reacted at room temperature for 24 hours. 100 μL of the reaction product obtained above was added to 360 μL each of HSA and keyhole limpet hemocyanin (KLH) (manufactured by Pierce) dissolved in PBS to a concentration of 10 mg / mL, and the mixture was stirred at room temperature for 4 hours. Reacted. After completion of the reaction, dialysis was performed against PBS for 2 days, and hapten-binding proteins as immunogens were collected. These are called Q3GA-HSA and Q3GA-KLH, respectively.

Immunization In this example, BALB / C strain mice (Blb / c, female, 6 weeks old) were used for immunization.

  Suspensions prepared by suspending each immunogen prepared above in Freund's complete adjuvant (FCA, Sigma-Aldrich) so that the dose of the immunogen at a single dose is 60 μg / mouse. The mice were injected intraperitoneally (first time). In addition, a suspension prepared by suspending each immunogen in Freund's incomplete adjuvant (FIA, Sigma-Aldrich) is used once every 2 weeks from the first administration. The mice were repeatedly injected 4-5 times (2-6 times) so that the concentration was 2 μg / mouse. After the final administration, a PBS solution was administered from the orbital vein so that the concentration was 50 μg / mouse to prepare immunized mice. Mouse antibody production in the immunization process was confirmed by the ELISA method.

Cell fusion
Three days after the orbital administration of the PBS solution, the spleen was removed from the immunized mouse, and the spleen cells were collected.

  Using polyethylene glycol having an average molecular weight of 1500, splenocytes and a mouse myeloma cell line (P3-X63-Ag8-U1) were prepared according to a conventional method (for example, see Nature, Vol. 256, pp495-497 (1975)). Hybridomas were obtained by cell fusion with Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) containing% fetal bovine serum (FBS).

Hybridomas were selectively grown in hypoxanthine / aminopterin / thymidine (HAT) medium using 96-well plates (Nunc). All cell cultures were performed in a CO ₂ incubator (CO ₂ concentration: 5% by volume, temperature: 37 ° C., humidity: 95%). In the following culture, unless otherwise indicated, the culture was performed under the same conditions as described above.

Cell culture and cloning The culture supernatant was collected from the hybridoma in which selective growth was confirmed. The culture supernatant was subjected to primary screening of the desired antibody by ELISA. 250 ng of Q3GA-HSA was used as the solid phase antigen. As the antibody solution, cell culture supernatant was used. A peroxidase-labeled anti-mouse IgG antibody was used as the second antibody.

  From the results according to the ELISA method, hybridomas that provided 14-well culture supernatants with confirmed antibody titers were selected, transferred to 24-well plates, and further cultured in HT medium containing 15% FCS. Secondary screening was performed in the same manner by ELISA, and one well in which the most prominent antibody production was observed was selected. The hybridoma in this well was cloned by limiting dilution, repeatedly screened, and when the same antibody titer was observed in all wells in which colonies were formed, this was judged as a clone. Thus, the desired monoclonal antibody-producing clone named “14A2” was obtained. Hereinafter, the monoclonal antibody produced by the 14A2 strain is referred to as “14A2 antibody”.

The present inventors suspended the selected cell line in 1 mL of a solution of FCS: dimethylsulfoxide = 9: 1 (volume ratio) at a concentration of 1 × 10 ⁷ cells / mL after centrifugation, − It is pre-frozen at 80 ° C, transferred to liquid nitrogen, and stored in a state that can be sold for long-term storage.

  To 50 mL of the culture supernatant, 15.65 g of ammonium sulfate was added little by little while stirring under ice cooling. After the addition of the whole amount, stirring was continued for another 5 minutes, and then left on ice for 1 hour. Centrifugation was carried out for 10 minutes at a rotation / minute, and the precipitated protein was dissolved in a small amount (about 1 mL) of PBS. To this, 1 mL of a saturated ammonium sulfate solution was added over several minutes and left on ice for 30 minutes. Again, the precipitated protein obtained by centrifugation at 10,000 rpm for 10 minutes was dissolved in a small amount of PBS. In the subsequent experiments, the ammonium sulfate fraction thus obtained was used as a 14A2 antibody (solution). This can be further purified by the following method.

Antibody Purification The selected 14A2 antibody culture supernatant is subjected to affinity chromatography using a protein A binding gel (Protein A Sepharose 4FF, Pharmacia), and the monoclonal antibody (14A2 antibody) is purified by the following procedure.

  The column packed with protein A binding gel is equilibrated with binding buffer (1.5 M glycine and 3 M NaCl, pH 8.9). The culture supernatant is diluted about 3 times with binding buffer and then applied to the equilibrated column. While monitoring the absorbance at 280 nm of the eluate from the column using an absorptiometer, the column was washed with a binding buffer until the elution of impurities was completed. After washing, elution buffer (100 mM citric acid, pH 4) is applied to the column (linear flow rate: about 20 cm / hour) to recover the IgG-containing eluate (monoclonal antibody solution). About the collect | recovered IgG containing eluate, the light absorbency of 280 nm is measured using an absorptiometer, and the density | concentration of an antibody is determined by converting the measured light absorbency by an extinction coefficient.

Antibody cross-reactivity
For the 14A2 antibody, the ELISA was performed using each dilution series of Q3GA-HSA and HSA, and the cross-reactivity (specific reactivity of the 14A2 antibody) between the 14A2 antibody and these protein antigens was evaluated. .

  More specifically, protein antigens diluted to various concentrations were immobilized on ELISA plates (Nunc). After blocking the uncoated part of the well with a blocking agent (“Block Ace”, manufactured by Dainippon Pharmaceutical Co., Ltd.), a certain amount of horseradish veroxidase-labeled anti-mouse IgG antibody and tetramethylbenzidine (TMB) coloring reagent BD Bioscience After that, the absorbance at 450 nm was measured with a microplate reader (Bio-Rad model 450).

  The results are shown in FIG. Figure 1 shows the absorbance at 450 nm (OD450 nm) on the vertical axis and the antibody diluted concentration (mg / mL) on the horizontal axis, and Q3GA-HSA at each concentration of 14A2 antibody (indicated by black circles in the figure) and 5 is a graph plotting results obtained by measuring the binding ability to HSA (indicated by white circles in the figure) by absorbance measurement.

  As is clear from the results shown in FIG. 1, the 14A2 antibody reacted strongly with Q3GA-HSA but did not react with HSA.

Inhibition against antibodies Next, the competitive inhibition of the 14A2 antibody (specificity of antibodies) by these competitors was examined by competitive ELISA using various quercetin analogs and related compounds as competitors.

  In the competitive ELISA method, various quercetin analogs and related compounds (competitors) diluted to various concentrations are added to the 14A2 antibody solution (according to the primary antibody dilution in the ELISA method), and the primary antibody is similarly prepared. The reaction was performed, and the degree to which each competitor inhibited the reactivity of 14A2 to solid phase antigen (Q3GA-HSA immobilized on ELISA plate (Nunc)) was evaluated.

  The following were tested as quercetin analogs and related compounds (competitors).

Quercetin
Q3GA (quercetin-3-glucuronide as hapten)
Q4'GA (Quercetin-4'-glucuronide, an isomer of Q3GA and one of the major metabolites found in the body, similar to Q3GA)
Isorhamnetin (isorhamnetin, 3-methylated form of quercetin)
Quercetin-3-O-sulfate (Quercetin-3-O-sulfate, sulfate conjugate of quercetin)
Rutin (Rutin, quercetin glycoside)
Hyperoside (Hyperoside, quercetin glycoside)
Q3G (isoquercitrin, quercetin glycoside)
(-)-Epicatechin
(-)-Epicatechin Gallate
(-)-Epigallocatechin
(-)-Epigallocatechin Gallate
α-tocopherol (vitamin E)
Ascorbic acid (Vitamin C)
GSH (glutathione)
GSSG (Glutathione)
β-carotene (carotenoids).

The results are shown in FIGS. Each figure shows B / B0 on the vertical axis, competitor concentration (competitor (μM or mM)) on the horizontal axis, and the inhibitory effect of each competitor's 14A2 antibody on the horizontal axis using commercially available graph software. It is the graph plotted as a logarithmic axis. Each curve in each figure uses the following competitors.
Curve (1) in Figure 2: Quercetin Curve (2) in Figure 2: Q3G
Curve (3) in Figure 2: Q4'GA
Curve (4) in Figure 2: isorhamnetin
Curve (5) in Figure 2: Quercetin-3-O-sulfate
Curve (1) in Figure 3: Quercetin Curve (2) in Figure 3: Q3G
Curve (3) in Figure 3: Rutin
Curve (4) in Figure 3: Hyperside
Curve (1) in Figure 4: (-)-Epicatechin
Curve (2) in Figure 4: (-)-Epicatechin Gallate
Curve (3) in Figure 4: (-)-Epigallocatechin
Curve (4) in Figure 4: (-)-Epigallocatechin Gallate
Curve (1) in Fig. 5: α-tocopherol
Curve (2) in Fig. 5: Ascorbic acid
Curve (3) in Figure 5: GSH
Curve (4) in Figure 5: GSSG
Curve (5) in Fig. 5: β-carotene
Here, B / B0 represents [ODs−ODc] / ODs (ODs: absorbance when no competitor is added and Odc: absorbance when the competitor is added).

  From the results shown in Figure 2-5, the following is clear.

  (1) The binding of 14A2 antibody is inhibited by Q3GA and Q4′GA, which is a 4′-position conjugate, and it can be seen that the inhibitory effect is concentration-dependent between approximately 200 nM and 50 μM. This shows that the 14A2 antibody shows cross-reactivity with these antibodies (see FIG. 2).

  (2) The reactivity of the 14A2 antibody is not inhibited by quercetin (aglycone), quercetin sulfate conjugate and methylated product in the above concentration range. Thus, it can be seen that the 14A2 antibody does not cross-react with these in a concentration range between 200 nM and 50 μM (see FIG. 2).

  (3) Quercetin glycosides are recognized by the 14A2 antibody because they inhibit the reactivity of the 14A2 antibody (see FIG. 3).

  (4) Catechins, vitamins, and glutathione do not inhibit the reactivity of the 14A2 antibody even at a high concentration of 0.25 mM (see FIG. 4).

  As described above, it was revealed that the monoclonal antibody of the present invention specifically recognizes quercetin to which a sugar chain is bound.

  (5) From the above, it is considered that the measurement sensitivity of quercetin glycoside in the competitive ELISA method is about 0.1 to 10 μM.

  Since the blood quercetin concentration after ingestion of dietary quercetin in vivo is usually several μM, it is considered that quantification of human blood quercetin is sufficiently possible by the ELISA method using the antibody of the present invention. .

Examination of tissue localization by immunostaining method Immunohistochemical staining using human atherosclerotic lesion tissue sections was performed as follows. Immunohistochemical staining was performed according to the method shown in Kawai et al., J Biol Chem. (2003) 278, 21040-9. That is, paraffin-embedded sections were deparaffinized with xylene and then hydrophilized by immersing in ethanol and PBS, and then derived from the same animal species as the secondary antibody animal species in the same manner as in the ELISA method. Blocking was performed with PBS containing serum (4 ° C., overnight), washed with PBS, and the primary antibody diluted with PBS containing 1% BSA was reacted at room temperature for 1 hour. After washing, an anti-mouse IgG biotinylated antibody (DAKO) was used as a secondary antibody and allowed to act at room temperature for 1 hour in the same manner. Further, after washing, an ABC (avidin-biotin complex) reagent (Vector) was allowed to act at room temperature for 50 minutes. After washing, a color reaction was carried out using ABC-AP kit (Vector).

  Macrophage staining was performed using 0.5 μg / mL CD68 antibody (Dako) as the primary antibody. For hematoxylin-eosin (H & E) staining, deparaffinized sections were treated with a hematoxylin solution for 10 minutes, washed with running water, and then treated with ammonia. After washing with running water, it was treated with eosin hydrochloride for 2 minutes. In the absorption test by coexisting with Q4'GA, Q4'GA was added to the primary antibody solution (14A2 antibody solution) so as to be 100 μM, and pretreated overnight at 4 ° C was used as the primary antibody solution for staining. . After staining, the sample was washed with PBS, immersed in ethanol and xylene in sequence, dehydrated, and sealed to obtain a permanent specimen.

  The obtained results are shown in FIG.

  In FIG. 6, A-D are photographs showing stained images of tested arteriosclerotic lesion tissue sections, and E-F are photographs showing stained images of normal arterial site tissue sections as controls. Each stained image was taken using a phase contrast microscope (Nikon Corp.) (magnification × 4). Each photograph A-F in the figure shows the following.

A: Stained image of lesion tissue section stained with hematoxylin-eosin (H & E),
B: Stained image of a lesion tissue section using a monoclonal antibody (Dako) that recognizes macrophage marker CD68 as a primary antibody,
C: For a lesion tissue section, a stained image obtained by performing the immunostaining method using 14A2 antibody as a primary antibody,
D: Stained image obtained by an absorption test (performed in the same manner as C above) in the presence of Q4'GA, which is an antibody recognition substance,
E: H & E stained image obtained in the same manner by using a normal tissue section instead of the lesion tissue section in A above, and
F: Stained image obtained in the same manner by using a normal tissue section instead of a lesion tissue section in C.

From the results shown in FIG. 6, the following is clear. That is,
(1) Staining consistent with the localization of arteriosclerotic lesions in which foamed macrophages accumulated in FIGS. 6A and 6B are accumulated is observed in staining using 14A2 antibody (see C in FIG. 6).

  (2) The 14A2 antibody has the property of binding significantly to Q3GA and Q4′GA glucuronide conjugates. In the staining image in the presence of Q4'GA, which is one of the recognition compounds of this antibody (see Fig. 6D), the staining image is consistent with the localization of the atherosclerotic lesion observed in Fig. 6C. Disappearance confirms the recognition specificity in this staining. In other words, in the presence of Q4'GA, which is the quercetin glucuronide, 14A2 antibody binds to the coexisting Q4'GA and staining of the lesion is inhibited, so quercetin glucuronide is specifically stained in this lesion. It is thought.

  (3) In addition, positive staining with 14A2 antibody is not observed in stained images of normal arterial sites where arteriosclerosis is not observed (E and F in FIG. 6). This indicates that quercetin metabolites accumulate in vascular injury sites where arteriosclerosis can develop in human blood vessels and may exhibit their functions including antioxidant properties.

  The antibody of the present invention specifically recognizes a quercetin metabolite such as a glucuronide conjugate, which is a main in vivo metabolite of quercetin, and has an antioxidant property derived from quercetin, for example, by quantification of quercetin in the body. Diagnosis is possible, and it can also be used for immunological evaluation of the pharmacokinetics of quercetin, for example, elucidation of the relationship of quercetin to the anti-atherosclerotic effect. The antibody of the present invention is also useful for quantification of quercetin (glycoside) present in the form of glycoside in plants as food.

3 is a graph showing the results of determining the reactivity between the antibody of the present invention (14A2 antibody) and an antigen according to an immunodetection method (ELISA method). 3 is a graph showing the results of determining the action of a competitor that inhibits the reactivity between the antibody of the present invention (14A2 antibody) and an antigen according to a competitive immunodetection method (ELISA method). 3 is a graph showing the results of determining the action of a competitor that inhibits the reactivity between the antibody of the present invention (14A2 antibody) and an antigen according to a competitive immunodetection method (ELISA method). 3 is a graph showing the results of determining the action of a competitor that inhibits the reactivity between the antibody of the present invention (14A2 antibody) and an antigen according to a competitive immunodetection method (ELISA method). 3 is a graph showing the results of determining the action of a competitor that inhibits the reactivity between the antibody of the present invention (14A2 antibody) and an antigen according to a competitive immunodetection method (ELISA method). 2 is an electron micrograph showing an immunohistochemically stained image of an arteriosclerotic lesion tissue section and a normal tissue section reacted with the antibody of the present invention (14A2 antibody) and the like.

Claims (7)

An anti-quercetin monoclonal antibody that recognizes a glycoside containing a glucuronide conjugate of quercetin and does not substantially cross-react with quercetin and its methyl conjugate and sulfate conjugate. An anti-quercetin monoclonal antibody produced from a fusion cell of a mouse spleen cell immunized with an antigen containing quercetin-3-glucuronide as a hapten and a syngeneic mouse myeloma cell. A fused cell line of a mouse spleen cell immunized with an antigen having quercetin-3-glucuronide as a hapten and a syngeneic mouse myeloma cell, which has an ability to produce an anti-quercetin monoclonal antibody. Culturing the cell line according to claim 3 and collecting an anti-quercetin monoclonal antibody that recognizes quercetin glucuronide conjugate and glycoside and does not substantially cross-react with quercetin and its methyl conjugate and sulfate conjugate. A method for producing an anti-quercetin monoclonal antibody. A detection reagent for immunological detection of quercetin glycoside, comprising the anti-quercetin monoclonal antibody according to claim 1 or 2. A method comprising the step of contacting a sample containing quercetin glycoside with the monoclonal antibody according to claim 1 or 2 to generate an immune complex, wherein the quercetin glycoside in the sample is immunologically characterized in that To detect automatically. 7. The detection method according to claim 6, wherein the immunological detection of quercetin glycoside is performed by sandwich ELISA.

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