JP2008267941A - Method for measuring molecular weight of hyaluronan, method for diagnosing disease, and kit for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily measuring the molecular weight of hyaluronan in a sample with high sensitivity. <P>SOLUTION: The method for measuring the molecular weight of hyaluronan includes steps of separating the sample containing hyaluronan by electrophoresis and transferring the sample to a transfer film; bringing hyaluronan transferred to the transfer film, into contact with hyaluronan bonding protein to form a complex of hyaluronan and hyaluronan binding protein; reacting a substrate with a chemiluminescent enzyme bound beforehand to the hyaluronan binding protein or a chemiluminescent enzyme directly or indirectly bound to the hyaluronan binding protein, to detect a complex by a chemiluminescence method. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for measuring the molecular weight of hyaluronan, a method for diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan, and a kit for these.

  Hyaluronan is one of the biological linear polymers in which many disaccharides consisting of N-acetylglucosamine and glucuronic acid are unit sugars (units), and these are linearly linked. The main component of extracellular matrix between tissue cells It is an ingredient. Hyaluronan has extremely high water retention, and in addition to its structural function as a component of the extracellular matrix, it maintains the hydrophilic environment of cells and maintains a barrier function that prevents it from drying out in the skin exposed to the outside air. It is known to have a function. It is known that these water retention functions depend on the molecular weight of hyaluronan (Non-patent Document 1: Lee et al.).

  In addition, it is known that hyaluronan has various physiological activities when the molecular weight is lowered. For example, it is known that medium to low molecular weight hyaluronan of tens of thousands of daltons induces inflammatory nuclear factor NF-κB and cytokines and enhances cell migration ability (Non-Patent Document 2: Noble PW). .). It is known that the oligosaccharide hyaluronan having a lower molecular weight has a function of promoting cell growth and inducing new blood vessels (Non-patent Document 3: Joddar B. et al.). Furthermore, as will be described later, it is known that there is a correlation between the molecular weight of hyaluronan and diseases such as rheumatism, arthropathy and cancer. Thus, the molecular weight of the hyaluronan function in vivo is deeply related, and techniques for measuring the molecular weight of hyaluronan are indispensable for the study of this function.

  However, conventionally, when measuring the molecular weight of hyaluronan in a living body, samples that can be used for the measurement have been limited to those containing abundant hyaluronan such as joint fluid and skin. The reason is as follows. That is, hyaluronan does not have a specific absorption wavelength or fluorescence wavelength like protein and nucleic acid, and hyaluronan has no antigenicity and thus cannot produce an antibody. For this reason, the conventional measurement methods used for measuring the molecular weight of proteins and nucleic acids such as column chromatography and electrophoresis cannot be applied to the molecular weight measurement of hyaluronan.

  In order to detect hyaluronan, for example, in column chromatography, a chemical colorimetric method is applied to each fractionated fraction, or a multiwell assay system using a hyaluronan-binding protein (also referred to as HABP) is used. It is possible to use. Alternatively, in order to detect hyaluronan, high-performance liquid chromatography can use a differential refractometer or UV (wavelength 210 nm).

  However, other than the method using HABP, there is no specificity for hyaluronan and the lower limit of detection is about several μg / mL. Therefore, in order to detect hyaluronan from a biological sample, hyaluronan must be highly purified. Moreover, although the multi-well assay system using HABP has high sensitivity and specificity, it requires processing by column chromatography at the previous stage. At this time, there is a limit to the column that can be used regardless of whether open column chromatography or high performance liquid column chromatography is used, and a polymer having a molecular weight exceeding 3 million which is present in a living body is currently available. It is difficult to analyze.

  As described above, in order to study the function of hyaluronan in each tissue organ in the living body, it is necessary to measure the molecular weight of hyaluronan in a small amount of sample. What is required for the measurement is a specific and high sensitivity to hyaluronan. Hong Gee Lee and Mary K. Cowman transferred hyaluronan to a positive charge membrane after separation by electrophoresis on an agarose gel, formed a complex of hyaluronan and HABP, and detected it by silver staining. A molecular weight measurement method was reported (Non-Patent Document 1: Lee et al.). This method has high specificity to hyaluronan and can measure high molecular weight hyaluronan as it exists in the living body. However, as described in this document, the detection method requires several μg of hyaluronan per sample as a sensitivity, and it cannot be said that measurement in a very small amount is possible in most biological samples. For example, since the hyaluronan concentration in blood is several hundred ng to several μg / mL, it is difficult to measure the molecular weight of hyaluronan by the method described in this document.

  Moreover, Lest et al. Has reported a method of using Azure A when measuring the molecular weight of glycosaminoglycan containing hyaluronan (Non-patent Document 4: Lest et al.). However, in this document, it is not intended to specifically detect each saccharide, and unlike other glycosaminoglycans, hyaluronan is smeared and the molecular weight cannot be measured.

Hong Gee Lee et al., ANALYTICAL BIOCHEMISTRY, 1994, 219, pp278-287 Noble PW., Matrix Biology, 2002, 21 (1), pp25-29 Joddar B., Ramamurthi A., Biomaterials, 2006, 27, pp2994-3004 Chris H. A. van de Lest et al., ANALYTICAL BIOCHEMISTRY, 1994, 221, pp356-361 Fraser & Laurent "HA turnover and metabolism" The Biology of Hyaluronan, Chiba Foundation Symposium, 1989, 143, pp41-59 Jiang D. et al, NATURE MEDHICINE, 2005, vol.11, no.11, pp1173-1179 Dahl LB et al, Ann.Rheum.Dis., 1985, 44, pp817-822 Wang PM, Lai-Fook SJ., HYALURONAN, Woodhead publishing Limited, 2002, vol 2, pp231-238 Yada T et al, HYALURONAN, Woodhead publishing Limited, 2002, vol 2, pp209-216 Holmess MW et al, Biochem J, 1988, 250, pp435-441 Tanimoto K., et al, J Dent Res, 2004, 83 (1), pp40-44 Hopwood J, Dorfman A, Pediatr Res, 1978, 12, pp52-56 Allerton SE et al, Cancer Res, 1970, 30, pp679-683 Horai T et al, Cancer, 1981, 48, pp2016-2021 Roboz J et al, Cancer Res, 1985, 45, pp1850-1854 Kawai T et al, Cancer, 1985, 56, pp567-574 Takeuchi J et al, Cancer Res, 1976, 36, pp2133-2139 Takeuchi J et al, Cancer, 1981, 47, pp2030-2035 Kojima J et al, Cancer Res, 1975, 35, pp542-547 Arnold F et al, Int J Clin Exp Microcirc, 1987, 5, pp381-386

  An object of this invention is to provide the method of measuring the molecular weight of hyaluronan in a sample simply and with high sensitivity. Another object of the present invention is to provide a method capable of diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan even from a small amount of sample, and a kit for use in the method.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have determined that the molecular weight of hyaluronan can be measured by using electrophoresis in the measurement of the molecular weight of a trace amount of hyaluronan in a sample and further using chemiluminescence as a detection method. Was able to be measured easily and with high sensitivity, and the present invention was completed. Furthermore, by measuring the hyaluronan molecular weight in a sample, it was found that a disease, disorder or condition associated with a change in hyaluronan molecular weight can be diagnosed or predicted from a minute sample of a subject, and the present invention has been completed.

That is, according to one aspect of the present invention, there is provided a method for measuring the molecular weight of hyaluronan,
Providing a sample comprising hyaluronan;
Providing a hyaluronan-binding protein having a specific binding ability to hyaluronan;
Separating the sample by electrophoresis and transferring it to a transfer film;
Contacting the hyaluronan transferred to the transfer membrane with the hyaluronan binding protein to form a complex of hyaluronan binding protein and hyaluronan;
Chemiluminescence by reacting the enzyme for chemiluminescence previously bound to the hyaluronan binding protein or the enzyme for chemiluminescence bound directly or indirectly to the hyaluronan binding protein with a substrate of the enzyme And detecting the complex by a method.

Also, according to another aspect of the present invention, a method for diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan comprising:
Measuring the molecular weight of hyaluronan contained in a sample obtained from a subject using the method,
Determining based on the measured hyaluronan molecular weight that the subject suffers from or is likely to suffer from the disease, disorder or condition.

  Furthermore, according to another aspect of the present invention, there is provided a kit for measuring the molecular weight of hyaluronan, comprising a hyaluronan-binding protein having a specific binding ability to hyaluronan and bound with a chemiluminescent enzyme. A kit is provided. Furthermore, according to another aspect of the present invention, there is provided a kit for measuring the molecular weight of hyaluronan, the hyaluronan binding protein having a specific binding ability to hyaluronan, and the direct or indirect connection with the hyaluronan binding protein. And a chemiluminescent enzyme capable of being specifically bound to each other. Furthermore, according to another aspect of the present invention, there is provided the above kit for diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan.

  According to the present invention, as will be described in detail below, the molecular weight of hyaluronan in a sample can be measured easily and with high sensitivity. Further, according to the present invention, it becomes possible to diagnose or predict a disease, disorder or condition associated with a change in the molecular weight of hyaluronan even from a small amount of sample. Moreover, according to this invention, the kit for using for these is provided.

  Embodiments of the present invention will be described below. However, the following description is for specifically explaining the present invention, and is not intended to limit the technical scope of the present invention.

As described above, according to one aspect of the present invention, there is provided a method for measuring the molecular weight of hyaluronan,
Providing a sample comprising hyaluronan;
Providing a hyaluronan-binding protein having a specific binding ability to hyaluronan;
Separating the sample by electrophoresis and transferring it to a transfer film;
Contacting the hyaluronan transferred to the transfer membrane with the hyaluronan binding protein to form a complex of hyaluronan binding protein and hyaluronan;
Chemiluminescence by reacting the enzyme for chemiluminescence previously bound to the hyaluronan binding protein or the enzyme for chemiluminescence bound directly or indirectly to the hyaluronan binding protein with a substrate of the enzyme And detecting the complex by a method.

  In the present invention, a sample containing hyaluronan is a measurement target. The sample to be measured is not particularly limited as long as it contains hyaluronan. In the present invention, hyaluronan is a polysaccharide containing D-glucuronic acid and DN-acetylglucosamine which are alternately glycosidically bonded, or a derivative thereof, or a salt thereof. In particular, in hyaluronan, the bond between position 1 of D-glucuronic acid and position 3 of DN-acetylglucosamine is a β1-3 bond, position 1 of DN-acetylglucosamine and 4 of D-glucuronic acid. The bond to the position is preferably a β1-4 bond.

The derivatives include those in which at least one of D-glucuronic acid and / or DN-acetylglucosamine in the constituent elements is an unsaturated sugar thereof. In particular, the derivatives include those in which the non-reducing terminal sugar in the constituent is the unsaturated sugar. In particular, the above derivatives include those in which D-glucuronic acid located at the non-reducing end is an unsaturated sugar in which the 4- and 5-position carbons are unsaturated. The derivatives include those in which at least one hydroxyl group in the constituent element is protected by a protecting group. The protecting group is preferably hydrolyzable, and may be, for example, an acyl group (RCO— (wherein R represents lower alkyl, preferably C _{1 to} C ₆ alkyl).

  In the present invention, hyaluronan may be a salt. Specifically, the salt includes alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, tri (n-butyl) amine salt, triethylamine salt, pyridine salt, amino acid salt and the like. Of the amine salt.

  In the present invention, the weight average molecular weight of hyaluronan is preferably 10,000 to 6,000,000, and more preferably 10,000 to 4,000,000. This is because the molecular weight distribution of hyaluronan distributed in the living body falls within this range, as will be described later. In addition, in this specification, a weight average molecular weight shall mean the conversion value from the intrinsic viscosity by a viscosity method.

  In the present invention, the sample to be measured may be a sample derived from a living body such as an animal or a plant, or may be an industrially produced sample. In particular, the sample to be measured is preferably an animal (preferably human) derived sample. This is because the method for measuring the molecular weight of hyaluronan according to the present invention can be applied to a diagnostic method and a prediction method as described later. When the sample to be measured is derived from an animal, it is not limited to a biomaterial containing abundant hyaluronan such as joint fluid or skin, and any biomaterial containing hyaluronan can be used as the sample. In particular, samples are serum, blood vessel, lymph, lymphatic vessel, lymph node, thymus, skin, cartilage, synovial fluid, synovial membrane, umbilical cord, cerebrospinal fluid, CSF, urine, kidney, uterus, liver, alveoli, alveoli It is preferably derived from at least one selected from fluid, lung surfactant, liver, ocular sclera and vitreous fluid, and malignant tumor tissue and inflamed tissue generated in these organs. As will be described later, according to the present invention, the molecular weight of hyaluronan can be measured with high sensitivity even for biomaterials that have been difficult to measure with conventional techniques.

  When using a sample derived from a living body, it is preferable to use a sample pretreated so as to be suitable for electrophoresis. First, when using a sample derived from a living body, it is preferable to degrade the protein in the sample by an enzyme treatment using a proteolytic enzyme. As a proteolytic enzyme, proteinase, actinase, collagenase and the like can be used. Furthermore, it is preferable to perform a degreasing treatment with a lipase, a polar or nonpolar organic solvent or the like as a pretreatment of the sample as necessary. As the polar or nonpolar organic solvent, acetone, hexane or the like can be used. It is preferable to heat for a short time after the enzyme treatment (for example, at 100 ° C. for 5 minutes). When insoluble matter is generated, it is preferable to remove it by centrifugation. The sample after these treatments, for example, is preferably added with 3 times the amount of ethanol and centrifuged, and the resulting precipitate is redissolved in distilled water or the like to obtain a sample to be measured.

  In the present invention, a hyaluronan-binding protein having a specific binding ability to hyaluronan is used. In the present invention, the hyaluronan-binding protein is not particularly limited as long as it has a specific binding ability to hyaluronan. That is, in the present invention, any protein having a hyaluronan-binding domain can be used as the hyaluronan-binding protein. Specifically, hyaluronan-binding proteins include aggrecan, versican, brevican, neurocan, LYVE-1, CD44, TSG-6, RHAMM, GHAP (Glial hyaluronate binding protein) and hyaluronectin (Hyaluronectin), type VI collagen, And proteins having these hyaluronan binding domains.

  As the hyaluronan-binding protein, a hyaluronan-binding protein to which a chemiluminescent enzyme is bound in advance (that is, a hyaluronan-binding protein labeled with a chemiluminescent enzyme) can be used. As will be described later, this enables specific detection of hyaluronan to be performed with fewer steps.

  The enzyme for chemiluminescence is not particularly limited as long as it can be used in a normal chemiluminescence method. For example, peroxidase (for example, horseradish peroxidase), alkaline phosphatase, or the like can be used as the chemiluminescent enzyme.

  When a hyaluronan binding protein to which a chemiluminescence enzyme is not bound in advance is used as the hyaluronan binding protein, it is necessary to specifically bind the chemiluminescence enzyme to the hyaluronan binding protein. In this case, a hyaluronan binding protein to which a labeling substance is bound is used as the hyaluronan binding protein, and a chemiluminescent enzyme to which a labeling substance binding substance having a specific binding ability to the labeling substance is bound. Further use is preferable. Thereby, the specific recognition of the hyaluronan binding protein by the chemiluminescent enzyme can be performed more efficiently.

  Biotin and avidin (such as streptavidin) can be used as a combination of the labeling substance and the labeling substance-binding substance used for the specific recognition. In particular, biotin and avidin are preferably used as a combination thereof. Specifically, biotinylated hyaluronan binding protein and avidin chemiluminescence enzyme are combined as a combination of a hyaluronan binding protein to which a labeling substance is bound and a chemiluminescence enzyme to which a labeling substance binding substance is bound. It is preferable to use it.

  As the hyaluronan binding protein, a protein to which neither the chemiluminescent enzyme nor the labeling substance is bound can be used. In this case, it is preferable to use a chemiluminescent enzyme to which a HABP binding substance having specific binding ability to HABP is bound. For example, a chemiluminescent enzyme to which an anti-HABP antibody is bound can be used as such a chemiluminescent enzyme. Alternatively, an HABP-binding substance having a specific binding ability to HABP is used, and a chemiluminescent enzyme to which the HABP-binding substance binding substance having a specific binding ability to the HABP-binding substance is further bound Can be used. For example, an anti-HABP antibody (primary antibody) and an antibody that specifically recognizes the primary antibody (secondary antibody) as such chemiluminescent enzyme to which the HABP binding substance and the HABP binding substance binding substance are bound ) Can be used.

  In the present invention, a chemiluminescent substrate is further used. As the chemiluminescent substrate, various commercially available substrates can be used depending on the chemiluminescent enzyme. For example, use TMB (3,3'5,5'-tetramethylbenzidine) for peroxidase and BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium) for alkaline phosphatase. Can do.

  The molecular weight measurement method according to the present invention includes a step of separating the sample by electrophoresis and transferring it to a transfer film.

  A sample for electrophoresis can be prepared by adding approximately the same volume of a glycerin aqueous solution (for example, 40% by volume) to a measurement target sample containing hyaluronan. According to the present invention, 0.5 ng of hyaluronan can be detected per lane. For this reason, in consideration of workability and the like, the amount of hyaluronan in the sample applied to the electrophoresis gel is preferably 1 to 50 ng / lane, more preferably 1 to 20 ng / lane. When a gel for electrophoresis having a general size is used, the amount of sample applied per lane is preferably 1 to 50 μL. For this reason, according to the present invention, the amount of hyaluronan contained in the sample to be measured is preferably 20 ng / mL to 50 μg / mL, more preferably 100 ng / mL to 20 μg / mL. In addition, before adding glycerol aqueous solution to a measuring object sample, it is also possible to concentrate a sample under reduced pressure as needed.

  In Non-Patent Document 1 (Lee et al.), The recommended lower limit of detection of hyaluronan is 4 μg, and in Non-Patent Document 4 (Lest et al.), The molecular weight of hyaluronan cannot be measured. On the other hand, surprisingly, according to the present invention, it was possible to measure several ng of hyaluronan, which has a sensitivity about 1000 times that of the prior art. As a result, the molecular weight of hyaluronan can be measured even for a specimen having a relatively low content of hyaluronan such as blood.

  For example, the hyaluronan content in each tissue of a rat frequently used as an experimental animal is as follows. Brain: 74 μg / g, Lung: 34 μg / g, Kidney: 30 μg / g, Intestinal tract: 44 μg / g, Liver: 4 μg / g, Lymph: 1-47 μg / g, Plasma: 0.02-0.03 μg / g (Non-patent literature) 5: Fraser & Laurent). With this hyaluronan content, in order to measure the hyaluronan molecular weight by the conventional measurement method, it is necessary to collect several tissues, and it was difficult to examine individual differences in the hyaluronan molecular weight. However, according to the present invention, it is possible to sufficiently measure a tissue piece from one individual. In addition, when using body fluids, a sufficient amount of hyaluronan could not be obtained unless the animals were sacrificed and collected by conventional methods. However, according to the present invention, the hyaluronan molecular weight can be measured with an amount that can be continuously collected while keeping one individual alive. Furthermore, if this is applied to humans, it becomes possible to measure the hyaluronan molecular weight with a tissue piece to the extent obtained by biopsy or the like.

  The concentration of hyaluronan can be measured by any method, but it is preferably measured by a method having high specificity to hyaluronan and excellent sensitivity. As a method for measuring the concentration of hyaluronan, for example, the method of measuring the hyaluronan content in a sample by inhibiting the binding of hyaluronan binding protein (HABP) to a multiwell plate on which hyaluronan is immobilized (HA / HABP binding antagonistic inhibition method) Can be mentioned.

  Further, in order to determine the molecular weight of hyaluronan contained in the sample to be measured, it is preferable to use a hyaluronan sample solution whose molecular weight is known in advance as a standard sample. The standard sample can be prepared in the same manner as the sample to be measured. The applied amount of the standard sample may be about 1 ng as the amount of hyaluronan per lane. If it is desired to quantify the concentration of hyaluronan in the sample to be measured, it is also possible to prepare several levels of the applied amount of the standard sample.

  For example, an agarose gel or a polyacrylamide gel can be used as the gel for electrophoresis, but the gel is not limited to these as long as it can be electrophoresed. In particular, the gel for electrophoresis is preferably an agarose gel. These gels can be prepared by a general method. For example, when an agarose gel is used as an electrophoresis gel, first, the agarose for electrophoresis is heated in an electrophoresis buffer (for example, 40 mM Tris, 5 mM sodium acetate, 0.9 mM EDTA, pH 8.0, also referred to as TAE). Dissolve. At this time, the concentration of agarose is preferably 0.5% by weight to 0.75% by weight. However, the concentration of agarose can be adjusted according to the expected hyaluronan molecular weight. Further, the agarose solution dissolved by heating is poured into the assembled gel plate, and the sample comb is inserted and cooled and hardened in a refrigerator or the like. The size of the gel is arbitrary, but can be, for example, 10 cm × 10 cm in length and width and 2 mm in thickness. Capillary electrophoresis can also be used.

  Separation by electrophoresis can be performed, for example, according to the following procedures and conditions. That is, a sample comb is extracted from the prepared gel, set in a migration tank, and an electrophoresis buffer such as TAE is injected. Further, an electrophoresis sample and a molecular weight standard sample are injected into the well. For example, the electrophoresis can be performed at a constant voltage and about 2.5 V per 1 cm length in the migration direction of the electrophoresis gel. In the case of this voltage, the electrophoresis time is preferably 10 cm in length, a gel concentration of 0.75% by weight for about 5 hours, and a gel concentration of 0.5% by weight for about 3.5 hours.

Transfer of hyaluronan separated by electrophoresis to a transfer film can be performed, for example, according to the following procedure and conditions. That is, the transfer film is immersed in an electrophoresis buffer such as TAE, and the gel after electrophoresis is placed thereon. Further, three or four filter papers (Whatman 3MM, etc.) cut into a gel size are placed in a buffer solution for electrophoresis such as TAE, and the gel and the transfer film are sandwiched between the top and bottom. This is set, for example, in a semi-dry type blotter. The transfer can be performed, for example, at 100 mA per 100 cm ^{2 of} gel for 2 hours. Since hyaluronan is an anionic polysaccharide, any film having positive ions such as a positive charge film (Amersham Bioscience, Hybond N +, etc.), a cellulose film or the like can be used as a transfer film.

  In addition, as a pretreatment for the transfer, the gel after electrophoresis is preferably treated with iron ions for the purpose of partially reducing the molecular weight of hyaluronan. Thereby, transfer to a transfer film can be performed more efficiently. The iron ion treatment can be performed, for example, according to the following procedure and conditions. That is, the gel after electrophoresis is immersed in an iron ion solution and shaken for 30 minutes. Thereafter, the iron ion solution is discarded, and the mixture is shaken twice in a buffer for electrophoresis for 15 minutes and washed. As an iron ion solution, a solution obtained by further adding 12.5 mM L-ascorbic acid and 250 mM ferrous sulfate to a buffer for electrophoresis such as TAE can be used.

  Further, as a post-treatment of the transfer, it is preferable to perform a blocking treatment on the transfer film before the treatment with the hyaluronan binding protein. Thereby, it can prevent that a hyaluronan binding protein adsorb | sucks nonspecifically to a transcription | transfer film | membrane. The blocking process can be performed, for example, according to the following procedure and conditions. That is, a blocking agent such as skim milk is dissolved in, for example, 50 mM Tris-HCl, 0.1 M NaCl, pH 7.5 (also referred to as TBS), and if necessary, blocking salmon DNA or the like heated at 100 ° C. for about 5 minutes immediately before use. The transferred transfer film is brought into contact with the blocking solution obtained by further adding the agent, and shaken overnight at room temperature. The membrane after blocking is preferably washed twice for 10 minutes each with, for example, TBS before treatment with the hyaluronan-binding protein.

  The molecular weight measurement method according to the present invention includes a step of bringing hyaluronan transferred to the transfer film into contact with the hyaluronan binding protein to form a complex of hyaluronan binding protein and hyaluronan. As described above, a hyaluronan-binding protein labeled with a chemiluminescent enzyme, a biotinylated hyaluronan-binding protein, or a hyaluronan-binding protein to which a chemiluminescent enzyme and a labeling substance are not bound is used as the hyaluronan-binding protein. be able to.

Specifically, for example, Block Ace ^R (Dainippon Sumitomo Pharma Co., Ltd.) in which biotinylated hyaluronan-binding protein (also referred to as b-HABP) is diluted with phosphate buffered saline (also referred to as PBS) or 1/10. To 0.1 μg / mL. Immerse the membrane in 10 mL b-HABP solution per piece and shake for 1 hour at room temperature.

  In one aspect, the molecular weight measurement method according to the present invention comprises detecting the complex by a chemiluminescence method by reacting a chemiluminescent enzyme previously bound to the hyaluronan-binding protein with a substrate of the enzyme. Including the steps of: That is, when the enzyme is previously bound to the hyaluronan-binding protein, a complex of the chemiluminescent enzyme, hyaluronan-binding protein, and hyaluronan is formed by treatment of the transfer membrane with the hyaluronan-binding protein. Thereby, hyaluronan can be detected by chemiluminescence.

  In another aspect, the molecular weight measurement method according to the present invention includes a chemiluminescence method in which a chemiluminescent enzyme directly or indirectly bound to the hyaluronan-binding protein is reacted with a substrate of the enzyme. Detecting the complex. That is, when the enzyme is not previously bound to the hyaluronan binding protein, a step of directly or indirectly binding the enzyme to a complex of hyaluronan binding protein and hyaluronan is required.

  In this case, for example, a hyaluronan binding protein to which a labeling substance is bound and a chemiluminescence enzyme to which a labeling substance binding substance is bound, such as biotinylated hyaluronan binding protein and avidin chemiluminescence enzyme. By using the combination, the enzyme can be directly bound to the complex of hyaluronan-binding protein and hyaluronan. Further, for example, an HABP binding substance and an HABP binding substance, such as an anti-HABP antibody (primary antibody) and an enzyme for chemiluminescence to which an antibody (secondary antibody) that specifically recognizes the primary antibody is bound. By using a combination with a binding substance, the enzyme can be indirectly bound to a complex of hyaluronan-binding protein and hyaluronan.

This can be done by sequentially bringing the transfer film into contact with a solution containing a substance that specifically binds. Specifically, for example, the chemiluminescent enzyme can be directly bound to the hyaluronan-binding protein by the following procedure and conditions. That is, the transfer film treated with b-HABP is washed three times for 15 minutes each with 0.05% Tween 20 or PBS (also referred to as PBS-Tween) added with a surfactant equivalent thereto. And 0.05 [mu] g / mL by dissolving avidin-conjugated horseradish peroxidase (the referred to as Avi-HRP) in the same solvent as b-HABP (PBS or 1/10 BlockAce ^R, etc.), at room temperature immersed membrane therein Shake for 1 hour.

  Detection by the chemiluminescent method can be performed by reacting a chemiluminescent enzyme with a substrate of the enzyme. Detection by the chemiluminescence method can be performed, for example, according to the following procedures and conditions. That is, the membrane is washed three times for 15 minutes each with PBS-Tween or the like. A transparent wrap is placed on a cassette for X-ray film, and a film is placed thereon. Chemiluminescence treatment is performed using a commercially available chemiluminescence reaction substrate solution (PIERCE Supersignal, etc.), and photography is performed using an X-ray film (Fuji Film, RX-U, etc.).

According to the molecular weight measurement method of the present invention, the molecular weight of hyaluronan in a sample derived from a living body can be measured with high sensitivity. That is, according to the present invention, it is possible to measure the molecular weight of hyaluronan even for a specimen that contains only a trace amount of hyaluronan, which has been difficult to measure, and the present invention is therefore capable of measuring changes in the molecular weight of hyaluronan. It can be applied to methods of diagnosing or predicting related diseases, disorders or conditions. That is, according to another aspect of the present invention, a method for diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan comprising:
Measuring the molecular weight of hyaluronan contained in a sample obtained from a subject using the method,
Determining based on the measured hyaluronan molecular weight that the subject suffers from or is likely to suffer from the disease, disorder or condition.

  Since the molecular weight of hyaluronan changes when suffering from a specific disease, disorder or condition, the increase or decrease in the molecular weight of hyaluronan in a sample derived from a living body is measured using the measurement method according to the present invention, and the measured value is used as an index. It is possible to diagnose or predict a disease, disorder or condition.

  The subject to be diagnosed or predicted is not particularly limited as long as it is an animal in which a relationship between a specific disease, disorder or condition and a change in the molecular weight of hyaluronan is observed, but a human is particularly preferable. Further, the sample obtained from the subject is not particularly limited as long as it is a biomaterial in which a change in the molecular weight of hyaluronan is recognized in a specific disease, disorder or condition. With respect to the obtained sample, the molecular weight of hyaluronan contained in the sample can be measured by the molecular weight measurement method described above.

  The disease, disorder or condition that can be diagnosed or predicted is not particularly limited as long as it is a disease, disorder or condition associated with a change in hyaluronan molecular weight. Specifically, diseases, disorders or conditions that can be diagnosed or predicted include aging, rheumatism, arthropathy, cancer, dermatitis, CPD (chronic obstructive respiratory disorder), pulmonary fibrosis, hepatitis, cirrhosis, nephritis. , Allergic enteritis, and cervical ripening at birth (Non-patent document 6: Jiang D. et al, Non-patent document 7: Dahl LB et al, Non-patent document 8: Wang PM et al, Non-patent Reference 9: Yada T et al)

  For example, it is known that the molecular weight of hyaluronan in the affected joint decreases due to rheumatism, and returns to a normal molecular weight as the symptoms recover (Non-Patent Document 10: Holmess MW et al). There is individual variation in the degree of molecular weight reduction, and this seems to reflect individual differences in the degree of symptoms. Therefore, treatment suitable for the individual can be selected by measuring the molecular weight. The joint fluid can hardly be collected by a healthy person, and it is difficult to determine the molecular weight of hyaluronan contained in the joint fluid of a healthy person. However, according to the present invention, the molecular weight of hyaluronan can be determined even from a very small amount of synovial fluid, and the molecular weight of hyaluronan contained in synovium and cartilage collected by biopsy can also be measured.

  In addition, it is known that the molecular weight of hyaluronan decreases as a result of having arthropathy (non-patent document 11: Tanimoto K. et al). This is largely due to the action of active oxygen at the site of inflammation in the affected area, and is therefore considered to reflect the degree of symptoms. Measuring the molecular weight, like rheumatism, is a way to know the degree of individual symptoms of the patient.

  Furthermore, when cancer is affected, the amount of hyaluronic acid produced increases inside and outside the cancer tissue. High molecular weight hyaluronan is known to promote the invasion of cancer cells into surrounding tissues, and low molecular weight hyaluronic acid is thought to promote neovascularization and contribute to the nutritional supplementation of cancer tissues ( Non-patent document 12: Hopwood J et al, Non-patent document 13: Allerton SE et al, Non-patent document 14: Horai T et al, Non-patent document 15: Roboz J et al, Non-patent document 16: Kawai T et al, Non-patent document 17: Takeuchi J et al, Non-patent document 18: Takeuchi J et al, Non-patent document 19: Kojima J et al, Non-patent document 20: Arnold F et al). Therefore, by measuring the molecular weight of local hyaluronan inside and outside the cancer tissue, the malignancy of the cancer tissue can be monitored. For example, compare the molecular weight of hyaluronan between the peripheral region of cancer and cancer tissue, and predict that hyaluronanase activity will increase if hyaluronan molecular weight is reduced in the peripheral region and that the resulting low molecular weight hyaluronan will promote angiogenesis. Can do. Further, if a large amount of high molecular weight hyaluronan is observed in the peripheral portion, it can be confirmed that the cancer cell has high activity of invasion into surrounding tissues.

  Furthermore, according to other aspects of the present invention, kits for measuring the molecular weight of hyaluronan and kits for diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan are provided. The kit according to the present invention comprises a reagent particularly suitable for the molecular weight measurement method or diagnosis / prediction method of hyaluronan. With this kit, the hyaluronan molecular weight in a sample can be measured easily and with high sensitivity, and diagnosis or prediction can be performed with a smaller amount of sample from a subject.

  In one embodiment, the kit according to the present invention has a specific binding ability to hyaluronan and a hyaluronan-binding protein to which a chemiluminescent enzyme is bound (for example, a hyaluronan binding labeled with a chemiluminescent enzyme). Sex protein).

  In another aspect, the kit according to the present invention includes (1) a hyaluronan-binding protein having a specific binding ability to hyaluronan, and (2) a specific binding directly or indirectly to the hyaluronan-binding protein. And a chemiluminescent enzyme that can be produced.

  In such an embodiment, it is preferable that the chemiluminescent enzyme is bound to a HABP binding substance having a specific binding ability to the hyaluronan binding protein. For example, the kit preferably includes (1) a hyaluronan binding protein and (2) a chemiluminescent enzyme to which an anti-HABP antibody is bound.

  Alternatively, in such an embodiment, the hyaluronan binding protein is bound to a labeling substance, and the chemiluminescent enzyme is bound to a labeling substance binding substance having a specific binding ability to the labeling substance. Preferably it is. For example, the kit preferably contains (1) a biotinylated hyaluronan binding protein and (2) an avidinized chemiluminescent enzyme.

  Alternatively, in this aspect, the kit further includes a HABP-binding substance having a specific binding ability with a hyaluronan-binding protein, and the chemiluminescent enzyme has a specific binding ability with a HABP-binding substance. It is preferable that a HABP-binding substance having a binding property is bound. For example, in the kit, (1) a hyaluronan binding protein, (2) an anti-HABP antibody (primary antibody), and (3) an antibody (secondary antibody) that specifically recognizes the primary antibody are bound. It preferably contains a chemiluminescent enzyme.

  The hyaluronan-binding protein, labeling substance, labeling substance-binding substance, chemiluminescent enzyme and the like in these kits can be selected in the same manner as the molecular weight measurement method according to the present invention described above.

  The kit according to the present invention preferably further comprises a substrate for the enzyme. Moreover, the kit concerning this invention can use the buffer solution normally required in a measurement, a molecular weight standard sample, etc. as the component, unless the desired effect of this invention is impaired as needed.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited by these description.

[1. Sample preparation]
As a biological sample, a mouse cervical lymph node fragment was used. The biological sample was subjected to enzyme treatment with a proteolytic enzyme (pronase). Further, degreasing treatment with acetone or the like was performed. After the enzyme treatment, the mixture was heated at 100 ° C. for 5 minutes, and insoluble matters were removed by centrifugation.

[2. Measurement of hyaluronan concentration]
Three times the amount of ethanol was added to the obtained enzyme-treated solution and centrifuged, and the precipitate was redissolved in distilled water to obtain a sample to be measured. The concentration of hyaluronan contained in this solution was 1.4 to 9.8 μg / mL.

[3. Preparation of electrophoresis sample]
Based on the obtained hyaluronan concentration, the same volume of 40% glycerin was added to 0.5 to 3.7 μL of the enzyme treatment solution (equivalent to about 5 ng of hyaluronan) to prepare a sample for electrophoresis.

[4. Preparation of gel for electrophoresis]
An appropriate amount of agarose for electrophoresis was taken and dissolved by heating in TAE. The agarose concentration was 0.5%. The agarose solution was poured into the assembled gel plate, and a sample comb was inserted and cooled and hardened in a refrigerator or the like. The size of the gel was 10 cm × 10 cm in length and width, and the thickness was 2 mm.

[5. Electrophoresis]
A sample comb of the obtained gel was extracted and set in an electrophoresis tank to inject TAE. The prepared electrophoresis sample and molecular weight standard sample were injected into the wells. The electrophoresis was performed at a constant voltage and 2.5 V per 1 cm length in the migration direction of the electrophoresis gel. The electrophoresis time was 3.5 hours.

[6. Iron ion treatment]
The gel after electrophoresis was immersed in TAE containing 12.5 mM L-ascorbic acid and 250 mM ferrous sulfate, and shaken for 30 minutes. Thereafter, the iron ion solution was discarded, and it was newly shaken in TAE twice for 15 minutes and washed.

[7. Transfer to positive charge film]
A positive charge membrane (Amersham Bioscience Hybond N +) was immersed in TAE, and a gel after iron ion treatment was placed thereon. Further, three or four filter papers (Whatman 3MM) cut into a gel size were stacked and dipped in TAE, and the gel and the membrane were sandwiched between the top and bottom. This was set in a semi-dry type blotter. The transfer was performed at 100 mA per 100 cm ^{2 of} gel for 2 hours.

[8. blocking]
Skimmed milk was dissolved in TBS to 10%, salmon DNA heated at 100 ° C. for 5 minutes just before use was added to 0.2 mg / mL, and the transferred membrane was immersed in this. Shake overnight at room temperature.

[9. HABP processing]
The blocked membrane was washed twice with TBS for 10 minutes each. Biotinylated hyaluronan binding protein (Seikagaku Corporation) was dissolved in PBS to 0.1 μg / mL. The membrane was immersed in 10 mL of biotinylated HABP solution per plate and shaken for 1 hour at room temperature.

[10. Avidin-HRP treatment]
The membrane was washed three times for 15 minutes each with PBS supplemented with 0.05% Tween20. Avidin-conjugated horseradish peroxidase (Invitrogen) was dissolved in PBS to 0.05 μg / mL, and the membrane was immersed in this and shaken at room temperature for 1 hour.

[11. Chemiluminescence treatment]
The membrane is washed 3 times for 15 minutes each with PBS-Tween. A transparent wrap was placed on a cassette for X-ray film, and a film was placed thereon. Chemiluminescence treatment was performed using a commercially available chemiluminescent reaction substrate solution (SuperSignal, PIERCE), and photography was performed using an X-ray film (Fuji Film, RX-U).

[result]
FIG. 1 shows the results of measuring the molecular weight of hyaluronan contained in mouse cervical lymph nodes by the molecular weight measurement method according to the present invention. As shown in FIG. 1, it was possible to measure that the hyaluronan molecular weight of the cervical lymph node taken from five mice was about 800,000 from a sample containing about 5 ng of hyaluronan.

The result of having measured the molecular weight of the hyaluronan contained in a mouse cervical lymph node by the molecular weight measuring method according to the present invention is shown.

Claims (10)

A method for measuring the molecular weight of hyaluronan,
Providing a sample comprising hyaluronan;
Providing a hyaluronan-binding protein having a specific binding ability to hyaluronan;
Separating the sample by electrophoresis and transferring it to a transfer film;
Contacting the hyaluronan transferred to the transfer membrane with the hyaluronan binding protein to form a complex of hyaluronan binding protein and hyaluronan;
Chemiluminescence by reacting the enzyme for chemiluminescence previously bound to the hyaluronan binding protein or the enzyme for chemiluminescence bound directly or indirectly to the hyaluronan binding protein with a substrate of the enzyme Detecting the complex by a method.   2. The method of claim 1, wherein the enzyme is pre-bound to the hyaluronan binding protein.   The method of claim 1, wherein the enzyme is not pre-bound to the hyaluronan binding protein and the detecting comprises binding the enzyme directly or indirectly to the complex. A method of diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan comprising:
Measuring the molecular weight of hyaluronan contained in a sample obtained from a subject using the method according to claim 1;
Determining based on the measured hyaluronan molecular weight that the subject is suffering from or likely to suffer from the disease, disorder or condition.   The disease, disorder or condition is aging, rheumatism, arthropathy, cancer, dermatitis, CPD (chronic obstructive respiratory disorder), pulmonary fibrosis, hepatitis, cirrhosis, nephritis, allergic enteritis, and cervical at birth The method according to claim 4, which is selected from the group consisting of partial ripening. A kit for measuring the molecular weight of hyaluronan,
A kit comprising a hyaluronan-binding protein having a specific binding ability to hyaluronan and bound with a chemiluminescent enzyme. A kit for measuring the molecular weight of hyaluronan,
A hyaluronan-binding protein having a specific binding ability to hyaluronan;
A kit comprising a chemiluminescent enzyme capable of specifically binding directly or indirectly to the hyaluronan-binding protein.   The kit according to claim 6 or 7, further comprising a substrate for the enzyme.   The kit according to any one of claims 6 to 8, for diagnosing or predicting a disease, disorder or condition associated with a change in the molecular weight of hyaluronan.   The disease, disorder or condition is aging, rheumatism, arthropathy, cancer, dermatitis, CPD (chronic obstructive respiratory disorder), pulmonary fibrosis, hepatitis, cirrhosis, nephritis, allergic enteritis, and cervical at birth The kit according to any one of claims 6 to 9, which is selected from the group consisting of partial ripening.