JP2007078705A - Method and kit for quantitation of heparan sulfate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a quantitation method, capable of specifying diabetic patients who although not affected by diabetic early-stage nephropathy by classification by the amount of albumin in a urine specimen, but shifting to diabetic early-stage nephropathy or diabetic patients who are affected by diabetic early-stage nephropathy and shifting diabetic nephropathy, at as an early a stage as possible. <P>SOLUTION: In a heparan sulfate quantitation method, a competing antigen-antibody reaction, between a labeled heparan sulfate and a heparan sulfate in a specimen, is made to occur to an anti-heparan sulfate antibody fixed to a solid carrier, and the labeled heparan sulfate bonded to the anti-heparan sulfate antibody in the solid carrier is detected. In a method for quantifying a heparan sulfate in a specimen, a competing antigen-antibody reaction between a heparan sulfate made to fix to a solid carrier or particulate solid carriers, and a heparan sulfate in a specimen is made to occur in an anti-heparan sulfate antibody, and the anti-heparan sulfate antibody bonded to the heparan sulfate on the solid carrier or aggregated particulate solid carriers are detected. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a means for quantifying heparan sulfate. The present invention is useful in grasping the pathological condition of diabetic nephropathy.

  Diabetes is known to cause a number of complications, and one of these fatal complications is “diabetic nephropathy” that causes severe renal failure. . In healthy kidneys, the negative charge in the heparan sulfate sugar chain of heparan sulfate proteoglycan present in the glomerular basement membrane functions as a “charge barrier” for proteins in the blood, thereby causing glomeruli such as proteins. It is protected against deposition and leakage into urine (Proc. Natl. Acad. Sci. USA 76: 1303, 1979).

  However, in diabetic nephropathy, the glomerular basement membrane is often damaged and no longer serves as a “charge barrier”. Conventionally, as a diagnostic method for diabetic nephropathy, albumin leaked through the `` charge barrier '' and leaked into the urine was calibrated, and the calibrated value was compared with the normal value of healthy individuals to identify diabetic early nephropathy. A method has been used.

  Further, as a method for measuring heparan sulfate, a method using anti-heparan sulfate antibody JM403 has been known. However, it is known that this anti-heparan sulfate antibody has specificity to hyaluronic acid in addition to heparan sulfate. (Kidney International, 41 (1992) pp 115-123), because of the lack of accuracy in the determination of heparan sulfate, it is not practical as a specific method for determining heparan sulfate alone. The use was impossible.

  Diabetic patients have a high probability of suffering from diabetic nephropathy, and depending on the amount of albumin in urine, (1) diabetes (high blood glucose level / creatinine correction (CR) urinary albumin amount less than 12 mg / gCR: hereinafter also referred to as DM1), (2) Diabetic early nephropathy (high blood glucose level / creatinine corrected urinary albumin amount 12 mg / gCR to 200 mg / gCR: hereinafter also referred to as DM2), (3) diabetic nephropathy (high blood glucose level / creatinine corrected) Urinary albumin amount 200 mg / g CR or more: hereinafter also referred to as DM3).

  In the DM1 stage, kidney abnormalities are not detected, and in the DM2 stage, there are cases where the kidneys have mild abnormalities but the disease is restored to the DM1 state by appropriate treatment. However, at the stage of DM3, the kidney has serious damage, and even if appropriate treatment is performed, there is almost no recovery from the disease state. Thus, since the prognosis when diabetic nephropathy develops in earnest is extremely poor, it is necessary to diagnose the transition to diabetic nephropathy as early as possible.

  However, the amount of albumin in a urine sample, which has been conventionally used as an index for diagnosis of diabetic nephropathy, varies even in healthy individuals, and from this amount of albumin alone, diabetes (DM1) to diabetic early nephropathy (DM2), Furthermore, it is difficult to accurately grasp the pathology of diabetic patients who are shifting to diabetic nephropathy (DM3).

  In addition, even if appropriate treatment is started at the time when a definitive diagnosis of diabetic early nephropathy or diabetic nephropathy is made from the change in the amount of albunmin in the urine sample, diabetic nephropathy is in the DM3 stage. Most cases have progressed, the therapeutic effect does not appear, and there are many cases that progress to severe nephropathy and further renal failure.

  Therefore, the problem to be solved by the present invention is not diabetic early nephropathy in the classification according to the amount of albumin in the urine sample, but is a diabetic patient who is shifting to diabetic early nephropathy, or early diabetic nephropathy, The purpose is to find a means for identifying a diabetic patient who is moving to diabetic nephropathy as early as possible as a quantitative means.

    In order to solve the above-mentioned problems, the present inventor focused on the metabolic abnormality of heparan sulfate existing in the basement membrane prior to the structural change of the glomeruli of the kidney in diabetic nephropathy. We conducted an intensive study. As a result, in the urine of diabetic nephropathy patients, heparan sulfate is quantitatively and / or qualitatively changed as this nephropathy progresses, and this change is performed by performing a specific quantitative method. The present invention has been completed by finding that it can be detected. In addition, “quantitative and / or qualitative change of heparan sulfate” means, for example, a change in the amount of specific heparan sulfate, molecular weight Examples thereof include a change, a change in the amount of sulfate group, or a change in the amount of heparan sulfate having a sulfate group at a specific site.

  That is, the present invention provides the following (1) to (3) heparan sulfate quantification method (hereinafter also referred to as the present invention quantification method) and a quantification kit using the method.

(1) A labeled heparan sulfate antibody and a heparan sulfate in a specimen are competitively antigen-antibody reacted with an anti-heparan sulfate antibody fixed on a solid phase carrier, and the label is bound to the anti-heparan sulfate antibody on the solid phase carrier. A method for quantifying heparan sulfate, wherein heparan sulfate is detected.

(2) Anti-heparan sulfate antibody bound to heparan sulfate on the solid phase carrier by competitively reacting heparan sulfate immobilized on the solid phase carrier and heparan sulfate in the sample with an antigen-antibody reaction against the anti-heparan sulfate antibody A method for quantifying heparan sulfate in a specimen, wherein the antibody is detected.

(3) Anti-heparan sulfate antibody is subjected to an antigen-antibody reaction competitively with heparan sulfate immobilized on a fine particle solid phase carrier and heparan sulfate in a specimen to aggregate the solid phase carrier. A method for quantifying heparan sulfate in a specimen by detecting the degree of the above.

  In the quantification method of the present invention, “quantification” means, for example, measurement and identification of the amount of specific heparan sulfate in which any residue of a constituent sugar of heparan sulfate has undergone some modification. It is.

  The quantification method of the present invention uses an antigen-antibody reaction between heparan sulfate and an anti-heparan sulfate antibody, particularly in a sample such as a urine sample or a blood sample, and thus is easy to operate. It is possible to screen. In addition, since it is possible to quantify the measured value even for a large number of specimens, heparan sulfate can be accurately quantified by using this heparan sulfate quantification method.

  The “urine sample” means urine or raw urine or a liquid component obtained by a process for removing blood cell components from the urine, but in the present invention, it is preferable to use urine as collected as the urine sample. . In addition, the term “detection of heparan sulfate” includes as a technical concept the determination of heparan sulfate in a specimen unless otherwise specified.

  According to the present invention, means for identifying a diabetic patient who is transitioning to diabetic nephropathy as early as possible, that is, a quantitative method for accurately quantifying heparan sulfate in a specimen, and a quantitative kit for performing the quantitative method Is provided.

Embodiments of the present invention will be described below.

The quantification method of the present invention is a method for quantifying heparan sulfate shown in the following (1) to (3).
(1) A labeled heparan sulfate antibody and a heparan sulfate in a specimen are competitively antigen-antibody reacted with an anti-heparan sulfate antibody fixed on a solid phase carrier, and the label is bound to the anti-heparan sulfate antibody on the solid phase carrier. A method for quantifying heparan sulfate, wherein heparan sulfate is detected.

(2) Anti-heparan sulfate antibody bound to heparan sulfate on the solid phase carrier by competitively reacting heparan sulfate immobilized on the solid phase carrier and heparan sulfate in the sample with an antigen-antibody reaction against the anti-heparan sulfate antibody A method for quantifying heparan sulfate in a specimen, wherein the antibody is detected.

(3) Anti-heparan sulfate antibody is subjected to an antigen-antibody reaction competitively with heparan sulfate immobilized on a fine particle solid phase carrier and heparan sulfate in a specimen to aggregate the solid phase carrier. A method for quantifying heparan sulfate in a specimen by detecting the degree of the above.

  The heparan sulfate in the present invention is a cell membrane or basement membrane in the state of proteoglycan bound to a protein in the lung, liver, kidney, brain, spleen, aorta, etc. in mammals (also referred to as “heparan sulfate proteoglycan” in this specification). Is a sulfated polysaccharide. The basic sugar chain structure has a repetitive structure of D-glucosamine and hexuronic acid (D-glucuronic acid and L-iduronic acid) common to heparin, the N-position sulfate group, the 6-position sulfate group and / or D-glucosamine. Alternatively, although it is a substitution product of the sulfate group at the 2-position of hexuronic acid, the content of sulfate group, L-iduronic acid, and N-sulfated glucosamine is low and anticoagulant activity is extremely low compared to heparin.

  In addition, as long as the antibody used in the quantification method of the present invention is an anti-heparan sulfate antibody capable of recognizing heparan sulfate, regardless of whether it is a monoclonal antibody or a polyclonal antibody, heparan sulfate in a sample should be quantified. However, it is preferable to use an antibody that has specificity for heparan sulfate and does not show crossover property for hyaluronic acid. In addition, in order to obtain a more accurate result, it has specificity for a specific antigenic determinant in heparan sulfate (the antibody can recognize a specific antigenic determinant and trigger an antigen-antibody reaction). It is preferable to use a monoclonal antibody.

  A monoclonal antibody can be produced by employing a generally known method for selecting a desired antibody-producing clone from a hybridoma of an immune cell using heparan sulfate as an immunogen. Thus, the quantification method of the present invention is provided.

  In the progression to diabetic nephropathy that can be detected by applying the quantification method of the present invention, the glucosamine residue containing the N-sulfate group of heparan sulfate present in the glomerular basement membrane has a particularly strong tendency to decrease ( Diabetologia.38,161-172 (1995)). Therefore, the anti-heparan sulfate antibody used in the quantification method of the present invention is preferably an antibody having at least a glucosamine residue containing the N-sulfate group of this heparan sulfate as an antigenic determinant.

  In particular, when a monoclonal antibody is used as the anti-heparan sulfate antibody, it is particularly preferable to select a monoclonal antibody having a glucosamine residue containing the N-sulfate group of heparan sulfate as an antigenic determinant. Examples of such monoclonal antibodies include anti-heparan sulfate antibody 10E4 and HepSS-1 (both manufactured by Seikagaku Corporation).

  For example, in the case where a monoclonal antibody having a glucosamine residue containing an N-sulfate group of heparan sulfate, such as these anti-heparan sulfate antibodies 10E4, as an antigenic determinant is used as one of the quantification means of heparan sulfate, The boundary value is preferably set to 3 EU / mg CR or more, 40 EU / mg CR or less, preferably 5 EU / mg CR or more, and 30 EU / mg CR or less (meaning of EU described here is described in Examples). .

  As an aspect of the quantification method of the present invention, for example, (1) an antigen-antibody reaction between an anti-heparan sulfate antibody fixed to a solid phase carrier (described later), labeled heparan sulfate and heparan sulfate in a sample is performed. A method used as its principle, (2) a method using as its principle an antigen-antibody reaction between heparan sulfate immobilized on a solid phase carrier and heparan sulfate and anti-heparan sulfate antibody in a specimen, and (3) heparan By contacting the specimen with a particulate solid phase carrier (described later) to which sulfuric acid is fixed, and further bringing the anti-heparan sulfate antibody into contact with this particulate solid phase carrier to aggregate the particulate solid phase carriers, A method for detecting heparan sulfate in a specimen is preferred and preferred.

  Each element [for example, solid phase carrier (including particulate solid phase carrier), labeled heparan sulfate, anti-heparan sulfate antibody, etc.] that can be used in the quantification method of the present invention using these antigen-antibody reactions as its principle is: The specific detection method can be appropriately selected depending on the specific detection method, and is not particularly limited. However, typical examples of each element are specifically exemplified here.

  Examples of the solid phase carrier to which the anti-heparan sulfate antibody or heparan sulfate is fixed include, for example, a microplate, a bead, a tube, a membrane, a gel, a particulate solid phase carrier [for example, gelatin particles, kaolin particles, synthetic polymer particles (latex particles, etc.) Among these solid phase carriers, microplates, beads, tubes, or particulate solid phase carriers are preferably used. In consideration of accurate quantification and convenience in carrying out the measurement, it is preferable to use a microplate as a solid phase carrier.

  For example, as a method for detecting heparan sulfate using an antigen-antibody reaction using a microplate to which an anti-heparan sulfate antibody is immobilized, the following are described: “1) Heparan sulfate labeled with a specimen on a microplate to which an anti-heparan sulfate antibody is immobilized. A competitive antigen-antibody reaction that detects heparan sulfate in a sample using binding of heparan sulfate in a sample and labeled heparan sulfate to a competitive anti-heparan sulfate antibody, or 2 ) Contact the sample with the microplate to which the anti-heparan sulfate antibody is fixed, bind the heparan sulfate in this sample to the anti-heparan sulfate antibody on the microplate, and then contact the labeled heparan sulfate with the microplate. On the microplate against heparan sulfate derived from the specimen bound to the anti-heparan sulfate antibody on the microplate As a principle of the detection method, an inhibitory antigen-antibody reaction in which labeled heparan sulfate is bound to the anti-heparan sulfate antibody in an inhibitory manner to detect heparan sulfate in a specimen can be used.

  The detection method of the embodiment using the microplate to which the anti-heparan sulfate antibody mentioned here is fixed can be mentioned as a very preferable detection embodiment in order to facilitate simultaneous detection of a large number of specimens.

  As a method for fixing an anti-heparan sulfate antibody to a solid phase carrier such as a microplate, the anti-heparan sulfate antibody is fixed to a solid phase carrier according to a commonly used method such as a physical adsorption method, a covalent bond method, or a comprehensive method. be able to. In general, it is preferable to fix the anti-heparan sulfate antibody to the solid phase carrier by a physical adsorption method due to the simplicity of the operation, but the fixing method that can be adopted in this physical adsorption method is not limited.

  Furthermore, a method using a solid phase carrier such as a microplate to which heparan sulfate is fixed can be mentioned as one of typical detection modes. For example, after adding a specimen to a microplate to which heparan sulfate is fixed, the heparan sulfate fixed to the microplate and the heparan sulfate in the specimen are reacted competitively with the anti-heparan sulfate antibody. The anti-heparan sulfate antibody that reacts with the heparan sulfate adhering to the heparan sulfate and detects the anti-heparan sulfate antibody bound to the heparan sulfate using a labeled secondary antibody (having specificity for the anti-heparan sulfate antibody). Examples include a method using a competitive or inhibitory antigen-antibody reaction, such as a method for detecting heparan sulfate.

The labeled heparan sulfate that can be used in these series of detection methods is obtained by binding a labeling substance to heparan sulfate. As this labeling substance, for example, one substance of a specific binding pair (for example, avidin such as biotin and streptavidin, or lectin and sugar chain); FITC, phycoerythrin, europium, phycocyanin, rhodamine, Texas red, umbelliferone, Fluorescent substances such as tricolor, cyanine, 7-amino-4-methylcoumarin-3-acetic acid (AMCA); enzymes such as alkaline phosphatase, β-galactosidase, peroxidase, glucose oxidase; dinitrofluorobenzene, AMP (adenosine monophosphate) Acid), haptens such as 2,4-dinitroaniline; isotopes such as ¹²⁵ I, ¹³¹ I and ³ H can be used, and although not particularly limited, one of the above-mentioned specific binding pairs It is preferable to select Properly, in particular, it is preferable to select one of the substances of a specific binding pair consisting of biotin and avidin.

  In addition, the method for binding these labeling substances to heparan sulfate can follow conventional methods. For example, as a method of labeling heparan sulfate with biotin, for example, EDC (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide) dissolved in heparan sulfate solution with MES (2-morpholinoethanesulfonic acid) buffer or the like And a method of reacting by adding a biotin-hydrazide solution. In this binding method, unreacted biotin can be removed by a conventional method such as dialysis, ethanol fractionation or ultrafiltration, and biotin-hydrazide-labeled heparan sulfate can be easily obtained. This is preferable.

  Further, the method for quantifying the labeling substance of heparan sulfate to which the labeling substance is bound can be performed using a method already established as a method for detecting each labeling substance. For example, as a method for detecting heparan sulfate labeled with biotin, avidin having a high affinity for biotin (labeled: preferably labeled with a specific enzyme that is easy to detect typified by peroxidase, for example) A method of indirectly detecting heparan sulfate by reacting biotin bound to heparan sulfate is mentioned.

  When the above peroxidase is used as a labeling substance for avidins such as streptavidin, the substrate to be colored by this peroxidase is usually a coloring substance such as tetramethylbenzidine (TMB) used for this purpose, and a peroxidase. It is preferable to use hydrogen peroxide water or the like as the substrate.

  In addition, the secondary antibody having specificity for the anti-heparan sulfate antibody used for detecting the above-described anti-heparan sulfate antibody is not particularly limited, but if the anti-heparan sulfate antibody is derived from a rabbit, it is an antibody against rabbit immunoglobulin. In the case of mouse origin, an antibody against mouse immunoglobulin and the like can be mentioned, and it is appropriately selected depending on the origin and type of anti-heparan sulfate antibody which is a primary antibody. Examples of the substance for labeling the secondary antibody include the substance group exemplified as the substance for labeling the above-described heparan sulfate, and among them, enzymes are generally used. A substance for labeling these secondary antibodies and a method for detecting the labeling substance can be appropriately selected as necessary. As a particularly preferred combination of anti-heparan sulfate antibody-labeled secondary antibody, a combination of mouse IgM antibody 10E4 and peroxidase-labeled anti-mouse IgM antibody can be exemplified.

  In the quantification method of the present invention, a container-like solid phase carrier such as these microplates, or other forms other than the mode using a solid phase carrier of a size that can be visually observed can be used. A typical example of such an embodiment is a method using as an index an agglutination reaction based on the antigen-antibody reaction of the particulate solid phase carrier described above.

  For example, a fine solid phase carrier such as latex particles which are synthetic polymer particles is used, a specimen is brought into contact with the fine solid phase carrier to which heparan sulfate is fixed, and an anti-heparan sulfate antibody is further applied to the fine solid phase carrier. Regarding the agglutination reaction between latex particles via anti-heparan sulfate antibody caused by contact, by detecting the degree to which this agglutination reaction is inhibited by heparan sulfate in the sample by contact with the sample, It is possible to detect heparan sulfate.

  The quantification method of the present invention is a means for identifying a diabetic patient who is transitioning to diabetic nephropathy as early as possible, that is, a quantification method capable of accurately quantifying heparan sulfate in a sample, and the quantification method. It is also possible to apply to a quantification kit.

  In healthy kidneys, the negative charge in the heparan sulfate sugar chain of heparan sulfate proteoglycan present in the glomerular basement membrane functions as a “charge barrier” for proteins in the blood, thereby causing glomeruli such as proteins. Protects against deposition and leakage into urine.

  And the amount of normal form heparan sulfate in urine samples tends to decrease as diabetic patients move to diabetic nephropathy. Using the quantification method of the present invention, it is possible to detect the increase or decrease in the urine sample of this normal form of heparan sulfate, and to identify the state of the diabetic patient using the detected value as an index, and diabetic nephropathy The transition stage to can be detected.

  That is, by performing the quantification method of the present invention, the above-mentioned DM1 diabetic patient is shifted to DM2 diabetic early nephropathy and further to DM3 diabetic nephropathy, in other words, a conventional urine sample. It is possible to detect a transition stage between diabetic nephropathy pathological classifications (DM1 to DM3) defined by the amount of intermediate albumin, that is, “transition stage to diabetic nephropathy”.

  If the quantification method of the present invention detects that the amount of heparan sulfate in the normal form quantified from the urine sample is less than the normal value, the diabetic patient is shifting to diabetic nephropathy This indicates the state of the diabetic patient.

  For example, if a boundary value of heparan sulfate in a urine sample is set, and the quantification method of the present invention quantifies that the detected amount of heparan sulfate is less than this set value, then “the diabetic patient is diabetic nephropathy On the contrary, if it is greater than the set value, it is detected that the patient is not in the transition stage to diabetic nephropathy, and the transition stage to diabetic nephropathy is detected. Is possible.

  Furthermore, the quantification method of the present invention can be used alone as a means for specifying the state of a diabetic patient. For example, the quantification value is combined with another indicator such as albumin level in an existing urine sample. By using it, it is possible to further improve its reliability.

  The kit for performing the above-described quantification method of the present invention is a kit capable of quantifying heparan sulfate in a specimen.

  The kit for carrying out the quantification method of the present invention is not particularly limited as long as it is a kit for simply carrying out the quantification method of the present invention described above. Specifically, this kit includes at least an anti-heparan sulfate antibody and a labeled heparan sulfate or heparan sulfate fixed to a solid phase (hereinafter, these heparan sulfates are referred to as “competitive heparan sulfate”). However, the embodiment of the kit including the solid phase carrier to which the anti-heparan sulfate antibody is fixed is the most preferable embodiment.

  Furthermore, the kit for carrying out the quantification method of the present invention most preferably includes, in addition to the above-described elements, (1) a reagent containing labeled heparan sulfate obtained by labeling heparan sulfate with a labeling substance as a competitive heparan sulfate, and (2) This kit includes a reagent for detecting a labeled substance bound to the labeled heparan sulfate.

  Of course, it is preferable that the heparan sulfate in the reagent, including the labeled heparan sulfate in this kit, is a constant amount and is present uniformly in the reagent system. In addition, the type of the labeled substance in the labeled heparan sulfate in the kit of this embodiment and the type of the detection substance corresponding thereto can be selected according to the explanation in the above-described quantification method of the present invention.

  In addition, the kit for carrying out such a quantification method of the present invention includes a buffer solution, distilled water, ultrapure water, or the like contained in a normal quantification kit as long as the desired effect of the present invention is not impaired as necessary. In this case, the component can be used.

  The kit for carrying out the quantification method of the present invention described above can also take a form as a diagnostic kit for detecting the transitional stage of diabetic nephropathy and specifying the state of a diabetic patient. The diagnostic kit may be transferred to normal values obtained by quantitatively determining heparan sulfate in urine samples from healthy individuals (including diabetic patients who do not suffer from diabetic nephropathy) and diabetic nephropathy Quantifying and comparing heparan sulfate in urine specimens of diabetic patients with diabetes to identify changes in heparan sulfate in urine specimens of diabetic patients, and the transition stage of diabetic patients to diabetic nephropathy It is a kit that can detect the condition of a diabetic patient.

  The elements that can be included in the kit for detecting the transitional stage of diabetic nephropathy and identifying the state of the diabetic patient are basically the same as those of the above-described quantification kit of the present invention, but because urine is used as a specimen. Of course, it is preferable to include an element such as a urine pretreatment reagent which is particularly necessary for the urine.

   Hereinafter, although the present invention is explained more concretely using an example etc., the technical scope of the present invention should not be limited by this example etc.

Analysis of antigenic determinant of anti-heparan sulfate antibody 10E4 The antigenic determinant of 10E4 was analyzed by examining the change in reactivity of anti-heparan sulfate monoclonal antibody 10E4 with the change of sulfate group in heparan sulfate molecule. It was. That is, on a plate to which the anti-heparan sulfate antibody 10E4 is fixed, (1); heparan sulfate derived from bovine kidney at various concentrations [Seikagaku Corporation, Lot. No. S94Z02: HS (BK)], (2) The modified heparan sulfate derived from bovine kidney (CDSNAc-HS) obtained by completely desulfating the heparan sulfate of (1); (3); heparan sulfate obtained by reintroducing a sulfate group at the N-position of CDSNAc-HS of (2); The modified product (CDSNS-HS) and the heparan sulfate (NDSNAc-HS) derived from the above bovine kidney in which only the N-position sulfate group in (4); (3) was completely desulfated were added. Simultaneously, biotin-labeled heparan sulfate was added and reacted with the anti-heparan sulfate antibody 10E4 immobilized on the plate by a competitive antigen-antibody reaction.

  Heparan sulfate that did not react with the anti-heparan sulfate antibody 10E4, each heparan sulfate modified product, and biotin-labeled heparan sulfate were removed by washing. Subsequently, biotin was detected using peroxidase-labeled streptavidin as described later. As described later, the enzyme activity of peroxidase was quantified using a TMB solution containing hydrogen peroxide as a chromogenic substrate and finally using the absorbance at 450 nm as an index (FIG. 1). In FIG. 1, the vertical axis represents the absorbance (O.D.), and the horizontal axis represents the amount of heparan sulfate and the amount of each modified heparan sulfate.

  From this result, it was found that the reactivity of the anti-heparan sulfate antibody 10E4 depends on the presence or absence of an N-sulfate group in the glucosamine residue of heparan sulfate. That is, it was revealed that the antigenic determinant of the anti-heparan sulfate antibody 10E4 has a structure containing at least the N-sulfate group in the glucosamine residue of heparan sulfate. Further, as a result of examining the reactivity of the anti-heparan sulfate antibody 10E4 to biotin-labeled hyaluronic acid produced by the same method as that of heparan sulfate, it was also found that the anti-heparan sulfate antibody 10E4 does not show cross-reactivity to hyaluronic acid. It was.

Preparation of calibration curve (1) Preparation of biotin-labeled heparan sulfate Heparan sulfate (Seikagaku Corporation) was dissolved in 0.1M 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to give 10 mg / mL. A 1.0 mL heparan sulfate solution was prepared. To this heparan sulfate solution, 25 μL of biotin-LC-hydrazide (manufactured by PIERCE) prepared to 20 mM with dimethyl sulfoxide (DMSO) was added. Subsequently, 12.5 μL of EDC (manufactured by PIERCE) adjusted to 100 mg / mL with 0.1 M MES buffer (pH 5.5) was added thereto and stirred well, followed by stirring at room temperature for 16-24 hours. After completion of the reaction, this reaction product was sufficiently removed from the biotin with a microdialysis apparatus (manufactured by BIO-TECK) using PBS (-) as a dialysate in a dialysis membrane having a molecular weight of 3,500 cutoff. After completion of dialysis, biotin-labeled heparan sulfate was prepared at a concentration of 5 mg / mL and stored frozen.

  (2) Preparation of anti-heparan sulfate antibody-coated plate Anti-heparan sulfate antibody 10E4 (Seikagaku Corporation) was added to phosphate buffered saline (pH 7.2 to 7.5, free of divalent ions such as calcium ions: hereinafter, PBS (Also described as (-)), dilute to 20 μg / mL, add 50 μL of this solution to each well of a NUNQUI immunoplate (trade name: Maxisorp, NUNK), and store at 4 ° C. for 14-18 hours To uniformly coat. This plate was washed twice with PBS (−), and 3% bovine serum albumin (BSA) (available from Seikagaku Corporation) was used as a blocking substance for blocking wells that were not coated with anti-heparan sulfate antibody 10E4. ) -Containing PBS (−) solution was added and allowed to stand at room temperature for 2 hours. After standing, this plate was washed 3 times with a washing solution (PBS (-) containing 0.05% Tween 20) to obtain a desired anti-heparan sulfate antibody-coated plate.

  (3) Preparation of calibration curve After washing, 100 μL of PBS (−) containing 0.05% Tween 20 and 1% BSA (hereinafter referred to as reaction solution) is added to each well of the anti-heparan sulfate antibody-coated plate prepared in (2). Added one by one. Subsequently, 10 μL of heparan sulfate standard solution (derived from bovine kidney, manufactured by Seikagaku Corporation) (each concentration of 25 to 800 μg / mL) was added to each well of this plate. Furthermore, 100 μL of the biotin-labeled heparan sulfate prepared in (1) above, diluted to the optimal concentration in the reaction solution, was added to each well and left at 37 ° C. for 60 minutes for antigen-antibody reaction (the heparan sulfate standard solution). The reaction solution was used as a solvent).

  After completion of this reaction, each well was washed three times with the washing solution, and then 100 μL of peroxidase-labeled streptavidin (HRP-labeled streptavidin: manufactured by VECTOR) diluted 1000 times with the reaction solution was added to each well, and this was added at 37 ° C. And left to react for 30 minutes.

  After completion of the reaction, the plate was washed 3 times with the washing solution, 100 μL of TMB solution (manufactured by Moss) was added as a substrate for peroxidase, and the mixture was reacted at 37 ° C. for 15 minutes to develop color. The reaction was stopped by adding 100 μL of 1N-HCl to the plate, and the absorbance at a wavelength of 450 nm (control wavelength: 630 nm) of the colored liquid resulting from the decomposition of TMB was measured with a well reader (SK-601, sold by Seikagaku Corporation). A calibration curve was created (FIG. 2).

  Since heparan sulfate is rich in variety, 1 μg of heparan sulfate derived from bovine kidney (Seikagaku Corporation, Lot. No. S94Z02) is defined as 1 EU as a unique definition. Expressed in equivalent heparan sulfate equivalents.

  In addition, when mouse IgM was fixed to the plate instead of anti-heparan sulfate antibody, no reaction was observed, and this reaction was not a non-specific reaction but a reaction specific to anti-heparan sulfate antibody (10E4). It was shown (FIG. 2: control).

Detection of the transitional stage of diabetic nephropathy in diabetic patients by quantitative determination of heparan sulfate in the sample Heparan sulfate present in this urine sample using urine collected from healthy subjects (27 cases) and diabetic patients (68 cases) Was quantified by the method described above.

  That is, diabetic patients were classified into three groups DM1, DM2, and DM3 according to urinary albumin concentration (creatinine correction value: ACR; mg / g · CR). DM1 has ACR <12, and the ACR is a group of diabetic patients at the same level as healthy people. DM2 is 12 ≦ ACR <200, which is a diabetic early nephropathy patient group. DM3 is 200 ≦ ACR and is a diabetic nephropathy patient group.

  FIG. 3 is a comparison of heparan sulfate levels in urine samples between groups. In urine samples, there is a clear difference in heparan sulfate levels between diabetic patients (DM1) and healthy individuals. (Heparan sulfate level in urine specimens of diabetic patients is clearly lower than that of healthy individuals).

  Furthermore, urine samples of healthy subjects (83 cases), DM1 (40 cases), DM2 (41 cases) and DM3 (43 cases) were collected as specimens, and the albumin concentration in these specimens (creatinine correction value: ACR; mg / (gCR) and heparan sulfate concentration (creatinine correction value: mg / gCR) were plotted, and the distribution region was analyzed (FIG. 4). The concentration of albumin in urine specimens of healthy people and DM1 patients is at the same level. Further, the distribution of heparan sulfate in the urine sample of DM1 patients has a wide distribution from the same level as that of a healthy person to a low level of about 1/10. It is considered that the tendency of the heparan sulfate concentration in the urine sample to decrease with the progress of the pathological condition of diabetic nephropathy is already observed at the DM1 stage. And it is thought that the DM1 patient with a low heparan sulfate concentration in the urine sample has a high risk of progression to diabetic early nephropathy (DM2).

  Therefore, if 5EU / mg CR is defined as a boundary value, and if it is classified as less than or equal to the boundary value, and the boundary value is negative and the boundary value is positive, the rate of positive with the progression of diabetic nephropathy Increased (FIG. 5). Therefore, it was suggested that 5EU / mg CR as a boundary value in this way is preferable in carrying out the detection method of the present invention based on heparan sulfate in the urine specimen quantified using the quantification method of the present invention. .

  Furthermore, when categorizing the duration of diabetes associated with the onset of diabetic nephropathy, the percentage of positives increased with the duration of disease (FIG. 6). In light of the results shown in Fig. 6, there is a general trend toward an increase in the number of patients diagnosed with diabetic nephropathy in about 5 to 10 years, and about 15 years for diabetic nephropathy. Considered together, this result shows that it is possible to grasp the progression stage of the pathological condition of diabetic nephropathy and identify the state of diabetic patients by the present invention specifying method based on the quantification method of the present invention. This shows the usefulness of the quantification method of the present invention and the specific method of the present invention.

Kit according to the present invention An example of the composition and usage of the kit according to the present invention will be described below. The kit in a present Example consists of the following elements 1-8.

1. 1. Immunoplate to which anti-heparan sulfate antibody 10E4 is fixed (blocked with a blocking agent): 1 sheet Standard heparan sulfate solution (25, 50, 100, 200, 400, 800 EU / mL, 0.1 mL each): 1 vial each Biotin-labeled heparan sulfate solution (optimum concentration, 15 mL): 1 vial 4. Peroxidase-labeled streptavidin solution (1 μg / mL, 15 mL): 1 vial Tetramethylbenzidine solution (1.25 mM TMB, 2.21 mM hydrogen peroxide, 1% DMSO, 0.08 M acetate buffer, pH 4.9, 15 mL): 1 vial Reaction solution (1% BSA, PBS containing 0.05% Tween 20 (−), 40 mL): 1 vial Washing solution (PBS (-) (500 mL) containing 0.05% Tween 20): 1 vial Stop solution (1N HCl (15 mL)): 1 vial

  When using the kit according to the present invention, each well of the immunoplate to which the anti-heparan sulfate antibody 10E4 had been fixed was washed in advance with a washing solution, and 100 μL of the reaction solution was dispensed into each well. In this kit for detecting the transitional stage of diabetic nephropathy, 10 μL each of heparan sulfate standard solution of each concentration or sample urine of diabetic patients is added, and 100 μL of biotin-labeled heparan sulfate solution is added to each well. It was allowed to stand at 37 ° C. for 60 minutes.

  Thereafter, in this kit, each well was washed three times with a washing solution, and then 100 μL of a peroxidase-labeled streptavidin solution was added to each well, which was allowed to stand at 37 ° C. for 30 minutes for reaction. After standing, each well was washed three times with a washing solution, and then 100 μL of tetramethylbenzidine solution was added to each well and reacted at 37 ° C. for 15 minutes for color development.

After color development, 100 μL of a reaction stop solution was added to each well to stop the reaction, and the absorbance at 450 nm (control wavelength 630 nm), which is the coloring wavelength of the reaction product of TMB and H ₂ O ₂ by peroxidase, was measured with a well reader (SK601, Quantitative analysis by Seikagaku Corporation.

It is drawing which showed the recognition rate of the anti- heparan sulfate antibody 10E4 in heparan sulfate and various modifications. It is a calibration curve created using a heparan sulfate standard solution. It is drawing which shows the comparison between each group which made the heparan sulfate level in a urine sample the albumin density | concentration in a urine sample as a parameter | index. It is drawing which contrasts the albumin density | concentration in a urine sample, and a heparan sulfate concentration, and shows the distribution area | region. It is a drawing in which 5EU / mg CR is assumed to be a boundary value, and the correlation with the symptoms of diabetic nephropathy is examined by classifying it below this boundary value or above the boundary value. FIG. 5 is a drawing in which 5EU / mg CR is assumed to be a boundary value, and the correlation between the onset of diabetic nephropathy and the disease duration is examined by classifying it below this boundary value or above the boundary value.

Claims (17)

Anti-heparan sulfate antibody immobilized on a solid phase carrier is subjected to a competitive antigen-antibody reaction between labeled heparan sulfate and heparan sulfate in a sample, and labeled heparan sulfate bound to the anti-heparan sulfate antibody on this solid phase carrier A method for quantifying heparan sulfate to be detected. The method for quantifying heparan sulfate according to claim 1, wherein the labeling substance in the labeled heparan sulfate is a substance selected from the group consisting of one substance of a specific binding pair, a fluorescent substance, an enzyme, a hapten, and a radioisotope. The method for quantifying heparan sulfate according to claim 2, wherein the labeling substance in the labeled heparan sulfate is one substance of a specific binding pair. The method for quantifying heparan sulfate according to claim 3, wherein one substance of the specific binding pair is biotin. Anti-heparan sulfate antibody bound to heparan sulfate on the solid phase carrier is detected by a competitive antigen-antibody reaction between heparan sulfate immobilized on the solid phase carrier and heparan sulfate in the sample against the anti-heparan sulfate antibody. A method for determining heparan sulfate in a specimen. Anti-heparan sulfate antibody is subjected to a competitive antigen-antibody reaction between heparan sulfate immobilized on a particulate solid support and heparan sulfate in a specimen to cause aggregation of the particulate solid support. A method for quantifying heparan sulfate in a sample by detection. The method for quantifying heparan sulfate according to any one of claims 1 to 6, wherein the anti-heparan sulfate antibody is an anti-heparan sulfate antibody having at least a glucosamine residue containing an N-sulfate group in heparan sulfate as an antigenic determinant. Method for quantifying heparan sulfate comprising the following steps: (1) A labeled heparan labeled with a sample and biotin on a microplate to which an anti-heparan sulfate antibody having at least a glucosamine residue having an N-sulfate group as an antigenic determinant is fixed. Anti-heparan sulfate antibody that has been brought into contact with sulfuric acid and adhered to the microplate is subjected to a competitive antigen-antibody reaction with heparan sulfate and labeled heparan sulfate in the contacted specimen, and (2) labeled heparan sulfate bound to the microplate. (3) A step of quantifying heparan sulfate in the specimen by binding peroxidase-labeled avidin and measuring peroxidase activity in the microplate. The method for quantifying heparan sulfate according to any one of claims 1 to 8, wherein the anti-heparan sulfate antibody is a monoclonal antibody. The quantification kit for performing the quantification method according to any one of claims 1 to 4, comprising a solid phase carrier to which at least an anti-heparan sulfate antibody is fixed. The quantification kit according to claim 10, wherein the anti-heparan sulfate antibody is an anti-heparan sulfate antibody having at least a glucosamine residue containing an N-sulfate group in heparan sulfate as an antigenic determinant. The quantification kit according to claim 10 or 11, wherein the anti-heparan sulfate antibody is an anti-heparan sulfate antibody in which no cross-reactivity to hyaluronic acid is observed. The quantification kit according to any one of claims 10 to 12, wherein the anti-heparan sulfate antibody is a monoclonal antibody. The quantification kit for performing the quantification method according to any one of claims 1 to 4, comprising heparan sulfate labeled with a labeling substance. The quantification kit according to claim 14, wherein the labeled heparan sulfate is labeled heparan sulfate labeled with a substance selected from the group consisting of one substance of a specific binding pair, a fluorescent substance, an enzyme, a hapten, and a radioisotope. The quantification kit according to claim 14, wherein the labeled heparan sulfate is labeled heparan sulfate labeled with one substance of a specific binding pair. The quantification kit according to claim 16, wherein one substance of the specific binding pair is biotin.