JP5143621B2 - Method for measuring molecular weight distribution of hyaluronic acid in biological fluid samples derived from mammals - Google Patents

Description

  The present invention relates to a method for measuring the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a mammal, a method for determining rheumatoid arthritis using the method, and an experimental set for these methods.

  Rheumatoid arthritis (RA), whose number of patients has been increasing in recent years, is a chronic inflammatory joint disease that causes joint destruction and dysfunction due to long-term morbidity. Joint tissue is composed of various extracellular matrices. Among them, hyaluronic acid (HA) is a polysaccharide consisting of repeating N-acetylglucosamine and D-glucuronic acid disaccharides, and has a protective effect on joints. It is an important joint component.

  In general, it is known that the concentration and molecular weight of HA in synovial fluid of RA patients are lower than that in healthy individuals, but the correlation between the pathology of RA and the mechanism of HA metabolism in synovial fluid is unknown. The current situation is that there are a lot of points and little is clarified. Although it is expected that the balance between HA synthetase and degrading enzyme will be lost in the joints of RA patients, the effects are hardly elucidated.

  Non-patent document 1 is known as an example of measuring HA molecular weight in serum for the first time, as an example of measuring HA molecular weight in serum for the first time. Non-Patent Document 2 describes a method for quantifying the HA molecular weight in human serum using a radioisotope. Non-Patent Document 3 describes a quantification method using hyaluronic acid binding protein (HABP).

Biochem. J., 236, 521-525 (1986) Int. J. Cancer, 44, 445-448 (1989) Res. Commun. Mol. Pathol. Pharmacol., 99 (2), 207-216 (1998)

  However, the prior art described in the above literature has room for improvement in the following points.

  First, if the analysis is limited to the concentration of HA, it is possible to apply the existing method. However, HA in the serum of RA patients moves from the joint to the blood as the disease progresses, and the concentration is increased. The molecular weight changes in a complex manner. In other words, since HA in blood reflects various sites in RA pathology and the process of progression, it is not enough to simply measure HA concentration and molecular weight in pathological analysis of RA patients. It is extremely important to accurately grasp the molecular weight distribution.

  Secondly, in order to correlate and analyze the behavior of HA in the serum of individual patients and the pathology of RA, it is necessary to appropriately collect and examine the patient's blood over a long period of time. However, since the amount of blood that can be collected at one time from an individual patient is naturally limited, it is necessary to obtain information on HA kinetics from a small amount of patient serum. That is, it is desired to develop a method for measuring the molecular weight and molecular weight distribution of HA in patient serum with high sensitivity even when there is a quantitative restriction on the amount of sample collected at one time.

  However, in Non-Patent Document 1, when measuring the HA molecular weight in serum, 50 to 500 mL of RA patient serum is pooled and used, and it is not a method that can be applied to small-scale analysis. The method described in Non-Patent Document 2 is a method for quantification using a radioisotope, and therefore requires a special facility, and requires 2.5 mL of serum before concentration. Furthermore, the method described in Non-Patent Document 3 employs a quantitative method using hyaluronic acid binding protein (HABP), but requires 3 mL of serum. Thus, a method capable of efficiently measuring not only the HA concentration but also the molecular weight of HA and the distribution pattern of the molecular weight from a small amount of serum of 1 mL or less has not been found so far.

  The present invention has been made in view of the above circumstances, and provides a technique for efficiently measuring the distribution pattern of the molecular weight of hyaluronic acid from a small amount of a biological fluid sample derived from a mammal of 1 mL or less without using a radioisotope. For the purpose.

  According to the present invention, there is provided a method for measuring the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a mammal, the step of molecular weight fractionating a biological fluid sample, and each fraction containing hyaluronic acid that has been subjected to molecular weight fractionation. And a step of quantifying the amount of hyaluronic acid in each of the concentrated fractions.

  According to this method, a step of molecular weight fractionating a biological fluid sample, a step of concentrating each fraction containing hyaluronic acid that has been molecular weight fractionated, and a step of quantifying the amount of hyaluronic acid in each of the concentrated fractions Therefore, even if the concentration of hyaluronic acid is reduced during molecular weight fractionation, the concentration of hyaluronic acid can be increased by concentration. It becomes possible to quantify with. Therefore, according to this method, it is possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a small amount of a biological fluid sample derived from a mammal of 1 mL or less without using a radioisotope.

  Further, according to the present invention, there is provided a determination method for analyzing a biological fluid sample derived from a mammal and determining whether the biological fluid sample is collected from a mammal suffering from rheumatoid arthritis disease. , Measuring the molecular weight distribution of hyaluronic acid in a mammal-derived biological fluid sample using the measurement method described above, and the measured molecular weight distribution and a biological fluid sample derived from a normal mammal of the same kind as the mammal described above Comparing the molecular weight distribution of hyaluronic acid in the blood, and determining whether the biological fluid sample was collected from a mammal suffering from rheumatoid arthritis disease based on the comparison result of the molecular weight distribution of hyaluronic acid A determination method is provided.

  According to this method, since the molecular weight distribution of hyaluronic acid in a sample of a biological fluid derived from a mammal is measured using the measurement method described above, a small amount of biological fluid derived from a mammal of 1 mL or less is used without using a radioisotope. It becomes possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a sample. Therefore, by comparing the molecular weight distribution measured for a biological fluid sample derived from a mammal as a subject and the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a normal mammal of the same species as the subject mammal, It is possible to determine whether or not a biological fluid sample derived from a mammal as a subject has been collected from a mammal suffering from rheumatoid arthritis disease.

  Further, according to the present invention, a biological fluid sample derived from a mammal is analyzed to determine which type of biological fluid sample was collected from a mammal suffering from rheumatoid arthritis disease. A method for measuring a molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a mammal using the measurement method described above, a plurality of molecular weight distributions measured and a plurality of the same species as the mammal described above Based on the results of comparing the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a mammal suffering from a type of rheumatoid arthritis disease and the comparison result of the molecular weight distribution of hyaluronic acid, And determining which type of rheumatoid arthritis disease is collected from a mammal suffering from the above.

  According to this method, since the molecular weight distribution of hyaluronic acid in a sample of a biological fluid derived from a mammal is measured using the measurement method described above, a small amount of biological fluid derived from a mammal of 1 mL or less is used without using a radioisotope. It becomes possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a sample. Therefore, in the biological fluid sample derived from a mammal suffering from multiple types of rheumatoid arthritis diseases of the same type as the subject mammal and the molecular weight distribution measured for the biological fluid sample derived from the mammal being the subject. By comparing the molecular weight distribution of hyaluronic acid, it is determined whether a biological fluid sample derived from the subject mammal is collected from a mammal suffering from rheumatoid arthritis disease of multiple types Is possible.

  Further, according to the present invention, an experimental set comprising a reagent and a column for measuring the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a mammal, comprising a proteolytic enzyme, a size exclusion chromatography column, An experimental set comprising a hyaluronic acid binding protein is provided.

  According to this experimental set, a biological fluid sample pretreated with a proteolytic enzyme was subjected to molecular weight fractionation using a size exclusion chromatography column, and then each fraction containing hyaluronic acid fractionated by molecular weight was studied. It can be concentrated by drying under reduced pressure using a suitable device such as a centrifugal evaporator provided in the chamber, etc., and the amount of hyaluronic acid in each concentrated fraction can be quantified using hyaluronic acid binding protein. .

  Therefore, according to this experimental set, even when the concentration of hyaluronic acid is reduced during molecular weight fractionation using a size exclusion chromatography column, the concentration of hyaluronic acid is reduced using an appropriate device such as a centrifugal evaporator. Since the concentration can be increased by drying and concentrating, the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity using a hyaluronic acid binding protein. Therefore, according to this experimental set, it is possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a small amount of a biological fluid sample derived from a mammal of 1 mL or less without using a radioisotope.

  The present invention also includes a reagent and a column for analyzing a biological fluid sample derived from a mammal and determining whether the biological fluid sample is collected from a mammal suffering from rheumatoid arthritis disease. An experimental set of proteolytic enzymes, size exclusion chromatography columns, hyaluronic acid binding proteins, biological fluid samples from normal mammals of the same kind as mammals or hyaluronic acid obtained from the biological fluid samples And an experimental set comprising molecular weight distribution data.

  According to this experimental set, a biological fluid sample pretreated with a proteolytic enzyme was subjected to molecular weight fractionation using a size exclusion chromatography column, and then each fraction containing hyaluronic acid fractionated by molecular weight was studied. It can be concentrated by drying under reduced pressure using a suitable device such as a centrifugal evaporator provided in the chamber, etc., and the amount of hyaluronic acid in each concentrated fraction can be quantified using hyaluronic acid binding protein. .

  Therefore, according to this experimental set, even when the concentration of hyaluronic acid is reduced during molecular weight fractionation using a size exclusion chromatography column, the concentration of hyaluronic acid is reduced using an appropriate device such as a centrifugal evaporator. Since the concentration can be increased by drying and concentrating, the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity using a hyaluronic acid binding protein. Therefore, according to this experimental set, it is possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a small amount of a biological fluid sample derived from a mammal of 1 mL or less without using a radioisotope.

  And according to this experimental set, the biological fluid sample derived from a normal mammal of the same species as the subject mammal or the molecular weight distribution data of hyaluronic acid obtained from the biological fluid sample is included. Even so, the molecular weight distribution data of hyaluronic acid in a biological fluid sample derived from a normal mammal of the same species as the subject mammal can be obtained. Therefore, by comparing the molecular weight distribution measured for a biological fluid sample derived from a mammal as a subject and the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a normal mammal of the same species as the subject mammal, It is possible to determine whether or not a biological fluid sample derived from a mammal as a subject has been collected from a mammal suffering from rheumatoid arthritis disease.

  Further, according to the present invention, a biological fluid sample derived from a mammal is analyzed, and it is determined which type of the biological fluid sample is collected from a mammal suffering from rheumatoid arthritis disease. A set of experiments and reagents, including a proteolytic enzyme, a size exclusion chromatography column, a hyaluronic acid binding protein, and multiple types of rheumatoid arthritis disease similar to mammals An experimental set comprising a biological fluid sample derived from a mammal or molecular weight distribution data of hyaluronic acid obtained from the biological fluid sample is provided.

  According to this experimental set, a biological fluid sample pretreated with a proteolytic enzyme was subjected to molecular weight fractionation using a size exclusion chromatography column, and then each fraction containing hyaluronic acid fractionated by molecular weight was studied. It can be concentrated by drying under reduced pressure using a suitable device such as a centrifugal evaporator provided in the chamber, etc., and the amount of hyaluronic acid in each concentrated fraction can be quantified using hyaluronic acid binding protein. .

  Therefore, according to this experimental set, even when the concentration of hyaluronic acid is reduced during molecular weight fractionation using a size exclusion chromatography column, the concentration of hyaluronic acid is reduced using an appropriate device such as a centrifugal evaporator. Since the concentration can be increased by drying and concentrating, the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity using a hyaluronic acid binding protein. Therefore, according to this experimental set, it is possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a small amount of a biological fluid sample derived from a mammal of 1 mL or less without using a radioisotope.

  According to this experimental set, a biological fluid sample derived from a mammal suffering from multiple types of rheumatoid arthritis diseases of the same type as the subject mammal, or hyaluronic acid obtained from the biological fluid sample Since molecular weight distribution data is included, in any case, hyaluronic acid molecular weight distribution data in biological fluid samples from mammals suffering from multiple types of rheumatoid arthritis disease of the same type as the subject mammal Is obtained. Therefore, in the biological fluid sample derived from a mammal suffering from multiple types of rheumatoid arthritis diseases of the same type as the subject mammal and the molecular weight distribution measured for the biological fluid sample derived from the mammal being the subject. By comparing the molecular weight distribution of hyaluronic acid, it is determined whether a biological fluid sample derived from the subject mammal is collected from a mammal suffering from rheumatoid arthritis disease of multiple types Is possible.

  According to the present invention, it is possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a small amount of mammal-derived biological fluid sample of 1 mL or less.

<Explanation of terms>
(I) Hyaluronic acid In the present specification and claims, “hyaluronic acid” is a kind of glycosaminoglycan (mucopolysaccharide), and it is scientifically called a hyaluronan. Means. Hyaluronic acid has a structure in which disaccharide units of N-acetylglucosamine and glucuronic acid (GlcNAcβ1-4GlcAβ1-3) are linked.

(Ii) Rheumatoid arthritis In the present specification and claims, “rheumatoid arthritis (RA)” means that autoimmunity mainly affects the joints of the limbs, thereby causing joint pain and joint deformation. It means inflammatory autoimmune disease that occurs. Rheumatoid arthritis is sometimes called “rheumatoid arthritis”. The Japanese translation of Rheumatoid Arthritis was decided to be “Rheumatoid Arthritis” at the 6th Annual Meeting of the Japanese Association of Rheumatology (1962). A statement has been announced. Accordingly, in 2006, the name of the specific disease by the Ministry of Health, Labor and Welfare was changed to “Rheumatoid arthritis”.

(Iii) Others In the present specification and claims, “lower limit numerical value to upper limit numerical value” means not less than the lower limit numerical value and not more than the upper limit numerical value.

  Hereinafter, embodiments of the present invention will be described in detail.

<Embodiment 1: Measuring method of molecular weight distribution of hyaluronic acid>
The present inventors diligently studied to develop a method for efficiently measuring not only the HA concentration but also the molecular weight and the molecular weight distribution pattern using a small amount of serum. As a result, the present inventors have found a method for measuring the molecular weight and molecular weight distribution of hyaluronic acid in serum using a small amount of serum.

  The measurement method according to this embodiment thus completed is a method for measuring the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a mammal. In this measurement method, a biological fluid sample is subjected to molecular weight fractionation, then each fraction containing hyaluronic acid that has been subjected to molecular weight fractionation is concentrated, and then the amount of hyaluronic acid in each concentrated fraction is quantified.

That is, in other words, this embodiment relates to a method for measuring the molecular weight and molecular weight distribution of hyaluronic acid in serum, including the following steps.
Step 1: Molecular weight fractionation of serum sample Step 2: Concentrate each fraction containing hyaluronic acid molecular weight fractionated in Step 1 Step 3: Quantify the hyaluronic acid concentrated in Step 2 Step 4: Obtained hyaluronic acid Reconstruct molecular weight distribution from acid content

  The method according to the present embodiment includes a step of molecular weight fractionating a biological fluid sample, a step of concentrating each fraction containing hyaluronic acid that has been subjected to molecular weight fractionation, and quantifying the amount of hyaluronic acid in each of the concentrated fractions. Process, so that the concentration of hyaluronic acid can be increased by concentration even when the concentration of hyaluronic acid is reduced during molecular weight fractionation. It becomes possible to quantify with high sensitivity. Therefore, according to this method, it is possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a small amount of a biological fluid sample derived from a mammal of 1 mL or less without using a radioisotope.

  The biological fluid sample described above is not particularly limited, and any biological fluid sample derived from a mammal can be used. Specific examples of the biological fluid (also called body fluid) sample are roughly divided into an intracellular fluid (ICF) and an extracellular fluid (ECF), but an extracellular fluid that can be easily collected is preferable. This extracellular fluid includes blood and lymph, tissue fluid that fills between cells outside the blood vessels, and body cavity fluid in the body cavity. In the present specification and claims, serum obtained from blood is also included in the biological fluid sample. The biological fluid sample mentioned above is particularly preferably blood (serum) or joint fluid because it is considered to change more complicatedly reflecting various sites and progression processes in the pathology of RA. In the following description, the description will be focused on serum among biological fluid samples.

  Here, the amount of the biological fluid sample (serum) collected from the mammal is preferably 1 mL or less per time, considering the suppression of the mental and physical burden of the mammal of the subject, and 500 μL or less. It is more preferable that it is 200 μL or less. Until now, it was difficult to measure the molecular weight distribution of hyaluronic acid on the basis of such a small amount of biological fluid sample (serum), but the present inventors have found that the molecular weight fractionation Even when the concentration of hyaluronic acid is reduced at the time, the concentration of hyaluronic acid can be increased by concentration, so that the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity. Based on this idea, various combinations of processes were tried and errored, and the molecular weight distribution of hyaluronic acid could be measured based on a small amount of biological fluid sample (serum) of 1 mL or less.

  The above molecular weight is an academically defined molecular weight (molecular weight) or relative molecular mass (relative molecular mass) concept (a value representing the relative mass of a molecule, and is similar to the atomic weight of 12C). Any type of molecular weight can be used as long as it is included in a dimensionless amount defined as 12 times the ratio to mass. That is, the above-mentioned molecular weight may be any of a viscosity average molecular weight, a weight average molecular weight, and a number average molecular weight, but it is particularly preferable to use a viscosity average molecular weight that is generally used and excellent in versatility. In the following description, when it is simply described as molecular weight, it will be described as meaning the viscosity average molecular weight.

  Hereinafter, the steps included in the method of the present embodiment will be described.

(I) Pretreatment step The method according to this embodiment may further include a step of pretreatment with a proteolytic enzyme prior to molecular weight fractionation of the biological fluid sample. That is, serum can be fractionated by molecular weight fractionation as it is, or it can be mixed with hyaluronic acid in serum by pretreatment of biological fluid sample serum or serum dilution with a proteolytic enzyme in advance. Since protein can be decomposed, the recovery rate of hyaluronic acid can be improved and the influence of contaminants (proteins) can be removed, and a more preferable measurement result of the molecular weight distribution of hyaluronic acid can be obtained.

  Here, the above-mentioned proteolytic enzyme is not particularly limited as long as it is capable of degrading proteins bound to hyaluronic acid in serum and does not contain hyaluronic acid degrading enzyme. Any proteolytic enzyme can be used. Specific examples of the above-mentioned proteolytic enzymes are preferably serine proteinases, metalloproteinases, thiol proteinases, aspartic proteinases and the like because they are easily available and proteolytic. Among these proteolytic enzymes, serine proteinase, particularly proteinase K, is preferably used because it is particularly excellent in availability and proteolytic properties.

  At this time, the pretreatment with proteinase K and other proteolytic enzymes is carried out with 0.02 w / v% of proteolytic enzymes so that the proteolytic reaction proceeds sufficiently and there is no adverse effect due to too much proteolytic enzymes. It is preferable to add ˜1 w / v%, and it is particularly preferable to add 0.05 w / v% to 0.2 w / v% by volume of proteolytic enzyme.

  At this time, pretreatment with proteinase K and other proteolytic enzymes is preferably performed within a range of 20 ° C. to 60 ° C. so that the activity of the proteolytic enzymes is increased and the proteolytic enzymes are not inactivated. It is particularly preferable to carry out within the range of from 50 ° C to 50 ° C.

  In addition, this pretreatment is preferably performed within a range of 6 hr to 48 hr, particularly preferably within a range of 12 hr to 24 hr so that the proteolytic reaction proceeds sufficiently and the proteolytic enzyme is not inactivated. . The pretreatment reaction solution preferably has a pore size of 0.1 μm to 1 μm, particularly preferably a pore size of 0.2 μm in order to remove precipitates and the like due to the pretreatment reaction before proceeding to the next molecular weight fractionation step. It is desirable to perform centrifugal filtration with a membrane filter of ˜0.6 μm.

(Ii) Molecular Weight Fractionation Step In the step 1 of the present embodiment, the molecular weight fractionation method of HA in a serum sample is not particularly limited as long as it can fractionate HA in serum based on the difference in molecular weight. Any conventionally known molecular weight fractionation method can be used. Specific examples of this molecular weight fractionation method are preferably size exclusion chromatography, field flow fractionation, electrophoresis, ultracentrifugation, and the like because of their ease of execution and fractionation performance. Among these molecular weight fractionation methods, a method using high-performance liquid chromatography is preferable because of particularly excellent ease of execution and fractionation performance, and size exclusion chromatography is most preferable. When performing size exclusion chromatography, if necessary, two or more types of size exclusion chromatography columns (for example, Shodex OHpak SB-806HQ, Asahipak GS) are used in order to obtain good separation from high molecules to low molecules. -820HQ or the like) may be connected in series.

  Moreover, in the above-mentioned step 1, when using size exclusion chromatography, the mobile phase is not particularly limited as long as it can separate hyaluronic acid by molecular weight, and can generally be used for size exclusion chromatography. A mobile phase can be used. Specific examples of the mobile phase described above include water, phosphate buffer, sodium chloride-added phosphate buffer, and acetate buffer because of excellent availability, mobility and separation properties, and the ability to denature hyaluronic acid. Citrate buffer, borate buffer, lithium chloride aqueous solution, sodium chloride aqueous solution, potassium chloride aqueous solution, sodium nitrate aqueous solution, sodium carbonate aqueous solution, sodium acetate aqueous solution, ammonium acetate aqueous solution, ammonium formate aqueous solution, ammonium bicarbonate aqueous solution, tris buffer A solution obtained by adding an organic solvent miscible with water, such as ethanol, methanol, acetonitrile, etc., at an arbitrary ratio can be used. Among these mobile phases, it is preferable to use a phosphate buffer solution, an aqueous ammonium acetate solution, an aqueous ammonium formate solution, and an aqueous ammonium bicarbonate solution because it is particularly excellent in mobile characteristics and separation characteristics. Further, by using an ammonium acetate aqueous solution, an ammonium formate aqueous solution, or an ammonium bicarbonate aqueous solution as the mobile phase described above, it is possible to volatilize and remove the salt in the fraction containing HA in the concentration operation of Step 2.

  Here, when HPLC is performed for molecular weight fractionation, the fraction size for performing molecular weight fractionation is improved in accuracy of molecular weight fractionation, and the operation is saved, and each fraction is further reduced. In order to obtain a sufficient amount of hyaluronic acid exceeding the detection limit, fractionation is preferably performed at a size of 50 μL to 500 μL / fraction, particularly preferably at a size of 200 μL to 300 μL / fraction. However, when HPLC is performed for molecular weight fractionation, a sufficient amount of hyaluronic acid does not elute at the beginning and end of the elution time, so fractionation may be omitted for these. it can.

(Iii) Concentration step In the step 2 of the present invention, the method for concentrating the fraction containing HA is not particularly limited as long as it can stably concentrate HA in the fraction obtained by molecular weight fractionation. Any known concentration method can be used. Specific examples of the above-described concentration method include ease of operation and stability of hyaluronic acid, shortening the concentration time, and further reducing the cost of concentration. It is preferable to use a drying method, a salting-out method, a solvent precipitation method, an isoelectric point precipitation method, or the like. Among these concentration methods, since the ease of operation and the stability of hyaluronic acid are particularly excellent, it is particularly preferable to use a vacuum drying method or a freeze drying method.

  Here, in the case of evaporating to dryness with a centrifugal evaporator, it is necessary to evaporate and dry sufficiently to remove moisture and further heat it more than necessary to prevent denaturation of hyaluronic acid. It is preferable to evaporate and dry within the range of, and it is particularly preferable to evaporate and dry at 50 ° C to 70 ° C. Further, it is preferable to evaporate and dry within a range of 1 hr to 10 hr in order to sufficiently evaporate and dry to remove water, and further heat it more than necessary to prevent denaturation of hyaluronic acid. It is particularly preferred to evaporate to dryness within the range.

(Iv) Quantification step Furthermore, in the above-mentioned step 3, the method for quantifying the HA concentrated in step 2 is not particularly limited as long as HA can be quantified with high sensitivity, and any conventionally known quantification method can be used. Can be used. Specific examples of the above-described quantification method include a method using hyaluronic acid binding protein (HABP), a post-column fluorescence HPLC method (microorganism-derived hyaluronic acid-degrading enzyme) because of its excellent sensitivity and quantification and easy operation. Is used to digest the HA to disaccharide units to obtain unsaturated disaccharide HA.After separating the unsaturated disaccharide HA and contaminants on the HPLC column, the unsaturated disaccharide HA is derivatized with a fluorescent dye to increase the fluorescence intensity. Measurement method), pre-column fluorescence HPLC method (method of digesting HA to disaccharide units using microorganism-derived hyaluronic acid-degrading enzyme, derivatizing with fluorescent dye, and separating and quantifying with HPLC column), anti-HA antibody It is preferable to use the immunological method used. Among these quantification methods, it is particularly preferable to use a method using HABP or a post-column fluorescence HPLC method because it is particularly excellent in sensitivity and quantification.

(V) Molecular Weight Distribution Reconstruction Step Further, as a method for determining the molecular weight and molecular weight distribution of HA in serum from the amount of HA in the fraction obtained by molecular weight fractionation in Step 4 of the present invention, the molecular weight of HA in serum is as follows. If distribution can be expressed appropriately, it will not specifically limit, Arbitrary well-known arithmetic methods and statistical methods can be used. As a specific example of the method for determining the molecular weight distribution described above, it is excellent in quantification of the molecular weight of HA in serum, is easy to measure and calculate, can easily draw a graph, and can intuitively grasp the molecular weight distribution. The molecular weight of each fraction was determined by measuring the HA concentration of fractions obtained by fractionating HA standards of various molecular weights, and based on this, the molecular weight and molecular weight were determined from the HA concentration profile of the serum-derived sample fraction. One method is to determine the distribution.

  At this time, the HA standard solution preferably has an HA concentration in the range of about 100 ng / mL to 2,000 ng / mL so that sufficient detection and quantification is possible, and the HA concentration is about 300 ng / mL to 1,000 ng. It is particularly preferable to prepare a standard solution within the range of / mL and draw a calibration curve. In addition, in order to save more time than necessary while being capable of sufficient detection and quantification, 3 to 10 standard solutions with appropriate intervals within a viscosity average molecular weight range of about 10,000 to 10 million. It is particularly preferable to prepare a standard curve of 4 to 5 points and draw a calibration curve while leaving an appropriate interval within a range of viscosity average molecular weight of about 50,000 to 5,000,000.

  However, it is not always necessary to obtain the molecular weight of each fraction by measuring the HA concentration of the fraction obtained by fractionating the HA standard products of various molecular weights each time. If the HA concentration of the fraction obtained by fractionation is measured and recorded, the molecular weight and molecular weight distribution can be obtained from the HA concentration profile of the serum-derived sample fraction from the next time by using the measurement result. You may use it. The record of measuring the HA concentration of the fractions obtained by fractionating the HA standard products of various molecular weights may be described as numerical data or graph data on paper, and the numerical data or graph data is recorded on the recording medium. May be stored electromagnetically.

Hereinafter, the effect of this embodiment is demonstrated.
The measuring method according to the present embodiment is a method for measuring the molecular weight distribution of hyaluronic acid in mammal-derived serum, and includes a step of fractionating serum in molecular weight and each fraction containing hyaluronic acid that has been subjected to molecular weight fractionation. Concentrates the concentration of hyaluronic acid even if the concentration of hyaluronic acid is reduced during molecular weight fractionation because it includes a step of concentrating and a step of quantifying the amount of hyaluronic acid in each concentrated fraction Therefore, the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity. Therefore, according to this method, it is possible to efficiently measure the distribution pattern of the molecular weight of hyaluronic acid from a small amount of mammal-derived serum of 1 mL or less without using a radioisotope. That is, according to this method, the molecular weight and molecular weight distribution of hyaluronic acid in serum can be measured with a serum volume of 1 mL or less.

  In addition, the measurement method of the present embodiment may further include a step of pretreating with the proteolytic enzyme in advance before the above-described serum is subjected to molecular weight fractionation. In this way, since the protein mixed with hyaluronic acid in the serum can be degraded, the recovery rate of hyaluronic acid can be improved and the influence of contaminants (proteins) can be removed. The measurement result of the molecular weight distribution of hyaluronic acid is obtained.

  In the measurement method of the present embodiment, the molecular weight fractionation step described above may include a step of molecular weight fractionation of serum by size exclusion chromatography. In this way, HA in serum can be fractionated with high accuracy by a difference in molecular weight by a simple operation.

  In the measurement method of the present embodiment, the concentration step described above may include a step of drying each fraction containing hyaluronic acid having a molecular weight fractionated under reduced pressure. In this way, HA in the fraction obtained by molecular weight fractionation by a simple operation can be stably concentrated.

  Further, in the measurement method of the present embodiment, the step of quantifying may include a step of quantifying hyaluronic acid in each concentrated fraction using a hyaluronic acid binding protein. In this way, the fractionated and concentrated HA can be quantified with high sensitivity by a simple operation.

  In addition, the measurement method of the present embodiment may further include a step of calculating the molecular weight distribution of hyaluronic acid in serum based on the amount of hyaluronic acid determined for each fraction. In this way, the molecular weight distribution of HA in serum can be properly grasped by a simple arithmetic method.

<Embodiment 2: Method for determining whether or not the patient has rheumatoid arthritis disease>
The determination method according to the present embodiment is a determination method for analyzing serum derived from a mammal and determining whether the serum is collected from a mammal suffering from rheumatoid arthritis disease. In this determination method, the molecular weight distribution of hyaluronic acid in the above-mentioned mammal-derived serum is measured using the measurement method according to Embodiment 1, and then the measured molecular weight distribution and normal of the same kind as the above-described mammal are used. We compared the molecular weight distribution of hyaluronic acid in serum derived from mammals, and then, based on the comparison result of the molecular weight distribution of hyaluronic acid, whether the above-mentioned serum was collected from mammals suffering from rheumatoid arthritis disease Judge whether or not.

  According to this method, in order to measure the molecular weight distribution of hyaluronic acid in mammal-derived serum using the measurement method described above, hyaluronic acid can be obtained from a small amount of mammal-derived serum of 1 mL or less without using a radioisotope. It is possible to efficiently measure the molecular weight distribution pattern. Therefore, by comparing the molecular weight distribution measured for the serum from the mammal as the subject and the molecular weight distribution of hyaluronic acid in the serum from the normal mammal of the same species as the subject mammal, It is possible to determine whether or not serum derived from a mammal has been collected from a mammal suffering from rheumatoid arthritis disease.

  Here, in the comparison result of the molecular weight distribution of hyaluronic acid, the determination step described above includes the first peak in the molecular weight range of 50,000 to 300,000 and the second peak in the molecular weight range of 500,000 to 2,000,000. It is preferable to include a step of determining whether or not the above-mentioned serum is collected from a mammal suffering from rheumatoid arthritis disease based on whether or not the presence or absence matches.

  As will be described in the Examples below, the present inventors have made it possible to analyze the molecular weight distribution of hyaluronic acid in serum collected from mammals that has been made possible for the first time by the measurement method of Embodiment 1, and thus the present inventors Discovered that the molecular weight distribution of hyaluronic acid in serum collected from can have a first peak in the range of 50,000 to 300,000 and a second peak in the range of 500,000 to 2 million doing. Whether or not the first peak and the second peak are the same is useful in determining whether the serum is collected from a mammal suffering from rheumatoid arthritis disease. It has been suggested. Therefore, in this way, whether the serum was collected from a mammal suffering from rheumatoid arthritis disease based on whether the presence or absence of these first and second peaks coincided It is thought that it can be determined whether or not.

  In addition, in the above-described determination step, in the comparison result of the molecular weight distribution of the hyaluronic acid, in addition to whether or not the first peak and the second peak are the same, Preferably, the method includes determining whether the above-mentioned serum is collected from a mammal suffering from rheumatoid arthritis disease based on the ratio of the height of the first peak and the second peak.

  As will be described in the Examples below, the present inventors have made it possible to analyze the molecular weight distribution of hyaluronic acid in serum collected from mammals that has been made possible for the first time by the measurement method of Embodiment 1, and thus the present inventors Discovered that the molecular weight distribution of hyaluronic acid in serum collected from can have a first peak in the range of 50,000 to 300,000 and a second peak in the range of 500,000 to 2 million doing. The ratio of the heights of these first and second peaks (the absolute value of the HA concentration) is then used to determine whether the serum was collected from a mammal suffering from rheumatoid arthritis disease. Has been suggested to be useful. Therefore, in this way, in addition to whether or not the first peak and the second peak are the same, based on the ratio of the heights of the first peak and the second peak. Thus, it can be determined whether the serum was collected from a mammal suffering from rheumatoid arthritis disease.

  The index that can be used to determine whether or not the above-described serum is collected from a mammal suffering from rheumatoid arthritis disease is limited to the above-described index related to the molecular weight distribution of hyaluronic acid in the serum. Needless to say, the accuracy of determination of the presence or absence of rheumatoid arthritis disease can be improved by combining with other indicators. For example, the average concentration of hyaluronic acid in serum (concentration measured without molecular weight fractionation) may be combined, or may be combined with subjective knowledge such as interviews and observations by medical staff such as doctors. Good.

<Embodiment 3: Method for determining which type of rheumatoid arthritis disease is affected>
The determination method according to the present embodiment analyzes a serum derived from a mammal, and determines whether the serum is collected from a mammal suffering from rheumatoid arthritis disease of a plurality of types. This is a determination method. In this determination method, the molecular weight distribution of hyaluronic acid in the above-mentioned mammal-derived serum is measured using the measurement method according to Embodiment 1, and then the measured molecular weight distribution and a plurality of the same species as the above-mentioned mammal are measured. Comparing the molecular weight distribution of hyaluronic acid in sera from mammals suffering from a type of rheumatoid arthritis disease, and subsequently comparing the molecular weight distribution of hyaluronic acid to Determine which type of rheumatoid arthritis disease was collected from the mammal.

  According to this method, in order to measure the molecular weight distribution of hyaluronic acid in mammal-derived serum using the measurement method described above, hyaluronic acid can be obtained from a small amount of mammal-derived serum of 1 mL or less without using a radioisotope. It is possible to efficiently measure the molecular weight distribution pattern. Therefore, the molecular weight distribution measured for serum from the subject mammal and the molecular weight of hyaluronic acid in the serum from mammals suffering from multiple types of rheumatoid arthritis disease of the same type as the subject mammal By comparing the distributions, it is possible to determine which type of serum from the mammal as the subject has been collected from a mammal suffering from rheumatoid arthritis disease.

  Here, the above-described determination step includes the first peak in the molecular weight range of 50,000 to 300,000 and the second peak in the molecular weight range of 500,000 to 2,000,000 in the comparison result of the molecular weight distribution of the hyaluronic acid. Preferably, the method includes a step of determining which type of biological fluid sample is collected from a mammal suffering from rheumatoid arthritis disease among a plurality of types based on whether the presence or absence of peaks is consistent. .

  As will be described in the Examples below, the present inventors have made it possible to analyze the molecular weight distribution of hyaluronic acid in serum collected from mammals that has been made possible for the first time by the measurement method of Embodiment 1, and thus the present inventors Discovered that the molecular weight distribution of hyaluronic acid in serum collected from can have a first peak in the range of 50,000 to 300,000 and a second peak in the range of 500,000 to 2 million doing. Whether or not the first peak and the second peak coincide with each other is determined whether the serum is collected from a mammal suffering from any type of rheumatoid arthritis disease. It is suggested that it may be useful in determining whether or not. Therefore, in this way, based on whether or not the first peak and the second peak coincide with each other, the serum suffers from any type of rheumatoid arthritis disease among a plurality of types. It can be judged whether it has been collected from a mammal.

  In addition, in the above-described determination step, in the comparison result of the molecular weight distribution of the hyaluronic acid, in addition to whether or not the first peak and the second peak are the same, Determining, based on the ratio of the height of the first peak and the second peak, whether the above-mentioned serum was collected from a mammal suffering from rheumatoid arthritis disease of a plurality of types It is preferable.

  As will be described in the Examples below, the present inventors have made it possible to analyze the molecular weight distribution of hyaluronic acid in serum collected from mammals that has been made possible for the first time by the measurement method of Embodiment 1, and thus the present inventors Discovered that the molecular weight distribution of hyaluronic acid in serum collected from can have a first peak in the range of 50,000 to 300,000 and a second peak in the range of 500,000 to 2 million doing. And the ratio of the height of these first peak and second peak (the absolute value of the HA concentration) is such that the serum has any type of rheumatoid arthritis disease among a plurality of types. It has been suggested that it may be useful in determining whether it was collected from Therefore, in this way, in addition to whether or not the first peak and the second peak are the same, based on the ratio of the heights of the first peak and the second peak. Thus, it is considered that it is possible to determine which of a plurality of types of serum was collected from a mammal suffering from rheumatoid arthritis disease.

  Here, the multiple types of rheumatoid arthritis diseases are not particularly limited, and are classified into different types of rheumatoid arthritis diseases based on some physiological findings, pathological findings, and clinical findings. Any type can be used as long as it can be used.

  For example, depending on the progress of rheumatoid arthritis disease (for example, reduction of cartilage inside the joint, progress of pain, etc.), the rheumatoid arthritis disease is (i) early, (ii) middle, (iii) late, ( iv) You may classify into the last four. Or, according to sex, age, etc. of patients with rheumatoid arthritis disease, (i) young male type, (ii) young female type, (iii) old male type, (iv) old female type May be. And (i) the first peak of hyaluronic acid increases in the early stage of rheumatoid arthritis disease, (ii) the second peak of hyaluronic acid increases in the middle period, and (iii) the first peak of hyaluronic acid in the later stage. If the peak decreases and (iv) the second peak of hyaluronic acid decreases at the end stage, serum hyaluron collected from the mammal of the subject in light of such variation in the molecular weight distribution of hyaluronic acid The molecular weight distribution of the acid may be analyzed to determine the type of the subject rheumatoid arthritis disease. In any case, such a suitable classification will be clarified by future research and development in the fields of medicine, pharmacy, biochemistry, etc., and the determination method according to the present embodiment, which is an idea of the present inventors, is provided. It is assumed that it will be used suitably.

  The index that can be used to determine whether the above-mentioned serum is collected from a mammal suffering from rheumatoid arthritis disease of a plurality of types is the molecular weight of hyaluronic acid in the above-mentioned serum Needless to say, the present invention is not limited to the distribution-related index, and can be combined with another index to increase the accuracy of determination of the type of rheumatoid arthritis disease. For example, the average concentration of hyaluronic acid in serum (concentration measured without molecular weight fractionation) may be combined, or may be combined with subjective knowledge such as interviews and observations by medical staff such as doctors. Good.

<Embodiment 4: Experimental set for measurement of molecular weight distribution of hyaluronic acid>
The experimental set of this embodiment is an experimental set including a reagent and a column for measuring the molecular weight distribution of hyaluronic acid in a biological fluid sample derived from a mammal, and includes a proteolytic enzyme, a size exclusion chromatography column, Hyaluronic acid binding protein.

  According to this experimental set, serum pre-treated with a proteolytic enzyme was subjected to molecular weight fractionation using a size exclusion chromatography column, and each fraction containing hyaluronic acid fractionated with molecular weight was then analyzed in a laboratory or the like. Using a suitable device such as a centrifugal evaporator, the mixture is concentrated by drying under reduced pressure or the like, and the amount of hyaluronic acid in each concentrated fraction can be quantified using hyaluronic acid binding protein.

  Therefore, according to this experimental set, even when the concentration of hyaluronic acid is reduced during molecular weight fractionation using a size exclusion chromatography column, the concentration of hyaluronic acid is reduced using an appropriate device such as a centrifugal evaporator. Since the concentration can be increased by drying and concentrating, the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity using a hyaluronic acid binding protein. Therefore, according to this experimental set, it is possible to efficiently measure the molecular weight distribution pattern of hyaluronic acid from a small amount of mammal-derived serum of 1 mL or less without using a radioisotope.

<Embodiment 5: Experimental set for determining morbidity of rheumatoid arthritis disease>
The experimental set according to the present embodiment is an experimental set including a reagent and a column for analyzing a serum derived from a mammal and determining whether the serum is collected from a mammal suffering from rheumatoid arthritis disease. Proteolytic enzyme, size exclusion chromatography column, hyaluronic acid binding protein, serum from normal mammal of the same species as mammals, or molecular weight distribution data of hyaluronic acid obtained from the serum, Including.

  According to this experimental set, serum pre-treated with a proteolytic enzyme was subjected to molecular weight fractionation using a size exclusion chromatography column, and each fraction containing hyaluronic acid fractionated with molecular weight was then analyzed in a laboratory or the like. Using a suitable device such as a centrifugal evaporator, the mixture is concentrated by drying under reduced pressure or the like, and the amount of hyaluronic acid in each concentrated fraction can be quantified using hyaluronic acid binding protein.

  Therefore, according to this experimental set, even when the concentration of hyaluronic acid is reduced during molecular weight fractionation using a size exclusion chromatography column, the concentration of hyaluronic acid is reduced using an appropriate device such as a centrifugal evaporator. Since the concentration can be increased by drying and concentrating, the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity using a hyaluronic acid binding protein. Therefore, according to this experimental set, it is possible to efficiently measure the molecular weight distribution pattern of hyaluronic acid from a small amount of mammal-derived serum of 1 mL or less without using a radioisotope.

  According to this experimental set, serum from normal mammals of the same species as the subject mammal or molecular weight distribution data of hyaluronic acid obtained from the serum is included. The molecular weight distribution data of hyaluronic acid in serum derived from normal mammals of the same species as the sample mammal can be obtained. Therefore, by comparing the molecular weight distribution measured for the serum from the mammal as the subject and the molecular weight distribution of hyaluronic acid in the serum from the normal mammal of the same species as the subject mammal, It is possible to determine whether or not serum derived from a mammal has been collected from a mammal suffering from rheumatoid arthritis disease.

<Embodiment 6: Experimental set for determining which type of rheumatoid arthritis disease is affected>
The experimental set according to the present embodiment is for analyzing mammal-derived serum to determine which type of mammal from which rheumatoid arthritis disease is collected among a plurality of types. An experimental set comprising reagents and columns, derived from a mammal suffering from multiple types of rheumatoid arthritis disease of the same type as mammals, including proteolytic enzymes, size exclusion chromatography columns, hyaluronic acid binding proteins, and mammals And molecular weight distribution data of hyaluronic acid obtained from that serum.

  According to this experimental set, serum pre-treated with a proteolytic enzyme was subjected to molecular weight fractionation using a size exclusion chromatography column, and each fraction containing hyaluronic acid fractionated with molecular weight was then analyzed in a laboratory or the like. Using a suitable device such as a centrifugal evaporator, the mixture is concentrated by drying under reduced pressure or the like, and the amount of hyaluronic acid in each concentrated fraction can be quantified using hyaluronic acid binding protein.

  Therefore, according to this experimental set, even when the concentration of hyaluronic acid is reduced during molecular weight fractionation using a size exclusion chromatography column, the concentration of hyaluronic acid is reduced using an appropriate device such as a centrifugal evaporator. Since the concentration can be increased by drying and concentrating, the amount of hyaluronic acid in each concentrated fraction can be quantified with high sensitivity using a hyaluronic acid binding protein. Therefore, according to this experimental set, it is possible to efficiently measure the molecular weight distribution pattern of hyaluronic acid from a small amount of mammal-derived serum of 1 mL or less without using a radioisotope.

  According to this experimental set, serum from mammals suffering from multiple types of rheumatoid arthritis diseases of the same type as the subject mammal or molecular weight distribution data of hyaluronic acid obtained from the serum are obtained. In any case, molecular weight distribution data of hyaluronic acid in sera from mammals suffering from multiple types of rheumatoid arthritis diseases of the same type as the subject mammal can be obtained. Therefore, the molecular weight distribution measured for serum from the subject mammal and the molecular weight of hyaluronic acid in the serum from mammals suffering from multiple types of rheumatoid arthritis disease of the same type as the subject mammal By comparing the distributions, it is possible to determine which type of serum from the mammal as the subject has been collected from a mammal suffering from rheumatoid arthritis disease.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

  For example, in the above embodiment, the biological fluid sample is serum, but it is not particularly limited. For example, blood, lymph, tissue fluid that fills between cells outside the blood vessels, and body cavity fluid in the body cavity may be used. Even in this case, blood and lymph fluid, tissue fluid filling between cells outside the blood vessels, and body cavity fluid in the body cavity mutually exchange components in the living body, so that the hyaluron is similar to the above embodiment. It is envisaged that it will be possible to determine the molecular weight distribution of the acid and to determine the presence or type of chronic rheumatic disease.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Example 1>
25 mg of proteinase K (manufactured by Roche Diagnostics) was dissolved in 2.5 mL of distilled water to prepare an enzyme solution. 180 μL of RA patient serum (case 1, HA concentration: about 320 ng / mL) was collected, 18 μL of enzyme solution was added, and the mixture was allowed to stand at 40 ° C. for 16 hours. After completion of the reaction, the enzyme was boiled at 100 ° C. for 10 minutes to completely deactivate the enzyme.

  The reaction solution was subjected to centrifugal filtration with a membrane filter having a pore size of 0.45 μm, and fractionated by size exclusion chromatography (SEC).

Separation condition column of SEC: Shodex OHpak SB-806HQ and Asahipak GS-820HQ connected in series Column temperature: 40 ° C
Flow rate: 0.6 mL / min Mobile phase: 50 mM sodium phosphate buffer (pH 6.0)
Injection volume: 100 μL
Analysis time: 60 min
Fractionation: 270 μL / fraction

  Each fraction obtained by fractionation by SEC was evaporated to dryness at 60 ° C. for 3 hr by a centrifugal evaporator.

  The dried solid product of each fraction was redissolved with 55 μL of a reaction buffer solution of a hyaluronic acid measurement kit (manufactured by Seikagaku Biobusiness, Inc., a method using HABP), and the HA concentration was quantified according to the protocol of the hyaluronic acid measurement kit.

  A SEC chromatogram prepared from the obtained HA concentration and the elution time of each fraction is shown in FIG.

  Using HA standard solutions with a concentration of about 500 ng / mL and viscosity average molecular weights of about 100,000, 400,000, 900,000, and 2.1 million, the same operation as for serum samples was performed, and the peak top of the chromatogram of each HA standard solution A calibration curve of viscosity average molecular weight prepared from the elution time is shown in FIG.

  A molecular weight distribution chart reconstructed by converting the horizontal axis of the chromatogram of the serum sample from the elution time to the viscosity average molecular weight using the calibration curve of the viscosity average molecular weight is shown in FIG. The result was a broad molecular weight distribution with peaks in the viscosity average molecular weights of 200,000 and 1 million.

<Example 2>
Proteinase K (manufactured by Roche Diagnostics) was dissolved in 2.5 mL of distilled water to prepare an enzyme solution. 180 μL of RA patient serum (Case 2, HA concentration: about 360 ng / mL) was collected, 18 μL of enzyme solution was added, and the mixture was allowed to stand at 40 ° C. for 16 hours. After completion of the reaction, the enzyme was boiled at 100 ° C. for 10 minutes to completely deactivate the enzyme.

  The reaction solution was subjected to centrifugal filtration with a membrane filter having a pore size of 0.45 μm, and fractionated by SEC under the same conditions as in Example 1.

  Each fraction obtained by fractionation by SEC was evaporated to dryness at 60 ° C. for 3 hr by a centrifugal evaporator.

  Chondroitinase ACII Arsulo (Seikagaku Biobusiness Co., Ltd., a microorganism that degrades HA to unsaturated disaccharides) prepared to 1 mU / mL with 50 mM phosphate buffer (pH 6.0) was added to the dried product of each fraction. 30 μL of hyaluronic acid-degrading enzyme) was added, and the mixture was allowed to stand in a 40 ° C. water bath for 3 hours to carry out the decomposition reaction of HA. The amount of unsaturated disaccharide HA was measured using a post-column fluorescence HPLC method.

HPLC condition column: CAPCELL PAK NH2 UG80 (250 mm × 1.5 mm ID)
Column temperature: 30 ° C
Eluent: 0.15M-KH2PO4
Eluent flow rate: 0.1 mL / min
Injection volume: 3 μL
Reaction liquid: 0.125M-sodium tetraborate / 1M-NaOH / 0.5M 2-cyanoacetamide Reaction liquid flow rate: 0.04 mL / min each
Reaction temperature: 130 ° C
Detection: Fluorescence EX. 346 nm EM. 410nm
Analysis time: 20 min
Unsaturated disaccharide HA preparation: HA preparation having a known concentration (viscosity average molecular weight 2.1 million) obtained by digestion with chondroitinase ACII Arsulo (0.5, 1.0, 2.5, 5.0, 10ng / 3μL)

  From the obtained unsaturated disaccharide HA concentration and elution time of each fraction, a SEC chromatogram was prepared.

  Using HA standard solutions with a concentration of about 500 ng / mL and viscosity average molecular weights of about 100,000, 400,000, 900,000, and 2.1 million, the same operation as for serum samples was performed, and the peak top of the chromatogram of each HA standard solution A calibration curve of viscosity average molecular weight was prepared from the elution time. FIG. 4 shows a molecular weight distribution chart reconstructed by converting the horizontal axis of the chromatogram of the serum sample from the elution time to the viscosity average molecular weight using the calibration curve of the viscosity average molecular weight. The result was a broad molecular weight distribution with peaks in the viscosity average molecular weights of 200,000 and 1 million.

<Example 3>
180 μL of RA patient serum (Case 3, HA concentration: about 770 ng / mL) was collected and processed in the same manner as in Example 1, and SEC chromatograms were prepared from the HA concentration and elution time of each fraction. A molecular weight distribution chart reconstructed by converting the horizontal axis of the chromatogram from the elution time to the viscosity average molecular weight is shown in FIG. The result was a molecular weight distribution having a peak in the vicinity of a viscosity average molecular weight of 100,000 to 200,000, but no peak was observed in the vicinity of a viscosity average molecular weight of 1,000,000 as in Case 1. This suggested that the HA viscosity average molecular weight in blood may reflect the joint condition in RA.

<Example 4>
180 μL of RA patient serum (case 4, HA concentration: about 400 ng / mL) was collected and treated in the same manner as in Example 1. However, the mobile phase of SEC was a 50 mM ammonium acetate solution. FIG. 5 shows a molecular weight distribution chart in which a SEC chromatogram was prepared from the HA concentration of each fraction and the elution time, and the horizontal axis of the serum sample chromatogram was converted from the elution time to the viscosity average molecular weight. The result was a molecular weight distribution having a peak in the vicinity of a viscosity average molecular weight of 200 to 300,000. Although there is no clear peak even in the vicinity of a viscosity average molecular weight of 1,000 to 2,000,000, a shoulder is observed, and it can be seen that a polymer HA is also present. Thus, it can be measured even when the mobile phase is changed to an ammonium acetate solution.

<Discussion>
All of Examples 1 to 4 have succeeded in graphing the molecular weight distribution of hyaluronic acid in serum using only 180 μL of RA patient serum. That is, in any of Examples 1 to 4, hyaluronic acid was removed from a small amount of serum from RA patients of 1 mL or less by a simple operation without using laboratory reagents and experimental equipment that are difficult to handle safely such as radioisotopes. The molecular weight distribution pattern can be measured efficiently.

  Moreover, the ratio of the presence or absence or height (absolute value of HA concentration) of the first peak and the second peak is changed depending on the case of Examples 1 to 4, and the HA viscosity average molecular weight in blood is changed. It is suggested that the distribution (pattern) may reflect the state of the joint in RA. That is, by analyzing the distribution (pattern) of HA viscosity average molecular weight in blood, it can be determined whether or not the subject is an RA patient. If the subject is an RA patient, what type of RA patient is used? It can be said that there is a possibility that it can be judged whether there is.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

  For example, in the above embodiment, the type of mammal used as a patient is human, but it is not limited to humans, and other mammals (mouse, rat, pig, rabbit, cow, goat, dog, cat, etc.) Needless to say, monkeys, etc.). However, it is preferable that the subject of measurement or determination for actual medical treatment is a human or a primate.

  Further, in the above examples, the viscosity average molecular weight was used as the molecular weight, but it is not intended to be limited to human beings in particular. Or if it can mutually convert, it can use suitably for the measurement of the molecular weight distribution of a hyaluronic acid similarly to a viscosity average molecular weight, or determination of the presence or absence or type | mold of a chronic rheumatic disease.

  According to the present invention, it is possible to obtain information on the blood kinetics of HA using a small amount of RA patient blood. As a result, the patient's blood is appropriately collected over a long period of time, and the progress of RA pathology Can be examined over time, and can greatly contribute to the diagnosis and treatment of RA patients and the elucidation of the onset mechanism of RA.

2 is a chromatogram created in Example 1. 2 is a calibration curve created in Example 1. 2 is a molecular weight distribution chart reconstructed in Example 1. 2 is a molecular weight distribution chart reconstructed in Example 2. 4 is a molecular weight distribution chart reconstructed in Example 3. 6 is a molecular weight distribution chart reconstructed in Example 4.

Claims (7)

A method for measuring the molecular weight distribution of hyaluronic acid in mammal-derived serum ,
Prior to molecular weight fractionation of the serum, pre-treatment with a proteolytic enzyme;
Molecular weight fractionation of the serum by size exclusion chromatography based on viscosity average molecular weight ;
Concentrating each fraction containing hyaluronic acid fractionated by molecular weight by drying under reduced pressure ;
Quantifying the amount of hyaluronic acid in each of the concentrated fractions using hyaluronic acid binding protein ;
Calculating the molecular weight distribution of hyaluronic acid in the serum based on the amount of hyaluronic acid in each of the quantified fractions;
Including a measuring method. A determination method for analyzing serum derived from a mammal and determining whether the serum is collected from a mammal suffering from rheumatoid arthritis disease,
Measuring the molecular weight distribution of hyaluronic acid in serum derived from the mammal using the measurement method according to claim 1 ;
Comparing the measured molecular weight distribution and the molecular weight distribution of hyaluronic acid in serum from a normal mammal of the same species as the mammal;
Determining whether the serum is collected from a mammal suffering from rheumatoid arthritis disease based on the comparison result of the molecular weight distribution of the hyaluronic acid;
Only including,
In the determination result, the comparison result of the molecular weight distribution of the hyaluronic acid indicates whether the first peak in the molecular weight range of 50,000 to 300,000 and the second peak in the molecular weight range of 500,000 to 2,000,000 are present. A determination method comprising a step of determining whether or not the serum is collected from a mammal suffering from rheumatoid arthritis disease based on whether or not it has done . In the determination result, in the comparison result of the molecular weight distribution of the hyaluronic acid, in addition to whether or not the first peak and the second peak are coincident, the second molecular weight distribution in the measured molecular weight distribution is determined. 3. The determination of claim 2 , comprising determining whether the serum has been collected from a mammal suffering from rheumatoid arthritis disease based on the ratio of the height of one peak and the second peak. Method. A determination method for analyzing serum derived from a mammal and determining whether the serum was collected from a mammal suffering from rheumatoid arthritis disease of a plurality of types,
Measuring the molecular weight distribution of hyaluronic acid in serum derived from the mammal using the measurement method according to claim 1 ;
Comparing the measured molecular weight distribution and the molecular weight distribution of hyaluronic acid in serum from a mammal suffering from the multiple types of rheumatoid arthritis disease of the same species as the mammal;
Determining whether the serum was collected from a mammal suffering from rheumatoid arthritis disease of a plurality of types based on the comparison result of the molecular weight distribution of the hyaluronic acid;
Only including,
In the determination result, the comparison result of the molecular weight distribution of the hyaluronic acid indicates whether the first peak in the molecular weight range of 50,000 to 300,000 and the second peak in the molecular weight range of 500,000 to 2,000,000 are present. A determination method comprising a step of determining whether the serum is collected from a mammal suffering from rheumatoid arthritis disease of a plurality of types based on whether or not the serum has been used. In the determination result, in the comparison result of the molecular weight distribution of the hyaluronic acid, in addition to whether or not the first peak and the second peak are coincident, the second molecular weight distribution in the measured molecular weight distribution is determined. Determining whether the serum was collected from a mammal suffering from rheumatoid arthritis disease of a plurality of types based on the ratio of the height of one peak and the second peak The determination method according to claim 4 . An experimental set comprising a reagent and a column for analyzing serum from a mammal to determine whether the serum has been collected from a mammal suffering from rheumatoid arthritis disease,
With proteolytic enzymes,
A size exclusion chromatography column;
Hyaluronic acid binding protein;
Serum from a normal mammal of the same species as the mammal or hyaluronic acid molecular weight distribution data obtained from the serum ,
Only including,
Before the mammal-derived serum is subjected to molecular weight fractionation, it is pretreated with the proteolytic enzyme in advance,
The serum is molecular weight fractionated by the size exclusion chromatography,
The amount of hyaluronic acid in each fraction concentrated by vacuum drying is quantified by the hyaluronic acid binding protein,
The molecular weight distribution calculated based on the amount of hyaluronic acid in each quantified fraction and the molecular weight distribution of hyaluronic acid in serum from normal mammals of the same species as the mammal are compared,
In the comparison result of the molecular weight distribution of the hyaluronic acid, whether or not the first peak in the molecular weight range of 50,000 to 300,000 and the second peak in the molecular weight range of 500,000 to 2,000,000 match. Based on, it is determined whether the serum was collected from a mammal suffering from rheumatoid arthritis disease,
Experimental set. An experimental set comprising reagents and columns for analyzing sera from mammals to determine which of a plurality of types of sera was collected from mammals suffering from rheumatoid arthritis disease. And
With proteolytic enzymes,
A size exclusion chromatography column;
Hyaluronic acid binding protein;
And molecular weight distribution data of hyaluronic acid obtained from the serum or the serum from a mammal suffering from rheumatoid arthritis disease plurality of types of said mammal same kind,
Only including,
Before the mammal-derived serum is subjected to molecular weight fractionation, it is pretreated with the proteolytic enzyme in advance,
The serum is molecular weight fractionated by the size exclusion chromatography,
The amount of hyaluronic acid in each fraction concentrated by vacuum drying is quantified by the hyaluronic acid binding protein,
Calculated based on the amount of hyaluronic acid in each quantified fraction, the molecular weight distribution of hyaluronic acid in the serum,
The molecular weight distribution calculated based on the amount of hyaluronic acid in each of the quantified fractions and the hyaluronic acid concentration in serum from mammals suffering from the above-mentioned multiple types of rheumatoid arthritis diseases of the same species as the mammal Molecular weight distributions are compared,
In the comparison result of the molecular weight distribution of the hyaluronic acid, whether or not the first peak in the molecular weight range of 50,000 to 300,000 and the second peak in the molecular weight range of 500,000 to 2,000,000 match. Based on the above, it is determined whether the serum is collected from a mammal suffering from rheumatoid arthritis disease of which type among a plurality of types,
Experimental set.

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