JP2000039435A - Periodontal pocket elastase measuring reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply, accurately measure an elastase originated by granulocyte in a periodontal pocket collecting sample in a short time, by using a periodontal pocket content diluent liquid and granulocytic elastase measuring reagent. SOLUTION: Since white blood cell or particularly granulocyte is early wetted to a gingival inner edge of a periodontal pocket prior to a periodontal change, a diagnosis of a periodontal disease can be rapidly and objectively diagnosed by measuring an elastase originated by the granulocyte. That is, a pediodontal pocket content diluent liquid and granylocytic elastase measuring reagent are used. Gingival groove liquid as a measuring sample, periodontal pocket plaque or the like as a measuring sample is collected by a manual scaler or the like, dissolved, suspended and diluted with diluent liquid. The dilutent liquid is buffer composition and contains, for example, phosphoric acid, tris-hydrochloric acid or the like. As an immulonogical measuring method of the elastase originated by the granulocyte, a particle slide aggregation method, particle turbidimetric method, a solid-phase immunological marker antibody analysis method, an immunochromatography or the like is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring periodontal pocket elastase which is useful for early diagnosis of periodontal disease such as alveolar pyorrhea.

[0002]

BACKGROUND OF THE INVENTION Alveolar pyorrhea affects more than 75% of adults and is considered as a national disease, and its prevention and treatment are regarded as important.

[0003] There are 20 teeth in children and 28 teeth in adults,
In healthy teeth, the roots of the teeth are firmly held in the alveolar bone of the chin bone through a strong muscular periodontal ligament, and the outside thereof is protected by gums.

The constricted part where the gums and teeth join is called a periodontal pocket, and the plaque accumulated in the periodontal pocket becomes tartar and causes dental caries and inflammation. If it becomes chronic or progressive, it will result in alveolar purulence.

[0005] When periodontal tissue is infected with periodontopathogenic bacteria,
Granulocytes such as neutrophils gather due to infiltration of leukocytes and try to stop the infection.However, if appropriate treatment and treatment are neglected, the gingival inner epithelium is gradually destroyed, and epithelial attachment to the tooth shifts to the apical direction. As a result, the periodontal pockets become deeper, forming a so-called cul-de-sac.

[0006] In order to treat the above-mentioned pathological periodontal pocket, various treatment methods such as surgical operation such as gingival resection, gingival exfoliation and curettage as well as calculus removal, root lubrication, washing, drug application, disinfection, and the like are available. Yes, which treatment method to use depends on the symptoms, but in any case, early diagnosis and early treatment are the most important and effective.

Methods for diagnosing periodontal disease include measuring the gingivitis index by visual observation by a dentist, measuring the depth of the periodontal pocket with a periodontal probe, and measuring the periodontal pocket temperature with a measuring needle having a temperature sensing element. There are methods for measurement, measurement of alveolar bone resorption by radiography, measurement of periodontal pathogenic bacteria, and the like.

[0008]

However, in these methods, there are individual differences in the criterion of the dentist, the measurement takes a long time, the diagnosis cost is high, and the inflammation site of periodontal disease is high. There is a problem that it is difficult to determine whether it is an active period or a stationary period.

[0009]

Prior to the periodontal lesion, the present inventors focused on the early infiltration of leukocytes, especially granulocytes, into the gingival inner edge of the periodontal pocket, and measured granulocyte-derived elastase. As a result, periodontal disease can be diagnosed quickly and objectively, and the present invention has been completed. That is, the present invention relates to a periodontal pocket elastase measurement reagent comprising a periodontal pocket contents diluent and a granulocyte elastase measurement reagent.

As a measurement sample, gingival crevicular fluid, periodontal exudate, periodontal pocket plaque and the like can be used, and these are collected, for example, with a hand scaler, and dissolved, suspended, and diluted with a diluent.

The diluent has a buffer composition commonly used, for example, phosphoric acid, Tris-HCl, glycine-NaOH
Etc., containing 0.15 M NaCl at pH 7-9,
Proteins such as BSA, casein, etc. can be added at from 0.5 to 0.5 (w / v)%.

The leukocyte components infiltrating periodontal tissue include:
45-65% neutrophils, 0-5% acid-fast spheres, 0-0.5% basophils, 3-10% monocytes, 20-4 lymphocytes
Neutrophils, acid-reactive spheres, and basophils, which are present in 0% each and have granules in cells, are called granulocytes. Therefore, granulocytes occupy 45 to 70% of leukocytes and contain a large amount of enzymes in cells. In particular, elastase has a strong proteolytic enzyme activity and has a large content. Can be determined.

[0013] Granulocytes phagocytose periodontopathogenic bacteria and the like, and repeatedly carry out enzymatic treatments such as elastase in vacuoles, but eventually die, and cell contents leak out. When the amount becomes large, it becomes pus, but flows into gingival crevicular fluid and periodontal exudate at an early stage. Granulocytes are activated in response to stimuli such as complement, antigen-antibody complex, endotoxin, and cytokines, and release granule components. Among these granule components, neutral protease and active oxygen have destructive and bactericidal effects on foreign substances, and granulocyte elastase is the most abundant neutral protease released and has strong enzyme activity. .

Granulocyte elastase has low substrate specificity and can easily decompose almost all biological constituent proteins such as elastin, collagen, proteoglycan, fibronectin, etc., so that α1-plasmin inhibitor, α2
-It is controlled by an inhibitor such as macroglobulin so as not to exert elastase activity more than necessary. On the other hand, the active oxygen itself has a strong bactericidal action, but also has a function of inactivating these inhibitors to maintain granulocyte elastase activity. As a result, a strong infection-protecting function is exerted in a very limited area only around the activated granulocytes.

[0015] In order to measure elastase in granulocytes mixed into gingival crevicular fluid or the like by infiltrating into periodontal tissue, granulocytes are hemolyzed and destroyed by a hemolytic agent, and the contents are released. Examples of the hemolytic agent include dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, saponin, lecithin, cholic acid, sodium dodecyl sulfate, polyoxyethylene octylphenyl ether, and polyoxyethylene sorbitol ester. The hemolytic agent is used as an aqueous solution having a concentration of 0.001 to 5 (W / V)%, preferably 0.05 to 1 (W / V)%. Alternatively, other general hemolysis means, for example, a method using ultrasonic waves or the like may be used.

Elastase derived from granulocyte has a molecular weight of 295
In the plasma and in body tissues, it is immediately inactivated by binding to inhibitors such as α1-plasmin inhibitor and α2-macroglobulin. Elastase-α1-
Plasmin inhibitor complex and elastase-α2-
The abundance ratio of the macroglobulin complex is 9: 1, and almost all are complexes with α1-plasmin inhibitor. α1-plasmin inhibitor has a molecular weight of 51,000
Therefore, the molecular weight of the elastase-α1-plasmin inhibitor complex is 80,500. Therefore, in order to quantitatively measure granulocyte elastase, it is desirable to keep the molecular weight of the measurement object constant.

Therefore, it is preferable to add α1-plasmin inhibitor to the diluent. Since α1-plasmin inhibitor is contained in a large amount in animal plasmas of rabbits, sheep, goats, cows, horses, etc., these animal plasmas may be added in an amount of 0.05 to 2 (V / V)%. Furthermore, since granulocyte elastase is easily adsorbed on the wall surface of a glass container, it is necessary to use a plastic container as a dilution container.

As a means for measuring elastase derived from granulocytes, it is desirable to use an immunological technique from the viewpoint of specificity and measurement accuracy. As the antibody against elastase, a polyclonal antibody obtained by purifying elastase by a known method, or using commercially available purified neutrophil-derived elastase and immunizing rabbits, goats, guinea pigs, chickens and the like may be used. In addition, anti-elastase monoclonal antibody produced from a hybridoma obtained by cell fusion of anti-elastase antibody-positive splenocytes obtained by immunizing a mouse and myeloma cells of the mouse with polyethylene glycol, one or two or more kinds are combined. May be used. For both polyclonal and monoclonal antibodies, self-prepared products or commercially available products can be used.

The immunoassay may be performed by insolubilizing an anti-elastase antibody in carrier particles such as latex particles, metal colloid particles, and liposomes. Enzyme immunoassay, chemiluminescence assay, fluorescence immunoassay using a solid-phased antibody insolubilizing the elastase antibody and a labeled antibody in which a labeling substance such as an enzyme, a chemiluminescent substance, a fluorescent substance, or a radioactive substance is bound to an anti-elastase antibody. Method, a solid-phase immunolabeling antibody analysis method such as a radioimmunoassay method, or an immunochromatography method can be used.

For example, in the particle slide agglutination method using latex, carrier particles are 3 μm or less, preferably 0.1 μm or less.
As a method for insolubilizing the anti-elastase antibody using latex particles of 2 to 1.0 μm, a general physical adsorption method or a chemical bonding method using a water-soluble carbodiimide or a bifunctional reagent is used. F (a) from which the Fc portion of the anti-elastase antibody was removed by pepsin treatment
b ′) 2 antibody fragments can also be used. When an elastase-containing sample and an anti-elastase antibody-sensitized latex test solution are mixed on a slide plate, an aggregate is formed by an antigen-antibody reaction between elastase and anti-elastase,
The presence or absence of aggregation is visually confirmed, and the presence or absence of elastase in the measurement sample is determined.

In the particle turbidimetric method using latex,
1.6 μm or less as carrier particles, preferably 0.05 to
Using 0.5 μm latex particles, the anti-elastase antibody can be insolubilized in the same manner as described above. Perform the measurement using an automatic analyzer or latex agglutination analyzer,
The measurement wavelength depends on the particle size of the latex, but is 500 to 95.
0 nm, preferably 550-800 nm.

Examples of the enzyme immunoassay include a solid-phased antibody in which an anti-elastase antibody is insolubilized in the wells or beads of a 96-well or split flat bottom tray for microtiter, and an enzyme such as peroxidase or alkaline phosphatase. A complex proportional to the amount of elastase in the measurement sample is formed by a sandwich EIA method using the labeled anti-elastase antibody bound to, and the amount of enzyme in the complex is determined using hydrogen peroxide and orthophenylene in the case of peroxidase. Diamine is used in combination as a substrate, and in the case of alkaline phosphatase, color is developed using 4-nitrophenyl phosphate as a substrate, and optical measurement is performed.

Further, the immunochromatographic measurement reagent is constituted by sequentially providing a sample addition section, a particle-labeled anti-elastase antibody containing section, a detection section and an absorption section in a developing and moving direction on a chromatographic medium.

As a chromatographic medium, a granulocyte elastase-α1-plasmin inhibitor complex in a diluted sample obtained by diluting the contents of a periodontal pocket as a substance to be detected, and a particle-labeled antibody impregnated in a particle-labeled antibody-containing portion are used. Any material may be used as long as it is a porous material that can evolve and move in the direction of the detection unit in synchronization with the elastase antibody and the antigen-antibody reaction,
The material is selected by a method of immobilizing an anti-elastase antibody on the detection part. For example, when an anti-elastase antibody is directly immobilized and used as an immobilized antibody, a porous nitrocellulose membrane or an activated nylon membrane is preferable because the antibody can be easily bound and immobilized. When an anti-elastase antibody is used as an antibody-immobilized latex adsorbed or chemically bonded to carrier particles such as latex, indirectly immobilized on a chromatographic medium, a glass fiber having a pore diameter that prevents the development and movement of the antibody-immobilized latex is used. , Woven fabric, non-woven fabric and the like are used, and borosilicate glass fiber is particularly preferable.

The chromatographic medium may be composed entirely of the same porous material, and the optimal material is arranged for each section such as a sample addition section, a section containing a particle-labeled anti-elastase antibody, a detection section, and an absorption section. Then, it may be configured to be able to continuously deploy and move between these

The sample addition section is provided at one end of the chromatographic medium, and then forms a section containing a particle-labeled anti-elastase antibody. As a particle-labeled antibody, an anti-elastase antibody may be adsorbed or chemically bonded to a colored latex, a dye-containing liposome, or a metal colloid particle to amplify and visualize the reaction. The presence or absence of the color of the particle-labeled antibody is relative to the color of the chromatographic medium, and when the chromatographic medium is white, the color of the labeled antibody is necessary for visualization determination,
By staining the chromatographic medium with black or a relatively dark color, white particle-labeled antibodies can also be used. Anti-elastase antibodies for particle-labeled antibodies can be used with either monoclonal antibodies or polyclonal antibodies, but monoclonal antibodies cause secondary aggregation of antigen-antibody reaction conjugates between elastase and particle-labeled antibodies. Therefore, the detection sensitivity is high, and even if a high concentration of elastase is present, it can be developed and moved in the chromatographic medium, and the detection concentration range is widened.

The particle-labeled anti-elastase antibody is impregnated with a protein component, a sugar, a surfactant and the like so that it can be developed and moved in the chromatographic medium, impregnated in the chromatographic medium, dried and stored.

Next, when a nitrocellulose membrane, an activated nylon membrane, or the like is used as a chromatographic medium, the detection section spots the anti-elastase antibody solution on the downstream side of the developing and moving direction of the particle-labeled anti-elastase antibody containing section. Alternatively, the anti-elastase antibody solution may be applied in a width of 1 to 2 mm in the width direction of the chromatographic medium, and a plurality of anti-elastase antibody solutions may be applied at intervals of several mm. The active portion other than the portion where the anti-elastase antibody is immobilized is BSA, prior to the formation of the particle-labeled anti-elastase antibody-containing portion.
Block with a protein such as casein to prevent non-specific reactions. When glass fiber is used as the chromatographic medium, the anti-elastase antibody-sensitized latex may be similarly impregnated on the downstream side in the developing and moving direction of the particle-labeled anti-elastase antibody-containing part, or may be placed in the width direction of the chromatographic medium. Apply and impregnate with a width of ~ 2mm, and a few meters
A plurality of lines may be formed at intervals of m. Anti-elastase antibodies for forming these detection parts are polyclonal antibodies,
Any of monoclonal antibodies may be used.

Further, the absorbing portion is provided on the downstream side in the developing and moving direction of the detecting portion in order to promote the developing and moving of the substance to be detected, elastase-α1-plasmin inhibitor complex and the particle-labeled antibody, in the chromatographic medium. A woven fabric, a nonwoven fabric, a water-absorbing pad, and the like are arranged in contact to form a water-absorbing portion.

[0030]

Diluent obtained by adding 0.5 (V / V)% sheep plasma and 0.01 (V / V)% dodecyltrimethylammonium bromide to 0.15 M NaCl-20 mM phosphate buffer (pH 8.5). Is placed in a dilution container, and the contents of the periodontal pocket collected with a manual scaler are added thereto, followed by stirring to dissolve and dilute. When leukocytes are present in the contents of the periodontal pocket, the contents in the leukocytes are released by the hemolytic action of the surfactant. If granulocytes such as neutrophils are present in leukocytes, enzymes such as elastase are eluted from granules in these granulocytes.

7 μl of the diluted sample of the contents of the periodontal pocket was placed on a slide reaction plate, and 0.1 (W / V)% BSA-
0.15 M NaCl-0.1 M glycine-NaOH (p
H8.5) (Glycine buffer solution for reaction) was added in an amount of 100 μl, and the mixture was stirred uniformly. Then, 50 μl of an anti-elastase antibody-sensitized latex test solution was added, and the slide reaction plate was rotated and swung to obtain elastase and anti-elastase in the diluted sample. The antibody-sensitized latex test solution forms an aggregate by an antigen-antibody reaction, and the presence or absence of aggregation can be visually determined in about 2 minutes. If agglutination was observed, the presence of granulocyte elastase was evident, and granulocytes were present in the contents of the periodontal pocket, or granules were released from granulocytes infiltrating periodontal tissue. It is determined that the disease is positive. If no aggregation is observed, the presence of granulocyte elastase is denied, and it is determined that periodontal disease is negative.

In the case of measurement by the latex turbidimetric method, 5 μl of the aforementioned periodontal pocket elastase diluent is added to 200 μl of the glycine buffer for reaction and stirred, and then 40 μl of the anti-elastase antibody-sensitized latex test solution is added and stirred. Then, the elastase in the diluted sample and the anti-elastase antibody sensitized to the anti-elastase antibody-sensitized latex undergo an antigen-antibody reaction, and the antibody-sensitized latex aggregates according to the elastase concentration. After adding and stirring the antibody-sensitized latex test solution, the absorbance is measured at an appropriate measurement wavelength, for example, 660 nm, and after several minutes, the absorbance is measured at the same wavelength to calculate the absorbance difference.

An elastase standard solution is measured in advance by the same operation to prepare a standard curve, and the elastase concentration in the diluted sample is calculated from the standard curve.

In the case of the solid-phase immunolabeled antibody analysis method, an aliquot of the diluted sample is added to a well to which a mouse anti-elastase monoclonal antibody has been adsorbed, subjected to a primary reaction at room temperature for 30 minutes, and then to a peroxidase-labeled anti-elastase polyclonal antibody. The antibody was added and a secondary reaction was performed at room temperature for 30 minutes.
After washing with a washing solution, a substrate solution consisting of orthophenylenediamine and hydrogen peroxide was added, and the mixture was reacted at room temperature for 15 minutes, and then washed.
The reaction is stopped by adding M sulfuric acid, and the absorbance at 490 nm is measured using an immunoreader.

The standard solution is measured in the same manner as above to prepare a standard curve, and the elastase concentration in the diluted sample is calculated in the same manner as in the case of the latex turbidimetric method described above.

In the case of the immunochromatographic reagent, the contents of the periodontal pocket are diluted by the same operation as described above, and the sample addition section of the chromatographic medium is inserted into the dilution container. The elastase in the diluted sample develops and moves in the chromatographic medium by capillary action, reaches the particle-labeled anti-elastase antibody-containing portion, and causes an antigen-antibody reaction with the anti-elastase antibody labeled with, for example, red colored latex particles. Further, it reaches an upper detecting unit. Since the anti-elastase antibody is immobilized on the chromatographic medium in the detection section,
The arriving antigen-antibody complex of elastase and the red latex-labeled anti-elastase antibody reacts with the immobilized anti-elastase antibody by an antigen-antibody reaction and binds to the immobilized anti-elastase antibody. The phased part is colored red. Further, as time elapses, excess particle-labeled antibodies move upward and are absorbed by the absorption section, leaving a red colored band only in the detection section. Thereby, the presence of elastase is confirmed in the diluted sample, and it is determined that granulocytes have infiltrated into the periodontal part.

By forming three bands of the immobilized anti-elastase antibody on the detection section, when the amount of elastase in the diluted sample is large, the band of the immobilized anti-elastase antibody on the downstream side is developed together with the development movement. And two or three colored bands appear in proportion to the amount of elastase. Therefore, the amount of elastase is proportional to the number of colored bands, and the degree of infiltration of granulocytes in the periodontal region can be determined.

[0038]

Example 1 A commercially available polystyrene latex particle having a particle size of 0.2 μm was sensitized by physically adsorbing a sheep anti-elastase antibody (Binding Site) by an ordinary method. 0.1% B
0.2 M glycine-NaCl buffer containing SA (pH
7.2) (hereinafter referred to as BSA-GSB)
An anti-elastase antibody sensitized latex test solution was obtained.

0.1 (W / V)% BSA-0.15MN
aCl-0.1 M glycine-NaOH (pH 8.5)
A diluent is prepared by adding 0.5 (V / V)% sheep plasma and 0.01 (V / V)% dodecyltrimethylammonium bromide to (glycine buffer for reaction). 10 μl of a standard solution dilution series obtained by diluting neutrophil-derived elastase (Cosmo Bio) with the diluent was collected in each optical reaction vessel, 200 μl of glycine buffer for reaction was added and stirred, and then the anti-elastase antibody Add 40 μl of the sensitized latex test solution, stir, measure the absorbance at 1 minute and 5 minutes after the start of the reaction at a wavelength of 660 nm, calculate the absorbance difference, and plot the relationship between each concentration of the standard solution and each absorbance difference. Together with the three-dimensional log-logit formula

The standard curve shown in FIG. 1 was created.

0.3 ml of the diluting solution is placed in each well of a split-type microtiter tray, and the contents of the periodontal pocket collected by a manual scaler are added thereto, followed by stirring to dissolve and dilute. If there are multiple sites suspected of periodontal disease, wipe the tip of the scaler with absorbent cotton, collect the contents sequentially from another periodontal pocket, dissolve similarly,
Dilute. When leukocytes are present in the contents of the periodontal pocket, the contents in the leukocytes are released by the hemolytic action of the surfactant. If granulocytes such as neutrophils are present in leukocytes, enzymes such as elastase are eluted from granules in these granulocytes.

The diluted sample of each periodontal pocket content was measured in the same manner as the above-mentioned standard solution, and the elastase concentration was calculated from the absorbance difference of each diluted sample at 660 nm for 1 minute and 5 minutes using the standard curve. .

Example 2 A mouse anti-elastase monoclonal antibody (Organon Technica) was adsorbed and fixed to each well of a flat-bottomed microtiter tray, washed and blocked with a BSA solution to prepare an immobilized antibody tray. Horseradish peroxidase was labeled with an anti-elastase polyclonal antibody (Binding Site) by an ordinary method.

3 μl of the diluted sample of Example 1 was added to a well of a flat-bottomed microtiter tray in which the elastase monoclonal antibody was insolubilized, and 0.1 (W / V)%
100 μl of PBS containing BSA was added, and a primary reaction was performed at room temperature for 30 minutes. Next, 100 μl of the labeled antibody solution was added, and a secondary reaction was performed at room temperature for 30 minutes. After washing, a substrate solution (orthophenylenediamine / hydrogen peroxide) was added, and the mixture was reacted at room temperature for 15 minutes. Then, the reaction was stopped with a 3N sulfuric acid solution.
The absorbance was measured at 490 nm and the difference from the absorbance of the zero blank was calculated.

The absorbance of the elastase standard solution was measured in the same manner as described above, the difference between the absorbance of the blank was calculated, and the relationship between each concentration of the standard solution and the absorbance difference was plotted. By log-logit formula of dimension

The standard curve shown in FIG. 2 was created. The elastase concentration was calculated from the absorbance difference of the diluted sample using the standard curve.

Example 3 An anti-elastase monoclonal antibody (Organon Technica) was applied to red colored particles of styrene-methacrylic acid copolymer latex (Nippon Synthetic Rubber Co., Ltd.) having a particle size of 0.27 μm in the same manner as in Example 1 above. Sensitized, 0.6 (W / V)
% Polyvinyl alcohol-BSA-GSB,
A red latex-labeled anti-elastase antibody was obtained.

A nitrocellulose membrane (Sartorius) having a pore size of 8 μm was cut into a size of 80 mm long × 60 mm wide, and a 2 mg / ml solution of an IgG fraction of an anti-elastase polyclonal antibody (Binding Site) was passed through a 1 mm diameter nozzle. Using a microcontroller controlled micro-injector capable of injecting the correct amount of reagent, a 1 mm wide line was drawn at 37 mm, 39 mm, and 42 mm from one end (lower end) in the length direction of the nitrocellulose membrane by 60 mm. After applying three of them so as to draw in the width direction of the to form a detecting portion, and drying at room temperature for 1 hour,
The nitrocellulose membrane was immersed in a 2 (W / V)% BSA solution at room temperature for 30 minutes to block extra binding sites. The nitrocellulose membrane was thoroughly rinsed with purified water and then dried at 30 ° C. for 30 minutes.

Next, a solution prepared by dissolving 60% (W / V)% saccharose in purified water was applied to one surface of the nitrocellulose membrane on the side where the detection portion was formed by an airbrush equipped with a microprocessor control system. It was applied to form an undercoat layer.

Further, the red latex-labeled anti-elastase antibody was applied with the airbrush at a width of 10 mm in the width direction at a distance of 7 mm from the lower end of the nitrocellulose membrane on which the undercoat layer had been formed. Direct application was performed to form a colored particle-labeled anti-elastase antibody-containing portion, and the resultant was dried at 30 ° C. for 30 minutes.
A range of 7 mm from the lower end of the nitrocellulose membrane was used as a sample addition section.

This nitrocellulose membrane is 6 mm
Cut in the length direction at the width of the acrylic resin support (6x
80 mm) with a double-sided adhesive tape, and a water absorption pad for immunochromatography 6 × 10 mm (Pall Corporation) is adhered above the detection unit formed on the nitrocellulose membrane (downstream in the developing movement direction) to form an absorption unit. A measurement reagent was prepared.

When the sample addition section of the immunochromatographic measurement reagent was inserted into the well of the split type microtiter tray containing the diluted sample of the periodontal pocket contents collected and diluted in the same manner as in Example 1, the sample was diluted. Components such as granulocyte elastase and the like in the sample begin to expand and move upward in the nitrocellulose membrane, which is a chromatographic medium, by dissolving the undercoat layer with water while soaking in the sample.

When a component such as elastase in the diluted sample reaches the colored particle-labeled anti-elastase antibody-containing portion, if elastase is present, elastase and the anti-elastase antibody labeled with red latex undergo an antigen-antibody reaction, and The antibody moves further upward while forming an antibody complex. Since the anti-elastase antibody labeled with red latex is a monoclonal antibody, it binds by reacting with elastase and an antigen-antibody antibody, but elastase bound to the anti-elastase monoclonal antibody is secondary to other surrounding anti-elastase monoclonal antibodies. Because of the inability to react, it develops and moves within the chromatographic medium without the formation of large aggregates.

When the elastase antigen-antibody complex reaches the detection section, the antigen-antibody complex of elastase-red latex-labeled anti-elastase antibody is sequentially added to the anti-elastase polyclonal antibody immobilized on the detection section by the antigen-antibody reaction. It is captured and a first colored band is expressed at the upstream side of the anti-elastase antibody immobilization site. If elastase is not present in the periodontal pocket content dilution sample,
No antigen-antibody reaction occurs and no colored band is observed.
When elastase is present in a relatively large amount, the first anti-elastase immobilization site is saturated, and the antigen-antibody complex that could not be captured is captured by the second anti-elastase antibody immobilization site. Two colored bands develop. Furthermore, when elastase is present in a large amount, the second anti-elastase antibody immobilized site is saturated, and a third colored band is developed. After about 15 minutes, the transfer of the red latex-labeled anti-elastase antibody to the water-absorbing pad on the upper end was completed, and the band stained red in the detection part was visually observed and judged.

[0053]

As described above, according to the present invention, granulocyte-derived elastase among leukocytes in a periodontal pocket sample is hemolyzed, diluted and detected and measured with an immunological assay reagent. Periodontal pocket elastase can be measured relatively easily and accurately in a short time.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing one embodiment of the present invention.

FIG. 2 is an explanatory view showing another embodiment of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/531 G01N 33/577 B 33/577 A61C 19/04 Z F-term (Reference) 2G045 AA25 BB29 BB40 BB42 CA21 CB01 CB05 CB06 CB21 DA20 FA18 FB01 FB03 FB06 FB07 FB11 GA05 GC12 4B063 QA01 QA07 QA19 QQ02 QQ36 QQ96 QR48 QR84 QS03 QS20 QS33 QS36 QX01 4C052 AA20 NN01 NN11

Claims (15)

[Claims] 1. A reagent for measuring periodontal pocket elastase, comprising a diluent for periodontal pocket contents and a reagent for measuring granulocyte elastase. 2. The reagent for measuring periodontal pocket elastase according to claim 1, wherein the contents of the periodontal pocket are gingival crevicular fluid, periodontal exudate, and periodontal pocket plaque. 3. The diluent for periodontal pocket contents is a hemolytic agent, α
The reagent for measuring periodontal pocket elastase according to claim 1, comprising at least one of a 1-plasmin inhibitor. 4. The reagent for measuring periodontal pocket elastase according to claim 3, wherein the hemolytic agent is a surfactant. 5. The reagent for measuring periodontal pocket elastase according to claim 1, wherein the reagent for measuring granulocyte elastase uses particles sensitized with an anti-elastase antibody. 6. The reagent for measuring periodontal pocket elastase according to claim 5, wherein the carrier particles of the anti-elastase antibody-sensitized particles are any of bacterial cells, metal colloid particles, latex particles, and synthetic particles. 7. The reagent for measuring periodontal pocket elastase according to claim 5, wherein the reagent for measuring granulocyte elastase is one of a visual slide aggregation determination reagent and a particle aggregation turbidimetric method. 8. The reagent for measuring periodontal pocket elastase according to claim 1, wherein the reagent for measuring granulocyte elastase uses an immobilized anti-elastase antibody and a labeled anti-elastase antibody. 9. The reagent for measuring periodontal pocket elastase according to claim 5, wherein the anti-elastase antibody is a polyclonal antibody, one or more monoclonal antibodies. 10. The reagent for measuring periodontal pocket elastase according to claim 8, wherein the label is an enzyme. 11. The reagent for measuring periodontal pocket elastase according to claim 1, wherein the reagent for measuring granulocyte elastase is a reagent for measuring immunochromatographic elastase. 12. The reagent for measuring periodontal pocket elastase according to claim 11, wherein a sample addition section, a section containing a particle-labeled anti-elastase antibody, a detection section, and an absorption section are sequentially provided in the developing medium in the chromatographic medium. 13. The periodontal pocket elastase according to claim 12, wherein the particle-labeled anti-elastase antibody is one of an anti-elastase antibody-sensitized colored particle, a liposome containing an anti-elastase antibody-sensitized coloring substance, and a metal colloid particle-labeled anti-elastase antibody. Measurement reagent. 14. The reagent for measuring periodontal pocket elastase according to claim 12, wherein the detection portion has one or more anti-elastase antibody immobilized sites. 15. The reagent for measuring periodontal pocket elastase according to claim 13, wherein the labeled anti-elastase antibody is an anti-elastase monoclonal antibody, and the immobilized anti-elastase antibody is another anti-elastase monoclonal antibody or anti-elastase polyclonal antibody.