CN113767287A

CN113767287A - Absorption pad for immunochromatography diagnostic kit and immunochromatography diagnostic kit

Info

Publication number: CN113767287A
Application number: CN202080030364.8A
Authority: CN
Inventors: 堀井厚志; 松瀬武志; 佐藤润一; 原雄一
Original assignee: Asahi Kasei Corp
Current assignee: Asahi Kasei Corp; Asahi Chemical Industry Co Ltd
Priority date: 2019-05-14
Filing date: 2020-05-13
Publication date: 2021-12-07
Also published as: JPWO2020230826A1; KR20210107781A; TW202109044A; WO2020230826A1

Abstract

Providing: an absorbent pad for an immunochromatographic diagnostic kit, which has excellent reproducibility of test results and does not cause reverse flow. The present invention relates to an absorbent pad for an immunochromatographic diagnostic kit, which is characterized by comprising carboxymethylated cellulose fibers, wherein the average degree of substitution of hydroxyl groups in glucose units constituting the carboxymethylated cellulose fibers is 0.05 to 1.5, and an immunochromatographic diagnostic kit comprising the absorbent pad.

Description

Absorption pad for immunochromatography diagnostic kit and immunochromatography diagnostic kit

Technical Field

The present invention relates to an absorbent pad for an immunochromatographic diagnostic kit. More particularly, the present invention relates to an absorbent pad for an immunochromatographic diagnostic kit, which has excellent reproducibility of test results and does not cause a reverse flow.

Background

In recent years, there have been developed simple test reagents, diagnostic reagents, and diagnostic kits for performing various tests in a short time, such as the presence or absence of infection with pathogens such as viruses and bacteria, the presence or absence of pregnancy, the presence or absence of tumor markers, the presence or absence of specific materials in foods, and the presence or absence of harmful substances such as residual pesticides. These methods utilize specific reactions between each test substance and a substance capable of reacting specifically with the test substance. In particular, many immunological assays utilizing an antigen-antibody reaction between an antigen and an antibody have been developed, such as immunochromatography, turbidimetric immunoassay, enzyme immunoassay, chemiluminescence assay, radioimmunoassay, and assay utilizing surface plasmon resonance. These measurement methods are used for examinations of diseases and the like in hospitals, medical facilities, and the like, food examinations in food companies, and the like. Wherein, the immunochromatography assay has the following characteristics: it is applied to the execution of a very large number of examinations because it requires no special equipment, and knowledge, is easy to handle, is low in cost, and can be diagnosed quickly. In recent years, pregnancy test drugs, HIV test drugs, and the like are sold by general drugstores, and measurement is possible by general consumers, and further, not only qualitative inspection for inspecting the presence or absence of a test substance but also quantitative inspection of a measured amount and the like can be performed.

As the measurement principle of the immunochromatographic assay, there are a method called a sandwich method and a method called a competition method. In addition, as measurement formats, there are methods called a Flow Through (Flow Through) type and a Lateral Flow (Lateral Flow) type. Various substances can be detected as a substance to be tested in a sample, and a typical example is measurement for detecting an antigen by a sandwich method, and the following operations are sequentially performed.

(1) An antibody that specifically binds to an antigen as a detection target substance is immobilized on a predetermined portion of a chromatographic medium such as a chromatographic medium membrane, and a reaction site called a test line (hereinafter referred to as "TL") is formed at an arbitrary position of the chromatographic medium.

(2) Preparing a detection reagent, coating the detection reagent on a binding pad and the like, drying to form a part containing the detection reagent, and combining the part with the chromatography medium to form an immunochromatographic diagnostic kit; the detection reagent is obtained by loading an antibody capable of specifically binding to a substance to be detected on a label such as an enzyme, a label, a fluorescent label, or a magnetic particle.

(3) The antigen and the detection reagent are developed on the chromatographic medium by dropping the specimen itself containing the antigen or a solution obtained by diluting the specimen with an arbitrary liquid on a specific position of the immunochromatographic diagnostic kit, for example, a sample pad.

By these operations, the label is captured by the antibody immobilized on the chromatographic medium via the antigen at the reaction site, and the signal of the label is detected, thereby performing diagnosis using the immunochromatographic diagnostic kit.

In clinical practice, it has been strongly required to accurately determine, in particular, a disease in an examination by the immunochromatography. For example, there are several examination items useful for diagnosing acute coronary syndrome. In acute coronary syndrome, acute myocardial infarction is a disease in which a coronary artery is blocked by a thrombus to cause necrosis of myocardial tissue, and the success or failure of reperfusion at an early stage of onset has a great influence on prognosis. Therefore, a diagnosis with high accuracy becomes particularly important. In this case, it is necessary to quantitatively detect a marker required for diagnosis, and it is needless to say that the variations in the color intensity among immunochromatographic diagnostic kit products are reduced as much as possible in the immunochromatographic diagnostic kit used for the diagnosis. If an immunochromatographic diagnostic kit having a large variation in color intensity is used, misdiagnosis may naturally occur, and the disease may be further life-threatening due to misdiagnosis. In view of these facts, minimizing the variation among immunochromatographic diagnostic kit products is one of the major issues in the development of immunochromatographic diagnostic kits.

The variation is considered to be caused by the influence of each member constituting the kit, but particularly, since the absorbent pad is a portion responsible for liquid absorption, the variation in the color intensity is caused by a problem such as background coloring.

Here, "background" means: the labeled reagent blocks pores of the chromatography medium film or causes coloring of the chromatography medium film by nonspecific adsorption. When the background is poor, the contrast between the chromatographic medium film and the Test Line (TL) is poor, or a line in which the TL color development is difficult to observe becomes difficult, and it is difficult to make a judgment in the medical field. Further, when the background is poor, it becomes difficult to determine negative TL (i.e., no color development). These phenomena may lead to misdiagnosis.

In addition to the background coloring, one of the causes of the problem of misdiagnosis is the background coloring due to the backflow of the labeling reagent after the determination. Generally, the judgment time of an immunochromatographic diagnostic kit depends on the respective immunochromatographic diagnostic kits, but in a very busy clinical site, the judgment time cannot be accurately judged in general. Here, "the countercurrent flow of the labeling reagent" means: by drying the chromatographic medium, the labeled reagent temporarily adsorbed by the absorbent pad is developed on the chromatographic medium in the reverse direction in reverse flow. This backflow may contaminate the coloration of the background and thus the TL, thereby possibly causing misdiagnosis.

Conventionally, cotton, nonwoven fabric, filter paper, and the like made of fibers such as cellulose fibers, glass fibers, and pulp have been used as an absorbent pad used in an immunochromatographic test kit. However, the absorbent pad made of only these materials is affected by the structure, constituent materials, and production method thereof, and thus has a problem that the water absorption is insufficient and the absorption amount varies or a reverse flow is caused.

In order to solve these problems, an absorbent pad using superabsorbent polymer particles is studied in the following patent document 1, and an absorbent pad obtained by spraying silica particles or the like onto a nonwoven fabric material such as cellulose fibers is studied in the following patent document 2. However, in the case of these absorbent pads, it is difficult to make the fiber density uniform and the spray amount uniform, and even if the water absorption property is improved, the water absorption rate is likely to vary among products, so that variation among products is not improved at present.

Further, problems in the manufacturing process of the kit due to the conventional absorbent pad have also been reported. For example, the absorbent pad made of glass fiber may scatter as dust when the absorbent pad is cut in the production process of the immunochromatographic diagnostic kit. The inhalation of the glass fiber dust may cause inflammation of the bronchus, and therefore, the use thereof is not recommended in some cases from the viewpoint of health hazards to the manufacturing personnel. Further, there are many reports on failures such as blade breakage of the dicing die. For these reasons, it is desirable to avoid the use of an absorbent pad made of glass fiber as much as possible.

As described above, there is a strong demand for an absorbent pad that minimizes variation and prevents backflow.

Documents of the prior art

Patent document

Patent document 1: japanese patent No. 6134615

Patent document 2: japanese patent laid-open publication No. 2012-189346

Disclosure of Invention

Problems to be solved by the invention

In view of the above-described conventional techniques, an object of the present invention is to provide an absorbent pad for an immunochromatographic diagnostic kit, which has excellent reproducibility of test results and does not cause a reverse flow.

Means for solving the problems

The present inventors have intensively studied and repeated experiments to solve the above problems, and as a result, have unexpectedly found that: the present invention has been completed by the fact that the absorbent pad contains specific carboxymethylated cellulose fibers, whereby reproducibility is improved and the reverse flow is suppressed.

Namely, the present invention is as follows.

[1] An absorbent pad for an immunochromatographic diagnostic kit, comprising carboxymethylated cellulose fibers, wherein the average degree of substitution of hydroxyl groups in glucose units constituting the carboxymethylated cellulose fibers is 0.05 to 1.5.

[2] The absorbent pad for an immunochromatographic diagnostic kit according to the above [1], wherein the capture and release index of the pad is 0.5 or less.

[3]According to the above [1]]Or [2]]The absorbent pad for an immunochromatographic diagnostic kit, wherein the weight per unit area of the pad is 10g/m²Above and 400g/m²And a thickness of 0.03mm to 10.00 mm.

[4]According to the above [1]]～[3]The absorbent pad for an immunochromatographic diagnostic kit according to any one of the above, wherein the pad has a liquid absorption volume of 5g/100cm when impregnated with physiological saline²Above and 50g/100cm²The following.

[5] The absorbent pad for an immunochromatographic diagnostic kit according to any one of the above [1] to [4], wherein the pad is in the form of a single-layer sheet or a laminated sheet.

[6] An immunochromatographic diagnostic kit comprising the absorbent pad for an immunochromatographic diagnostic kit according to any one of the above [1] to [5 ].

ADVANTAGEOUS EFFECTS OF INVENTION

The immunochromatographic diagnostic kit using the absorbent pad for an immunochromatographic diagnostic kit of the present invention has excellent reproducibility of test results and does not cause reverse flow.

Drawings

Fig. 1 is a sectional view of an immunochromatographic diagnostic kit as an embodiment of the present invention.

Detailed Description

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

The absorbent pad for an immunochromatographic diagnostic kit of the present embodiment is an absorbent pad for an immunochromatographic diagnostic kit comprising carboxymethylated cellulose fibers, and the average substitution degree of hydroxyl groups in glucose units constituting the carboxymethylated cellulose fibers is 0.05 or more and 1.5 or less.

In the immunochromatography-based test method carried out using the immunochromatography diagnostic kit, there are generally two types of methods: the adsorption pad for an immunochromatographic diagnostic kit of the present embodiment may be used in any of these methods, and is not limited to these in the meaning of a system of an immunochromatographic diagnostic kit in which water is absorbed.

The carboxymethylated cellulose fibers contained in the absorbent pad for an immunochromatographic diagnostic kit of the present embodiment have an average degree of substitution of hydroxyl groups in the glucose units constituting the cellulose at the time of carboxymethylation of 0.05 to 1.5. If the average substitution degree is too high, a water mass is formed in the front portion of the absorbent pad when the developed liquid is absorbed. This prevents a sufficient amount of developing solution from being absorbed, and a development failure occurs, thereby causing inspection variation. The upper limit of the average degree of substitution is preferably 1.3 or less, and more preferably 0.9 or less. More preferably 0.6 or less. When the average substitution degree is 0.05 or more, a sufficient liquid absorption amount can be secured. The lower limit of the average degree of substitution is preferably 0.1 or more, and more preferably 0.2 or more.

At the time of carboxymethylation, the carboxymethyl group of the carboxymethylated cellulose fiber is terminated with a sodium salt, but then, a part of the carboxymethyl group can be converted into a proton type by acid treatment. The method for partially protonating carboxymethylated cellulose fibers is not particularly limited, but protonation is preferably performed by immersing in acetic acid, hydrochloric acid, or nitric acid adjusted to a predetermined concentration using an alcohol-containing solvent, and protonation is more preferably performed by immersing in acetic acid adjusted to a predetermined concentration using an alcohol-containing solvent.

The capture and release index of the absorbent pad for an immunochromatographic diagnostic kit comprising carboxymethylated cellulose fibers of the present embodiment is preferably 0.5 or less. In the present specification, "capture and release index" means: an index that measures the balance of the property of the absorbent pad to absorb the imparted liquid and the property of the absorbent pad to retain the imparted liquid is calculated by the following formula:

capture and release index ═ (100 × C ÷ D) ÷ T

{ formula (II), (III): liquid absorption amount (g/100 cm)²) D: weight per unit area (g/m)²) T: release time (minutes) }. When the capture and release index is too large, the labeled reagent temporarily absorbed by the absorbent pad flows back on the chromatographic medium, or a detection deviation occurs. The upper limit of the capture and release index is more preferably 0.3 or less, and still more preferably 0.1 or less. The lower limit is preferably 0.0001 or more, and more preferably 0.001 or more. The acquisition and release index can be adjusted by controlling the average degree of substitution, average fiber diameter, fiber length, etc. of the aforementioned carboxymethylated cellulose fibers and/or the morphology of the aforementioned absorbent pad comprising carboxymethylated cellulose fibers, etc.

In the present specification, the term "release time" means: an absorbent pad having a width of 4mm × a length of 25mm was stacked on a chromatography medium having a width of 4mm × a length of 25mm without variation, and the value of the time required for the physiological saline to start transferring from the absorbent pad to the chromatography medium when 20. mu.L of the physiological saline was slowly and non-exhaustively dropped onto the absorbent pad was expressed in units of minutes. In the measurement of the "release time", the size of the absorbent pad and the amount of physiological saline may be different from those described above as long as the correlation between the size of the absorbent pad and the amount of physiological saline is the same as that described above.

The average fiber diameter of the carboxymethylated cellulose fibers is preferably 1 μm or more and 100 μm or less. If the average fiber diameter is too large, a water mass is formed in the front portion of the absorbent pad when absorbing the developed liquid. This prevents a sufficient amount of developing solution from being absorbed, and a development failure occurs, thereby causing inspection variation. The upper limit of the fiber diameter is preferably 80 μm or less, and more preferably 70 μm or less. When the average fiber diameter is 1 μm or more, a sufficient liquid suction amount can be secured. The lower limit of the average fiber diameter is preferably 3 μm or more, and more preferably 5 μm or more. The average fiber diameter was determined by using an average fiber diameter measured by a scanning electron microscope (SEM, JEOL) with N being 10.

The absorbent pad for an immunochromatographic diagnostic kit comprising carboxymethylated cellulose fibers of the present embodiment may be in the form of a woven fabric, a knitted fabric or a nonwoven fabric, and for example, in the form of a nonwoven fabric, the weight per unit area (g/m)²) (i.e., weight (g) of nonwoven fabric/area (m) of nonwoven fabric²) Preferably 10g/m²Above and 400g/m²The following. When the weight per unit area is too low, a sufficient amount of liquid absorption cannot be obtained as an absorbent pad for immunochromatographic diagnostic reagents. The lower limit of the weight per unit area is more preferably 20g/m²More preferably 30g/m or more²The above. On the other hand, if the weight per unit area is too high, flexibility is impaired, and handling in the manufacture of an immunochromatographic diagnostic kit is significantly impaired, resulting in a variation in diagnostic results. The upper limit of the weight per unit area is more preferably 350g/m²Hereinafter, it is more preferably 300g/m²Hereinafter, it is more preferably 250g/m²Hereinafter, more preferably 200g/m²Hereinafter, more preferably 190g/m²Hereinafter, more preferably 180g/m²The following.

The thickness of the absorbent pad for an immunochromatographic diagnostic kit comprising carboxymethylated cellulose fibers of the present embodiment is preferably 0.03mm to 10.00mm, for example, when the absorbent pad is in the form of a fabric or a sheet. If the thickness of the pad is too large, for example, when the pad is used in an immunochromatographic diagnostic kit, the pad is difficult to be housed in a case, and as a result, variations in diagnostic results become large. The upper limit of the thickness of the mat is more preferably 9.00mm or less, and still more preferably 8.00mm or less. On the other hand, when the sheet is too thin, a sufficient amount of liquid absorption cannot be secured as an absorbent pad of the immunochromatographic diagnostic kit. The lower limit of the pad thickness is more preferably 0.05mm or more, and still more preferably 0.10mm or more. In this specification, "thickness" of the pad means: a value obtained by measuring the load at 1.96kPa in a thickness test according to JIS-L1096.

The amount of liquid absorbed when the absorbent pad for immunochromatographic diagnostic kit of the present embodiment is impregnated in physiological saline is preferably 5g/100cm²Above and 50g/100cm²The following. When the amount of the liquid-absorbed solution is too large, the structure collapses after the liquid absorption to cause poor development when the reagent is used in an immunochromatographic diagnostic kit. The upper limit of the liquid absorption amount is preferably 50g/100cm²Hereinafter, more preferably 30g/100cm²The following. The liquid absorption amount is less than 5g/100cm²In the case, the desired effect cannot be exhibited, and the backflow may occur. The lower limit of the liquid absorption amount is preferably 5g/100cm²Above, more preferably 10g/100cm²The above.

The cellulose fiber (before the carboxymethylation) which is a raw material of the carboxymethylated cellulose fiber is not particularly limited, and examples thereof include: the cellulose fibers known as cuprammonium rayon, viscose rayon, lyocell, cotton, pulp, and polynosic fiber are preferably cuprammonium rayon and viscose rayon, and more preferably cuprammonium rayon. Some of the raw materials for cellulose fibers have a low degree of polymerization, and in such raw materials, fibers are easily decomposed if the degree of substitution is increased, and it is necessary to keep the degree of substitution as low as possible.

The cellulose fiber may be either a long fiber or a short fiber. The long fibers are preferably continuous long fibers. In the present specification, the long fiber means a fiber having a fiber length of 10mm or more, and the fiber length is preferably 20mm or more, more preferably 50mm or more, and further preferably a continuous long fiber. Since the short-fiber sheet is short fiber, if the degree of substitution is increased, the fiber is easily separated and the degree of substitution needs to be suppressed as low as possible; on the other hand, when the continuous long fiber sheet is used, there is an advantage that the sheet is not easily disintegrated even at a relatively high substitution degree, and a wide range of substitution degrees can be selected.

The form of the absorbent pad for an immunochromatographic diagnostic kit comprising carboxymethylated cellulose fibers of the present embodiment is preferably a form of a cotton, a woven, a knitted, or a nonwoven fabric, more preferably a woven or nonwoven fabric of carboxymethylated regenerated cellulose fibers, and still more preferably a form of a nonwoven fabric of carboxymethylated regenerated cellulose fibers. If the nonwoven fabric is used, the fibers are entangled even in a gelled state when wet, so that high liquid absorbency can be obtained while maintaining the strength when wet, and the absorbent pad can be suitably used as an absorbent pad for an immunochromatographic diagnostic kit.

In the absorbent pad for an immunochromatographic diagnostic kit comprising carboxymethylated cellulose fibers of the present embodiment, fibers other than natural or regenerated cellulose fiber sources, for example, synthetic fibers such as polyester fibers, polypropylene fibers, nylon fibers, etc., may be contained within a range in which the desired effect is not impaired; and bioabsorbable fibers such as polyglycolic acid, polylactic acid, and polymalic acid. Such a raw material is preferably 50% or less, more preferably 30% or less, as long as the desired effects are not impaired. Such fibers may be long fibers or short fibers. The long fiber is preferably a continuous long fiber.

The absorbent pad for an immunochromatographic diagnostic kit of the present embodiment may be one in which sheets are further laminated according to various applications. When the nonwoven fabric is laminated, various known methods such as hot embossing, a heat fusion method such as ultrasonic fusion bonding, a mechanical entanglement method such as needle punching or water jet, a method using an adhesive such as a hot melt adhesive or a urethane adhesive, and extrusion lamination can be used, and there are no particular limitations thereon.

The sheet material used for laminating the sheets is not limited at all, and may be a bioabsorbable polymer such as cellulose, a thermoplastic resin, polyglycolic acid, polylactic acid, and polymalic acid. The thermoplastic resin may be a polyolefin resin, a polyester resin, or a polyamide resin, and specifically, the following may be mentioned: high-pressure low-density polyethylene of homopolymers or copolymers of α -olefins such as ethylene, propylene, 1-butene, 1-hexene, 4-methyl-1-pentene and 1-octene, linear low-density polyethylene (LLDPE), high-density polyethylene, polypropylene (propylene homopolymer), polypropylene random copolymer, poly-1-butene, poly-4-methyl-1-pentene, polyolefin such as ethylene/propylene random copolymer, ethylene-1-butene random copolymer and propylene-1-butene random copolymer, polyester (polyethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, etc.), polyamide (nylon-6, nylon-66, polyhexamethylene isophthalamide, etc.), polyvinyl chloride, polyimide, polyamide, polyethylene terephthalate, polyethylene naphthalate, etc.), polyethylene terephthalate, etc, Ethylene/vinyl acetate copolymers, polyacrylonitrile, polycarbonate, polystyrene, ionomers, mixtures thereof, and the like. In particular, when used in the present embodiment, any one or a mixture of polyethylene terephthalate, polybutylene terephthalate, polypropylene, and polyethylene is suitable from the viewpoint of stability against heat and moisture, and versatility.

In general, most of thermoplastic resins are hydrophobic, and even if the structure is formed into a nonwoven fabric, there is little water absorption. In the present embodiment, it can be seen that: the variation in water absorption can be reduced by hydrophilizing a nonwoven fabric composed of fibers of a hydrophobic thermoplastic resin. The method of hydrophilization is not particularly limited, and examples of physical processing methods include hydrophilization by corona treatment or plasma treatment, and examples of chemical processing methods include: introduction of surface functional groups such as sulfonic acid groups and carboxylic acid groups by oxidation treatment; a known method is a method of impregnating and applying a treatment agent such as a water-soluble polymer such as polyvinyl alcohol (PVA), a polyester resin, polystyrene sulfonic acid, or polyglutamic acid, and/or a nonionic surfactant, an anionic surfactant, a cationic surfactant, or a surfactant such as a zwitterionic surfactant, to a nonwoven fabric by a padding method, a spray method, or the like. In consideration of the water absorption required for the absorbent pad, an appropriate hydrophilization processing method and conditions, such as the amount of the treating agent and the amount of the functional group introduced, can be selected.

When a nonwoven fabric is used for laminating the sheets, the method for producing the nonwoven fabric is not particularly limited, and examples thereof include: spunbond nonwoven fabrics, meltblown nonwoven fabrics, wet nonwoven fabrics, dry pulp nonwoven fabrics, flash spun nonwoven fabrics, open fiber nonwoven fabrics, etc., and particularly, as nonwoven fabrics composed of ultrafine fibers, meltblown nonwoven fabrics and flash spun nonwoven fabrics are preferable.

As the nonwoven fabric used for lamination of the sheets, there can be suitably used: the weight per unit area is 20g/m²Above and 200g/m²A nonwoven fabric having a thickness of 0.06mm to 1.20mm and an average fiber diameter of 0.7 μm to 5.0 μm.

The following shows an example of a method for producing an absorbent pad for an immunochromatographic diagnostic kit comprising carboxymethylated cellulose fibers according to the present embodiment, but the method is not limited thereto.

The structure of natural or regenerated cellulose fibers was stirred at 35 ℃ for 30 minutes while maintaining the alkaline state in an aqueous solution of sodium hydroxide containing alcohol. Then, after the reagent in the reaction vessel is discharged, sodium monochloroacetate containing alcohol is added and stirred for 1 to 12 hours at a temperature of 30 to 55 ℃. The degree of substitution at this time is controlled by the bath ratio of the reaction solution to the structure, the temperature, and the time. In addition, other reaction conditions may be appropriately changed in consideration of production cost and the like. The pH of the resulting structure is adjusted to 6.0 to 8.0 with an aqueous ethanol solution containing acetic acid, and then alcohol substitution is performed with 70 wt%, 90 wt%, and 100 wt% ethanol. Since the resin hardens when a small amount of water is contained, alcohol substitution can be reliably performed and morphological stability can be maintained by gradually increasing the alcohol concentration. Then, the sheet was immersed in an acid-containing ethanol solution adjusted to a predetermined concentration, stirred for 1 hour, alcohol-substituted with 70 wt%, 90 wt%, and 100 wt% ethanol, and dried to obtain a protonated sheet structure. Further, the sheet-like mat of the present embodiment was obtained by immersing the mat in an aqueous ethanol solution containing calcium hydroxide adjusted to a predetermined concentration, stirring the solution for 1 hour, performing alcohol substitution with 70 wt%, 90 wt%, and 100 wt% ethanol, and drying the resulting solution.

The "diagnostic method" carried out using the immunochromatographic diagnostic kit using the absorbent pad for an immunochromatographic diagnostic kit of the present embodiment refers to various diagnoses carried out in vitro using the immunochromatographic diagnostic kit. The diagnostic subject is not particularly limited, and can be used for various examinations of the diagnostic subject such as human, animal, food, plant, and other environmental examinations. In a general diagnostic procedure, a sample to be tested is collected from a test subject, and if necessary, subjected to pretreatment such as extraction or filtration, dropped on a sample pad, and waits for a specific time from the start of the test, and the presence or absence of a test subject substance causes different color development to determine the diagnostic result. Of course, the method is not limited to this step, and can be used for diagnosis based on the same steps and principles. Preferably, the sample to be tested is filtered in advance to remove unnecessary foreign matter or foreign matter, thereby further speeding up the diagnosis and improving the diagnosis accuracy.

The immunochromatographic diagnostic kit of the present embodiment is used for easily detecting the presence or absence of a substance to be detected in various samples. The type of diagnostic kit includes a lateral flow type and a flow through type. The labeling agent and the absorbent pad are not particularly limited as long as they are used, and a lateral flow type is preferable. In addition, the side flow type includes a dip stick type and a cassette type, but the type thereof is not particularly limited. The constitution of the diagnostic kit is not particularly limited, and may be any constitution commonly used in this field. The type of the member is not particularly limited as long as it is used in this field, and examples thereof include: FIG. 1 shows (a) a sample pad, (b) a conjugate pad (containing an antibody-sensitized labeled reagent), (e) a chromatographic medium film, (f) an absorbent pad, and (g) a backing paper. Further, a part of these components may be omitted as necessary.

As shown in fig. 1, (f) the absorption pad is a portion which finally absorbs the specimen as the measurement object in the immunochromatography, (c) is a Test Line (TL), and (d) is a control line (CT). As described above, examples of the conventional general absorbent pad include: cellulose filter paper, glass fiber, and the like.

The sample pad is a portion that first receives a sample to be measured in immunochromatography. Examples of typical sample pads include: cellulose filter paper, glass fiber, acrylic fiber, nylon fiber, various fabrics, and the like. In addition, pretreatment may be performed as necessary. For example, a treatment may be performed in advance with a buffer solution, a surfactant, a protein, a reagent for trapping foreign matter in a sample to be tested, an antiseptic, an antibacterial agent, an antioxidant, a moisture absorbent, and the like.

The conjugate pad is a portion in which labeled particles such as an antibody-sensitized labeling reagent are dried and immobilized in advance. Typical examples of the bonding pad include: glass fiber, acrylic fiber, PET fiber monomer or composite non-woven fabric, woven fabric and the like. In addition, pretreatment may be performed as necessary.

The labeling reagent immobilized on the conjugate pad means: a granular substance which is insoluble in water, a buffer solution, etc. and which is loaded with a pigment, a dye, etc. The raw material constituting the particles is not particularly limited, and examples of such a labeling agent include: metal colloid particles such as gold colloid, platinum colloid, silver colloid, and selenium colloid, colored latex particles obtained by coloring styrene-based latex such as polystyrene latex, acrylic latex, and the like, colored silica particles obtained by coloring silica composed of a three-dimensional structure containing a silicon atom and an oxygen atom, colored cellulose particles obtained by coloring cellulose, a labeling agent obtained by directly granulating a coloring component such as carbon black, magnetic particles, and the like. In addition, the labeling reagent may be a fluorescent particle.

The labeling reagent is required to carry a substance capable of specifically binding to a test substance such as an antibody, and the method of carrying the substance is not particularly limited. Examples thereof include: a load by physical adsorption, a load by covalent bond, a load by a combination thereof, and the like. The kind and amount of the substance to be supported are not particularly limited. As the kind of the substance to be supported, an antibody is most general and preferable. In addition, as a method of supporting, from the viewpoint of easiness, a support by physical adsorption is preferable, and from the viewpoint of stability, performance and the like, a support by covalent bond is preferable.

The object that can be diagnosed by the immunochromatographic diagnostic kit of the present embodiment is not particularly limited, and specific examples thereof include the following: tumor markers, hormones, infectious diseases, autoimmunity, plasma proteins, TDM, coagulation, fibrinolysis, amino acids, peptides, proteins, genes, cells, and the like. More specifically, there may be mentioned: CEA, AFP, ferritin, beta 2 microglobulin, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1.2, fecal occult blood, urinary beta 2 microglobulin, PIVKA-2, urinary BTA, insulin, E3, HCG, HPL, LH, HCV antigen, HBs antibody, HBc antibody, HBe antigen, HBe antibody, HTLV-1 antibody, HIV antibody, Toxoplasma antibody, syphilis, ASO, influenza A antigen, influenza A antibody, influenza B antigen, influenza B antibody, rotavirus antigen, adenovirus antigen, rotavirus adenovirus antigen, group A streptococcus, group B streptococcus, candida antigen, CD bacteria, cryptococcus antigen, cholera bacteria, meningitis antigen, granulocyte elastase, helicobacter pylori antibody, O157 antigen, leptospira antibody, Aspergillus antigen, MRSA, MRRF, total IgE, total serum albumin, PSA, and HBT, urine BTA antibody, LE assay (lupus erythematosus assay), CRP, IgG, IgA, IgM, IgD, transferrin, urine albumin, urine transferrin, myoglobin, C3. C4, SAA, LP (a), α 1-AC, α 1-M, haptoglobin, micro transferrin, APR Score (Acute Phase Reactant Score), FDP, D-dimer, plasminogen, AT3, α 2PI, PIC, PAI-1, protein C, blood coagulation factor XIII, type IV collagen, hyaluronic acid, GHbA1C, other various antigens, various antibodies, various viruses, various bacteria, various amino acids, various peptides, various proteins, various DNAs, various cells, various allergens, various residual pesticides, various harmful substances.

The chromatography medium used in the immunochromatographic diagnostic kit is not particularly limited, and various types of chromatography media generally used can be used. More specifically, there may be mentioned: a nitrocellulose membrane.

An example of a method for producing the immunochromatographic diagnostic kit will be described below.

A dispersion of a labeled reagent adjusted to a specific concentration is prepared, and a buffer solution and an antibody are added thereto, and the mixture is stirred for a certain period of time while adjusting the temperature, so that the labeled reagent adsorbs the antibody. After the stirring for a certain period of time, a blocking agent (blocking agent) is further added, and the mixture is stirred for a certain period of time while the temperature is adjusted, thereby blocking the colored cellulose particles. As the blocking agent, various blocking agents can be used depending on the composition of the substance to be examined, the specimen, or a solution for diluting them. To wash the labeled reagent after antibody adsorption/blocking, centrifugation was performed, the supernatant containing the remaining antibody and blocking agent was separated from the settled particles, and the supernatant was removed by decantation. A liquid such as a buffer is added to the settled particles, and dispersion treatment is performed by ultrasonic waves or the like as necessary. By performing a series of operations of sedimentation by the centrifugal separation, removal of the supernatant, and addition of the liquid, a dispersion containing the particles subjected to the antibody adsorption/blocking at a specific concentration is prepared by washing the particles a necessary number of times. If necessary, a protein, a surfactant, and sugar such as sucrose or trehalose are added to the dispersion, and the resulting solution is applied to a bonding pad made of glass fiber in a predetermined amount and dried to prepare a portion containing a detection reagent. Further, if necessary, a buffer solution, a surfactant, a protein, a reagent for trapping foreign matter in a sample to be tested, a preservative, an antibacterial agent, an antioxidant, a moisture absorbent, and the like are applied to the regenerated cellulose continuous long fiber nonwoven fabric and dried to prepare a sample pad. Further, a chromatographic medium made of a chromatographic medium film having an antibody immobilized at a specific position and an absorbent pad made of cellulose filter paper for absorbing a sample to be examined were prepared. These were fixed to a sheet having an adhesive portion called a Backing sheet (Backing sheet), and cut into a specific size, thereby producing an immunochromatographic diagnostic kit.

Examples

The present invention will be described more specifically with reference to examples and comparative examples, but the present invention is not limited to these examples.

< determination of average degree of substitution >

(i) Acidity and basicity

About 1g of the sample (anhydride) was precisely weighed into a 300mL Erlenmeyer flask, and dissolved by adding about 200mL of water. To this, 5mL of 0.05 mol/L sulfuric acid was added by a pipette, boiled for 10 minutes, cooled, added with phenolphthalein indicator, and titrated with 0.1 mol/L potassium hydroxide (SmL). A null test (BmL) was also performed, calculated by the following formula (1):

basicity { (B-S) × f }/weight of anhydrous sample (g) … formula (1)

{ formula (1): 0.1 mol/L potassium hydroxide titer }.

Here, when the value of (B-S). times.f is (-) the alkalinity can be read as the acidity.

(ii) Average degree of substitution

Precisely measuring 0.5-0.7 g of a sample (anhydrous substance), wrapping the sample in filter paper and ashing the filter paper in a porcelain crucible. After cooling, it was transferred to a 500mL beaker, approximately 250mL of water was added, 35mL of 0.05 mol/L sulfuric acid was further added with a pipette, and boiled for 30 minutes. It was cooled, phenolphthalein indicator was added, the excess acid was back-titrated with 0.1 mol/L potassium hydroxide, and the degree of substitution was calculated by the following formulas (2) and (3):

a ═ a (a xf-b xf 1)/weight of sample anhydride (g) -basicity (or + acidity) … formula (2)

Degree of substitution (162 × a)/(10000-80 × a) … formula (3)

{ formula (I), wherein A: amount of 0.05 mol/L sulfuric acid consumed by bound base in sample 1g (mL), a: amount of 0.05 mol/L sulfuric acid used (mL), f: titer of 0.05 mol/L sulfuric acid, b: titration amount (mL) of 0.1 mol/L potassium hydroxide, f 1: 0.1 mol/L potassium hydroxide titer }, and the average value thereof (N ═ 3 or more) was defined as the average degree of substitution.

< amount of liquid absorption C (g/100 cm)²))＞

The liquid absorption of the sheet-like structure was measured under conditions allowing free morphological changes. Specifically, the sheet-like structure was cut into 5cm × 5cm and placed in a petri dish. Then, 50 times the weight of the sample in physiological saline was heated to 37 ℃, added thereto, and allowed to stand in a thermostat at 37 ℃ for 30 minutes. Then, the liquid absorption amount was calculated from the weight before and after incubation according to the following formula (4):

liquid absorption amount C (g/100 cm)²) (B-a) × 4 … formula (4)

{ formula (I), wherein A: weight (g) in dry state before impregnation, B: weight (g) after 30 minutes incubation.

< weight per unit area D (g/m)²)＞

To the area of 0.5m²The nonwoven fabric was dried at 105 ℃ until a constant weight was obtained, and then placed in a thermostatic chamber at 20 ℃ and 65% RH for 16 hours or more, and the weight of the nonwoven fabric per unit area was measured.

< thickness >

The method comprises the following steps: the value obtained by measuring the load at 1.96kPa in a thickness test according to JIS-L1096.

< Release time (min) >

A plurality of test pieces each having an absorbent pad of 4mm in width by 25mm in length stacked on a chromatographic medium of 4mm in width by 25mm in length without any deviation were prepared, and physiological saline was slowly and without omission added to the absorbent pad of each test piece every 20. mu.L. When the time T1 (min) elapsed since the end of the addition of the physiological saline, the absorbent pad was removed from the chromatographic medium for one set of test pieces, and the surface state of the chromatographic medium was visually observed. At this time, when a color change was observed on the surface of the chromatographic medium due to wetting with physiological saline, the release time of the absorbent pad was T1 (min). On the other hand, in the case of a portion where no color change was observed, when the time T2 (min) (T1 < T2, where) elapsed since the end of the addition of the physiological saline, the color change on the surface of the chromatographic medium was judged by the same method as described above for a set of test pieces different from the test piece confirmed after the elapse of the time T1 (min). The above operation was repeated until a color change due to wetting was observed on the surface of the chromatographic medium, and the release time was determined. When no color change on the surface of the chromatographic medium due to wetting with a liquid was observed at any time from the completion of the addition of the physiological saline to the elapse of 900 minutes, the release time of the absorbent pad was set to 900 minutes.

< background of immunochromatographic diagnostic kit (BG staining) >)

The immunochromatographic diagnostic kit cut to an appropriate width is put in a plastic case. Next, 66mM PBS, pH7.4, containing 1 wt% BSA was prepared as a negative sample. Using the diagnostic kit containing the obtained shell, 100. mu.L of a negative specimen was dropped on the sample dropping part of the diagnostic kit, and after 5 minutes, the 1-point color development strength (in mABS) between TL and CL was measured using an immunochromatographic reader C10066-10 manufactured by Hamamatsu Photonics corporation. Here, when the color intensity is 20mABS or more, BG coloration is determined. The reason why 20mABS or more is used here is that if 20mABS or more is used, coloring can be reliably confirmed by visual observation.

< countercurrent confirmation of immunochromatographic diagnostic kit >

The confirmation of the reverse flow was performed by the same method as the above-described background confirmation experiment. The immunochromatographic diagnostic kit cut to an appropriate width was put in a plastic housing, and then, 66mM PBS containing 1% by weight BSA, pH7.4, was prepared as a negative specimen. Using the diagnostic kit comprising the obtained casing, 100. mu.L of a negative specimen was dropped on the sample dropping part of the diagnostic kit, and after 20 minutes, the color development intensity (in mABS) at 1 point between TL and CL was measured. Here, the case where the color intensity was 20mABS or more was judged to be BG-colored (judged as x). The non-colored state was recorded as good.

< deviation of color development intensity of Test Line (TL) >)

For the measurement of TL coloration intensity, 100. mu.L of the positive specimen was dropped into the sample dropping part of the diagnostic kit in the same manner as described above, and the coloration intensity (mABS) of the test line after 5 minutes was measured by the immunochromatographic reader. The measurement was performed 30 times in total, and the average color intensity (mABS) was taken as TL color intensity.

Further, the standard deviation of the TL developing intensity was simultaneously obtained, and the index% CV representing the reproducibility was calculated by the following formula (5):

% CV (standard deviation of TL developed intensity/TL developed intensity) × 100 … formula (5).

Reference example 1 (samples 1 to 15)

Regenerated cellulose continuous long fiber sheet-like structures (cuprammonium fiber sheet-like structures) (samples 1 to 12), cuprammonium short fiber nonwoven fabrics (sample 13), lyocell short fiber nonwoven fabrics (sample 14), and rayon short fiber nonwoven fabrics (sample 15) (width 20cm, weight per unit area, thickness 0.2 to 7.0 mm): 100g of the mixture was placed in a reaction vessel, and then an aqueous ethanol solution containing sodium hydroxide (water: 875g, ethanol 875g, NaOH: 162.5g) was added thereto, followed by stirring at 35 ℃ for 30 minutes. Subsequently, after the reagents in the reaction vessel were discharged, an ethanol aqueous solution containing sodium monochloroacetate (300 g of water, 960g of ethanol, 122.5g of sodium monochloroacetate) was added thereto, and the mixture was stirred at 30 ℃ or 50 ℃ for 1 to 12 hours. Then, drying is performed to obtain a carboxymethylated sheet-like structure. The sheet-like structure obtained above was adjusted to pH6.0 to 8.0 with an aqueous ethanol solution containing acetic acid (acetic acid: 37.5g, distilled water: 375g, ethanol: 875g), washed 1 time with 1375g of a 70 wt% aqueous ethanol solution, 1 time with 1250g of a 90 wt% aqueous ethanol solution, and alcohol-exchanged 2 times with 1250g of 100 wt% ethanol. Then, the sheet-like structure was immersed in 1250g (1 to 100 wt%) of an ethanol solution containing acetic acid or 2 to 5 wt% of an ethanol aqueous solution containing hydrochloric acid, stirred for 1 hour, washed 1 time with 1375g of a 70 wt% ethanol aqueous solution, 1 time with 1250g of a 90 wt% ethanol aqueous solution, subjected to 2 alcohol substitution with 1250g of 100 wt% ethanol, dried, and then placed in a hot air dryer at 105 ℃ for 6 hours or at 120 ℃ for 3 hours to obtain a sheet-like structure. The average degree of substitution, trapping and releasing indexes, weight per unit area, thickness, and liquid absorption of the obtained sheet-like structures (samples 1 to 15) are shown in table 1 below. These samples 1 to 15 were used as absorbent pads.

[ preparation of antibody-sensitized gold colloidal particles ]

To 2500. mu.L of gold colloidal particle suspension (product of Takara Shuzo Co., Ltd., average particle diameter 40nm, particle concentration 0.006 wt%, average particle diameter 40nm), 600. mu.L of phosphate buffer (50mM, pH7.0) was added, and 200. mu.L of a 0.1% aqueous solution of an anti-hCG-. alpha.mouse antibody (product of Fitzgerald Co., Ltd., monoclonal antibody) was further added, followed by stirring with a vortex. Subsequently, the mixture was stirred at 25 ℃ for 10 minutes while adjusting the temperature. To the suspension, 300. mu.L of a 1% PEG aqueous solution and 600. mu.L of a 10% BSA aqueous solution (pH9.0, containing 50mM boric acid) were added and stirred with swirling. Then, a centrifugation operation (10000g, 30 minutes) was performed to remove the supernatant. 11000. mu.L of a 1% BSA aqueous solution (containing 0.05% PEG, 150mM NaCl, pH8.2, and 20mM Tris) was added to the residue, and the mixture was stirred with a vortex. Then, a centrifugation operation (10000g, 30 minutes) was performed to remove the supernatant. To the residue, 900. mu.L of a 1% BSA aqueous solution (containing 0.05% PEG, 150mM NaCl, pH8.2, and 20mM Tris) was added, and sonication was performed for 30 seconds.

[ impregnation and drying of the labeling reagent into the conjugate pad ]

A bonding pad made of glass fiber (manufactured by Ahlstrom corporation, #8951) was cut into a shape having a height of 10mm and a length of 300 mm. Then, 1020. mu.L of the antibody-sensitized gold colloidal particle dispersion was applied uniformly, and the resultant was dried at 50 ℃ for 60 minutes.

[ pretreatment of sample pad ]

A sample pad (C048, Millipore) was immersed in a large excess of PBS buffer (66mM, pH7.4) containing 2.0 wt% BSA (Sigma-Aldrich Co., Ltd., A7906) and 2.0 wt% Tween-20 (registered trade mark), and after removing the excess liquid, the pad was dried at 50 ℃ for 60 minutes, and then cut into a shape having a height of 18mM and a length of 300 mM.

[ preparation of nitrocellulose Membrane coated with Capture antibody ]

A nitrocellulose membrane (SHF 0900425, manufactured by Millipore) was cut into a shape having a height of 25mm and a length of 300 mm. A PBS solution (66mM, pH7.4) containing 0.1% by weight of an anti-hCG-. beta.mouse antibody (manufactured by MedixBiochemica, Inc., 6601) was applied to a portion having a height of 7mM at a ratio of 0.1. mu.l/mM using a liquid application apparatus (300 DS manufactured by Wucang high tech Co., Ltd.). Next, a PBS solution (66mM, pH7.4) containing 0.1% by weight of an anti-mouse-rabbit antibody (manufactured by Daco, Z0259) was applied to a portion having a height of 12mM at a rate of 0.1. mu.l/mM. Subsequently, the mixture was dried at 37 ℃ for 30 minutes.

[ preparation of Immunochromatographic diagnostic kit ]

The prepared nitrocellulose membranes coated with the capture antibody, the absorbent pads (samples 1 to 15) manufactured in reference example 1, the binding pad containing the labeling reagent, and the sample pad were bonded to a cardboard liner (AR 9020, manufactured by Adhesives research). Then, the sheet was cut into a width of 5mm by a cutter to obtain an immunochromatographic diagnostic kit having a width of 5mm and a height of 60 mm.

[ examples 1 to 15]

[ evaluation of the Performance of the immunochromatographic diagnostic kit ]

Samples 1 to 15 shown in table 1 below were used as absorbent pads and evaluated. In the performance evaluation using an absorbent pad having a width of 5mm and a thickness of 0.2mm or more of the immunochromatographic diagnostic kit, the development potential amount of the developing solution was about 80 to 120. mu.L, and therefore 100. mu.L of the developing solution was actually developed and evaluated. In addition, when the width of the reagent kit is 2.5mm or the thickness of the absorbent pad is less than 0.2mm, the development energy of the developing solution is about 30 to 80. mu.L, and thus 50. mu.L of the developing solution is actually developed and evaluated. The evaluation results are shown in table 2 below. Any of the diagnostic kits had a small variation and no adverse current occurred. In addition, any diagnostic kit has less defibering, and can ensure safety in the process.

[ Table 1]

[ Table 2]

[ sample 16]

A regenerated cellulose continuous long fiber sheet-like structure (cuprammonium fiber sheet-like structure) (width 20cm, 100 g/m) was prepared²Weight per unit area, thickness 0.9 mm).

[ sample 17]

A regenerated cellulose continuous long fiber sheet-like structure (cuprammonium fiber sheet-like structure) (width 20cm, 100 g/m) was prepared²Basis weight, thickness 1.0mm), and the sheet-like structure was obtained by the same method as in reference example 1 except that the amount of monochloroacetic acid used was increased and the degree of substitution was increased.

[ sample 18]

A regenerated cellulose continuous long fiber sheet-like structure (cuprammonium fiber sheet-like structure) (width 20cm, 300 g/m) was prepared²Basis weight and thickness 4.0mm) was processed in the same manner as in reference example 1 to obtain a sheet-like structure.

[ sample 19]

A rayon staple fiber sheet-like structure (width 20cm, 300 g/m) was prepared²Basis weight and thickness of 12.0mm) was carried out in the same manner as in reference example 1, except that the sheet-like structure was obtained.

[ sample 20]

A sheet-like structure of rayon staple fibers (width 20cm, 430 g/m) was prepared²Basis weight and thickness 6.7mm) were processed in the same manner as in reference example 1 to obtain a sheet-like structure.

[ comparative examples 1 to 7]

[ evaluation of the Performance of the immunochromatographic diagnostic kit ]

The evaluation was performed using samples 16 to 20 shown in table 3 below. The above samples 16 to 20 and an absorbent pad (74 g/m) made of glass fiber were used²Weight per unit area and thickness of 0.5mm), and cellulose absorbent pad (180 g/m)²Weight per unit area and thickness of 0.51mm) was evaluated in the same manner as in example 1. The evaluation results are shown in table 4 below. As a result, the sample 16 which had not been carboxymethylated was caused to flow backward, and the gel of the absorbent pad was disintegrated and the variation became large during the development of the sample 17 whose average degree of substitution exceeded the range of the present application. The capture and release index of the sample 18 is too high, and thus the deviation of the inspection results becomes large. Sample 19 was too thick, and therefore, flexibility was impaired, and the adhesive could not be reliably bonded, and the variation was large. Sample 20 had an excessively large weight per unit area, so that flexibility was impaired, handling property was significantly lowered, and reliable adhesion was not achieved, and variation was large. The glass fiber-made absorbent pad and the conventionally used cellulose-made absorbent pad are subjected to a reverse flow. In addition, when glass fibers are used, the amount of detached fibers is large, and it is difficult to ensure safety in the process.

[ Table 3]

[ Table 4]

Industrial applicability

The immunochromatographic diagnostic kit using the absorbent pad for an immunochromatographic diagnostic kit of the present invention has little variation in color development intensity and excellent reproducibility. Furthermore, since the reverse flow can be prevented, the absorbent pad can be suitably used as an absorbent pad for various immunochromatographic diagnostic kits.

Description of the reference numerals

(a) Sample pad

(b) Combined pad (containing antibody sensitization labeling reagent)

(c) Test Line (TL)

(d) Control line (CT)

(e) Chromatographic medium film sample pad

(f) Absorption pad

(g) Lining paper

Claims

1. An absorbent pad for an immunochromatographic diagnostic kit, comprising carboxymethylated cellulose fibers, wherein the average degree of substitution of hydroxyl groups in glucose units constituting the carboxymethylated cellulose fibers is 0.05 to 1.5.

2. The absorbent pad for an immunochromatographic diagnostic kit according to claim 1, wherein the capture and release index of the pad is 0.5 or less.

3. The absorbent pad for immunochromatographic diagnostic kit according to claim 1 or 2, wherein the weight per unit area of the pad is 10g/m²Above and 400g/m²And a thickness of 0.03mm to 10.00 mm.

4. The absorbent pad for an immunochromatographic diagnostic kit according to any one of claims 1 to 3,wherein the pad has a liquid absorption amount of 5g/100cm when impregnated in a physiological saline solution²Above and 50g/100cm²The following.

5. The absorbent pad for an immunochromatographic diagnostic kit according to any one of claims 1 to 4, wherein the pad is in the form of a single-layer sheet or a laminated sheet.

6. An immunochromatographic diagnostic kit comprising the absorbent pad for an immunochromatographic diagnostic kit of any one of claims 1 to 5.