JPH07151754A - Specific bond assaying reagent and measuring method using it - Google Patents

Abstract

PURPOSE:To provide a method by which a patient can be diagnosed in an earlier stage by using his fresh blood, blood serum, or blood plasma and can be cured in an early stage even when an immunological colloidal particle labeling or a reactive reagent for micro-particle labeling chromatography are used and, at the same time, to quickly and accurately perform inspections, especially, in the field of emergency medical treatment. CONSTITUTION:A specific bond assaying reagent containing a colloidal particle or micro-particle labeling bonding material and buffer solution contains heparin in an amount which is sufficient to substantially prevent an undesirable nonspecific bonding reaction. In addition, an agglutination reaction assaying reagent containing immobilized carrier particles and a buffer solution contains heperin in an amount which is sufficient to substantially prevent an undesirable nonspecific bonding reaction. In addition, when measurement is performed by using these reagents, the measurement is performed under the presence of heperin in a sufficient amount which is sufficient to substantially prevent an undesirable nonspecific bonding reaction.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a specific binding assay method using a reagent labeled with colloidal particles or microparticles, in which measurement is carried out using fresh blood or serum or plasma as an analytical sample. Even if there is, there is no serious problem such as the occurrence of false positive reactions that are thought to be due to non-specific binding reactions,
Yet it provides an easy way. The present invention is a specific binding assay method using a colloidal particle-labeled or microparticle-labeled reagent, in which colloidal particle-labeled or microparticle-labeled reagent in contact with fresh blood or serum or plasma as an analysis sample, By adding and blending heparin,
It is a quick and accurate assay.

In particular, the present invention relates to an immunological assay method in which the principle of chromatography is combined with a colloidal particle label or a microparticle label, together with a reagent containing blood or serum or plasma as an analysis sample and heparin, together with the colloidal particle label. Alternatively, the microparticle-labeled binding substance is chromatographically transferred and reacted with a specific binding substance such as an antigen or antibody immobilized on a chromatography carrier with or without an analyte in an analytical sample. The present invention relates to a method for specifically detecting an antibody, an antigen, etc. in an analysis sample using a colloidal particle label or a microparticle label, and a reagent used therefor. The present invention also provides a fresh blood or serum or an immunological agglutination assay method using an insoluble carrier particle (hereinafter, abbreviated as immobilized carrier particle) reagent on which a specific binding substance such as an antigen or an antibody is immobilized. The corresponding antibody or antigen in the sample is measured by reacting an analytical sample that is plasma with a reagent containing immobilized carrier particles and heparin, and observing the agglutination reaction or agglutination inhibition reaction based on the antigen-antibody reaction that occurs. Method, and a reagent used therefor.

[0003]

BACKGROUND OF THE INVENTION There is a need for a simple, highly reliable, non-hazardous, highly sensitive method for detecting substances present in liquids at low concentrations. Detecting low concentrations of a substance can be very difficult in the field of clinical testing, where the amount of sample that can normally be used in the test is very limited.

However, analytical systems having high specificity and applicable to detection of a wide range of substances have been studied, and analytical methods utilizing specific binding reactions have been developed. The specific binding assay thus developed has proved to be very useful for various clinical applications. Among such specific binding assays, particularly immunological assays are antigen-antibody which is a specific binding reaction between an immunoglobulin (antibody) and a substance having a specific antigenic determinant (antigen). It utilizes a reaction. The antibody selectively binds to a ligand substance showing antigenicity. That is, an antibody specifically reacts with a ligand substance having antigenicity, and makes it possible to distinguish the ligand from other substances including those having similar properties. Examples of the immunological assay method include a radioimmunoassay method, a fluorescent immunoassay method, a luminescent immunoassay method, an enzyme immunoassay method, and a fine particle agglutination assay method such as latex, and various blood or serum or plasma are to be measured. It is used in a wide range of fields such as human clinical examination and diagnosis, and animal disease diagnosis.

In recent years, this immunological assay method has been widely used and applied to human clinical examinations and diagnoses, animal disease diagnosis, or research in many fields. In this immunological assay method such as enzyme immunoassay method or radioimmunoassay method, an antigen, an antibody or the like is immobilized on a solid support such as a microplate such as polystyrene, and the solid support is used as an analysis target. By contacting with various analysis samples such as blood or serum containing antigens, etc., the antigens, antibodies, etc. fixed on the solid support can be specifically bound to the antibodies, antigens, etc. in the analysis sample. The free binding sites present on the surface of the support and the analyte in blood, serum, or plasma that is an analytical sample binds nonspecifically, which causes a measurement error. For this reason, usually, an antigen or an antibody is immobilized on a plate, and then a solid support is coated with a blocking agent to block the free sites, thereby preventing non-specific adsorption and performing the measurement. As typical blocking agents used for this measurement, bovine serum albumin, egg albumin, gelatin, skim milk, polyvinyl alcohol and the like have been conventionally known.

By the way, recently, as a method for quickly, highly accurately and simply measuring a relatively small amount of an analyte in an analytical sample such as blood or serum, a reagent labeled with colloidal particles or microparticles is used. A specific binding assay method utilizing PEG has been developed, but when such a colloidal particle-labeled or microparticle-labeled reagent is used, the assay can be performed easily, quickly, with high sensitivity and by visual detection. Because it can be easily done, it is not necessary to use various measuring instruments, uniform processing of many samples in a short time, and no need for skilled technical operation It is bathing. Especially, the combination of chromatographic principle and colloidal particle labeling has been proposed as a method which enables rapid, highly sensitive and easy visual detection of an extremely small amount of an analyte such as blood, serum or plasma with a simple operation. Immunoassays are gaining attention. In this method, a colloidal particle-labeled or microparticle-labeled reagent is chromatographically transferred together with an analysis sample, and an antibody, an antigen, etc. in the analysis sample is measured by detecting the labeling reagent on the chromatography medium. .

[0007] However, in the medical field using immunological colloidal particle-labeled or microparticle-labeled chromatographic reaction reagents, in order to diagnose earlier and realize early treatment, or at bedside, etc. Furthermore, it is required to measure a smaller amount of antibody or antigen immediately using an analysis sample such as blood or serum. Particularly in recent years, there is an increasing need for immediate examination in the field of emergency medicine. However, in such a situation, it was found that a false-positive reaction occurs and a problem occurs when the measurement is performed using fresh blood, serum or plasma.
Furthermore, regardless of how the analytical sample was obtained, and whether the measurement personnel were excellent in the procedure,
It is required to obtain accurate and accurate measurement results immediately.

Further, conventionally, an antigen-antibody reaction has occurred between an insoluble carrier particle having an antigen or an antibody immobilized thereon by physical adsorption or formation of a covalent bond on the insoluble carrier particle and a corresponding antibody or antigen in a specimen such as serum or urine. An immunological measurement reagent for measuring a corresponding antibody or antigen in a sample by observing an agglutination reaction or an agglutination inhibition reaction based on the agglutination reaction is known. And, the measuring method using the immobilized carrier particles can measure a relatively small amount of antibody or antigen contained in a sample quickly, highly accurately and easily, and in that case, it can be measured in a short time. It can be used for diagnosis and is widely used.

However, even when such an immunological agglutination reagent is used, a smaller amount of antibody or antigen is added to blood or serum in order to diagnose earlier and realize early treatment. There is a demand for quick and accurate measurement using an analytical sample. Conventionally, polyethylene glycol was added to the reagent for this immunological agglutination reaction in an attempt to improve the measurement sensitivity of the reagent (Japanese Patent Laid-Open No. 58-58).
However, there is a drawback that the specificity of the measurement is poor due to the reaction between polyethylene glycol and the sample or the reaction between polyethylene glycol and the immobilized carrier particles. Also add polyvinylpyrrolidone,
It has also been proposed to try to improve the measurement sensitivity by suppressing non-specific reactions. However, it has not been known that the occurrence of false positive reactions is reliably prevented when the measurement is performed using fresh blood or serum or plasma.

[0010]

Means for Solving the Problems As a result of intensive studies conducted by the present inventors to obtain an immunological reaction reagent capable of achieving the above object, the present invention comprises a colloidal particle labeling or microparticle labeling binding substance and a buffer solution. In the measurement reagent,
When heparin is added, the specificity is not impaired in clinical diagnosis, and there is no problem such as false positive even when fresh blood or serum or plasma is used as an analytical sample for urgent measurement. The present invention was started and the present invention was completed.

That is, the present invention provides a measurement reagent comprising a specific binding substance, for example, colloidal particles or microparticles on which an antigen or an antibody is immobilized, and a specific binding assay reagent containing heparin. I will provide a. It is also an object of the present invention to provide means for improving the assay method when colloidal particles or microparticles used as a label in a specific binding assay are used by utilizing the chromatographic transfer principle. To do. Furthermore, the present invention provides a specific binding agglutination assay reagent characterized by containing heparin in a measurement reagent comprising a specific binding substance, for example, insoluble carrier particles on which an antigen or antibody is immobilized and a buffer solution. Will also be provided.

When fresh blood, serum or plasma is used for the measurement, the immunoassay system using the colloidal particle-labeled or microparticle-labeled binding substance must absorb or block the non-specific reaction derived from the sample. I couldn't, and I couldn't trust the measurement results. In other words, until now, it has not been possible to prevent non-specific reactions caused by fresh blood or serum or plasma. On the other hand, in the present invention, fresh blood or serum or plasma can be used as an analytical sample by a simple method, and a non-specific reaction can be absorbed or blocked irrespective of which blood collection method they used. . In the present invention, substantially preventing an undesired non-specific binding reaction means that, for example, using fresh blood or serum or plasma within 3 hours or less after blood collection as an analysis sample, for example, When an immunochromatographic assay is performed, it refers to preventing false positive reactions that cause a serious problem in the assay result, and enabling accurate visual identification of the assay result.

In a preferred embodiment of the present invention, an immunological assay reagent comprising colloidal particles or microparticles immobilized with an antigen or antibody and a buffer solution substantially prevents an undesired nonspecific binding reaction. A sufficient amount of heparin. The assay reagent does not have to be a single liquid, and may be composed of a plurality of liquids and dispersion liquids. When the assay reagent is a single solution, the sample and the assay reagent are mixed and used, and when the assay reagent is composed of multiple solutions and dispersions, each component of the reagent and the sample are followed according to a certain operation procedure. Use with mixed. When the assay reagent is composed of a plurality of liquids and dispersions, heparin may be added to select a component containing heparin so that the reagent characteristics do not change during storage. If the assay reagent is a single solution, prepare two solutions, a solution in which immobilized colloid particles or microparticles are dispersed and a buffer solution containing heparin, and mix them into one solution immediately before use. It is preferable because it can prevent undesired reaction between the specific binding substance labeled with colloidal particles or microparticles and heparin during storage.
It is also possible to separately use solid heparin as a component of the assay reagent. In the present invention,
Heparin may be used as a component of the reagent as it is as a solid, but in order to carry out the assay operation easily, it is preferable to dissolve the heparin in a buffer solution before use.

Examples of heparin include those obtained from the liver, lung, or intestinal mucosa of healthy edible animals such as cattle, sheep, pigs and chickens. Those produced from bovine lung and porcine small intestinal mucosa can be preferably used. Heparin is D-glucosamine, D-glucuronic acid, L-
Mucopolysaccharide sulfate ester consisting of iduronic acid, usually sodium heparin, calcium heparin is available,
Heparin sodium is preferred because it is water-soluble. Heparin having a molecular weight of 6,000 daltons to 30,000 daltons is generally easily obtained and can be used in the present invention. However, heparin has a molecular weight of 7,000 daltons to 25,000.
Daltons can be used more preferably.

In the case of this immunological specific binding assay, the amount of heparin used has the effect of improving the sensitivity, the phenomenon that the labeled colloidal particles or microparticles react nonspecifically and the specificity decreases, and Can be determined in consideration of the phenomenon that the reagent viscosity increases and the assay becomes difficult, but the concentration of heparin relative to the total volume of the solution at the time of measurement should be sufficient to suppress non-specific binding reactions. It is necessary, for example, that the concentration of heparin relative to the total amount of liquid at the time of measurement is preferably set to a value larger than 0.05 mg / ml, for example, 0.06 mg / ml to 0.4 mg / ml
The range is preferably 0.08 mg / m
It is particularly preferable that it is in the range of 1 to 0.2 mg / ml.

The immunological specific binding assays of the present invention include three types, namely competitive binding assays, sandwich assays and agglutination assays, which are well known in the art. .
Further included are methods for coating the surface of microtiter wells with an antibody or antigen and conducting the reaction in solution in such wells. Attempts to measure aggregates with electronic devices such as lasers are well known. The colloidal particles that can be used in connection with the methods, kits and devices of the invention can be those commonly used in specific binding assays.
Especially well known are colloidal metal particles,
Especially the use of colloidal gold for carrying out immunological assays. Polymeric dye particles, which are also used as labels in specific binding assays such as those disclosed in US Pat. No. 4,166,105 to Hirschfeld and US Pat. No. 4,452,886 to Henry. Other colloidal particles or microparticles could also be used in this chromatographic transfer specific binding assay. Colloidal particle labels also include non-metal particles such as selenium, tellurium and sulfur,
It is to be understood that anything that gives a visible signal, without limitation, can be used for the purposes of the present invention.

Colloidal particles and microparticles suitable for use as the label of the present invention include those bound to the specific binding substance without interfering with the activity of the specific binding substance or other reagents or analytes. included. The particles must be detectable, but preferably produce a visually detectable signal even when present at relatively low concentrations. Colloidal metal particles are one particularly suitable for use as labels, including particles made of metal or metal compounds including metal oxides, metal hydroxides and salts. The particles may consist of pure metals or metal compounds, but also those with polymer cores coated with metals or metal compounds. Such particles are known to exhibit properties similar to particles made of pure metal or metal compounds. Suitable metals or metal compounds include, for example:
Metals such as platinum, gold, silver and copper, for example silver iodide, silver bromide, silver hydroxide, iron oxide, iron hydroxide or hydrated iron oxide, aluminum hydroxide or hydrated aluminum oxide, chromium hydroxide or Examples thereof include those selected from the group consisting of metal compounds such as hydrated chromium hydroxide, copper sulfate, mercury sulfate, and barium sulfate titanium dioxide. Preferred metal colloidal particles include particles made of gold, silver or iron oxide.

The colloidal metal particles can be manufactured according to methods generally known in the art. Fren's Nature, 241 , 20
(1973) discloses a method for producing gold sol particles of various sizes. The gold colloidal particles can be produced by a method in which a solution of gold chloride is heated to boiling and then mixed with a solution of sodium citrate to reduce gold chloride. As soon as the two solutions were mixed, the boiling solution turned light blue indicating the onset of nucleation, and shortly thereafter the blue turned red indicating the formation of monodisperse particles. The reduction of gold chloride is complete after boiling continues for a few more minutes. The size of the particles obtained can be adjusted by varying the concentration of the sodium citrate solution. Particles of other metals and metal compounds as well as particles with polymer nuclei could also be obtained by similar methods. The color of the visually detectable signal provided by the metal particle label is different depending on the type and particle size of the metal particles. For example, colloidal gold particles produce colors ranging from orange to red to magenta depending on the particle size of the sol.

The non-metal colloidal particles such as selenium, tellurium and sulfur can be prepared by the method described in JP-A-64-35372 or a modification thereof.
Among the non-metallic colloidal particles thus obtained, selenium colloidal particles are particularly preferable for use in the specific binding assay. As micro particles,
Examples thereof include fine particles composed of synthetic or natural polymers such as bentonite, latex, and others known in the art. Of these, those colored with a dye or the like can be preferably used.

The specific binding substance of the present invention and the colloidal particle or microparticle label can be bound by a method generally known in the art. The colloidal particle label or the like need not be directly bound to the specific binding substance, and may be attached by coating or pretreatment with another reagent. Leveling U.S. Pat. No. 4,313,734 discloses a method of coating metal sol particles with inert polymer and copolymer coatings. The metal sol may be contacted with the polymer or the sol may be placed in an environment containing one or more monomers to initiate the polymerization reaction. After coating with an inert polymer, the specific binding component can be attached to the coating material by adsorption or covalent bonding.

The chromatographic medium useful in the present invention is a chromatographic medium that exhibits capillary action and is capable of transferring non-immobilized reagents and reactive sample components to the chromatographic solvent by the selected chromatographic solvent. Substances. The chromatographic medium substance used in the present invention is preferably in the form of a strip, and further, those in which these strips are connected at a plurality of ends can be mentioned, but other forms including particles or gel substances in a chromatography column are mentioned. However, it is not limited to these. A wide range of chromatographic strip materials such as woven and non-woven fibrous materials used in paper chromatography can be used in the present invention, although microporous or fine granular thin layer chromatography can be used. Substances are particularly preferred because they enhance the speed and analytical power of the assay. Chromatographic substances that are inert and generally do not physically or chemically react with any of the sample components, reagents, colloidal particle labels, buffers or reaction products are preferably used. Others, such as those that react positively with interfering substances and give good assay results, are acceptable without departing from the scope.

The substrate material for the chromatogram which can be used in the present invention may be any solid such as glass or plastic, but a porous or fibrous matrix such as paper or nitrocellulose is preferable and thin. Examples thereof include those used for layer chromatography, and particularly suitable substrate materials for thin layer chromatography include materials for granular thin layer chromatography such as silica and fine granular cellulose. As the preferred non-granular porous material, microporous cellulose ester,
Examples thereof include cellulose esters with aliphatic carboxylic acids such as alkanecarboxylic acids having 1 to 7 carbon atoms such as acetic acid, propionic acid, butyric acid, and valeric acid. Particularly preferred are microporous materials made from nitrocellulose, where the nitrocellulose may include any nitrate ester of cellulose. Preferred chromatographic materials include a combination of nitrocellulose and the above carboxylic acid cellulose ester. Therefore, modifying the pure cellulose ester to give 6
Cellulose esters which contain 3 nitrate groups per carbon atom can also be used. As the substance for nitrocellulose chromatography, a commercially available product can be used.

Various chromatographic substrate materials include
As such, it can be used in a suitable form such as a film, a strip, a sheet or a strip. These substrate materials may also be coated and adhered laminates on suitable inert support materials such as paper, glass, plastics, metals or fabrics. Such a support material not only serves as a structural support for the chromatographic substrate material, but also serves to prevent evaporation of reagents and solvent materials during the assay process. The cover plate can also be made of such an inert material. Cover plates are not necessary for the practice of the invention, but are preferred to provide additional structural support and prevent evaporation of reagents and solvent substances during the assay process. Such cover plates may be transparent so that the progress of the assay may be viewed and may be provided with a port for the addition of sample material, chromatographic solvent or reagents.

The morphology and size of the chromatographic medium in which the assay is performed may be any, but those having a thickness in the range of about 0.01 mm to about 0.5 mm are preferably used, most preferably. In the form of strips about 0.1 mm thick,
It is not limited to this. The dimensions of the other parts of the strip can be varied over a wide range, but are preferably considerably smaller to reduce the time to assay progress and minimize the use of expensive reagents as well as chromatographic substrate materials. If the strip is very small, it may be attached to a suitable handle or holder to facilitate handling and observing the results. Strips having a width of about 3 mm to about 5 mm and a length of about 40 mm to about 100 mm are particularly suitable, although other sizes may be used for the purposes of the present invention. The pore size can be varied over a wide range, but the lower limit of the pore size will be limited by the size of the analyte, reagent and colloidal particle label to be moved. If the pore size is too small, the assay substance will either migrate slowly or not at all. On the other hand, the upper limit of the pore size is limited by the bond value. It is generally preferred that the chromatographic transfer be carried out promptly and that the assay be completed promptly. The chromatographic migration should not be too fast and the specific binding values should not be lost due to the lack of time for the reagents to specifically bind to each other. Therefore, the combination of pore size and substrate thickness and size can be varied depending on the properties of the chromatographic solvent, specific reagents, sample material and colloidal particle label to obtain the desired speed and analytical power. it can. In preparing the strip material, it is desirable to avoid irregularities in the material, or in the edges of the material, resulting in non-uniform flow in the chromatographic material.

Since the chromatographic medium of the device used in the present invention is preferably chemically inert, the immobilization is accomplished in any zone where it is desired to immobilize the specific binding substance against solvent transfer. In some cases, activation may be required. Various methods can be used to immobilize the reagents, depending on the particular chemistry of the chromatographic substrate material and reagents used. Examples of such a method include a covalent bond and a physical adsorption method. The covalent bond method is a method of immobilizing a chromatography medium and a reagent by covalently bonding them. The functional group involved in binding the reagent to the chromatographic substrate material by this method is α-
Examples thereof include amino group, ε-amino group, α-carboxyl group, β-carboxyl group, γ-carboxyl group, hydroxyl group, mercapto group, imidazole group and phenol group. These functional groups are usually converted into a more reactive functional group by an aldehyde, an acid halide, an acid azide, an acid anhydride, a diazonium group, an isocyanate, an active alkyl halide or the like for use.

Generally, when the chromatographic medium is nitrocellulose or mixed nitrocellulose esters, no special chemical coupling is required to immobilize the reagents. Various techniques can be used for other substances and reagents, including carbonyldiimidazole, glutaraldehyde, succinaldehyde, malonaldehyde, succinic acid, o-phenylene bismaleimide, epichlorohydrin, N-hydroxysuccinimide, Diisocyanate compound, diisothiocyanate compound, N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, [N- (m-maleimidobenzoyloxy) succinimide], N-succinimidyl 3- (2-pyridyldithio) Functionalization with substances such as propionate, or treatment with substances such as cyanogen bromide are included. Other suitable reactions include Schiff bases and aldehyde groups,
Treatment with borohydride to reduce carbonyl and amino groups may be mentioned.

Specific binding substances suitable for use in the present invention include those that are one of the specific binding partners consisting of ligand and receptor. A ligand and a receptor can be considered to be closely related to each other in that the receptor can specifically bind to the ligand and discriminate the ligand from substances having other similar properties. The methods, kits and devices of the present invention include those that are particularly useful in performing immunological assays in which the specific binding agents are antigens and antibodies. Further, specific binding substances such as avidin, biotin, streptapidine and antibiotin can also be labeled with colloidal particles or microparticles and used, and can be used in the chromatographic solvent transfer assay according to the present invention. The methods, kits and devices of the invention will also be useful in conducting other specific binding assays, including those involving receptors for hormones and other biologically active agents.

In the present invention, heparin may be contained in an agglutination assay reagent containing immobilized carrier particles and a buffer solution. The assay reagent does not have to be a single liquid and may be composed of a plurality of liquids and solids. When the assay reagent is a single liquid, the sample and the assay reagent are mixed and used, and when the assay reagent is composed of multiple liquids and solids, each component of the reagent and the sample are followed according to a certain operation procedure. Use with mixed. If the assay reagent consists of multiple liquids and solids,
The component containing heparin may be selected so that heparin is not added and the reagent properties do not change during storage. If the assay reagent is a single solution, prepare two solutions, a solution in which immobilized carrier particles are dispersed and a buffer solution in which heparin is added,
It is preferable to use it by mixing it with one solution immediately before use because it is possible to completely prevent nonspecific reaction between the immobilized carrier particles and heparin during storage. It is also possible to separately use the dried immobilized carrier particles and the solid heparin as one component of the assay reagent.

In the present invention, heparin can be used as a component of the reagent as it is as a solid, but it is preferable to use it by dissolving it in a buffer solution in advance in order to easily carry out the assay operation. The buffer used in the present invention can be used various buffers without limitation, such as phosphate buffer,
Buffers such as glycine-sodium hydroxide buffer, Tris-hydrochloric acid buffer, ammonium chloride-ammonia buffer, etc. are preferably used. The amount of heparin used considers the effect of improving sensitivity, the phenomenon that the immobilized carrier particles non-specifically aggregate to reduce the specificity, and the phenomenon that the reagent viscosity increases and stirring becomes difficult. The concentration of heparin relative to the total amount of liquid at the time of measurement must be sufficient to suppress non-specific binding reactions.For example, the concentration of heparin relative to the total amount of liquid at the time of measurement. And then 0.05 mg
It is preferable that the value be larger than / ml,
It is preferably in the range of 0.06 mg / ml to 0.4 mg / ml, and further 0.08 mg / ml to 0.2 mg
It is particularly preferable that it is in the range of / ml.

In the present invention, the immobilized carrier particles are prepared by immobilizing a specific binding substance such as an antigen or an antibody on the insoluble carrier particles. The insoluble carrier particles are insoluble carrier particles that are substantially insoluble in the liquid medium used for immobilization, storage and measurement, and fine particles having an average particle size of about 10 μm or less are preferably used. As these particles, various particles already used for specific binding reaction, for example, antigen-antibody reaction are known, and in the present invention, these known fine particles can be used without particular limitation. Particularly preferred examples are those obtained by emulsion polymerization such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer. Silane coupling to fine particles of organic polymer substances such as organic polymer latex particles, fine particles of gelatin, bentonite, agarose, cross-linked dextran, etc., or inorganic oxides such as silica, silica-alumina, and alumina, or inorganic oxides thereof. Examples thereof include inorganic particles having a functional group introduced by an operation such as treatment, and particles of biological origin such as human O-type red blood cells and sheep red blood cells.

Antibodies useful in carrying out the immunological assays of this invention include those that specifically react with a variety of analytes for which detection in biological fluids is desired. Such antibodies are preferably IgG, IgM, IgA, I
gE, IgD antibodies or mixtures thereof, which should be substantially free of association with antibodies capable of binding non-analyte molecules. The antibody may be polyclonal or monoclonal. As the antibody, known methods include mammals such as rabbits, guinea pigs, mice, goats, sheep and horses,
It may be obtained by immunizing birds such as chickens, ducks, geese, etc. with an antigen, a hapten, etc., and may be obtained by using biotechnology, for example, a technique such as a cell fusion method. Is purified before use. These can be commercially available,
The monoclonal antibody may also be mouse ascites fluid, tissue culture or obtained by other techniques known in the art.

Antigens and haptens useful in carrying out the immunoassays of the present invention can be selected whether natural or synthetic. In particular, a substance which, when present on the chromatographic strip substance of the present invention, exhibits an antigenic determinant site to which an antibody to be analyzed specifically reacts is mentioned. According to the present invention, hormones, vitamins, medicines, agricultural chemicals, metabolites, biological components, receptors, etc. can be measured. Synthetic antigens include those produced by conventional chemical synthesis and those produced by recombinant DNA technology.

Examples of the antigen include steroid hormones, peptide hormones and protein hormones, biological proteins, bacteria such as Mycobacterium tuberculosis and MRSA, α-fetoprotein, carcinoembryonic antigen (CEA), cancer antigens such as CA 19-9, and the like. Hepatitis B surface antigen (HBs antigen), rabies virus, influenza virus, rubella virus, AIDS
Examples include virus antigens such as viruses. In the present invention, the antigen or antibody immobilized on the insoluble carrier particles is not particularly limited, and known ones as described above can be used.

In the present invention, as the method for immobilizing the antigen or antibody on the insoluble carrier particles, the above-mentioned method can be used, and either physical adsorption or chemical covalent bond formation may be used. Many methods have already been proposed for forming a chemical covalent bond, and the immobilization method may be selected from known methods depending on the characteristics of the antibody to be immobilized. Generally, the antibody may be mixed with the insoluble carrier particles in the dispersion medium in the presence of a crosslinking agent, if necessary. Known crosslinking agents such as glutaraldehyde and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride can be used. The dispersion medium for immobilizing the antigen or antibody on the insoluble carrier particles is not particularly limited and a known dispersion medium is used, but when the above crosslinking agent is used, the components in the dispersion medium react with the crosslinking agent. It is necessary to use a dispersion medium that does not. The concentration of the insoluble carrier particles in the dispersion medium at the time of immobilization is not particularly limited. The concentration of the antigen or antibody is also not particularly limited. In the present invention, an immunological assay method using immobilized carrier particles, that is, an agglutination reaction based on an antigen-antibody reaction between an antigen or antibody on antibody-immobilized carrier particles and a corresponding antibody or antigen in a test sample As a method for observing the aggregation-inhibiting reaction, known methods such as visual observation and optical measurement can be used without particular limitation.

[0035]

EXAMPLES Next, the present invention will be described more specifically by showing examples. Example 1 A sandwich immunoassay method using a monoclonal antibody using a chromatographic principle was performed as follows. The measurement kit consists of 1) anti-HBs mouse monoclonal antibody solid-phase chromatography sheet 2) selenium colloid-labeled anti-HBs mouse monoclonal antibody reagent 3) selenium colloid-labeled anti-HBs mouse monoclonal antibody reagent solution.

(Preparation of selenium colloid) Boiling distilled water (2
To 00 ml) was added 3% selenium dioxide (2 ml) and freshly dissolved 1% ascorbic acid (4.5 ml).
The solution was refluxed for 10 minutes and then cooled to room temperature. The resulting brownish red solution was centrifuged at 2,500 xg for 20 minutes at 4 ° C. The pellets obtained were distilled water (2
It was resuspended in (00 ml).

(Preparation of selenium colloid-labeled anti-HBs mouse monoclonal antibody reagent) 10 mM Tris buffer (p
Purified anti-HBs mouse monoclonal antibody (1.0 mg / ml) manufactured by Dynabot Co. in H7.4) is added to 5 ml of the above selenium colloid so as to be 12 μg / ml, and the mixture is stirred at 25 ° C. for 20 minutes. Thereafter, polyethylene glycol (PEG: average molecular weight 20 k) was added to a final concentration of 0.
The mixture was added to be 5% and centrifuged at 8,000 xg and 4 ° C for 5 minutes to obtain a pellet. 2% FCS,
50% containing 2% alkaline treated casein and 0.5% PEG
It was suspended in 75 ml of mM Tris buffer (pH 7.4).
This suspension of selenium colloid-labeled antibody solution was freeze-dried to obtain a selenium colloid-labeled anti-HBs mouse monoclonal antibody reagent.

(Preparation of selenium colloid-labeled anti-HBs mouse monoclonal antibody reagent solution) A 10 mM Tris buffer solution (pH 7.4) was prepared and used as a solution.

(Anti-HBs Mouse Monoclonal Antibody Solid-Phase Chromatography Sheet) The test sheet (test strip) used nitrocellulose paper cut into a size of 0.3 cm × 3.0 cm as a support phase. 1 μl of a purified anti-HBs mouse monoclonal antibody solution (3.0 mg / ml) manufactured by Dynabot was spotted on a test strip and dried at room temperature to obtain a final solid-phase chromatography sheet.

(Measurement test) Heparin plasma,
EDTA plasma and serum were used. The heparin concentration in heparin plasma was at a normal blood sampling level. Each sample must be within 30 minutes, 1 hour, 3 hours,
After 5 hours and 1 day. The selenium colloidal particle-labeled antibody reagent was dissolved and prepared in an equal volume of the solution for selenium colloidal particle-labeled antibody reagent to obtain the selenium colloidal particle-labeled antibody reagent solution thus obtained. On the other hand, similarly, another selenium colloidal particle-labeled antibody reagent of the same lot is dissolved and prepared in an equal volume of the selenium colloidal particle-labeled antibody reagent solution, and heparin is added to the selenium colloidal particle-labeled antibody reagent solution thus obtained. An antibody reagent solution added so as to have a concentration of 0.2 mg / ml was prepared and used as a heparin-added selenium colloid particle-labeled antibody reagent solution.

Selenium colloid particle labeling antibody reagent solution or heparin-added selenium colloid particle labeling antibody reagent solution 25
μl is dropped into the test hole using a micropipette. Next, 25 μl of a positive control or a sample is added to each of the holes using a micropipette. The reagent solution was subjected to suspension treatment so as to be uniform before addition. After adding the sample, stir. The mixture is then pre-incubated at about 15 to about 30 ° C. for about 2 minutes or longer. Then, the antibody-immobilized chromatography sheet is dipped in each hole and incubated at about 15 to about 30 ° C. for about 15 minutes. Similarly, a test was conducted in which the incubation was performed for about 40 minutes and about 18 hours. After the incubation, remove the chromatography sheet from each hole and judge the result.

The assay results were judged by the presence or absence of red color derived from the selenium colloidal particles generated in the anti-HBs mouse monoclonal antibody-immobilized part (spot part) attached to the antibody-immobilized chromatography sheet. A test using 32 samples of heparin plasma, 39 samples of EDTA plasma, and 31 samples of serum was performed for each of heparin addition and non-heparin addition. The assay results are shown in Table 1.

[0043]

[Table 1]

[0044]

INDUSTRIAL APPLICABILITY According to the present invention, in a specific binding assay method using a colloidal particle-labeled or microparticle-labeled reagent, even when measurement is performed using fresh blood or serum or plasma, There is no problem that a false positive reaction, which is considered to be caused by a non-specific binding reaction, occurs. Therefore, it enables quick and accurate examination in the field of emergency medicine. Also,
It will be possible to measure easily next to the bed in a short time. It is not necessary to control how the analytical sample was obtained, such as how it was obtained, and how long it took to collect the sample, and the test results can be obtained quickly and accurately regardless of how the analytical sample is diluted. Can be obtained. Further, according to the present invention, in an immunological agglutination assay method using an insoluble carrier particle reagent on which an antigen or an antibody is immobilized, even if fresh blood or serum or plasma is used, non-specific reaction can be performed rapidly and accurately. The test result can be obtained.

[Table 1]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/553 // C12N 15/02 C12P 21/08 9161-4B (C12P 21/08 C12R 1: 91)

Claims (18)

[Claims] 1. A specific binding assay reagent containing a specific binding substance labeled with colloidal particles or microparticles and an amount of heparin sufficient to substantially prevent undesired non-specific binding reactions. 2. The reagent according to claim 1, wherein the amount of heparin gives a value of at least 0.05 mg / ml per sample at the time of measurement. 3. The reagent according to claim 1, wherein the amount of heparin gives at least 0.08 mg / ml per sample at the time of measurement. 4. The amount of heparin provides at least 0.1 mg / ml per sample at the time of measurement.
Described reagents. 5. The reagent according to any one of claims 1 to 4, wherein the specific binding assay is an immunoassay utilizing an antigen-antibody binding reaction. 6. The reagent according to any one of claims 1 to 5, wherein the specific binding assay is an immunological chromatography assay. 7. The reagent according to claim 1, wherein the specific binding assay is a sandwich type immunochromatographic assay. 8. The specific binding substance is selected from the group consisting of an antibody, an antigen, and a hapten.
The reagent according to any one of 1. 9. The reagent according to claim 1, wherein the reagent can be measured by using fresh blood or fresh serum or plasma as an analysis sample. 10. The colloidal particles are dye polymer, sulfur,
Selenium, tellurium, gold, platinum, silver, copper, silver iodide, silver bromide, iron oxide, copper hydroxide, aluminum hydroxide, chromium hydroxide, copper sulfate, mercury sulfate, barium sulfate, titanium dioxide and iron hydroxide. The reagent according to any one of claims 1 to 9, which is selected from the group consisting of: 11. A method for measuring the presence or absence or the amount of an analyte in an analytical sample using a specific binding substance labeled with colloidal particles or microparticles, which comprises: A method comprising reacting a labeled specific binding substance in the presence of heparin in an amount sufficient to substantially prevent undesired non-specific binding. 12. The method according to claim 11, wherein the measuring method uses an immunochromatographic assay method. 13. An immunological agglutination assay comprising insoluble carrier particles having a specific binding substance immobilized thereon and a sufficient amount of heparin to substantially prevent an undesired non-specific binding reaction. reagent. 14. The reagent according to claim 13, wherein the amount of heparin gives a value of at least greater than 0.05 mg / ml per sample at the time of measurement. 15. The reagent according to claim 13, wherein the amount of heparin provides at least 0.08 mg / ml per sample at the time of measurement. 16. The reagent according to claim 13, wherein the amount of heparin gives at least 0.1 mg / ml per sample at the time of measurement. 17. The specific binding substance is selected from the group consisting of an antibody, an antigen, and a hapten.
The reagent according to any one of to 16. 18. A method for measuring the presence or absence or the amount of an analyte in an analytical sample, wherein insoluble carrier particles having a specific binding substance capable of specifically binding to the analyte immobilized thereon, and the analytical sample. And a reaction in the presence of heparin in an amount sufficient to substantially prevent undesired non-specific binding, and then the presence or absence or amount of the agglutination reaction or the aggregation inhibition reaction is measured.