JP5137880B2 - Method for producing dry particles with immobilized binding substance - Google Patents

Description

The present invention relates to a method for producing dry particles having a binding substance immobilized thereon and a method for drying particles having a binding substance immobilized thereon. Furthermore, the present invention relates to a dry particle immobilized with a binding substance, a method for measuring a test substance, and a kit including the dried particle immobilized with a binding substance, which can be carried out by the above method.

Until now, to identify diseases in clinical diagnosis, collect body fluid components such as blood (whole blood, plasma, serum, etc.) and urine as test samples and measure the concentration of the target component in the test sample Thus, a method using the measurement target component concentration as an index has been adopted. In such a clinical test mode, when the affinity of the measurement target component in the test sample is used, for example, when the measurement target component is an antigen or an antibody, the antibody strictly identifies and binds the antigen. An immunological measurement method is used as a quantitative method utilizing the properties.

Among immunological measurement methods, the method mainly used is ELISA (Enzyme-Linked ImmunoSorbent Assay). When applying ELISA to clinical diagnosis, the enzyme labeled antibody used for ELISA may be dried. As a method for drying an enzyme-labeled antibody, a freeze-drying method is generally used, and the performance (sensitivity) of the ELISA did not change significantly before and after the enzyme-labeled antibody was lyophilized.

On the other hand, an antibody or antigen is immobilized on an insoluble carrier particle, for example, latex instead of an enzyme, and reacted with the antigen or antibody as a test substance. From the decrease in the transmittance of the reaction mixture accompanying the progress of this reaction (latex aggregation), A method for measuring the concentration of a test substance is known. Further, latex particles in which a binding substance that binds to a test substance in this method is immobilized can be stored while maintaining the binding property by freeze-drying (see Patent Document 1).

JP-A-11-258241

Generally, unlike latex particles, when the fluorescent particles on which the binding substance is immobilized are dried, the binding property of the binding substance may decrease. Therefore, as a method for drying the fluorescent particles on which the binding substance is immobilized, it is preferable to employ a method that can suppress a decrease in binding property of the binding substance, that is, a high storage stability. However, no such method has been known so far.

Accordingly, an object of the present invention is to provide a method for producing dry particles having a binding substance immobilized thereon and a method for drying particles having a binding substance immobilized thereon, which can suppress a decrease in the binding property of the binding substance. It is in. Furthermore, the objective of this invention is providing the kit containing the dry particle which fix | immobilized the binding substance, the measuring method of a test substance, and the dry particle which fix | bonded the binding substance which can be implemented by the said method. .

As a result of diligent studies to solve the above problems, the present inventors surprisingly mixed particles having a binding substance immobilized thereon with a solution adjusted to a pH near the isoelectric point of the binding substance. After that, it was found that the bonding property of the binding substance can be stably maintained by drying the particles, and the present invention was completed.

Therefore, according to the present invention, the binding substance comprising: mixing the particle having the binding substance immobilized thereon with a solution adjusted to a pH near the isoelectric point of the binding substance and then drying the particle. A method for producing dry particles in which is immobilized is provided.

According to another aspect of the present invention, the binding includes mixing the particles having the binding substance immobilized thereon and a solution adjusted to a pH near the isoelectric point of the binding substance, and then drying the particles. Provided is a method for drying particles having an immobilized active substance.

Preferably, the particles are colored particles.
Preferably, the colored particles are fluorescent particles.
Preferably, the particles are polymer particles, magnetic particles, liposomes, or glass particles.
Preferably, the binding substance is an antigen, an antibody, or a complex thereof.
Preferably, the binding substance is binding to a test substance or a substance that is binding to the test substance.

According to another aspect of the present invention, there are provided dry particles obtained by immobilizing a binding substance, which can be obtained by the method of the present invention.

According to another aspect of the present invention, there is provided a method for measuring a test substance, comprising measuring the test substance using dry particles on which the binding substance of the present invention is immobilized.
Preferably, dry particles in which the binding substance of the present invention dispersed in an aqueous solution is immobilized and a test substance that binds to the binding substance, and a substance that binds to the test substance are immobilized. Contacting on a solid phase, and detecting the binding substance formed on the solid phase, the test substance, and a complex composed of the test substance and a substance capable of binding to the test substance.

According to another aspect of the present invention, there is provided a kit for carrying out the method for measuring a test substance of the present invention, comprising dry particles on which the binding substance of the present invention is immobilized.

According to the method of the present invention, it is possible to obtain dry particles in which a binding substance having high storage stability that can suppress a decrease in binding property of the binding substance is immobilized. If the dried particles to which the binding substance obtained by the method of the present invention is immobilized are used, the immunoassay method has a sensitivity of 80% or more compared to the case of using the particles to which the binding substance before drying is immobilized. Can be implemented. In addition, the method of the present invention requires a dedicated device and further uses a simple drying method such as a vacuum drying method without using a freeze-drying method that requires freeze-drying and at least two steps. Thus, dry particles having the binding substance immobilized thereon can be produced. Moreover, it is estimated that the method of this invention can obtain the dry particle which fix | immobilized the binding substance with high storage stability irrespective of the kind of binding substance.

The result of ELISA using the biotinylated antibody of Example 1 is shown. The result of ELISA using the fluorescent particle labeled antibody of Example 2 is shown.

Hereinafter, the present invention will be described in detail.
The method for producing a dry particle having a binding substance immobilized thereon according to the present invention comprises mixing the particle having the binding substance immobilized thereon with a solution adjusted to a pH near the isoelectric point of the binding substance, Including drying. Further, the method for drying the particles having the binding substance immobilized thereon according to the present invention comprises mixing the particles having the binding substance immobilized thereon with a solution adjusted to a pH in the vicinity of the isoelectric point of the binding substance. Including drying. Hereinafter, these will be described as “the method of the present invention”.

1. Particles The particles used in the method of the present invention (hereinafter also simply referred to as “particles”) are not particularly limited as long as they are particulate insoluble carriers, but are preferably labeled. The label of the particle may be either a label of the particle itself or a label obtained by performing a treatment such as binding a labeling substance to the particle. For example, the type of label is not particularly limited. For example, when the label is a colored label, particles can be easily detected by visual observation, a microscope, a digital microscope, a plate reader, or the like based on the wavelength of the color. can do. Therefore, the particles used in the method of the present invention are preferably colored particles, and more preferably fluorescent particles in consideration of color intensity. The material of the particles is not particularly limited, but for example, polymer particles, magnetic particles, glass particles, liposomes and the like are preferable as easily available materials. The case where the particles are fluorescent particles will be described below.

As fluorescent particles, fluorescently colored particles that can be normally used for immunoaggregation reactions can be used. For example, fluorescent polymer particles such as fluorescent polystyrene beads and fluorescent glass particles such as fluorescent glass beads can be used. . Specific examples of the material of the fluorescent particles include polymers using monomers such as styrene, methacrylic acid, glycidyl (meth) acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, and butyl methacrylate. There are synthetic polymer powders such as copolymers using two or more monomers, and latexes in which these are uniformly suspended are preferred. Other organic polymer powders and inorganic substance powders, microorganisms, blood cells, cell membrane fragments, liposomes and the like can be mentioned.

The average particle diameter of the particles varies depending on the particle material, the concentration range for quantifying the test substance, the measuring instrument, and the like, but a range of 0.001 to 10 μm (more preferably 0.001 to 1 μm) is preferable. Liposomes and microcapsules containing fluorescent dyes can also be used as fluorescent particles. Fluorescent color development is not particularly limited as long as it is excited by absorbing ultraviolet light or the like and is emitted when returning to the ground state. ), Blue (350-356 nm / 415-440 nm), red (535-580 nm / 575-605 nm), orange (540 nm / 560 nm), red orange (565 nm / 580 nm), crimson (625 nm / 645 nm), dark red ( Fluorescence development such as 660 nm / 680 nm) can be used. These fluorescent particles that emit fluorescence are available from, for example, Invitrogen, and are commercially available under the trade name FluoSpheres (registered trademark).

2. Binding substance The particles used in the method of the present invention are those obtained by immobilizing a binding substance. Specifically, in the particles, (a) a binding substance to the test substance, or (b) a binding substance to a substance having a binding property to the test substance is immobilized. Examples of the binding substance (a) to the test substance include an antibody to the test substance (antigen), an antigen to the test substance (antibody), an aptamer to the test substance (protein, low molecular weight compound, avidin, biotin, etc.) There is no particular limitation as long as the compound has an affinity for a substance. The binding substance for the substance having the binding property to the test substance (b) may be the test substance itself, or may be a compound having a site that recognizes the binding substance to the test substance (a). This corresponds to a compound in which a derivative of a test substance and a protein (for example, BSA) are bound. However, (a) the binding substance to the test substance and (b) the binding substance to the substance having the binding to the test substance are amphoteric electrolytes, that is, substances having an anionic functional group and a cationic functional group. It is.

Preferred examples of the binding substance to be used in the method of the present invention are antigens, antibodies, or complexes thereof, but are not limited thereto. For example, when the binding substance is an antibody, as an antibody having specificity for the test substance, for example, an antiserum prepared from the serum of an animal immunized with the test substance, an immunoglobulin purified from the antiserum A fraction, a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with the test substance, or a fragment thereof [for example, F (ab ′) 2, Fab, Fab ′, or Fv] is used. be able to. These antibodies can be prepared by a conventional method. Furthermore, the antibody may be modified as in the case of a chimeric antibody, or may be a commercially available antibody or an antibody prepared from animal serum or culture supernatant by a known method.

The antibody can be used regardless of the animal species or subclass. For example, antibodies that can be used in the present invention include antibodies derived from organisms such as mice, rats, hamsters, goats, rabbits, sheep, cows, and chickens that can cause an immune reaction, specifically mouse IgG, mouse IgM. Rat IgG, rat IgM, hamster IgG, IgM rabbit IgG, rabbit IgM, goat IgG, goat IgM, sheep IgG, sheep IgM, bovine IgG, bovine IgM, avian IgY, etc., applicable to both polyclonal or monoclonal is there. A fragmented antibody is a molecule derived from a complete antibody having at least one antigen-binding site, and specifically, Fab, F (ab ′) 2, and the like. These fragmented antibodies are molecules obtained by enzyme or chemical treatment or using genetic engineering techniques. Specific examples of antibodies used in the method of the present invention include an anti-hTSH monoclonal antibody that recognizes human thyroid stimulating hormone (hTSH) as an antigen and an anti-hCG monoclonal antibody that recognizes human chorionic gonadotropin (hCG) as an antigen. However, it is not limited to these.

3. Method for immobilizing binding substance on particles Methods for immobilizing binding substances such as antigens and antibodies on particles include, for example, JP 2000-206115 A and FluoSpheres (registered trademark) polystyrene by Molecular Probes. Any known method described in the protocol attached to Microsphere F8813 and the like for preparing a reagent for immunoagglutination reaction can be used. In addition, as a principle for immobilizing an antibody as a binding substance on a particle, any of physical adsorption and chemical bonding by covalent bond can be adopted. As a blocking agent that covers the particle surface that is not coated with the antibody after the antibody is fixed to the particle, known substances such as BSA (bovine serum albumin), skim milk, casein, soybean-derived component, fish-derived component, polyethylene glycol, etc. In addition, a commercially available blocking agent for immune reaction containing these substances or substances having the same properties as these can be used. These blocking agents can be subjected to pretreatment such as partial modification with heat, acid, alkali, or the like, if necessary.

A specific method for immobilizing the antibody on the particles is exemplified below. An antibody solution adjusted to a concentration of 0.01 to 20 mg / mL is added to and mixed with the liquid dispersed so that the solid content concentration of the particles becomes 0.1 to 10%. Stirring is continued at a temperature of 4-50 ° C. for 5 minutes to ˜48 hours. Subsequently, the particles and the solution are separated by centrifugation or other methods to sufficiently remove the antibody that is not bound to the particles contained in the solution. Thereafter, the operation of washing the particles with a buffer solution is repeated 0 to 10 times. After the operation of mixing the particles and the antibodies and binding the antibodies to the particles, components that do not participate in the antigen-antibody reaction, preferably proteins, more preferably BSA (bovine serum albumin), block ace, skim milk, casein, etc. It is desirable to protect the part of the particle surface where the antibody is not bound by using a blocking agent.

When immobilizing antigens or antibodies on the particles, stabilizers can be added as necessary. Stabilizers are not particularly limited as long as they stabilize antigens and antibodies, such as synthetic or natural polymers such as sucrose and polysaccharides, and commercially available products such as Immunoassay Stabilizer (ABI) can also be used. It is.

4). Solution adjusted to a pH near the isoelectric point of the binding substance A solution adjusted to a pH near the isoelectric point of the binding substance used in the method of the present invention is near the isoelectric point of the binding substance. Although it will not be restrict | limited especially if it is a solution which has pH of this, Preferably, it is the buffer solution (Buffer) adjusted to pH near the isoelectric point of a binding substance. As the buffer solution, those known so far such as acetate buffer solution, phosphate buffer solution, citrate buffer solution, succinate buffer solution, tris-HCl buffer solution, EDTA buffer solution and the like can be used without limitation.

The isoelectric point should be widely interpreted as having a generally known meaning. For example, after the binding substance is ionized, the charge average (or electrophoretic mobility) of the whole substance is 0. It can be called a pH value. The isoelectric point of the binding substance can be determined, for example, by isoelectric focusing. In isoelectric focusing, when a binding substance is added to an electrophoresis gel with a pH gradient and an electric field is applied under low ionic strength, the binding substance moves toward the same pH as its isoelectric point (pI). Move and eventually settle and concentrate. The isoelectric point of the binding substance can be known by confirming the pH at which the binding substance exists after isoelectric focusing.

A solution adjusted to a pH near the isoelectric point of the binding substance can be obtained by adjusting the pH by a generally known method if the isoelectric point of the binding substance can be known. For example, when using anti-hTSH 5409 SPTNE-5 of Medix biochemica as a binding substance, the isoelectric point of anti-hTSH 5409 SPTNE-5 is 5.8 to 6.3, so MES with pH adjusted to around 5.8 to 6.3 A buffer solution, specifically, a MES buffer solution adjusted to pH 6.0 can be used as a solution adjusted to a pH near the isoelectric point of the binding substance. Similarly, when using anti-hCG 5008 SP-5 from Medix biochemica as the binding substance, the isoelectric point of anti-hCG 5008 SP-5 is 6.8 to 8.1, so the pH was adjusted to around 6.8 to 8.1. A Tris-HCl buffer, specifically, a Tris-HCl buffer adjusted to a pH of 8.2 can be used as a solution adjusted to a pH near the isoelectric point of the binding substance. The solution adjusted to a pH near the isoelectric point of the binding substance may be one in which a stabilizer such as sucrose is dissolved in addition to the solute for adjusting the pH.

The pH near the isoelectric point includes a pH slightly higher or lower than the isoelectric point of the binding substance to such an extent that the binding property of the binding substance is not affected. 7, more preferably isoelectric point ± 0.5, more preferably isoelectric point ± 0.3, still more preferably isoelectric point ± 0.2, particularly preferably isoelectric point ± 0.1. is there.

5. Mixing and drying of the particles having the binding substance immobilized thereon and a solution adjusted to a pH in the vicinity of the isoelectric point of the binding substance; and the particles having the binding substance immobilized thereon and the isoelectric point of the binding substance There is no particular limitation on the mixing of the solution adjusted to the pH of, for example, a few seconds to several times after contacting the particles with the binding substance immobilized thereon and the solution adjusted to the pH near the isoelectric point of the binding substance. It may be left for a period of time, may be stirred by a stirring device such as a stirrer or vortex, manually, or a combination thereof.

There is no particular limitation on the method for drying the particles having the binding substance immobilized thereon after mixing the particles having the binding substance immobilized thereon with a solution adjusted to a pH near the isoelectric point of the binding substance. For example, after separating the particles and the solution using a device such as a pipette or an aspirator, or after immersing the particles in the solution, the particles or the solution containing the particles is removed from the atmosphere. Alternatively, a method of drying at an appropriate temperature and time using a dryer such as a vacuum dryer or a thermostat can be applied. Specifically, a part of the solution containing the particles having the binding substance immobilized thereon and adjusted to a pH near the isoelectric point of the binding substance is taken on a culture plate, and then using a vacuum dryer or a thermostat, It can be dried for several minutes to several hours at several to several tens of degrees Celsius.

6). Dry particles with immobilized binding substance Mixing particles with immobilized binding substance and a solution adjusted to a pH near the isoelectric point of the binding substance, and then drying the particles Thus, dry particles having the binding substance immobilized thereon are obtained. The dry particles on which the binding substance thus obtained is immobilized are also within the scope of the present invention.

The dry particles to which the binding substance is immobilized are not particularly limited as long as the particles to which the binding substance is immobilized are dried and the binding property of the binding substance is maintained. It is preferable from the viewpoint of storage stability that the particles are substantially free of moisture. The degree of binding of the binding substance in the dry particles to which the binding substance is immobilized is 80% or more, preferably 85% of the binding degree of the binding substance in the particles to which the undried binding substance is fixed. % Or more.

7). Method for measuring test substance using dry particles to which binding substance is immobilized The test substance can be measured using dry particles to which binding substance obtained by the method of the present invention is immobilized. . Such a method for measuring a test substance is also within the scope of the present invention.

The test substance measurement method of the present invention is interpreted as the broadest concept including detection of the presence or absence of the test substance and measurement (ie, quantification) of the test substance, and is generally known. A measurement method based on specific affinity, for example, an immunological measurement method is included without limitation. Hereinafter, the sandwich method and the competitive method will be described as specific embodiments of the method for measuring a test substance of the present invention, but the present invention is not limited thereto.

(Sandwich method)
The sandwich method is not particularly limited. For example, the test substance can be measured by the following procedure. First, a binding substance (for example, an antibody) that has a binding property to a test substance (for example, an antigen) is immobilized on a particle, for example, a fluorescent particle. In parallel, a substance (for example, an antibody) that binds to the test substance at a site different from the binding substance is immobilized on a solid phase (for example, a plate). Hereinafter, the substance immobilized on the solid phase is referred to as a solid phase immobilized substance. Next, a test sample (or an extract thereof) that may contain a test substance and fluorescent particles on which the binding substance is immobilized are brought into contact on a solid phase. When the test substance is present in the test sample, an antigen-antibody reaction occurs between the test substance and the binding substance and between the test substance and the solid-phase immobilized substance. This antigen-antibody reaction can be performed in the same manner as a normal antigen-antibody reaction. As a result, when the test substance is present in the test sample, an immune complex composed of the binding substance, the test substance, and the solid phase-immobilized substance is formed. After the completion of the immune complex formation reaction, the fluorescent particles that have not formed the immune complex are removed and washed. Next, the concentration of the test substance can be measured by detecting the degree of formation of the immune complex as the fluorescence intensity. The fluorescence intensity and the test substance concentration have a positive correlation.

(Competitive law)
The competitive method is not particularly limited, but for example, the test substance can be measured by the following procedure. The competition method is known as a technique for detecting an antigen of a low molecular weight compound that cannot be assayed by the sandwich method.

First, a binding substance (for example, an antibody) having a binding property to a test substance (antigen) is immobilized on a particle, for example, a fluorescent particle. In parallel, a substance having an epitope for the binding substance similar to the test substance itself or the test substance, which is binding to the binding substance, is immobilized on a solid phase (for example, a substrate). Hereinafter, the substance immobilized on the solid phase is referred to as a solid phase immobilized substance. Next, when the test sample (or its extract) that may contain the test substance and the fluorescent particles immobilized with the binding substance are brought into contact with the solid phase, if the test substance is not present in the test sample, By the binding substance and the solid phase-immobilized substance, an antigen-antibody reaction occurs on the solid phase, and an immune complex is formed. On the other hand, when the test substance exists, the test substance binds to the binding substance, so that the subsequent antigen-antibody reaction between the binding substance and the solid-phase immobilized substance is inhibited, and the antigen-antibody reaction The combination by does not occur. After the reaction between the binding substance and the test substance or the solid phase-immobilized substance is completed, the fluorescent particles that have not formed the immune complex are removed. Next, the concentration of the test substance can be measured by detecting the degree of formation of the immune complex as the fluorescence intensity.

A specific example of the method for measuring a test substance of the present invention is a method based on the above sandwich method, in which a dry particle in which a binding substance dispersed in an aqueous solution is immobilized, and a test substance having a binding property to the binding substance are used. Contacting a substance having a binding property to the test substance on an immobilized solid phase, and binding to the binding substance, the test substance and the test substance formed on the solid phase Detecting a complex of a substance.

The test sample is not particularly limited as long as it is a sample that may contain a test substance that is a substance to be measured. For example, a biological sample, particularly a body fluid of an animal (particularly human) (Eg, blood, serum, plasma, spinal fluid, tears, sweat, urine, pus, runny nose, or sputum) or excrement (eg, stool), organs, tissues, mucous membranes and skin, and squeezes that may contain them Examples include over-analytes (swabs), gargle, or animals and plants themselves or their dried bodies.

In the method for measuring a test substance of the present invention, the test sample is used as it is or in the form of an extract obtained by extracting the test sample with an appropriate extraction solvent, and further, the extract is diluted appropriately. It can be used in the form of a diluted solution obtained by diluting with an agent, or in a form obtained by concentrating the extract by an appropriate method. As a solvent for extraction, a solvent (for example, water, physiological saline, or buffer solution) used in a usual immunological measurement method, or an antigen-antibody reaction is directly performed by diluting with the solvent. Water miscible organic solvents that can be used can also be used.

The test sample used for the test substance measurement method of the present invention is preferably blood, more preferably plasma or serum. Specific examples of the test sample used in the test substance measurement method of the present invention include, but are not limited to, a 50% blood component-containing aqueous solution in which a blood component is diluted with water.

The solid phase used for the test substance measurement method of the present invention is not particularly limited, but preferably has no label similar to particles. For example, polymers using monomers such as styrene, methacrylic acid, glycidyl (meth) acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, butyl methacrylate, or two or more monomers are used. And a substrate made of a material such as glass or glass.

The method for measuring a test substance of the present invention can take various forms other than those described above. For example, a test substance or a substance having an epitope of the test substance is immobilized on a particle as a binding substance, and the test is performed on a solid phase. A system that immobilizes a substance having a binding property to the substance can also be used.

The buffer solution that can be used in the method for measuring a test substance of the present invention may be any buffer solution that has a buffer capacity under pH conditions under which a normal antigen-antibody reaction is performed. -11, more preferably 6-10 are used. Examples of buffers having a buffer capacity under this pH condition include, for example, normal biochemical experiments such as phosphate buffer, Tris buffer, MES buffer, HEPES buffer, glycine buffer, and citrate buffer. Any buffer can be used as long as it can be used in the above.

As the measurement of the test substance, conventionally used fluorescence measurement method (FIA), enzyme color development method (EIA), radioactivity measurement method (RIA), luminescence method (CLEIA), electrochemiluminescence method (ECLIA), magnetic It can be applied to various techniques such as immunoassay (MIA). In addition, non-electrochemical methods such as surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, measurement technology using a functionalized surface from gold colloidal particles to ultrafine particles are preferably used. Can do.

For example, when the method for measuring a test substance of the present invention employs a system in which the test substance is measured by detecting the fluorescence intensity, the detection of the fluorescence intensity is not particularly limited, but, for example, an apparatus capable of detecting the fluorescence intensity Specifically, the fluorescence intensity can be detected using a microplate reader, an SPF biosensor, or the like. The detection of the fluorescence intensity is usually completed after a certain time after the antigen-antibody reaction, for example, several minutes to several hours. By detecting the degree of formation of the immune complex as fluorescence intensity, the concentration of the test substance can be quantified from the relationship between the fluorescence intensity and the concentration of the test substance.

A surface plasmon fluorescence (SPF) biosensor is, for example, an optical waveguide formed of a material that transmits excitation light of a predetermined wavelength, as described in JP-A-2008-249361. A metal film formed on one surface of the optical waveguide, a light source that generates a light beam, and an incident angle that causes the light beam to pass through the optical waveguide and generates surface plasmons at the interface between the optical waveguide and the metal film. A sensor including an optical system to be incident and fluorescence detection means for detecting fluorescence generated by excitation by an evanescent wave enhanced by the surface plasmon.

8). Kit According to another aspect of the present invention, a kit for measuring a test substance is provided. The kit of the present invention is not particularly limited as long as it includes dry particles having a binding substance immobilized thereon.

The following examples further illustrate the present invention, but are not intended to limit the scope of the present invention.

[Example 1]
1. reagent
Medix biochemica Anti-h TSH 5407 SPTN-1
Medix biochemica Anti-h TSH 5409 SPTNE-5 (isoelectric point: 5.8 to 6.3)
Medix biochemica Anti-h alpha subunit 6601 SPR-5
Medix biochemica Anti-h hCG 5008 SP-5 (isoelectric point: 6.8 to 8.1)
PIERCE Blocke BSA in PBS
PIERCE SuperSignal ELISA FemtoMaximum Sensitivity Substrate
PIERCE Streptavidin, Horseradish Peroxidase Conjugated
PIERCE Ez-link NHS-PEO4-Biotin
Thermo Scientific nunc FLUORONUNC PLATE
Scrip Laboratories cTSH

2. machine
YAMATO Vacuum Drying Oven DP43
perkin elmar ARVO

3. Preparation of antibody plate
Medix biochemica Anti-h TSH 5407 SPTN-1 and Medix biochemica Anti-h alpha subunit 6601 SPR-5 antibody were diluted with carbonate buffer (pH 9.6) to 10 μg / mL to prepare an antibody solution. This was dispensed to Thermo Scientific nunc FLUORONUNC PLATE at 100 μL / well and allowed to stand overnight at room temperature. The antibody solution in the plate was removed and washed twice with PBST. PIERCE Blocker BSA in PBS was diluted 10-fold with PBS, dispensed at 300 μL / well, and allowed to stand at room temperature for 4 hours. PIERCE Blocker BSA in PBS diluted 10-fold was removed and stored at 4 ° C. As a result, an hTSH antibody 5407 solid-phased plate and an hCG antibody 6601 plate were prepared.

4). Antigen preparation
TSH was adjusted with a 1% BSA-PBS solution to 2.5 × 10 ⁻¹⁰ pM using CALBIOCHEM Cat 869006 Thyroid Stimulating Hormone, Human Pituitary, and Lodination Grade sold by Merck.

For hCG, Cat.NO.PA1180XC Human Chorionic Gonadortopin (hCG) sold by Acris was used and adjusted with a 1% BSA-PBS solution to 2.5 × 10 ⁻¹⁰ pM. In addition, a 1% BSA-PBS solution was used as a 0 pM concentration.

5. Production of biotinylated antibody
PIERCE Ez-link NHS-PEO4-Biotin is attached to Medix biochemica Anti-h TSH 5409 SPTNE-5 and Medix biochemica Anti-h hCG 5008 SP-5 using PIERCE Ez-link NHS-PEO4-Biotin. Then, biotinylated hTSH antibody 5409 and biotinylated hCG antibody 5008 were prepared.

6). Drying of biotinylated antibody and preparation of biotinylated antibody solution The biotinylated hTSH antibody 5409 and biotinylated hTSH antibody 5008 prepared in 1 above were respectively added to MES buffer (pH 6.0) and Tris-HCl (pH 8.2). The sucrose was dissolved in a solution added to 5%, added to the plate, and dried for 1 hour at room temperature using Vacuum Drying Oven DP43. This was dissolved in a 1% BSA-PBS solution to obtain a biotinylated antibody solution.

7). Assay: 2.5 × 10 ⁻¹⁰ pM antigen and 0 pM antigen prepared in 4 above were dispensed into wells of hTSH antibody 5407 solid phase plate and hCG antibody 6601 plate, respectively, followed by biotinylated hTSH antibody 5409 solution or biotin. The hCG antibody 5008 solution was dispensed in 50 μL aliquots. After shaking at 700 rpm for 4 hours, washing was performed 4 times with PBST. PIERCE Streptavidin, Horseradish Peroxidase Conjugated was diluted with pure water to a concentration of 0.1 μg / mL, and dispensed at 100 μL into the wells. After shaking for 30 minutes at 700 rpm, washing was performed 6 times using PBST. According to the protocol of PIERCE SuperSignal ELISA Femto Maximum Sensitivity Substrate, light was emitted within the plate, and measurement was performed using perkin elmar ARVO to measure the amount of luminescence.

[Example 2]
1. Preparation of fluorescent particle-labeled antibody Using a molecular probe FluoSpheres (registered trademark) polystyrene microsphere F8813, according to the attached protocol, Medix biochemica Anti-h TSH 5409 SPTNE-5 and Medix biochemica Anti-h hCG 5008 SP- 5 was labeled to obtain a fluorescent particle labeled hTSH5409 solution and a fluorescent particle labeled hCG5008 solution.

2. Add sucrose to 5% of MES buffer (pH 6.0) and Tris-HCl (pH 8.2) for dry fluorescent particle-labeled hTSH antibody 5409 and fluorescent particle-labeled hCG antibody 5008, respectively. It was dissolved in the prepared solution, added to each plate, and dried for 1 hour at room temperature using Vacuum Drying Oven DP43 to obtain a dried fluorescent particle-labeled antibody.

3. Preparation and assay of fluorescent particle-labeled antibody The dried fluorescent particle-labeled antibody was dissolved in a 1% BSA-PBS solution to obtain a fluorescent particle-labeled antibody solution. Each 50 μL of diluted antigen was dispensed onto the antibody plate prepared in [Example 1] above, and 50 μL of fluorescent particle-labeled hTSH5409 solution and fluorescent particle-labeled hCG antibody 5008 solution were dispensed. After shaking at 700 rpm for 4 hours, washing was performed 4 times with PBST. Measurement was carried out using Perkinelmer ARVO to measure the amount of fluorescence. As a result, it was clarified that when the solution was dried using a solution having substantially the same pH as the isoelectric point of each antibody, it was least affected by drying.

[result]
The results of Example 1 are shown in FIG. 1, and the results of Example 2 are shown in FIG. When the label is biotin (Example 1; FIG. 1), the binding ability of the antibody to the antigen is maintained at 80% or more regardless of the pH of the solution. However, when the label is fluorescent particles (Example 2; FIG. 2), when the antibody-immobilized fluorescent particles and a solution adjusted to a pH near the isoelectric point of the antibody are mixed and dried, The binding to the antigen was 80% or more. However, when the fluorescent particles on which the antibody was immobilized and the solution adjusted to a pH higher or lower than the isoelectric point of the antibody were mixed and dried, the binding of the antibody to the antigen The property was about 50 to 75%.

Claims (9)

After mixing the prepared solution to a pH in the range of isoelectric point ± 0.5 of the fluorescent particles bound substance immobilized and the binding material comprises drying the fluorescent particles, a binding agent A method for producing immobilized dry fluorescent particles. After mixing the prepared solution to a pH in the range of isoelectric point ± 0.5 of the fluorescent particles bound substance immobilized and the binding material comprises drying the fluorescent particles, a binding agent A method for drying immobilized fluorescent particles. The method according to claim 1 or 2 , wherein the fluorescent particles are polymer particles, magnetic particles, liposomes, or glass particles. The method according to any one of claims 1 to 3 , wherein the binding substance is an antigen, an antibody, or a complex thereof. The method according to any one of claims 1 to 4 , wherein the binding substance is binding to a test substance or a substance that is binding to the test substance. The dry fluorescent particle which fixed the binding substance obtained by the method of any one of Claims 1-5 . A method for measuring a test substance, comprising measuring the test substance using dry fluorescent particles on which the binding substance according to claim 6 is immobilized. The dry fluorescent particles having the binding substance according to claim 6 immobilized in an aqueous solution immobilized thereon and a test substance having binding property to the binding substance, and a substance having binding property to the test substance are immobilized. Contact on the solid phase, and detecting the binding substance formed on the solid phase, the test substance, and a complex comprising the test substance and a substance capable of binding to the test substance. Item 8. A method for measuring a test substance according to Item 7 . The kit for implementing the measuring method of the test substance of Claim 7 or 8 containing the dry fluorescent particle which fix | immobilized the binding substance of Claim 6 .

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