JP2010181155A - Method of manufacturing substance fixing substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of simply manufacturing a substance fixing substrate capable of enhancing the S/N ratio when adapted to immunoassay, and to provide the substance fixing substrate obtained thereby. <P>SOLUTION: In the method of manufacturing the substance fixing substrate, a substrate is coated with a solution containing the substance interacting with a substance to be examined and a blocking agent at a molar ratio (substance/blocking agent) of 0.2-2.5. The substance to be examined is an antigen and the substance interacting with the substance to be examined is an antibody. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a substance-immobilized substrate, wherein a solution containing a substance that interacts with a test substance and a blocking agent in a predetermined ratio is applied onto the substrate. Furthermore, this invention relates to the substance fixed board | substrate obtained by this method.

  At present, many measurements using intermolecular interactions such as immune reactions are carried out in clinical examinations and the like, but in any technique, a substrate having a surface on which a physiologically active substance is immobilized is important. The function of the biosensor is realized by immobilizing various biocatalyst components and biochemical binding reaction components on the sensor surface and detecting the interaction with the substance to be analyzed by the sensor. The component immobilized on the sensor surface must stably retain the activity necessary for reaction with the analyte. Also, the sensor surface needs to be stable so as not to bring unnecessary signals to the sensor.

  Immobilization of a substance on the sensor surface is roughly classified into immobilization by physical adsorption and immobilization by chemical bonding reaction. Examples of immobilization by physical adsorption include protein adsorption on the surface of a hydrophobic resin. Antibody-immobilized plates used in ELISA are usually prepared by this method. The antibody is immobilized by physical adsorption, then treated with a blocking agent, and used for detection.

  In immobilization by a chemical bonding reaction, a functional group introduced on the substrate surface is chemically bonded to a functional group of a substance to be immobilized. This immobilization can achieve a strong bond by covalent bond, thereby reducing the possibility of liberation. However, since it involves a chemical reaction, there is a risk that it may involve a structural change of the substance to be immobilized, and that the reaction for immobilization is relatively complicated. In addition, since functional groups that can be introduced on the substrate surface or functional groups that can be used for bonding in the substance to be immobilized are limited, it is difficult to apply to a wide range of substances.

  Hereinafter, description will be made by taking surface plasmon resonance (SPR) as an example. A commonly used measurement chip is composed of a transparent substrate (eg, glass), a deposited metal film, and a thin film having a functional group capable of immobilizing a physiologically active substance thereon, and the metal surface is interposed through the functional group. Immobilize physiologically active substances. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte. The thin film having a functional group capable of immobilizing a physiologically active substance is composed of a functional group that binds to a metal, a linker having a chain length of 10 or more, and a compound having a functional group that can bind to a physiologically active substance. A measurement chip in which a substance is immobilized has been reported (see Patent Document 1).

  As a method for reducing non-specific binding due to physical adsorption of a physiologically active substance, so-called blocking with bovine serum albumin or the like has been used in immunochemical analysis using a plastic plate. In the analysis method using a metal surface, when a DNA sensor using SPR is prepared by immobilizing DNA having a thiol end, and adding 6-hydroxy-1-heptanethiol, nonspecificity is obtained. It has been reported that the target DNA can be detected with high sensitivity without causing adsorption (see Patent Document 2). It has also been reported that reaction non-specific binding with streptavidin can be suppressed by simultaneously mixing thiolated biotin and 11-hydroxy-1-undecanethiol (see Non-Patent Document 1). .

Japanese Patent No. 2815120 U.S. Patent No. 5942397

J. Am. Chem. Soc., 1999, 121, 6469-6478

  However, the methods for producing the substrates described in Patent Documents 1 and 2 and Non-Patent Document 1 are complicated in operation, and the S / N (S / N) of immunological measurement methods using substrates obtained by these methods is also included. There is a problem that the signal / noise ratio is low.

  Therefore, an object of the present invention is to provide a method for easily producing a substance-immobilized substrate capable of improving the S / N ratio when applied to an immunological measurement method, and a substance-immobilized substrate obtained by the method.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present application are obtained by a simple method of applying a solution containing a substance that interacts with a test substance and a blocking agent in a predetermined ratio on a substrate. The material-immobilized substrate was found to improve the S / N ratio when applied to an immunological measurement method, and the present invention was completed.

  Therefore, according to the present invention, a solution containing a substance that interacts with a test substance and a blocking agent in a molar ratio (substance / blocking agent) of 0.2 to 2.5 is applied on the substrate. A method for manufacturing a substance-fixing substrate is provided.

  In a preferred embodiment of the method for producing a substance-immobilized substrate of the present invention, the test substance is an antigen, and the substance that interacts with the test substance is an antibody.

  In a preferred embodiment of the method for producing a substance-immobilized substrate of the present invention, the test substance is a hormone, and the substance that interacts with the test substance is a hormone receptor.

  In a preferred embodiment of the method for producing a substance-fixed substrate of the present invention, the substrate is a metal, plastic, or glass.

  In a preferred embodiment of the method for producing a substance-immobilized substrate of the present invention, the blocking agent is at least one selected from the group consisting of globulin, albumin, casein, gelatin, skim milk, polyvinyl alcohol, and polyvinylpyrrolidone.

  According to another aspect of the present invention, there is provided a substance fixed substrate obtained by the method for manufacturing a substance fixed substrate of the present invention.

  According to the method for producing a substance-immobilized substrate of the present invention, a substance-immobilized substrate capable of realizing an immunological measurement method having a higher S / N ratio than that of the conventional method can be easily produced. As a result, the immunological measurement method and biosensor using the substance-immobilized substrate of the present invention can quantify the antigen or antibody that is the test substance with higher sensitivity than the conventional method.

FIG. 1 shows the relationship between antibody / BSA mole ratio and S / N ratio. In the figure, plots of antibody / BSA (molar ratio) of 2, 1 and 0.5 are examples, and plots of 10, 3 and 0.1 are plots of comparative examples.

Hereinafter, the present invention will be described in detail.
In the method for producing a substance-fixed substrate of the present invention, a solution containing a substance that interacts with a test substance and a blocking agent in a molar ratio (substance / blocking agent) of 0.2 to 2.5 is applied on the substrate. Features. The substance-fixed substrate obtained by the method for producing a substance-fixed substrate of the present invention is also within the scope of the present invention.

  The substance-immobilized substrate of the present invention can be used without limitation for general immunoassay methods including ELISA as a detection method. Moreover, the substance-fixed substrate of the present invention can be used, for example, as a biosensor substrate. The biosensor referred to in this specification is to be interpreted in the broadest sense, and refers to a sensor that measures and detects a target substance by converting an interaction between biomolecules into a signal such as an electrical signal. means. A normal biosensor is composed of a receptor site for recognizing a chemical substance to be detected and a transducer site for converting a physical change or chemical change generated therein into an electrical signal. In the living body, there are enzymes / substrates, enzymes / coenzymes, antigens / antibodies, hormones / receptors and the like as substances having affinity for each other. Biosensors use the principle that one of these substances having affinity with each other is immobilized on a substrate and used as a molecular recognition substance, whereby the other substance to be matched is selectively measured.

  Examples of the biosensor using the substance-immobilized substrate of the present invention include a surface plasmon resonance (SPR) biosensor and a surface plasmon fluorescence (SPF) biosensor. Concerning the configuration of the SPR biosensor, for example, Japanese Patent Publication No. 6-27703, PR 4, page 48 to page 14, line 15 and FIG. 1 to FIG. 8, US Pat. No. 6,829,073, column 6, line 31 to column 7 Line 47 and FIGS. 9A and 9B. The configuration of the SPF biosensor is described in, for example, Japanese Patent Application Laid-Open No. 2008-249360, page 7, line 5 to page 9, line 36 and FIGS. 1 to 3.

1. Substrate The substrate used in the method for producing a substance-fixed substrate of the present invention is not particularly limited as long as it can fix a test substance. As a material for the substrate, for example, metal, glass, plastic, and the like are preferable, metal is more preferable, and free electron metals such as gold, silver, copper, aluminum, and platinum are more preferable. The material of the substrate can be used alone or in combination. In consideration of adhesion to the substrate, an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal. The film thickness and structure of the substrate and other matters relating to the manufacture of the substrate can be determined by adjusting those by a person skilled in the art according to ordinary methods.

  When the substance-fixed substrate of the present invention is used in, for example, an ELISA using a microplate reader, a substrate having no metal surface or metal film, for example, a substrate made of an insoluble polymer such as glass or plastic as described above is used. Can be used.

2. Substance that interacts with test substance The substrate-immobilizing substance used in the method for producing a substance-immobilized substrate of the present invention is not particularly limited as long as it interacts with the test substance. Enzymes, microorganisms, nucleic acids, low molecular weight organic compounds, non-immune proteins, immunoglobulin binding proteins, sugar binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, polypeptides or oligopeptides with ligand binding ability, etc. Is mentioned. “Interact with a test substance” is not particularly limited as long as it is an interaction in the meaning normally used in the art. For example, it physically or chemically or biochemically adsorbs or binds to a test substance. It is preferable to specifically adsorb or bind to the test substance.

  Examples of immune proteins include antibodies and haptens that use the test substance as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, and IgD can be used. Specifically, when the test substance is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigen 26, 86, 55, 111, 157 and the like can be used.

  The hormone receptor is not particularly limited as long as it is a protein that recognizes and specifically binds a hormone as a test substance. Hormone is to be interpreted in a generally known meaning, and refers to, for example, a physiologically active substance synthesized and secreted in a specific organ in a living body. Hormones are usually responsible for information transmission by circulating through the body fluid (blood) and acting on the target organ. In general, the secretion form of hormones is called endocrine, and the organs that secrete hormones are called endocrine organs. Specific examples of hormones include gonadal stimulating hormones such as chorionic gonadotropin, inhibin, parathyroid hormone (PTH), calcitonin, thyroid stimulating hormone releasing hormone (TRH), thyroid stimulating hormone (TSH), antidiuretic hormone (ADH), etc. Protein hormones (simple peptides, glycoproteins); thyroid hormones such as thyroxine; amine and amino acid derivative hormones such as adrenal medullary hormones such as epinephrine and norepinephrine; Female hormones such as steroid hormones such as electrolyte corticoid (aldosterone) and glucocorticoid (cortisol).

  The enzyme is not particularly limited as long as it shows activity against a test substance or a substance metabolized from the test substance, and various enzymes such as oxidoreductase, hydrolase, isomerase, A desorption enzyme, a synthetic enzyme, etc. can be used. Specifically, if the test substance is glucose, glucose oxidase can be used, and if the test substance is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.

  The non-immune protein is not particularly limited, and for example, avidin (streptavidin), biotin or a receptor can be used. As the immunoglobulin-binding protein, for example, protein A or protein G, rheumatoid factor (RF) and the like can be used. Examples of sugar-binding proteins include lectins. Examples of the fatty acid or fatty acid ester include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.

  When the substance that interacts with the test substance is a protein or nucleic acid such as an antibody or enzyme, the immobilization is shared with the functional group on the substrate surface using the amino group, thiol group, etc. of the substance that interacts with the test substance. This can be done by combining them.

  When the substrate is a metal substrate, the substance that interacts with the test substance may be immobilized on the metal substrate via a self-assembled monolayer, for example. A self-assembled monolayer typically includes an alkanethiol compound and a gold surface that react with an alkanethiol compound when left in contact with the gold surface to form an Au-S bond, It is known as a highly monolayered monolayer formed on the gold surface by the interaction between long chains. Surface plasmon resonance / fluorescence and Quartz Crystal Microbalance (QCM) ) Etc. are widely used in the field.

（式中、Qは金属基板への結合基を示し、Rは置換されていてもよい炭素数３〜２０のアルキル基を示し、Xは水素原子又は官能基を示す。） The component that forms the self-assembled monolayer can be represented by, for example, the following formula (1).

(In the formula, Q represents a bonding group to the metal substrate, R represents an optionally substituted alkyl group having 3 to 20 carbon atoms, and X represents a hydrogen atom or a functional group.)

  In the above formula (1), not only a thiol group (—SH) but also a disulfide group (—S—S—), a sulfide group (—S—) and the like can be preferably used as the bonding group to the metal substrate. . Examples of the compound represented by the formula (1) include 1-undecanethiol.

  By forming the self-assembled monolayer on the metal substrate, the substance can be stably fixed without being directly affected by the state of the metal substrate surface. Immobilizing a substance on the surface of a self-assembled monolayer formed on a metal substrate stably captures the test substance via the substance and minimizes the influence of the metal substrate. Can contribute.

  Preferred specific examples of the test substance and the substance that interacts with the test substance in the method for producing the substance-immobilized substrate of the present invention include an antigen (test substance) and an antibody (substance that interacts with the test substance), a hormone (test substance), and a hormone. Examples include receptors (substances that interact with a test substance), but the present invention is not limited thereto.

  The antibody is not particularly limited. For example, antiserum prepared from the serum of an animal immunized with an antigen recognized by the antibody, an immunoglobulin fraction purified from the antiserum, and immunized with the antigen Monoclonal antibodies obtained by cell fusion using animal spleen cells, or fragments thereof [eg, F (ab ′) 2, Fab, Fab ′, or Fv] can be used. These antibodies can be prepared by a conventional method. Further, 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.

  Fragmented antibodies can be used regardless of the animal species or subclass. For example, antibodies that can be used in the present invention are antibodies derived from monkeys, mice, rats, rabbits, goats, sheep, etc., specifically mouse IgG, mouse IgM, rat IgG, rat IgM, rabbit IgG, Rabbit IgM, goat IgG, goat IgM, sheep IgG, sheep IgM, etc. are applicable to both polyclonal and monoclonal. Examples of antibody subclasses include mouse IgG1 and mouse IgG2a, but are not limited thereto. 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.

  The human chorionic gonadotropin (hCG) and anti-hCG monoclonal antibody shown in the Examples can be related to a plateau and an antibody, or can be related to a hormone and a hormone receptor.

3. Blocking agent In the method for producing a substance-immobilized substrate of the present invention, a substance that interacts with a test substance is applied and immobilized on the substrate together with a blocking agent. The blocking agent used in the present invention is not particularly limited as long as it is a substance that suppresses nonspecific adsorption of the labeling substance to the immobilized surface. For example, globulin, albumin, casein, gelatin, skim milk, polyvinyl alcohol, polyvinyl Although pyrrolidone etc. can be mentioned, albumin is preferable and bovine serum albumin (Bovine serum albumin; BSA) is more preferable. Any one of these blocking agents may be used alone, or two or more of these blocking agents may be used in combination.

  A conventional method for producing a substance-immobilized substrate for measuring a test substance (hereinafter also referred to as a conventional method) is a method in which a substance that interacts with a test substance is applied to the substrate, and then the substance that interacts with the test substance from the substrate. Then, after the substrate is washed or without washing the substrate, the blocking agent is applied to the substrate as it is, and then the blocking agent is removed from the substrate. However, the method for producing a substance-immobilized substrate of the present invention is a simple method in which several steps in the conventional method are omitted by simultaneously applying a substance that interacts with a test substance and a blocking agent to the substrate. In addition, by setting the molar ratio of the substance that interacts with the test substance applied to the substrate and the blocking agent to a predetermined ratio, the obtained substance-fixed board is compared with the substance-fixed board manufactured by the conventional method. In the measurement of substances, the S / N ratio was improved, and as a result, a highly sensitive measurement could be realized. These findings are found for the first time by the present inventors.

  In the present invention, the molar ratio of the substance that interacts with the test substance to be applied on the substrate and the blocking agent (substance / blocking agent) is, for example, 0.2 to 2.5, and preferably the lower limit is 0. .2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5, and the upper limit is 2.5 or 2.0, particularly preferably 0.5 to 2.0. The substance-immobilized substrate obtained by adopting these molar ratios improves the S / N ratio of the measurement system when measuring the test substance as compared with the control (substance-immobilized substrate manufactured by the conventional method). .

4). Substance Fixed Substrate In the present invention, a solution containing a substance that interacts with a test substance and a blocking agent is applied directly on the substrate or via a self-assembled monolayer on the substrate. The solvent used for dissolving the substance that interacts with the test substance and the blocking agent is not particularly limited as long as it stably dissolves the substance that interacts with the test substance and the blocking agent. For example, water, buffer solution , Saline, water-miscible organic solvent, and the like, but preferably saline is used.

  Application of a solution containing a substance that interacts with a test substance and a blocking agent to the substrate is not particularly limited. For example, the above solution is added to the substrate and allowed to stand at 0 to 50 ° C. for several minutes to several hours. It is carried out by doing. In addition, you may block the site | part which the substance which interacts with a test substance, and a blocking agent were not fixed after the said application | coating, for example using further blocking agents, for example, BSA, block ace, skim milk, casein. Furthermore, in this case, with or without removing the blocking agent, it is possible to stabilize the immobilization of synthetic or natural polymers such as polyethylene glycol and polysaccharides, surfactants, and commercially available Immunoassay Stabilizer (ABI). A stabilizing agent may be added. These operations are preferably performed by taking measures for preventing evaporation, for example, by sealing a container including a substrate. The obtained substance-fixing substrate is preferably subjected to drying at 0 to 50 ° C. for several minutes to several hours using a dryer or the like.

  The substance-immobilized substrate obtained as described above can be used, for example, as an ELISA plate or a biosensor measurement chip to measure a test substance that interacts with a substance immobilized on the substance-immobilized substrate. it can. As used herein, “measuring a test substance” means an event that should be interpreted in the broadest concept, such as detecting the presence or absence of the test substance, measuring the amount of the test substance, that is, quantifying the test substance. It is.

  Specifically, the substance immobilized on the surface of the ELISA plate or biosensor measurement chip by contacting the test substance with the ELISA plate or biosensor measurement chip. Test substances that interact with can be measured.

  A test substance is usually contained in a test sample. 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.

  The test sample is used as it is or in the form of an extract obtained by extraction using an appropriate extraction solvent, and further in the form of a diluted solution obtained by diluting the extract with an appropriate diluent, or The extract is used in a concentrated form 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.

  When the test substance is an antigen, the antigen is not particularly limited as long as it can be specifically recognized by the substance (antibody) immobilized on the substance-immobilized substrate of the present invention. It may be recognized by the antibody, or may be recognized by the antibody via a part of an antigen, that is, an epitope. In addition to the site recognized by the antibody, the antigen may have a site (epitope) recognized by one or more antibodies.

  The measurement of the test substance is conventionally performed by fluorescence measurement method (FIA), enzyme color development method (EIA), radioactivity measurement method (RIA), luminescence method (CLEIA), electrochemiluminescence method (ECLIA), magnetic immunity The measurement can be performed using various methods such as a measurement method (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.

  The substance-immobilized substrate of the present invention can also be included in a test substance measurement kit for measuring the above-described test substance.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.

1. Preparation of anti-hCG antibody-modified fluorescent particles (diameter: 500 nm) as detection labels
An hCG antibody (Anti-hCG 5008 SP-5, Medix Biochemica) was prepared at 2 mg / mL using 50 mM MES-NaOH (pH 6.0). 250 μL of a 2 mg / mL hCG antibody solution was dispensed into a 2 mL eppen. 250 uL of 2% solids fluorescent particles (500 nm diameter fluorescent particle solution (Calboxylate-Modified Microsphere, Invitrogen, # F8812, 2% solid) was added. The mixture was stirred with an orbital shaker (1200 rpm, 25 ° C., 15 min). The eppen was placed in an ultrasonic cleaner so that it was immersed in the water in the tank (100% output, 1 min), and 5 μL of 10 mg / mL EDC aqueous solution was added. The eppen was placed in an ultrasonic cleaner so that the entire solution was immersed in the water in the tank (output 100%, 1 min.) 25 μL of 2 mol / L Glycine aqueous solution was added to the eppen (final concentration 100 mM). (1200rpm, 25 ° C, 30min) The eppen was centrifuged (15000rpm = 20400xg, 4 ° C, 20min) to settle the particles, and the supernatant was pipetted without breaking the pellet. 500 μL of PBS (pH 7.4) (GIBCO) was added to the eppen containing the pellet. Resuspend the pellet in the eppen using a wave washer and repeat this 3 times, add 0.5 mL of 1% BSA in PBS (pH 7.4) and resuspend using an ultrasonic washer. The resulting solution was stored refrigerated as a 1% solid solution.

2. Tip creation
hCG antibody (Anti-h-alpha subunit 6601 SPR-5, Medix Biochemica) and BSA in a molar ratio of 10: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1:10 The antibody was prepared to 10 μg / mL with 150 mM NaCl. 100 μL of the antibody solution (10 μg / mL, 150 mM NaCl) was dispensed into each well of a well plate having a gold film deposited on the bottom so as not to touch the wall. In order to prevent evaporation, each well was covered with a plate seal and allowed to stand at room temperature for 1 hour. The antibody solution was removed by discarding and tapping on a Kim towel. Blocking Casein in TBS (Pierce) 100uL was placed in a reservoir and dispensed with an 8-unit automatic pipettor (1250uL tip). Covered with a plate seal to prevent evaporation. The mixture was allowed to stand at room temperature for 1 hour with light shielding. The solution was removed by discarding and tapping on a Kim towel. Immunoassay Stabilizer (ABI) 100 uL was dispensed in the same manner as above. The plate seal was covered to prevent evaporation. The mixture was allowed to stand at room temperature for 1 hour with light shielding. The solution was removed. The antibody-immobilized chip was obtained by drying overnight at room temperature in a dryer. Further, only the hCG antibody was prepared to 150 μg / mL with 150 mM NaCl, and the subsequent operation was performed in the same manner as described above to obtain an antibody-immobilized chip as a control.

3. Assay Evaluation Antigen (hCG) was prepared to the following concentrations using 1% BSA / PBS: 0, 1.8E-13M, 1.8E-12M, 1.8E-11M, 1.8E-10M, 1.8E-9M. hCG antibody (Anti-hCG 5008 SP-5, Medix Biochemica) -bound Yellow-Green particles (500 nm) were diluted to 0.02% solid with 1% BSA / PBS. Into a 1.5 mL microtube, 350 uL of the antigen solution and 1 above. 350 uL of the fluorescent particle antibody prepared in (1) was added. Incubation was performed at 1200 rpm and 25 ° C. for 10 minutes. The background of the wells was measured with ARVO (yellow-green 0.1s; Perkin Elmer). 60 uL of reaction solution was added to each chip so as not to touch the wall (n = 3). Covered with a plate seal and incubated at 700 rpm, 25 ° C. for 4 hours. The reaction solution was removed by tapping on a Kim towel. Washed 4 times with 100 uL of PBS-T (PBS phosphate buffer + 0.05% Twenn20). The reflected fluorescence was measured with ARVO (yellow-green 0.1s). The S / N ratio was defined by the fluorescence signal (S) at each concentration and the fluorescence signal (N) ratio at 0 M antigen.

4). Measurement Results The results of measuring the antibody-immobilized chip are summarized in Table 1, including both the examples and comparative examples. Similarly, FIG. 1 is a graph showing the S / N ratio of antibody-immobilized chips prepared by changing various antibody / BSA molar ratios. As shown in Table 1 and FIG. 1, the antibody-immobilized chips having antibody / BSA molar ratios of 2, 1, and 0.5 as examples had improved S / N ratio and detection sensitivity compared to the control. . In contrast, the antibody-immobilized chips with antibody / BSA molar ratios of 10, 3 and 0.1 as comparative examples had the same or lower S / N ratio and detection sensitivity than the control. .

Claims (6)

  A method for producing a substance-immobilized substrate, comprising: applying a solution containing a substance that interacts with a test substance and a blocking agent at a molar ratio of 0.2 to 2.5 (substance / blocking agent) on the substrate.   The method for producing a substance-immobilized substrate according to claim 1, wherein the test substance is an antigen and the substance that interacts with the test substance is an antibody.   The method for producing a substance-immobilized substrate according to claim 1, wherein the test substance is a hormone, and the substance that interacts with the test substance is a hormone receptor.   The manufacturing method of the substance fixed board | substrate of any one of Claims 1-3 whose board | substrate is a metal, a plastics, or glass.   The method for producing a substance-immobilized substrate according to any one of claims 1 to 4, wherein the blocking agent is at least one selected from the group consisting of globulin, albumin, casein, gelatin, skim milk, polyvinyl alcohol, and polyvinylpyrrolidone. .   A substance-fixed substrate obtained by the method for manufacturing a substance-fixed substrate according to claim 1.

Images