JP2008190946A - Immunoassay using protein adsorption inhibitor, and substrate and kit for immunoassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a means using a blocking agent that is not derived from a living body, in order to greatly reduce the nonspecific adsorption of a unreacted material, in an immunoassay that uses a specific immunoreaction. <P>SOLUTION: In immunoassay, for example in an oxygen immunoassay, an inorganic particle material which is not derived from a living body and/or an organic inorganic complex particle material which is not derived from a living body are used as a nonspecific adsorbing inhibitor (blocking agent) for inhibiting nonspecific adsorption of the unreacted material, and a substrate or a kit used therefor is provided. Preferably, the inorganic particle or the organic inorganic complex particles are constituted mainly of silicon dioxide whose particle size is in a range of 0.1-500 nm. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention is characterized by significantly reducing nonspecific adsorption of unreacted substances using a specific nonspecific adsorption inhibitor (blocking agent) in an immunoassay utilizing a specific immune reaction. The present invention relates to an immunoassay method, and a substrate and kit used therefor.

The immunoassay is a method for qualitatively quantifying a small amount of a low molecular weight substance using the specificity of an antigen-antibody reaction. Examples of immunoassay include radioimmunoassay (RIA method) using a radioisotope as a labeling reagent, enzyme immunoassay method (EIA method) using an enzyme, electrochemiluminescence immunoassay method (ECL method) using a metal complex, etc. There is. In these measurement methods, the labeling reagent and the method for detecting the label are different, but the antibody to which the labeling reagent is immobilized is used to immunize the antigen to be measured and other substances (hereinafter collectively referred to as antigen in the present invention). They are common in that they are bound by reaction and the concentration of the bound labeled antibody is measured by various detection methods.

For example, an enzyme immunoassay is widely known as a method for detecting and measuring an antigen using the color of an enzyme in an antigen-antibody reaction. The method of immobilizing an antibody on a plate or the like in the EIA method is known as an ELISA method (Enzyme Linked Immuno Sorbent Assay), and is widely used for various analyzes because it is safe and convenient. Yes.

In these immunoassay methods, for example, ELISA, when a specific substance is detected in the presence of biomolecules such as proteins and lipids, nonspecific adsorption of the coexisting substance to the surface of a solid phase such as a plate is It interferes with specific adsorption as a ground, preventing high sensitivity. In order to solve this problem, water-soluble polymers such as albumin, casein, and gelatin have conventionally been used as blocking agents for suppressing / inhibiting nonspecific adsorption of coexisting substances in immunoassays.

However, it is difficult to completely prevent nonspecific adsorption to the solid phase surface even if the blocking operation by the conventional method is performed, and nonspecific adsorption to the blocking agent cannot be ignored. When a blocking agent is used, there is a problem in that the blocking operation affects the immune reaction due to contamination with immunoglobulin, biotin, etc., and there are limitations on the animal species of the antibody and the enzyme coloring system. In the case of a blocking agent derived from a living body, since a substance obtained from an animal tissue is used, there are increasing concerns from the viewpoint of stability, price, and safety represented by mad cow disease problems. Therefore, development of a high-performance blocking agent that is not derived from a living body has been desired.

An improved method has also been proposed for blocking agents derived from living bodies. For example, Patent Document 1 proposes to use polymer particles with low protein adsorption as a carrier. Specifically, as a means to suppress non-specific adsorption, a general biological material (blocking agent) such as albumin or casein is bound to the latex particle surface, and a higher effect than before is proposed. . However, this does not change the use of biomaterials and has not led to a radical solution.
JP 2001-21563 A

Regarding blocking agents that are not derived from living bodies, for example, block copolymers containing a polyethylene glycol segment (Patent Document 2) and non-crosslinked polymers based on polyethylene glycol chain segments (Patent Document 3) have been proposed. However, the effect of suppressing nonspecific adsorption is not always sufficient.
JP 2006-226882 A International Publication No. 2005/010529 Pamphlet

An object of the present invention is to provide a method / means using a blocking agent that is not derived from a living body in order to significantly reduce non-specific adsorption of unreacted substances in an immunoassay utilizing a specific immune reaction. It is in.

In the process of studying a method for suppressing specific adsorption of proteins by closely arranging inorganic particle materials surface-modified with a sterically hindered organic polymer, such as colloidal silica, on an ELISA plate, In addition to colloidal silica, rather than colloidal silica that is not particularly modified on the surface, the specific adsorption of lipophilic proteins is suppressed due to the hydrophilicity of the OH group on the silica surface, which is detected. The inventors have found that the background is low and have reached the present invention.

That is, the present invention provides non-specific adsorption inhibition for inhibiting non-specific adsorption of unreacted substances in non-biological-derived inorganic particle material and / or non-biological-derived organic-inorganic composite particle material in immunoassay. It is an immunoassay characterized by being used as an agent (blocking agent) (Invention of Claim 1).

In the present invention, the particle diameter of the inorganic particles or organic-inorganic composite particles is preferably in the range of 0.1 to 500 nm (the invention of claim 2). More preferably, it is 1-150 nm. The inorganic particles or organic-inorganic composite particles are preferably composed mainly of silicon dioxide (invention of claim 3). In addition, the present invention can be suitably applied to enzyme immunoassay among immunoassays (invention of claim 4).

Another aspect of the present invention is a substrate for use in an immunoassay, wherein at least a part of the surface thereof is coated with a non-biologically derived inorganic particle material and / or a non-biologically derived organic-inorganic composite particle material. This is a substrate for immunoassay (invention of claim 5).

In still another aspect of the present invention, a non-biological-derived inorganic particle material and / or a non-biological-derived organic-inorganic composite particle material for coating at least a part of the surface of a substrate used in an immunoassay method Is a kit for immunoassay containing one of its constituents (Invention of claim 6).

According to the present invention, there are provided simple means and methods for greatly reducing non-specific adsorption of unreacted substances in various immunoassays utilizing specific immune reactions. In particular, the present invention can be suitably used in enzyme immunoassay methods such as ELISA. Therefore, in the fields of clinical examination, pathological research, agriculture / biotechnology, etc., examination / detection / measurement using antigen-antibody reaction is possible. Therefore, a simple and highly sensitive method / means is provided.

In the immunoassay method, the present invention provides a non-specific adsorption inhibitor for inhibiting non-specific adsorption of unreacted substances by using non-biological-derived inorganic particle material and / or non-biological-derived organic-inorganic composite particle material ( It is used as a blocking agent. In the present invention, the immunoassay is a method for qualitatively quantifying a small amount of a low molecular weight substance by utilizing the specificity of an antigen-antibody reaction. Examples of immunoassay include radioimmunoassay (RIA method) using a radioisotope as a labeling reagent, enzyme immunoassay method (EIA method) using an enzyme, electrochemiluminescence immunoassay method (ECL method) using a metal complex, etc. There is. The present invention is particularly preferably applied to enzyme immunoassay.

The enzyme immunoassay is widely known as a method for detecting and measuring an antigen by utilizing the color of an enzyme in an antigen-antibody reaction. There are many known methods for enzyme immunoassay including direct method, indirect method, competitive method, sandwich method, and other modified methods. Strictly speaking, an enzyme immunoassay method in which an antibody is immobilized on a substrate such as a plastic plate is referred to as an ELISA method (Enzyme Linked Immuno Sorbent Assay). It is sometimes used synonymously with the sandwich method of sandwiching and quantifying with different types of antibodies. The ELISA method is a combination of an immune reaction, ie, an antigen-antibody reaction, and an enzyme substrate reaction. That is, the antigen captured by the antigen-antibody reaction is detected by using a color reaction with an enzyme and a color substrate. At this time, as means for relating the two reactions, there are a sandwich method using an antibody-enzyme complex and a so-called competition method using an antigen-enzyme complex. In the present invention, the enzyme immunoassay includes all the above methods.

In a normal enzyme immunoassay, a chromogenic material such as fluorescence is used as a chromogenic substrate in order to cause a chromogenic reaction between the enzyme and the chromogenic substrate. For example, in the case of an antibody labeled with alkaline phosphatase, when p-nitrophenyl phosphate as a substrate is allowed to act, p-nitrophenol is liberated and color develops. Therefore, the amount of color development is determined by measuring the wavelength at 405 nm with an absorptiometer.

In the present invention, as a non-specific adsorption inhibitor (blocking agent) for inhibiting non-specific adsorption of unreacted substances of immune reaction, non-biological-derived inorganic particle material or non-biological-derived organic-inorganic composite particle material Is used. Fine particles are used as the inorganic particles or organic-inorganic composite particles, but those having a particle size in the range of 0.1 to 500 nm are preferable. More preferably, it is 1-150 nm.

Examples of the inorganic particles include particles such as CaO, SiO ₂ , P ₂ O ₅ , MgO, K ₂ O, Na ₂ O, TiO _2, apatite, titanium apatite, glass, and ceramics. Organic-inorganic composite particles are obtained by chemically bonding various organic compounds on the surface of the inorganic particles. Specific examples of the organic compound include an amino group, a carboxyl group, a hydroxyl group, a sulfonic acid group, an amide group, a sulfonamide group, an epoxy group, an ester bond, a sulfonic acid ester group, an ether bond, an amide bond, a urethane bond, and fluorine. Examples thereof include an organic compound having at least one atom or a polymer of the organic compound.

As the inorganic particles or organic-inorganic composite particles, those composed mainly of silicon dioxide as an inorganic substance are preferable. Of these, colloidal silica (colloidal silica or colloidal silicic acid) is preferable. Colloidal silica usually refers to a colloidal suspension of SiO ₂ having OH groups on its surface by hydration in water. Generally, it is produced by adding hydrochloric acid to an aqueous solution of sodium silicate. However, in the present invention, those dispersed in a non-aqueous solution and those in the form of fine powder made by a gas phase method are also included. The particle diameter is usually many nm to several μm.

The organic / inorganic composite particles are preferably organic composite silicate particles obtained by chemically combining colloidal silica and an organic substance. As the organic substance, hydrophilic compounds such as acrylic acid monomers and polymers, aliphatic diamine compounds and polyamine compounds, polyethylene glycols, acrylamide and derivatives thereof are preferable.

Another aspect of the present invention is a substrate for use in an immunoassay, wherein at least a part of the surface thereof is coated with a non-biologically derived inorganic particle material and / or a non-biologically derived organic-inorganic composite particle material. This is a substrate for immunoassay.

In still another aspect of the present invention, a non-biological-derived inorganic particle material and / or a non-biological-derived organic-inorganic composite particle material for coating at least a part of the surface of a substrate used in an immunoassay method Is a kit for immunoassay containing one of its constituents. The immunoassay kit means, for example, a combination of instruments and reagents for performing the ELISA method.

In the present invention, the substrate means a substrate such as a plate for fixing an antibody or the like used in immunoassay. For example, an immunoassay plate composed of a plastic (polypropylene, polystyrene, etc.) plate having 4 holes, 6 holes, 24 holes, 96 holes, or the like. In the present invention, at least a part of the surface of the substrate is coated with the non-biologically derived inorganic particle material and / or the non-biologically derived organic-inorganic composite particle material. For example, a plate in which a particle material such as colloidal silica is adhered to and attached to the bottom of a 96-well plate is used as a plate for immunoassay.

The plate may be provided in a form in which a part of its surface is previously coated with a non-biological-derived inorganic particle material and / or a non-biological-derived organic-inorganic composite particle material, or with other constituent materials A kit for immunoassay may be formed in combination, and it may be configured such that a particle material such as colloidal silica is adhered and pasted on the bottom surface of a 96-well plate on the spot at the time of examination / measurement.

The technique for improving the detection sensitivity of the present invention, that is, the technique for suppressing or inhibiting the adsorption of unreacted substances by increasing the hydrophilicity of the substrate will be described as follows by taking a so-called sandwich method as an example.
(1) An antibody against a specific antigen is immobilized on a substrate (antibody immobilization). The substrate used may be one generally used in the ELISA method, and examples thereof include polypropylene, polystyrene, and gold thin film. (2) A non-biologically derived inorganic particle material and / or a non-biologically derived organic-inorganic composite particle material is attached to an immunoassay substrate on which the antibody is immobilized. (3) An antigen in a specimen is captured by an antibody immobilized on a substrate. (4) An enzyme-labeled antibody, that is, a secondary antibody having a recognition site different from that of (1) is bound to an antigen, and then the substrate and the enzyme in the enzyme-labeled antibody are reacted to form a colored product. Measure absorbance.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to reference examples, examples, and test examples, but the present invention is not limited thereto.

[Synthesis Example 1: Synthesis of SiO ₂ -MPS]
EtOH dispersion containing 2.1 g of colloidal silica (catalyst chemical industry) with a particle size of 125 nm in a 200 ml three-necked flask equipped with a reflux tube, thermometer and 50 ml dropping funnel and containing a stirrer piece 20 ml and 1.0 g of 3-methacrylic acid propyltriethoxysilane (MPS) manufactured by Chisso Corporation were added. After stirring at 55 ° C. for 24 hours, the reaction solution was centrifuged to remove the supernatant. Next, 20 ml of EtOH was added to the remaining residue and re-dispersed, followed by a washing operation of centrifuging to remove the supernatant five times. The residue after washing was dried under reduced pressure to prepare 1.953 g of colloidal silica (composite 1: SiO ₂ -MPS) whose surface was modified with propyl methacrylate.

[Synthesis Example 2: Synthesis of SiO ₂ —NH ₂ ]
Synthetic product 1 (200 mg) and ethylenediamine (50 mg) were mixed in an eggplant-shaped flask to which 20 ml of EtOH was added, stirred for 24 hours at room temperature, and then the reaction solution was centrifuged to remove the supernatant. Next, 20 ml of EtOH was added to the remaining residue for redispersion, and centrifugation was performed to remove the supernatant five times. The residue obtained by washing was dried under reduced pressure to prepare 185 mg of ethylenediamine-modified colloidal silica (Compound 2: SiO ₂ —NH ₂ ).

[Synthesis Example 3: Synthesis of SiO ₂ -PAAm]
Synthetic product 1 (200 mg) and polyallylamine (200 mg) were mixed in an eggplant-shaped flask to which 20 ml of EtOH was added, stirred for 24 hours at room temperature, and then the reaction solution was centrifuged to remove the supernatant. Next, 20 ml of EtOH was added to the remaining residue for redispersion, and centrifugation was performed to remove the supernatant five times. The residue obtained by washing was dried under reduced pressure to prepare 207 mg of polyallylamine-modified colloidal silica (Synthesis 3: SiO ₂ -PAAm).

[Synthesis Example 4: Synthesis of SiO ₂ -PEG]
Particle size 120
nm colloidal silica 500
1 mg of polyethylene glycoloxyamidopropyltriethoxysilane with an average molecular weight of 2000 was mixed in an eggplant-shaped flask containing 50 ml of dehydrated EtOH, stirred at room temperature for 24 hours, centrifuged the reaction solution and supernatant Was removed. Next, 50 ml of EtOH was added to the remaining residue and re-dispersed, and then a washing operation for removing the supernatant by centrifuging was performed five times. The residue after washing was dried under reduced pressure to prepare 473 mg of polyethylene glycol-modified colloidal silica (Compound 4: SiO ₂ -PEG).

[Synthesis Example 5: Synthesis of SiO ₂ -PAc]
Compound 1 (250 mg) and 2.0 g of acrylic acid were mixed in an eggplant type flask to which 100 ml of ion-exchanged water was added, and AAP (2-2'-azobis-2-diaminopropane dihydrochloride was used as the polymerization initiator. 40 mg was added and reacted at 70 ° C. for 6 hours, and then the reaction solution was centrifuged to remove the supernatant. The remaining residue was redispersed by adding 100 ml of ion-exchanged water, and the washing operation for removing the supernatant by centrifuging was performed 8 times. The residue after washing was dried under reduced pressure to prepare 244 mg of polyacrylic acid-modified colloidal silica particles (Compound 5: SiO ₂ -PAc).

[Synthesis Example 6: Synthesis of SiO ₂ -PNIPAM]
Add synthetic compound 1 (250 mg), N-isopropylacrylamide (NIPAM) 500 mg, polymerization initiator azobisisobutyronitrile (20 mg), and EtOH 50 ml to an eggplant-shaped flask, and stir at 70 ° C. for 6 hours. The supernatant was removed by centrifuging the solution. The remaining residue was redispersed by adding 50 ml of EtOH, and the washing operation for removing the supernatant by centrifuging was performed 8 times. The residue after washing was dried under reduced pressure to prepare 253 mg of N-isopropylacrylamide-modified colloidal silica (Compound 6: SiO ₂ -PNIPAM).

[test]
In general, protein adsorption is more likely to occur as the interface becomes more hydrophobic. Therefore, the blocking agent used for the purpose of suppressing the non-specific adsorption of protein to the substrate fulfills its function by imparting hydrophilicity to the substrate. On the other hand, one of the indicators for judging hydrophilicity is a method for obtaining the contact angle between water and the interface. Therefore, the contact angle between water and a substrate coated with colloidal silica or the like was measured, and the hydrophilicity of the substrate was evaluated, and the protein adsorption inhibition effect was simply evaluated.

[Test Example 1: Measurement of contact angle]
A polystyrene coated substrate was prepared by dropping 15 μl of a 0.5% ethyl acetate solution of polystyrene (polymerization degree 2000) on a 1.8 × 1.8 mm glass substrate and performing spin coating at 3000 rpm for 30 seconds. On this polystyrene-coated substrate, 10 μl of a 10 mg / ml ethanol dispersion such as colloidal silica was dropped and spin-coated at 3000 rpm for 30 seconds to prepare a coated substrate such as colloidal silica. In addition, the colloidal silica etc. to be used are the chemically modified particles (composite 2 to compound 6) of colloidal silica shown in the above Synthesis Examples 2 to 6, and two kinds of unmodified colloidal silica particles (particles) having different particle sizes. A total of 7 types having a diameter of 120 nm and 60 nm) were used. The contact angle to water of the prepared 7 kinds of colloidal silica coated substrates was measured. The results are shown in Table 1.

[Comparative Example 1]
A polystyrene coated substrate was prepared by dropping 15 μl of a 0.5% ethyl acetate solution of polystyrene (degree of polymerization 2000) onto a 1.8 × 1.8 mm glass substrate and performing spin coating at 3000 rpm for 30 seconds. The polystyrene-coated substrate was immersed in a 0.5% bovine serum albumin (BSA) in PBS and dip-coated at 40 ° C. for 1 hour to prepare a BSA-coated substrate. In addition, an untreated substrate (glass) and a polystyrene coat substrate (PS) were used as substrates for comparison. The contact angles with respect to water of three types of substrates prepared as comparative examples were measured. The results are shown in Table 2.

From the measurement results of the contact angle, it was confirmed that the substrate coated with colloidal silica or the like had a significant decrease in contact angle compared to the substrate coated with polystyrene. In addition, it was revealed that the hydrophilicity of the substrate surface was improved to the same level or higher compared to the substrate treated with BSA, which is a general blocking agent.

[Test Example 2: Evaluation of the amount of protein (antibody) adsorbed nonspecifically on the substrate]
Blocking: A 96-well plate molded product (Nunc) with 0.1 mg / ml colloidal silica / phosphate buffer (PBS) (pH 7.4) dispersion in 8 wells at a rate of 300 μl / well. The solution was dispensed and allowed to stand at 40 ° C. for 1 hour to block colloidal silica on the substrate. After completion of the reaction, the reaction mixture was washed three times with a washing solution (PBS-Tween) made of PBS to which Tween 20 (polyoxyethylene (20) sorbitan monolaurate) was added to 0.05%. The colloidal silica used was a total of seven types of chemically modified particles shown in Synthesis Examples 2 to 6 (Synthetic Product 2 to Synthetic Product 6) and two types of unmodified particles (particle size 125 nm and 60 nm) having different particle sizes. It was examined using. For comparison, 0.5% bovine serum albumin (BSA) / PBS-Tween was dispensed at 300 μl / well, and blocking and washing were performed in the same manner.

Non-specific adsorption treatment: Peroxidase-labeled anti-goat IgG (Invitrogen) or peroxidase-labeled anti-human albumin IgG (Bethyl) in 8 PBS-Tween solutions (1000,
500, 250, 100, 50, 25, 10, 0 ng / ml) was dispensed in an amount of 100 μl into 8 wells blocked with each particle, and allowed to stand for 1 hour to immobilize anti-human albumin IgG onto the plate. Adsorbed. After the reaction, it was washed 5 times with PBS-Tween.

Coloring reaction: 0.5 mg / ml citrate phosphate buffer solution (containing 0.03% hydrogen peroxide) (pH 4.5) of o-phenylenediamine (OPD) manufactured by Wako Pure Chemical Industries, Ltd. was dispensed at 100 μl / well, and placed on the plate. The peroxidase in the adsorbed antibody was reacted for 30 minutes. The reaction was stopped by dispensing 2N sulfuric acid at 50 ul / well, and the absorbance (492 nm) of the colored solution in each well was measured with a plate reader. The results are shown in Tables 3 and 4.

From the results of Test Example 2, when various colloidal silicas were used as blocking agents, the absorbance was equal to or less than that of BSA (bovine serum albumin), which is commonly used as a blocking agent. Accordingly, it is considered that nonspecific adsorption between the plate and the peroxidase-labeled antibody (protein) was suppressed as in BSA. In particular, unmodified colloidal silica (60 nm) having a particle size of 60 nm showed a very good adsorption suppression effect.

[Test Example 3]
Primary antibody treatment: A 96-well plate molded product (Nunc) was dispensed with 100 ng / well of rabbit IgG / PBS made of MP Biomedicals at a concentration of 1000 ng / ml as the primary antibody into all wells used in the experiment. The reaction was carried out at 40 ° C. for 1 hour to immobilize the primary antibody. After completion of the reaction, it was washed 3 times with PBS-Tween.

Blocking treatment: Colloidal silica or the like / PBS dispersion prepared to 0.1 mg / ml was dispensed at 300 μl / well into wells treated with the primary antibody and reacted at 40 ° C. for 1 hour. After completion of the reaction, the plate was washed 3 times with PBS-Tween. In addition, the colloidal silica to be used is a total of 7 types including the chemically modified particles shown in Synthesis Examples 2 to 6 (Synthetic Product 2 to Synthetic Product 6) and two types of unmodified particles (particle size 125 nm and 60 nm) having different particle sizes. Each was blocked for 8 wells. Also, as a comparison 0.5%
BSA / PBS-Tween was dispensed at 300 μl / well, and the same reaction and washing were used.

Antigen treatment: 8 types of PBS-Tween solutions of goat anti-rabbit IgG (ROCKLAND) (1000,
500, 250, 100, 50, 25, 10, 0 ng / ml) was dispensed into each blocking 8 well, 100 μl each, and allowed to stand for 1 hour to react with the immobilized rabbit IgG. After the reaction, the solution in the well was discarded and the plate was washed 3 times with PBS-Tween.

Secondary antibody treatment: Peroxidase-labeled anti-goat IgG (Invitrogen) PBS-Tween solution (150 ng / mL) was dispensed at 100 μl / well and allowed to react at room temperature for 1 hour. After the reaction, it was washed 5 times with PBS-Tween.

Coloring reaction: 0.5 mg / ml citrate phosphate buffer solution (containing 0.03% hydrogen peroxide) (pH 4.5) of o-phenylenediamine (OPD) manufactured by Wako Pure Chemical Industries, Ltd. was dispensed at 100 μl / well. The reaction was stopped for 10 minutes with the peroxidase immobilized on the solution and 2N sulfuric acid was dispensed at 50 ul / well. The absorbance (492 nm) of the colored solution in each well was measured with a plate reader. The measurement results are shown in Table 5.

[Test Example 4]
Primary antibody treatment: A 96-well plate molded product (Nunc) was dispensed with 1000 ng / ml Bethyl anti-human albumin IgG / PBS as a primary antibody at a rate of 100 μl / well to all wells used in the experiment. The reaction was carried out at 40 ° C. for 1 hour to immobilize the primary antibody. After completion of the reaction, it was washed 3 times with PBS-Tween.

Blocking treatment: 0.1 mg / ml colloidal silica or the like / PBS dispersion was dispensed at 300 μl / well into wells treated with the primary antibody and reacted at 40 ° C. for 1 hour. After completion of the reaction, the plate was washed 3 times with PBS-Tween. In addition, the colloidal silica to be used is a total of 7 types including chemically modified particles shown in Synthesis Examples 2 to 6 (Synthetic Product 2 to Synthetic Product 6) and two types of unmodified particles having different particle sizes (particle size 125 nm and 60 nm). It was examined using. For comparison, 0.5% BSA / PBS-Tween was dispensed at 300 μl / well, and the reaction and washing were performed in the same manner.

Antigen treatment: 8 types of PBS-Tween solution of human albumin (manufactured by Wako Pure Chemical Industries) (1000,500,250,100,50,25,10,0
ng / ml) was dispensed into 8 wells treated with 100 μl each, and allowed to stand for 1 hour to react with the immobilized anti-human albumin IgG. After the reaction, the solution in the well was discarded and the plate was washed 3 times with PBS-Tween.

Secondary antibody treatment: Peroxidase-labeled anti-human albumin IgG (Bethyl) PBS-Tween solution (75 ng / mL) was dispensed at 100 μl / well and allowed to react at room temperature for 1 hour. After the reaction, it was washed 5 times with PBS-Tween.

Coloring reaction: 0.5 mg / ml citrate phosphate buffer solution (containing 0.03% hydrogen peroxide) (pH 4.5) of o-phenylenediamine (OPD) manufactured by Wako Pure Chemical Industries, Ltd. was dispensed at a volume of 100 μl / well. The mixture was reacted with peroxidase immobilized on 10 minutes, and 2N sulfuric acid was dispensed at 50 ul / well to stop the reaction. The absorbance (492 nm) of the colored solution in each well was measured with a plate reader. The measurement results are shown in Table 6.

As a result of confirming the blocking effect of various colloidal silicas by ELISA, it was confirmed that the absorbance was the same or decreased as compared with BSA as a general blocking agent. The decrease in absorbance means that unreacted substances in the antigen-antibody reaction are non-specifically adsorbed on the substrate and the background is improved. Therefore, it was confirmed that colloidal silica has an excellent effect of suppressing nonspecific adsorption as compared with BSA.

Claims (6)

In immunoassay, non-biologically derived inorganic particle material and / or non-biologically derived organic-inorganic composite particle material is used as a nonspecific adsorption inhibitor (blocking agent) for inhibiting nonspecific adsorption of unreacted substances. An immunoassay characterized in that it is used. The immunoassay method according to claim 1, wherein the particle diameter of the inorganic particles or the organic-inorganic composite particles is in the range of 0.1 to 500 nm. The immunoassay method according to claim 1 or 2, wherein the inorganic particles or organic-inorganic composite particles are composed mainly of silicon dioxide. The immunoassay according to any one of claims 1 to 3, wherein the immunoassay is an enzyme immunoassay. A substrate for use in an immunoassay, wherein at least a part of the surface thereof is coated with a non-biologically derived inorganic particle material and / or a non-biologically derived organic-inorganic composite particle material. A liquid containing a non-biologically derived inorganic particle material and / or a non-biologically derived organic-inorganic composite particle material for coating at least a part of the surface of a substrate used in an immunoassay is one of its constituent elements. Kit for immunoassay.