JP4432252B2 - Method for producing protein-adsorbing carrier and measuring method using the carrier - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention is a carrier or container used for chromatography, electrophoresis, immunoassay, etc. used as separation analysis / separation purification means in the fields of biochemistry, clinical chemistry, pharmaceutical chemistry, etc. The present invention relates to a method for producing a carrier or a container capable of suppressing the adsorption, or a carrier or container for specifically binding any target substance.
[0002]
[Prior art]
In the fields of chemistry, biochemistry, medicine, and the like such as chromatography, gel electrophoresis, and heterogeneous immunoassay, separation analysis and separation purification are widely performed using a carrier. These utilize the interaction between specific functional groups on the carrier, specific sites in the protein immobilized on the carrier by adsorption, etc., and the target component (substance). For example, it is frequently used for separation analysis and separation purification of trace components in biological samples.
[0003]
In the process of separation analysis, the target substance and the labeling substance that binds the enzyme etc. to the antibody or antigen to generate the signal related to the target component may be adsorbed non-specifically to the carrier or container. Such non-specific adsorption is a major obstacle when aiming at high sensitivity of separation analysis. In particular, when the target component is a component that exists only in a very small amount, the amount of the target component that can be actually analyzed is reduced by the non-specific adsorption as described above, and the target component is non-specific. Signal from the adsorbed labeling substance increases, and the S / N ratio in the separation analysis deteriorates.
[0004]
Also, in the separation and purification process, the target component may be adsorbed to the carrier or the container in the same manner as described above. become.
[0005]
For the purpose of reducing nonspecific adsorption as described above, for example, in separation analysis using a carrier such as heterogeneous immunoassay, an antigen or antibody is immobilized on the carrier, and then the hydrophobic adsorption point on the carrier is immunized. Blocking with a protein unrelated to the reaction is generally performed. Besides using protein, for example, a blocking method using polyvinyl alcohol (see JP-A-4-19561) or a blocking method using 2-methacryloyloxyethylsulfoylcholine polymer (JP-A-7-1995). -83923) is also reported. On the other hand, in order to reduce the nonspecific adsorption of the target component and the labeling substance to the container (reaction container) for performing an immune reaction, an adsorption inhibitor is also added to the reaction solution (Japanese Patent Laid-Open No. 11). -248706).
[0006]
In order to reduce non-specific adsorption to the carrier, it is said that it is effective to increase the hydrophilicity on the carrier surface and increase the steric repulsion between the carrier and the adsorbing component. Protein is composed of about 20 kinds of amino acids, and in aqueous solution, the hydrophobic amino acid side chain is arranged on the inner side to reduce the contact area with water molecules to form a core, and the dissociative amino acid side chain and polar amino acid side Although the chain is arranged outside and takes a three-dimensional structure in contact with the surrounding water, a part of the surrounding water binds to the protein as hydration water to form a hydrated layer. Therefore, blocking the surface of the carrier with a protein is considered to be effective as a method for preventing nonspecific adsorption of a target component or the like because it newly imparts hydrophilicity to the carrier.
[0007]
[Problems to be solved by the invention]
However, in the conventional blocking method in which the protein is simply adsorbed on the carrier, adsorption denaturation occurs due to the protein adsorbing on the carrier, resulting in a new interaction with the target component. Non-specific adsorption may be induced. Therefore, in order to further increase the sensitivity of the separation analysis using the carrier, it has been necessary to reduce the amount of nonspecific adsorption of the target component and the labeling substance on the carrier more than before. As described above, a method of using a synthetic polymer instead of protein for blocking is also known, but this method has a problem that the preparation of the synthetic polymer is complicated and expensive.
[0008]
In addition, in order to improve the analytical sensitivity or improve the recovery rate of the target component even when the target component such as a biological component is separated and analyzed or separated and purified using chromatography or gel electrophoresis, There is a need to reduce non-specific adsorption of components and labeling substances to carriers and containers. For this reason, in chromatography and electrophoresis, a hydrophilic polymer is used as a carrier substrate (Y. Kato et al, J. Chromatogr., Vol. 266, p385, 1983), or a carrier or container is used as a hydrophilic polymer. However, when the target component is a trace component in a biological sample, depending on the type, there may be no effect.
[0009]
Therefore, the object of the present invention is to prevent or reduce non-specific adsorption of target components and labeling substances in separation analysis and / or separation purification, thereby improving the measurement sensitivity of separation analysis or improving the recovery rate in separation purification. An object of the present invention is to provide a method for manufacturing a carrier and a container.
[0010]
[Means for Solving the Problems]
The invention of claim 1 of the present invention made to achieve the above object is a method for producing a carrier or a container. In producing a carrier or container having reduced nonspecific adsorption by adsorbing a protein to the carrier or container. And irradiating the carrier or the container with microwaves while immersed in the protein solution.
[0011]
Further, the invention of claim 2 of the present invention made to achieve the above object is a method for producing a carrier or a container, wherein a protein or a container is adsorbed to the carrier or container to produce a carrier or container having reduced nonspecific adsorption. In this case, the protein solution is irradiated with microwaves, and then the carrier or container is immersed in the protein solution.
[0012]
The invention of claim 3 of the present invention made to achieve the above object relates to the invention of claim 1 or 2, wherein the protein solution contains serum albumin. The invention of claim 4 of the present application relates to the invention of claim 1 or 2, characterized in that the protein solution is a solution containing serum albumin and having a pH adjusted to pH 3.0 to 6.5. .
[0013]
In order to achieve the above object, the invention of claim 5 of the present invention is directed to producing a carrier or container with reduced non-specific adsorption by adsorbing a protein to the carrier or container, and containing serum albumin. And dipping the carrier or container in a protein solution adjusted to pH 3.0 to 6.5.
[0014]
The invention of claim 6 relates to the invention of claims 1 to 5, characterized in that a ligand is adsorbed to the carrier or container prior to the adsorption of the protein. Hereinafter, the present invention will be described in detail.
[0015]
The present invention relates to a method for producing a carrier or the like by adsorbing a protein to the surface of a carrier or a container, thereby increasing the hydrophilicity of the surface and thereby preventing or reducing nonspecific adsorption of a target component or labeling substance. It is. Here, as a protein to be adsorbed on the surface, preferably serum albumin, more preferably serum albumin acid-denatured under acidic conditions is denatured by microwave, adsorbed to a carrier or container, and then denatured by microwave. It is characterized in that it is adsorbed on a carrier or a container, or denatured after adsorbing on a carrier or a container. Another aspect of the present invention is characterized in that a protein, preferably serum albumin, is acid-denatured under acidic conditions and adsorbed on a carrier or a container.
[0016]
Although there is no division in the past that has not used microwave technology, it has been known that microwaves are irradiated to an insoluble particle carrier on which an immunologically active component is immobilized to shorten the particles. A technique for uniformly dispersing with time (see JP-A-5-18972), or a technique for accelerating the reaction by irradiating microwaves during antigen-antibody reaction (see JP-A-6-43161), This technology only uses the shaking and stirring effects of microwaves. In addition, as a technique that utilizes the denaturation effect of microwave proteins, there is a technique (see Japanese Patent Application Laid-Open No. 8-33386) that quickly fixes a tissue when preparing a tissue specimen. No mention is made of the effect of suppressing or reducing nonspecific adsorption to a carrier or a container. The present invention has the effect of efficiently exposing the hydrophilic interface of the protein to the surface by microwave irradiation or acid modification, and the orientation of the modified protein by microwave irradiation when the modified protein is adsorbed on the carrier or container. It was completed based on the knowledge that the effect of adjusting (the direction of the hydrophilic interface) can be obtained.
[0017]
The carrier or container produced according to the present invention has limited protein adsorption, and not only proteins such as serum albumin adsorbed on the surface in the production process, but also proteins in separation analysis and separation purification operations, that is, target components, labels It suppresses or reduces the adsorption of substances and further contaminating proteins other than these.
[0018]
In the present invention, the carrier or the container is, for example, a polymer or copolymer such as styrene, ethylene glycol, acrylic acid or methacrylic acid, one formed using an inorganic material such as silica or ferrite, and the like. A carrier or a container into which a reactive functional group such as an ion exchange group such as a sulfopropyl group or a diethylaminoethyl group, a tosyl group, a tresyl group, or an epoxy group is introduced. There is no restriction | limiting in the shape of a support | carrier and a container, For example, a plate shape, bead shape, fine particle shape, gel shape, a tube shape, and a cup shape can be illustrated.
[0019]
For example, in immunoassay, a ligand such as an antigen or antibody is bound to a carrier or container, and then another protein, that is, a target component (component to be measured) or a labeling substance (for example, a detectable fluorescent substance or enzyme) is added. It is necessary to prevent non-specific adsorption of the bound antigen or antibody) to the carrier or the like. In order to use the carrier or container of the present invention for such immunoassay or the like, the above ligand may be bound prior to the adsorption of the protein. Here, the ligand means a substance that can specifically bind to a target substance when performing analysis or purification using the carrier or container produced according to the present invention. In addition to the antigen and antibody described above, for example, Examples include receptors, enzymes, avidin, biotin, streptavidin, nucleic acids and the like.
[0020]
Microwaves are electromagnetic waves having a frequency of 300 MHz to 300 GHz. Such microwaves are used as various communication means, or for cooking microwave ovens and thermal therapy utilizing the heating effect. In the present invention, the microwave applied to the carrier, the container, or the protein solution is not limited as long as it has an oscillation frequency and output that can affect the rotational energy region of water molecules. Specifically, the frequency and output of the microwave are an oscillation frequency of 1000 to 10000 MHz and an output of about 100 to 1000 W. For example, a microwave oscillated from a magnetron built in a microwave oven for cooking has a frequency of 2450 MHz and an output of about 200 to 600 W, and can be used to implement the present invention.
[0021]
In the present invention, examples of the protein adsorbed on the carrier or container include bovine serum albumin (hereinafter BSA), human serum albumin (hereinafter HSA), ovalbumin, casein, gelatin, γ-globulin, immunoglobulin and the like. It is particularly preferable to use a protein solution containing at least BSA or HSA, because it has a high adsorptivity to a container and can be easily improved in hydrophilicity by microwave denaturation or acid denaturation. Further, if the carrier or container of the present invention is used particularly for separating and analyzing human-derived components, it is preferable to use a protein solution containing BSA that has a low possibility of affecting the measurement system. The protein as described above may be used in a state in which it is dissolved in various buffer solutions used in, for example, a blocking operation in normal immunoassay, for example, a phosphate buffer solution, a Tris-HCl buffer solution, a carbonate buffer solution or the like. The protein concentration in this case can be exemplified as about 0.1 to 10% by weight.
[0022]
The pH of the protein solution to be irradiated with microwaves is preferably set to a pH that can minimize the amount of nonspecific adsorption of proteins such as target components to the carrier and container after production. Such a pH value can be obtained by conducting a preliminary study in accordance with the denaturation characteristics of the protein to be adsorbed depending on the isoelectric point or pH.
[0023]
The present invention uses a protein solution containing at least serum albumin, without irradiating microwaves, simply acidifying the pH of the protein solution to acid-denaturate serum albumin and adsorbing it to a carrier or container, as described above. To achieve an effect. Even in the case of microwave irradiation, if the protein solution containing at least serum albumin is acidified and acid-denatured, non-specific adsorption compared to the case of only microwave irradiation. It is possible to make the effect of reducing the above remarkable. The effect of such acid modification is because the hydrophilicity at the molecular interface is enhanced by acid modification of serum albumin. When serum albumin is acid-denatured, the pH of the protein solution is preferably adjusted to a range of 3.0 to 6.5, particularly preferably a pH of 3.0 to 4.0.
[0024]
Microwave irradiation of the protein solution may be performed for about 10 to 100 seconds while maintaining the temperature of the protein solution in the range of 0 to 37 ° C. For example, it is performed for 30 seconds or more at room temperature using a cooking microwave oven. When irradiating microwaves, the output is relatively high, so it is important to prevent bumping of the protein solution. For this purpose, for example, the protein solution is preferably ice-cooled. As a result, the effects of the present invention can be achieved without causing modification due to bumping or temperature rise. For more detailed irradiation conditions, perform preliminary studies in advance according to the type of protein to be adsorbed, and set so that the amount of nonspecific adsorption of proteins such as target components to the carrier and container after production can be minimized. Is preferred.
[0025]
In carrying out the present invention, microwaves may be irradiated while the carrier or container is immersed in the protein solution, or after irradiating the protein solution with microwaves, the carrier or container is immersed in the protein solution. You may do it. When irradiating microwaves with the carrier or container immersed in the protein solution, the carrier or container is immersed in the protein solution at the same time as irradiating the microwave, and the protein denatured by the microwave is applied to the carrier or container. For example, after adsorbing and adsorbing a protein to a carrier or a container, the adsorbed protein can be denatured by microwave irradiation. However, as is clear from the following examples, it is preferable to immerse the carrier or container in the protein solution, previously adsorb the protein to the carrier or container, and then irradiate the microwave. In this case, the microwave irradiation is particularly preferably performed after the protein adsorption to the carrier or the container is saturated.
[0026]
The present invention uses microwave irradiation and / or acid denaturation to adsorb the denatured protein on the surface of a carrier or container, thereby suppressing or reducing nonspecific adsorption of other proteins. To do. Therefore, as described above, when a ligand is previously bound to a carrier or a container, the ligand does not lose its reactivity with a specific binding target substance by microwave irradiation and / or acid denaturation. It is important to make it. On the other hand, as shown in the following examples, when microwaves are applied to a carrier or container to which a ligand is bound, the binding site of the ligand faces outward (solution side) due to the molecular orientation effect, and the effective ligand amount That is, there is an effect of microwave irradiation that has not been known so far, that is, the amount of ligand that can truly bind to the binding target substance increases. Therefore, for carriers and containers preliminarily bound with a ligand, the ligand may be denatured by microwave irradiation and the reactivity may be reduced, the ligand may be acid-denatured and the reactivity may be lowered, and the microwave irradiation In order to compare and consider the possibility that the amount of effective ligand increases due to the molecular orientation effect of the ligand, a preliminary experiment is performed according to the description of Examples 1 to 3 below, and any method of the present invention is adopted based on the result. It is preferable to determine whether. As a result of the preliminary experiment, when the ligand is denatured in the protein solution adjusted to be acidic and loses the reactivity, a protein solution not adjusted to acid is used to avoid this, and / or When the ligand is denatured by microwave irradiation and loses the reactivity, it is preferable to immerse a carrier or container in the solution after irradiating the protein solution with microwave.
[0027]
The method of previously binding the ligand to the carrier or the container may be a method of simply adsorbing the ligand or a method of binding by chemical bonding. Further, instead of directly binding the carrier or container to the ligand, a substance that can specifically bind to the ligand may be bound by the above-described method, and the ligand may be bound via this substance.
[0028]
The carrier and container of the present invention produced as described above can be used for various applications. For example, in the separation / purification step, the sample can be used for a sample to be used in the step or a container, a tube or the like for containing a component obtained by separation / purification. In chromatography and the like, it can be used as a carrier gel or, in some cases, as a column for holding it. Moreover, it can also be set as the disposable pipette tip for handling a protein sample, the centrifuge tube for performing centrifugation, etc.
[0029]
In the step of separation analysis, for example, after a protein is adsorbed on a carrier or container in which a first specific binding substance for a target component is previously bound, a second specific binding substance (labeling substance) for the sample and the target component is used. Mixing, washing the carrier and container, and labeling in the complex of the first specific binding substance-target component-second specific binding substance (labeling substance) formed on the carrier or container. In addition to so-called sandwich type immunoassay such as detection, it can be used for affinity analysis represented by so-called competitive type immunoassay and nucleic acid analysis. The labeling substance here is, for example, an antigen, an antibody, a receptor, an enzyme or the like to which a label such as a fluorescent dye, a luminescent substance, an enzyme, a light-absorbing substance, or a compound having a dinitrophenyl group is bound, and includes a protein. The effects of the carrier and container of the present invention can be sufficiently exhibited.
[0030]
DETAILED DESCRIPTION OF THE INVENTION
Examples will be shown below to describe the present invention in more detail. However, these examples show one embodiment of the present invention and do not limit the present invention.
[0031]
Example 1
The carrier of the present invention was produced by the following two production methods.
[0032]
(Method 1)
About 1000 carriers made of ethylene-vinyl acetate copolymer (particle diameter: about 1.6 mm, hereinafter referred to as ferrite beads) coated with ferrite on the surface were taken in seven 15 ml polypropylene centrifuge tubes. Contains 0.5% protein (BSA, HSA, gelatin, casein, bovine γ-globulin, goat immunoglobulin G or ovalbumin, one of a total of 7 proteins) and 0.1% sodium azide A 0.1 M Tris-HCl buffer solution pH 8.0 was prepared, 10 ml was added to each centrifuge tube containing ferrite beads, gently mixed by inversion, and allowed to stand at 4 ° C. for 12 hours or more.
[0033]
(Method 2)
Three ferrite beads identical to those used in method 1 were placed in a 2 ml microtube, and 200 μl of the same protein solution as in method 1 (a protein solution containing one of a total of seven types) was added. After pre-ice-cooling the microtube for 10 minutes, the cap is opened, and in the ice-cooled state, the cooking microwave oven (MR-M25, turntable method, oscillation frequency 2450 MHz, rated high-frequency output 500 W, manufactured by Hitachi, Ltd.) The microwave was irradiated for 30 seconds exactly at “strong”. The tube was taken out, the cap was closed, and the mixture was shaken and stirred for 6 hours in a 37 ° C. incubator and stored at 4 ° C.
[0034]
Three each of the carriers produced in the method 1 or 2 are put into a 2 ml microtube, and the protein solution is completely removed by suction with an aspirator, and then 0.1 ml phosphorus containing 1 ml of 0.1% BSA. Washed twice with acid buffer (pH 7.4). 100 μl of an alkaline phosphatase-labeled anti-α-fetoprotein antibody solution (manufactured by Tosoh Corporation) diluted to 3.7 μg / ml with 0.1 M phosphate buffer (pH 7.4) containing 0.1% BSA was added. Shake for 10 minutes at ° C.
[0035]
After the alkaline phosphatase-labeled anti-α-fetoprotein antibody solution was removed by suction, the carrier was washed three times with 1 ml of 0.1 M NaCl solution containing 0.044% Triton X-100. After this solution is removed by suction, the carrier is transferred to a 12 × 75 mm glass tube, and 100 μl of chemiluminescence substrate (CSPD, 0.2 mM solution, Tropix) for alkaline phosphatase activity measurement is dispensed and immediately detected for luminescence. The amount of luminescence was counted with a device (trade name LUMAT LB9501, manufactured by Bertoold). The photometric time was 20 seconds immediately after setting to the detector.
[0036]
FIG. 1 shows the measurement results of luminescence. In all seven types of proteins, the amount of non-specific adsorption was reduced by microwave irradiation. The amount of non-specific adsorption after microwave irradiation was the lowest for BSA and casein, and BSA showed the highest reduction rate of non-specific adsorption by microwave irradiation.
[0037]
Example 2
The carrier of the present invention was produced by the following three production methods.
[0038]
(Method 1)
About 1000 ferrite beads are placed in a 15 ml polypropylene centrifuge tube, and a total of 14 protein solutions with different pHs (0.1 M phosphate buffer containing 0.5% BSA and 0.1% sodium azide) are used. 10 ml of a solution (pH 3.0 to 7.5) or 0.1 M Tris-HCl buffer (pH 8.0 to 11.2) containing 0.5% BSA and 0.1% sodium azide After gently mixing by inversion, the mixture was allowed to stand at 4 ° C. for 12 hours or more.
[0039]
(Method 2)
Three ferrite beads were placed in a 2 ml microtube, and 200 μl of the same protein solution (14 types in total) as in Method 1 was added. After pre-ice-cooling the microtube for 10 minutes, the cap is opened, and in the ice-cooled state, the cooking microwave oven (MR-M25, turntable method, oscillation frequency 2450 MHz, rated high-frequency output 500 W, manufactured by Hitachi, Ltd.) The microwave was irradiated for 30 seconds exactly at “strong”. The tube was taken out, the cap was closed, and the mixture was shaken and stirred for 6 hours in a 37 ° C. incubator and stored at 4 ° C.
[0040]
(Method 3)
First, without adding a carrier, only 10 ml of the same protein solution (total 14 types) as in Method 1 is taken into a 15 ml polypropylene centrifuge tube and pre-cooled with ice for 30 minutes. In the state of the above, it was put in the microwave oven for cooking and irradiated with microwaves for 30 seconds precisely at “strong”. Thereafter, about 1000 ferrite beads were added to each protein solution, the cap was closed, and the mixture was allowed to stand at 4 ° C. for 12 hours or longer and stored.
[0041]
Three each of the carriers produced as described in methods 1 to 3 are placed in a 2 ml microtube, and the protein solution is completely removed by suction with an aspirator, and then 0.1 ml of 0.1 M phosphorus containing 1 ml of 0.1% BSA. Washed twice with acid buffer (pH 7.4). 100 μl of an alkaline phosphatase-labeled anti-α-fetoprotein antibody solution (manufactured by Tosoh Corporation) diluted to 3.7 μg / ml with 0.1 M phosphate buffer (pH 7.4) containing 0.1% BSA was added. Shake for 10 minutes at ° C.
[0042]
After the alkaline phosphatase-labeled anti-α-fetoprotein antibody solution was removed by suction, the carrier was washed three times with 1 ml of 0.1 M NaCl solution containing 0.044% Triton X-100. After this solution is removed by suction, the carrier is transferred to a 12 × 75 mm glass tube, and 100 μl of chemiluminescence substrate (CSPD, 0.2 mM solution, Tropix) for alkaline phosphatase activity measurement is dispensed and immediately detected for luminescence. The amount of luminescence was counted with a device (trade name LUMAT LB9501, manufactured by Bertoold). The photometric time was 20 seconds immediately after setting in the detector.
[0043]
FIG. 2 shows the measurement results of luminescence. In the carrier produced by the method 1 without irradiation with microwaves, the nonspecific adsorption amount of the protein (labeling substance, ie, alkaline phosphatase-labeled anti-α-fetoprotein antibody) to the carrier has a pH of pH 3.0 to 6.5. In particular, when a protein solution having a pH of 3.0 to 4.0 was used, the amount was significantly reduced as compared with the case of using a protein solution having a pH of 7.0 to 8.0. The carrier manufactured by irradiating microwaves by method 2 or method 3 is different from the carrier manufactured by irradiating microwaves by method 1 except for the one manufactured at pH 3.0 in method 2. Was able to produce a carrier with a low amount of non-specific adsorption of protein regardless of the pH of the protein solution.
[0044]
The carrier produced using a protein solution having a pH of 3.5 to 6.0 in Method 2 or the carrier produced using a protein solution having a pH of 3.0 to 4.0 in Method 3 is obtained by using Method 1 In comparison with a carrier produced using a protein solution having a pH of 7.0 to 8.0, ie, a carrier produced conventionally, the amount of nonspecific adsorption of the protein is clearly reduced. By using a protein solution having a pH of 4.0 in method 2, a carrier having the lowest nonspecific adsorption amount could be produced. In addition, it was also clarified that in the carrier produced by Method 2, the fluctuation width between measurements of the nonspecific adsorption amount of the protein is smaller than the fluctuation between measurements of the carrier produced by other methods.
[0045]
Example 3
(1) Preparation of anti-thyroid stimulating hormone antibody-immobilized ferrite beads
Using a glass container with a screw cap of 100 ml, 0.1 M Tris-HCl buffer solution (pH 8) containing 0.1% sodium azide and containing 500 μg of anti-thyroid stimulating hormone antibody Fab ′ fraction (manufactured by Tosoh Corporation). 50 ml of 0) was prepared. About 4000 ferrite beads were added to this antibody solution, stirred at 33 ° C. for 1 hour, and bound by physical adsorption. The solution before and after mixing was subjected to high-speed gel permeation chromatography to determine the amount of antibody bound to the carrier. As a result, the entire amount of antibody bound to the beads.
[0046]
The supernatant was removed by aspiration, and the carrier was taken out and divided into two 50 ml centrifuge tubes. One carrier was washed twice with 40 ml of 0.1 M Tris-HCl buffer (pH 8.0), and the other carrier was washed twice with 40 ml of 0.1 M phosphate buffer (pH 4.0). The same buffer solution (each having the same pH) containing 0.5% BSA and 0.1% sodium azide was dispensed and allowed to stand at 4 ° C. for 24 hours or more.
[0047]
Each of the two kinds of carriers treated as described above was divided into two 50 ml centrifuge tubes, each of which was about 1000, and a carrier was produced based on the method 1 or method 2 described above. That is, as method 1, the carriers in two centrifuge tubes, one by one, were gently mixed by inversion and allowed to stand at 4 ° C. for 12 hours or more to prepare a carrier. Then, as method 2, each of the carriers in the two centrifuge tubes, one by one, is pre-ice-cooled for 30 minutes, and then in the ice-cooled state in the cooking microwave oven, “strongly” for 30 seconds exactly. The mixture was irradiated with waves and then stirred with a roller (9 rpm) for 6 hours at room temperature, and stored in the same solution at 4 ° C.
[0048]
(2) One-step sandwich analysis of thyroid stimulating hormone
Immunoassay was performed using 12 of each of the two types of carriers produced in (1) above. Each carrier was put into an analysis container dedicated to a fully automatic enzyme immunoassay device (trade name AIA-21, manufactured by Tosoh Corporation), and after removing the protein solution by suction, 200 μl of 0.1 M Tris-HCl buffer (pH 8.0) Washed once with an alkaline phosphatase-labeled antithyroid stimulating hormone antibody (Tosoh Corp.) diluted to 3.7 μg / ml with 0.1 M phosphate buffer (pH 7.4) containing 0.1% BSA. ) Was dispensed in a volume of 100 μl. On the other hand, thyroid stimulating hormone (TSH) standard solution (0 and 50.1 μIU / ml) (manufactured by Tosoh Corp.) was put in a dedicated sample cup and prepared as described above, and the analysis was carried out. Automatic measurement was performed with the container set in the apparatus.
[0049]
After dispensing of the sample, an antigen-antibody reaction was performed at 37 ° C. for 40 minutes, and after B / F washing, 220 μl of 1 mM 4-methylumbelliferyl phosphate solution, which is a fluorescent substrate for alkaline phosphatase activity measurement, was added, A rate assay was performed for 300 seconds immediately after the addition of the substrate, and the result was converted to a concentration.
[0050]
3 and 4 are diagrams showing measurement results for a standard solution containing 0 μIU / ml and 50.1 μIU / ml TSH, respectively. The measurement result of the 0 μIU / ml standard solution is the nonspecific adsorption of the protein, that is, the target component (TSH) or the labeling substance (alkaline phosphatase-labeled antithyroid stimulating hormone antibody) to the carrier to which the antibody is bound and the analysis container containing the antibody It reflects the quantity. Regarding this measurement result, the carrier produced by irradiating microwaves (Method 2) has a nonspecific adsorption amount of pH 8 compared to the carrier produced without irradiating microwaves (Method 1). It was reduced to 75% when using a protein solution of 0.0, and 63% when using a protein solution with a pH of 4.0.
[0051]
The measurement result of the 50.1 μIU / ml standard solution mainly reflects the amount of the sandwich immune complex formed on the carrier, not the nonspecific adsorption amount of the protein. With regard to this result, the carrier produced by irradiation with microwaves (method 2 carrier) has a pH of 8.0 compared to the carrier produced without irradiation with microwaves (method 1 carrier). Even when the solution was used, the value was 78% higher. This is considered to be a result of the increase in the amount of effective antibody, that is, the amount of antibody that can bind to the antigen, due to the molecular orientation effect of the microwave, with the antigen binding site of the antibody molecule facing the solution. Even in the case of a carrier produced by microwave irradiation (Method 2), when a protein solution having a pH of 4.0 is used, the enzyme activity is about 50% of that produced using a protein having the same pH of 8.0. The reason is that the microwave orientation effect on the ligand (antibody) bound to the carrier is dependent on pH.
[0052]
FIG. 5 shows the result of obtaining the lower detection limit value by the 2SD method for the measured values shown in FIGS. 3 and 4. In the carrier produced by irradiating microwaves (Method 2), when a protein solution having a pH of 8.0 is used, compared to the carrier produced without irradiating microwaves (Method 1), about 50% That is, it can be seen that a sensitivity increase of about twice has been achieved.
[0053]
In addition, from the present Example, even if a carrier or container containing a conductive material such as ferrite is irradiated with microwaves, the characteristics of the conductive material (magnetism, etc.) and a ligand that is preliminarily bonded to the carrier, etc. It can also be seen that there is no adverse effect on the reactivity of.
[0054]
Example 4
Blocking of DEAE-5PW packing material for ion exchange chromatography and preparation of packed column
Commercially available ion exchange gel (trade name DEAE-5PW, manufactured by Tosoh Corporation) was taken in two 50 ml polypropylene centrifuge tubes, each 4 g in dry weight, with 0.5% BSA and 0.1% on one side. 20 ml of 100 mM Tris-HCl buffer solution (pH 8.0) containing% sodium azide, and 20 ml of 100 mM phosphate buffer solution (pH 4.0) containing 0.5% BSA and 0.1% sodium azide Then, gently mixed by inversion so as not to foam. After stirring for 18 hours at 9 rpm at room temperature with a roller-type stirrer, 10 ml each of the gel suspensions in the two centrifuge tubes are taken into separate polypropylene tubes with caps (14 ml size). It was immersed in a 300 ml polypropylene beaker containing for 10 minutes and cooled on ice.
[0055]
After inversion and mixing again, the cap was removed and the beaker was returned to the beaker. The beaker was immediately placed in the microwave oven for cooking using the turntable method, and microwaves were irradiated with “strong” for exactly 30 seconds. The tube was taken out, a cap was attached, and the mixture was stirred for 6 hours at 9 rpm at room temperature with a roller stirrer.
[0056]
Two types of carriers manufactured without microwave irradiation (method 1 carrier), two types of carriers manufactured by microwave irradiation (method 2 carrier), and 0.1% azide of the gel. A total of 5 types of control carriers suspended in 100 mM Tris-HCl buffer (pH 8.0) containing sodium chloride were packed in a 4.6 mm ID × 3.5 cm stainless steel column at a flow rate of 4 ml / min. The filling solution is a buffer used in the process of producing each carrier, and the control carrier is a 100 mM Tris-HCl buffer (pH 8.0) containing 0.1% sodium azide.
[0057]
After the packing, in order to dissociate the BSA bonded to the ion exchange group of each carrier, 20 mM Tris-HCl buffer (pH 8.5) containing 1 M NaCl is supplied to each column for 1 hour at a flow rate of 0.5 ml / min. Washed.
[0058]
In order to observe the non-specific adsorption of the protein to the carrier prepared as described above, BSA having a high adsorptivity with the carrier substrate was used as a sample under the following conditions, and the recovery rate was determined. It was measured. The sample BSA is the protein itself adsorbed on the carrier. However, since the carrier was sufficiently washed with a high salt concentration solution after the production as described above, there is no problem and the adsorptivity to the carrier is correctly reflected. It is thought that.
[0059]
(Analysis conditions)
Sample: 250 μg / ml BSA solution,
Injection volume: 20 μl,
Eluent;
A: 20 mM Tris-HCl buffer (pH 8.5),
B: 20 mM Tris-HCl buffer (pH 8.5) containing 0.5 M sodium chloride,
Elution conditions;
A to B linear gradient (5 minutes),
Flow rate: 1.0 ml / min,
Detection: Absorbance measurement in UV (280 nm).
[0060]
The state in which the column is removed from the flow path, that is, without using any column, the peak area obtained by simply injecting only the sample into the flow path system is 100, and the column is incorporated in the flow path, that is, each column The percentage of the peak area obtained by passing the sample through was obtained as the recovery rate and is shown in FIG. In other words, the recovery rate decreases by the amount of protein (BSA) adsorbed on the carrier in the column, and the higher the recovery rate, the lower the nonspecific adsorption of protein (BSA).
[0061]
In a column packed with a carrier produced by irradiating microwaves by method 2, the recovery rate is 86% when a protein solution having a pH of 8.0 is used, and the protein solution having a pH of 4.0 is used. The recovery rate was 88%, significantly exceeding the control carrier recovery rate of 76%. Among the results regarding the carrier produced by microwave irradiation according to Method 2, the result of the carrier using a protein having a pH of 4.0 was obtained by using the protein solution at pH 4.0 without irradiating the microwave in Method 1. The reason why the recovery rate was almost the same as that of the carrier produced in this way is considered that protein (BSA) is adsorbed to the end fitting other than the carrier. That is, when a protein solution having a pH of 4.0 is used, the carrier produced by either method 1 or method 2 has substantially achieved a recovery rate of 100%, and 12% It is considered that the reduction in the recovery rate is caused mainly by protein adsorption to the liquid contact part in the column centering on end fitting.
[0062]
【The invention's effect】
The surface of the carrier or container provided by the present invention is adsorbed with a protein that has been modified by microwave irradiation and / or acid treatment and has improved hydrophilicity. Compared with a carrier or a container produced by a method for adsorbing (so-called ordinary blocking method), nonspecific adsorption of protein can be further suppressed or reduced. As a result, when the carrier or container of the present invention is used, measurement sensitivity and recovery rate in separation analysis and separation purification can be improved.
[0063]
The above effect can be achieved only by acid-denaturing serum albumin when a solution containing at least serum albumin is used as a protein to be adsorbed on a carrier or a container, but in addition to acid denaturation, microwave irradiation is performed. According to the embodiment, additional effects described later can also be achieved. In other words, irradiation with microwaves after binding ligands such as antibodies can direct the antigen binding site of ligand molecules to the solution side due to the molecular orientation effect of the microwaves, increasing the amount of effective ligands. In addition, it is possible to further improve the measurement sensitivity in separation and purification and further improve the recovery rate in separation and purification.
[0064]
For example, the effect of immunoassay by binding an antibody or antigen, which is a ligand for the target component, to the carrier or container of the present invention is specifically as follows. First, since the non-specific adsorption of the target component, such as the antigen or antibody, and the labeling substance (labeled antibody, labeled antigen, etc.) bound to the label to the protein carrier can be reduced, the background for detecting the label The signal can be reduced. This contributes to improvement in measurement sensitivity. In addition, after binding antibodies and antigens to a carrier or container and then irradiating with microwaves in a protein solution, the molecular orientation effect of the microwaves is not only the protein adsorbed on the surface but also the ligand. It extends to antibodies and antigens, and as a result, the antigen-binding sites of antibodies and antigens become more oriented on the solvent side, and the amount of effective solid phase antibody increases. This contributes to the improvement of the detection limit as well as the improvement of measurement sensitivity.
[Brief description of the drawings]
FIG. 1 is a graph showing the inhibitory effect of non-specific protein adsorption of the carrier produced in Example 1. FIG. The results for BSA, HSA, gelatin, casein, bovine γ-globulin, goat immunoglobulin G and ovalbumin are shown in order from the left in the figure.
FIG. 2 is a graph showing the effect of suppressing nonspecific adsorption of protein by the carrier produced in Example 2. FIG.
FIG. 3 shows the results of immunoassay performed using the carrier produced in Example 3.
4 is a diagram showing the results when immunoassay was performed using the carrier produced in Example 3. FIG.
FIG. 5 is a diagram showing the results when immunoassay was performed using the carrier produced in Example 3.
FIG. 6 is a diagram showing the results when ion exchange chromatography was performed using the carrier produced in Example 4.

Claims (5)

  A carrier comprising: irradiating a carrier or container with microwaves while being immersed in a protein solution in producing a carrier or container having reduced nonspecific adsorption by adsorbing a protein to the carrier or container. Or the manufacturing method of a container.   The method according to claim 1, wherein the protein solution is a solution containing serum albumin.   The method according to claim 1 or 2, wherein the protein solution is a solution containing serum albumin and having a pH adjusted to pH 3.0 to 6.5. In producing a carrier or container having reduced nonspecific adsorption by adsorbing protein to the carrier or container, the carrier or container is added to a protein solution containing serum albumin and having a pH adjusted to pH 3.0 to 4.0. A method for producing a carrier or a container, comprising immersing the container. The production method according to any one of claims 1 to 4 , wherein a ligand is bound to the carrier or container prior to the adsorption of the protein.

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