JP2009216613A - Creation method of solid phase for biochemical analysis, and apparatus for biochemical analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a creation method of a solid phase for biochemical analysis and an apparatus for biochemical analysis can solidify accurately a biochemical material such as an antigen or an antibody, suppress nonspecific adsorption of a material to be reacted specifically to a solid phase surface, and obtain a high signal/noise ratio even when a blocking process is eliminated. <P>SOLUTION: A method for forming the solid phase for biochemical analysis by using the apparatus for analysis constituted of a resin material includes processes for: preparing a mixed solution obtained by mixing a biochemical material comprising an antigen or an antibody to a water-soluble resin having a photoreactive functional group on a side chain; forming a coating layer of the mixed solution on the surface of the apparatus by allowing the mixed solution to adhere onto the inner surface of the apparatus; and forming a water-insoluble hardened film containing the biochemical material by hardening the coating layer by light irradiation. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a solid phase for biochemical analysis and an instrument for biochemical analysis.

  In biochemical measurement by a conventional solid phase method typified by immunoassay, an immunological substance such as an antibody or antigen is immobilized on a plastic substrate such as polystyrene by using nonspecific adsorption, and then solidified. A method of quantifying a target molecule in a sample solution by using an immune reaction, and finally adding a molecule labeled with a radioisotope, enzyme, fluorescent substance, etc. to the target molecule. Is generally done.

 Immunoassay is used not only in the field of research and development but also in medical fields such as clinical tests, and if the measurement result is false, it may cause a serious situation involving human life. Measurement sensitivity and reproducibility are particularly important.

 However, in the method using non-specific adsorption, the amount of immunizing substance to be immobilized is small and sufficient measurement sensitivity cannot be obtained, or the amount of immobilized phase is not stable, resulting in increased signal variability. And a problem that noise occurs due to adsorption of a substance to be reacted with the solid surface in the steps after the solid phase adsorption.

  As a measure for reducing noise and preventing nonspecific adsorption of a substance that reacts specifically with the solid surface, a general immunoassay method includes a step called blocking. Blocking is a step of masking a non-solid phase portion by bringing a protein, such as skim milk or albumin, that is easily adsorbed and does not affect the system into contact with the solid phase surface. Techniques relating to solid phase immunoassay have been disclosed (see, for example, Patent Documents 1 and 2). Moreover, the technique which suppresses nonspecific adsorption | suction by adding surfactant is also disclosed (for example, refer patent document 3).

However, since the effect of blocking is strongly affected by the type of blocking agent used, the type of solvent, the pH of the solution, the reaction temperature, and the reaction time, enormous examination of conditions is necessary to obtain the expected effect. Even if the conditions are established once, it is difficult to obtain reproducibility because slight changes in those factors greatly affect the results.
Japanese translation of PCT publication No. 2002-508843 JP 2007-225603 A Japanese Patent Laid-Open No. 08-240590

  The present invention has been made in view of the above circumstances, and is capable of immobilizing a biochemical substance such as an antigen or an antibody with high accuracy and capable of reacting specifically with a solid surface. To provide a method for preparing a solid phase for biochemical analysis that can suppress non-specific adsorption and obtain a high signal / noise ratio even if the blocking step is omitted, and a biochemical analysis instrument obtained by this method. .

（７）前記抗原又は抗体からなる生化学物質が、固相免疫測定法における固相化物質である（１）ないし（６）のいずれかに記載の生化学分析用固相の作成方法。
（８）（１）ないし（７）のいずれかに記載の生化学分析用固相の作成方法によって、容器内面に生化学分析用固相を有していることを特徴とする生化学分析用器具。 Such a problem can be solved by the present invention described in (1) to (8) below.
(1) A method for producing a solid phase for biochemical analysis using an analytical instrument composed of a resin material,
A mixed solution adjusting step of preparing a mixed solution in which a biochemical substance composed of an antigen or an antibody is mixed with a water-soluble resin having a photoreactive functional group in a side chain;
A coating layer forming step of attaching the mixed solution to the inner surface of the device and forming a coating layer of the mixed solution on the surface of the device;
A cured film forming step of curing the coating layer by light irradiation to form a water-insoluble cured film containing a biochemical substance;
A method for producing a solid phase for biochemical analysis, comprising:
(2) The water-soluble resin having a photoreactive functional group in the side chain includes at least one selected from saponified polyvinyl acetate, polyvinyl pyrrolidone, and polyethylene glycol. How to create a solid phase for chemical analysis.
(3) The method for preparing a solid phase for biochemical analysis according to (2), wherein the saponified product of polyvinyl acetate is obtained by saponifying 20 to 100 mol% of the whole polyvinyl acetate.
(4) The method for producing a solid phase for biochemical analysis according to any one of (1) to (3), wherein the photoreactive functional group contains a nitrogen atom.
(5) The method for producing a solid phase for biochemical analysis according to any one of (1) to (4), wherein the photoreactive functional group has an azide group.
(6) Preparation of the solid phase for biochemical analysis according to any one of (1) to (5), wherein the water-soluble resin having a photoreactive functional group is represented by the following formula (I): Method.

(7) The method for producing a solid phase for biochemical analysis according to any one of (1) to (6), wherein the biochemical substance comprising the antigen or antibody is a solid phased substance in a solid phase immunoassay.
(8) For biochemical analysis, characterized by having a solid phase for biochemical analysis on the inner surface of the container by the method for producing a solid phase for biochemical analysis according to any one of (1) to (7) Instruments.

  According to the present invention, a biochemical substance such as an antigen or an antibody can be solid-phased accurately, and non-specific adsorption of a substance that reacts specifically with the solid-phase surface is suppressed, It is possible to provide a method for preparing a solid phase for biochemical analysis in which a high signal / noise ratio can be obtained even if the blocking step is omitted, and a biochemical analysis container obtained by this method.

  Hereinafter, the method for preparing a solid phase for biochemical analysis of the present invention (hereinafter, simply referred to as “method for preparing a solid phase”) will be described in detail.

  First, a mixed solution preparation process for preparing a mixed solution in which a biochemical substance is mixed with a water-soluble resin having a photoreactive functional group in a side chain, which is important in the solid phase preparation method of the present invention, will be described.

  In biochemical analysis, it is important that the solid-phased biochemical substance maintains the biochemical activity. However, in the solid phase preparation method of the present invention, a water-soluble resin is used for preparing the mixed solution. Thus, it is one of the features that a mixed solution is obtained while obtaining good compatibility with the biochemical substance and at the same time maintaining the activity of the biochemical substance.

  In addition, by immobilizing the above-mentioned mixed solution, that is, the biochemical substance and the water-soluble resin in a mixed state, the biochemical substance can be immobilized in comparison with the conventional method using non-specific adsorption. The amount can be stabilized.

  Furthermore, the present invention is also capable of rapid curing by suppressing the structural change of the biochemical substance, that is, the decrease in biochemical activity, by using light irradiation in the step of forming the mixed solution into a water-insoluble cured film. Is one of the features.

  In addition, since the water-soluble resin becomes a hydrophilic film that reduces non-specific adsorption of proteins after being cured in water-insoluble, a substance that reacts with the solid-phase surface in the steps after the solid-phase is based. It also has a feature that it is difficult to adsorb to the material and a blocking step is unnecessary.

  The method for mixing the biochemical substance with the water-soluble resin is not particularly limited. After preparing the water-soluble resin solution, an arbitrary amount of biochemical substance is added, or a pre-adjusted biochemical substance solution is prepared. A water-soluble resin may be added thereto.

  The amount of the biochemical substance in the above mixed solution is not particularly limited, and it is sufficient to select a concentration at which a necessary signal can be obtained according to the antibody titer and purpose of the biochemical substance to be used. In the immunoassay method, it is preferable to select in the range of 0.01 μg / mL or more and 10 μg / mL or less because a higher signal can be obtained.

  Examples of the photoreactive functional group include a functional group containing a nitrogen atom, a functional group containing a sulfur atom, a functional group containing a bromine atom, a functional group containing a chlorine atom, or a functional group containing neither of these atoms. It is done. Among these, a functional group containing a nitrogen atom is preferable.

  Specific examples include a functional group containing an azide group, a functional group containing a diazo group, and a functional group containing a diazide group. Among these, a functional group containing an azide group is preferable. Thereby, it can be made to react with a practical wavelength of 230-500 nm, and the formability of a film | membrane can be improved with the further outstanding resolution.

More specifically, it is preferable to use one represented by the following formula (II), which is easy to give to a water-soluble resin and at the same time has excellent reactivity at a wavelength of 230 to 500 nm as described above. Therefore, a good solid surface for biochemical analysis can be obtained.

  Examples of water-soluble resins include saponified polyvinyl acetate, polyvinyl pyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate, And a copolymer of monomers constituting them, and a copolymer of 2-methacryloyloxyethyl phosphorylcholine and another monomer (for example, butyl methacrylate). Among these, a structure comprising at least one selected from saponified products of polyvinyl acetate, polyvinyl pyrrolidone, and polyethylene glycol and the reactive group is preferable. As a result, it has excellent compatibility with biochemical substances, and also has an excellent effect of suppressing nonspecific adsorption of substances that react specifically with the solid phase surface by biochemical reaction in the steps after solid phase immobilization. Solid phase surfaces for biochemical analysis can be constructed.

Here, the quart of polyvinyl acetate refers to, for example, polyvinyl alcohol or a copolymer of vinyl alcohol and another compound. Furthermore, for example, vinyl alcohol and modified vinyl acetate saponified products such as hydrophilic group modification, hydrophobic group modification, anion modification, cation modification, amide group modification or reactive group modification such as acetoacetyl group are included.
In addition, water-soluble resin means what can melt | dissolve 1.0g or more with respect to 100g of 25 degreeC water here.

  The average degree of polymerization of the water-soluble resin is not particularly limited, but is preferably 100 or more and 10,000 or less, particularly preferably 200 or more and 5,000 or less. When the average degree of polymerization is 100 or more, a solid-phased film can be formed uniformly, and when the average degree of polymerization is 10,000 or less, the viscosity of the mixed solution suitable for workability can be obtained. .

  When the saponified product of polyvinyl acetate is used, the saponification degree of the saponified product of polyvinyl acetate is preferably 20 mol% or more and 100 mol% or less, particularly 50 mol% or more and 95 mol% or less. preferable. When the degree of saponification of the polyvinyl acetate is within the above range, the compatibility with the biochemical substance and the effect of suppressing nonspecific adsorption of the substance that reacts specifically with the solid surface are particularly excellent.

  It is preferable to use the water-soluble resin prepared using a solvent such that the viscosity at 20 ° C. is 1 mPa · s or more and 10 mPa · s or less, preferably 2 mPa · s or more and 7 mPa · s or less. . The solvent used at that time is preferably water, but in order to increase the solubility, an organic solvent that does not destroy the characteristics of the biochemical substance can be used by mixing with water.

  Next, the process of hardening the said coating layer is demonstrated in detail.

  The water-soluble resin coating layer formed on the surface of the biochemical analysis instrument is cured to obtain the water resistance of the solid phase surface required for the solid phase biochemical measurement method.

  The curing method is not particularly limited, and can be achieved by reacting a functional group having a side chain of the water-soluble resin. It can be cured, and when it has a vinyl group, it can be cured by radiation.

A light source in the case of curing by light irradiation is not particularly limited, illumination can also be used 5.0 mW / cm ² of about ultra-high pressure mercury lamp or 0.1 mW / cm ² about UV lamps. Curing by light irradiation can be controlled by illuminance and irradiation time, so when using a light source with low illuminance, the irradiation time can be lengthened, and when a highly reactive photosensitive group is selected, it is cured under a fluorescent lamp. It is also possible.
For example, when using an extra-high pressure mercury lamp with 5.0 mW / cm ² irradiation 1 to 10 seconds, when using a UV lamp 0.1 mW / cm ² be sufficiently cured by irradiation of 3 to 10 minutes I can do it.

  Next, a biochemical analysis instrument (hereinafter, simply referred to as “analysis instrument”) obtained by the solid phase preparation method of the present invention will be described in detail.

  The analytical instrument of the present invention is made of a resin material, whereby the analytical instrument can be made into a disposable type, and various shapes can be easily formed.

  Examples of the resin material include polypropylene resins, polyethylene resins, polyolefin resins such as ethylene-propylene copolymers, polystyrene resins such as polystyrene and acrylonitrile-butadiene-styrene resins, polycarbonate resins, polyethylene terephthalate resins, and polymethyl methacrylate. Methacrylic resin such as resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, polyetherimide resin, fluorine resin such as polytetrafluoroethylene, Examples thereof include acrylic resins such as polymethylpentene resin and polyacrylonitrile, and fiber-based resins such as propionate resin. Among these, polystyrene resins are preferable in terms of moldability and transparency required for biochemical analysis instruments, and they may be colored by compounding pigments according to purposes such as fluorescence measurement and luminescence measurement.

  The weight average molecular weight of the resin material is not particularly limited, but is preferably 10,000 to 500,000, and particularly preferably 20,000 to 100,000. When the weight average molecular weight is within the above range, the moldability of the device is excellent.

The weight average molecular weight is, for example, G.M. P. C. It can obtain | require in conversion of styrene using.
Additives such as hydrocarbon-based, fatty acid amide-based lubricants, phenol-based, and amine-based antioxidants are added to the resin material as long as the object of the present invention is not impaired for the purpose of improving moldability and weather resistance. can do.

For example, injection molding, blow molding, or injection blow molding can be used as the method for producing the analytical instrument of the present invention from the above resin material.
Examples of the shape of the analytical instrument include containers such as a multi-well plate and a cuvette, and even a sheet-like molded product can be used by being installed in an environment such as a container bottom. Among these, a 6 to 1536 hole multiwell plate used for immunoassay is preferable. Thereby, the precision of evaluation and research using immunoassay can be improved.

Examples of the biochemical substance include natural or synthetic peptides, proteins, enzymes, saccharides, lectins, viruses, bacteria, nucleic acids, DNA, RNA, immune substances,
Among these, antigens (eg, including recombinant antigens) antibodies, IgG. IgM. Immunoglobulins such as IgE: complement, CRP. Plasma proteins such as ferritin, α1 microglobulin, β2 microglobulin and their antibodies: tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA19-9, CA-125 and Their antibodies: hormones such as luteinizing hormone (LH), follicle stimulating hormone (FSH), human ciliary gonadotropin (hCG), estrogen, insulin and their antibodies: HBV-related antigens (HBs.HBe.HBc). HIV. Substances related to virus infection such as ATL and their antibodies: bacteria such as diphtheria, botulinum, mycoplasma, syphilis treponema and their antibodies: protozoa such as toxoplasma, trichomonas, leishmania, tribanosome, malaria parasite and their antibodies: Drugs such as antiepileptic drugs such as phenytoin and phenobarbital, cardiovascular drugs such as quinidine and digoxinine, antiasthma drugs such as theophylline, antibiotics such as chloramphenicol and gentamicin and their antibodies: other enzymes, bacterial cells By using exotoxins (such as styrelidin O), it can be suitably used for clinical diagnosis and biochemical experiments using solid-phase immunoassay.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

(Example)
A commercially available polystyrene 96-well ELISA plate (MS-8896F, manufactured by Sumitomo Bakelite Co., Ltd.) was used as an analytical instrument composed of a resin material.
Next, as a water-soluble resin having a photoreactive functional group in the side chain, polyvinyl alcohol having an azide group in the side chain (AWP manufactured by Toyo Gosei Kogyo Co., Ltd., average polymerization degree 1800 of the water-soluble resin, photoreactive functional group) The base modification rate of 0.6 mol% was dissolved in pure water in a glass apparatus shielded from light with an aluminum foil to prepare a 0.7 wt% solution.
Anti-rat albumin antibody (manufactured by Cosmo Bio) was dissolved in the above solution so as to be 1.0 μg / mL to obtain a mixed solution.
Dispense the above-mentioned mixed solution onto the above-mentioned ELISA plate at 200 μL / well, turn the ELISA plate upside down, discard the solution thoroughly, and dry overnight at room temperature to form a coating layer of the mixed solution. Formed.
Furthermore, a water-insoluble cured film was formed by irradiating UV light of 250 nm with a UV lamp for 0.1 mW / cm ² × 30 seconds to obtain a container having a solid phase for biochemical analysis of the present invention.

(Comparative Example 1)
The same procedure as in Example 1 was performed except that polyvinyl alcohol having no photoreactive functional group in the side chain was used as the water-soluble resin (Polyvinyl Alcohol saponification degree 78-82 mol%, average polymerization degree 1800, manufactured by Wako Pure Chemical Industries, Ltd.). A container was obtained.

(Comparative Example 2)
A container was obtained in the same manner as in Example 1 except that the step of forming a cured film using a UV lamp was omitted from the process of Example 1.

(Comparative Example 3)
A container was obtained in the same manner as in Example 1 except that the water-soluble resin was not dissolved in the mixed solution and only the anti-rat albumin antibody was dissolved.

  The following ELISA evaluation was performed on the containers of Examples and Comparative Examples 1 to 3. The obtained results are shown in Table 1.

1. ELISA evaluation without blocking step Washed 3 times with 300 μL / well of washing solution (pH 7.4 phosphate buffer containing 0.05% Tween 20) using a plate washer (Cerawasher Model MW-96R manufactured by Biotech). Later, rat albumin (manufactured by Cosmo Bio) diluted with phosphate buffer solution at pH 7.4 into 4 levels of concentration series of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL The solution was dispensed into each of the Examples and Comparative Examples 1 to 3 at 100 μL / well in 24 wells at each of the above four levels, and allowed to stand at room temperature for 1 hour.

Thereafter, the plate washer was washed 3 times with 300 μL / well of the above washing solution, and then a peroxidase-labeled anti-rat albumin antibody (manufactured by Cosmo Bio) diluted to 2 μg / mL with a phosphate buffer of pH 7.4 was added at 200 μL / well. The well was dispensed and allowed to stand at room temperature for 1 hour.
Further, after washing 3 times with 300 μL / well of the washing solution in the same manner as described above, the color was developed using a color development kit (Peroxidase color development kit T manufactured by Sumitomo Bakelite Co., Ltd.), and a wavelength of 450 nm using a plate reader (Spectra Fluor manufactured by TECAN). Absorbance was measured at.

2. ELISA evaluation with blocking step Washed 3 times with 300 μL / well of washing solution (pH 7.4 phosphate buffer containing 0.05% Tween 20) using a plate washer (Cerawasher Model MW-96R manufactured by Biotech). Thereafter, a blocking agent (skim milk prepared to 5% with a phosphate buffer solution of pH 7.4) was dispensed at 200 μL / well and allowed to stand at room temperature for 2 hours.

  After washing 3 times with 300 μL / well of washing solution as above, dilute with phosphate buffer solution at pH 7.4 to a concentration series of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL. The rat albumin solution (manufactured by Cosmo Bio) was dispensed at 100 μL / well for each of the 24 wells at a concentration of 24 for each of Example and Comparative Example 1-3, and allowed to stand at room temperature for 1 hour.

  Thereafter, after washing three times with 300 μL / well of the washing solution in the same manner as described above, peroxidase-labeled anti-rat albumin antibody (manufactured by Cosmo Bio) diluted to 2 μg / mL with a phosphate buffer of pH 7.4 was separated at 200 μL / well Poured and allowed to stand at room temperature for 1 hour.

Further, after washing 3 times with 300 μL / well of the washing solution in the same manner as described above, the color was developed using a color development kit (Peroxidase color development kit T manufactured by Sumitomo Bakelite Co., Ltd.), and a wavelength of 450 nm using a plate reader (Spectra Fluor manufactured by TECAN). Absorbance was measured at.
The results are shown in Table 1.

  As is clear from the results in Table 1, in Examples using the method for preparing a solid phase for biochemical analysis of the present invention, an absorbance correlated with the concentration of rat albumin was obtained regardless of the presence or absence of the blocking step. In addition, the value (CV value) of each well of 24 wells was stable.

  On the other hand, in the comparative example which does not use the method for preparing the solid phase for biochemical analysis of the present invention, in Comparative Example 1 and Comparative Example 2, the correlation with the concentration of rat albumin is low regardless of the presence or absence of the blocking step. In Comparative Example 3 in which a correlation was obtained, correlation was obtained by ELISA evaluation, but the sensitivity was lower than that of the Example, and the variation in the values (CV values) of 24 wells at each concentration was large.

  From these results, in Comparative Example 1 and Comparative Example 2, it was estimated that the water-soluble resin had disappeared in the washing step together with the rat albumin antibody because no cured film was formed, and obtained by ELISA evaluation without the blocking step. The signal produced was presumed to be due to nonspecific adsorption of peroxidase-labeled anti-rat albumin antibody.

  In addition, although the ELISA evaluation including the blocking step of Comparative Example 3 corresponds to a general ELISA measurement, the container using the method for preparing a solid phase for biochemical analysis of the present invention is sensitive and accurate. Both proved to be excellent and the blocking step could be omitted.

Claims (8)

A method of creating a solid phase for biochemical analysis using an analytical instrument composed of a resin material,
A mixed solution adjusting step of preparing a mixed solution in which a biochemical substance composed of an antigen or an antibody is mixed with a water-soluble resin having a photoreactive functional group in a side chain;
A coating layer forming step of attaching the mixed solution to the inner surface of the device and forming a coating layer of the mixed solution on the surface of the device;
A cured film forming step of curing the coating layer by light irradiation to form a water-insoluble cured film containing a biochemical substance;
A method for producing a solid phase for biochemical analysis, comprising:   2. The biochemical analysis according to claim 1, wherein the water-soluble resin having a photoreactive functional group in the side chain contains at least one selected from saponified polyvinyl acetate, polyvinyl pyrrolidone, and polyethylene glycol. How to make a solid phase.   The method for producing a solid phase for biochemical analysis according to claim 2, wherein the saponified product of polyvinyl acetate is saponified by 20 to 100 mol% of the whole polyvinyl acetate.   The method for producing a solid phase for biochemical analysis according to any one of claims 1 to 3, wherein the photoreactive functional group contains a nitrogen atom.   The method for producing a solid phase for biochemical analysis according to any one of claims 1 to 4, wherein the photoreactive functional group has an azide group. The method for producing a solid phase for biochemical analysis according to any one of claims 1 to 5, wherein the water-soluble resin having a photoreactive functional group is represented by the following formula (I).

  The method for producing a solid phase for biochemical analysis according to any one of claims 1 to 6, wherein the biochemical substance comprising the antigen or antibody is a solid phase substance in a solid phase immunoassay.   A biochemical analysis instrument comprising a biochemical analysis solid phase on an inner surface of a container according to the method for producing a solid phase for biochemical analysis according to any one of claims 1 to 7.