JP2018136179A - Measurement reagent with absorbed protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement reagent that reduces an amount of nonspecific adsorption to a carrier of a marker in a heterogeneous assay method, avoids a false high value in low concentration measurement, and is good in repeatability.SOLUTION: An insoluble carrier supporting one or more reactive specific components to catch a measurement object by specific reaction and an association component of bovine serum albumin (molecular weight fraction within a molecular weight range 300,000-1,000,000) is used as a measurement reagent of a heterogeneous assay method, which reduces an amount of nonspecific adsorption to a carrier of a marker to avoid a false high value and thus improves repeatability.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a measurement reagent that is suitable for heterogeneous assay (heterogeneity measurement method), reduces the amount of nonspecific adsorption of a labeled body to a carrier, and avoids the generation of false high values in low concentration measurement.

  As an immunoassay method, a heterogeneous method using an insoluble carrier carrying a substance that specifically binds to an analyte such as an antibody or an antigen is one of the methods for measuring an analyte in a biological sample with high sensitivity. Assay methods are known. This method requires B / F separation (separation of the label bound to the carrier and the unbound label), the carrier after the immune reaction is washed with a washing solution, and the label contained in the immune complex on the carrier The measurement object is quantified based on the signal generated from the body label. Since the measurement target cannot be measured with high sensitivity if the label is non-specifically adsorbed on the carrier, usually a substance that specifically binds to the target is supported on the carrier, and then the protein on the surface of the carrier is not affected by the immune reaction. A method of coating a non-specific adsorption site (hereinafter, this operation is sometimes referred to as blocking) is employed.

  Patent Document 1 discloses a carrier particle for immunological agglutination reaction (average) using bovine serum albumin (hereinafter also referred to as BSA) that has been heat-denatured to have a high molecular weight (fractionation by molecular weight is not performed). The method of blocking the particle size (300 nm) improves the coverage of the carrier and suppresses the nonspecific aggregation reaction between the carrier particles compared to the conventional method of supporting with non-heat-denatured (mostly monomeric) BSA. The method is disclosed.

  However, in the case of a heterogeneous assay method that requires high sensitivity of measurement, the slight non-specific adsorption of the label to the carrier often affects the measurement performance, and the blocking method of Patent Document 1 is used. However, the non-specific adsorption reaction to the carrier of the labeled substance cannot be completely suppressed, and the measured value of the low concentration sample suddenly becomes a false high value (there is a risk that the negative sample is erroneously determined to be positive). The fatal problem was not solved, and the suppression of non-specific adsorption reaction was insufficient.

JP-A-10-197530

  Provided is a highly reproducible measurement reagent that is suitable for heterogeneous assay methods, reduces the amount of non-specific adsorption of the labeled substance to the carrier, and avoids the occurrence of false high values in low concentration measurement.

  As a result of intensive studies, the present inventors have determined that when bovine serum albumin used for carrier blocking is denatured to obtain an association component, components that are present without being associated (including monomers) and components that have a low degree of association are obtained. By using the optimal molecular weight fraction that has been removed, the reproducibility of low-level measurements in heterogeneous assays has been improved. Especially, it is a fatal problem for measurement reagents in which the measurement values in the low-concentration range suddenly become falsely high. Has been found to be able to be avoided, and the present invention has been completed.

The present invention made to solve the above-mentioned problems and achieve the object includes the following inventions:
That is, the first aspect of the present invention is:
It is an insoluble carrier carrying one or more reaction-specific components for capturing a measurement object by a specific reaction and an association component of bovine serum albumin (molecular weight fraction falling in the molecular weight range of 300,000 to 1,000,000). .

Next, the second aspect of the present invention is as follows.
A measurement reagent which is a suspension in which the above-described insoluble carrier is suspended in a diluent, and a measurement reagent obtained by lyophilizing the suspension.

Next, the third aspect of the present invention is as follows.
A sample is added to the above-described measurement reagent, the measurement target in the sample is captured by a reaction-specific component supported on an insoluble carrier, unreacted substances are removed, a label is added, and unreacted substances are further removed. Then, it is a measuring method for quantifying the measurement object captured by the reaction-specific component.

Next, the fourth aspect of the present invention is as follows.
A sample is added to the above-described measurement reagent, the measurement object in the sample is captured by a reaction-specific component supported on an insoluble carrier, a label is added, unreacted substances are removed, and then the reaction-specific component is added. This is a measurement method for quantifying a captured measurement object.

Next, the fifth aspect of the present invention provides
Heat treating bovine serum albumin at 50-60 ° C. for 10 minutes-24 hours;
Fractionating the molecular weight fraction of the heat-treated bovine serum albumin so as to fall within a molecular weight range of 300,000 to 1,000,000;
Adjusting the molecular weight fraction of bovine serum albumin to a concentration of 0.1 to 5.0% (w / v), and providing an insoluble carrier carrying one or more reaction-specific components for capturing the measurement object by a specific reaction. Dipping step;
Diluting the insoluble carrier after immersion with a diluent;
A method for producing a measurement reagent containing

  According to the present invention, insolubility carrying one or more reaction-specific components for capturing a measurement object by a specific reaction and an association component of bovine serum albumin (molecular weight fraction falling in the molecular weight range of 300,000 to 1,000,000). By using the carrier as a measurement reagent for the heterogeneous assay method, it is possible to reduce the amount of non-specific adsorption of the label to the carrier, avoid a false high value, and improve the reproducibility of the measurement.

It is a figure which shows the chromatogram at the time of fractionating the association component of heat-denatured BSA used for the measuring reagent of this invention. It is a figure which shows the result of having measured the zero concentration thyroid stimulating hormone (TSH) standard solution 60 times using the measuring reagent (Control) of Example 1 and Comparative Example 1. FIG.

  The present invention is described in further detail below.

  Examples of the reaction-specific component in the present invention include antibodies, antigens, receptors, enzymes, avidin, biotin, streptavidin, nucleic acids and the like.

  The association component of bovine serum albumin in the present invention refers to the bovine serum albumin by physical treatment such as heating, pressurization, ultraviolet irradiation, ultrasonic irradiation, or acid, alkali, surfactant, organic solvent, metal ion. Components that have been modified by chemical treatment to add bovine serum albumin and further, unmodified bovine serum albumin (including monomer) is cross-linked with a cross-linking agent without physical or chemical treatment. Refers to a component having a high molecular weight. In general, denaturation by heating is preferable in that the aggregate can be easily obtained. Usually, bovine serum albumin whose concentration is adjusted to 0.1 to 10% (w / v) is 50 to 60 ° C., preferably 50 When heated at ˜56 ° C. for 10 minutes to 24 hours, preferably 1 hour to 12 hours, a mixture of associative components of the intended molecular weight is obtained. Although there is a difference depending on the grade of bovine serum albumin, a mixture of aggregate components usually contains a monomer (a component that does not associate even when heated), or a dimer-trimer low-association component, and has a molecular weight In order to obtain an associative component having a molecular weight range of 300,000 to 1,000,000, preferably a molecular weight range of 400,000 to 600,000, more preferably a molecular weight range of 400,000 to 520,000 due to the wide range. It is desirable to perform fractionation by gel permeation chromatography or the like. The molecular weight fraction of the associating component supported on the carrier may be narrow or wide as long as it is within the above molecular weight range. In particular, components having low association degree (dimer to trimer) including monomers are removed. Thus, it can be suitably used if fractionated.

  Examples of the shape of the insoluble carrier in the present invention include a particulate shape, a tube shape, a cup shape, and a plate shape, but a particulate shape, particularly a spherical particle carrier is preferable. The material of the insoluble carrier is not particularly limited as long as it is a carrier used for heterogeneous measurement, such as an inorganic substance such as glass or silica, a plastic made of various synthetic polymer compounds, or a cellulose derivative. As an example, polyethylene, polypropylene, ethylene-vinyl acetate copolymer, nylon, acrylic resin, methacrylic resin, polystyrene, styrene-acrylic copolymer, styrene-butadiene copolymer, polyvinyl chloride, or these are the main components. A carrier made of a thermoplastic resin such as a copolymer or a mixture, and further, an ion exchange group such as a carboxyl group, a sulfopropyl group and a diethylaminoethyl group, and a reactive functional group such as a tosyl group, a tresyl group and an epoxy group were introduced into these carriers. Things can be raised. There is no restriction | limiting in particular also about the average particle diameter of a support | carrier, For example, the support | carrier of a particle diameter of 0.01-10,000 micrometers can be used.

  The insoluble carrier in the present invention may be a carrier that does not contain a magnetically sensitive component, but a carrier (magnetic particle) that contains a magnetically sensitive component is preferred in terms of facilitating subsequent removal of unreacted components. Examples of the magnetically sensitive component include soft ferrites such as manganese-zinc ferrite and magnetite containing iron trioxide as a main component. The magnetically sensitive component may be uniformly contained inside by mixing at the time of producing the carrier made of the above-mentioned thermoplastic resin, or may be contained in one layer of the carrier having a multilayer structure, You may make it contain by making it carry | support by the surface by adsorption | suction or heat fusion. The content (supported amount) of the magnetically sensitive component on the carrier can be determined as appropriate depending on the strength of the magnetic field applied to flow the carrier and the degree of fluidity of the carrier. It is -80% (w / v), and 5-50% (w / v) is preferable. The magnetically sensitive component itself may be used as the carrier itself or the core of the carrier.

  The method for supporting bovine serum albumin on the carrier is a method in which the reaction-specific component for capturing the measurement object by a specific reaction is supported on the carrier by a supporting method such as a chemical binding method or a physical adsorption method, and then the bovine serum A method in which the association component of albumin is supported by a physical adsorption method on a non-specific adsorption site remaining on the surface of the carrier, or after the reaction-specific component is supported by a chemical bonding method using a functional group on the surface of the carrier, the bovine Examples include a method of supporting an association component of serum albumin directly by a chemical bonding method using a functional group remaining on the surface of the carrier or via a cross-linking agent. It may be supported by an optimum method as appropriate. The concentration of the association component added to the insoluble carrier is preferably 0.1 to 5% (w / v), more preferably 0.5 to 3% (w / v).

  The form of the measurement reagent of the present invention is used as it is in a state in which one or more reaction-specific components for capturing a measurement object by a specific reaction and an insoluble carrier carrying an association component of bovine serum albumin are suspended in a diluent. Alternatively, the suspension may be lyophilized for use.

  In addition, the above-mentioned diluent is a protein such as mammalian normal serum protein, albumin, collagen, gelisate, skim milk, lactic acid fermented product, gelatin or a degradation product thereof, or D-mannitol, sucrose, myo-inositol, trehalose, Contains sugars such as β-cyclodextrin, glucose, lactose, fructose, cellulose, raffinose, maltose, galactose, and xylose. The concentration is not particularly limited, but is preferably 0.1 to 50% (w / v), more preferably 1 to 15% (w / v) for proteins, and preferably 1 to 10% (for sugars). w / v), more preferably 1 to 5% (w / v). In addition to protein or sugar, depending on the purpose, protective agents such as BSA or casein, antioxidants such as ascorbic acid and vitamin E, binders such as carboxymethyl cellulose, wetting agents such as cellulose and polyethylene glycol, polyvinylpyrrolidone, etc. Suspending agents, emulsifiers such as alkyl sulfonic acids, solubilizing agents such as glycerin, buffers such as phosphates and tris-hydroxylamine hydrochloride, isotonic agents such as D-sorbitol and sodium chloride, magnesium chloride, A surfactant such as zinc chloride, Triton X-100, or Tween 20 may be added.

  The measurement method using the measurement reagent of the present invention is, for example, the first reaction-specific component in which the measurement reagent and sample are added to a measurement container and stirred, and the measurement object in the sample is supported on an insoluble carrier. The second reaction in which the insoluble carrier is washed with a washing solution and specifically reacts with the measurement target after removing contaminant components in the sample that have been trapped in the sample and not captured (first B / F separation). A labeled body in which a labeled substance is bound to a specific component is added and stirred, and the labeled body is reacted with the measurement target captured on an insoluble carrier to form a sandwich complex (first reaction-specific component-measurement). (Object-labeled body) and unreacted substances are removed (second B / F separation), and then the measurement object is converted based on the signal generated from the labeled object of the label contained in the sandwich complex. Measurement methods for quantification are included.

  In addition, the measurement reagent and the sample are added to the measurement container and stirred, the measurement object in the sample is captured by the reaction-specific component supported on the insoluble carrier, and the label is further bound to the measurement object. Is added and stirred to remove unreacted substances (B / F separation), and then the measurement object is quantified based on the signal generated from the label of the labeled body captured by the reaction-specific component supported on the insoluble carrier. There are also measuring methods to do.

  Examples of the label used in the present invention include those obtained by labeling a reaction-specific component that specifically reacts with an object to be measured with a label, or those obtained by labeling the object to be measured. Examples include alkaline phosphatase, peroxidase, β-D-galactosidase, glucose oxidase, luciferase and other luminescent materials such as acridinium derivatives and luminol derivatives, and fluorescent materials such as fluorescein derivatives and rhodamine derivatives. There is no particular limitation as long as it is used for a method or a luminescence or fluorescence immunoassay. The form of the labeled reagent may be used in the same manner as the carrier reagent, or may be used after lyophilization. Further, the label reagent may be mixed with the carrier reagent and lyophilized in the same container, or the label reagent and the carrier reagent may be lyophilized in separate containers. Also good.

  The method for producing a measuring reagent of the present invention is, for example, a polypropylene bovine serum albumin solution whose concentration is adjusted to 0.1 to 10% (w / v) with a buffer such as Tris-HCl buffer or phosphate buffer. A high temperature liquid chromatograph in which a column for gel permeation chromatography is set after the step of heat treatment in a warm bath at 50 to 60 ° C. for 10 minutes to 24 hours, and the heated bovine serum albumin solution is returned to room temperature. The step of separating the molecular weight fraction of the associative component of bovine serum albumin using a graph so as to fall within the molecular weight range of 300,000 to 1,000,000, and the reaction-specific component was used when supported on an insoluble carrier. One or more in which the solution of the association component is adjusted to a concentration of 0.1 to 5.0% (w / v) with a buffer solution having the same composition as the buffer solution, and the measurement target is captured by a specific reaction. A step of blocking the insoluble carrier by immersing the insoluble carrier carrying the reaction-specific component in a solution of the association component at 25 to 37 ° C. for about 1 to 12 hours; And a step of diluting with a buffer containing 50% (w / v) protein or 1-10% (w / v) sugar.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, these Examples do not limit this invention.

Example 1
A 0.05 mol / L Tris hydrochloric acid buffer solution (pH 8.0) containing 1.0% BSA (fraction V, manufactured by Sigma) was heated in a warm bath at 50 ° C. for 7 hours to obtain a solution containing an association component of BSA. . This was treated with a 0.22 μm filter, separated using a TSK gel G3000SW _XL gel permeation chromatography column, and then detected with an ultraviolet absorption detector (detection wavelength 280 nm). The average molecular weight was about 480,000 (molecular weight 400). , 5,000 to 520,000 fractions) containing a BSA association component (fractionated by the broken line in FIG. 1). The protein concentration was measured using BCA Protein Assay Kit (Pierce).

  Next, 0.01 mol / L MES of magnetic fine particles (material: magnetic material-containing vinyl polymer coated fine particles, average particle size 2.1 μm, type of magnetic material: magnetite, amount of magnetic material: 30 to 50%) as an insoluble carrier. Adjust the slurry concentration to 1% (w / v) in buffer (pH 6.0), add antithyroid stimulating hormone antibody (hereinafter referred to as anti-TSH antibody) to a concentration of 0.2 mg / mL, The anti-TSH antibody was physically adsorbed on the magnetic microparticles by incubating at 37 ° C. for 1 hour. After washing the magnetic fine particles, the BSA-associated components are blocked (physically adsorbed) by incubating in the aforementioned BSA 1.0% associating component solution at 37 ° C. for 7 hours, and anti-TSH antibody-immobilized magnetic fine particles are obtained. And diluted with 0.05 mol / L Tris hydrochloric acid buffer (pH 8.0) containing 5% bovine serum to prepare a diluted solid phase suspension and lyophilized to obtain a measurement reagent.

(Comparative Example 1)
Non-associated BSA for blocking magnetic particles (non-heated, monomer, as well as some dimers and trimers) (fraction of molecular weight 66,000 (main fraction) to 200,000) A diluted solid phase suspension was prepared in the same manner as in Example 1 except that it was performed in a 1.0% solution, and lyophilized to obtain a measurement reagent.

(Evaluation by chemiluminescent enzyme immunoassay)
A conjugate of anti-TSH antibody and alkaline phosphatase diluted with 0.05 mol / L Tris hydrochloric acid buffer (pH 8.0) containing 5% bovine serum is lyophilized and labeled antibody reagent for detection (hereinafter labeled antibody) A zero-concentration standard solution for TSH measurement (hereinafter referred to as TSH standard solution) diluted with 0.03 mol / L Tris-HCl buffer (pH 8.0) to 5% bovine serum. It was.

  FIG. 2 is a diagram showing the results of measuring a TSH standard solution 60 times by a chemiluminescent enzyme immunoassay using the lyophilized product prepared in Example 1 and the lyophilized product prepared in Comparative Example 1.

The measurement was performed according to the following procedure.
The measurement reagent was dissolved in pure water containing a surfactant and a zero-concentration TSH standard solution, and then subjected to an immunoreaction at 37 ° C. for 5 minutes to perform the first B / F separation. Next, a labeled antibody reagent dissolved in pure water containing a surfactant was added, and an immunoreaction was performed at 37 ° C. for 3 minutes, and a second B / F separation was performed. Thereafter, a chemiluminescent substrate for alkaline phosphatase (DIFURAT, 3- (5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo [3,2,0] hept-1-yl) phenyl phosphorus Acid ester disodium salt) and a chemiluminescence auxiliary reagent were added, and the luminescence intensity (count (s) / second (cps)) was measured. The results are shown in Table 1.

全６０回測定の中で、比較例１の測定試薬では、３７回目（３６８ｃｐｓ）と４３回目（１，３７５ｃｐｓ）にバックグラウンド平均値（７４．９ｃｐｓ）のそれぞれ約５倍と約１８倍の非特異吸着量に相当するカウント数（偽高値）が得られた。それに比べて、実施例１の測定試薬では、バックグラウンド平均値は４０．６ｃｐｓと比較データに比べ５４％にまで低減して偽高値は検出されず、ＴＳＨ標準溶液における測定の同時再現性（ＣＶ％）は比較データの２３４．７％に比べて１０．７％と大幅に改善されることがわかった。

Among the total 60 measurements, the measurement reagent of Comparative Example 1 was about 5 times and about 18 times the background average value (74.9 cps) at the 37th (368 cps) and 43rd (1,375 cps), respectively. A count number (false high value) corresponding to the amount of specific adsorption was obtained. In contrast, in the measurement reagent of Example 1, the background average value was 40.6 cps, which was reduced to 54% compared with the comparison data, and no false high value was detected. Simultaneous reproducibility of the measurement in the TSH standard solution (CV %) Was significantly improved to 10.7% compared to 234.7% in the comparative data.

Claims (9)

  An insoluble carrier carrying one or more reaction-specific components for capturing a measurement object by a specific reaction and an association component of bovine serum albumin having a molecular weight fraction falling in the molecular weight range of 300,000 to 1,000,000.   The insoluble carrier according to claim 1, wherein the reaction-specific component is an antibody or an antigen.   The insoluble carrier according to claim 1, wherein the insoluble carrier is a magnetic particle.   The insoluble carrier according to any one of claims 1 to 3, wherein the measurement object is thyroid stimulating hormone (TSH).   A measurement reagent which is a suspension in which the insoluble carrier according to any one of claims 1 to 4 is suspended in a diluent.   The measurement reagent according to claim 5, wherein the suspension is freeze-dried. A measurement method using the measurement reagent according to claim 5 or 6,
A sample is added to the measurement reagent, a measurement target in the sample is captured by a reaction-specific component, an unreacted substance is removed, a label is added, and an unreacted substance is further removed. The said measuring method which quantifies the said measuring object capture | acquired by the component. A measurement method using the measurement reagent according to claim 5 or 6,
A sample is added to the measurement reagent, the measurement object in the sample is captured by a reaction-specific component, a label is added, an unreacted material is removed, and the measurement object captured by the reaction-specific component is The measuring method for quantification. Heat treating bovine serum albumin at 50-60 ° C. for 10 minutes-24 hours;
Fractionating the molecular weight fraction of the heat-treated bovine serum albumin so as to fall within a molecular weight range of 300,000 to 1,000,000;
Adjusting the molecular weight fraction of bovine serum albumin to a concentration of 0.1 to 5.0% (w / v), and providing an insoluble carrier carrying one or more reaction-specific components for capturing the measurement object by a specific reaction. Dipping step;
Diluting the insoluble carrier after immersion with a diluent;
A method for producing a measurement reagent comprising:

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