JP6409016B2 - Measurement error reduction method in latex immunoaggregation - Google Patents

Description

  The present invention relates to a method for reducing a measurement error derived from a sample in a latex immunoaggregation method using latex particles carrying a specific affinity substance for a substance to be measured. In particular, the present invention relates to a method for reducing measurement error derived from a blood sample, including a step of performing a latex immunoagglutination reaction in the presence of imidazole.

As a method for measuring a substance to be measured in a biological sample, a latex immunoaggregation method (also referred to as a latex immunoagglutination turbidimetric method, hereinafter sometimes referred to as LTIA) is frequently used in the field of clinical examination. LTIA uses, for example, latex particles carrying an antibody against a substance to be measured, and optical means for determining the degree of aggregation (turbidity) of latex particles caused by binding of antigen as a substance to be measured and antibody-carrying latex particles. It is a measurement method to detect by such as.
Here, since a biological sample is a measurement object, a measurement error due to the influence of various interfering substances derived from the sample becomes a problem. In particular, blood components of patients receiving intravenous fat emulsion components and patients with hyperlipidemia contain fat components, and these effects cause errors in measured values.
For example, Patent Documents 1 to 3 are known as methods for avoiding the influence of a fat component in a method for measuring a component in a blood sample using insoluble carrier particles.

Patent Document 1 discloses a method of performing an antigen-antibody reaction in the presence of albumin, an enzyme having lipase activity, and a nonionic surfactant in order to avoid the influence of chyle in a test sample such as serum or plasma. It is disclosed. According to this method, the structure of fat in the sample is changed by the surfactant, the core part is decomposed by lipase to become fatty acid, and the fatty acid is adsorbed to albumin, thereby interfering with antigen-antibody reaction by chyle. Is avoided.
Patent Document 2 discloses a method of eliminating an error caused by the presence of lipid in a serum sample by adding lipase to a specimen in the measurement of testosterone and estradiol using magnetic particles.
However, both Patent Document 1 and Patent Document 2 are methods for decomposing lipids that are present in a sample and cause measurement errors with lipase, and it is necessary to stably maintain the lipase activity in the constituent reagents.
Patent Document 3 discloses a method of using a polynuclear phenol ethoxylate together with a secondary linear alcohol ethoxylate that is a nonionic surfactant in order to remove turbidity due to lipid components of blood samples such as serum and plasma. . However, the description is only about the removal of turbidity by the fat component of the sample, and there is no specific description about the immunoagglutination measurement method using insoluble carrier particles such as latex.

JP 2006-71754 A JP2013-200208A JP-A-10-213582

  An object of the present invention is to avoid the influence of measurement errors derived from blood samples in latex immunoagglutination.

The present inventors have conducted various studies in order to avoid the influence of measurement errors derived from blood samples in the latex immunoagglutination method. And, by variously changing the buffer component in the liquid phase in which the sample and latex particles carrying a specific affinity substance for the substance to be measured are brought into contact, it is found that the influence of measurement error can be unexpectedly avoided, The present invention has been completed.
That is, the present invention has the following configuration.
<1>
A method for reducing measurement errors derived from blood samples in latex immunoagglutination,
The said reduction method including the process of contacting the said sample and the latex particle which carries the specific affinity substance with respect to a measuring object substance in presence of imidazole or its derivative (s), or these salts in a liquid phase.
<2>
The method according to <1>, wherein the blood sample is serum or plasma.
<3>
The method according to <1> or <2>, wherein the concentration of imidazole or a derivative thereof or a salt thereof when measuring the agglutination reaction is 37.5 mM or more and 375 mM or less.
<4>
The method according to any one of <1> to <3>, wherein the latex immunoaggregation method is a method based on the homogeneous method.
<5>
A method of measuring a substance to be measured in a blood sample by latex immunoagglutination,
The said measuring method including the process which the latex particle which carries the specific affinity substance with respect to a measuring object substance in the presence of imidazole or its derivative (s), or these salts in a liquid phase is contacted.
<6>
The method according to <5>, wherein the blood sample is serum or plasma.
<7>
<5> or <6> The method according to <5> or <6>, wherein the concentration of imidazole or a derivative thereof or a salt thereof when measuring the aggregation reaction is 37.5 mM or more and 375 mM or less.
<8>
The method according to any one of <5> to <7>, wherein the latex immunoaggregation method is a method based on the homogeneous method.
<9>
A method of measuring a substance to be measured in a blood sample by latex immunoagglutination,
The said measuring method including the following processes.
(1) Step of contacting the sample with imidazole or its derivative or a salt thereof in the liquid phase (2) After the step (1), latex particles carrying a specific affinity substance for the substance to be measured Step <3> of measuring the aggregation reaction between the substance to be measured and the latex particles after the steps (3) and (2) of adding into the liquid phase
<9> The method according to <9>, wherein the blood sample is serum or plasma.
<11>
The method according to <9> or <10>, wherein the concentration of imidazole or a derivative thereof or a salt thereof in the step (3) of measuring the aggregation reaction of latex particles is 37.5 mM or more and 375 mM or less.
<12>
The method according to any one of <9> to <11>, wherein the latex immunoaggregation method is a method based on the homogeneous method.
<13>
A reagent kit for measuring a substance to be measured in a blood sample by latex immunoagglutination, comprising:
(1) A first reagent containing a buffer containing imidazole or a derivative thereof or a salt thereof (2) A second reagent <14> containing latex particles carrying a substance having a specific affinity for the substance to be measured
<13> The substance to be measured in the blood sample is contained in such a form that the concentration of imidazole or a derivative thereof or a salt thereof can be adjusted to 37.5 mM or more and 375 mM or less when measured by latex immunoagglutination. The reagent kit according to 1.
<15>
The reagent kit according to <13> or <14>, wherein the latex immunoaggregation method is a method based on the homogeneous method.
<16>
A liquid reagent for measuring a substance to be measured in a blood sample by latex immunoagglutination, which comprises imidazole or a derivative thereof or a salt thereof.
<17>
<16> The substance to be measured in the blood sample is contained in such a form that the concentration of imidazole or a derivative thereof or a salt thereof can be adjusted to 37.5 mM or more and 375 mM or less when measured by latex immunoagglutination. A liquid reagent according to 1.
<18>
The liquid reagent according to <16> or <17>, wherein the latex immunoaggregation method is a method based on the homogeneous method.
<19>
An agent for reducing measurement error derived from a blood sample in latex immunoagglutination, comprising imidazole or a derivative thereof or a salt thereof as an active ingredient.

  According to the present invention, accurate measurement can be performed by reducing measurement errors derived from blood samples in latex immunoagglutination.

(Latex immunoaggregation method)
LTIA (latex immunoaggregation method) is a method for measuring a substance to be measured using latex particles with a specific affinity substance immobilized on the substance to be measured such as an antigen or an antibody, and is widely used in the field of clinical tests. Has been. As a method for measuring an antigen as a measurement target substance by LTIA, latex particles to which an antibody against the measurement target substance is immobilized are reacted with an antigen as a measurement target substance to form a sandwich-type immune complex, and immune The method of measuring the substance to be measured (antigen) from the degree of aggregation of the latex particles associated with the formation of the complex, and the latex particles to which the antigen is immobilized compete with the antigen in the sample (measuring substance). The method can be roughly divided into methods for measuring a substance to be measured (antigen) based on the degree of inhibition of aggregation of latex particles accompanying inhibition of immune complex formation by inhibiting the formation of immune complexes between particles and antibodies.

(sample)
The sample in the present invention is a blood sample, and examples thereof include whole blood, serum, and plasma.

(Reduction of measurement error)
In LTIA, some component contained in the blood sample may cause agglomeration that should not occur in the latex particles on which the specific affinity substance for the measurement target substance is immobilized (positive measurement error) or agglomeration that should occur. Often occurs (negative measurement error). These are called non-specific reactions and are known to cause various measurement errors.
In the present invention, “reducing measurement error” refers to bringing the positive or negative measurement error of the measurement value close to the original measurement value (true value).
In the present invention, a measurement error derived from a blood sample refers to a measurement error due to some non-specific reaction caused by a component in the blood sample. Examples of a component in the blood sample that causes a non-specific reaction include chyle and simple. Lipids such as lipids and neutral fats can be mentioned.
In particular, the present invention reduces measurement errors that occur in blood samples derived from individuals with chyle findings (individuals with a high fat state), individuals instilled with a fat emulsion, and the like.

(Fat emulsion)
Examples of the fat emulsion include an oil-in-water type fat emulsion obtained by emulsifying fat and oil with an emulsifier and emulsifying the average particle size of fat particles to 1 μm or less, preferably 0.5 μm or less, more preferably 0.3 μm or less. Various fat emulsions used for infusion (intravenous injection) can be used, and commercially available products include, for example, Intralipos (registered trademark, manufactured by Otsuka Pharmaceutical Factory), Intrafat (registered trademark, manufactured by Nippon Pharmaceutical Co., Ltd.) ), Intralipid (registered trademark, manufactured by Fresenius Kirby Japan), and the like. The components described in the package inserts are refined soybean oil, refined egg yolk lecithin, concentrated glycerin, and sodium hydroxide (pH adjuster).

A fat emulsion is prepared by adding fats and oils to a solution in which an emulsifier is dispersed in water, stirring to prepare a crude emulsion, and then emulsifying the crude emulsion by a conventional method such as a high-pressure emulsification method. Can be prepared. When the emulsion is prepared by the high pressure emulsification method, for example, using an emulsifying machine such as a Manton Gaulin homogenizer, the crude emulsion 20~700Kg / cm ² about conditions, carried out by passing 5 to 50 times. In order to obtain a stable and fine-grain emulsion upon emulsification, it is preferable to carry out emulsification in the presence of glucose and / or glycerin. According to this method, an emulsion having an average particle diameter of 0.17 μm or less can be easily prepared. be able to. Moreover, you may emulsify by adding an emulsification adjuvant etc. as needed.

  As the oil and fat, any oil and fat can be used as long as it is an edible oil. For example, vegetable oil (for example, soybean oil, cottonseed oil, safflower oil, corn oil, coconut oil, perilla oil, sesame oil, etc.), fish oil (for example, Cod liver oil etc.), medium chain fatty acid triglycerides [e.g. panacetate (trade name), ODO (trade name) etc.] and chemically synthesized triglycerides [e.g. 2-linoleoyl-1,3-dioctanoylglycerol (8L8), 2 -1 type, or 2 or more types of fats and oils selected from [Chemically defined triglycerides] such as linoleoyl-1,3-didecanoylglycerol (10L10). Moreover, any emulsifier can be used as an emulsifier as long as it is used in pharmaceutical preparations. For example, egg yolk phospholipid, hydrogenated egg yolk phospholipid, soybean phospholipid, hydrogenated soybean phospholipid, and nonionic interface One or more emulsifiers selected from activators [for example, Pluronic F68, HCO-60 (both are trade names), etc.] are preferably used.

  Particularly preferred is a fat emulsion in which soybean oil is used as the fat and oil, egg yolk phospholipid is used as the emulsifier, and the average particle size of the fat particles is adjusted to 0.3 μm or less, more preferably 0.17 μm or less. The composition of the fat emulsion is not particularly limited, but preferred examples include fats and oils of about 0.1 to 30 w / v%, preferably about 1 to 20 w / v%, more preferably about 2 to 10 w / v%, and emulsifiers. A fat emulsion comprising about 01 to 10 w / v%, preferably about 0.05 to 5 w / v%, more preferably about 0.1 to 1 w / v%, and an appropriate amount of water.

  The above fat emulsion may contain a saccharide such as a reducing sugar. Examples of reducing sugars include glucose, fructose, maltose and the like, and these reducing sugars may be used in combination of two or more. Furthermore, you may use the mixture which added sorbitol, xylitol, and / or glycerol to these reducing sugars. The addition of these saccharides may be performed after preparing the emulsion or may be performed when preparing the emulsion. Preferred examples of fat emulsions containing these saccharides include fats and oils of about 0.1 to 30 w / v%, preferably about 1 to 20 w / v%, more preferably about 2 to 10 w / v%, emulsifier 0.01 About 10 to 10 w / v%, preferably about 0.05 to 5 w / v%, more preferably about 0.1 to 1 w / v%, sugars about 1 to 60 w / v%, preferably about 5 to 40 w / v% More preferably, a fat emulsion comprising about 10 to 30 w / v% and an appropriate amount of water can be mentioned.

  The fat emulsion may be added with an anti-coloring agent (for example, thioglycerol, dithiothreitol, etc.) in order to prevent coloring of the fat emulsion during sterilization and storage, and to stabilize the pH. Therefore, a buffer [for example, L-histidine, tris (hydroxymethyl) aminomethane, etc.] may be added. The addition amounts of these anti-coloring agents and buffering agents are usually about 1% or less, respectively. Furthermore, in the present invention, vitamins (for example, vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, etc.) may be added to the fat emulsion. These additives may be added after preparing the emulsion, or may be added when preparing the emulsion.

(Measuring method)
As a measurement method of LTIA, a test substance can be measured by optically or electrochemically observing the degree of aggregation that has occurred. Examples of the optical observation method include methods (endpoint method, rate method, etc.) of measuring scattered light intensity, absorbance, or transmitted light intensity with an optical instrument. The measured value such as absorbance obtained by measuring the sample was compared with the measured value such as absorbance obtained by measuring the standard substance (sample whose concentration of the measurement target substance is known), and was included in the sample. Calculate the concentration (quantitative value) of the analyte. In addition, the measurement of absorbance, such as transmitted light or scattered light, may be one-wavelength measurement or two-wavelength measurement (difference or ratio by two wavelengths). The measurement wavelength is generally selected from 500 nm to 800 nm.

  The measurement of the substance to be measured in the sample of the present invention may be performed by a method, or may be performed using an apparatus such as a measuring apparatus. The measuring device may be a general-purpose automatic analyzer or a dedicated measuring device (dedicated machine). Moreover, it is preferable to implement this measurement by the method performed by several operation steps, such as a 2 step method (2 reagent method).

(Substance to be measured)
The substance to be measured in the present invention is not particularly limited as long as it is a substance measured by an immunological measurement method, for example, various substances such as proteins (antigens, haptens, antibodies), carbohydrates, lipids, nucleic acids, chemical substances ( Hormones, drugs) and the like. Especially, what uses an antigen as a measuring object substance is preferable, and a protein antigen is more preferable. Specifically, for example, soluble IL-2 receptor (sIL-2R), CRP, fibrin and fibrinogen degradation products, D dimer, soluble fibrin (SF), lipoprotein (a) (Lp (a)), matrix metalloprotease -3 (MMP-3), prostate specific antigen (PSA), IgG, IgA, IgM, IgE, IgD, anti-streptolysin O, rheumatoid factor, transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, α2- Macroglobulin, hemopexin, antithrombin-III, α-fetoprotein, CEA (calcinoembryonic antigen), ferritin, HBs-Ag (hepatitis B envelope antigen), Anti-HBs (anti-hepatitis B envelope antibody), HBe-Ag (hepatitis B e antigen), Ant -HBe (hepatitis B e antibody), Anti-HBc but (anti hepatitis B core antibody) and the like, but is not limited thereto.

(Specific affinity substance)
In the present invention, specific affinity substances for the measurement target substance supported on latex include proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, haptens, etc. Although there is no restriction | limiting in particular, The antibody or antigen which can be used for the immunoassay using an immune reaction is mentioned.

(antibody)
The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. More preferably, it is a monoclonal antibody.
As the antibody of the present invention, a functional fragment of an antibody having antigen-antibody reaction activity can be used in addition to the whole antibody molecule. In addition to those obtained through the immunization process for general animals (mouse, goat, sheep, etc.), the amino acid sequence of the animal species is different from the animal immunized with the immunogen (substance to be measured) by genetic recombination technology. It may be an antibody (such as a chimeric antibody, a humanized antibody, or a fully humanized antibody). Examples of the functional fragment of the antibody include F (ab ′) ₂ , Fab ′, and single chain antibody (scFv), which are fragments having antigen-antibody reaction activity. Functional fragments of these antibodies can be produced by treating the antibody obtained as described above with a proteolytic enzyme (for example, pepsin or papain).

(Latex particles)
The latex particles used in the present invention are not particularly limited as long as they are latex particles generally used as an immunological measurement reagent. Latex particles can be obtained by polymerizing or copolymerizing various monomers. Examples of the monomer include polymerizable monomers having a phenyl group such as styrene, α-methylstyrene, o-methylstyrene, p-methylstyrene, p-chlorostyrene, 4-vinylbenzoic acid, divinylbenzene, and vinyltoluene. A polymerizable monomer having a phenyl group and a sulfonate, such as a monomer, a styrene sulfonate, a divinylbenzene sulfonate, an o-methyl styrene sulfonate, and a p-methyl styrene sulfonate, 1-vinyl naphthalene, Polymerizable unsaturated aromatics such as polymerizable monomers having a naphthyl group such as 2-vinylnaphthalene, α-naphthyl (meth) acrylate, β-naphthyl (meth) acrylate, such as (meth) acrylic acid, Polymerizable unsaturated carboxylic acids such as itaconic acid, maleic acid, fumaric acid such as methyl (meth) acrylate, (meth T) Ethyl acrylate, (meth) acrylic acid-n-butyl, (meth) acrylic acid-2-hydroxyethyl, (meth) acrylic acid glycidyl, ethylene glycol di- (meth) acrylic acid ester, (meth) acrylic Polymerizable unsaturated carboxylic acid esters such as tribromophenyl acid, (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide, N-methylol- (meth) acrylamide, methylenebis (meth) acrylamide, butadiene, isoprene, acetic acid Examples thereof include unsaturated carboxylic acid amides such as vinyl, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, and vinyl bromide, polymerizable unsaturated nitriles, vinyl halides, and conjugated dienes. These monomers are appropriately selected depending on the required surface properties, specific gravity and the like, and can be used alone or in combination of two or more.
The average particle size of the latex particles is preferably 0.02 to 1.6 μm, more preferably 0.1 μm to 0.4 μm in consideration of the concentration of the substance to be measured in the sample or the detection sensitivity of the measuring instrument. Is appropriately selected. Latex particles to be used can be appropriately selected from materials and particle sizes in order to obtain desired performance such as sensitivity improvement, and those having different materials and particle sizes can also be used in combination. Further, the concentration of the latex particles at the time of measuring the agglutination reaction in the present invention is not particularly limited, and can be appropriately set according to desired sensitivity and performance.

(Latex particles carrying specific affinity substances)
The specific affinity substance for the substance to be measured can be immobilized and supported on latex particles by a known method such as a physical adsorption (hydrophobic bond) method, a chemical bond method, or a combination thereof.

  In the case of the physical adsorption method, according to a known method, a measurement is performed by mixing a substance having a specific affinity for a measurement target substance and latex particles in a solution such as a buffer solution and bringing them into contact with each other, or dissolving in a buffer solution or the like. A specific affinity substance for the target substance can be brought into contact with a carrier.

  When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with known methods described in ed. “Shinsei Kagaku Kenkyu Ken 1 Protein IV”, published by Tokyo Kagaku Dojin, 1991, etc., a specific binding substance for a substance to be measured and a carrier are mixed with glutaraldehyde, carbodiimide, imide ester or maleimide. Mixed with, and brought into contact with, a bivalent cross-linking reagent such as a specific binding substance for the substance to be measured, and each of the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, etc. It can be performed by reacting with a crosslinking reagent.

  In order to form a sandwich, a plurality of specific affinity substances for specific substances carried by latex particles are preferable. For example, when the specific affinity substance is a monoclonal antibody, a plurality of monoclonal antibodies having different recognition sites are used. For example, when the specific affinity substance is a polyclonal antibody, it may be a polyclonal antibody derived from one type of antiserum, or may be derived from a plurality of types of antisera. Moreover, you may use combining a monoclonal antibody and a polyclonal antibody.

  In addition, if it is necessary to perform treatment in order to suppress natural aggregation of latex particles, non-specific reaction, etc., the surface of latex particles has bovine serum albumin (BSA), casein, gelatin, egg white albumin or a salt thereof, etc. The carrier may be subjected to a blocking process (masking process) by a known method such as contact with a protein, a surfactant, skim milk powder or the like to coat the protein.

(Imidazole)
In the present invention, imidazole or a derivative thereof or a salt thereof is present or contained in a measurement reaction solution or a measurement reagent, thereby measuring a measurement target substance in a sample.
Here, the imidazole or a derivative thereof may be a compound having an imidazole skeleton, such as imidazole, 1-methylimidazole, 1-ethylimidazole, 1-propylimidazole, 1-butylimidazole, 1-phenylimidazole, 1 -Vinylimidazole, 1-allylimidazole, 1-benzyl-2-methylimidazole, 1-benzyl-2-formylimidazole, 1-benzyl-4-hydroxymethylimidazole, 1-benzyl-5-hydroxymethylimidazole, (2-hydroxyethyl) -imidazole, 1- (2-hydroxyethyl) -2-methylimidazole, 2-methylimidazole, 2-ethylimidazole, 2-propylimidazole, 2-butylimidazole, 2-phenylimidazole Dazole, 2-formylimidazole, 2-hydroxymethylimidazole, 2-methyl-1-vinylimidazole, 2-butyl-4-formylimidazole, 2-butyl-4-hydroxymethylimidazole, 2-butyl-4-chloro -5-formylimidazole, 2-hydroxymethyl-1-benzylimidazole, 2-hydroxymethyl-2-methylimidazole, 2-ethyl-4-methylimidazole, 4-butylimidazole, 4-formylimidazole, 4-formyl -1-methylimidazole, 4-formyl-1-trisylimidazole, 5-formyl-1-methylimidazole, 4-formyl-5-methylimidazole, 4-hydroxymethylimidazole hydrochloride, methylimidazole-4- Rubokishireto, ethyl imidazole-4-carboxylate, may be mentioned 1,2-dimethylimidazole, or 1,2,4 known avian methylimidazole ones.
The salt of imidazole or a derivative thereof is a chemically acceptable salt. In addition to potassium salt, magnesium salt, lithium salt, calcium salt or zinc salt, adipate, alginate, citrate, aspartate , Benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, Sesquiki (fumarate), hydrochloride, dihydrochloride, trihydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactic acid Salt, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropiate Phosphate, picrate, pivalate, propionate, succinate, sulfate, bis (tartrate), tartrate, (L) tartrate, bis ((L) tartrate), (D) Tartrate, bis ((L) tartrate), (DL) tartrate, bis ((DL) tartrate), meso tartrate, bis (meso tartrate), thiocyanate, phosphate, glutamate, carbonic acid From hydrogen salt, bis ((D) tartrate), bis (bromide), bis (sulfate), bis (phosphate), tris (hydrochloride), p-toluenesulfonate, and undecanoate A salt selected from the group consisting of:

In the present invention, a commercially available product can be used as it is for imidazole or a derivative thereof or a salt thereof (hereinafter sometimes referred to as imidazole). The concentration of imidazole, etc. is not limited as long as it does not exert a strong influence on the main reaction such as antigen-antibody reaction in the step of measuring the agglutination reaction after mixing the sample and the measurement reagent, 37.5 mM or more, 45 mM or more, 52.5 mM or more, 60 mM or more, 67.5 mM or more, 75 mM or more. As an upper limit, it is 750 m or less, and 675 mM or less, 600 mM or less, 525 mM or less, 450 mM or less, 375 mM or less, 300 mM or less, 225 mM or less is mentioned.
As a preferable concentration range, in addition to the range of 37.5 to 750 mM, a combination of the above upper limit and the lower limit may be mentioned. For example, 37.5 to 675 mM, 37.5 to 600 mM, 37.5 to 525 mM, 37.5 ˜450 mM, 37.5˜375 500 mM, preferably 37.5˜300 mM, more preferably 37.5˜225 mM, 45˜225 mM, 52.5˜225 mM, 60˜225 mM, 67.5˜225 mM Further, a range of 75 to 225 mM is particularly preferable.
Further, there is no problem even if the concentration of imidazole or the like is contained at 375 mM or more, but a sufficient effect can be obtained even at 375 mM. Furthermore, the imidazole may be used alone or a plurality of imidazoles may be used in combination. Moreover, when using several imidazole etc. together, the density | concentration which combined both should just be said density | concentration range.
The “buffer solution containing imidazole” contained in the measurement reagent of the present invention may be an imidazole buffer solution or a buffer solution other than an imidazole buffer solution containing imidazole or the like. It is also possible to use a combination of a buffer solution other than the imidazole buffer solution and an imidazole buffer solution.

  Further, when the measurement method and the measurement reagent of the present invention are a two-step method (two-reagent method) or a multi-step method (multi-reagent method), the ratio of each addition amount when measuring the measurement target substance What is necessary is just to determine the density | concentration of imidazole or its derivative (s), or these salts so that the density | concentrations, such as imidazole, in the measurement reaction liquid after this mixing may become said range when it mixes. For example, in the case of the two-step method (two-reagent method), if the concentration of imidazole and the like in the measurement reaction liquid after mixing falls within the above concentration range, imidazole and the like are contained in both the first reagent and the second reagent. May be.

(contact)
When a sample containing a substance to be measured in the presence of imidazole or the like is brought into contact with latex particles carrying a substance having a specific affinity for the substance to be measured, the sample and a substance having a specific affinity for the substance to be measured are carried. It is sufficient that imidazole or the like is contained in the reaction solution for reacting the latex particles to be reacted, and typically, the following (1) to (4) are mentioned.
(1) A mode in which the sample is mixed with a buffer containing imidazole, and then mixed with a reagent containing latex particles carrying a specific affinity substance for the substance to be measured.
(2) A mode in which the sample, a buffer containing imidazole and the like, and a reagent containing latex particles carrying a specific affinity substance for the substance to be measured are mixed simultaneously.
(3) A mode in which the sample is mixed with a reagent containing latex particles carrying a specific affinity substance for the substance to be measured, and then a buffer solution containing imidazole or the like is added to the mixture and mixed (4) The target to which the sample is added after mixing the reagent containing latex particles carrying a specific affinity substance for the substance to be measured and the buffer containing imidazole etc.

(Reagent for measurement)
The measuring reagent of the present invention is composed of two or more constituent reagents, and at least one constituent reagent contains latex particles carrying a specific affinity substance for the substance to be measured, and the same constituent reagent as the constituent reagent and / Or different constituent reagents include imidazole and the like. The measuring reagent of the present invention is a liquid reagent.

  Of these, the measuring reagent of the present invention is preferably a two-reagent type comprising a first reagent and a second reagent. For example, the first reagent of the two-reagent type includes a buffer solution containing imidazole or the like, and the second reagent includes latex particles carrying a specific affinity substance for the measurement target substance.

  Further, when the measurement reagent of the present invention is a three-reagent type composed of a first reagent, a second reagent, and a third reagent, for example, the first reagent extracts a measurement target substance from a sample diluted with the sample. The second reagent contains a buffer containing imidazole, and the third reagent contains latex particles carrying a specific affinity substance for the substance to be measured.

Imidazole and the like may be included in any or all of the constituent reagents as long as they can exhibit the effect of reducing measurement errors derived from blood samples in the mixed liquid state at the time of measurement and do not affect the stability of the constituent reagents. .
Therefore, in the two-reagent type and the three-reagent type, imidazole or the like may be contained in all constituent reagents, or may be contained in all constituent reagents not containing latex particles.
The concentration in each constituent reagent such as imidazole may be contained in a form that can be adjusted to 37.5 mM or more and 375 mM or less in the mixed state of the reagent and the sample at the time of measurement, and varies depending on each reagent type.

(Reagent kit)
The reagent kit of the present invention includes at least the following (1) and (2)
(1) A first reagent including a buffer containing imidazole or the like (2) An element of a second reagent including latex particles carrying a substance having a specific affinity for a substance to be measured is included. The concentration in the buffer such as imidazole may be included in a form that can be adjusted to 37.5 mM or more and 375 mM or less in the mixed state of the reagent and the sample at the time of measurement, and varies depending on each reagent type. For example, when the first reagent and the second reagent are 3: 1 and the collected sample is a trace amount, the concentration of imidazole or the like in the buffer solution as the first reagent is preferably 50 mM or more and 500 mM or less.
Further, the reagent kit of the present invention can contain, in addition to the above-described measurement reagent, instructions for use, sample collection tools (collection pipette, syringe, cotton swab, filtration filter, etc.), sample diluent, and sample extract.

(Measurement error reducing agent)
The measurement error reducing agent of the present invention is a drug for reducing measurement error derived from a blood sample in latex immunoagglutination, and contains at least imidazole or the like as an active ingredient. A reagent containing imidazole or the like in the measurement reagent can be used as it is.

(Homogeneous method)
In the present invention, the homogeneous method is a measurement that specifically detects a reaction proceeding by a measurement target substance in a mixed solution (reaction solution) of a sample and a reagent solution without performing B / F (binding / non-binding) separation. This is a measurement method called in contrast to the heterogeneous measurement method, in which the main reaction is detected after the excess components not involved in the measurement reaction are completely washed and removed by the B / F separation operation. That means. Therefore, “the latex immunoaggregation method is a method based on the homogeneous method” in the present invention means that in the following steps (1) to (3), (1) After the steps (2) and (1) of contacting with imidazole and the like, after the steps (3) and (2) of adding latex particles carrying a specific affinity substance for the substance to be measured into the liquid phase The step (3) of measuring the agglutination reaction between the substance to be measured and the latex particles is “in the middle of the step (2) or after the step (2) without passing through a washing / separation step. It means “a step of measuring the aggregation reaction between the substance to be measured and the latex particles”.

(Other reagent components)
The reagent of the present invention may contain a polymer such as polyethylene glycol, polyvinyl pyrrolidone, or phospholipid polymer as a component that enhances the formation of aggregates of insoluble carrier particles. In addition, as a component for controlling the formation of aggregates, one or more commonly used components such as proteins, amino acids, saccharides, metal salts, surfactants, reducing substances and chaotropic substances may be included, or a combination of a plurality of components may be included. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to these and is not interpreted.

[Test Example 1] Confirmation of measurement error reduction effect due to addition of imidazole of the present invention Confirmation of influence of measurement error due to lipid in blood sample, and examination of measurement error reduction effect due to addition of buffer type, imidazole of the present invention, etc. I did it.
1. Reagent (1) First reagent The following components (concentration indicates concentration in the reagent; the same shall apply hereinafter) are included.
-Buffers shown in Table 1-500 mM NaCl
・ 1.0% BSA
・ 0.05% Proclin 300
(2) Second reagent Equal amounts of the following two types of antibody-sensitized latex particle solutions were mixed, and the absorbance at a wavelength of 600 nm was 5.0 Abs. With a 5 mM MOPS buffer (pH 7.0). The second reagent was diluted so that
(I) 92212 antibody-sensitized latex particle solution 1.0% latex solution with an average particle size of 0.3 μm (5 mM Tris buffer (hereinafter referred to as Tris-HCl or simply Tris) (pH 8.5) in an equal amount of 5 mM Add 92212 antibody solution diluted to 0.36 mg / mL with Tris-HCl (pH 8.5)
Stir for 2 hours. Thereafter, an equal amount of 0.5 mM BSA-containing 5 mM Tris-HCl (pH 8.5) was added and stirred at 4 ° C. for 1 hour to prepare a 92212 antibody-sensitized latex particle solution.
(Ii) 92204R antibody-sensitized latex particle solution A 92204R antibody-sensitized latex particle solution was prepared in the same manner as (i) above using latex having an average particle size of 0.3 μm.

2. Test sample Serum A randomly extracted and mixed (pooled serum A)

3. Sample preparation method (The concentration of intralipos indicates the concentration in the sample before mixing with the reagent. The same applies hereinafter.)
1/10 amount of a fat emulsion for intravenous use (10% Intralipos Infusion (Otsuka Pharmaceutical)) mixed with an equal amount of physiological saline (Otsuka Pharmaceutical) in 9/10 amount of the test sample, 0.5% Intra A lipos sample was prepared.
1/10 volume of physiological saline was added to 9/10 volume of the test sample to prepare a 0% Intralipos sample.
0.5% Intralipos 1/5 amount was added to the test sample 4/5 amount to obtain a 0.1% Intralipos sample.

4). Measurement Method The first reagent and the second reagent were combined, and the soluble interleukin 2 receptor concentration in the sample containing the fat emulsion for vein was measured using Hitachi 7180 automatic analyzer. Specifically, 150 μL of the first reagent was added to 5 μL of the sample and kept at 37 ° C. for 5 minutes, and then 50 μL of the second reagent was added and stirred. The change in absorbance associated with the formation of aggregates was measured over 5 minutes at a main wavelength of 570 nm and a subwavelength of 800 nm, and the change in absorbance was applied to a calibration curve obtained by measuring a standard substance having a known concentration, and soluble interleukin-2 receptor The concentration was calculated (common to Test Examples 1 to 3).
The measured value of the 0% intralipos sample was taken as 100%, and the relative value of the measured value of each concentration of intralipos sample was calculated (common in Test Examples 1 and 2).

5. Measurement conditions The parameter conditions of the Hitachi 7180 automatic analyzer are shown below.
(1) Liquid volume Sample-first reagent-second reagent; 5 μL-150 μL-50 μL (2) Analytical method Two-point end method (photometric point 19-34)
(3) Measurement wavelength Main wavelength 570nm / Sub wavelength 800nm
(4) Calibration spline

6). Measurement result When the measured value of the 0% Intralipos sample is 100% and the measured value of the 0.1% Intralipos sample is less than 85% or more than 115%, the influence of the presence of Intralipos has occurred. It was judged. The results are shown in Table 1.

7). Discussion When only Tris or HEPES was used as a buffering agent in the first reagent, there was an effect due to the presence of intralipos, but when only imidazole buffer was used or when Tris or HEPES and imidazole buffer were combined Intralipos coexistence did not occur.

[Test Example 2] Relationship between imidazole concentration and measurement error reduction effect The imidazole concentration in the immune agglutination reaction solution was changed, and the relationship with the measurement error reduction effect was examined.
1. Reagent (1) First reagent The same as Test Example 1 except that the buffer shown in Table 1 was replaced with the buffer shown in Table 2.
(2) Second reagent Same as Test Example 1.

2. Test sample (1) Randomly extracted and mixed serum B (pooled serum B)
(2) Randomly extracted and mixed serum C (pooled serum C)
(3) Randomly extracted and mixed serum D (pooled serum D)

3. Sample Preparation Method The preparation method of the 0.5% Intralipos sample, 0% Intralipos sample, and 0.1% Intralipos sample is the same as Test Example 1. The 0.05% intralipos sample was prepared by adding 1/10 amount of 0.5% intralipos sample to 9/10 amount of the test sample.

4). 4. Measurement method and Measurement conditions Same as Test Example 1.

6). Measurement result 0% Intralipos sample measured value is 100%. If the measured value of 0.05% or 0.1% Intralipos sample is 85% or more and 115% or less, the influence of intralipos coexistence occurs. Not (+).
The case where no effect occurred in any of the pooled sera B to D was designated as ++, the case where no effect occurred in any two of the sera was designated as ++, and the case where no effect occurred in any one of the serums was designated as +. The results are shown in Table 2.

7). Discussion When the concentration of the imidazole buffer is in the range of 50 mM to 300 mM, it was possible to avoid the influence of Intralipos.

[Test Example 3] Examination of cause of measurement error In order to confirm whether the measurement error is based only on a blood sample containing lipid or a reagent containing latex, a reagent not containing latex is used as the second reagent. And examined.
1. Reagent (1) First Reagent Same as the first reagent in Test Example 1 except that the buffer in Table 1 is replaced with 100 mM Tris (pH 7.0). (2) Second Reagent Saline

2. Test sample Pooled serum B, pooled serum C, pooled serum D of Test Example 2

3. Sample Preparation Method Same as Test Example 2.

4). 4. Measurement method and Measurement conditions Same as Test Example 1.

6). Measurement Results and Discussion The measured absorbance is 0 to 1.2 mAbs. Met. Since no increase in absorbance dependent on intralipos concentration was observed, the effect on the measured value was not the turbidity of the blood sample due to the addition of intralipos, but the non-latency of latex particles due to contact with the blood sample containing intrapolis. Presumably due to specific aggregation.

  In latex immunoaggregation, a blood sample is contacted with a latex sample carrying a specific affinity substance for a substance to be measured in the presence of imidazole or a derivative thereof or a salt thereof in a liquid phase. It is possible to provide a method for reducing the derived measurement error. Therefore, a sample containing a large amount of lipid such as hyperlipidemia can be accurately measured by latex immunoagglutination.

Claims (5)

A method for reducing measurement errors derived from blood samples in latex immunoagglutination,
In the liquid phase, in the presence of imidazole or a derivative thereof or a salt thereof, the step of contacting the sample with latex particles carrying a specific affinity substance for the substance to be measured.
A method for reducing the measurement error. The method of claim 1, wherein the blood sample is serum or plasma. The method according to claim 1 or 2, wherein the concentration of imidazole or a derivative thereof or a salt thereof when measuring the aggregation reaction is 37.5 mM or more and 375 mM or less. The method according to any one of claims 1 to 3, wherein the latex immunoaggregation method is a method based on the homogeneous method. An agent for reducing measurement error derived from a blood sample in latex immunoagglutination, comprising imidazole or a derivative thereof or a salt thereof as an active ingredient.