JP2012251789A - IMMUNOLOGICAL MEASURING METHOD FOR HbA1c - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method that is applicable to immuno-chromatography as a simple measurement system and immunologically and accurately measures HbA1c.SOLUTION: There is provided an immuno-chromatography method for HbA1c including processes (A), (B), (C), and (D) as follows: the process (A) of treating a measurement sample containing red blood cells with a surface active agent having a property of exposing an N-terminal of a hemoglobin β chain on a protein surface; the process (B) of bringing the measurement sample obtained in the process (A) into contact with cyclic polysaccharides in a water insoluble state; the process (C) of bringing the measurement sample obtained in the process (B) into contact with an antibody recognizing the N-terminal of hemoglobin labeled with particles or an antibody recognizing other than the N-terminal of the hemoglobin label with the particles; and the process (D) of detecting an immune complex of the antibody recognizing the N-terminal of hemoglobin labeled with particles and/or the antibody recognizing other than the N-terminal of the hemoglobin label with the particles, and the HbA1c.

Description

  The present invention relates to a simple and accurate immunoassay method and reagent for HbA1c.

  The value of glycated hemoglobin (glycohemoglobin) in which sugar is bound to hemoglobin in blood, particularly hemoglobin A1c in which the N-terminal valine residue of the hemoglobin β chain is glycated (hereinafter sometimes referred to as “HbA1c”) is 1 Since it reflects the average blood glucose level of ˜2 months, it is widely used as an index suitable for diabetes diagnosis and diabetes follow-up.

  Known methods for measuring HbA1c include HPLC, capillary electrophoresis, enzymatic methods, immunological measurement, and the like. In immunoassay, HbA1c is measured using an antibody specific for the glycated portion of HbA1c (hemoglobin β-chain N-terminus), but the glycated portion is not present on the surface of the hemoglobin protein. It has been found that it is buried in the hemoglobin protein and exists in a place where the antibody is difficult to bind under physiological conditions. From such properties, a technique for exposing the epitope to the surface of the hemoglobin protein has been developed in order to efficiently react with an antibody that recognizes the glycation site as an epitope (Patent Document 1). .

  As a means for exposing the epitope to the surface of hemoglobin protein, a method of treating hemoglobin using a component that exposes the N-terminus of hemoglobin β chain to the protein surface (hereinafter sometimes referred to as “N-terminal exposing agent”) or hemoglobin can be used. A method for treating hemoglobin by adsorbing to latex particles has been reported. Examples of the exposure method using an N-terminal exposure agent include a method in which guanidine, thiocyanic acid, or lithium thiocyanate is individually mixed with a glycated hemoglobin-containing sample and exposed, or a combination of oxidizing agents such as thiocyanic acid and ferricyanide. , A method of exposing using a combination of guanidine and a nonionic surfactant and / or nitrite, an ionic surfactant, a nonionic surfactant or the like (Patent Documents 1 to 7) .

JP 61-172064 A Japanese Patent Laid-Open No. 01-155268 Japanese Patent Laid-Open No. 03-51759 Japanese Patent Laid-Open No. 06-11510 Japanese Patent Laid-Open No. 2001-33442 JP 2007-163182 A International Publication 2010-067612 Pamphlet

  However, with respect to the treatment method for exposing glycation site epitopes using these conventional N-terminal exposing agents, there are the following problems. For example, the method of exposing the N-terminus using guanidine described in Patent Document 1 requires heating and is complicated, and thiocyanic acid described in Patent Document 2 and Patent Document 3 is an environmentally harmful substance. In addition, oxidizing agents such as ferricyanide have a problem that they are easily colored and lack storage stability. Further, according to the study by the present inventors, some of the N-terminal exposing agents (chaotropic agents, reducing agents, organic solvents and surfactants) described in the prior art documents are used under the conditions described in the documents. However, it has been clarified that there are those that do not provide a sufficient epitope exposure effect. In such a case, if an N-terminal exposure agent is used at a high concentration in order to obtain a sufficient epitope exposure effect, dilution is required before immunological measurement, and the operation becomes complicated. It was also revealed that an N-terminal exposure agent that cannot be used for immunological measurement is included because the antigen-antibody reaction is inhibited even if the epitope exposure effect is sufficient. Furthermore, the present inventors used a measurement sample (hereinafter, also referred to as “specimen” or “blood sample”) after the treatment for exposing an epitope using a surfactant as an N-terminal exposure agent, as a colloidal gold particle. When applied to a measurement system using an immunochromatographic method (hereinafter sometimes referred to as “particle immunochromatographic method”) in which colored latex particles or colored latex particles are used as a labeling substance for detection, it is labeled with particles depending on the type of N-terminal exposure agent used. The present inventors have found a new problem in that accurate measurement cannot be performed because the produced antibodies (hereinafter sometimes referred to as “particle-labeled antibodies”) aggregate and do not develop on the membrane. The aggregation of the particle-labeled antibody was observed with various surfactants, and it was difficult to predict or avoid it only from attributes such as the properties and chemical structure of the surfactant. Therefore, an object of the present invention is to expose a glycation site (hemoglobin β chain N-terminus) of HbA1c that can be applied to a novel immunological measurement method of HbA1c, specifically, a particle immunochromatography method which is a simple measurement system. It is to provide an immunological measurement method of HbA1c using the treatment and the treatment method.

  The present inventors have reexamined an N-terminal exposing agent that can efficiently and easily expose the glycation site of HbA1c and does not inhibit the antigen-antibody reaction between the anti-HbA1c antibody and HbA1c, and achieves both at the same time. N-terminal exposure agent that can be obtained. However, even with these N-terminal exposing agents, the problem that particle-labeled antibodies were aggregated and could not be measured accurately remained, so further investigation was repeated. As a result, when the blood sample treated with the N-terminal exposing agent was contacted with the cyclic polysaccharide in a water-insoluble state and then reacted with the particle-labeled antibody, the particle-labeled antibody was not agglutinated between the membranes. It was found that the above problems can be solved. That is, the present inventors are able to easily and accurately measure HbA1c by particle immunochromatography without exposing the saccharification site to a detectable level, without inhibiting the antigen-antibody reaction, and without aggregation between the labeled antibodies. The method was found and the present invention was completed.

That is, the present invention relates to the following.
[1] An immunochromatographic measurement method for HbA1c, comprising the following steps (A), (B), (C) and (D).
(A) A measurement sample containing red blood cells is treated with a surfactant having the property of exposing the N-terminus of hemoglobin β chain to the protein surface. (B) The measurement sample obtained in step (A) is water-insoluble. The step (C) of contacting with the cyclic polysaccharide in the state of (C) The measurement sample obtained in the step (B) is an antibody that recognizes the N-terminus of hemoglobin labeled with particles or other than the N-terminus of hemoglobin labeled with particles Step (D) for contacting with any antibody that recognizes the antibody that recognizes the N-terminus of hemoglobin labeled with particles and / or other than the N-terminus of hemoglobin labeled with particles formed in step (C) The step of detecting an immune complex between the antibody to be treated and HbA1c [2] The cyclic polysaccharide is an α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, Roxyalkylated (α, β or γ) cyclodextrins, sulfoalkylated (α, β or γ) cyclodextrins, monochlorotriazinyl (α, β or γ) cyclodextrins, cluster cyclodextrins, modified cluster cyclodextrins and cyclodextrins The immunochromatographic measurement method according to [1], selected from the group consisting of amylose.
[3] The surfactant having the property of exposing the N-terminus of the hemoglobin β chain to the protein surface is selected from the group consisting of sucrose monolaurate, an anionic surfactant, a cationic surfactant and an amphoteric surfactant. [1] The immunochromatographic measurement method.
[4] The immunochromatographic measurement method according to [1], wherein the particles are gold colloid particles or latex particles.
[5] The immunochromatographic measurement method according to [1], wherein the cyclic polysaccharide in a water-insoluble state is chemically bonded to a solid phase or is a water-insoluble polymer of the cyclic polysaccharide.
[6] The immunochromatographic measurement method according to [1], wherein the solid phase is a sample pad in an immunochromatographic test strip.
[7] A test strip for immunochromatographic measurement of HbA1c, comprising a sample pad containing a cyclic polysaccharide in a water-insoluble state or a water-insoluble polymer of cyclic polysaccharide.

  According to the immunological measurement method of the present invention, a glycated portion in HbA1c in a blood sample can be efficiently exposed by a simple treatment in a short time, and there is no inhibition of the antigen-antibody reaction, and the particle-labeled antibody can be used. HbA1c can be accurately measured by the immunoassay to be detected. That is, according to the present invention, there is no need to dilute a sample in which the glycation site of HbA1c is exposed with an N-terminal exposing agent before measurement, and various factors that cannot be predicted only from attributes such as the chemical structure of the surfactant. It is possible to improve the aggregation of the particle-labeled antibodies caused by.

It is a schematic structure diagram of an immunochromatographic device having one antibody application part. It is a correlation diagram of the measurement result of the conventional method (enzyme method) and the measuring method of the present invention. It is a schematic structure figure of the immunochromatography device which has two antibody application parts.

  The sample used in the present invention is a test sample for measuring HbA1c, and examples thereof include blood samples such as blood and red blood cell fractions. Moreover, the measurement object of the present invention is HbA1c in blood.

The immunological measurement method of HbA1c of the present invention is as follows:
(A) A measurement sample containing red blood cells is treated with a surfactant having the property of exposing the N-terminus of hemoglobin β chain to the protein surface. (B) The measurement sample obtained in step (A) is water-insoluble. The step (C) of contacting with the cyclic polysaccharide in the state of (C) The measurement sample obtained in the step (B) is an antibody that recognizes the N-terminus of hemoglobin labeled with particles or other than the N-terminus of hemoglobin labeled with particles Step (D) for contacting with any antibody that recognizes the antibody that recognizes the N-terminus of hemoglobin labeled with particles and / or other than the N-terminus of hemoglobin labeled with particles formed in step (C) And detecting an immune complex between the antibody and HbA1c.

  Step (A) is a step of exposing the epitope in HbA1c with an N-terminal exposing agent. The epitope exposed by the step (A) is, for example, a glycated N-terminal region of hemoglobin β chain, and more preferably a region containing a peptide in which the N-terminal valine of hemoglobin β chain is glycated.

As the N-terminal exposing agent used in the step (A), a non-ionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and the glycated N-terminal region of the above-described hemoglobin β chain Can be selected as long as it does not inhibit the antigen-antibody reaction between an antibody that recognizes the N-terminus of hemoglobin or an antibody that recognizes other than the N-terminus of hemoglobin and HbA1c. It is. These N-terminal exposure agents may be used alone or in combination of two or more.
Nonionic surfactants include sucrose monolaurate.
As the anionic surfactant, anionic surfactants other than bile salts can be used, alkyl sulfates such as sodium lauryl sulfate, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, Alkylbenzenesulfonates such as sodium dodecylbenzenesulfonate, acylamino acid salts such as lauroylmethylalanine, N-lauroylsarcosine sodium salt, sodium dialkylsulfosuccinate, β-naphthalenesulfonic acid formalin condensate sodium salt, special polycarboxylic acid type polymer Surfactant is mentioned. Here, the bile salt refers to, for example, sodium cholate, sodium deoxycholate, and the like.
Examples of the cationic surfactant include higher alkylamines and quaternary ammonium salts, and quaternary ammonium salts are preferable in terms of the effect of epitope exposure. Examples of the quaternary ammonium salt include mono long chain alkyltrimethylammonium salt, dilong chain alkyldimethylammonium salt, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and decalinium chloride. Among them, the mono-long chain alkyl trimethylammonium salts, C ₈ -C ₂₂ alkyl trimethylammonium chloride, bromide C ₈ -C ₂₂ alkyl trimethylammonium, and the like. The di-long chain alkyl dimethyl ammonium salts, di chloride (C ₈ -C ₂₂ alkyl) dimethylammonium, Nioikaji (C ₈ -C ₂₂ alkyl) dimethyl ammonium and the like. Specific examples of more preferred quaternary ammonium salts include dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, bromide Hexadecyltrimethylammonium, octadecyltrimethylammonium bromide, di (dodecyl) dimethylammonium chloride, di (tetradecyl) dimethylammonium chloride, di (hexadecyl) dimethylammonium chloride, di (octadecyl) dimethylammonium chloride, di (dodecyl) dimethyl bromide Ammonium, di (tetradecyl) dimethylammonium bromide, di (hexadecyl) dimethylammonium bromide, bromide (Octadecyl) dimethylammonium, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, dequalinium chloride and the like.
As the amphoteric surfactant, amphoteric surfactants excluding bile acid derivatives can be used, and those having a betaine structure are particularly suitable. Examples of amphoteric surfactants having a betaine structure include sulfobetaine 12-16, lauryl betaine, 2-alkyl-2-carboxymethyl-N-hydroxyethylimidazolium betaine, lauryl dimethylamine oxide, amide dimethyl dimethylaminoacetate laurate Is mentioned. Here, bile acid derivatives include, for example, 3-[(3-colamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate. (CHAPSO) refers to a compound bonded to a zwitterionic group such as sulfobetaine through an amide bond formed by an acyl group derived from a bile acid.

  The N-terminal exposing agent is preferably used as an aqueous solution containing the N-terminal exposing agent (hereinafter sometimes referred to as “specimen pretreatment liquid”). The concentration of the N-terminal exposure agent in the N-terminal exposure agent-containing specimen pretreatment solution is 0.01 to 10% by mass (hereinafter simply expressed as%) from the viewpoint of the epitope exposure effect and the maintenance of the antigen-antibody reactivity of the antibody. ), More preferably 0.05 to 5%, and particularly preferably 0.1 to 3%. The sample pretreatment liquid can be used by dispensing into a tube or the like. Alternatively, it can be impregnated and dried in a membrane or the like and incorporated into a part of an immunochromatography device as a pad. . The pad needs to be arranged so as to come into contact with the sample liquid before the sample pad and the particle-labeled antibody-containing pad described later.

A hemolytic agent may be added to the sample pretreatment solution, but the N-terminal exposure agent may also serve as a hemolytic agent. The sample pretreatment solution may optionally contain a preservative such as sodium azide, a chelating agent such as EDTA, a metal ion such as NaCl or MgCl ₂ , and suppress nonspecific reaction during subsequent immunochromatographic measurements. For the purpose, proteins such as BSA and casein may be included. The sample pretreatment liquid may contain a surfactant for the purpose of improving the development of the measurement sample and the particle-labeled antibody on the membrane at the time of immunochromatography measurement, and the N-terminal exposing ability of the surfactant. Does not matter. The surfactant may be a nonionic surfactant.

  The pH of the sample pretreatment liquid is preferably 4.0 to 9.0, more preferably 5.0 to 9.0, and even more preferably 6.0 to 8.0. In order to adjust to these pH, you may add a buffer agent suitably. Examples of the buffer include citric acid, phthalic acid, acetic acid, succinic acid, cacodylic acid, maleic acid, imidazole, collidine, phosphoric acid, universal buffer such as Johnson-Lindsay, 2-morpholinoethanesulfonic acid (hereinafter referred to as “MES”). ), Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (hereinafter referred to as “Bis-Tris”), piperazine-N, N′-bis (2-ethanesulfonic acid) (hereinafter referred to as “PIPES”) Good buffering agents such as

  In order to process a measurement sample containing red blood cells using the sample pretreatment solution, the sample pretreatment solution may be added to the blood sample solution and stirred. The temperature is preferably 1 ° C to 50 ° C, more preferably 10 ° C to 40 ° C, and further preferably 15 ° C to 25 ° C. The treatment time is preferably 10 seconds to 30 minutes, and more preferably 30 seconds to 5 minutes.

  The glycated site of the glycated hemoglobin (HbA1c) in the blood sample is efficiently exposed on the hemoglobin protein surface by the treatment (step (A)).

  Next, in the present invention, the sample is brought into contact with a cyclic polysaccharide in a water-insoluble state (step (B)). The step (B) needs to be performed before the measurement sample treated with the sample pretreatment liquid comes into contact with the particle-labeled antibody for detection.

  The cyclic polysaccharide used in the step (B) includes a cyclic oligosaccharide and a cyclic polysaccharide. As the cyclic oligosaccharide, cyclodextrin or modified cyclodextrin is preferable. More specifically, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyalkylated (α, β or γ) cyclodextrin, sulfoalkylated (α, β or γ) cyclodextrin, monochlorotriazinyl (Α, β or γ) cyclodextrin (monochlorotriazinyl group may be abbreviated as “MCT”) and the like. Examples of the cyclic polysaccharide include cluster cyclodextrin, modified cluster cyclodextrin, cycloamylose and the like. Of these, cyclodextrin or modified cyclodextrin is preferred.

  The cyclic polysaccharide is used in a water-insoluble state. The water-insoluble state refers to a state in which when the cyclic polysaccharide comes into contact with water, the cyclic oligosaccharide molecule or the cyclic polysaccharide molecule constituting the cyclic polysaccharide is not dissolved and diffused in water. Specific examples of the water-insoluble state include, for example, a state in which a cyclic polysaccharide is immobilized by a chemical bond on a membrane or the like usually used in an immunochromatography method, a state in which the cyclic polysaccharide itself forms a polymer, Examples include a state of being kneaded in a porous resin. The membrane or polymer can be used as, for example, a pad constituting a part of a device in an immunochromatography method. For example, it can be chemically bonded to the fibers of the sample pad (b) in FIG. 1, or a cyclic polysaccharide polymer processed into a sheet shape so as to be in contact with the upper part or the lower part of the sample pad (b). In the present invention, there is also provided a test strip for immunochromatographic measurement of HbA1c, comprising a sample pad containing the above-mentioned cyclic polysaccharide in a water-insoluble state or a water-insoluble polymer of cyclic polysaccharide. The

  As a method of chemically bonding a cyclic polysaccharide to a solid phase, for example, a cotton cloth is immersed in a cyclodextrin (MCT-β-CD) aqueous solution into which a reactive group has been introduced, followed by air drying and heat treatment (Matsuda et al., Hiroshima Prefectural Tobu Industrial Technology Center research report No.19 p.15-19, Fig. 1 (2006)), a method using a crosslinking agent, a method of chemically bonding a cyclodextrin prepolymer with ultraviolet rays, an isocyanate It can be produced by a conventional method such as a chemical bonding method using Polymers of cyclic oligosaccharides and cyclic polysaccharides are commercially available (for example, α-cyclodextrin polymer (code No. 037-21611), β-cyclodextrin polymer (code No. 034-21621), γ-cyclodextrin. A dextrin polymer (code No. 031-221631), both of which are Wako Pure Chemical Industries, Ltd.) can be used.

  The concentration of cyclic polysaccharide used (the amount used) is 1 to 20 times the amount of the N-terminal exposure agent (surfactant) in the measurement sample reaction solution from the point of obtaining accurate HbA1c measurement results. It is preferably 1 to 10 times, more preferably 1 to 5 times. The amount of cyclic polysaccharide bound to the membrane when the cyclic polysaccharide is chemically bound to a membrane, for example, was titled “Textile Finishing with MCT-β-Cyclodextrin (Acabado textil con MCT-β-ciclodextrina)” JP Moldenhauer, H.M. It can be calculated with reference to Reuscher's document (http://revistavirtualpro.com/files/TIE09_200704.pdf). In addition, when using a cyclic polysaccharide polymer (sheet-like or bead-like), considering the amount of N-terminal exposure agent in the measurement sample, when using a sheet-like cyclic polysaccharide polymer In the case of using a bead-like cyclic polysaccharide polymer, a suitable number of particles or the like is calculated or experimentally used.

  The temperature at which the measurement sample is brought into contact with the cyclic polysaccharide is not particularly limited, and is preferably 1 ° C to 50 ° C, more preferably 1 ° C to 30 ° C, and further preferably 15 ° C to 25 ° C. The contact time between the sample and the cyclic oligosaccharide or cyclic polysaccharide is not particularly limited, but is preferably within 30 minutes, particularly preferably within 5 minutes. Even when the sample passes through a membrane or the like in which the cyclic polysaccharide is chemically bonded in a very short time of about several seconds, the effect is sufficiently obtained.

  In the step (C) of the present invention, the measurement sample obtained in the step (B) is prepared by using an antibody that recognizes the N-terminus of hemoglobin labeled with particles or an antibody that recognizes other than the N-terminus of hemoglobin labeled with particles. In step (D), the antibody formed in step (C) recognizes the N-terminus of hemoglobin labeled with particles and / or the N-terminus of hemoglobin labeled with particles. This is a detection step of an immune complex between the antibody to be recognized and HbA1c. These steps (C) and (D) are carried out in the same manner as in the usual immunochromatographic measurement method.

When measuring by immunochromatography, HbA1c can be quantified by the following method using a blood component with known HbA1c concentration (%) as a standard product. This will be described with reference to FIG. First, the measurement sample solution processed in the step (A) of the present invention is dropped onto the sample pad (b). The dropped measurement sample solution is drawn into the particle-labeled antibody-containing pad (c) carrying the particle-labeled anti-hemoglobin antibody for detection by capillary action, and the hemoglobin (HbA1c) in the measurement sample passes through the pad. , HbA0, HbF, HbS, etc.) form a complex by reacting with the particle-labeled antibody and antigen-antibody. The complex develops on the antibody-immobilized membrane (d) on which the anti-HbA1c antibody is applied (immobilized) in a line shape. When the complex reaches the anti-HbA1c antibody application part (e), HbA1c in the measurement sample in the application part. And captures HbA1c by antigen-antibody reaction. Other hemoglobin moves to the water absorption pad (f) without being captured. HbA1c in the sample can be quantified by measuring the color intensity, reflection intensity, absorbance, etc. derived from the labeled particles in the line that has captured HbA1c with an immunochromatographic reader.
Since the binding ratio of HbA1c and other hemoglobin (HbA0, HbF, HbS, etc.) to the anti-hemoglobin antibody is the same as that in the sample, a sufficient amount of anti-HbA1c antibody is immobilized on the antibody-immobilized membrane. In this case, since the amount of detection particles bound to HbA1c captured by the anti-HbA1c antibody reflects the HbA1c ratio in the specimen, HbA1c (%) in the sample can be obtained from the above quantitative result.

  The antibody immobilized on the antibody-immobilized membrane may be an anti-HbA1c antibody alone, or an antibody different from the anti-HbA1c antibody that does not affect HbA1c measurement is immobilized independently of the HbA1c line. Also good. For example, when a substance that does not exist in the human body is labeled with particles and added to the particle-labeled antibody-containing pad (c) and an antibody that recognizes the substance is applied independently of the anti-HbA1c antibody, The line can be used as a control line. The strength of the control line can be used to ensure the validity of individual measurements, and can also be used to correct the HbA1c line strength as needed.

  Furthermore, when measuring by immunochromatography, HbA1c and hemoglobin A0 (hereinafter referred to as “HbA0”) in which the β-chain N-terminus of hemoglobin is not modified can be measured simultaneously. At this time, blood components containing known concentrations (μM) of HbA1c and HbA0 are used as standard products. Hereinafter, a description will be given with reference to FIG. First, an antibody-immobilized membrane (d) in which the anti-HbA1c antibody and the anti-HbA0 antibody are immobilized at different sites (anti-HbA1c antibody application part (e) and anti-HbA0 antibody application part (f)) is used. The sample liquid processed in the step (A) of the present invention is dropped onto the sample pad (a). The dropped sample liquid is developed on the antibody-immobilized membrane (d) by capillary action, and when it reaches the anti-HbA1c antibody application part (e), only HbA1c in the sample reacts and is applied to the anti-HbA0 antibody application part (f). When it reaches, only HbA0 in the sample reacts. Other hemoglobin moves to the water absorption pad (h) without reacting. HbA0 and HbA1c in the sample can be quantified by measuring the color intensity, reflection intensity, absorbance, etc. of both antibody coating portions with an immunochromatographic reader. Here, assuming that the quantitative value of HbA0 is A and the quantitative value of HbA1c is B, HbA1c (%) can also be obtained as HbA1c (%) = (B / (A + B)) × 100.

  Here, the anti-HbA1c antibody may be a monoclonal antibody or a polyclonal antibody, and for example, those described in patent documents (Patent Document 1, JP-A-6-66796) can be used.

  The detection label used in the immunochromatography method of the present invention is a fine particle that can be used as an immunological measurement reagent, and metal colloid particles or latex particles are preferable. Examples of the metal colloid particles include gold colloid particles, silver colloid particles, platinum colloid particles, iron oxide colloid particles, aluminum hydroxide colloid particles, and the like. In particular, gold colloid particles can be preferably used. These metal colloidal particles have an average particle diameter of 1 to 500 nm, more preferably in the range of 10 nm to 150 nm, more preferably 20 to 100 nm at which a particularly strong color tone is obtained. For example, when gold colloid particles are used as the metal colloid particles, the gold colloid particles can be prepared by a conventional method, for example, a method of reducing chloroauric acid with sodium citrate, or commercially available ones may be used. .

  The latex particles are not particularly limited as long as they are latex particles generally used as an immunological measurement reagent, and may be appropriately colored with a dye. Latex particles can be obtained by polymerizing or copolymerizing various monomers. Examples of monomers include polymerizable unsaturated aromatics such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene and vinyltoluene, for example, polymerization of (meth) acrylic acid, itaconic acid, maleic acid, fumaric acid and the like. Unsaturated carboxylic acids such as methyl (meth) acrylate, ethyl (meth) acrylate, (n-butyl) (meth) acrylate, 2-hydroxyethyl (meth) acrylate, glycidyl (meth) acrylate, ethylene Polymerizable unsaturated carboxylic acid esters such as glycol-di- (meth) acrylic acid ester, tribromophenyl (meth) acrylic acid, (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide, N-methylol- ( (Meth) acrylamide, methylenebis (meth) acrylamide, butaji Unsaturated carboxylic acid amides such as ethylene, isoprene, vinyl acetate, vinyl pyridine, N-vinyl pyrrolidone, vinyl chloride, vinylidene chloride, vinyl bromide, polymerizable unsaturated nitriles, vinyl halides, conjugated dienes, etc. Can be mentioned. These monomers are appropriately selected depending on the surface characteristics and specific gravity required as a labeling substance, and can be used alone or in combination of two or more.

  The shape of the latex particles is not particularly limited, but the average particle size is desirably large enough to allow the aggregates resulting from the aggregation reaction between the protein on the latex particle surface and the substance to be measured to be detected visually or optically. The average particle diameter is preferably 0.02 to 1.6 μm, particularly preferably 0.03 to 0.5 μm.

  The method for sensitizing the latex particles with the anti-HbA1c antibody is not particularly limited, and a known method can be used. For example, a method of physically adsorbing on the latex particle surface, a method of covalently bonding to the latex particle surface having a functional group, a method of sensitizing by immunological binding, and the like can be mentioned.

  In the immunochromatography method, the step (B) is preferably performed on a sample pad. That is, if the measurement sample solution obtained by immobilizing the cyclic oligosaccharide or cyclic polysaccharide on the sample pad and performing the step (A) is dropped on the sample pad, the step (B) is performed on the sample pad. The measurement sample solution subjected to the step (B) moves to the next immune reaction field by capillary action. The “particle-labeled antibody-containing pad” containing the particle-labeled antibody is used after being placed in contact with the sample liquid after the sample pad.

  EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.

Reference Example 1: Preparation of anti-hemoglobin antibody and anti-HbA1c antibody (I) Material and method (1) Preparation of purified HbA0 and purified HbA1c HbA0 and HbA1c are described in non-patent literature (Melisenda J. McDonald et al, JBC, 253 (7 ), 2327-2332, 1978), and purified from human erythrocyte lysate by ion exchange chromatography using Bio-Rex70 (Bio-Rad), and used in subsequent experiments.

(2) Preparation of various peptides and glycated peptides Peptides of two types of amino sequences (VHLTC (SEQ ID NO: 1) and VHLTPEEKYYC (SEQ ID NO: 2): the alphabet indicates one letter of amino acids) are used with an automatic peptide synthesizer It was synthesized and purified by the Fmoc method. The purity of each peptide was confirmed to be 95% or higher by HPLC. Moreover, it confirmed that the molecular weight of each peptide was the same as a theoretical value by mass spectrometry (MALDI-TOF method).
The two types of peptides were saccharified by the method described in Patent Document 1, and glycated peptides (f-VHLTC and f-VHLTPEEKYC: f means fructosylation) were purified. That is, each peptide of each sequence was reacted with glucose in anhydrous pyridine to synthesize a glycated peptide and purified by HPLC. The molecular weight of each glycated peptide was confirmed by mass spectrometry (MALDI-TOF method) to be the same as the theoretical value, that is, the molecular weight obtained by adding 162 to the molecular weight of each peptide.

(3) Preparation of Peptide Binding Protein and Glycated Peptide Binding Protein Peptide binding protein and glycated peptide binding protein are prepared by using the peptide or glycated peptide prepared in (2) above as carrier protein ovalbumin ( OVA). That is, a peptide (VHLTC) dissolved in a concentration of 5 mg / mL in 150 mmol / L NaCl-containing 20 mmol / L phosphate buffer (pH 7.2) (hereinafter sometimes collectively referred to as “PBS”), or a glycated peptide (F-VHLTC) was mixed with maleimide active ovalbumin (manufactured by PIERCE) dissolved in purified water at a concentration of 5 mg / mL at a volume ratio of 1: 1, and then 2 while gently rotating the reaction vessel at room temperature. Prepared by incubating for hours and dialyzed against PBS before use (VHLTC-OVA, f-VHLTC-OVA).

(4) Preparation of anti-hemoglobin antibody and anti-HbA1c antibody As the anti-hemoglobin antibody, a mouse monoclonal antibody prepared by a conventional method using the purified HbA0 of (1) above as an immunogen was used. The monoclonal antibody has a property of reacting with both purified HbA1c and purified HbA0 in an antigen-immobilized ELISA.
As the anti-HbA1c antibody, a mouse monoclonal antibody prepared by the method described in Patent Document 1 was used. That is, the glycated peptide (f-VHLTPEEKYYC) synthesized in the above (2) was bound to squash hemocyanin and used as an immunogen. In the hybridoma screening, a strain that reacted with purified HbA1c in an antigen-immobilized ELISA but did not react with purified HbA0 was selected. Through cloning, a monoclonal antibody that finally reacted with purified HbA1c in an antigen-immobilized ELISA and did not react with purified HbA0 was obtained.

Example 1: Confirmation of N-terminal exposure of hemoglobin and inhibition of antigen-antibody reaction by N-terminal exposure agent candidate (I) Materials and methods (1) N-terminal exposure rate confirmation test of N-terminal exposure agent candidate by competitive ELISA method (a ) After glycated peptide-binding protein (f-VHLTC-OVA) was diluted with PBS to a concentration of 1 μg / mL, 50 μL / well was dispensed into a 96-well microplate and allowed to stand at 4 ° C. overnight.
(B) The microplate of (a) above was washed 3 times with PBS containing 0.05% Tween 20 (registered trademark) (hereinafter referred to as “PBST”) 400 μL / well, and then a blocking solution (1% bovine serum albumin (BSA) PBST (hereinafter referred to as “BSA-PBST”) was dispensed at 100 μL / well on a microplate and allowed to stand at room temperature for 1 hour to prepare an f-VHLTC-OVA immobilized plate.
(C) Blood collected by an EDTA blood collection tube was centrifuged at 10 ° C. and 3000 rpm for 5 minutes to separate blood cells, and the blood cells were frozen and thawed at −70 ° C. or lower to lyse red blood cells to obtain hemolyzed red blood cells. (When hemolyzed erythrocytes were used as samples in the subsequent tests, they were prepared in the same manner). The N-terminal exposure agent candidates listed in Tables 1 and 2 are prepared using a 5 mmol / L MES buffer solution (pH 6.0) containing the concentrations shown in Tables 1 and 2 as a sample pretreatment solution, The treatment liquid was well mixed at a volume ratio of 1:50 and allowed to stand at 25 ° C. After 5 minutes, the mixture was diluted 32 times with 5 mmol / L MES (pH 6.0) to obtain a sample solution. As a negative control, a 5 mmol / L MES buffer solution (pH 6.0) not containing the N-terminal exposure agent candidates listed in Tables 1 and 2 was used as a sample pretreatment solution, and hemolyzed erythrocytes were prepared in the same manner as described above. And a negative control sample solution was prepared.
(D) After washing the microplate of (b) three times with PBST, each sample solution of (c) was dispensed into the microplate in a volume of 25 μL / well. Subsequently, 25 μL / well of anti-HbA1c antibody diluted to 1.0 μg / mL with BSA-PBST was dispensed onto a microplate and shaken at room temperature for 1 hour.
(E) After washing the microplate of (d) three times with PBST, a solution obtained by diluting Polyclonal Goat Anti-mouse Immunoglobulins / HRP (manufactured by Dako Denmark A / S) 5000 times with BSA-PBST at 50 μL / well Each was dispensed into microplates and shaken at room temperature for 1 hour.
(F) After washing the microplate of (e) three times with PBST, a citrate buffer solution containing orthophenylenediamine hydrochloride (Tokyo Kasei Co., Ltd.) at a concentration of 2 mg / mL and hydrogen peroxide at a concentration of 0.02% ( 50 μL / well of pH 5.0) was dispensed on a microplate and allowed to stand at room temperature for 10 minutes.
(G) The reaction was stopped by dispensing 1.5 N sulfuric acid at 50 μL / well onto the microplate of (f), and then the absorbance at 492 nm was measured with a plate reader.

(2) Calculation of N-terminal exposure rate The competitive ELISA method of (1) is a competitive inhibition method using a glycated peptide immobilized on a plate and HbA1c in a sample solution. In the case of a negative control in which the N-terminus of the hemoglobin is not exposed because the N-terminal exposure agent is not present in the sample pretreatment solution, competition between the glycated peptide and HbA1c does not occur in the measurement system. Become. On the other hand, competition increases as the degree of N-terminal exposure by the N-terminal exposure agent increases, and the measured absorbance decreases. From the absorbance measured in (1) and (g), the exposure rate (degree of competition) of each N-terminal exposure agent candidate is calculated using the following formula, and the N-terminal exposure effect of each N-terminal exposure agent candidate is calculated. It was used as an index. It can be said that the greater the exposure rate, the higher the N-terminal exposure effect. In this example, the case where the exposure rate was 10% or more was used as a criterion for determining that “the N-terminal exposure effect was present”.
Exposure rate (%) = [(A−B) / A] × 100
A: Absorbance of negative control B: Absorbance of each sample solution

(3) Anti-antibody reaction inhibition rate confirmation test of N-terminal exposure agent candidate by ELISA method In the procedure (c) of (1) competitive ELISA method, an N-terminal exposure agent candidate at the concentrations shown in Table 1 and Table 2 The procedure was the same as (1) above, except that the 5 mmol / L MES buffer solution (pH 6.0) was used as it was as a sample solution without mixing with hemolyzed erythrocytes.

(4) Calculation of inhibition rate of antigen-antibody reaction against anti-HbA1c antibody When N-terminal exposure agent candidate inhibits antigen-antibody reaction between anti-HbA1c antibody and glycated peptide immobilized on plate, measured with increasing degree of inhibition Absorbance decreases. From the measured absorbance, the antigen-antibody reaction inhibition rate of each N-terminal exposure agent candidate was calculated using the following formula, and used as an index of the antigen-antibody reaction inhibition effect of each N-terminal exposure agent candidate. It can be said that the greater the inhibition rate, the higher the antigen-antibody reaction inhibition effect. In this example, in consideration of the high concentration of the N-terminal exposure agent candidate, a case where the inhibition rate considered that the antigen-antibody reaction is established from a practical viewpoint is less than 70% is referred to as “antigen-antibody reaction”. It was set as a criterion for determining that “do not inhibit”.
Inhibition rate (%) = [(C−D) / C] × 100
C: Absorbance of negative control D: Absorbance of each sample solution

(II) Results (1) Comparison of N-terminal exposure rate and antigen-antibody reaction inhibition rate of each N-terminal exposure agent candidate N of each N-terminal exposure agent candidate calculated by (I) (2) and (4) The end exposure rate and the antigen-antibody reaction inhibition rate are shown in the respective columns of Tables 1 and 2.

  Among the surfactants tested as N-terminal exposure agent candidates, those determined to have “the N-terminal exposure effect” based on the above exposure rate criteria are sucrose among sucrose fatty acid ester types in nonionic surfactants. For monolaurate and anionic surfactant, sulfate type, sulfonate type, carboxylic acid type, polycarboxylic acid polymer type, aromatic sulfonic acid formalin condensate, for cationic surfactant, ammonium type, benzalkonium type The amphoteric surfactant was a surfactant belonging to the category of betaines (sulfobetaine type, alkylbetaine type, amide betaine type, etc.) excluding bile acid derivatives (where bile acid derivatives are derived from bile acids) And a zwitterionic group such as sulfobetaine via an amide bond formed by the acyl group of This result was the same even when tested with anti-HbA1c antibodies other than those used in this example. The surfactant categories in Tables 1 and 2 are generally used classifications, for example, “Surfactant Analysis Study Group” (1987), “New Surfactant Analysis Method”, Koshobo, Tokiyuki Yoshida et al. Ed. (2000), “New Edition Surfactant Handbook”, Engineering Books Co., Ltd., Japan Patent Office Homepage, “Standard Technology Collection (Agricultural Chemicals Technology) Database: Surfactant, http://www.jpo.go.jp” Based on the classification described in “/shiryou/s_sonota/hyoujun_gijutsu/nouyaku/0005.html” and the like, it is classified in consideration of product information such as catalogs of commercial products.

Among the surfactants tested as N-terminal exposure agent candidates, those determined as “does not inhibit the antigen-antibody reaction” based on the inhibition rate criterion are the interfaces determined as having the “N-terminal exposure effect”. It was an activator (sucrose monolaurate, anionic surfactant, cationic surfactant, amphoteric surfactant).
Based on the above, the surfactant group determined as “having an N-terminal exposure effect” and determined as “does not inhibit the antigen-antibody reaction” in this example was selected as the “N-terminal exposure agent of the present invention”.

Of the components other than the surfactants tested as N-terminal exposure agent candidates, the criteria for determining the exposure rate and inhibition rate of this example were simultaneously met with a chaotropic agent (guanidine hydrochloride, guanidine hydrochloride, sodium nitrite, Polyoxyethylene sorbitan monolaurate (Tween 20 (registered trademark) mixture). Urea, which is widely used as a protein denaturant, did not inhibit the antigen-antibody reaction, but did not show any N-terminal exposure effect. When the reducing agents L-cysteine and dithiothreitol were used, an N-terminal exposure effect was observed, but at the same time, the antigen-antibody reaction was greatly inhibited. When dimethyl sulfoxide, dimethylformamide, and 2-butanol, which are organic solvents, were used, the antigen-antibody reaction was not inhibited while the N-terminal exposure effect was negligible.
Based on the above, the chaotropic agent was selected as the “N-terminal exposure agent of the present invention”.

  In the N-terminal exposure rate confirmation test by the competitive ELISA method of this example, it was confirmed by the following that the decrease in absorbance was not due to inhibition of the antigen-antibody reaction by the N-terminal exposure agent. The sample pretreatment solution containing each selected N-terminal exposure agent of the present invention was directly diluted 32-fold with 5 mmol / L MES buffer (pH 6.0) without adding hemolyzed erythrocytes. N-terminal exposure agent but no hemolyzed erythrocytes) and compared to negative control (no N-terminal exposure agent but hemolyzed erythrocytes). When any of the selected N-terminal exposure agents of the present invention was used, the effect on the N-terminal exposure was sufficiently small, and there was no inhibition of the antigen-antibody reaction. In this test, the concentration of the selected N-terminal exposure agent of the present invention was changed to a 32-fold diluted concentration of (I) (3) above, and the antigen-antibody reaction inhibition rate confirmation test by the ELISA method of this example was performed. It corresponds to a thing.

  Further, instead of f-VHLTC-OVA, a plate immobilized with hemoglobin and an anti-hemoglobin antibody described later were used and selected in the same manner as in Examples (I) 1 (3) and (4). The presence or absence of antigen-antibody reaction inhibition by each N-terminal exposure agent of the present invention was confirmed, and it was determined that any N-terminal exposure agent did not inhibit the antigen-antibody reaction. As described above, the sample is detected by the sandwich method with the anti-HbA1c antibody or the like without damaging the recognition site of the anti-hemoglobin antibody by the selected N-terminal exposure agent of the present invention or without inhibiting the antigen-antibody reaction with the anti-hemoglobin antibody. It was confirmed that the hemoglobin contained therein could be detected.

Comparative Example 1: Measurement of HbA1c by particle immunochromatography (1)
(I) Materials and Methods (1) Untreated Sample Pad As an untreated sample pad, a cellulose membrane (Millipore, Surewick (registered trademark) C083) was used.

(2) Preparation of gold colloid particle-labeled antibody-containing pad The absorbance at 527 nm is 1.0 O.D. D. Anti-hemoglobin antibody was added to a gold colloid particle aqueous solution (particle size 50 nm, pH 9.0) at a final concentration of 1.5 μg / mL and stirred at room temperature for 10 minutes. Further, BSA was added to the gold colloid-antihemoglobin antibody mixed solution so as to have a final concentration of 0.3%, and the mixture was stirred at room temperature for 5 minutes for blocking treatment. Subsequently, the mixed solution was centrifuged at 10,000 rpm for 45 minutes at 10 ° C. to obtain a sediment (gold colloid particle-labeled anti-hemoglobin antibody). The resulting gold colloid particle-labeled hemoglobin antibody has an absorbance at 4.01 nm at 531 nm. D. / ML was suspended in 20 mmol / L PBS (pH 7.0) containing 1.3% (w / v) casein to prepare a colloidal gold particle labeled antibody solution. A glass fiber sheet (Nippon Pole Co., No. 8964) was immersed in the gold colloid particle-labeled antibody solution, drained to such an extent that the liquid did not drip, and then dried with a drier to obtain a gold colloid particle-labeled antibody-containing pad. .

(3) Preparation of latex particle-labeled antibody-containing pad 1% (w / v) red latex suspension (polystyrene latex particles, particle size 150 nm, 20 mmol / L Tris buffer (pH 8.5)) Hemoglobin antibody was added to a final concentration of 0.25 mg / mL and stirred at 4 ° C. for 2 hours. Further, BSA was added to the latex-anti-hemoglobin antibody mixed solution so as to have a final concentration of 1%, and the mixture was stirred at 4 ° C. for 1 hour for blocking treatment to obtain a latex particle-labeled anti-hemoglobin antibody. The obtained latex particle-labeled anti-hemoglobin antibody was dialyzed three times using 100-fold amount (v / v) of 20 mmol / L phosphate buffer (pH 7.0), and then the final concentration of casein was applied to the latex particles. It added so that it might become 1.3% (w / w), and it was set as the latex particle labeled antibody liquid. A glass fiber sheet (Nippon Pole Co., No. 8964) was immersed in the gold colloid particle-labeled antibody solution, drained to such an extent that the liquid did not drip, and then dried with a drier to obtain a gold colloid particle-labeled antibody-containing pad. .

(4) Preparation of antibody-immobilized membrane To 10 mmol / L phosphate buffer (pH 7.0), 1.0 mg / mL anti-HbA1c antibody and 2.5% (w / v) sucrose were added. And an anti-HbA1c antibody immobilization reagent (hereinafter sometimes referred to as “A1c antibody”). The anti-A1c antibody was applied in a line shape on a nitrocellulose membrane (Millipore, HF180) (FIG. 1 (e)) and dried with a dryer to obtain an antibody-immobilized membrane (FIG. 1 (d) )). Hereinafter, the line on which the antibody for A1c is applied (FIG. 1 (e)) may be referred to as “A1c line”.

(5) Preparation of immunochromatographic device The antibody-immobilized membrane (d) prepared in (4) above is attached to the plastic adhesive sheet (a), and the untreated sample pad (b), (2) or (3) above is used. The two types of produced particle-labeled antibody-containing pads (c) and water-absorbing pads (Whatman, BTS-SP300) (f) were arranged as shown in FIG. That is, in the antibody-immobilized membrane (d) prepared in (4) above, the particle-labeled antibody-containing pad (c) prepared in (2) or (3) in the vicinity of the upstream end in the developing direction of the measurement sample. Any one of these was placed, and the untreated sample pad (b) was placed so as to partially overlap the particle-labeled antibody-containing pad (c). Further, a water absorbing pad (f) was disposed at the downstream end in the development direction of the measurement sample of the antibody-immobilized membrane (d) prepared in (4) so as to partially overlap the membrane. After placing each member, it was further covered with a transparent plastic seal (g). The bonded sheet was cut to a width of 6 mm to obtain a test strip. The outer dimension of the test strip was 6 mm × 70 mm (width × length). At the time of measurement, it was housed and mounted in a plastic dedicated housing (having a sample addition window portion and a detection window portion, which housing is not shown in FIG. 1) to form an immunochromatography device.

(6) Preparation of Specimen Solution The N-terminal exposure agent selected in Example 1 was added at 10 mmol /% containing 0.3% BSA and 0.05% Procrine (registered trademark) 300 so that the concentrations shown in Table 3 were obtained. Dissolved in L Tris buffer (pH 7.2) (hereinafter sometimes referred to as “BSA-Tris”) to prepare a sample pretreatment solution. Subsequently, 1 μL of hemolyzed erythrocyte was added to 500 μL of each sample pretreatment solution and mixed well to obtain a sample solution.

(7) Measurement 30 seconds after the mixing, 150 μL of the sample solution was dropped on the sample pad of the immunochromatography device prepared in (5) above, and the accumulation of each particle-labeled antibody on the A1c line (hereinafter referred to as “A1c detection line Sometimes called “formation”). Further, 15 minutes after dropping, the test strip is taken out from the housing, and in the vicinity of the contact portion between the end of the particle-labeled antibody-containing pad (c) and the antibody-immobilized membrane (d) in the side view shown in the upper part of FIG. Aggregation of colloidal gold particle-labeled antibody or latex particle-labeled antibody at “label pad contact” was visually observed.

(II) Results Table 3 shows the results. As the N-terminal exposing agent selected in Example 1, sucrose monolaurate (nonionic surfactant), sodium lauryl sulfate (anionic surfactant), lauryltrimethylammonium bromide (cationic surfactant), lauryl A sample solution obtained by treating hemolyzed erythrocytes with a sample pretreatment solution containing betaine (amphoteric surfactant) and guanidine hydrochloride (chaotropic agent) was dropped onto an untreated sample pad and measured, and the following results were obtained. .

(1) When detected with colloidal gold particle-labeled antibody Except in the case of a sample liquid containing sodium lauryl sulfate, the formation of the A1c detection line is poorly reproducible. In the case of a sample liquid containing guanidine hydrochloride, the A1c detection line Was not formed. Further, except for the sample solution containing sodium lauryl sulfate, aggregation of the colloidal gold particle labeled antibody at the label pad contact was confirmed. The degree of aggregation of colloidal gold particle-labeled antibody was greatest when guanidine hydrochloride or lauryltrimethylammonium bromide was used as the N-terminal exposure agent, but clearly when sucrose monolaurate or laurylbetaine was used. It could be confirmed.

(2) When Detected with Latex Particle Labeled Antibody In the case of the sample liquid containing guanidine hydrochloride, the A1c detection line was not formed, and the formation of the A1c detection line was poorly reproducible even with other N-terminal exposure agents. In addition, in the case of the sample solution containing any N-terminal exposing agent, aggregation of latex particle labeled antibody at the label pad contact was confirmed. The degree of aggregation of the latex particle-labeled antibody is greatest when guanidine hydrochloride or lauryltrimethylammonium bromide is used as the N-terminal exposure agent, and then when laurylbetaine or sodium lauryl sulfate and sucrose monolaurate are used. It was in order.

  As described above, even when sodium lauryl sulfate that did not show aggregation of colloidal gold particle labeled antibody was used as the N-terminal exposure agent, it was selected in Example 1 because the aggregation of latex particle labeled antibody was confirmed. When a sample solution containing an N-terminal exposure agent is to be widely applied to the particle immunochromatography, the particle-labeled antibody may be aggregated regardless of the category of the N-terminal exposure agent, and the measurement system by the particle immunochromatography may not be established. confirmed.

  In Table 3, “-” does not recognize the aggregation of the particle-labeled antibody or formation of the A1c detection line, and “+”, “++”, and “++++” indicate the degree of aggregation of the particle-labeled antibody depending on the number of +. Or the formation of the A1c detection line was clear. “±” indicates that the reproducibility of the A1c detection line formation was poor.

Comparative Example 2: Confirmation of improvement effect of particle-labeled antibody aggregation by cyclic polysaccharide (1)
(I) Materials and methods (1) Preparation of cyclic polysaccharide aqueous solution 5 mmol / L MES buffer (pH 6.0) containing any of the cyclic polysaccharides shown in Table 4 and containing the concentrations shown in Table 4, A cyclic polysaccharide aqueous solution was prepared. Also, calixarenes having a structure similar to a cyclic polysaccharide in which sugars are cyclically bonded (phenols are cyclically bonded) were prepared in the same manner as described above and tested as reference conditions.

(2) Production of immunochromatographic device Untreated sample pad described in Comparative Example 1 (I) (1), gold colloid particle-labeled binding antibody-containing pad described in (2), and antibody described in (4) Using the immobilized membrane, an immunochromatographic device was produced in the same manner as described in (5).

(3) Preparation of specimen liquid BSA-Tris was prepared by adding lauryltrimethylammonium bromide (N-terminal exposure agent selected in Example 1; cationic surfactant) to 0.75% (w / v). Dissolved and used as a sample pretreatment solution. Subsequently, 1 μL of hemolyzed erythrocyte was added to 500 μL of the sample pretreatment solution and mixed well to obtain a sample solution. 30 seconds after the mixing, 50 μL of the cyclic polysaccharide aqueous solution prepared in the above (1) was mixed with 80 μL of the sample solution to obtain a cyclic polysaccharide-containing sample solution, which was immediately used for measurement.
Separately, α-cyclodextrin was dissolved in BSA-Tris so as to be 50 mmol / L, and the N-terminal exposure agent and hemolyzed erythrocyte were not added, and used as a control sample solution as it was.

(4) Measurement 150 μL of each cyclic polysaccharide-containing specimen solution was dropped onto the sample pad of the immunochromatography device prepared in (2), and the formation of the A1c detection line was confirmed. Further, 15 minutes after the dropping, the test strip was taken out of the housing, and the aggregation of the colloidal gold particle labeled antibody at the label pad contact was visually confirmed. In addition, the calixarenes tested as reference conditions were not subjected to the operation after dropping to the sample pad because the sample solution became cloudy when mixed with the sample solution containing lauryltrimethylammonium bromide.

(II) Results Table 4 shows the results. When any cyclic polysaccharide-containing sample liquid is measured, the formation of the A1c detection line is confirmed, but the formed detection line is not clear as compared to the case where the cyclic polysaccharide-free sample liquid is measured. The reproducibility was still poor. In addition, the aggregation of colloidal gold-labeled antibody at the contact point of the label pad was improved compared to the measurement of the sample liquid without addition of cyclic polysaccharide in any cyclic polysaccharide, but A1c detection was performed. Due to the poor line formation, the effect was not sufficient in practice.
In addition, when measuring a sample solution for control that does not contain an N-terminal exposure agent and hemolyzed erythrocytes, it is aggregated at a label pad contact, although the degree is smaller than when a sample solution containing no cyclic polysaccharide is measured. Was confirmed. Conventionally, cyclodextrin is known for its effect of stabilizing the dispersion of fine particles. Therefore, the aggregation of colloidal gold particle-labeled antibody occurs in the measurement of a control sample liquid that is a mixture of cyclodextrin and colloidal gold particle-labeled antibody. However, it was unexpectedly found that the colloidal gold particle-labeled antibody may be aggregated. From the above, it was confirmed that the measurement could not be applied to the particle immunochromatography method simply by making the cyclic polysaccharide coexist with the N-terminal exposing agent.
In addition, instead of the cyclic polysaccharide, a non-cyclic sugar aqueous solution was prepared using glucose, which is a constituent sugar of cyclodextrin, and was operated in the same manner as in this comparative example, but the formation of the particle-labeled antibody aggregation and the A1c detection line was This was the same as in the case where no cyclic polysaccharide was added. From the above, it was confirmed that the cyclic polysaccharide is effective in improving particle-labeled antibody aggregation and A1c detection line formation.

  In Table 4, “+” and “++” indicate that the degree of particle-labeled antibody aggregation is large or the formation of the A1c detection line was clear depending on the number of +. “±” indicates that the reproducibility of the line formation was poor.

Comparative Example 3: Confirmation of improvement effect of particle-labeled antibody aggregation by cyclic polysaccharide (2)
(I) Materials and methods (1) Preparation of cyclodextrin-immersed sample pad In purified water, α-cyclodextrin (Nacalai Tesque, 10005-82) or monochlorotriazinized β-cyclodextrin (Cyclochem, CAVASOL (registered trademark)) W7 MCT) was dissolved to 10% (w / v) to prepare an aqueous solution of cyclic polysaccharide. After immersing the untreated sample pad of Comparative Example 1 (I) (1) in the cyclic polysaccharide aqueous solution for 10 minutes, draining it to such an extent that the liquid does not drip, and then drying under reduced pressure at room temperature. A sample pad was used.

(2) Preparation of immunochromatographic device Comparative Example 1 (I) The untreated sample pad described in (1) or the two types of cyclodextrin-immersed sample pads prepared in (1) above, and Comparative Example 1 (I) (2) Using the gold colloid particle-labeled bound antibody-containing pad described in 1 and the antibody-immobilized membrane described in Comparative Example 1 (I) (4), an immunochromatography is performed in the same manner as in Comparative Example 1 (I) (5). A device was fabricated. Depending on the type of sample pad incorporated in each device, device A (untreated sample pad), device B (α-cyclodextrin soaked sample pad), device C (monochlorotriazinized β cyclodextrin soaked sample pad) (Table) 5)

(3) Preparation and Measurement of Specimen Solution BSA- is prepared so that lauryltrimethylammonium bromide (N-terminal exposure agent selected in Example 1; cationic surfactant) is 0.75% (w / v). Dissolved in Tris to prepare a sample pretreatment solution. Subsequently, 1 μL of hemolyzed erythrocyte was added to 500 μL of the sample pretreatment solution and mixed well to obtain a sample solution. The sample solution was measured in the same manner as in Comparative Example 1 (I) (7).

(II) Results Table 5 shows the results. In the device A (untreated sample pad), the formation of the A1c detection line was not observed at all, and the agglomerates of colloidal gold particle-labeled antibodies were confirmed in a dense state at the label pad contact, and the results of Comparative Examples 1 and 2 were reproduced. did. As described above, when the sample liquid containing the N-terminal exposing agent is applied to the particle immunochromatography as it is, aggregation of the particle-labeled antibody occurs and the measurement system is not established. In contrast, Device B (α-cyclodextrin soaked sample pad) and Device C (monochlorotriazinized β cyclodextrin soaked sample pad) had poor reproducibility of A1c detection line formation, but gold at the label pad contact point. The aggregation of colloidal particle-labeled antibodies was clearly improved with respect to device A in both devices B and C. One of the reasons why the formation of the A1c detection line is not improved while the aggregation of the colloidal gold particle labeled antibody is improved is considered to be the influence of the viscosity of the cyclodextrin solution. That is, the cyclodextrin contained in the sample pad in the dry state is redissolved by adding the sample solution, leaks from the sample pad, reaches the antibody-immobilized membrane, and develops on the membrane together with the sample solution. Therefore, the development of the sample liquid on the membrane becomes uneven or hindered, and the sample liquid (particularly the immune complex of HbA1c and particle-labeled antibody) cannot reach the A1c line uniformly and sufficiently. It is possible that
As described above, it is confirmed that the cyclic polysaccharide has an effect of suppressing or improving the aggregation of the colloidal gold particle-labeled antibody by the N-terminal exposure agent, and the particle immunochromatography is caused by its high viscosity. It proved to be an obstacle to application to the law.

  In Table 5, “-” does not recognize the aggregation of the particle-labeled antibody or formation of the A1c detection line, and “+”, “++”, and “++++” indicate the degree of aggregation of the particle-labeled antibody depending on the number of +. Or the formation of the A1c detection line was clear. “±” indicates that the reproducibility of the line formation was poor.

Example 2: Confirmation of Improvement Effect of Particle Labeled Antibody Aggregation by Cyclic Polysaccharide Using Method of the Present Invention (I) Material and Method (1) Preparation of Cyclodextrin Chemical Binding Sample Pad 4% of sodium carbonate in purified water (W / v), monochlorotriazinylated β-cyclodextrin (Cyclochem, CAVASOL (registered trademark) W7 MCT) was dissolved to 10% (w / v). After immersing the untreated sample pad of Comparative Example 1 (I) (1) in the liquid for 10 minutes, the liquid was drained to such an extent that the liquid did not drip, and then dried under reduced pressure at room temperature. Subsequently, after heating at 150 ° C. for 10 minutes, it was immersed in purified water to wash and remove unreacted raw materials, and further dried at 70 ° C. for 30 minutes to obtain a cyclodextrin chemical bond sample pad.

(2) Preparation of immunochromatographic device The untreated sample pad described in Comparative Example 1 (I) (1) or the cyclodextrin chemical binding sample pad prepared in (1) above and Comparative Example 1 (I) (2) or comparison Comparative Example 1 (I) (5) using the two types of particle-labeled bound antibody-containing pads described in Example 1 (I) (3) and the antibody-immobilized membrane described in Comparative Example 1 (I) (4) An immunochromatographic device was prepared in the same manner as described in 1).

(3) Preparation and measurement of sample solution As the N-terminal exposure agent selected in Example 1, sucrose monolaurate, sodium lauryl sulfate, lauryltrimethylammonium bromide, laurylbetaine, guanidine hydrochloride were used, and Comparative Example 1 (I ) A sample solution was prepared in the same manner as in (6). The measurement was performed in the same manner as in Comparative Example 1 (I) (7).

(II) Results Table 6 shows the results.
(1) When detected with colloidal gold particle labeled antibody Except when guanidine hydrochloride was used as the N-terminal exposure agent, the formation of the A1c detection line was confirmed clearly and with good reproducibility. In addition, the aggregation of colloidal gold-labeled antibody at the label pad contact was remarkably improved except for the case where guanidine hydrochloride was used as the N-terminal exposure agent, and was not visually confirmed.

(2) When detected with a latex particle-labeled antibody Except when guanidine hydrochloride was used as the N-terminal exposure agent, the formation of the A1c detection line was confirmed clearly and with good reproducibility. Further, the aggregation of the latex particle-labeled antibody at the label pad contact was remarkably improved by sodium lauryl sulfate, lauryltrimethylammonium bromide, and laurylbetaine, and was not visually confirmed. With sucrose monolaurate, aggregation was reduced compared to using an untreated sample pad, but it was sometimes observed slightly. In the case of guanidine hydrochloride, aggregation of the latex particle-labeled antibody was still confirmed.

From the above, immunochromatographic devices that store and mount test strips incorporating cyclodextrin chemical bond pads as sample pads are non-ionic surfactants, anionic surfactants, cationic surfactants, amphoteric as N-terminal exposure agents. Any of the surfactants can be used in the sample pretreatment, and any of the colloidal gold particles and latex particles used as the detection label particles was effective in improving aggregation.
In addition, when guanidine hydrochloride is used as the N-terminal exposure agent, one of the reasons why the effect of improving the particle aggregation and the formation of A1c line was lower than that when the surfactant was used as the N-terminal exposure agent was N It is conceivable that the amount (concentration) of guanidine hydrochloride required for terminal exposure is higher than the amount (concentration) of the surfactant group. From this, it is presumed that the effect of the present invention can be obtained by adjusting the amount ratio of the cyclic polysaccharide to the guanidine hydrochloride in the sample liquid to an appropriate range.

  In Table 6, “-” indicates no aggregation of the particle-labeled antibody or formation of an A1c detection line, and “+” and “++” indicate that the degree of aggregation of the particle-labeled antibody is large depending on the number of “+” or A1c detection. It shows that the formation of the line was clear.

Example 3: Quantitative measurement of HbA1c using the method of the present invention (I) Material and method (1) Production of immunochromatographic device Example 2 (I) Cyclodextrin chemical binding sample pad described in (1), comparative example Using the colloidal gold particle-labeled bound antibody-containing pad according to 1 (I) (2) and the antibody-immobilized membrane according to Comparative Example 1 (I) (4), the method described in Comparative Example 1 (I) (5) An immunochromatographic device was produced in the same manner as the method.

(2) Comparative measurement Blood collected by an EDTA blood collection tube was centrifuged at 3000 rpm for 5 minutes at 10 ° C. to separate red blood cells. Enzymatic HbA1c measurement reagent “Nordia (registered trademark) N HbA1c” (Sekisui Medical Co., Ltd.), “Nordia (registered trademark) HbA1c pretreatment solution for HbA1c” (Sekisui Medical Co., Ltd.), and general-purpose automatic analyzer “BM9130” (JEOL Ltd.) and HbA1c (%) (HbA1c concentration relative to the total hemoglobin concentration in the specimen) was measured according to the package insert.

(3) Preparation of specimen liquid BSA-Tris was prepared by adding lauryltrimethylammonium bromide (N-terminal exposure agent selected in Example 1; cationic surfactant) to 0.75% (w / v). Dissolved and used as a sample pretreatment solution. 1 μL of red blood cells obtained in (2) above was added to 500 μL of the sample pretreatment liquid and mixed well to prepare a sample liquid.

(4) Measurement 30 seconds after the mixing, 150 μL of the sample solution was dropped on the sample pad of the immunochromatography device prepared in (1), and the A1c line absorbance (reflected light) 10 minutes later was measured by Rapid Pier (Hamamatsu Photonics). ).

(II) Results Table 7 and FIG. 2 show the results of measurement of erythrocytes for 10 persons using the immunochromatographic device of the present invention and Nordia (registered trademark) N HbA1c. In the measurement using the immunochromatography device incorporating the cyclodextrin chemical bonding pad of the present invention, the absorbance depending on the concentration of HbA1c (%) measured using Nordia N HbA1c was measured (correlation coefficient r = 0.964). ). From the above, it was confirmed that according to the method of the present invention, the HbA1c concentration relative to the total hemoglobin concentration in the specimen can be measured.

Reference Example 2: Preparation of anti-HbA0 specific antibody (I) Material and method (1) Material Anti-HbA0 antibody is prepared by using the peptide binding protein (VHLTC-OVA) prepared in Reference Example 1 (I) (3) as an immunogen. A mouse monoclonal antibody prepared by a conventional method was used. In the hybridoma screening, a strain that reacted with purified HbA0 in an antigen-immobilized ELISA but did not react with purified HbA1c was selected. The clone selected in the screening was cloned, and the hybridoma producing the anti-HbA0 monoclonal antibody was incorporated into the National Institute of Advanced Industrial Science and Technology (November 28, 2008, 1-1-1 Higashi 1-chome, Tsukuba, Ibaraki, Japan). Deposited at the center 6). The deposit number is as follows.
Antibody number: 85201
Accession Number: FERM BP-11187

  Each of the N-terminal exposing agents listed in Tables 1 and 2 was prepared in the same manner as in Example 1 (I) 1 (3) and (4) using the purified HbA0-immobilized plate and the anti-HbA0 antibody. As a result, it was determined that any of the N-terminal exposure agents did not inhibit the antigen-antibody reaction. As described above, the recognition site of the anti-HbA0 antibody is not impaired by the selected N-terminal exposure agent of the present invention, or the antigen-antibody reaction with the anti-HbA0 antibody is not inhibited, and the sample is isolated in the sample by the sandwich method with the anti-hemoglobin antibody. It was confirmed that HbA0 was detectable.

Example 4: Simultaneous detection of HbA1c and HbA0 using the method of the present invention (1) Preparation of antibody-immobilized membrane Anti-HbA0 antibody prepared in Reference Example 2 against 10 mmol / L phosphate buffer (pH 7.0) Was added at 1.0 mg / mL and sucrose at 2.5% (w / v) to obtain an anti-HbA0 antibody immobilization reagent (hereinafter referred to as A0 antibody). As the antibody for A1c, the antibody for A1c described in Reference Example 1 (I) (4) was used. The two types of antibody immobilization reagents are applied to a nitrocellulose membrane (Millipore, HF180) in the order of the A1c antibody (FIG. 3 (e)) and the A0 antibody (FIG. 3 (f)) in the order of development. The antibody-immobilized membrane was applied in a line shape with an interval between each other and dried with a dryer (FIG. 3D). Hereinafter, the line to which the antibody for A1c is applied (FIG. 3 (f)) may be referred to as “A0 line”.

(2) Production of immunochromatographic device An immunochromatographic device was produced in the same manner as in Example 3 except that the antibody-immobilized membrane produced in (1) was used as the antibody-immobilized membrane.

(3) Preparation of specimen liquid Lauryltrimethylammonium bromide was dissolved in BSA-Tris so as to be 0.75% (w / v) to obtain a specimen pretreatment liquid. Subsequently, 1 μL of hemolyzed erythrocyte was added to 500 μL of the sample pretreatment solution and mixed well to obtain a sample solution.

(4) Measurement 30 seconds after the mixing, 150 μL of the sample solution is dropped onto the sample pad of the immunochromatography device prepared in (2) above, and the accumulation of the particle-labeled antibody on the A1c line or A0 line (hereinafter referred to as “A1c or The formation of the A0 detection line ”may be confirmed. Further, 15 minutes after dropping, the test strip is taken out from the housing, and in the vicinity of the contact portion between the end of the particle-labeled antibody-containing pad (c) and the antibody-immobilized membrane (d) in the side view shown in the upper part of FIG. Aggregation of colloidal gold particle-labeled antibody or latex particle-labeled antibody at “label pad contact” was visually observed.

(II) Results In the measurement using the immunochromatographic device incorporating the cyclodextrin chemical bond pad of the present invention, formation was successfully confirmed in both the A1c detection line and the HbA0 detection line. Also, no aggregation of colloidal gold particles at the label pad contact was observed. As described above, in the method of the present invention, HbA1c and HbA0 can be simultaneously detected by particle immunochromatography, and a method of directly measuring HbA0 and calculating HbA1c (%), which has not been achieved in the past, can be easily carried out. Became possible.

Claims (7)

An immunochromatographic measurement method for HbA1c, comprising the following steps (A), (B), (C) and (D).
(A) A measurement sample containing red blood cells is treated with a surfactant having the property of exposing the N-terminus of hemoglobin β chain to the protein surface. (B) The measurement sample obtained in step (A) is water-insoluble. The step (C) of contacting with the cyclic polysaccharide in the state of (C) The measurement sample obtained in the step (B) is an antibody that recognizes the N-terminus of hemoglobin labeled with particles or other than the N-terminus of hemoglobin labeled with particles Step (D) for contacting with any antibody that recognizes the antibody that recognizes the N-terminus of hemoglobin labeled with particles and / or other than the N-terminus of hemoglobin labeled with particles formed in step (C) Detecting an immunocomplex of an antibody that binds to HbA1c   Cyclic polysaccharides are α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyalkylated (α, β or γ) cyclodextrin, sulfoalkylated (α, β or γ) cyclodextrin, monochlorotriazinyl The immunochromatographic measurement method according to claim 1, wherein the method is selected from the group consisting of (α, β or γ) cyclodextrin, cluster cyclodextrin, modified cluster cyclodextrin and cycloamylose.   The surfactant having the property of exposing the N-terminus of hemoglobin β chain to the protein surface is selected from the group consisting of sucrose monolaurate, anionic surfactant, cationic surfactant and amphoteric surfactant. Item 4. The immunochromatographic measurement method according to Item 1.   The immunochromatographic measurement method according to claim 1, wherein the particles are gold colloid particles or latex particles.   The immunochromatographic measurement method according to claim 1, wherein the cyclic polysaccharide in a water-insoluble state is chemically bonded to a solid phase or a water-insoluble polymer of the cyclic polysaccharide.   The immunochromatographic measurement method according to claim 1, wherein the solid phase is a sample pad in an immunochromatographic test strip.   A test strip for immunochromatographic measurement of HbA1c, comprising a sample pad containing a cyclic polysaccharide in a water-insoluble state or a water-insoluble polymer of cyclic polysaccharide.

Images