JP7436838B2 - Method for measuring hemoglobin A1c using antibody-dendrimer complex - Google Patents

Description

The present invention relates to a means for measuring a specific component in blood using an artificial material, and more specifically, it relates to a means for measuring hemoglobin A1c using an antibody-dendrimer complex.

Hemoglobin A1c (HbA1c) in blood is widely used as a diagnostic marker for diabetes, which is less affected by diet and exercise before blood collection.

Methods for measuring hemoglobin A1c in blood are broadly divided into HPLC methods, immunological methods, and enzymatic methods. As an aspect of the immunological method, measurements using insoluble carrier particles such as latex or colloidal gold are performed. There is.

For example, a latex reagent for measuring hemoglobin A1c sensitizes the surface of latex particles in the first reagent to hemoglobin in blood, and aggregation of the latex particles caused by an anti-hemoglobin A1c antibody contained in the second reagent. The blood hemoglobin A1c is generally measured using this method.

On the other hand, a dendrimer is a nano-sized particle having a dendritic polymer structure from the center (D. A. Tomalia et al., Polym. J., 17, 117 (1985)).

Patent Document 1 discloses an embodiment in which an antibody is supported on a dendrimer.

Patent Document 2 discloses an assay for detecting biological signals from a dendrimer carrying an antibody.

Non-Patent Document 1 discloses an immunoassay in which nanoparticles are synthesized using a dendrimer as a template, and antibodies are supported on the nanoparticles.

Non-Patent Document 2 discloses an ELISA method using a single dendrimer as a sensitizer.

Special Publication No. 1987-501876 Special Publication No. 10-507778

Xiaomei Pei et al., RSC Adv., 2013, 3, 16410-16415 Arai, Tsukada et al., “Biological sample analysis” Vol.35, No 3 (2012), p216-224

In the latex reagent for hemoglobin A1c measurement, when performing aggregation of latex particles by the above-mentioned antigen-antibody reaction, in order to promote the aggregation, for example, "anti-hemoglobin A1c antibody is maintained in a state where it can bind to hemoglobin A1c". In addition, an antibody-antibody complex (generally a complex of multiple anti-hemoglobin A1c antibodies and an anti-anti-hemoglobin A1c antibody that binds to a site other than the antigen-binding site of the anti-hemoglobin A1c antibody) is prepared in advance and added. Things are being done. However, the above-mentioned antibody-antibody complexes have poor storage stability, and fluctuations in measured values are particularly marked due to increases in storage temperature.Furthermore, although the aggregation promoting effect of each lot is excellent, there is variation in performance between lots. The problem is that it is recognized.

The present inventor focused on a complex of an antibody and a dendrimer, which is a synthetic nanoparticle, and used it in a measurement assay using insoluble carrier particles for hemoglobin A1c, thereby solving the problems associated with the use of the antibody-antibody complex described above. The present invention was completed by discovering that it is possible to solve the problem.

That is, the present invention first provides a dendrimer complex in which the whole or part of an antibody against hemoglobin A1c is bound to a dendrimer in a state capable of binding to hemoglobin A1c, to an insoluble carrier particle sensitized with a hemoglobin component in a blood sample. This is a measurement method (hereinafter also referred to as the measurement method of the present invention) in which hemoglobin A1c in the blood sample is measured using the aggregation of the insoluble carrier particles as an indicator by contacting the blood sample with the blood sample in an aqueous liquid.

Second, the present invention is directed to a dendrimer in which the whole or part of an antibody against hemoglobin A1c is bonded to hemoglobin A1c in an aqueous solution to an insoluble carrier particle sensitized with a hemoglobin component in a blood sample. This is a method of using a dendrimer complex (hereinafter also referred to as a method of using the present invention), in which the insoluble carrier particles are brought into contact with the bound dendrimer complex to cause aggregation of the insoluble carrier particles.

Thirdly, the present invention provides a content of an insoluble carrier particle for sensitizing a hemoglobin component in a blood sample, and an antibody against hemoglobin A1c, in whole or in part, bound to a dendrimer in a state capable of binding to hemoglobin A1c. This is a hemoglobin A1c measurement kit (hereinafter also referred to as the kit of the present invention), which contains a dendrimer complex comprising:

By the measuring method of the present invention, the method of use of the present invention, and the kit of the present invention (hereinafter also referred to as "the present invention" including all these categories), hemoglobin A1c in a blood sample can be measured by agglutination of insoluble carrier particles. It is possible to prevent disturbances in measured values when measuring as an index. In particular, it is possible to prevent disturbances in measured values when the reagent is stored at room temperature or above. Note that room temperature is approximately 25°C, but in practical terms, reagents should be placed in an environment (non-refrigerated environment) that is not subject to ``refrigerated storage'' (approximately 2-10°C), which is the principle of reagent storage. This means the risk of temperature rise. Such non-refrigerated storage may include the above-mentioned "room temperature or higher." For example, problems with transportation of reagents, carelessness in handling reagents, etc. are cited as reasons for the above-mentioned non-refrigerated storage. In addition, since a dendrimer complex with a relatively large dendrimer, which is a homogeneous artificial product, is used as an agglutination-promoting component, the performance between lots is improved compared to when an antibody, which is a pure natural product, is used as an agglutination-promoting component. Not only is there little variation, but it is also advantageous in terms of cost.

(1) Insoluble carrier particles The "insoluble carrier particles" in the present invention are not particularly limited as long as they can be used as immunoassay reagents. The "insolubility" of "insoluble carrier particles" refers to the property with respect to specimens and solvents used in ordinary clinical tests. Examples of the "insoluble carrier particles" include latex particles, colloidal gold particles, silica particles, and gelatin particles. Among these, latex particles are fine particles of polymer contained in latex, and colloidal gold particles are fine particles of gold contained in colloidal gold.

A typical example of the insoluble carrier particles used in the present invention is latex particles. Latex, also called a polymer emulsion, is a product in which a polymer is dispersed in an aqueous solvent such as water, where the aqueous solvent becomes the continuous phase and polymer particles in the shape of true spheres or near spheres form the discontinuous phase. . As mentioned above, latex particles are polymer particles that form the discrete phase of the latex.

A further typical example is colloidal gold particles. Gold colloid is a dispersion of fine particles made of bonded gold atoms, and is synthesized by a method such as reducing tetrachloroauric (III) acid in a liquid. In other words, Au ³⁺ ions are reduced to gold atoms, some of them combine to reach a supersaturated state, and then nuclear particles of 1 nm or less are generated. Unbonded gold particles combine with these one after another to form particles. Synthesized through growth. By sufficiently stirring during the synthesis process, it is possible to make the particle size uniform. Although a stabilizer such as citric acid is added to prevent fine particles (colloidal gold particles) from agglomerating, they still have the potential to cause aggregation. The color of gold colloids is due to surface plasmon resonance, and gold colloids with monodisperse particle sizes have absorption at a single wavelength. The particle size of the gold colloid particles in the gold colloid is smaller than that of the latex particles, and the specific surface area relative to unit weight is larger.

(2) Sensitization of the hemoglobin component in the blood specimen by insoluble carrier particles In the present invention, it is necessary to sensitize the hemoglobin component in the blood specimen to the insoluble carrier particles. Then, the hemoglobin component sensitized to the insoluble carrier particles is subjected to the aggregation step of the insoluble carrier particles using hemoglobin A1c contained in the hemoglobin component using a predetermined antibody-dendrimer complex.

The blood specimen used in the present invention is a blood sample separated from a living body of a mammal, preferably a human, and must contain the hemoglobin component of the blood of the specimen source, such as a human. Therefore, the blood sample is preferably a whole blood sample or a hemolyzed sample.

In the present invention, "sensitization" refers to the act of attaching a hemoglobin component of a blood sample, which is an antigen, to an insoluble carrier particle, or a state in which it is attached, and has the same meaning as "supporting." Furthermore, "adsorption" is also included in "sensitization." Sensitization of the hemoglobin component to the insoluble carrier particles can be carried out by bringing the insoluble carrier particles into contact with the hemoglobin component of the blood sample or its diluted product (including hemolysate). For example, when the blood sample is a whole blood sample, the whole blood sample is subjected to hemolysis treatment such as mixing with a hypotonic solution, and the resulting hemolysate or its diluted product is brought into contact with insoluble carrier particles to obtain the desired Insoluble carrier particles sensitized with hemoglobin components can be obtained. In the above-mentioned contact step, the whole blood sample before hemolysis and the insoluble carrier particles are prepared separately, and after the hemolysis treatment, the lysed hemolysate and the insoluble carrier particles or their dispersion are mixed, and the hemoglobin component and the insoluble carrier are mixed. It is possible to carry out this process by making the particles coexist in the liquid phase. It is also possible to perform the hemolysis/sensitization step by directly mixing the whole blood sample and insoluble carrier particles or a dispersion thereof. In this direct mixing mode, it is also possible to use the dispersion of insoluble carrier particles itself as a hypotonic solution for hemolysis, and after the above-mentioned direct mixing, a hypotonic solution for hemolysis is separately added to carry out the hemolysis/sensitization process. It is also possible to do so. The hypotonic solution for hemolysis or the dispersion solvent for insoluble carrier particles is not particularly limited as long as it satisfies the predetermined objectives such as hemolysis, dispersion, and both hemolysis and dispersion, and water, physiological saline, Aqueous solvents such as various buffers are suitable. Examples of such buffers include, but are not limited to, buffers commonly used in reagents and the biochemical field, such as glycine buffer, borate buffer, phosphate buffer, and Good's buffer. isn't it. Furthermore, additives such as BSA, gum arabic, surfactants, choline, chelating agents, preservatives, citrate, betaine, and ω-aminocarboxylic acids may also be added in a qualitative or quantitative manner that does not substantially impair the effects of the present invention. Additions can be made within reasonable limits. The pH of the aqueous solvent is preferably within a range that does not interfere with the antigen-antibody reaction, specifically about 4-9, particularly preferably about 6-9.

A hemolyzed sample is a blood sample obtained by subjecting a whole blood sample to hemolytic treatment as described above, and the step of sensitizing hemoglobin components to insoluble carrier particles using this sample is performed in the same manner as above. This can be carried out by mixing carrier particles or a dispersion thereof to make the hemoglobin component and insoluble carrier particles coexist in the liquid phase.

(3) Dendrimer complex The dendrimer complex used in the present invention is "a dendrimer complex in which the whole or part of an antibody against hemoglobin A1c is bound to a dendrimer in a state capable of binding to hemoglobin A1c" (hereinafter also referred to as dendrimer complex). ).

Dendrimer is a tree-like multi-branched polymer with a regular branching structure, and each molecule has a nanometer-scale structure with a clear spatial configuration, and the branched chains extend radially from the center while repeating branching. There is. Generation is a concept that corresponds to the degree of polymerization of linear polymers in dendrimers, and those in which one layer of building block molecules is bonded around a core molecule are called "first generation," and those in which two layers are bonded are called "generation." Those with n-layer bonding are called "nth generation". Only the core molecule is considered to be the "0th generation". As the generation increases, the molecular size of the dendrimer increases, and the number of end groups increases exponentially. Methods for producing dendrimers are already known, and various methods are known. Typical manufacturing methods include the "divergent method," in which molecules that serve as building blocks are bonded radially, and the "convergent method," in which dendrons, which form the partial structure of the dendrimer, are synthesized from the outside, and finally the dendrons are bonded to the core molecule. Examples include [“Dendrimers and Other Dendritic Polymers”, J.M.J.Frechet and D.A. Tomalia (Eds.), (John Wiley & Sons, 2011)]. In addition, various manufacturing methods are provided. Moreover, dendrimers are also commercially available, and it is also possible to manufacture dendrimer composites using commercially available products. The type of dendrimer used in the present invention is not particularly limited, and examples thereof include polyamide amine dendrimer, polypropylene imine dendrimer, benzyl ether type dendrimer, polyethylene glycol linear dendrimer, and the like. Furthermore, the number of generations of the dendrimer used in the present invention can be 1 to 5 generations, preferably 2, 3, or 4 generations.

In the dendrimer complex, the whole or part of an antibody against hemoglobin A1c is bound to the above-mentioned dendrimer in a state capable of binding to hemoglobin A1c. The antibody against hemoglobin A1c may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Furthermore, the class of the antibody is not limited, and may be any of IgA, IgG, IgM, IgD, and IgE as long as it has the ability to bind to hemoglobin A1c. Furthermore, antibodies derived from animals other than humans can be used as long as they have the ability to bind to hemoglobin A1c. Further, the antibody may be the whole antibody or only a portion thereof, but it is necessary that the antibody contains a portion that binds to hemoglobin A1c, specifically, a variable region. It is presumed that the binding property of the dendrimer complex to hemoglobin A1c can be achieved by including, on the dendrimer surface, the whole or part of the antibody oriented so that the part of the antibody that binds to hemoglobin A1c is exposed. be done. There are no particular limitations on the method of binding the whole or part of the dendrimer to the antibody, but for example, it can be produced by adding an activated terminal group capable of binding to an antibody to the terminal group of the dendrimer and binding the antibody to this. be able to. At this time, avoiding the part that binds to hemoglobin A1c of the whole or part of the antibody to be bound, and separately adding an activated terminal group that has binding affinity to the activated terminal group added to the above-mentioned dendrimer, Although it is possible to bond activated end groups to each other, in consideration of economic efficiency, etc., the addition process of activated end groups is performed without limiting the position, and the activated end groups are added to the desired position with a reasonable probability. ``Probabilistic processing'' that expects the addition of ``probabilistic processing'' is practical. These methods are disclosed in, for example, Patent Documents 1 and 2, and are well-known techniques. The number of whole or partial antibodies against hemoglobin A1c that can be bound to one dendrimer can be one, but by using two or more antibodies, the aggregation reaction of insoluble carrier particles can be carried out efficiently. It is possible to cause Hereinafter, the above-mentioned "whole or part of the antibody" will also be referred to as "antibody element."

(4) Contact between the dendrimer complex and insoluble carrier particles sensitized with a hemoglobin component The dendrimer complex of the present invention and insoluble carrier particles sensitized with a hemoglobin component in a blood sample (hereinafter also referred to as sensitized carrier particles) The contacting in (2) is carried out in an aqueous liquid. The contacting step in the aqueous liquid is performed subsequent to the step of preparing the sensitized carrier particles, that is, the step of sensitizing the hemoglobin component in the blood sample to the insoluble carrier particles. Therefore, the aqueous solution may be the "blood sample or diluted product thereof" in the above-mentioned sensitized carrier particle preparation process. As mentioned above, the blood sample is preferably a whole blood sample or a hemolyzed sample. The dilution solvent of these dilutions is used as a hypotonic solution for hemolysis or a dispersion solvent for insoluble carrier particles, and is used for specific purposes such as hemolysis, dispersion, and both hemolysis and dispersion. There is no particular limitation as long as it is satisfactory, and aqueous solvents such as water, physiological saline, and various buffer solutions are suitable. Examples of such buffers include, but are not limited to, buffers commonly used in reagents and the biochemical field, such as glycine buffer, borate buffer, phosphate buffer, and Good's buffer. isn't it. Furthermore, additives such as BSA, gum arabic, surfactants, choline, chelating agents, preservatives, citrate, betaine, and ω-aminocarboxylic acids may also be added in a qualitative or quantitative manner that does not substantially impair the effects of the present invention. Additions can be made within reasonable limits. The pH of the aqueous solvent is preferably within a range that does not interfere with the antigen-antibody reaction, specifically about 4-9, particularly preferably about 6-9.

In addition to the embodiment of the above-mentioned "blood sample or its dilution," the prepared sensitized carrier particles may be separately dispersed in another aqueous liquid and the above-mentioned contact step may be performed in the dispersion. It is possible. The other aqueous liquids used here are similar to the diluent solvents used in the diluent described above.

(5) Measurement of hemoglobin A1c The measurement of hemoglobin A1c using the measurement method or measurement kit of the present invention is preferably the "agglutination method" in which the aggregation of insoluble carrier particles is used as an indicator of antigen-antibody reaction, as described above. It is. Examples of the aggregation method include a slide test method, an optical measurement method, a microtiter method, and a filter separation method. These methods are also methods for measuring insoluble carrier particles aggregated by the method of use of the present invention.

According to the present invention, the measurement of hemoglobin A1c, which is an important indicator of diabetes, is performed using the aggregation of insoluble carrier particles as an indicator, by suppressing variations in measurement values due to temperature rise in non-refrigerated environments and lot differences in the reagent itself. A means by which this can be done is provided.

1 is a drawing showing the results of measuring hemoglobin A1c of a human whole blood sample using the measuring method of the present invention.

[Dendrimer composite]
In producing the dendrimer complex, it is necessary to bind an antibody element against hemoglobin A1c to the dendrimer in a state capable of binding to hemoglobin A1c. "Binding in a state capable of binding to hemoglobin A1c" means that the total or partial amount of the "antibody element against hemoglobin A1c" bound to the dendrimer is bound in such a way that the ability to bind to hemoglobin A1c is maintained. It is realized by This ability to bind to hemoglobin A1c is presumed to be due to the fact that the portion that binds to hemoglobin A1c is oriented toward the outside of the dendrimer. Although it is preferable that the degree of such orientation is high, from the viewpoint of economic efficiency, etc., the activation end group is added to the antibody element without limiting the position, and the desired orientation is achieved with a reasonable probability. It is practical to use a "stochastic process" in which the addition of an activated end group is expected to occur at a position.

The addition of the antibody element against hemoglobin A1c to the dendrimer preferably utilizes or introduces an activated end group on the surface of the dendrimer, and chemically bonds the activated end group with the binding target of the antibody element. This is realized by As the binding target in the antibody element for hemoglobin A1c, predetermined amino acid residues originally present in the antibody element may be used as they are, or may be used after being subjected to a predetermined treatment. Alternatively, it may be an amino acid residue that has been partially specifically substituted. Further, other activated end groups capable of chemically bonding with the activated end groups introduced onto the surface of the dendrimer are introduced into all or part of the binding targets of the antibody, and these activated end groups are bonded to each other. It is also possible to chemically bond them.

The activated terminal group on the surface of the dendrimer or the activated terminal group to be introduced is not particularly limited, but may include an amino group, a carboxyl group, a hydroxy group, a sulfhydryl (SH-) group, an azide group, an alkynyl group, an aldehyde group, Examples include maleimide groups. The introduction of activated end groups usually involves the introduction of cross-linkers or spacers carrying these activated end groups onto the surface of the dendrimer. For example, N-hydroxy ester (NHS ester) cross-linking agents such as sulfo-SMCC, Disuccinimidyl suberate (DSS), Bis (sulfosuccinimidyl) suberate (BS3), imido ester cross-linking agents, maleimide cross-linking agents, haloacetyl cross-linking agents, pyridyl sulfide cross-linking agents, etc. Bridge agent etc. are exemplified.

The binding target on the antibody side is not particularly limited, but includes amino acid residues, such as side chain amino groups of lysine residues or sulfhydryl groups. Side chain amino groups of lysine residues are abundant on the surface of antibody proteins and are highly reactive, making it easy to form chemical bonds, but on the other hand, it is difficult to control the binding site. accompanied by. Therefore, when the side chain amino group of a lysine residue is used as a binding target, the above-mentioned "stochastic treatment" is suitable. When using a sulfhydryl group as a binding target for an antibody, the disulfide bond between the chains of the antibody can be cleaved with a reducing agent, or a primary amine (e.g., Although it is necessary to introduce a sulfhydryl group into the side chain amino group of the lysine residue mentioned above, it is easier to achieve a more homogeneous dendrimer complex than when using the side chain amino group of the lysine residue. . Furthermore, a cross-linker or spacer is also introduced to the binding target on the antibody side, and a chemical bond is formed between the cross-linker or spacer and the activated end group of the above-mentioned dendrimer to produce a dendrimer complex. You can also. In this case, as a cross-linker or spacer used as a binding target on the antibody side, 2-iminothiolane hydrochloride (Traut's reagent) or the like can be used. Note that the progress of the dendrimer complex reaction can be controlled by performing the inactivation treatment according to the activated terminal group on the dendrimer side or the antibody side.

The binding density of the antibody elements in the dendrimer complex can be controlled by adjusting the ratio (molar ratio) of the above-mentioned dendrimer and antibody elements subjected to the binding reaction. The preferred ratio is dendrimer:antibody from 1:1 to 1:5 (molar ratio).

[Insoluble carrier particles]
Even if the insoluble carrier particles used in the present invention are in a freeze-dried state before use, they are used as a dispersion of insoluble carrier particles at least when used. For example, latex particles are used in the form of latex, which is a system in which latex particles are dispersed in an aqueous phase, and colloidal gold particles are used in the form of colloidal gold, which is a system in which fine gold particles are dispersed in an aqueous phase. used.

The average particle diameter of the insoluble carrier particles is not particularly limited as long as it can be used in an immunoassay reagent. For example, latex particles can be selected from a wide range of average particle diameters (mode diameters) of approximately 0.01-1 μm. Gold colloid particles can be selected from a wide range of average particle diameters (mode diameters) of 0.005-0.1 μm.

Among the insoluble carrier particles, the type of latex is not limited as long as it can sensitize the hemoglobin component of blood. Typical examples include latex for physical adsorption such as polystyrene latex, high-density polystyrene latex, extremely low carboxylic acid-modified latex, latex with localized hydrophilic groups, and colored latex thereof, but other latexes, such as This does not exclude the use of chemical bonding latexes such as carboxylic acid-modified latexes, amino-modified latexes, hydroxy-modified latexes, glycidyl-modified latexes, aldehyde-modified latexes, amide-modified latexes, etc., and magnetic latexes.

[Measurement of hemoglobin A1c]
The measurement method of the present invention is "a measurement method in which a dendrimer complex is brought into contact with sensitized carrier particles in an aqueous liquid, and hemoglobin A1c in a blood sample is measured using the aggregation of the sensitized carrier particles as an indicator", The outline is as described above.

The aggregation of sensitized carrier particles due to the specific binding of hemoglobin A1c sensitized to sensitized carrier particles in an aqueous solution and the antibody element against hemoglobin A1c bound to the dendrimer complex is used as an indicator. Hemoglobin A1c can be measured. The amount of the dendrimer complex added at this time is not limited as long as the insoluble carrier can be agglomerated to an extent that allows measurement of hemoglobin A1c in the blood sample, and the amount of the dendrimer complex added is not limited. It is preferable to set the range within which the aggregation promoting effect of the insoluble carrier particles commensurate with the increase, and those skilled in the art can easily select the range according to specific conditions. For example, when using a dendrimer complex as the second reagent as shown in Examples, the mixing ratio with the first reagent, which is a dispersion of insoluble carrier particles, is 3:1 (first reagent: second reagent = 3:1). (capacity ratio), the concentration of the dendrimer complex in the second reagent is approximately 0.01-0.5 mg/mL. Although the concentration of the insoluble carrier particles in the first reagent in this example is assumed to be 0.1% by mass of the entire first reagent, it is not limited to this.

In particular, in the measurement method of the present invention, even when the dendrimer composite is placed in a non-refrigerated environment, that is, at room temperature or higher, stable data similar to that obtained when stored in a refrigerated environment can be obtained. can be obtained. In the measurement, for example, a quantitative value of hemoglobin A1c in the blood sample can be obtained by creating a calibration curve using a standard hemoglobin A1c and applying the measured values in the blood sample to this. This quantitative value can be used as basic data for diagnosing diabetes in the sample donor. This basic data is applied to the existing standard for diabetes diagnosis based on the quantitative value of hemoglobin A1c, and a diagnosis of diabetes in the sample donor is made. If a diagnosis of diabetes is made, depending on the degree of diagnosis, guidance on exercise therapy and diet therapy, or prescription of antidiabetic drugs will be given. Examples of therapeutic drugs for diabetes include oral hypoglycemic drugs such as biguanides, thiazolidines, DPP-4 inhibitors, sulfonylureas, rapid-acting insulin secretagogues, α-glucosidase inhibitors, and SGLT2 inhibitors.

[Measurement kit]
As described above, the measurement kit of the present invention is "a hemoglobin A1c measurement kit containing an insoluble carrier particle content and a dendrimer complex." The content of the insoluble carrier particles is, for example, "latex" in the case of latex particles, and "gold colloid" in the case of fine gold particles. Usually, the contents of the insoluble carrier particles are configured as a "first reagent" and the dendrimer complex is configured as a "second reagent." In addition to these, a hypotonic solution for hemolysis, a diluent, etc. may be contained.

The measurement method of the present invention can be easily carried out using the measurement kit of the present invention. It will also necessarily involve the use of the present invention.

Examples of the present invention are disclosed below, but these are not intended to limit the scope of the present invention.

[Production example] Measurement element (1) First reagent (unsensitized latex dispersion)
Uncolored polystyrene latex particles (0.12 μm, manufactured by Fujikura Kasei Co., Ltd.) whose surface functional group is a sulfo group (-SO ₃ ) were added to a buffer solution containing 10 mmol HEPES to a concentration of 0.1% by mass, and hydroxylated. A first reagent was prepared by adjusting the pH to 7.9 with an aqueous sodium solution. Note that the mass % of the polystyrene latex particles mentioned above is expressed as a percentage of the mass (g) of the latex particles (dry solid content) as polymer nanoparticles in 100 g of the buffer solution. The meaning of mass % with respect to this latex particle is the same herein.

(2) Second reagent (antibody dilution solution: for standards and comparative examples)
20 g/L sodium chloride and 0.2% Tween 20 were added to a buffer solution containing 10 mmol HEPES, and the pH was adjusted to 7.0. To an appropriate amount of this buffer, add 0.05 mg/mL of anti-human hemoglobin A1c (HbA1c) mouse monoclonal antibody (manufactured by Fujikura Kasei Co., Ltd.) and 0.2 mg/mL of anti-mouse IgG goat polyclonal antibody (manufactured by Nippon Biotest Co., Ltd.). A second reagent was prepared by adding hydroxypropyl cellulose in an amount of 1% by mass based on the total amount of the second reagent.

(3) Second reagent (dendrimer complex: for example)
5 mg of third generation polyamide amine (PAMAM) dendrimer (manufactured by Sigma-Aldrich) was dissolved in PBS, 20 molar equivalents of dendrimer was added to sulfo-SMCC (manufactured by Thermo Fisher Scientific), and the mixture was inverted at room temperature for 1 hour. Mixed. After mixing, the mixture was desalted using PD-10 (manufactured by GE Healthcare) to prepare a maleimidized dendrimer.

Separately, 5 mg of anti-human hemoglobin A1c mouse monoclonal antibody (manufactured by Fujikura Kasei Co., Ltd.) was dissolved in PBS, 10 molar equivalents of 1-iminothiolane hydrochloride (Traut's reagent) (manufactured by Thermo Fisher Scientific) were added, and 1 Mix by inverting for an hour. After mixing, the mixture was desalted using PD-10 (manufactured by GE Healthcare) to prepare a thiolated antibody.

6.45 nmol of this thiolated antibody was fractionated, 3.23 nmol of maleimidized dendrimers of each generation mentioned above was added dropwise, mixed, and mixed by inversion at room temperature for 1 hour, so that the molar ratio of antibody/dendrimer was 2: A dendrimer composite of Example 1 was prepared (Example 1).

In place of the anti-HbA1c mouse monoclonal antibody, anti-mouse IgG goat polyclonal antibody, and hydroxypropyl cellulose in the second reagent (antibody dilution solution) in (2) above, the above dendrimer complex was added at a concentration of 0.05 mg/mL. was added to prepare the second reagent for the example.

(4) Standard measurement using an automatic analyzer Purified human with HbA1c value adjusted to 0% by mass, 5.0% by mass, 8.6% by mass, 12.0% by mass, 15.8% by mass of the entire solution Using a solution of HbA1c (standard reagent) as a specimen, 6 μL of the sample and 150 μL of the first reagent, which had been strictly refrigerated, were mixed, reacted for 5 minutes at 37°C, and then mixed with the second reagent (conventional method). 50 μL of Comparative Example 1) and this method (Example 1)) were added, and the amount of change in absorbance over 5 minutes was measured. The measurement was performed using a Hitachi automatic analyzer 7180 using a two-point end method with a main wavelength of 660 nm and a sub wavelength of 800 nm. The results are shown in Table 1 and were used as calibration data.

[Test Example] Measurement of Blood Sample (1) Measurement of Human Whole Blood Sample 10 μL of human whole blood sample collected using an EDTA-2K vacuum blood collection tube was collected and hemolyzed with 500 μL of pure water to prepare a hemolyzed sample. A total of 40 hemolyzed samples obtained in this way were measured for HbA1c (%) using the conventional method (Comparative Example 1) and the present method (Example 1) based on the above calibration data. We confirmed the correlation. As a result, it was revealed that Example 1 showed almost the same measured values as Comparative Example 1, and both showed equivalent sensitivity (FIG. 1).

(2) Confirmation of thermal stability The conventional product (Comparative Example 1) and the present invention product (Example 1) were stored in a thermostat at 37°C. The calibration curve measured on the first day was used as the initial value, and fluctuations in the calibration data were checked every week after storage in a 37° C. incubator.

As a result, the reaction of the conventional product (Comparative Example 1) was significantly reduced in the above calibration data, whereas the product of the present invention (Example 1) maintained fluctuations within ±10% of the initial value until the third week. It was revealed that the product had excellent stability even at temperatures considerably higher than room temperature (Table 2).

Claims (10)

A dendrimer complex in which the whole or part of an antibody against hemoglobin A1c is bound to a dendrimer in a state capable of binding to hemoglobin A1c is brought into contact with insoluble carrier particles sensitized to the hemoglobin component in a blood sample in an aqueous liquid. and measuring hemoglobin A1c in the blood sample using the aggregation of the insoluble carrier particles as an indicator. The measuring method according to claim 1, wherein the insoluble carrier particles are latex particles. The measuring method according to claim 1 or 2, wherein the antibody against hemoglobin A1c is a monoclonal antibody against hemoglobin A1c. The measuring method according to any one of claims 1 to 3, wherein the dendrimer generation number of the dendrimer complex is 2, 3, or 4. The measuring method according to any one of claims 1 to 4, wherein the blood sample is a whole blood sample or a hemolyzed sample. The measuring method according to any one of claims 1 to 5, wherein the aqueous liquid is a blood sample or a diluted product thereof. A dendrimer complex in which the whole or part of an antibody against hemoglobin A1c is bound to a dendrimer in a state capable of binding to hemoglobin A1c against insoluble carrier particles sensitized with hemoglobin components in a blood sample in an aqueous liquid. A method of using a dendrimer complex, which comprises bringing the insoluble carrier particles into contact with each other to cause aggregation. 8. The method of use according to claim 7, wherein the insoluble carrier particles are latex particles. Contains insoluble carrier particles for sensitizing hemoglobin components in a blood sample, and a dendrimer complex in which the whole or part of an antibody against hemoglobin A1c is bound to a dendrimer in a state capable of binding to hemoglobin A1c. Hemoglobin A1c measurement kit. The measurement kit according to claim 9, wherein the insoluble carrier particles are latex particles.

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