JP6614826B2 - Labeled antibody, its production method and immunological assay - Google Patents

Description

  The present invention relates to a labeled antibody that can be used for, for example, immunological measurement, a production method thereof, and an immunological measurement method.

  Since innumerable chemical substances exist in the living body, it is extremely important to analyze a specific trace component in the living body qualitatively and quantitatively. In the fields of medicine, pharmaceuticals, health food, biotechnology, environment, etc., drugs and foods that act only on specific parts (chemical substances) in the living body, analyzers and diagnostic agents that detect slight changes in the living body, It has been developed with the above technology.

  One of the analysis techniques is an immunoassay. This is also called an immunological measurement method, and is a method for qualitatively and quantitatively analyzing a trace component by utilizing a specific reaction between an antigen and an antibody, which is one of immune reactions. Antigen-antibody reaction is widely used in the above field because of its high sensitivity and selectivity of reaction. There are various measurement methods for immunoassays depending on the measurement principle. For example, enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), latex agglutination (LIA, PA), immunochromatography ( ICA), hemagglutination method (HA), hemagglutination inhibition method (HI) and the like.

  In the immunoassay, an antigen or antibody is detected qualitatively or quantitatively from a change (a change in the concentration of the antigen, antibody or complex) when the antigen and the antibody react to form a complex. When these are detected, the detection sensitivity is increased by binding a labeling substance to the antibody, antigen or complex. Therefore, it can be said that the labeling ability of the labeling substance is an important factor affecting the detection ability in the immunoassay. Also in the immunoassay exemplified above, as labeling substances, red blood cells (in the case of HA), latex particles (in the case of LIA), fluorescent dyes (in the case of FIA), radioactive elements (in the case of RIA), enzymes (in the case of EIA), Chemiluminescent materials (in the case of CLIA) are used.

  By the way, when colored fine particles are used as the labeling substance, detection can be confirmed by visual observation without using a special analyzer, so that simpler measurement is expected. As such colored fine particles, for example, Patent Document 1 proposes a colored latex composed of gold nanoparticles bonded to the surface of polymer latex particles. By bonding gold nanoparticles to the surface of the polymer latex particles, the gold nanoparticles themselves serve as a colorant to improve visual judgment and detection sensitivity. On the other hand, the gold nanoparticles themselves also bind to antigens or antibodies. Since it is excellent, it is said that a sufficient amount of antigen or antibody can be bound even if gold nanoparticles are bound to a sufficiently dark color.

  The colored latex is one in which gold nanoparticles are bonded to the surface of the latex by irradiating a dispersion of HAuCl, which is a precursor of styrene-acrylic acid copolymer latex and gold nanoparticles, with gamma rays. However, in the above colored latex, since gold nanoparticles are bound only to the surface of the latex, the amount of gold nanoparticles that exhibit surface plasmon absorption is limited and the gold nanoparticles are easily detached. As a result, the visibility and sensitivity as an immunological measurement reagent may not be sufficient. Moreover, since electromagnetic radiation such as gamma rays is irradiated, the latex may be damaged. Furthermore, in the specification of Patent Document 1, preferred ranges of the latex diameter and the gold nanoparticle diameter are disclosed. However, it is not clear whether these preferred ranges have been verified in the examples, and the preferred range is defined. There is no basis for.

  Patent Document 2 discloses polymer latex particles coated with metallic gold, and suggests application to reagents that can be used in microscopy and immunoassay methods. However, the polymer latex particles coated with metal gold do not disclose the material and particle size of the polymer latex particles. Furthermore, there is no verification of the effect as a reagent that can be used in immunoassay methods. Therefore, the effect as a reagent in metal gold and polymer latex particles is unknown.

  Against this background, the present inventors previously proposed a resin-metal complex having a specific structure as a labeling substance that enables highly sensitive immunological measurement (Japanese Patent Application No. 2014-136355, Application 2014-136356, Japanese Patent Application 2014-136357).

  By the way, an antibody bound to latex particles has a drawback that aggregation easily occurs. Therefore, for example, Patent Document 3 proposes that in an immunoassay composition using latex particles as a label, the pH is adjusted to 9.0 to 9.8 in order to prevent spontaneous aggregation of latex particles bound to an antibody. Has been.

JP 2009-168495 A Japanese Patent Laid-Open No. 3-206959 JP 2007-315883 A

  In order to use a resin-metal complex as a labeling substance in an immunological assay, it is necessary to stably bind to a ligand such as an antibody. However, when a ligand is labeled with a resin-metal complex, even if a stable binding state can be formed, excellent detection sensitivity is not always obtained. In particular, the detection sensitivity may be greatly reduced depending on the metal species constituting the metal particles of the resin-metal composite.

  Accordingly, an object of the present invention is to provide a labeled antibody that is labeled with a resin-metal complex and that can be subjected to highly sensitive immunological measurement.

  As a result of intensive studies, the present inventors have found that the above problem can be solved by binding a resin-metal complex having a specific structure to an antibody under specific conditions, and have completed the present invention. .

That is, the present invention is a method for producing a labeled antibody for producing a labeled antibody,
The method includes a step of obtaining a labeled antibody by mixing a resin-metal complex having a structure in which a plurality of metal particles are immobilized on resin particles with the antibody under a condition of pH 2-7.

The method for producing a labeled antibody of the present invention comprises a step of treating the labeled antibody under conditions within the range of pH 2-9.
May further be included.

  In the method for producing a labeled antibody of the present invention, the metal particles may be gold or gold alloy particles.

In the method for producing a labeled antibody of the present invention, the metal particles in the resin-metal complex are:
First particles having a portion exposed outside the resin particles;
Second particles entirely encapsulated in the resin particles;
May contain,
Among the first particles and the second particles, at least some of the particles may be three-dimensionally distributed in the surface layer portion of the resin particles.

  In the method for producing a labeled antibody of the present invention, the resin particles may be polymer particles having a structure having a substituent capable of adsorbing metal ions.

  In the method for producing a labeled antibody of the present invention, the average particle diameter of the metal particles may be in the range of 1 to 80 nm.

  In the method for producing a labeled antibody of the present invention, the resin-metal complex may have an average particle size in the range of 100 to 1000 nm.

  In the method for producing a labeled antibody of the present invention, the antibody may be an anti-influenza virus antibody.

  The labeled antibody of the present invention is produced by any one of the above-described methods for producing a labeled antibody.

  The immunological measurement method of the present invention is characterized by using the labeled antibody.

  According to the method for producing a labeled antibody of the present invention, by mixing the resin-metal complex with the antibody under the condition of pH 2 to 7, aggregation of the labeled antibody is suppressed, and highly sensitive immunological measurement. Can be produced. The labeled antibody obtained by the present invention is a material having excellent durability, visibility, visual judgment, and detection sensitivity, for example, immunology such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, HI, etc. It can be advantageously used in the measurement.

It is a schematic diagram which shows the structure of the cross section of the resin-metal composite_body | complex which concerns on one embodiment of this invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate.
[Production of labeled antibody]
The method for producing a labeled antibody according to the present embodiment produces an antibody labeled with a resin-metal complex. Here, the “antibody” is not particularly limited, and examples thereof include polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments [for example, H chain, L chain, Fab , F (ab ′) ₂ etc.] can be used. Further, IgG, IgM, IgA, IgE, or IgD may be used as the immunoglobulin. The animal species that produce the antibody may be humans or animals other than humans (eg, mice, rats, rabbits, goats, horses, etc.). Specific examples of the antibody include anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-HCV antibody, anti-IgG antibody, and anti-human IgE antibody.

  In the method for producing a labeled antibody of the present embodiment, the resin-metal complex used as a label has a structure in which a plurality of metal particles are immobilized on resin particles. Details of the resin-metal composite will be described later.

The method for producing the labeled antibody of the present embodiment comprises at least the following step A;
Step A) includes a step of obtaining a labeled antibody by mixing and binding the resin-metal complex with the antibody under conditions within the range of pH 2 to 7, and preferably further includes Step B;
Step B) A step of treating the labeled antibody under conditions within the range of pH 2 to 9 can be included.

<Process A>
In step A, the labeled antibody is obtained by mixing the resin-metal complex with the antibody under the condition of pH 2-7. In step A, it is preferable to contact the antibody in a state where the solid resin-metal complex is dispersed in the liquid phase. In this case, the binding between the resin-metal complex and the antibody is within the range of pH 2 to 7 from the viewpoint of uniformly contacting the resin-metal complex and the antibody while maintaining the dispersion of the resin-metal complex and the activity of the antibody. It is preferably carried out under the above conditions, and more preferably under acidic conditions, for example, in the range of pH 2.5 to 5.5. If the conditions for binding the resin-metal complex and the antibody are less than pH 2, the antibody may be altered and deactivated due to strong acidity. If the pH exceeds 7, the aggregation occurs when the resin-metal complex and the antibody are mixed. Dispersion becomes difficult. However, if the antibody is not inactivated by strong acidity, the treatment can be performed even at a pH of less than 2.

  Step A is preferably performed in a binding buffer adjusted to a pH in the range of 2-7. For example, a predetermined amount of the resin-metal complex is mixed with the binding buffer adjusted to the above pH and mixed thoroughly. As the binding buffer, for example, a boric acid solution adjusted to a predetermined concentration can be used. The pH of the binding buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide.

  Next, a labeled antibody-containing liquid can be obtained by adding a predetermined amount of the antibody to the obtained mixed liquid, sufficiently stirring and mixing. The labeled antibody-containing liquid obtained in this way can be used to separate only the labeled antibody as a solid part by solid-liquid separation means such as centrifugation.

<Process B>
In step B, the labeled antibody obtained in step A is treated under conditions within the range of pH 2 to 9, thereby performing blocking that suppresses nonspecific adsorption to the labeled antibody. In this case, the labeled antibody that has been separated by the solid-liquid separation means is dispersed in the liquid phase under the condition of pH 2-9. This blocking condition is, for example, in the range of pH 2 to 9 from the viewpoint of maintaining the activity of the antibody and suppressing the aggregation of the labeled antibody, and from the viewpoint of suppressing non-specific adsorption of the labeled antibody, further acidic conditions, For example, it is preferably within the range of pH 2-6. If the blocking condition is less than pH 2, the antibody may be altered and deactivated due to strong acidity. If the blocking condition exceeds pH 9, the labeled antibody will aggregate and dispersion will be difficult.

  It is preferable to perform the process B using the blocking buffer solution (Blocking Buffer) adjusted to the conditions within the range of pH 2-9. For example, a blocking buffer adjusted to the above pH is added to a predetermined amount of labeled antibody, and the labeled antibody is uniformly dispersed in the blocking buffer. As the blocking buffer, for example, a protein solution that does not bind to the detection target is preferably used. Examples of proteins that can be used in the blocking buffer include bovine serum albumin, ovalbumin, casein, and gelatin. More specifically, it is preferable to use a bovine serum albumin solution adjusted to a predetermined concentration. The pH of the blocking buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. A known method can be used for dispersing the labeled antibody. For example, it is preferable to use a dispersing means such as ultrasonic treatment or stirring with a rotator. In this way, a dispersion in which the labeled antibody is uniformly dispersed is obtained.

  As described above, a dispersion of labeled antibody is obtained. From this dispersion, only the labeled antibody can be collected as a solid part by solid-liquid separation means such as centrifugation. Moreover, arbitrary processes, such as a washing process and a preservation | save process, can be implemented as needed. Hereinafter, the cleaning process and the storage process will be described.

(Cleaning process)
In the washing treatment, a washing buffer is added to the labeled antibody collected by the solid-liquid separation means, and the labeled antibody is uniformly dispersed in the washing buffer. For dispersion, for example, a dispersion means such as ultrasonic treatment is preferably used. The washing buffer is not particularly limited, but for example, a Tris buffer solution, a glycinamide buffer solution, an arginine buffer solution, or the like having a predetermined concentration adjusted within a pH range of 8 to 9 may be used. it can. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid or sodium hydroxide. The washing treatment of the labeled antibody can be repeated a plurality of times as necessary.

(Save process)
In the storage treatment, a storage buffer is added to the labeled antibody separated by the solid-liquid separation means, and the labeled antibody is uniformly dispersed in the storage buffer. For dispersion, for example, a dispersion means such as ultrasonic treatment is preferably used. As the storage buffer, for example, a solution obtained by adding a predetermined concentration of an anti-aggregation agent and / or a stabilizer to the washing buffer can be used. As the aggregation inhibitor, for example, saccharides typified by sucrose, maltose, lactose, trehalose, polyhydric alcohols typified by glycerin, polyvinyl alcohol, and the like can be used. The stabilizer is not particularly limited, and for example, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin can be used. In this way, the labeled antibody can be stored.

  In each of the above steps, a surfactant or a preservative such as sodium azide or paraoxybenzoate can be used as necessary.

<Labeled antibody>
A labeled antibody can be produced as described above. The labeled antibody of the present embodiment can be used for various immunological measurements as with conventional labeled antibodies. For example, an immunological measurement can be performed by mixing a sample containing an analyte and a labeled antibody, reacting them, and measuring the color developed by the sample visually or using an analytical instrument. Therefore, the labeled antibody of the present embodiment can be preferably used as a labeled antibody in immunological measurements such as flow-through measurement and lateral flow measurement (immunochromatography).

<Resin-metal composite>
Next, the resin-metal complex used as a label in the method for producing a labeled antibody of the present embodiment will be described in detail. FIG. 1 is a schematic cross-sectional view of a resin-metal composite used as a label in the present embodiment. The resin-metal composite 100 includes resin particles 10 and metal particles 20.

  In the resin-metal composite 100, the metal particles 20 are dispersed or immobilized on the resin particles 10. In the resin-metal composite 100, a part of the metal particles 20 is three-dimensionally distributed in the surface layer portion 60 of the resin particle 10, and a part of the three-dimensionally distributed metal particles 20 is partially. The resin particles 10 are exposed outside, and the remaining part is encapsulated in the resin particles 10.

  Here, the metal particles 20 include metal particles completely encapsulated in the resin particles 10 (hereinafter also referred to as “encapsulated metal particles 30”), portions embedded in the resin particles 10, and outside the resin particles 10. Metal particles having an exposed portion (hereinafter also referred to as “partially exposed metal particles 40”) and metal particles adsorbed on the surfaces of the resin particles 10 (hereinafter also referred to as “surface-adsorbed metal particles 50”). Exists. The partially exposed metal particles 40 and the surface-adsorbed metal particles 50 correspond to “first particles” in the present invention, and the encapsulated metal particles 30 correspond to “second particles” in the present invention.

  For example, when the resin-metal complex 100 is used for immunological measurement, the antibody is immobilized on the partially exposed metal particles 40 or the surface-adsorbed metal particles 50 and used. At this time, the antibody is immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, while not immobilized on the encapsulated metal particles 30. However, since all of the metal particles 20 including the encapsulated metal particles 30 exhibit localized surface plasmon absorption, not only the partially exposed metal particles 40 and the surface adsorbed metal particles 50 but also the encapsulated metal particles 30 are immunological. Contributes to improved visibility as a target for measurement. Further, the partially exposed metal particles 40 and the encapsulated metal particles 30 have a large contact area with the resin particles 10 as compared with the surface-adsorbed metal particles 50, and also have a physical adsorption force such as an anchor effect due to the embedded state. Strong and difficult to desorb from the resin particles 10. Therefore, the durability and stability as an immunological measurement label using the resin-metal complex 100 can be improved.

  The inner metal particles 30 are all covered with the resin constituting the resin particles 10 on the entire surface. Further, the partially exposed metal particles 40 are those in which 5% or more and less than 100% of the surface area is covered with the resin constituting the resin particles 10. From the viewpoint of durability for each of the above uses, the lower limit is preferably 20% or more of the surface area, and more preferably 30% or more. Further, the surface-adsorbed metal particles 50 are covered with the resin constituting the resin particles 10 with more than 0% and less than 5% of the surface area thereof.

  In addition, the loading amount of the metal particles 20 (the total of the encapsulated metal particles 30, the partially exposed metal particles 40 and the surface-adsorbed metal particles 50) on the resin-metal composite 100 is based on the weight of the resin-metal composite 100. It is preferable that it is 5 wt%-70 wt%. If it is this range, the resin-metal composite body 100 is excellent in the visibility as a labeling substance, visual judgment property, and detection sensitivity. When the loading amount of the metal particles 20 is less than 5 wt%, the amount of antibody immobilized becomes small, and the detection sensitivity tends to decrease. More preferably, the supported amount of the metal particles 20 is 15 wt% to 70 wt%.

  Further, 10 wt% to 90 wt% of the metal particles 20 are preferably the partially exposed metal particles 40 and the surface adsorbed metal particles 50. If it is this range, since the fixed quantity of the antibody on the metal particle 20 can fully be ensured, the sensitivity as a labeling substance is high. More preferably, 20 wt% to 80 wt% of the metal particles 20 are the partially exposed metal particles 40 and the surface adsorbed metal particles 50, and the surface adsorbed metal particles 50 are more preferably 20 wt% or less from the viewpoint of durability. .

  Further, 60 wt% to 100 wt% of the metal particles 20 are present in the surface layer portion 60, and 5 wt% to 90 wt% of the metal particles 20 existing in the surface layer portion 60 are partially exposed metal particles 40 or surface adsorbed metal particles 50. It is preferable that the amount of the antibody immobilized on the metal particles 20 can be sufficiently secured, so that the sensitivity as a labeling substance is increased. In other words, it is preferable that 10 wt% to 95 wt% of the metal particles 20 existing in the surface layer portion 60 are the encapsulated metal particles 30.

  Here, the “surface layer portion” means a range of 50% of the particle radius in the depth direction from the surface of the resin particle 10. The “three-dimensional distribution” means that the metal particles 20 are dispersed not only in the surface direction of the resin particles 10 but also in the depth direction.

The resin particles 10 are preferably polymer particles having a substituent in the structure capable of adsorbing metal ions. In particular, nitrogen-containing polymer particles are preferable. Nitrogen atoms in the nitrogen-containing polymer are preferable because they easily chemisorb anionic metal ions, which are excellent in visibility and facilitate the immobilization of antibodies, and are precursors of metal particles such as gold and palladium. In the present embodiment, metal ions adsorbed in the nitrogen-containing polymer are reduced to form metal nanoparticles, so that part of the generated metal particles 20 becomes encapsulated metal particles 30 or partially exposed metal particles 40. . Also, like acrylic acid polymer, carboxylic acid and the like can adsorb cationic metal ions, so it is easy to adsorb cationic metal ions that are precursors of metal particles such as silver, nickel, copper, etc. The metal particles 20 such as nickel and copper can be formed, and an alloy with the metal such as gold and palladium can be formed.
On the other hand, in the case of resin particles other than the nitrogen-containing polymer having a substituent capable of adsorbing metal ions, such as polystyrene, it is difficult to adsorb the metal ions inside the resin. As a result, most of the generated metal particles 20 become surface-adsorbed metal particles 50. As described above, since the surface-adsorbed metal particles 50 have a small contact area with the resin particles 10, the adhesive force between the resin and the metal is small and the influence of the metal particles 20 being detached from the resin particles 10 tends to be large.
The nitrogen-containing polymer is a resin having a nitrogen atom in the main chain or side chain, and examples thereof include polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, and polyaniline. Preferable are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. Moreover, when it has a nitrogen atom in a side chain, for example, an acrylic resin, a phenol resin, an epoxy resin, etc. can be used widely.

  As a material of the metal particles 20, for example, silver, nickel, copper, gold, palladium or the like can be applied. These metals can be used as a simple substance or a composite such as an alloy. Preferably, gold and palladium are excellent in visibility and can be easily immobilized. These are preferable because they express absorption derived from localized surface plasmon resonance. More preferably, it is gold with good storage stability. In addition, gold or a gold alloy is most preferable as the material of the metal particles 20 from the viewpoint that excellent color developability can be obtained when the resin-metal complex 100 is bound to an antibody under the condition of pH 2-7. It is a metal species. Here, the gold alloy means, for example, an alloy made of gold and a metal species other than gold and containing 10% by weight or more of gold.

  Moreover, it is preferable that the average particle diameter of the metal particle 20 measured by scanning electron microscope (SEM) observation is 1-80 nm, for example. When the average particle diameter of the metal particles 20 is less than 1 nm or exceeds 80 nm, the localized surface plasmon is less likely to be expressed, and thus the sensitivity tends to decrease. The average particle diameter of the metal particles 20 is preferably 20 nm or more and less than 70 nm, and more preferably 22 nm or more and less than 50 nm.

  Moreover, the average particle diameter of the resin-metal composite body 100 is, for example, 100 to 1000 nm. When the average particle diameter is less than 100 nm, for example, when gold particles are used as the metal particles 20, the amount of gold particles tends to be reduced, so that coloring tends to be weaker than gold particles of the same size, 1000 nm. If it exceeds 1, the labeling substance or reagent tends to be clogged in the pores of a chromatographic medium such as a membrane filter, and the dispersibility tends to decrease. The average particle size of the resin-metal composite 100 is preferably 100 nm or more and less than 700 nm, and more preferably 340 nm or more and less than 650 nm. Here, the particle size of the resin-metal composite 100 means a value obtained by adding the length of the protruding portion of the partially exposed metal particle 40 or the surface-adsorbed metal particle 50 to the particle size of the resin particle 10, and laser diffraction. / Measured by scattering method, dynamic light scattering method, or centrifugal sedimentation method.

  The manufacturing method of the resin-metal composite 100 is not particularly limited. For example, a solution containing metal ions is added to a dispersion of resin particles 10 produced by an emulsion polymerization method, and the metal ions are adsorbed on the resin particles 10 (hereinafter referred to as “metal ion adsorption resin particles”). Furthermore, the metal ion adsorption resin particles are added to the reducing agent solution to reduce the metal ions to generate the metal particles 20, thereby obtaining the resin-metal composite 100.

Further, for example, when gold particles are used as the metal particles 20, examples of the solution containing metal ions include chloroauric acid (HAuCl ₄ ) aqueous solution. Moreover, you may use a metal complex instead of a metal ion.
Further, as a solvent for a solution containing metal ions, instead of water, water-containing alcohol or alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, hydrochloric acid, sulfuric acid, nitric acid An acid such as the above may be used.
Further, in the solution, if necessary, for example, water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, diisopropyl ether; alkylene glycol, polyalkylene glycol, these Additives such as various water-miscible organic solvents such as polyols such as monoalkyl ether or dialkyl ether, glycerol, etc .; ketones such as acetone and methyl ethyl ketone may be added. Such an additive is effective for accelerating the reduction reaction rate of metal ions and controlling the size of the metal particles 20 to be produced.

Moreover, a well-known thing can be used for a reducing agent. For example, sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid, sodium phosphinate, hydroquinone, hydrazine sulfate, formaldehyde And Rochelle salt. Of these, sodium borohydride, dimethylamine borane, and citric acid are preferable. A surfactant can be added to the reducing agent solution as necessary, and the pH of the solution can be adjusted. The pH can be adjusted with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.
Furthermore, the particle size of the metal particles to be formed can be controlled by adjusting the reduction rate of the metal ions depending on the temperature of the reducing agent solution.

  Further, when the metal ions in the metal ion adsorption resin particles are reduced to produce the metal particles 20, the metal ion adsorption resin particles may be added to the reducing agent solution, or the reducing agent may be added to the metal ion adsorption resin. Although it may be added to the particles, the former is preferable from the viewpoint of easy generation of the encapsulated metal particles 30 and the partially exposed metal particles 40.

  In order to maintain the dispersibility of the resin-metal composite 100 in water, for example, citric acid, poly-L-lysine, polyvinyl pyrrolidone, polyvinyl pyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 (Big Chemy Japan) A dispersant may be added. Furthermore, the dispersibility can be maintained by adjusting the pH with a buffer such as boric acid or phosphoric acid, an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium hydroxide.

  The resin-metal complex 100 having the above-described configuration is particularly suitable for immunology of EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA, HI, etc. by adsorbing antibodies on the surface of the metal particles 20. It can be preferably applied to measurement methods. In particular, it can be preferably applied as a labeling substance for immunological measurement or a reagent for immunological measurement excellent in visual determinability in a low concentration region (high sensitivity region). The form of the immunological measurement labeling substance or immunological measurement reagent is not particularly limited. For example, a dispersion in which the resin-metal complex 100 is dispersed in water or a pH-adjusted buffer. Can be used as

  EXAMPLES Next, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples. Unless otherwise specified in the following examples and comparative examples, various measurements and evaluations are as follows.

<Measurement of absorbance of resin-metal composite>
Absorbance of the resin-metal composite was measured by placing a resin-metal composite dispersion (dispersion medium: water) prepared at 0.01 wt% into an optical white glass cell (optical path length 10 mm), and an instantaneous multi-photometry system (Otsuka The absorbance at 570 nm was measured using MCPD-3700 manufactured by Denki Co., Ltd.

<Measurement of solid content concentration and metal loading>
1 g of the dispersion before concentration adjustment was placed in a magnetic crucible and heat-treated at 70 ° C. for 3 hours. The weight before and after the heat treatment was measured, and the solid content concentration was calculated by the following formula.

  Solid content concentration (wt%) = [weight after drying (g) / weight before drying (g)] × 100

The sample after the heat treatment was further heat-treated at 500 ° C. for 5 hours, the weight before and after the heat treatment was measured, and the metal loading was calculated from the following formula.
Metal loading (wt%) =
[Weight after heat treatment at 500 ° C. (g) / Weight before heat treatment at 500 ° C. (g)] × 100

<Measurement of average particle diameter of resin-metal composite>
Measurement was performed using a disk centrifugal particle size distribution meter (manufactured by CPS Instruments). The measurement was performed in a state where the resin-metal composite was dispersed in water.

<Measurement of average particle diameter of metal particles>
The average particle diameter of the metal particles is measured by dropping a resin-metal composite dispersion liquid onto a metallic mesh with a carbon support film, using a field emission scanning electron microscope (STEM; manufactured by Hitachi High-Technologies Corporation, SU -9000), the area average diameter of the metal particles was measured.

[Production Example 1]
<Synthesis of resin particles>
Aliquat 336 (manufactured by Aldrich) (1.00 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 2.00 g) were dissolved in 80 g of pure water, and then 2-vinylpyridine (2-VP, 9.90 g) and divinyl were dissolved. Benzene (DVB, 0.100 g) was added, and the mixture was stirred at 250 rpm and 60 ° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.100 g) dissolved in 9.00 g of pure water was added dropwise over 5 minutes, and the mixture was stirred at 250 rpm and 60 ° C. for 6 hours. As a result, resin particles A-1 having an average particle size of 0.36 μm were obtained. The A-1 was precipitated by centrifugation (9000 rpm, 10 minutes), the supernatant was removed, and then dispersed again in pure water to obtain a 2.1 wt% resin particle dispersion B-1.

<Synthesis of resin-gold composite>
A 30 mM chloroauric acid aqueous solution (106.6 g) was added to B-1 (19.09 g), and the mixture was allowed to stand at room temperature for 24 hours. Thereafter, the resin particles are precipitated by centrifugation (3000 rpm, 10 minutes), and the supernatant is removed to remove excess chloroauric acid, and then dispersed again in 40 g of pure water. -1 was prepared. The C-1 (20 g) was added dropwise to a 3.3 mM dimethylamine borane aqueous solution (600 ml) over 4 minutes, followed by stirring at 8 ° C. for 1 hour and further stirring at room temperature for 5 hours, so that the average particle size was 0. .38 μm resin-gold composite D-1 was obtained. The D-1 is precipitated by centrifugation (3000 rpm, 120 minutes), the supernatant is removed, an appropriate amount of pure water is added and dispersed again, and purified by ultrafiltration membrane to obtain 1 wt% resin-gold. A composite dispersion E-1 was obtained. The absorbance of the resin-gold complex F-1 in E-1 was 1.0 as a result of measurement according to the above method. Moreover, the average particle diameter of the gold particles in F-1 was 22.0 nm, and the amount of gold supported was 49.1 wt%. In this resin-gold composite, the gold particles are: encapsulated particles that are completely encapsulated in the resin particles; partially exposed gold particles that have a portion embedded in the resin particle and a portion exposed outside the resin particle; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.

[Production Example 2]
<Synthesis of resin particles>
Aliquat 336 [manufactured by Aldrich] (1.00 g) and polyethylene glycol methyl ether methacrylate (PEGMA, 10.00 g) were dissolved in 300 g of pure water, and then 4-vinylpyridine (4-VP, 48.00 g) and divinyl were dissolved. Benzene (DVB, 2.00 g) was added and stirred under a nitrogen stream at 150 rpm, 30 ° C. for 50 minutes, and then at 60 ° C. for 30 minutes. After stirring, 2,2-azobis (2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g) dissolved in 18.00 g of pure water was added dropwise over 2 minutes, and 3.5 rpm at 60 ° C. and 60 ° C. By stirring for a period of time, resin particles having an average particle diameter of 438 nm were obtained. After sedimentation by centrifugation (9000 rpm, 45 minutes) and removal of the supernatant, the operation of redispersing in pure water was performed three times, and then impurities were removed by dialysis. Thereafter, the concentration was adjusted to obtain a 10 wt% resin particle dispersion.

<Synthesis of resin-gold composite>
After adding 1233 ml of pure water to the resin beads (50 ml), a 30 mM chloroauric acid aqueous solution (100 ml) was added and left at room temperature for 24 hours. Thereafter, the resin particles were precipitated by centrifugation (3100 rpm, 30 minutes), and the operation of removing the supernatant was repeated three times to remove excess chloroauric acid. Thereafter, the concentration was adjusted to prepare a 2.5 wt% gold ion adsorption resin particle dispersion.

  Next, the 2.5 wt% gold ion adsorption resin particle dispersion (42.4 ml) is added to 1580 ml of pure water, and 528 mM dimethylamine borane aqueous solution (10 ml) is added over 2 minutes while stirring at 160 rpm at 3 ° C. After the dropwise addition, the mixture was stirred at room temperature for 2 hours to obtain a resin-gold composite having an average particle size of 448 nm. By precipitating the resin-gold complex by centrifugation (3100 rpm, 60 minutes), removing the supernatant, repeating the work of redispersing in pure water three times, purifying by dialysis, and adjusting the concentration, A 1 wt% resin-gold composite dispersion was obtained. The absorbance of the produced resin-gold composite was 0.99 as a result of measurement according to the above method. Further, the formed gold particles had an average particle diameter of 24.0 nm and a gold loading amount of 55.7 wt%. In this resin-gold composite, the gold particles are: encapsulated particles that are completely encapsulated in the resin particles; partially exposed gold particles that have a portion embedded in the resin particle and a portion exposed outside the resin particle; Surface adsorbed gold particles adsorbed on the surface of the resin particles, and at least some of the gold particles were three-dimensionally distributed in the surface layer portion of the resin particles.

[Reagents, etc.]
In Examples and Comparative Examples, the following reagents and the like were used.
Anti-influenza type A monoclonal antibody (7.15 mg / mL / PBS): Adtech Co., Ltd. Binding buffer a: 100 mM A boric acid solution was adjusted to pH≈3 with HCl.
Binding buffer b: 100 mM boric acid solution was adjusted to pH≈4 with HCl.
Binding buffer c: 100 mM boric acid solution was adjusted to pH≈5 with HCl.
Binding buffer d: 100 mM boric acid solution pH≈6.5
Binding buffer e: 100 mM boric acid solution was adjusted to pH ≈ 7.5 with NaCl.
Binding buffer f: 100 mM boric acid solution was adjusted to pH≈8.5 with NaCl.
Binding buffer g: 50 mM 2-morpholinoethanesulfonic acid solution pH≈3.8

Blocking buffer a: 1% by weight bovine serum albumin solution was adjusted to pH≈5 with HCl.
Blocking buffer b: A 1% by weight bovine serum albumin solution was adjusted to pH≈7 with HCl.
Blocking buffer c: 1% by weight bovine serum albumin solution was adjusted to pH≈8.5 with HCl.
Blocking buffer d: A 1% by weight bovine serum albumin solution was adjusted to pH≈9.5 with HCl.
Washing buffer: A 5 mM Tris solution was adjusted to pH≈8.5 with HCl.
Storage buffer: Sucrose was added to the wash buffer to a concentration of 10% by weight.
Influenza A positive control (APC): Influenza A virus inactivated antigen (manufactured by Adtech Co., Ltd.) was prepared by diluting 100 times with a sample treatment solution (manufactured by Adtech Co., Ltd.). The antigen concentration of APC corresponds to 5000 FFU / ml.
Negative control: Sample treatment solution (manufactured by Adtec Corporation)
AuNCP beads: Resin-gold composite obtained in Production Example 1 (1% by weight; average particle size 380 nm)

[Example 1]
(Joining process)
To a microtube [ibis (registered trademark; manufactured by ASONE) 2 mL], 0.1 mL of AuNCP beads as a resin-metal complex was added, and binding buffer a0.9 mL was added. After thoroughly mixing by inversion mixing, 100 μg of anti-influenza A monoclonal antibody is added, and the mixture is stirred at inversion for 3 hours at room temperature, and contains a labeled antibody containing an anti-influenza A monoclonal antibody labeled with a resin-metal complex A liquid A-1 was obtained.

(Block process)
Next, after the labeled antibody-containing solution A-1 is ice-cooled, it is centrifuged at 12000 rpm for 5 minutes, and the supernatant is removed. Then, the block buffer solution a1 mL is added to the solid residue, and it is applied for 10 to 20 seconds. Then, ultrasonic dispersion treatment was performed, and the mixture was further stirred at room temperature for 2 hours to obtain labeled antibody-containing liquid B-1.

(Cleaning process)
Next, after cooling the labeled antibody-containing solution B-1 with ice and centrifuging at 12000 rpm for 5 minutes and removing the supernatant, 1 mL of a washing buffer solution is added to the solid residue, and it is applied for 10 to 20 seconds. Then, ultrasonic dispersion treatment was performed. This operation was repeated three times to obtain a washing process.

(Save process)
Next, after cooling with ice, the mixture is centrifuged at 12000 rpm for 5 minutes, the supernatant is removed, 1 mL of a storage buffer is added to the solid residue, and ultrasonic dispersion treatment is performed for 10 to 20 seconds. To obtain labeled antibody-containing solution C-1.

[Example 2]
Labeled antibody-containing solutions A-2, B-2, and C-2 were obtained in the same manner as in Example 1 except that the binding buffer b was used instead of the binding buffer a in the binding step of Example 1. .

[Example 3]
Labeled antibody-containing solutions A-3, B-3, and C-3 were obtained in the same manner as in Example 1 except that the binding buffer c was used instead of the binding buffer a in the binding step of Example 1. .

[Example 4]
Labeled antibody-containing solutions A-4, B-4, and C-4 were obtained in the same manner as in Example 1 except that the binding buffer d was used instead of the binding buffer a in the binding step of Example 1. .

[Comparative Example 1]
When the binding buffer e was used instead of the binding buffer a in the binding step of Example 1, it was difficult to obtain a labeled antibody-containing solution because the resin-metal complex aggregated.

[Comparative Example 2]
When the binding buffer f was used in place of the binding buffer a in the binding step of Example 1, the resin-metal complex aggregated, making it difficult to obtain a labeled antibody-containing solution.

[Example 5]
In the blocking step of Example 1, labeled antibody-containing solutions B-5 and C-5 were obtained in the same manner as in Example 1 except that the blocking buffer b was used instead of the blocking buffer a.

[Example 6]
In the blocking step of Example 1, labeled antibody-containing liquids B-6 and C-6 were obtained in the same manner as in Example 1 except that the blocking buffer c was used instead of the blocking buffer a.

[Example 7]
Labeled antibody-containing solutions A-7, B-7, and C-7 were obtained in the same manner as in Example 1 except that the binding buffer g was used instead of the binding buffer a in the binding step of Example 1. .

<Evaluation method>
The evaluation was performed using a monochrome screen for influenza A evaluation (manufactured by Adtech), and the coloring levels after 5 minutes, 10 minutes and 15 minutes were compared. The color development level was determined using a color sample for gold colloid determination (manufactured by Adtech). In the screening evaluation, the influenza A positive control (APC) was used as the antigen. In the performance evaluation, the antigen used was a 2-fold dilution series (1-fold to 1024-fold dilution) of APC.

<Screening evaluation>
3 μL of the labeled antibody-containing solutions C-1 to 7 obtained in Examples 1 to 7 were placed in 7 wells of a 96-well plate, and 100 μL of APC was mixed therein. Next, 50 μL each was added to a monochrome screen for influenza A evaluation, and the color development level after 5 minutes, 10 minutes and 15 minutes was evaluated. The results are shown in Table 1. In addition, it means that a color development level is so high that a numerical value in Table 1 is large (color development is strong).

  From Table 1, it was confirmed that the antibody label-containing solution C-1 obtained in Example 1 exhibited the strongest color development and had excellent labeling performance.

<Performance evaluation>
3 μL each of the labeled antibody-containing solution C-1 obtained in Example 1 was placed in 12 wells of a 96-well plate, and APC was diluted 2 times (diluted 1 to 1024 times, APC × 1 to APC × 1024, respectively). 100 μL of each of the negative control and the negative control were mixed. Next, 50 μL was added to the influenza A evaluation monochrome screen, and the color development level after 5 minutes, 10 minutes and 15 minutes was evaluated. The results are shown in Table 2. In addition, it means that a color development level is so high that the numerical value in Table 2 is large (color development is strong).

  From Table 2, it was confirmed that the labeled antibody-containing solution C-1 obtained in Example 1 showed good color development even with a 256-fold diluted antigen, and had excellent labeling performance.

[Example 8]
Except that the resin-gold complex dispersion obtained in Production Example 2 was used, the same operation as in Example 1 was performed to obtain labeled antibody-containing liquids A-9, B-9, and C-9.
<Performance evaluation>
3 μL each of the labeled antibody-containing solution C-9 obtained in Example 8 was placed in 12 wells of a 96-well plate, and a 2-fold dilution series of APC (1-fold to 1024-fold dilution, APC × 1 to APC × 1024, respectively) 100 μL of each of the negative control and the negative control were mixed. Next, 50 μL was added to the influenza A evaluation monochrome screen, and the color development level after 5 minutes, 10 minutes and 15 minutes was evaluated. The results are shown in Table 3. In addition, it means that a color development level is so high that a numerical value in Table 3 is large (color development is strong).

  From Table 3 above, it was confirmed that the labeled antibody-containing solution C-9 obtained in Example 8 exhibited good color development with respect to the 256-fold diluted antigen.

  As mentioned above, although embodiment of this invention was described in detail for the purpose of illustration, this invention is not restrict | limited to the said embodiment.

DESCRIPTION OF SYMBOLS 10 ... Resin particle, 20 ... Metal particle, 30 ... Encapsulated metal particle, 40 ... Partially exposed metal particle, 50 ... Surface adsorption metal particle, 60 ... Surface layer part, 100 ... Resin-metal composite

Claims (10)

A method for producing a labeled antibody for producing a labeled antibody, comprising:
A label comprising a step of obtaining a labeled antibody by mixing a resin-metal complex having a structure in which a plurality of metal particles are immobilized on a resin particle with the antibody under a condition of pH 2-7 Antibody production method. Treating the labeled antibody under conditions in the range of pH 2-9,
The method for producing a labeled antibody according to claim 1, further comprising:   The method for producing a labeled antibody according to claim 1, wherein the metal particles are gold or gold alloy particles. The metal particles in the resin-metal composite are:
First particles having a portion exposed outside the resin particles;
Second particles entirely encapsulated in the resin particles;
Contains
4. The device according to claim 1, wherein at least some of the first particles and the second particles are three-dimensionally distributed in a surface layer portion of the resin particles. 5. A method for producing the labeled antibody according to 1.   The method for producing a labeled antibody according to any one of claims 1 to 4, wherein the resin particle is a polymer particle having a substituent capable of adsorbing a metal ion in its structure.   The method for producing a labeled antibody according to any one of claims 1 to 5, wherein the average particle diameter of the metal particles is in the range of 1 to 80 nm.   The method for producing a labeled antibody according to any one of claims 1 to 6, wherein an average particle diameter of the resin-metal complex is in a range of 100 to 1000 nm.   The method for producing a labeled antibody according to any one of claims 1 to 7, wherein the antibody is an anti-influenza virus antibody.   The labeled antibody manufactured by the manufacturing method of the labeled antibody of any one of Claim 1 to 8. An immunoassay method using the labeled antibody according to claim 9.

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