JP2023140980A - Production method of mark antibody, mark antibody and immunological measurement method - Google Patents

Abstract

To provide a mark antibody in which, an amount of antibodies coupled to a metal-resin composite body is sufficiently large, and by which sensitive immunological measurement can be performed.SOLUTION: There is provided a production method of a mark antibody configured so that, the antibody is adhered to a metal-resin composite body in a coupling step, by mixing, the antibody, and the metal-resin composite body having a structure in which, metal particles are stabilized to resin particles, in the presence of a coupling buffer solution with pH of 6-10 and including a good buffer whose pKa at 20°C is 7.5 or greater, where the good buffer may be preferably one or more kinds selected from: 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid (HEPES); N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS);N-cyclohexyl-3-aminopropanesulfonic acid (CAPS); 2-cyclohexylaminoethanesulfonic (CHES); and N-(tris(hydroxymethyl)methyl)-3-amino-2-hydroxypropanesulfonic acid (TAPSO).SELECTED DRAWING: None

Description

The present invention relates to a method for producing a labeled antibody that can be used, for example, in immunoassays, a labeled antibody, and an immunoassay method.

Immunoassay, also called immunoassay, is a method that qualitatively and quantitatively analyzes trace components by utilizing a specific reaction between an antigen and an antibody. Antigen-antibody reactions have high sensitivity and reaction selectivity, and are therefore widely used in fields such as medicine. Various immunoassay methods are known depending on the measurement principle, such as enzyme immunoassay (EIA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), and fluorescence immunoassay (CLIA). FIA), latex agglutination method (LIA, PA), immunochromatography method (ICA), hemagglutination method (HA), hemagglutination inhibition method (HI), and the like.

Immunoassays qualitatively or quantitatively detect antigens or antibodies based on changes (changes in the concentration of antigen, antibody, or complex) when the antigen and antibody react to form a complex. When detecting these, the detection sensitivity is increased by binding a labeling substance to the antibody, antigen, or complex. Therefore, the labeling ability of a labeling substance can be said to be an important factor that influences the detection ability in immunoassays.

Therefore, a metal-resin composite having a structure in which metal particles are immobilized on resin particles has been proposed as a labeling substance that enables highly sensitive immunological measurements (for example, Patent Documents 1 and 2).

WO2016/002742 WO2016/002743

In order to use a metal-resin complex as a labeling substance in an immunoassay, it is necessary to bind a sufficient amount of a ligand such as an antibody. If the amount of antibody bound to the metal-resin complex is small, excellent detection sensitivity cannot be obtained. However, metal-resin complexes dispersed in a buffer solution tend to be in an aggregated state, which may reduce the amount of antibody bound and greatly reduce detection sensitivity.

Therefore, an object of the present invention is to provide a labeled antibody that has a sufficiently large amount of antibody bound to a metal-resin complex and that enables highly sensitive immunological measurements.

As a result of extensive research, the present inventors have solved the above problem by binding a metal-resin complex, which has a structure in which metal particles are immobilized on resin particles, with an antibody under a specific binding buffer. The present invention was completed based on the discovery that the problem can be solved.

That is, the method for producing a labeled antibody of the present invention is a method for producing a labeled antibody, in which an antibody and a metal-resin composite having a structure in which metal particles are immobilized on resin particles are heated at 20°C. a binding step in which the antibody is attached to the metal-resin complex by mixing in the presence of a binding buffer in the pH range of 6 to 10 containing Good's buffer having a pKa of 7.5 or more;
This includes:

In the method for producing a labeled antibody of the present invention, the Good buffer is 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-3- Aminopropanesulfonic acid (TAPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 2-cyclohexylaminoethanesulfonic acid (CHES) and N-[tris(hydroxymethyl)methyl]-3-amino- It may be one or more selected from 2-hydroxypropanesulfonic acid (TAPSO).

In the method for producing a labeled antibody of the present invention, in the binding step, the ratio of the attached antibody to the total amount of antibody used may be 50% by weight or more.

In the method for producing a labeled antibody of the present invention, the metal particles may be silver, nickel, copper, gold, platinum, palladium, or an alloy containing any of these.

In the method for producing a labeled antibody of the present invention, the metal particles may include metal particles having a portion embedded within the resin particle and a portion exposed outside the resin particle.

In the method for producing a labeled antibody of the present invention, at least some of the metal particles may be three-dimensionally distributed in the surface layer 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 substituent in their structure capable of adsorbing metal ions.

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

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

The labeled antibody of the present invention is produced by any of the labeled antibody production methods described above.

The immunoassay method of the present invention uses the labeled antibody described above.

According to the method for producing a labeled antibody of the present invention, by using a specific binding buffer, aggregation of the metal-resin complex is suppressed, the amount of antibody attached is large, and highly sensitive immunological measurements are possible. Labeled antibodies can be produced. The labeled antibody obtained by the method of the present invention can be advantageously used in various immunological assays as a material with excellent durability, visibility, visual judgment, and detection sensitivity.

FIG. 1 is a schematic diagram showing a cross-sectional structure of a metal-resin composite used in an embodiment of the present invention. FIG. 2 is a cross-sectional view of an immunochromato strip prepared in an example.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate.

[Method for producing labeled antibody]
The method for producing a labeled antibody according to the present embodiment includes at least the following step A;
Step A) Antibodies and a metal-resin complex having a structure in which metal particles are immobilized on resin particles are mixed with a Good buffer (hereinafter referred to as "specific Good buffer") having a pKa of 7.5 or more at 20°C. a binding step in which the antibody is attached to the metal-resin complex by mixing in the presence of a binding buffer in the pH range of 6 to 10 containing
can include.

(antibody)
In this embodiment, the antibody is not particularly limited, and includes, for example, polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments that have antigen-binding ability [e.g., H chain, L chain, Fab, F(ab') ₂ , etc.] can be used. Further, the immunoglobulin may be any of IgG, IgM, IgA, IgE, and IgD. Antibody-producing animal species include humans as well as non-human animals (eg, mice, rats, rabbits, goats, horses, etc.). Specific examples of antibodies include anti-CRP antibody, anti-PSA antibody, anti-AFP antibody, anti-CEA antibody, anti-adenovirus antibody, anti-influenza virus antibody, anti-HCV antibody, anti-IgG antibody, anti-human IgE antibody, anti-human cardiac troponin. I antibodies, anti-SARS-COV-2 (COVID-19) antibodies, and the like.

(Metal-resin composite)
In this embodiment, the metal-resin composite used as a label has a structure in which metal particles are immobilized on resin particles. Details of this metal-resin composite will be described later.

<Step A: Bonding step>
In step A, the antibody is attached to the metal-resin complex by mixing the antibody and the metal-resin complex in the presence of a binding buffer in the pH range of 6-10. The bonding between the metal-resin complex and the antibody in this step is a so-called physical bond, and the antibody is bonded to the surface of the metal particles and/or the surface of the resin particles in the metal-resin complex through physical interaction. let Here, the bond due to physical interaction includes, for example, electrostatic bond, hydrogen bond, parent-hydrophobic interaction, and van der Waals force.

(Binding buffer)
In this step, a binding buffer containing a specific Good buffer and having a pH within the range of 6 to 10 is used. Good's buffer is a biochemically useful buffer that has acidic and basic functional groups, and several dozen types are known so far. Among them, in addition to the properties known for general Good buffers, specific Good buffers have the remarkable ability to effectively suppress aggregation of metal-resin complexes in buffer solutions due to their low ionic strength. play an action. In other words, it was confirmed that the specific Good buffer has an aggregation-inhibiting effect on the metal-resin complex, which is a different effect from the effect on other labeling substances. Therefore, by binding the antibody and the metal-resin complex using a binding buffer containing a specific Good buffer, it is possible to allow the antibody to act while the aggregation of the metal-resin complex is suppressed. Therefore, compared to, for example, a binding buffer using an inorganic buffer, the amount of antibody attached to the metal-resin complex can be significantly increased.

The specific good buffer may have a pKa of 7.5 or more at 20°C, but from the viewpoint of ensuring a sufficient amount of antibody adhesion, for example, 2-[4-(2-hydroxyethyl)-1- piperazinyl] ethanesulfonic acid [HEPES; pKa (20°C) 7.55], N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid [TAPS; pKa (20°C) 8.40], to N-cyclo Hexyl-3-aminopropanesulfonic acid [CAPS; pKa (20°C) 10.40], 2-cyclohexylaminoethanesulfonic acid [CHES; pKa (20°C) 9.5], N-[tris(hydroxymethyl)methyl ]-3-Amino-2-hydroxypropanesulfonic acid [TAPSO; pKa (20°C) 7.7] and the like are preferred.
Among the group of compounds known as Good buffers, those with a pKa of less than 7.5 at 20°C are effective in suppressing aggregation of metal-resin complexes, but they are outside the pH range suitable for antibody binding. In many cases, it is difficult to secure a sufficient amount of antibody attached.

In addition, by using a binding buffer containing a specific Good buffer, it is possible to prevent clogging of the test strip due to metal-resin complexes, regardless of the type of antibody, so false positives caused by negative samples in immunochromatography can be avoided. can be suppressed.

In this step A, it is preferable to use a binding buffer solution with a pH in the range of 6 to 10, more preferably in the range of pH 6 to 8. If the pH of the binding buffer is less than 6, the amount of antibody attached may decrease; if the pH exceeds 10, more ionic components will be added to adjust the pH, and the ionic strength of the buffer will increase, resulting in less adhesion of the antibody. - The effect of suppressing aggregation of the resin composite becomes poor. If the pH is within the range of 6 to 10, it becomes possible to bind a sufficient amount of antibody while suppressing aggregation of the metal-resin complex, and excellent detection sensitivity can be obtained in immunoassays.

The binding buffer used in Step A can be prepared according to a conventional method. For example, the binding buffer can be prepared by mixing an aqueous solution of a specific Good buffer with an alkaline solution of a predetermined concentration so that the pH is within the above range. Preferred examples of the alkaline solution include sodium hydroxide solution and potassium hydroxide. The pH of the binding buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, or the like.

(join operation)
In step A, a labeled antibody dispersion can be obtained by mixing the antibody and the metal-resin complex in the presence of a binding buffer and stirring thoroughly. In this step, it is sufficient to bind the antibody and the metal-resin complex in a binding buffer, so the metal-resin complex is dispersed in the binding buffer in advance and the antibody is added thereto. Alternatively, the antibody may be dispersed in a binding buffer in advance and the metal-resin complex may be added thereto. Dispersion can be carried out by a known method, but it is preferable to use a dispersion means such as ultrasonication, stirring with a rotator, or a vortex mixer. From the thus obtained labeled antibody dispersion, only the labeled antibody can be separated as a solid portion by solid-liquid separation means such as centrifugation.

(Antibody binding yield)
In this binding step, the proportion of antibody attached to the metal-resin complex (also referred to as "antibody binding yield") is preferably 50% by weight or more, and 70% by weight or more based on the total amount of antibody used. It is more preferable that If the proportion of the attached antibody is less than 50% by weight, it is not preferable because the expensive antibody will not be effectively utilized and will be wasted. In the present invention, by using a binding buffer containing a specific Good buffer, it is possible to increase the binding yield of antibodies to 50% by weight or more. It is possible to obtain higher detection sensitivity.

Further, in a reagent composition obtained by dispersing a metal-resin complex in a binding buffer, it is preferable that the non-aggregation ratio of the metal-resin complex is 50% or more. Here, the non-aggregation ratio refers to the ratio at which the metal-resin composite is dispersed as single particles without aggregation, and can be determined from the measurement results of the particle size distribution on a volume/mass basis.

By having a non-aggregation ratio of 50% or more, it is possible to increase the antibody binding yield when producing a labeled antibody and increase the amount of antibody attached to the metal-resin complex. Excellent detection sensitivity can be obtained when performing immunoassay. Such a high non-aggregation ratio is achieved by using a binding buffer containing a specific Good buffer for the metal-resin complex. On the other hand, if the non-aggregation ratio is less than 50%, it means that the proportion of aggregated particles in the metal-resin complex is relatively large, so the antibody binding yield and metal -The amount of antibody attached to the resin complex decreases, and the detection sensitivity when performing immunoassay using labeled antibodies becomes inferior.

The method for producing a labeled antibody of this embodiment can further include the following step B.

<Process B>
Step B is a step in which the labeled antibody is dispersed in a blocking buffer within the pH range of 2 to 9. In step B, blocking is performed to suppress nonspecific adsorption to the labeled antibody by dispersing the labeled antibody obtained in step A in a blocking buffer within the pH range of 2 to 9. . In this case, for example, a labeled antibody that has been separated by solid-liquid separation means is dispersed in a liquid phase under conditions within the pH range of 2 to 9. The blocking conditions are preferably within the range of pH 4 to 9 from the viewpoint of maintaining antibody activity and suppressing aggregation of the labeled antibody, and pH 5 to 9 from the viewpoint of suppressing nonspecific adsorption of the labeled antibody. It is preferably within the range of 9 to 9. If the blocking buffer has a pH of less than 2, the antibody may be denatured and inactivated due to strong acidity, and if the pH exceeds 9, the labeled antibody may aggregate and become difficult to disperse.

In step B, for example, a blocking buffer adjusted to the above pH range is added to a predetermined amount of labeled antibody obtained in step A, and the labeled antibody is uniformly dispersed in the blocking buffer. As the blocking buffer, it is preferable to use, for example, a solution of a protein that does not bind to the analyte. Examples of proteins that can be used in the blocking buffer include bovine serum albumin (BSA), 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, sodium hydroxide, or the like. Although known methods can be used to disperse the labeled antibody, it is preferable to use dispersion means such as ultrasonication and stirring with a rotator. In this way, a labeled antibody dispersion in which labeled antibodies are uniformly dispersed is obtained. From this labeled antibody dispersion, only the labeled antibody can be separated as a solid portion, for example, by solid-liquid separation means such as centrifugation. Moreover, arbitrary steps such as a cleaning treatment step and a preservation treatment step can be performed as necessary. The cleaning process and preservation process will be explained below.

(Cleaning process)
The washing process is a process in which a washing buffer is added to the labeled antibody separated by the solid-liquid separation means, and the labeled antibody is uniformly dispersed in the washing buffer. For dispersion, it is preferable to use dispersion means such as ultrasonication. The washing buffer is not particularly limited, but includes, for example, a Tris buffer (Tris hydroxymethylaminomethane buffer) with a predetermined concentration adjusted within the pH range of 8 to 9, a glycinamide buffer, Arginine buffer and the like can be used. The pH of the washing buffer can be adjusted using, for example, hydrochloric acid, sodium hydroxide, or the like. The labeled antibody washing process can be repeated multiple times as necessary.

(Preservation processing)
The preservation treatment is a treatment in which a preservation buffer is added to the labeled antibody fractionated by the solid-liquid separation means, and the labeled antibody is uniformly dispersed in the preservation buffer. For dispersion, it is preferable to use dispersion means such as ultrasonication. 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 above-mentioned washing buffer can be used. As the aggregation inhibitor, for example, sugars such as sucrose, maltose, lactose, and trehalose, and polyhydric alcohols such as glycerin and polyvinyl alcohol can be used. The stabilizer is not particularly limited, but for example, proteins such as bovine serum albumin, ovalbumin, casein, and gelatin can be used. In this way, the labeled antibody can be preserved.

In each of the above steps, a surfactant, a preservative such as sodium azide, or paraoxybenzoic acid ester may be used, if necessary.

[Labeled antibody]
A labeled antibody can be produced as described above. The labeled antibodies obtained by the method of the present invention can be used in various immunological assays in the same way as conventional labeled antibodies. For example, immunoassays can be performed by mixing a sample containing an analyte and a labeled antibody, allowing them to react, and measuring the resulting color visually or using an analytical instrument.

<Metal-resin composite>
Next, an example of a metal-resin complex used as a label in the method for producing a labeled antibody of this embodiment will be described in detail. The metal-resin composite has a structure in which metal particles are immobilized on resin particles.

FIG. 1 is a schematic cross-sectional view of a metal-resin composite 100 that can be used as a label in this embodiment. The metal-resin composite 100 includes resin particles 10 and metal particles 20.

(Structure of metal-resin composite)
In the metal-resin composite 100, metal particles 20 are dispersed or fixed in resin particles 10. Further, in the metal-resin composite 100, some of the metal particles 20 are three-dimensionally distributed in the surface layer 60 of the resin particles 10, and some of the three-dimensionally distributed metal particles 20 are partially distributed in the resin. It is preferable that the resin particles 10 are exposed outside the particles 10 and the remaining part is encapsulated in the resin particles 10. Here, the "surface layer part" means a range from the surface of the resin particle 10 to 50% of the particle radius in the depth direction. Moreover, "three-dimensionally distributed" 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.

Here, the metal particles 20 include metal particles completely encapsulated in the resin particles 10 (hereinafter also referred to as "encapsulated metal particles 30"), parts embedded in the resin particles 10, and parts exposed outside the resin particles 10. selected from metal particles having exposed parts (hereinafter also referred to as "partially exposed metal particles 40") and metal particles adsorbed on the surface of the resin particles 10 (hereinafter also referred to as "surface-adsorbed metal particles 50"). It may contain one or more types.

The entire surface of the encapsulated metal particle 30 is covered with the resin constituting the resin particle 10.
The partially exposed metal particles 40 have 5% or more and less than 100% of their surface area covered with the resin constituting the resin particles 10. From the viewpoint of durability as a label for immunological measurements, the lower limit is preferably 20% or more of the surface area, more preferably 30% or more.
More than 0% and less than 5% of the surface area of the surface adsorbed metal particles 50 is covered with the resin constituting the resin particles 10.

For example, when the metal-resin composite 100 is used for immunoassay, an antibody is immobilized on the surface of the resin particle 10, the partially exposed surface of the metal particle 40, or the surface of the surface-adsorbed metal particle 50. At this time, antibodies are immobilized on the partially exposed metal particles 40 and the surface-adsorbed metal particles 50, but not 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 Contributes to improved visibility as a measurement marker. Furthermore, the partially exposed metal particles 40 and the encapsulated metal particles 30 have a larger contact area with the resin particles 10 than the surface-adsorbed metal particles 50, and also have physical adsorption forces such as anchor effects due to the embedding state. It is strong and difficult to detach from the resin particles 10. Therefore, the metal-resin composite 100 can have excellent durability and stability as a label for immunoassay.

Further, the amount of metal particles 20 (total of encapsulated metal particles 30, partially exposed metal particles 40, and surface adsorbed metal particles 50) supported on the metal-resin composite 100 is based on the weight of the metal-resin composite 100. , preferably within the range of 5% to 70% by weight. Within this range, the metal-resin composite 100 has excellent visibility, visual judgment, and detection sensitivity as a labeling substance. When the amount of metal particles 20 supported is less than 5% by weight, the amount of immobilized antibodies tends to decrease, and detection sensitivity tends to decrease. The amount of metal particles 20 supported is more preferably within the range of 15% to 70% by weight.

Further, it is preferable that the partially exposed metal particles 40 and the surface-adsorbed metal particles 50 account for 10% to 90% by weight of the metal particles 20. Within this range, a sufficient amount of antibody immobilized on the metal particles 20 can be ensured, resulting in high sensitivity as a labeling substance. More preferably, 20% to 80% by weight of the metal particles 20 are partially exposed metal particles 40 and surface-adsorbed metal particles 50.

Further, it is preferable that 60% to 100% by weight of the metal particles 20 exist in the surface layer 60, and 5% to 90% by weight of the metal particles 20 present in the surface layer 60. However, partially exposed metal particles 40 or surface-adsorbed metal particles 50 are preferable because a sufficient amount of the antibody immobilized on the metal particles 20 can be ensured and the sensitivity as a labeling substance can be increased. In other words, it is preferable that the encapsulated metal particles 30 account for 10% to 95% by weight of the metal particles 20 present in the surface layer portion 60.

(resin particles)
It is preferable that the resin particles 10 are polymer particles having a structure containing a substituent capable of adsorbing metal ions. In particular, nitrogen-containing polymer particles are preferred. Nitrogen atoms in nitrogen-containing polymers have excellent visibility and are easy to chemisorb anionic metal ions, which are precursors of metal particles such as silver, nickel, copper, gold, platinum, and palladium, on which antibodies can be immobilized easily. preferred for. When the metal ions adsorbed in the nitrogen-containing polymer are reduced to form metal nanoparticles, some of the generated metal particles 20 become encapsulated metal particles 30 or partially exposed metal particles 40.
In addition, polymers with functional groups such as carboxylic acid, such as acrylic acid polymers, can adsorb cationic metal ions, so they can be used as precursors for metal particles such as silver, nickel, copper, gold, platinum, and palladium. Because it easily adsorbs cationic metal ions, it is possible to form metal particles 20 such as silver, nickel, copper, gold, platinum, palladium, etc., and it is possible to form an alloy with any of the above metals. is also possible.

The nitrogen-containing polymer is a resin having a nitrogen atom in its main chain or side chain, and includes, for example, polyamine, polyamide, polypeptide, polyurethane, polyurea, polyimide, polyimidazole, polyoxazole, polypyrrole, polyaniline, and the like. Preferred are polyamines such as poly-2-vinylpyridine, poly-3-vinylpyridine, and poly-4-vinylpyridine. Further, when the side chain has a nitrogen atom, a wide range of materials such as acrylic resin, phenol resin, epoxy resin, cellulose resin, and melamine resin can be used.

In addition, in the case of resin particles that do not have a substituent in their structure that can adsorb metal ions, such as unsubstituted polystyrene particles, irradiation of the particle surface with ultrasonic waves or ionizing radiation, metal plating of the resin particle surface, etc. This allows metal particles to be immobilized on the particle surface.

(metal particles)
Although the structure of the metal particles 20 is not particularly limited, it is preferable that the metal particles 20 exhibit color in the state of the metal-resin composite 100 immobilized on the resin particles 10, since visual determination can be easily made by immunoassay. As the material of the metal particles 20, for example, silver, nickel, copper, gold, platinum, and palladium can be used. These metals can be used alone or in composites such as alloys. Preferred are gold, platinum, and palladium, which have excellent visibility and allow easy immobilization of antibodies, and these materials develop color by absorption derived from localized surface plasmon resonance. More preferred are gold and platinum, which have good storage stability. Moreover, gold, platinum, a gold alloy, or a platinum alloy are the most preferred metal species from the viewpoint of obtaining excellent color development when combined with an antibody. Here, the gold alloy refers to an alloy consisting of, for example, gold and a metal species other than gold, and containing gold in an amount of 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more. Furthermore, a platinum alloy refers to an alloy consisting of, for example, platinum and a metal species other than platinum, and containing platinum in an amount of 10% by weight or more, preferably 50% by weight or more, and more preferably 60% by weight or more.

Further, the average particle diameter of the metal particles 20 measured by scanning electron microscopy (SEM) is preferably within the range of, for example, 1 to 80 nm. When the average particle diameter of the metal particles 20 is less than 1 nm or more than 80 nm, localized surface plasmons are less likely to occur, so sensitivity tends to decrease. The average particle diameter of the metal particles 20 is preferably in the range of 1 nm or more and less than 70 nm, more preferably in the range of 1 nm or more and less than 50 nm.

Further, the average particle diameter of the metal-resin composite 100 is, for example, within the range of 100 to 1000 nm. If the average particle diameter is less than 100 nm, for example, when gold particles or platinum particles are used as the metal particles 20, the amount of supported metal particles 20 tends to be smaller, so the coloring tends to be weaker than that of gold particles of the same size. If the wavelength exceeds 1000 nm, when used as a labeled antibody or reagent, it tends to clog the pores of a chromatographic medium such as a membrane filter, and its dispersibility tends to decrease. The average particle diameter of the metal-resin composite 100 is preferably in the range of 100 nm or more and less than 700 nm, more preferably in the range of 100 nm or more and less than 650 nm. Here, the particle diameter of the metal-resin 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 diameter of the resin particle 10, and It can be measured by / scattering method, dynamic light scattering method, or centrifugal sedimentation method.

The method for manufacturing the metal-resin 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 onto the resin particles 10 (hereinafter referred to as "metal ion adsorption resin particles"). Next, the metal-resin composite 100 can be obtained by adding metal ion-adsorbing resin particles to the reducing agent solution to reduce the metal ions and generate the metal particles 20. Here, when gold particles are used as the metal particles 20, it is preferable to use, for example, a chloroauric acid (HAuCl ₄ ) aqueous solution as the solution containing metal ions. Further, when platinum particles are used as the metal particles 20, it is preferable to use, for example, a chloroplatinic acid (H ₂ PtCl ₆ ) aqueous solution as the solution containing metal ions. Note that a metal complex may be used instead of the metal ion.

In addition, as a solvent for a solution containing metal ions, water-containing alcohols or alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, t-butanol, hydrochloric acid, sulfuric acid, nitric acid, etc. can be used instead of water. You may use the acid etc.

In addition, if necessary, water-soluble polymer compounds such as polyvinyl alcohol, surfactants, alcohols; ethers such as tetrahydrofuran, diethyl ether, and diisopropyl ether; alkylene glycol, Additives such as various water-miscible organic solvents such as alkylene glycol, their monoalkyl ethers or dialkyl ethers, polyols such as glycerin; and ketones such as acetone and methyl ethyl ketone may be added. Such additives are effective in accelerating the reduction reaction rate of metal ions and controlling the size of the metal particles 20 produced.

The reducing agent is not particularly limited, but examples include sodium borohydride, dimethylamine borane, citric acid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride, hydrazine sulfate, formaldehyde, sucrose, glucose, Preferably, ascorbic acid, sodium phosphinate, hydroquinone, hydrazine sulfate, formaldehyde, Rochelle's salt, etc. are used. Among these, sodium borohydride, dimethylamine borane, and citric acid are more preferred. A surfactant can be added to the reducing agent solution or the pH of the solution can be adjusted as necessary. The pH can be adjusted using 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.
Further, by adjusting the reduction rate of metal ions by adjusting the temperature of the reducing agent solution, the particle size of the metal particles 20 to be formed can be controlled.

Further, when reducing the metal ions in the metal ion adsorption resin particles to generate 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 particles. However, the former is preferable from the viewpoint of ease of generating the encapsulated metal particles 30 and the partially exposed metal particles 40.

In order to maintain the dispersibility of the metal-resin composite 100 in water, for example, citric acid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinyl alcohol, DISPERBYK194, DISPERBYK180, DISPERBYK184 (Big Chemie Japan Co., Ltd. You may also add a dispersant such as (manufactured by). Furthermore, 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 metal-resin composite 100 having the above configuration can be preferably applied to various immunoassay methods by adsorbing antibodies to the surfaces of the resin particles 10 and/or metal particles 20. In addition, it can be preferably applied as a material for a labeling substance for immunological measurement or a reagent for immunological measurement, which has excellent visual judgment in a low concentration range (high sensitivity range). There is no particular limitation on the form of the labeling substance for immunological assays or the reagent for immunological assays, but for example, the metal-resin complex 100 may be used as a dispersion liquid in water or a pH-adjusted buffer solution. You can also use

EXAMPLES Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples in any way. In the following Examples and Comparative Examples, unless otherwise specified, various measurements and evaluations are as follows.

<Absorbance measurement of metal-resin composite particles>
The absorbance of the metal-resin composite particles was measured by placing a metal-resin composite particle dispersion (dispersion medium: water) prepared at 0.01 wt% in a quartz glass cell (light path length 10 mm) and using a spectrophotometer (Shimadzu Corporation). The absorbance was measured at 570 nm in the case of the gold-resin composite and at 400 nm in the case of the platinum-resin composite using UV3600 (manufactured by Co., Ltd.).

<Measurement of solid content concentration and metal support amount>
1 g of the dispersion before concentration adjustment was placed in a porcelain crucible and dried at 70° C. for 3 hours. The weight before and after drying was measured, and the solid content concentration was calculated using the following formula.
Solid content concentration (wt%) =
[Weight after drying (g) / Weight before drying (g)] x 100
Further, the sample after the drying treatment was further heat-treated at 500° C. for 5 hours, the weight before and after the heat treatment was measured, and the amount of metal supported was calculated from the following formula.
Amount of metal supported (wt%) =
[Weight after heat treatment (g)/Weight before heat treatment (g)] x 100

<Measurement of average particle diameter and particle size distribution of resin particles and metal-resin composite particles>
The measurement was performed using a centrifugal sedimentation type particle size distribution analyzer (LUMiSizer610 manufactured by LUM GmbH). The measurements were performed with the particles dispersed in water or a solution.

<Measurement of average particle diameter of metal particles>
Image of a substrate prepared by dropping a metal-resin composite particle dispersion onto a metal mesh with a carbon support film, observed using a field emission scanning electron microscope (FE-SEM; manufactured by Hitachi High-Technologies, SU-9000) The area average diameter of 100 arbitrary metal particles was measured and defined as the average particle diameter.

[Preparation example 1]
<Synthesis of resin particles>
After dissolving trioctyl ammonium chloride (1.3 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) in 300 g of pure water, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added. The mixture was stirred at 30° C. for 50 minutes and then at 60° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.25 g) dissolved in 18.00 g of pure water was added dropwise and stirred at 60°C for 3.5 hours to form an average particle. Resin particles A-1 having a diameter of 349 nm were obtained. After precipitation by centrifugation (9000 rpm, 40 minutes) and removal of the supernatant, the mixture was redispersed in pure water, and impurities were removed by dialysis treatment. Thereafter, the concentration was adjusted to obtain a 10 wt % resin particle dispersion B-1.

[Preparation example 2]
<Synthesis of resin particles>
After dissolving trioctyl ammonium chloride (0.8 g) and polyethylene glycol methyl ethyl ether methacrylate (10.00 g) in 300 g of pure water, 2-vinylpyridine (48.00 g) and divinylbenzene (2.00 g) were added. The mixture was stirred at 30° C. for 50 minutes and then at 60° C. for 30 minutes under a nitrogen stream. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.25 g) dissolved in 18.00 g of pure water was added dropwise and stirred at 60°C for 3.5 hours to form an average particle. Resin particles A-2 having a diameter of 430 nm were obtained. After precipitation by centrifugation (9000 rpm, 40 minutes) and removal of the supernatant, the mixture was redispersed in pure water, and impurities were removed by dialysis treatment. Thereafter, the concentration was adjusted to obtain a 10 wt % resin particle dispersion B-2.

[Preparation example 3]
<Synthesis of gold-resin composite particles>
After adding 78.0 g of pure water to 30.0 g of resin particle dispersion B-1, 42.9 g of a 7 wt% chloroauric acid aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. This mixed solution was centrifuged (3000 rpm, 30 minutes) to precipitate the resin particles, and the supernatant was removed to remove excess chloroauric acid. Thereafter, the concentration was adjusted to obtain a 5 wt % gold ion adsorption resin particle dispersion C-3.

Next, 65 g of C-3 was added to 4725 g of pure water, and while stirring at 3°C, 15 g of a 528 mM dimethylamine borane aqueous solution was added dropwise over 2 minutes, followed by stirring at 3°C for 1 hour and at room temperature for 3 hours. Thus, gold-resin composite particles D-3 having an average particle diameter of 357 nm were obtained. After concentrating D-3 by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain 1 wt % gold-resin composite particle dispersion E-3. The absorbance of gold-resin composite particles F-3 in E-3 was 1.38. Further, the average particle diameter of the gold particles in F-3 was 21 nm, and the amount of gold supported was 48.0 wt%.

[Preparation example 4]
<Synthesis of platinum-resin composite particles>
After adding 85.0 g of pure water to 30.0 g of resin particle dispersion B-1, 31.0 g of a 7 wt% chloroplatinic acid aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. This mixed solution was centrifuged (3000 rpm, 30 minutes) to precipitate the resin particles, and the supernatant was removed to remove excess chloroplatinic acid. Thereafter, the concentration was adjusted to obtain a 5 wt % platinum ion adsorption resin particle dispersion C-4.

Next, 70.5g of C-4 was added to 4725g of pure water, and while stirring at 3°C, 137g of a 132mM dimethylamine borane aqueous solution was added dropwise over 20 minutes, followed by stirring at 3°C for 1 hour and at room temperature for 3 hours. By doing so, platinum-resin composite particles D-4 having an average particle diameter of 360 nm were obtained. After concentrating D-4 by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain 1 wt % platinum-resin composite particle dispersion E-4. The absorbance of platinum-resin composite particles F-4 in E-4 was 1.75. Further, the average particle diameter of the platinum particles in F-4 was 3.5 nm, and the amount of platinum supported was 39.1 wt%.

[Preparation example 5]
<Synthesis of platinum-resin composite particles>
After adding 85.0 g of pure water to 30.0 g of resin particle dispersion B-2, 31.0 g of a 7 wt% chloroplatinic acid aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. This mixed solution was centrifuged (3000 rpm, 30 minutes) to precipitate the resin particles, and the supernatant was removed to remove excess chloroplatinic acid. Thereafter, the concentration was adjusted to obtain platinum ion adsorption resin particle dispersion C-5 of 5 wt%.

Next, 70.5g of C-5 was added to 4725g of pure water, and while stirring at 3°C, 137g of a 132mM dimethylamine borane aqueous solution was added dropwise over 20 minutes, followed by stirring for 1 hour at 3°C and 3 hours at room temperature. As a result, platinum-resin composite particles D-5 having an average particle diameter of 445 nm were obtained. After D-5 was concentrated by centrifugation, it was purified by dialysis treatment and the concentration was adjusted to obtain a 1 wt % platinum-resin composite particle dispersion E-5. The absorbance of platinum-resin composite particles F-5 in E-5 was 1.75. Further, the average particle diameter of the platinum particles in F-5 was 3.7 nm, and the amount of platinum supported was 39.4 wt%.

[Preparation example 6]
<Preparation of binding buffer>
HEPES, TAPS, CAPS, 0.1 mol/L of 2-morpholinoethanesulfonic acid [MES; pKa (20 °C) 6.15] or 3-morpholinopropanesulfonic acid [MOPS; pKa (20 °C) 7.20] HEPES buffer, TAPS buffer, CAPS buffer was prepared by preparing a free acid solution and adjusting the buffer concentration and pH shown in Table 1 by mixing pure water and 0.1 mol/L sodium hydroxide solution with each solution. A buffer solution, MES buffer solution, and MOPS buffer solution were prepared.

[Example 1]
(Joining process)
After mixing 25 μg of anti-CRP antibody and 0.45 mL of 20 mM HEPES buffer (pH 7), 0.05 mL of 1 wt% gold-resin composite particle dispersion E-3 was added, and the mixture was stirred upside down for 2 hours at room temperature. A labeled antibody dispersion J-1 containing an anti-CRP antibody labeled with gold-resin composite particles F-3 was obtained.

(block process)
Next, labeled antibody dispersion J-1 was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed, and 0.5 mL of a 5 mM Tris aqueous solution (pH 5) containing 1 wt% BSA was added to the sediment. After sonic dispersion, the mixture was further stirred by inverting at room temperature for 2 hours to obtain labeled antibody dispersion K-1.

(Cleaning process)
Next, the labeled antibody dispersion K-1 was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed, and a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant was added to the sediment. .5 mL was added and subjected to ultrasonic dispersion. This operation was repeated three times to obtain a cleaning treatment.

(Preservation processing)
Next, after cooling on ice, centrifugation was performed at 3000 rpm for 5 minutes, the supernatant was removed, and the sediment was added to a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant and 10 wt% sucrose. ) was added and subjected to ultrasonic dispersion to obtain labeled antibody dispersion L-1.

(Conjugate pad preparation)
After centrifuging 0.12 ml of labeled antibody dispersion L-1 at 3000 rpm for 5 minutes and removing the supernatant, the precipitated sediment was mixed with an aqueous solution (pH 8.0) containing 5 wt% sucrose and 2.5 wt% BSA. .333 ml was added and subjected to ultrasonic dispersion to obtain labeled antibody dispersion M-1. After a glass fiber nonwoven fabric was uniformly impregnated with the labeled antibody dispersion M-1, it was dried at 50° C. for 1 hour to produce a conjugate pad N-1. At this time, the volume of labeled antibody dispersion M-1 was adjusted so that the content of gold-resin composite particles F-3 in conjugate pad N-1 was 3 μg per evaluation test using the immunochromatography method described below. did.

(Preparation of immunochromato strip)
An immunochromato strip having the structure shown in FIG. 2 was prepared. First, a test line 5 was drawn on a 25 mm wide nitrocellulose membrane 4 by applying an anti-CRP antibody. Further, an anti-mouse IgG antibody was applied to the downstream side of the test line 5 to draw a control line 6. After drying this nitrocellulose membrane 4 at 50°C for 1 hour, the laminate film 1, the conjugate pad N-1 as the conjugate pad 3, and the sample pad 2 (glass fiber nonwoven fabric) and absorbent pad 7 (cotton nonwoven fabric) were laminated. Finally, immunochromatography strip P-1 was prepared by cutting it to a width of 3.5 mm.

(Preparation of developing solution)
An aqueous solution (pH 7.1) containing 50 mM Tris, 150 mM NaCl, 1.0 wt% BSA, and 1.0 wt% PEG cetyl ether was prepared and designated as developing solution Q-1.

(Evaluation by immunochromatography)
Positive control (antigen concentration 312 ng/ml, 3.12 ng/ml) and negative control (antigen-free) sample solutions were prepared by diluting the CRP antigen using developer Q-1. 50 μl of the sample liquid was dropped onto sample pad 2 of immunochromato strip P-1. Thereafter, the color intensity of test line 5 after 30 minutes was measured using an immunochromatoreader (Hamamatsu Photonics C10066-10). In addition, it was visually determined whether the interface between the conjugate pad 3 and the membrane 4 and the downstream side thereof were clogged with gold-resin composite particles F-3. Table 1 shows the evaluation results by immunochromatography.

(Calculation of bound antibody amount)
The amount of antibody bound to the gold-resin composite particles F-3 was calculated using the supernatant liquid removed by centrifugation in the blocking step. To calculate the amount of bound antibody, measure the supernatant using a spectrophotometer, determine the amount of antibody contained in the supernatant from the absorbance at a wavelength of 280 nm, and subtract the amount of antibody in the supernatant from the 25 μg of antibody used in the binding step. It was calculated by Table 1 shows the calculated amounts of bound antibodies and antibody binding yields. Note that the binding yield means the weight ratio (%) of the attached antibody to the total amount of antibody used.

[Examples 2 to 5, Comparative Examples 1 to 5]
The binding process, blocking process, washing process, storage process, conjugate pad preparation, and immunochromatography strip were carried out in the same manner as in Example 1 except that the metal-resin composite particle dispersion and binding buffer shown in Table 1 were used. Preparation, immunochromatographic evaluation, and calculation of bound antibody amount and antibody binding yield were performed. Table 1 shows the calculation results of the bound antibody amount and antibody binding yield, and the immunochromatography evaluation results.

From the test results of Examples 1 to 5 and Comparative Examples 1 to 5, using a binding buffer containing Good's buffer with a pKa of 7.5 or more at 20°C increases the amount of bound antibody and improves immunochromatography. It can be seen that the clogging of the evaluation results is suppressed, the coloring intensity of the test line increases, and the sensitivity of immunochromatography is improved.

[Example 6]
(Joining process)
After mixing 25 μg of anti-influenza A virus antibody and 0.45 mL of 20 mM HEPES buffer (pH 7), 0.05 mL of 1 wt% gold-resin composite particle dispersion E-3 was added, and the mixture was incubated at room temperature for 2 hours. By performing inversion stirring, labeled antibody dispersion J-6 containing anti-influenza A virus antibody labeled with gold-resin composite particles F-3 was obtained.

(Block process, washing process, preservation process, conjugate pad preparation)
Conjugate pad N-6 was prepared by performing the blocking process, washing process, storage process, and conjugate pad preparation in the same manner as in Example 1 except for using labeled antibody dispersion J-6.

(Preparation of immunochromato strip)
An immunochromato strip having the structure shown in FIG. 2 was prepared. First, an anti-influenza A virus antibody was applied to a nitrocellulose membrane 4 with a width of 25 mm, and a test line 5 was drawn. Further, an anti-mouse IgG antibody was applied to the downstream side of the test line 5 to draw a control line 6. After drying this nitrocellulose membrane 4 at 50°C for 1 hour, the laminate film 1, the conjugate pad N-6 as the conjugate pad 3, and the sample pad 2 (glass fiber nonwoven fabric) and absorbent pad 7 (cotton nonwoven fabric) were laminated. Finally, immunochromato strip P-6 was prepared by cutting it into a 3.5 mm width.

(Preparation of developing solution)
An aqueous solution (pH 7.1) containing 50mM Tris, 150mM NaCl, 1.0wt% BSA, and 1.0wt% TritonX100 was prepared and designated as developing solution Q-6.

(Evaluation by immunochromatography)
Positive control (antigen concentration 1250 FFU/ml, equivalent to 20 FFU/ml) and negative control (antigen-free) sample solutions were prepared by diluting the inactivated influenza A virus antigen using developer Q-6. 50 μl of the sample solution was dropped onto sample pad 2 of immunochromato strip P-6. Thereafter, the color intensity of test line 5 after 30 minutes was measured using an immunochromatoreader (Hamamatsu Photonics C10066-10). In addition, it was visually determined whether the interface between the conjugate pad 3 and the membrane 4 and the downstream side thereof were clogged with gold-resin composite particles F-3. Table 2 shows the evaluation results by immunochromatography.

[Comparative example 6]
In the binding step of Example 6, the binding step, blocking step, washing treatment, storage treatment, Conjugate pad preparation, immunochromatography strip preparation, and immunochromatography evaluation were performed. Table 2 shows the immunochromatographic evaluation results.

From the test results of Example 6 and Comparative Example 6, by using a binding buffer containing Good's buffer with a pKa of 7.5 or higher at 20°C, clogging was suppressed in the immunochromatographic evaluation results, and the test line It can be seen that the color intensity increases, false positives with antigen-free negative control samples are suppressed, and the sensitivity of immunochromatography is improved.

[Reference test examples 1 to 4]
(Evaluation of degree of aggregation by mixing binding buffer and metal-resin composite particles)
After mixing 0.45 ml of the binding buffer shown in Table 3 and 0.05 ml of 1 wt% gold-resin composite particle dispersion E-3, the mixture was stirred upside down at room temperature for 2 hours, and the particle size distribution was measured. I did it. From the measurement results of the particle size distribution, the ratio at which the gold-resin composite particles F-3 were dispersed as single particles without agglomeration (non-aggregation ratio) was determined. The results are shown in Table 3.

From the test results of Reference Test Examples 1 to 4, it can be seen that in Reference Test Examples 1 to 3, which used a binding buffer containing Good's buffer with a pKa of 7.5 or more at 20°C, It can be seen that, compared to Test Example 4, aggregation of the gold-resin composite particles F-3 is suppressed. It is presumed that such aggregation-inhibiting effect leads to an increase in the amount of antibody attached, and also suppresses clogging in the immunochromatography strip, contributing to good immunochromatography evaluation results.

Although the embodiments of the present invention have been described above in detail for the purpose of illustration, the present invention is not limited to the above embodiments.

DESCRIPTION OF SYMBOLS 1... Laminate film, 2... Sample pad, 3... Conjugate pad, 4... Nitrocellulose membrane, 5... Test line, 6... Control line, 7... Absorption pad, 10... Resin particle, 20... Metal particle, 30... Encapsulation Metal particles, 40...Partially exposed metal particles, 50...Surface adsorbed metal particles, 60...Surface layer part, 100...Metal-resin composite

Claims (11)

A method for producing a labeled antibody, the method comprising:
An antibody and a metal-resin complex having a structure in which metal particles are immobilized on resin particles are bonded using a binding buffer within the pH range of 6 to 10, including Good's buffer having a pKa of 7.5 or more at 20°C. a binding step of attaching the antibody to the metal-resin complex by mixing in the presence of a liquid;
A method for producing a labeled antibody, comprising: The Good buffer includes 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), N- From cyclohexyl-3-aminopropanesulfonic acid (CAPS), 2-cyclohexylaminoethanesulfonic acid (CHES) and N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO). The method for producing a labeled antibody according to claim 1, wherein the labeled antibody is one or more selected types. 3. The method for producing a labeled antibody according to claim 1, wherein in the binding step, the proportion of the attached antibody to the total amount of antibody used is 50% by weight or more. 4. The method for producing a labeled antibody according to claim 1, wherein the metal particles are silver, nickel, copper, gold, platinum, palladium, or an alloy containing any of these. The method for producing a labeled antibody according to any one of claims 1 to 4, wherein the metal particles include metal particles having a portion embedded within the resin particle and a portion exposed outside the resin particle. . 6. The method for producing a labeled antibody according to claim 1, wherein at least some of the metal particles are three-dimensionally distributed in the surface layer of the resin particles. 7. The method for producing a labeled antibody according to claim 1, wherein the resin particles are polymer particles having a substituent in their structure capable of adsorbing metal ions. The method for producing a labeled antibody according to any one of claims 1 to 7, wherein the metal particles have an average particle diameter within a range of 1 to 80 nm. The method for producing a labeled antibody according to any one of claims 1 to 8, wherein the average particle diameter of the metal-resin composite is within the range of 100 to 1000 nm. A labeled antibody produced by the method for producing a labeled antibody according to any one of claims 1 to 9. An immunoassay method characterized by using the labeled antibody according to claim 10.

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