JPS62231171A - Labeling complex - Google Patents

Abstract

PURPOSE:To easily obtain a labeling complex having high sensitivity and high accuracy at a high yield by using water dispersion type high-molecular polymer particles as a carrier and conjugating both a labeling agent and immune reactant therewith to obtain the labeling complex. CONSTITUTION:The water dispersion type high-molecular polymer particles are prepd. by emulsion polymn. of 1 or >=2 monomers having unsatd. double bonds, for which commercially marketed water dispersion type high-molecular polymer particles are usable. Any labeling agents which are heretofore used for solid-phase immunoassy are usable as the labeling agent. There is no need for using the labeling agent at one molecule per molecule of the immune reactant and the labeling agent can be easily adjusted within a 0.01-1,000 molecule range. The labeling complex is obtd. by conjugating the immune reactant and the labeling agent with the water dispersion type high-molecular polymer particles, then separating and refining the labeling complex by an ordinary sepn. method.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is directed to antigens, haptens, or antibodies as analytes contained in a liquid sample, or when these analytes are attached to cells, tissues, or other substances. If included, labeled antigens used to quantify these analytes (
or hapten) or a method for producing an antibody.

(Prior art) The antigen (or hapten) or antibody is identified by utilizing the high specificity and detection sensitivity of the antigen (or hapten) antibody reaction,
Traditionally, solid-phase immunoassay (
immunoassay) is known.

This method requires labeling either the antigen (or hapten) or the antibody. Generally, these labeled antigens (or haptens) or antibodies (hereinafter referred to as labeled complexes) are made by binding a labeling agent to an antigen (or hapten) or antibody (hereinafter referred to as an immunoreactant). In order for such a labeling complex to have excellent practicality, high sensitivity, and high precision, the activity and function of the labeling agent and immunoreactant must be highly retained in the labeling complex. and that it can be stored for a long period of time.
It is strongly required that the product does not contain impurities such as unlabeled substances.

Conventionally, a crosslinking method has been known as a typical method for producing such a labeled complex. According to this crosslinking method, a labeling agent and an immunoreactant are dissolved in water, a dialdehyde such as glutaraldehyde is added to this aqueous solution, the labeling agent and the immunoreactant are mutually crosslinked, and then excess The labeled complex is obtained by removing the dialdehyde. In addition, as an improvement to this method, the labeling agent is treated with dialdehyde to give the labeling agent a free aldehyde group, and then the excess aldehyde is removed, and then the )! A labeled complex is obtained by binding an immunoreactant to a labeling agent using the +1 aldehyde group.

Furthermore, a method using a bifunctional reagent with high reaction specificity is also known. For example, bifunctional reagents such as N,N'-o-phenylene dimaleimide have reaction specificity in that they react quickly with thiol groups, but on the other hand, react with amino groups or hydroxyl groups usually very slowly. have Therefore, after reacting N,N"-0-phenylene dimaleimide with the immunoreactant having a thiol group, excess N,N'-0-phenylene dimaleimide was removed, and then the labeling agent having a thiol group was removed. A labeled complex can be obtained by reacting with.

As another method, a method using a reagent having two different functional groups with high reaction specificity, such as N-(metamareimidobenzoyloxy)succinimide, is also known.

However, in all of the conventional methods described above, there is a high probability that the immunoreactants are crosslinked with each other or the labeling agents are crosslinked with each other and do not contribute to the production of the labeling agent. Therefore, in addition to the yield of the obtained labeled complex being low, the activity of the labeling agent is also low because the binding density of the labeling agent on the immunoreactant is extremely low. Additionally, in order to directly bind the labeling agent to the immunoreactant, generally
Activity, function, specificity, etc. are likely to be lost. Furthermore, the types of immunoreactants and labeling agents that can be applied are limited, and
Purification of the resulting labeled complex is extremely complicated.

On the other hand, there are problems in its use depending on the labeling agent used. For example, a solid-phase radioimmunoassay method using a radioactive substance such as +51.1fflI, '+1.14C, etc. as a labeling complex is well known. Although this method is highly sensitive and has been widely put into practical use, its implementation is subject to strict regulations due to radioactive waste material disposal issues, and the shelf life of the labeling agent used is limited. It also has the practical disadvantage of being short.

In order to solve such problems, enzyme immunoassays that use enzymes as labeling agents and fluorescent immunoassays that use fluorescent substances, phosphorescent substances, atomic fluorescent substances, etc. are already known, but both of them are In addition to insufficient sensitivity and precision, the production yield is also low.
In addition, the purification process is also complicated.

(Purpose of the Invention) As a result of intensive research in order to solve the above-mentioned problems in conventional labeling complexes, the present inventors used water-dispersed polymer particles as a carrier, and added a labeling agent and an immunoreactant to the carrier. It has been discovered that by binding together to obtain a labeled complex, the above-mentioned problems can be solved at once, and a labeled complex with high sensitivity and high sensitivity can be obtained easily and in high yield, This led to the present invention.

(Structure of the Invention) The labeling complex according to the present invention uses water-dispersed polymer particles with a particle size of 0.01 to 3 μm as a carrier, and a labeling agent is bound to the carrier along with an antigen, hapten, or antibody. It is characterized by being

The water-dispersed polymer particles used in the present invention are usually prepared by emulsion polymerization of one or two monomers having unsaturated double bonds. Examples of such monomers include olefinic monomers such as ethylene and propylene;
Vinyl monomers such as vinyl acetate and vinyl chloride, styrene spinning wheels such as styrene, methylstyrene, and chlorostyrene (
1c, acrylic ester monomers such as methyl acrylate, methacrylic ester monomers such as methyl methacrylate, and diene monomers such as butadiene.

In addition, water-dispersible polymers are used for the purpose of imparting functional groups or ionic groups to homopolymer or copolymer particles of these monomers, or increasing the dispersion stability of the particles in an aqueous medium. For particle modification, acrylic acid, methacrylic acid, 2,
2. Monomers such as fluorinated methacrylic acid esters such as 2-1-lifluoroethyl methyl methacrylate, acrylonitrile, methacrylate trile, acrylamide, sodium styrene sulfonate, sulfopropyl (meth)acrylate sodium salt, N-vinylpyrrolidone, etc. It is also possible to copolymerize the iit-isomer with the iit-isomer.

Of course, commercially available water-dispersed polymer particles can also be suitably used. Such water-dispersed polymer particles include, for example, styrene or its derivatives, such as p
- Emulsions of water-dispersed polymer particles made of various styrene copolymers such as homopolymers and copolymers of chlorostyrene, styrene-butadiene copolymers, and styrene-acrylonitrile-butadiene copolymers may be mentioned. can. In addition, homopolymers consisting of long-chain alkyl esters of (meth)acrylic acid and their derivatives, and (
meth) methyl, ethyl acrylate, glycidyl (meth)
Copolymers with acrylates and the like can also be used in the present invention. A copolymer of the above-mentioned styrene or its derivative 4-isomer and (meth)acrylate ester or its m4-isomer may also be used.

Further, in addition to the above monomers, crosslinked water-dispersed polymer particles obtained by emulsion copolymerization of a polyfunctional monomer as a monomer component and an internal crosslinking agent can also be used. in general,
Copolymers with hydrophilic groups have water swelling properties and may have insufficient stability such as storage stability, pH resistance, and dispersion stability. By introducing a crosslinked structure, the water-dispersible polymer particles can be made non-swellable and the dispersion stability of the polymer particles in an aqueous medium can be increased. Furthermore, the glass transition of the resulting polymer particles can be improved. Temperature can be increased.

As such a polyfunctional monomer for internal crosslinking, for example, poly(meth)acrylate of aliphatic polyhydric alcohol is preferably used. Specific examples include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, and triethylene glycol di(meth)acrylate.
Preferably used are meth)acrylate, dipropylene glycol di(meth)acrylate, 1,3-butylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, tetramethylolmethanetetra(meth)acrylate, and the like.

Further, divinylbenzene, N,N'-methylenebisacrylamide, etc. can also be used as internal crosslinking agents.

The specific types of individual monomers are determined based on whether the obtained water-dispersed polymer particles are a labeled complex containing an immunoreactant and a labeling agent, and do not cause fusion or aggregation during use or storage. The glass is selected to have the required glass transition temperature so that it does not occur. The glass transition point of the polymer particles is preferably 10"C or higher, particularly room temperature or higher, taking into account the temperature at which the labeled complex is used.

Further, the water-dispersible polymer particles used in the present invention preferably have a particle diameter in the range of 0.01 to 3 μm. In particular, it is preferably in the range of 0.1 to 2 μm. When the particle size exceeds 3 μm, the particles tend to settle in the aqueous medium, and the activity of the resulting labeled complex decreases.On the other hand, when the particle size is smaller than 0.01 μm, the purified labeled complex becomes difficult to obtain. This is because it is complicated and difficult.

In the present invention, the immunoreactant and/or the labeling agent may be bonded to the water-dispersed polymer particles through a covalent bond, or as described later, the water-dispersed polymer particles may be bonded to the water-dispersed polymer particles via a so-called spacer group. When binding an immunoreactant and/or a labeling agent to the particles, the polymer particles need to have functional groups on their surfaces. Examples of such functional groups include carboxyl groups, hydroxyl groups, glycidyl groups, amino groups, polmyl groups, carbamoyl groups, isothiocyanate groups, azidocarbonyl groups, hydrazide groups, and acid anhydride groups. Therefore, in order to prepare polymer particles having these functional groups, monomers having carboxyl groups such as acrylic acid and methacrylic acid, such as hydroxyethyl acrylate, 2-
By emulsion copolymerizing a monomer having a hydroxyl group such as hydroxymethyl methacrylate, for example, a monomer having a glycidyl group such as glycidyl methacrylate, with other copolymerizable polymers as necessary, Water-dispersed polymer particles each having a carboxyl group, a hydroxyl group, and a glycidyl group can be obtained.

Further, after polymerizing the required monomer components, a functional group can be introduced into the obtained water-dispersed polymer particles. As a method for this purpose, for example, polymer particles containing carboxyl groups can be obtained by hydrolyzing polymer particles obtained by polymerizing an acrylic acid ester as a monomer component. In addition, in order to prepare water-dispersed polymer particles having an amino group or a hydrazide group, for example, a monomer having an amide group such as acrylamide, or a monomer having a methyl ester group such as methyl acrylate is used. It can be obtained by emulsion copolymerizing each monomer with another monomer, and then subjecting the amide group in the resulting copolymer to Hofmann degradation, or by reacting the methyl ester group with hydrazine.

Furthermore, in the labeling complex according to the present invention, when binding the immunoreactant and/or labeling agent to the water-dispersed polymer particles by a covalent bond, if necessary, the immunoreactant and/or labeling agent may be bonded to the water-dispersed polymer particles. In order to increase the degree of freedom on the polymer particles, the polymer particles and the immunologically active substance can be covalently bonded via a spacer group. The spacer group may be previously attached to the polymer particle and then the spacer group and the immunoreactant and/or the labeling agent may be attached, or the spacer group may be previously attached to the immunoreactant and/or the labeling agent. may be bonded to the polymer particles. Furthermore, if necessary, spacer groups can be bonded to all of the polymer particles, immunoreactant, and labeling agent in advance to bond them to each other.

The compound that can be used as the spacer group is at least a difunctional organic compound, and does not exclude polyfunctional polymers, but particularly difunctional polymers having a carbon chain group having 1 to 12 carbon atoms. Organic compounds are preferred. Specific examples of compounds that function as such spacer groups include hexamethylene diamine, dodecamethylene diamine,
Diamines such as glycylene diamine, aminoalkyl carboxylic acids such as glycine, β-aminopropionic acid, γ-aminobutyric acid, ε-aminocaproic acid, and ε-aminocaprylic acid, lysine, glutamic acid, β-alanine, arginine, glycyl Amino acids such as glycylglycine are preferably used, but are not limited thereto.

An immunoreactant and/or a labeling agent is directly covalently bonded to the water-dispersed polymer particles having the above-mentioned functional groups, or a spacer group is bonded to the polymer particles, and the spacer group is The method for covalently binding the immunoreactant and/or the labeling agent is not particularly limited, and any conventionally known method can be used. For example, one preferred method is to use a water-soluble carbodiimide as a binding reagent. For example, when using an aminoalkylcarboxylic acid as a spacer group, the aminoalkylcarboxylic acid is bonded to water-dispersed polymer particles using a water-soluble carbodiimide, and then the aminoalkylcarboxylic acid is bonded to the water-dispersed polymer particles. An immunoreactant and/or a labeling agent can be covalently attached to an aminoalkylcarboxylic acid in a similar manner using a water-soluble carbodiimide.

Examples of the water-soluble carbodiimide used in this method include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, l-cyclohexyl-
3-(2-morpholinoethyl)carbodiimide-meth-
Examples include p-toluenesulfonate. It is known in the art to use such water-soluble carbodiimides to covalently attach immunoreactants and/or labeling agents to polymer particles directly, with or without spacer groups. This can be done by the usual methods and conditions. For example, when using a spacer group,
An appropriate amount of the spacer group is added to the aqueous dispersion of polymer particles, for example, 0.01 to 10 mg per unit volume of the aqueous dispersion.
/ml and add water-soluble carbodiimide under normal conditions, such as maintaining pi (4 to 10, 5 to 6
The reaction may be carried out at a temperature of about 0"C for several minutes to several tens of hours, usually for about 1 to 5 hours. Next, the immunoreactant and/or label is added to the polymer particles to which the spacer group has been bonded in the same manner. All that is required is to combine the agents.

In addition, when the functional group is a hydroxyl group, the spacer group is bonded by the cyanogen bromide method, or by reacting with dialdehyde when it is an amino group to activate these functional groups, and then the above-mentioned method is used. In a similar manner, immunoreactants and/or labeling agents can be covalently attached to polymer particles.

It is also possible to bind the immunoreactant and/or labeling agent directly to the polymer particles.

Of course, in the present invention, the immunoreactant and the labeling agent may be bound by any conventionally known method other than the above-mentioned covalent bonding method, such as physical adsorption method or ionic bonding method. For example, to obtain a labeled complex using an adsorption method, a buffer solution or physiological saline solution containing the immunoreactant is prepared,
This is added to an aqueous dispersion of water-dispersible polymer particles, the immunoreactant is adsorbed onto the polymer particles, and the labeling agent is further adsorbed in the same manner. Even when using the ionic bond method,
Similar explorations can yield labeled complexes.

However, in the present invention, both the immunoreactant and the labeling agent are preferably immobilized on the water-dispersed polymer particles by a covalent bonding method.

In addition, in the present invention, the above methods may be arbitrarily combined. For example, a method in which an immunoreactant is bound to a polymer particle by a covalent bond method and then a labeled complex is bound by an ionic bond, or vice versa, as described above.
The immunoreactant and the labeling agent may be individually attached to the polymer particles using reagents with different reaction specificities.

In the present invention, as the labeling agent, any labeling agent conventionally used in solid-phase immunoassays can be used, and examples thereof include enzymes, fluorescent substances, dyes, and the like. Specifically, examples of enzymes include peroxidase, β-D-galactosidase, alkaline phosphatase, lysozyme, glucoamylase, glucose oxidase, glucose-6-phosphate dehydrogenase, urease, catalase, coenzyme NAD, etc. Examples of the phosphorescent substance and dye include fluorescein isocyanate, fluorescein, rhodamine, rhodamine, and acridine.

After binding the immunoreactant and the labeling agent to the water-dispersed polymer particles in this way, for example, membrane separation, filtration, etc.
The labeled complex according to the present invention can be separated and purified by conventional conventional separation methods such as centrifugation.

In the present invention, the amount of labeling agent bound to the water-dispersed polymer particles as a carrier does not need to be one molecule of labeling agent per molecule of immunoreactant, but it depends on the type and activity of the labeling agent. , usually 0.0 labeling agent per molecule of immunoreactant.
It can be set to 1 to 1000 molecules, and can be easily adjusted within this range. However, immunoreactant 1
If there is too much labeling agent per molecule, the blank value will become too high and the accuracy will decrease, while if there is too little, the sensitivity will also decrease, so it is preferable to use 0.000000 labeling agent per molecule of immunoreactant. A range of 1 to 100 molecules is preferred.

The labeled complex according to the present invention thus obtained may be stored by immersing it in water or by freeze-drying.

(Effects of the Invention) As described above, the labeling complex according to the present invention uses water-dispersed polymer particles as a carrier, and the immunoreactant and the labeling agent are both bonded to the carrier. In addition to being easy to purify, the resulting labeled complex has high sensitivity and precision, and furthermore, the sensitivity and precision can be easily adjusted. In addition, the activity of the immunoreactant is maintained at a high level, and the storage stability is also high, making it easy to use.

In particular, the labeling complex according to the present invention can be obtained by first binding only an antibody to water-dispersed polymer particles, then binding a labeling agent, as shown in the Examples described below.
By centrifugation, membrane separation, etc., the desired amount of immobilized antibody and labeling agent can be easily and freely adjusted.

(Examples) The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example (1) Preparation of carrier dispersion Preparation of carrier\dispersion A 2.2.2- Trifluoroethyl meccrylate 38
While stirring 2 g of acrylic acid and 348 g of distilled water at a temperature of 70°C under a nitrogen stream, an aqueous solution of 0.2 g of ammonium persulfate dissolved in log of water was added as a catalyst and polymerized for 8 hours to give an average Aqueous dispersion of water-dispersible polymer particles with a particle size of 0.21 μm (solid content 1000%)
) was obtained.

After washing the polymer particles in the order of alkali, acid, and distilled water, they were again dispersed in distilled water to a solid content concentration of 5% by weight to prepare carrier dispersion A.

Mix 100 ml of the above carrier dispersion A made by Dandansaka and Shokudara with 100 ml of ε-aminocaproic acid aqueous solution (0.02 M/A),
The pH was adjusted to 7.2 with an aqueous N sodium hydroxide solution.

Next, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (25 mg/ml) dissolved in borate buffer (pH 7,5,0, OIM/Li)
20 ml was added to the carrier dispersion containing ε-aminocaproic acid, and the mixture was reacted at room temperature for 3 hours with stirring. - After leaving it in the refrigerator for the night, the polymer particles were dissolved in borate buffer (p
Centrifugally washed three times with H7,5,0, OIM/l) to obtain water-dispersed polymer particles to which spacer groups were bonded,
This was added to the same boric acid buffer as above with a solid content concentration of 5% by weight.
A carrier dispersion B was prepared by re-dispersing the carrier dispersion liquid so that the carrier dispersion B was obtained.

Preparation of Carrier Molecular Liquid C Carrier dispersion C was prepared in exactly the same manner as in the preparation of carrier dispersion B, except that ε-aminocaproic acid was replaced with m-xylylenediamine.

Danpachi? # of D, 1 In the preparation of carrier dispersion A, 0.13 g of acrylic acid was mixed with 0.0 g of 3-sulfopropyl methacrylate sodium salt.
3g and triethylene glycol dimethacrylate-1-0,
A carrier dispersion containing polymer particles having an average particle size of 0.14 μm was prepared in exactly the same manner except that 1 g of the mixture was used.

In the preparation of carrier dispersion A, carrier dispersion E was prepared in the same manner as in the preparation of carrier dispersion B, using ε-aminocaproic acid as the spacer group.

8 Knees of Carrier Dispersion F Carrier dispersion F was prepared in the same manner as in the preparation of carrier dispersion B, using m-xylylenediamine as the spacer group.

(2) Production of labeled complex 15 ml of a dispersion of the carrier particles B adjusted to a solid content concentration of 1% by weight in a boric acid buffer (pH 7, 5, 0, OIM/A) was added to 1-ethyl- 3-(3-dimethyl7minopropyl)carbodiimide hydrochloride water? '8'e (5
+ng/ml) 0.3 ml was added and stirred well at 4°C for 3 hours, followed by centrifugal washing. Thereafter, the polymer particles were redispersed in the same buffer solution as above so that the solid content concentration was 1% by weight.

An aqueous solution in which 0.1 μ of insulin as an antigen and 8.9 μ of peroxidase (80 units/■) as a labeling agent were dissolved in 2 ml of the same buffer as above was added to the above dispersion, and stirred slowly at 4°C. At the same time, the reaction was carried out at night. After the reaction, the polymer particles were centrifugally washed and added to a phosphate buffer containing 0.9% by weight of sodium chloride (
pH? , Olo, 05M/N) to obtain a dispersion of the labeled complex I according to the present invention.

As a result of analysis by A211° spectrophotometry, it was confirmed that 30 protein components (insulin and peroxidase) were bound to this labeled complex per gram of dry polymer particles.

In addition, enzyme i
When the activity in producing purbrogalin 1 from t-pyrogallol is defined as 1 jP, the enzyme activity per 1 g of labeled complex was 492 units.

Preparation of peroxidase and IgG binding label complex Peroxidase 5
,0,0,3M/1), and 0.1 ml of an ethanol solution containing 1% by weight of 2,4-dinitrofluorohensene as a 7-mino group protecting agent was added thereto. Furthermore, in order to aldehyde the peroxidase, 1 ml of an aqueous sodium periodate solution (0.05M) was added and stirred at room temperature for 30 minutes. Next, an ethylene glycol solution (as an enzyme stabilizer) was added to this.
After adding 10ml of 0.16M/I and stirring at room temperature for 1 hour, sodium carbonate buffer (p119.5.0
．． Dialysis was carried out with OIM/β) at 4°C.

Next, a dispersion of the carrier particles C prepared by adjusting the peroxidase concentration to a solid concentration of 1% by weight was prepared.
0 ml and reacted overnight at 4°C, centrifuged, and the reacted polymer particles were added to BoM buffer (pH 7.5,
0, OIM, #) so that the solid content concentration was 1% by weight. Thereafter, in order to impart aldehyde groups to the particles, glutaraldehyde was dissolved in the dispersion of the polymer particles to a concentration of 1% by weight, and the mixture was reacted at 4°C for 3 hours with slow stirring.

After this, the polymer particles are centrifuged and the solid content concentration is 1%.
Borate buffer (p) so that! 7.5.0゜01
M/I! , contains 0.8% sodium chloride. ), 1 ml of IgG (1 mg/ml) was added thereto, and the mixture was reacted at 4°C for 4 hours with slow stirring.
Furthermore, in order to reduce excess aldehyde groups, 0.5 μm of sodium borohydride was added, and the reaction was allowed to proceed at 4° C. overnight. Thereafter, it was centrifuged and redispersed in the same buffer as above to obtain a dispersion of labeled complex 2 according to the present invention.

As a result of the same analysis as above, this labeled complex contained 26°5 and 1 g of peroxidase per 1 g of dry polymer particles.
It was confirmed that 37.1 mg of G was bound. In addition, the activity was 380 units per gram of labeled complex in the same manner as above.

The carrier particles F were soaked in a sodium carbonate buffer solution (pH 9.0.
It was dispersed in 0.1 M# so that the concentration was 1% by weight.

Fluorescein isothiocyanate as a labeling agent
Phosphate buffer (pH 7, 2') to give nv/ml
0.5 ml of the solution was added to 2 ml of the above carrier dispersion, and the mixture was stirred at room temperature for 2 hours.

Next, after centrifugally washing the polymer particles, the polymer particles were redispersed in a boric acid buffer (pH 8, O20, OIM/l) to a solid concentration of 1% by weight, and then heated at 4°C.
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (1/ml) at a temperature of 0
．． After 10 minutes, add 2 ml of borate buffer solution of goat anti-mouse immunoglobulin as antibody.
ml (1w/ml) was added, and the reaction was allowed to proceed at 4°C overnight. After this, centrifugal washing was performed, and phosphate buffer (pH 7, 4,
0.05 M/l, containing 0.9% sodium chloride. ) to obtain a solid content concentration of 1% by weight to obtain a dispersion of labeled complex 3 according to the present invention.

It was confirmed that 22 μ of fluorescein isothiocyanate and 30 μ of goat anti-mouse immunoglobulin were bound to this labeled complex per gram of dry polymer particles.

The carrier particles E were soaked in a borate buffer (pH 8,0,0, OI
It was dispersed at a concentration of 1% by weight in M/jl.

Anti-human IgG (rabbit) was added to borate buffer (pH 8,0
, 0.01 M/7 to give a concentration of 5 μ/ml, and 1 ml of the solution was added to 10 ml of the above dispersion.

Next, 0.5 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (51 μ/ml)
ml was added and reacted for 6 hours at a temperature of 1.4°C.

The amount of anti-human TgG (rabbit) bound was 21 μ/g of dispersion.

After washing this by centrifugation, it was redispersed in the same buffer solution as above so that the solid content concentration was 1% by weight.

Fluorescein isothiocyanate was dissolved in sodium carbonate buffer (pH 7.2) to a concentration of 1 mg/ml, and 0.1 ml of this was added to the dispersion solution above, and the mixture was reacted at room temperature for 2.5 hours with stirring. , centrifugal washing, and then redispersion in borate buffer (p) 17.5.0.OIM/I to a solid content concentration of 1% by weight to obtain labeled complex 4 according to the present invention. .

This labeling complex contains 6.0 mg of fluorescein isothiocyanate and anti-human Ig per micron of dry polymer particles.
It was confirmed that 21 mg of G (rabbit) was bound.

(3) Example of use in analysis Several glass pieces of solid phase preparation were heat-washed in 0.1M/J nitric acid for 3 hours, dried at 90°C in an open state, and then It was heated at a temperature of about 700°C for 3 hours.

This glass piece was refluxed with 10% by volume γ-aminopropyltriethoxysilane in toluene, and then washed with methanol to obtain an aminated glass piece. This is 1
After being immersed in a wt % aqueous glutaraldehyde solution for 2 hours with stirring, the glass pieces were thoroughly washed with water.

Concentration 1 in borate buffer (pH 7, 5, 0, OIM/β)
Rabbit (anti-guinea pig [gG)]
The glass pieces were immersed in a solution containing gG or rabbit (anti-lemoni gG) IgG, and allowed to react overnight at 4'C with gentle stirring.

In this way, rabbit (
A glass piece to which 0.07 μg of IgG (anti-guinea pig IgG) or 0.06 μg of rabbit (anti-human IgG) IgG was bound was obtained.

Test insulin standard solution using labeled complex 1 (0.01-1 mg/ml) 1
ml.

1 ml of an appropriately diluted anti-insulin serum (guinea pig) and 5 ml of phosphate buffer containing 0.1% by weight bovine serum albumin (ptl 7.2.0.05M/f) were mixed, and the mixture was incubated at room temperature for 1 hour. It was further incubated at 4°C for 4 hours.

To this was added a dispersion 1 of the labeled complex 1 at a concentration of 0.1% by weight.
ml was added and reacted at 4°C for 2 hours. After this, three pieces of the rabbit (anti-guinea pig t-!gG) IgG-conjugated glass pieces moistened with 0.5 ml of phosphate buffer were immersed,
Stir slowly at 4°C overnight.

After this, the rabbit (anti-guinea pig IgG) IgG
The bonded glass piece was washed with phosphate buffer and then wetted with 1 ml of phosphate buffer containing 0.1% by weight bovine serum albumin.

A substrate solution was prepared by mixing a 5-aminosalicylic acid solution (0.7 mg/ml) and a hydrogen peroxide solution (0.3% by weight) at a weight ratio of 100/1. 2 ml of this substrate solution was added to the above solution, and after reacting at room temperature for 1.5 hours, the absorbance of the supernatant was measured at 460 nm. The relationship between insulin concentration and absorbance at 460 nm is shown in FIG.

The rabbit (anti-human IgG ) Human IgG standard solution (0.01-2 p g/ml) on three IgG-binding glass pieces
After reacting 1 ml at 4°C for 2 hours, the glass piece was washed with phosphate buffer, and then added with 0.5 ml of phosphate buffer.
It was moistened with.

1 ml of the labeled complex 4 dispersed in a phosphate buffer containing 0.1% by weight of bovine serum albumin to a solid content concentration of 0.1% by weight was added to the glass piece, and the mixture was heated at 4°C.
After reacting for 1 hour, the glass plate was washed with water and subjected to fluorescence analysis. An interference filter 535 In was used in a Hitachi model 650-103 spectrofluorometer. human Ig
The relationship between G standard liquid level and relative fluorescence intensity is shown in Figure 2.

Comparative Example Insulin-peroxidase complex manufactured by Sigma (1-
Quantification was carried out in the same manner as in the quantitative test using labeled complex 1 described above, except that 2 mol peroxidase/1 mol insulin) was used. The relationship between insulin concentration and absorbance in 460 fins is shown in FIG. It is clear that the sensitivity is low because the amount of labeling substance peroxidase is smaller than that of insulin.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between insulin concentration and absorbance at 460 nm when insulin was quantified using the labeled complex according to the present invention, and FIG. Human 1gG when quantifying eye GC? It is a graph showing the relationship between J2 degrees and relative fluorescence intensity. FIG. 3 is a graph showing the relationship between the human IgG concentration and the absorbance at 460 m when GC was quantified using a commercially available insulin labeled with peroxidase as a labeled complex. Patent Applicant Nitto Electric Industry Co., Ltd. Agent Patent Attorney Ittsu Makino Department Fig. 100, Ta 1.0 fJ! Lζ style J hawk) Figure 2

Claims (4)

[Claims] (1) Water-dispersed polymer particles with a particle size of 0.01 to 3 μm are used as a carrier, and antigens or haptens,
Or a labeling complex characterized in that a labeling agent is bound together with the antibody. (2) The labeling complex according to claim 1, wherein the labeling agent is an enzyme. (3) The labeling complex according to claim 1, wherein the labeling agent is a fluorescent substance. (4) The labeling complex according to claim 1, wherein the labeling agent is a dye.