JPH09229937A - Immunological agglutination reagent and immunity analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunization agglutination reagent for measuring the amount of agglutination having sufficiently high sensitivity in practical use and physicochemical stability, which is not found in conventional liposome agglutination reagent and an immunity analysis method using the immunization agglutination reagent. SOLUTION: As the carrier of immunological agglutination reagent, the liposome, on which osmotic pressure is loaded by the substitution of the external water phase of the liposome with the aqueous solution having the osmotic pressure higher than the osmotic pressure of the hydration aqueous solution when the liposome is prepared by 120mOsm or more, is used. In details, the osmotic pressure of the hydration aqueous solution is made to be 0.5-30mOsm, the first substitution is performed so that the difference of the osmotic pressures of the hydration aqueous solution and the first substituted aqueous solution becomes 320mOsm or more and the small particle diameter and the high turbidity of the liposome are achieved. When the undesirable effect is observed after the first substitution, it is recommendable that the second substitution is performed with the second substitution aqueous solution having a osmotic pressure of 700mOsm or less.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoagglutination reagent for measuring the amount of agglutination generated in an antigen-antibody reaction,
More specifically, the concentration of the substance to be measured present in the sample to be measured can be measured in a wide range from the low concentration region to the high concentration region, and with simple operation, it can be measured visually or using an automatic analyzer with high sensitivity. The present invention relates to an immunoagglutination reagent that can be used and an immunoassay method using the same.

[0002]

2. Description of the Related Art An immunoassay utilizing an antigen-antibody reaction is
It is extremely important in clinical diagnosis of various diseases, and various immunoassay methods have been conventionally known.

Among them, an immunoassay using an immunoassay reagent in which an antigenic substance or an antibody is bound to a carrier is used in an immunoassay with an antibody or an antigenic substance contained in a subject.
Using the so-called immune agglutination reaction, which produces agglutination by antibody reaction, the amount of agglutinate produced is measured visually or by spectrophotometer as turbidity, and the result is prepared in advance as a standard. An immunoassay method for quantifying an antibody or an antigenic substance by checking it against a calibration curve is known.

The immunoassay reagent used in the immunoassay method for measuring such an amount of aggregation is called an immunoaggregation reaction reagent, and conventionally, synthetic latex particles such as polystyrene have been mainly used as a carrier for the reagent. It was being done. Since the surface of the conventional immunoagglutination reaction reagent using synthetic latex particles as a carrier is hydrophobic, the pH is 7 to 7.5 in a suitable aqueous solution for causing an antigen-antibody reaction. Is likely to occur, and so-called non-specific aggregation, which does not depend on the antigen-antibody reaction, occurs, so that there is a tendency to lack stability in analysis accuracy. For this reason, it has been proposed to add various additives to the immunoagglutination reaction reagent for measuring the amount of agglutination using synthetic latex particles as a carrier (for example, a special feature). Kaihei 3-142360, etc.).

In recent years, the immunoagglutination reaction reagent using such synthetic latex particles as a carrier has been applied to a general-purpose biochemical automatic analyzer to measure the agglutination amount of the antigen or antibody in the sample to be measured. However, there is a problem in that the immunoaggregation reaction reagent adheres to and contaminates the reaction cell of the automatic analyzer and affects the measurement accuracy.

In order to solve these problems, an immunoagglutination reagent for measuring the amount of agglutination using liposome as a carrier instead of the synthetic latex particle (for example,
JP-A-6-3358 and JP-A-6-23010) have been proposed.

[0007] The above-mentioned JP-A-6-3358 discloses an immunoaggregation reaction reagent in which an antibody is immobilized on the surface of a liposome.
Since non-specific adsorption of proteins is low because the liposome has a hydrophilic surface derived from phospholipids, it is possible to increase the sensitivity of the measurement reagent as compared with the immunoagglutination reagent using synthetic latex particles as a carrier. When a liposome is used as a carrier for an agglutination reagent for measuring the amount of agglutination generated in an antigen-antibody reaction, the turbidity of the carrier is so small that it cannot be measured with a latex agglutination reagent in a short wavelength range of about 340 nm. Therefore, the liposome agglutination reaction reagent can correspond to a wide range of concentrations of the substance to be measured as compared with the latex agglutination reaction reagent, and the liposome agglutination reaction reagent uses the liposome as a carrier. It is reported that it rarely adheres to the reaction cell of an automatic analyzer and contaminates the reaction cell.

Japanese Patent Laid-Open No. 23030/1994 discloses
It was pointed out that the liposome aggregation reaction reagent has insufficient measurement sensitivity for the measurement of the substance to be measured in a low concentration range because the turbidity of the liposome carrier itself is too small. In view of the lack of measurement sensitivity in the range, it has been proposed to increase the turbidity of the carrier and increase the sensitivity of the measurement reagent by including a specific water-soluble organic compound in the aqueous phase encapsulated in the liposome carrier. ing. However, when the concentration of the substance to be measured is lower, the measurement sensitivity is not yet satisfactory, and a measurement reagent having sufficient measurement sensitivity in a wide concentration range of the substance to be measured has not been provided yet.

In addition to the above two proposals for the liposome agglutination reaction reagent, JP-A-63-27480 also proposes a technique relating to a liposome agglutination reagent having high accuracy. In this publication, a method for forming antibody-sensitized polymerized liposomes and micelles using a polymerizable surfactant or a polymerizable lipid and an antibody chemically modified by a specific molecular chain, and further produced by the method. A method has been proposed in which an antigen-antibody aggregate is formed using antibody-sensitized polymerized liposomes and micelles, and the size of the particle size is determined by measuring the rate of change in relative absorbance with time, thereby measuring the concentration of antigen in the sample. ing.

In the proposal disclosed in the publication, an antibody-sensitized polymerized liposome is manufactured by a specific manufacturing method using a polymerizable compound for the purpose of improving the cryopreservation property of the liposome. It has been shown that liposomes can be cryopreserved and the antibody concentration in a test liquid can be quantitatively measured with high accuracy for a long period of time. However, the publication requires a measuring device with a special configuration having a laser light source in the device used for the measurement, and there is a disadvantage that a general-purpose measuring device such as a spectrophotometer or an automatic biochemical analysis device cannot be used. is there.

[0011]

Therefore, the object of the present invention is, in addition to the features of the liposome carrier, that the measurement in the short wavelength region is possible and the cell contamination of the measuring device is low, and the immunoaggregation reaction. A general-purpose measuring device can be used for the measurement, and the substance to be measured can be measured with a higher sensitivity than before in a wide range of low concentration to high concentration, especially in the low concentration range. It is intended to provide an immunoagglutination reaction reagent and an immunoassay method using the immunoagglutination reaction reagent.

[0012]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that the osmotic pressure of the outer aqueous phase of the liposome and the immunoaggregation reaction reagent using the liposome as a carrier. Focusing on the relationship with the measurement sensitivity, it is possible to measure in the short wavelength range, the cell contamination of the measuring device is low, and in addition, it has a sufficiently high sensitivity for measuring low concentration measured substances,
The inventors have invented an immune agglutination reagent for measuring the amount of agglutination produced by an immunological reaction between a substance to be measured in a subject and an immune agglutination reagent using a liposome as a carrier.

That is, the present invention relates to an immunoagglutination reagent for measuring the amount of agglutination caused by an immunological reaction with a substance to be measured in a subject, which is 120 mOsm or more from the osmotic pressure of the hydrated aqueous solution during liposome preparation. An immunoaggregation reaction reagent, characterized in that an osmotic pressure-loaded liposome in which the outer aqueous phase of the liposome is replaced with an aqueous solution of high osmotic pressure is used as a carrier.

Used in the immunoagglutination reagent of the present invention,
The liposome carrier loaded with an osmotic pressure is prepared by first substituting the outer aqueous phase of the liposome with a first displacement aqueous solution having an osmotic pressure 120 mOsm or more higher than the osmotic pressure of the hydration aqueous solution at the time of liposome preparation, and then subjecting the liposome to the first displacement. The aqueous phase can be obtained by second substitution with a second substitution aqueous solution having an osmotic pressure lower than that of the first substitution aqueous solution.

To prepare the immunoaggregation reaction reagent of the present invention, the osmotic pressure of the hydrated aqueous solution at the time of liposome preparation before substituting the outer aqueous phase is preferably 0.5 to 30 mOsm. To prepare the immunoaggregation reaction reagent of the present invention, the osmotic pressure of the second displacement aqueous solution is preferably 700 mOsm or less.

Further, the present invention is an immunoassay method characterized in that the immunoagglutination reaction reagent is allowed to act on a substance to be measured of a subject, and the amount of agglutination produced by an antigen-antibody reaction is measured by transmitted light.

As used herein, the term "immunologically active substance" refers to an antibody and / or an antigen.

As used herein, the term "polymerizable" refers to a state in which it has the ability to polymerize and has not yet been polymerized.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The constituent material of the liposome used in the immunoaggregation reaction reagent of the present invention is not particularly limited, but the phospholipids which are the main constituents of the liposome, the cholesterols which are the minor constituents, and the tocopherols. , Fatty acids, aliphatic amines, glyceroglycolipids, glycosphingolipids and the like. Further, other components such as a functionalizing agent may be added within a range that does not adversely affect the effects of the present invention.

Examples of the function-imparting agent include a film stabilizer, a hydrophilic modifier for the film surface, a curvature adjusting agent, an antioxidant, a charge-imparting agent and the like. Cholesterols or tocopherols function as a membrane stabilizer or a regulator of membrane permeability, and in the liposome loaded with osmotic pressure in the present invention, the composition can be appropriately selected and used. The alkyl group or acyl group constituting the phospholipid, fatty acid, or aliphatic amine has 6 to 2 carbon atoms.
4 straight or branched chains are available.

The phospholipid represented by the following formula (1) can be shown by giving a specific example of the phospholipid which is the main constituent of the liposome.

[0022]

Embedded image (However, X ¹ represents a phosphoric acid polar group, and R ¹ and R ² represent a fatty acid residue having 6 to 24 carbon atoms or a hydrocarbon group.) Examples of the phosphoric acid polar group represented by X ¹ include a phosphocholine group. , Phosphoethanolamine group, phospho-N
-Methylethanolamine group, phospho-N, N-dimethylethanolamine group, phosphoserine group, phosphoinositol group, phosphoinositol diphosphate group, phosphoglycerol group, phospho-O-aminoacylglycerol group, phosphoglycerophosphate group, phosphonic acid Alternatively, a phosphate group and the like can be mentioned.

Specific examples of the phospholipid that can be used in the present invention are enumerated. Natural phospholipids and derivatives thereof such as egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, and dimyristoylphosphatidylcholine. , Dipalmitoylphosphatidylcholine, distearylphosphatidylcholine, dioleylphosphatidylcholine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearylphosphatidylethanolamine,
Acyl group composition of dioleylphosphatidylethanolamine, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearylphosphatidic acid, dioleylphosphatidic acid, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearylphosphatidylglycerol, dioleylphosphatidylglycerol Examples of the synthetic phospholipids obtained by manipulating the above-mentioned phospholipids, phosphatidylserine, phosphatidylinositol, cardiolipin, sphingomyelin, ceramide phosphoryl ethanolamine, ceramide phosphoryl glycerol, and the like are included, but the present invention is not particularly limited thereto.

In addition to the specific examples of the phospholipid, compounds having a polymerizable group as X ¹ , R ¹ and R ² in the formula (1) can also be used in the present invention, and X ¹ is a polymerizable group. As a specific example of a group having, for example,
Phospho-N-polymerizable alkenylethanolamine group, phospho-N-polymerizable alkenoylethanolamine group, phospho-N-polymerizable alkenylserine group, phospho-N-polymerizable alkenoylserine group, phospho-polymerizable alkenoyl Examples include ester serine groups. In addition, Japanese Unexamined Patent Publication
A compound having a polymerizable polar group of phosphoric acid as disclosed in JP-A-157561 can also be used in the present invention.

Examples of the group in which R ^{1 and} / or R ² are polymerizable include, for example, vinylalkyl group, styrylalkyl group, (meth) acryloyloxyalkyl group, conjugated dienealkyl group, conjugated diynealkyl group. Groups. Specific examples of compounds having these groups include 2,4-octadecadienoic acid, 2,4-hexadecadienoic acid, 8,10,12-octadecatrienoic acid, and 9- (p-vinylbenzoyl). ) Nonanilic acid, 1,2
-Methacryloyl oxide decanoic acid, 10-undecic acid, 16-heptadecic acid, 22-tricosic acid, tricosa-10,12-diynoic acid, tricosa-2,4-diynoic acid, nonadeca-2,4-diynoic acid, etc. To be

The reason why the liposome used in the immunoaggregation reaction reagent of the present invention may contain a polymerizable group in its material is that the compound containing a polymerizable group is formed into a liposome and then polymerized. This is because by polymerizing the liposome membrane by the treatment, a polymerized liposome having physicochemical stability can be obtained. Examples of the method for polymerizing the polymerizable group include, for example, JP-A-3-291216 and JP-A-63-27.
4870, JP 61-155336, JP 61-155392, JP 62-1048.
It can be implemented by various methods shown in Japanese Patent Publication No. 44-44.

In the present invention, the antibody or antigen is immobilized on the surface of the liposome to obtain the immunoagglutination reagent of the present invention. The method of immobilizing this antibody or antigen on the liposome surface or the mode of immobilizing is not particularly limited, and examples thereof include a method using a crosslinking agent, a periodate method, a Passive method, and a physical adsorption method. Can be mentioned.

When a liposome and an antibody or an antigen are chemically bonded using a cross-linking agent, the functional groups that bond the two are amino group, imino group, carboxyl group, hydroxyl group, phenol group, thiol group, imide ester group, nitro group. Aryl halide group, thiophthalimide group, active halogen group, succinimide group, maleimide group, dithiopyridyl group, arylazide group or diazoalkane group and the like, particularly, succinimide group, dithiopyridyl group, maleimide group, arylazide group, An active halogen group and a diazoalkane group are mentioned as preferable groups.

In the method of immobilizing an antibody or an antigen on the surface of a liposome using a cross-linking agent having the above functional group, a preferable functional group capable of binding to an amino group of phospholipid (for example, an amino group of phosphatidylethanolamine). Examples include a method of using a cross-linking agent having a group such as a succinimidyl group at one end. More specifically, such a cross-linking agent includes a cross-linking agent having a succinimidyl group capable of binding to an amino group of a phospholipid at one end and a group capable of chemically binding to an antibody or an antigen at the other end. .
A known method can be applied to such a reaction, and examples of the cross-linking agent preferable in the present invention include the following bifunctional compounds.

Examples of the bifunctional compound include N-succinimidyl-3- (2-pyridyldithio) propionate, sulfosuccinimidyl-6- (3- (2-pyridyldithio) propionamide) hexanoate and succinimidyl-6-. (3- (2-Pyridyldithio) propionamido) hexanoate, N-succinimidyl-
4- (p-maleimidophenyl) butyrate, sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate, N-succinimidyl-4- (p-maleimidophenyl) acetate, N-succinimidyl-4-
(P-maleimidophenyl) propionate, 4-succinimidyloxycarbonyl-α-methyl-α- (2
-Pyridyldithio) -toluene, succinimidyl-4
-(N-maleimidoethyl) -cyclohexane-1-carboxylate, sulfosuccinimidyl-4- (N-
Maleimidoethyl) -cyclohexane-1-carboxylate, N- (γ-maleimidobutyryloxy) succinimide, N- (ε-maleimidocaproyloxy) succinimide, N-succinimidyl-6- (4′-azido-2′- Nitrophenylamino) hexanoate,
Sulfosuccinimidyl-2- (m-azido-O-nitrobenzamido) -ethyl-1,3'-dithiopropionate, N-succinimidyl- (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl-4
-(P-azidophenyl) butyrate, N-succinimidyl- (4-azidophenyl) -1,3'-dithiopropionate, sulfosuccinimidyl-2- (7-azido-4-methyliomarin-3) Acetamide) -ethyl-1,3′-dithiopropionate, dithiobis (succinimidyl propionate), ethylene glycol bis (succinimidyl succinate) and the like can be mentioned.

The method for immobilizing an antibody or antigen on the surface of the liposome used in the present invention through the amino group of phosphatidylethanolamine has been exemplified above. The immunoaggregation reaction reagent of the present invention can be obtained by this method. It is not particularly limited to the above.

In the liposome used in the present invention,
The composition of the inner aqueous phase is not particularly limited, but for the purpose of further improving the sensitivity as an immunoaggregation reaction reagent, a specific water-soluble organic compound or gelation treatment exemplified in JP-A-6-23010 It is also possible to include the compound. As a component included in the inner aqueous phase other than the compound disclosed in JP-A-6-23010, a physiologically active substance, an immunologically active substance, or an immunologically active substance, as long as it does not adversely affect the performance of the immune agglutination reagent or the stability of the liposome, It is also possible to freely add a pharmacologically active substance, an organic pigment, an inorganic pigment, a dye or the like.

The method for producing the liposome used in the immunoaggregation reaction reagent of the present invention from the above-mentioned liposome raw material is not particularly limited, but for example, "Liposome" issued by Nankodo Co., Ltd. (1988). As described in, a thin film method, an ultrasonic treatment method, an ethanol injection method, a French press method, an extrusion method, a dialysis method, a freeze-thaw method, a reverse phase evaporation method, a high pressure discharge type emulsifier, or the like can be used. is there.

Depending on the required particle size or qualities such as multilamellarity (lamellarity), the above method can be selected to prepare liposomes.

The hydration aqueous solution for hydrating the lipid that is the raw material of the liposome has a pH stability in order to keep the suspension state of the lipid or the lipid bilayer membrane during the process of preparing the liposome from the hydration of the lipid raw material. Property is required, and it is required to have an appropriate buffer capacity as a hydration aqueous solution. For example,
Preventing a decrease in pH due to the influence of carbon dioxide in the air is a very important point in industrial production of liposomes. In order to satisfy this pH stability, the hydration aqueous solution is required to contain a buffer.

On the other hand, the smaller the osmotic pressure of the liposome internal aqueous phase solution derived from the hydrated aqueous solution, the more the internal water phase and the external aqueous phase permeate under the load of osmotic pressure at the time of the first replacement of the external aqueous phase later. The pressure difference can be increased, and the size of the liposome can be reduced and the degree of turbidity can be increased. The reduction in the particle size and the increase in turbidity of the liposome increase the liposome surface area, that is, the amount of immunoaggregation reaction, and increase the turbidity of the liposome carrier, that is, the carrier of the immunoaggregating substance, which increases the immunoaggregation reaction field and the immunoaggregation reaction reagent. Carrier-It becomes possible to increase the turbidity of the aggregate consisting of the substance to be measured,
The measurement range of the test substance in the immunoaggregation reaction reagent can be broadened and the test substance at a low concentration can be measured, and the measurement sensitivity is increased.

As described above, on the one hand, from the viewpoint of pH stability in the liposome preparation step, it is required that the aqueous hydration solution contains a buffer, and on the other hand, the above-mentioned high performance of the immunoaggregation reaction reagent is required. From the viewpoint of achievement, it is required that the osmotic pressure of the aqueous phase in the liposome is smaller. However, these requirements are conflicting requirements as buffers generally have an osmotic pressure. The osmotic pressure of the hydrated aqueous solution used as the inner aqueous phase of the liposome, which is used when liposomes are prepared to satisfy these contradictory requirements, is preferably 0.1 to 100 mOsm, more preferably 0.5 to 30 mOsm, It is preferably 1 to 10 mOsm.

The buffer used in the hydrated aqueous solution includes boric acid, phosphoric acid, citric acid, acetic acid, phenolsulfonic acid,
Phenylacetic acid, phthalic acid, pentanoic acid, butanoic acid, propionic acid, lactic acid, pyruvic acid, acetoacetic acid, aconitic acid, ethylenediaminetetraacetic acid, cacodylic acid, tartaric acid, succinic acid, tetraoxalic acid, p-toluenesulfonic acid, pyrophosphoric acid , Acidic compounds such as maleic acid and carbonic acid, ammonia, choline, aminopyridine, 2-amino-2-methyl-1,3-propanediol, imidazole, ethanolamine, N-ethylmorpholine, ethylenediamine, diethanolamine, hydroxylamine, piperazine Basic compounds such as glycine, glycylglycine, N-2- (acetamido) -2-aminoethanolsulfonic acid, N-2- (acetamido) iminodiacetic acid,
N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, N, N-bis (2-hydroxyethyl) glycine, [bis- (2-hydroxyethyl) imino] -tris- (hydroxymethyl) -Methane, diethylenetriaminepentaacetic acid, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2- (N-
Morpholino) ethanesulfonic acid, 2-hydroxyethylimino-tris (hydroxymethyl) methane, nitrotriacetic acid, piperazine-N, N'-bis (2-ethane) sulfonic acid, N-tris (hydroxymethyl) methyl-2
-Aminoethanesulfonic acid, N-tris (hydroxymethyl) methylglycine, tris (hydroxymethyl) aminomethane, etc. It is possible to use a single compound or a plurality of compounds selected from the group of amphoteric compounds. It can be used depending on the method of preparing the solution.
The buffering agent that can be used in the present invention is not limited to the compounds listed here, and a water-soluble compound other than the buffering agent can be used within a range that does not adversely affect the particle size, turbidity, and stability of the liposome. In the above, addition and use are not limited at all.

Liposomes are prepared by using a lipid as a raw material of liposomes and the hydrated aqueous solution of the present invention according to a known predetermined procedure, and then the outer aqueous phase is separated by a known method such as gel filtration, ultrafiltration or dialysis. It is possible to produce a liposome loaded with an osmotic pressure by substituting the solution with a high osmotic pressure. The osmotic pressure may be applied either before or after immobilization of the antibody or antigen by the above method, and is not particularly limited.

In order to apply an osmotic pressure to the liposome, the osmotic pressure of the liposome outer aqueous phase (hereinafter referred to as the first displacement aqueous solution) to be replaced first is 120 mOsm or more higher than the osmotic pressure of the hydrated aqueous solution of the liposome. It is necessary to use a solution, preferably 160 mOsm or more, more preferably 320 mOsm or more. If the osmotic pressure of the first displacement aqueous solution is too high, the stability of the liposome is reduced, which is not preferable for industrial production. From this point, the difference between the osmotic pressure of the first substitution aqueous solution and the osmotic pressure of the hydrated aqueous solution of the liposome is appropriately 7,000 mOsm or less, preferably 5,000 mOsm or less, and more preferably 4,000 mOsm.
It is mOsm or less. By substituting the outer aqueous phase with the first displacement aqueous solution having such an osmotic pressure, the difference in osmotic pressure between the liposome inner aqueous phase and the outer aqueous phase is increased, and the liposome particle size reduction and high turbidity described above are achieved. The degree of progress can be greatly advanced.

For example, the first substitution of the liposome outer aqueous phase with the first substitution aqueous solution can reduce the particle size and increase the turbidity of the liposome loaded with osmotic pressure. If the particle size is about 200 nm, the average particle size will be 7
It becomes as small as 3.0% or less, and the turbidity becomes a large value of 150% or more before loading.

The method for measuring the osmotic pressure of the aqueous solution used in the present invention is not particularly limited, but it can be measured by appropriately using a method such as a freezing point depression method and a boiling point elevation method.

The average particle size of the liposomes loaded with osmotic pressure by the first displacement of the outer aqueous phase is 5 nm to 5 μm from the viewpoint of using the liposome as a carrier of an immunoactive substance in an immunoaggregation reaction reagent. Preferably,
The thickness is more preferably 10 to 600 nm, and most preferably 10 to 300 nm.

In the step of preparing the liposome used in the present invention, as the compound for adjusting the osmotic pressure of the aqueous solution, alkali metal hydrochlorides such as sodium chloride and potassium chloride, boric acid, phosphoric acid, sulfuric acid, phenylacetic acid, acetic acid, phenol are used. Sulfonic acid, citric acid, phthalic acid, pentanoic acid,
Butanoic acid, propionic acid, lactic acid, pyruvic acid, acetoacetic acid, aconitic acid, ethylenediaminetetraacetic acid, cacodylic acid, succinic acid, tartaric acid, tetraoxalic acid, p-toluenesulfonic acid, pyrophosphoric acid, maleic acid, aspartic acid, glutamic acid, Alkali metal salts of various acidic compounds such as tyrosine and cysteine, ammonia, choline,
Aminopyridine, 2-amino-2-methyl-1,3-propanediol, imidazole, ethanolamine, N
-Inorganic acid salts of various basic compounds such as ethylmorpholine, ethylenediamine, diethanolamine, hydroxylamine, piperazine, lysine, arginine, histidine and tris (hydroxymethyl) aminomethane,
Glycine, glycylglycine, N-2- (acetamido) -2-aminoethanolsulfonic acid, N-2- (acetamido) iminodiacetic acid, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, N, N-
Bis (2-hydroxyethyl) glycine, [bis- (2
-Hydroxyethyl) imino] -tris- (hydroxymethyl) -methane, diethylenetriaminepentaacetic acid,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2- (N-morpholino) ethanesulfonic acid, 2-hydroxyethylimino-tris (hydroxymethyl) methane, nitrotriacetic acid, piperazine-N,
Alkali metal or inorganic acid salts of various amphoteric compounds such as N'-bis (2-ethane) sulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid and N-tris (hydroxymethyl) methylglycine Is mentioned. One or more of the above osmotic pressure adjusting compounds can be used alone or in combination, and the use of other water-soluble compounds in a range that does not adversely affect the particle size, turbidity, and stability of liposomes is also not limited. Not something.

From the viewpoint of achieving high sensitivity, the osmotic pressure of the outer aqueous phase of the liposome, which is preferably loaded by the first substitution of the outer aqueous phase, causes the immunoaggregation reaction for reasons such as immunological reaction compatibility and biocompatibility. When the performance of the reagent is not favorable, the outer aqueous phase of the liposome can be replaced with a desired aqueous solution again by a known method such as gel filtration, ultrafiltration or dialysis as the second substitution. For example, when sodium chloride is used as the osmotic pressure adjusting substance of the first displacement aqueous solution and the osmotic pressure due to sodium chloride exceeds 700 mOsm, the immune agglutination reaction between the immunologically active substance and the test substance is significantly inhibited, resulting in a sufficient immunoagglutination reaction. There is a big problem that the performance as a reagent cannot be obtained.

When such an undesirable effect as an immunoaggregation reagent is observed, the inhibition of the first substitution aqueous solution of the outer aqueous phase is suppressed within a range that does not have a large adverse effect on the particle size and turbidity of the liposome carrier. It is possible to exchange the second substitution aqueous solution having a desired osmotic pressure as the second substitution. The osmotic pressure of the second displacement aqueous solution is 700 mOsm
The following is preferred, 500 mOsm or less is more preferred, and 250 mOsm to 400 mOsm is most preferred.
It is.

Next, the method of modifying the antibody or antigen to be immobilized on the liposome surface is not particularly limited,
For example, there may be mentioned a method in which the cross-linking agent is chemically bonded to an antigen or an antibody to introduce a functional group. In a buffer solution, the antibody or antigen to which a functional group is added is mixed with a cross-linking agent through the amino group of phosphatidylethanolamine, and a liposome having a functional group on the surface or a liposome loaded with osmotic pressure is mixed. The antibody or antigen can be immobilized by reacting with.

In the present invention, the antibody immobilized on the liposome surface may be a monoclonal antibody or a polyclonal antibody that recognizes the antigenic substance to be measured. The formulation of the liposome and the antibody to be immobilized on the liposome when used as an immunoaggregation reaction reagent is not particularly limited, but for example, a formulation for immobilizing a monoclonal antibody on the liposome surface or a polyclonal antibody on the liposome surface. Formulation, a formulation in which a monoclonal antibody and a polyclonal antibody are immobilized on the same liposome surface, a formulation in which a liposome immobilized with a monoclonal antibody and another liposome immobilized with a polyclonal antibody are mixed after the antibody is immobilized, and the like.

In the immunoaggregation reaction reagent in which the polyclonal antibody and the monoclonal antibody are used in combination, the monoclonal antibody having a strong affinity with the antigen can supplement the low affinity of the polyclonal antibody. At the same time, the polyclonal antibody having excellent aggregating property has a feature that the complexity of having to select and combine the monoclonal antibodies in order to enhance the aggregating activity can be avoided. As the antibody immobilized on the surface of the liposome, for example, the 1 gG fraction may be used as it is, or an F (ab ′) ₂ fragment thereof may be used.

In the present invention, the antigens immobilized on the liposome surface mainly include plasma proteins, viral proteins and the like. The antigenic substance to be measured applied to the immunoassay method using the immunoagglutination reagent of the present invention includes hormones (eg, insulin, HCG-β, growth hormone, TSH, LH, FSH, prolactin, thyroxine, triiodothyronine, Gastrin, glucagon,
Somatostatin, etc.), enzyme (eg, elastase, amylase, protease, lipase, ribonuclease, enolase, alkaline phosphatase, etc.), serum protein (eg, IgG, IgA, IgM, Ig)
E, IgD, CRP, macroglobulin, TBG, glycoprotein, glycolipid, apoprotein, etc.), tumor-associated antigen (for example, CEA, α-fetoprotein, ferritin, CA19-9, CAl 25, etc.), DNA-binding protein factor , Bacteria, viruses, protozoa and the like.
Of these, particularly suitable are serum proteins, tumor-associated antigens, viruses, etc., which require measurement of a large amount of sample to be measured in a short time.

The antibody substances to be measured include, for example, antibodies against viruses (eg, HIV, ATL, HB, etc.), AS
O, RF, various autoantibodies, antinuclear antibodies and the like.

The quantification of the target substance in the sample to be measured by the immunoassay method using the immunoagglutination reagent of the present invention is performed, for example, as follows. First, the immunoagglutination reagent of the present invention and the sample to be measured are treated with an appropriate buffer solution (for example, TES).
It mixes in a buffer solution) to induce an antigen-antibody reaction, thereby forming an aggregate of antibodies or antigen-immobilized liposomes. The amount of aggregation is optically measured by transmitted light of light or scattered light of light, for example, as a reduction of transmitted light depending on the amount of aggregation by a spectrophotometer or a general-purpose biochemical automatic analyzer. The amount of the substance to be measured in the sample can be measured by collating with the calibration curve or the like.

[0053]

EXAMPLES The following examples are for C-reactive proteins (hereinafter CR
(Abbreviated as P) The immunoaggregation reagent and the immunoassay method of the present invention are applied to the measurement of the antigen concentration. These are examples of the present invention and do not particularly limit the present invention.

The average particle size of the liposome was measured by a dynamic light scattering particle size distribution meter (NICOMP model 370HPL, manufactured by NOF Liposome Co., Ltd.) according to a predetermined procedure. The turbidity of liposomes was measured by measuring the phosphorus concentration derived from phospholipids, and measuring 34
Absorbance at 0 nm was used.

The osmotic pressure of each aqueous solution used in the present invention was measured using an Osmometer ONE-TEN (manufactured by Fiske Corp.).

[Reference Example 1-1]: Preparation of osmotic pressure 80 mOsm buffer solution The pH was adjusted to 7.5 with NaOH and the final osmotic pressure was adjusted to 8 with NaCl.
A 10 mM phosphate buffer adjusted to 0 mOsm was prepared.

Reference Example 1-2: Osmotic pressure 100 mOsm
Preparation of buffer pH to 7.5 with NaOH and final osmotic pressure to 1 with NaCl
A 10 mM phosphate buffer solution adjusted to 00 mOsm was prepared.

Reference Example 1-3: Osmotic pressure 200 mOsm
Buffer Preparation pH to 7.5 with NaOH and final osmolarity to 2 with NaCl.
A 10 mM phosphate buffer solution adjusted to 00 mOsm was prepared.

[Reference Example 1-4]: Osmotic pressure 300 mOsm
Buffer Preparation pH to 7.5 with NaOH and final osmotic pressure to 3 with NaCl.
A 10 mM phosphate buffer solution adjusted to 00 mOsm was prepared.

[Reference Example 1-5]: Osmotic pressure 350 mOsm
Buffer Preparation pH to 7.5 with NaOH and final osmotic pressure to 3 with NaCl.
A 10 mM phosphate buffer adjusted to 50 mOsm was prepared.

[Reference Example 1-6]: Osmotic pressure 400 mOsm
Buffer Preparation pH to 7.5 with NaOH and final osmolarity to 4 with NaCl.
A 10 mM phosphate buffer solution adjusted to 00 mOsm was prepared.

[Reference Example 1-7]: Osmotic pressure 500 mOsm
Buffer Preparation pH to 7.5 with NaOH and final osmolarity to 5 with NaCl.
A 10 mM phosphate buffer solution adjusted to 00 mOsm was prepared.

Reference Example 1-8: Osmotic pressure 1,000 mO
Preparation of sm buffer A 10 mM phosphate buffer was prepared by adjusting the pH to 7.5 with NaOH and the final osmotic pressure to 1,000 mOsm with NaCl.

[Reference Example 1-9]: Osmotic pressure 2,000 mO
Preparation of sm buffer A 10 mM phosphate buffer was prepared by adjusting the pH to 7.5 with NaOH and the final osmotic pressure to 2,000 mOsm with NaCl.

[Reference Example 1-10]: Osmotic pressure 80 mOsm
Buffer Preparation pH to 5.5 with NaOH and final osmotic pressure to 8 with NaCl.
A 10 mM phosphate buffer adjusted to 0 mOsm was prepared.

[Reference Example 1-11]: Osmotic pressure 100 mOs
m buffer preparation pH to 5.5 with NaOH and final osmolarity to 1 with NaCl
A 10 mM phosphate buffer solution adjusted to 00 mOsm was prepared.

[Reference Example 1-12]: Osmotic pressure 300 mOs
Preparation of buffer pH to 5.5 with NaOH and final osmolarity to 3 with NaCl.
A 10 mM phosphate buffer solution adjusted to 00 mOsm was prepared.

[Reference Example 1-13]: Osmotic pressure 350 mOs
Preparation of buffer pH to 5.5 with NaOH and final osmolarity to 3 with NaCl.
A 10 mM phosphate buffer adjusted to 50 mOsm was prepared.

[Reference Example 2-0]: Dipalmitoylphosphatidylcholine 37 mg (50 μmol), cholesterol 19 mg (50 μmol), 1,2-bis (hexadecanoyl) -sn-glycero-3-phospho-N-
(1-oxo-3- (2-pyridyldithio) propyl)
2 mg (2.5 μmol) of ethanolamine was dissolved in 20 ml of chloroform. The mixture was transferred to a 100 ml glass eggplant-shaped flask and the solvent was distilled off at room temperature using a rotary evaporator. Further, the solvent was completely distilled off while keeping the pressure under reduced pressure for 1 hour.

5 mM Tris (tris (hydroxymethyl) aminomethane hydrochloride) buffer solution (osmotic pressure 6 mO)
After adding 2 ml of sm, pH 7.5) and 2 g of glass beads, the raw material for lipid was suspended by vigorous shaking. The lipid suspension obtained by filtering off the glass beads was passed through a polycarbonate membrane filter incorporated in an extruder for sizing. A polycarbonate membrane filter having a diameter of 0.2 μm was finally passed through to complete the sizing to obtain a liposome suspension. The obtained liposomes had an average particle size of 209 nm and a turbidity of 0.699.

[Reference Example 2-1]: Preparation of liposome suspension (1) The liposome suspension obtained in [Reference Example 2-0] was dialyzed with a molecular weight cut off of 6,000 to 8,000. And transfer it to 100 times the volume of 300 mOsm buffer of [Reference Example 1-4].
It dialyzed for time and replaced the external water phase.

[Reference Example 2-2]: Preparation of Liposome Suspension (2) The buffer solution used in [Reference Example 2-1] was changed to the 100 mOsm buffer solution of [Reference Example 1-5], and [Reference Example] The outer aqueous phase was replaced in the same manner as in Example 2-1]. Further, the outer aqueous phase was replaced with the 100 mOsm buffer solution of [Reference Example 1-2] in the same manner.

[Reference Example 2-3]: Preparation of liposome suspension (3) The buffer solution used in [Reference Example 2-1] was changed to the 200 mOsm buffer solution of [Reference Example 1-3], and The outer aqueous phase was replaced in the same manner as in Example 2-1]. Further, the outer aqueous phase was replaced with the 100 mOsm buffer solution of [Reference Example 1-2] in the same manner.

[Reference Example 2-4]: Preparation of Liposomal Suspension (4) The same procedure was carried out by changing the buffer solution used in [Reference Example 2-1] to the 300 mOsm buffer solution of [Reference Example 1-4]. The external water phase was replaced by the method. Furthermore, 10 of [Reference Example 1-2] was prepared by the same method.
The outer aqueous phase was replaced with 0 mOsm buffer.

[Reference Example 2-5]: Preparation of Liposome Suspension (5) The buffer solution used in [Reference Example 2-1] was changed to the 350 mOsm buffer solution in [Reference Example 1-5], and [Reference Example] The outer aqueous phase was replaced in the same manner as in Example 2-1]. Further, the outer aqueous phase was replaced with the 100 mOsm buffer solution of [Reference Example 1-2] in the same manner.

[Reference Example 2-6]: Preparation of Liposome Suspension (6) The buffer solution used in [Reference Example 2-1] was changed to the 400 mOsm buffer solution of [Reference Example 1-6], and [Reference Example] The outer aqueous phase was replaced in the same manner as in Example 2-1]. Further, the outer aqueous phase was replaced with the 100 mOsm buffer solution of [Reference Example 1-2] in the same manner.

[Reference Example 2-7]: Preparation of liposome suspension (7) The buffer solution used in [Reference Example 2-1] was changed to the 500 mOsm buffer solution in [Reference Example 1-7], The outer aqueous phase was replaced in the same manner as in Example 2-1]. Further, the outer aqueous phase was replaced with the 100 mOsm buffer solution of [Reference Example 1-2] in the same manner.

[Reference Example 2-8]: Preparation of liposome suspension (8) The buffer solution used in [Reference Example 2-1] was changed to the 350 mOsm buffer solution in [Reference Example 1-5], The outer aqueous phase was replaced in the same manner as in Example 2-1]. Further, the outer aqueous phase was replaced with the 300 mOsm buffer solution of [Reference Example 1-4] in the same manner.

[Reference Example 2-9]: Preparation of liposome suspension (9) The buffer solution used in [Reference Example 2-1] was changed to the 500 mOsm buffer solution in [Reference Example 1-7], The outer aqueous phase was replaced in the same manner as in Example 2-1]. Further, the outer aqueous phase was replaced with the 300 mOsm buffer solution of [Reference Example 1-4] in the same manner.

[Reference Example 2-10]: Preparation of liposome suspension (10) The buffer solution used in [Reference Example 2-1] was changed to the 1,000 mOsm buffer solution in [Reference Example 1-8], [Reference Example 2-1]
The external water phase was replaced in the same manner as in. Further, the outer aqueous phase was replaced with the 300 mOsm buffer solution of [Reference Example 1-4] in the same manner.

[Reference Example 2-11]: Preparation of liposome suspension (11) The buffer solution used in [Reference Example 2-1] was changed to the 2,000 mOsm buffer solution in [Reference Example 1-9], [Reference Example 2-1]
The external water phase was replaced in the same manner as in. Further, the outer aqueous phase was replaced with the 300 mOsm buffer solution of [Reference Example 1-4] in the same manner.

Comparative Reference Example 2-1: Preparation of Liposome Suspension The buffer solution used in [Reference Example 2-1] was changed to the 80 mOsm buffer solution of [Reference Example 1-1], and [Reference Example 2]. The external water phase was replaced in the same manner as in [-1].

[Comparative Reference Example 2-2]: Preparation of Liposome Suspension The buffer solution used in [Reference Example 2-1] was changed to the 100 mOsm buffer solution of [Reference Example 1-2], and [Reference Example 2]. The external water phase was replaced in the same manner as in [-1].

[Reference Example 3-1]: Preparation of osmotic pressure 80 mOsm antibody solution In order to bind the bifunctional compound N-succinimidyl-3- (2-pyridyldithio) propionate (hereinafter abbreviated as SPDP) to the anti-CRP antibody, 30 mM SPDP
Then, 30 μl of the solution was added to 10 mg of the antibody, and the mixture was reacted at 25 ° C. for 30 minutes. Then 10m adjusted to osmotic pressure 80mOsm with NaCl
Using a Sephadex G-25 column equilibrated with M phosphate buffer (pH 6.0), unreacted SP from the antibody solution was used.
The DP was removed.

Dithiothreitol (hereinafter abbreviated as DTT) was added to the obtained antibody solution so that the final concentration was 10 mM, and the mixture was reacted at 25 ° C. for 30 minutes. Next, DTT was removed from the antibody solution using a Sephadex G-25 column equilibrated with 10 mM TES buffer solution (pH 7.5) whose osmotic pressure was adjusted to 80 mOsm with NaCl. The antibody solution thus obtained was adjusted with 10 mM TES buffer so that the antibody concentration was 3 mg / ml.

[Reference Example 3-2]: Osmotic pressure 100 mOsm
Preparation of Antibody Solution The osmotic pressure of each buffer solution was changed to 100 mOsm to prepare an antibody solution in the same manner as in [Reference Example 3-1].

[Reference Example 3-3]: Osmotic pressure 300 mOsm
Preparation of antibody solution The osmotic pressure of each buffer solution was changed to 300 mOsm, and an antibody solution was prepared in the same manner as in [Reference Example 3-1].

[Reference Example 3-4]: Osmotic pressure 350 mOsm
Preparation of Antibody Solution The osmotic pressure of each buffer solution was changed to 350 mOsm, and an antibody solution was prepared in the same manner as in [Reference Example 3-1].

[Example 1] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-1] was replaced with the 300 mOsm antibody solution prepared in [Reference Example 3-3]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 300 mOsm buffer prepared in [Reference Example 1-12] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-12] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent for CRP concentration measurement of Example 1.

Example 2 (1) Preparation of Antibody-Immobilized Liposome Suspension The liposome suspension prepared in [Reference Example 2-2] was replaced with the 350 mOsm antibody solution prepared in [Reference Example 3-4]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, to remove unreacted antibody, Sepharose C equilibrated with the 350 mOsm buffer prepared in [Reference Example 1-13] above.
An antibody-immobilized liposome suspension was obtained using an L4B column.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in (1) above.
The absorbance is measured at nm and diluted with the buffer solution prepared in the above [Reference Example 1-13] so that the absorbance becomes 2.4,
An immunoagglutination reaction reagent for measuring CRP concentration in Example 2 was obtained.

[Example 3] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-3] was replaced with the 100 mOsm antibody solution prepared in [Reference Example 3-2]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 100 mOsm buffer prepared in [Reference Example 1-11] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-11] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent of Example 3 for measuring CRP concentration.

Example 4 (1) Preparation of Antibody-Immobilized Liposome Suspension The liposome suspension prepared in [Reference Example 2-4] was replaced with the 100 mOsm antibody solution prepared in [Reference Example 3-2]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 100 mOsm buffer prepared in [Reference Example 1-11] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-11] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent of Example 4 for measuring CRP concentration.

Example 5 (1) Preparation of Antibody-Immobilized Liposome Suspension The liposome suspension prepared in [Reference Example 2-5] was replaced with the 100 mOsm antibody solution prepared in [Reference Example 3-2]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 100 mOsm buffer prepared in [Reference Example 1-11] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance at nm was measured and diluted with the buffer solution prepared in [Reference Example 1-11] so that the absorbance was 2.4 to obtain the immunoaggregation reaction reagent of Example 5 for measuring CRP concentration.

[Example 6] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-6] was replaced with the 100 mOsm antibody solution prepared in [Reference Example 3-2]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 100 mOsm buffer prepared in [Reference Example 1-11] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-11] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent of Example 6 for measuring CRP concentration.

[Example 7] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-7] was replaced with the 100 mOsm antibody solution prepared in [Reference Example 3-2]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 100 mOsm buffer prepared in [Reference Example 1-11] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-11] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent of Example 7 for measuring CRP concentration.

[Example 8] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-8] was replaced with the 300 mOsm antibody solution prepared in [Reference Example 3-3]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 300 mOsm buffer prepared in [Reference Example 1-12] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-12] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent of Example 8 for measuring CRP concentration.

[Example 9] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-9] was replaced with the 300 mOsm antibody solution prepared in [Reference Example 3-3]. The same amount was mixed and reacted at 4 ° C. for 3 days. Then, Sepharose CL4 equilibrated with 300 mOsm buffer prepared in [Reference Example 1-12] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-12] so that the absorbance would be 2.4 to obtain an immunoaggregation reaction reagent for measuring CRP concentration of Example 9.

[Example 10] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-10] was
An equal amount of the 300 mOsm antibody solution prepared in [Reference Example 3-3] was mixed and reacted at 4 ° C for 3 days. Then, to remove unreacted antibody, Sepharose C equilibrated with the 300 mOsm buffer prepared in [Reference Example 1-12].
An antibody-immobilized liposome suspension was obtained using an L4B column.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-12] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent of Example 10 for measuring CRP concentration.

[Example 11] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Reference Example 2-11] was
An equal amount of the 300 mOsm antibody solution prepared in [Reference Example 3-3] was mixed and reacted at 4 ° C for 3 days. Then, to remove unreacted antibody, Sepharose C equilibrated with the 300 mOsm buffer prepared in [Reference Example 1-12].
An antibody-immobilized liposome suspension was obtained using an L4B column.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance at nm was measured and diluted with the buffer solution prepared in [Reference Example 1-12] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent of Example 11 for measuring CRP concentration.

Comparative Example 1 (1) Preparation of Antibody-Immobilized Liposome Suspension The liposome suspension prepared in [Comparative Reference Example 2-1] was
The 80 mOsm antibody solution prepared in [Reference Example 3-1] was mixed in an equal amount and reacted at 4 ° C for 3 days. Then, Sepharose CL4 equilibrated with 80 mOsm buffer prepared in [Reference Example 1-10] to remove unreacted antibody.
Using the B column, an antibody-immobilized liposome suspension was obtained.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance was measured at nm and diluted with the buffer solution prepared in [Reference Example 1-10] so that the absorbance was 2.4 to obtain the immunoaggregation reaction reagent for CRP concentration measurement of Comparative Example 1.

[Comparative Example 2] (1) Preparation of antibody-immobilized liposome suspension The liposome suspension prepared in [Comparative Reference Example 2-2] was prepared as follows.
An equal amount of the 100 mOsm antibody solution prepared in [Reference Example 3-2] was mixed and allowed to react at 4 ° C for 3 days. Then, Sepharose C equilibrated with 100 mOsm buffer prepared in [Reference Example 1-11] to remove unreacted antibody.
An antibody-immobilized liposome suspension was obtained using an L4B column.

(2) Preparation of immunoaggregation reaction reagent 340 of the antibody-immobilized liposome suspension obtained in the above (1)
The absorbance at nm was measured and diluted with the buffer solution prepared in [Reference Example 1-11] so that the absorbance would be 2.4 to obtain the immunoaggregation reaction reagent for CRP concentration measurement of Comparative Example 2.

[Evaluation of CRP Concentration Measurement Sensitivity] Hitachi 715
Using a type 0 automatic analyzer (trade name: manufactured by Hitachi, Ltd.), the CRP concentration was measured under the following measurement conditions, and the results were obtained in Examples 1 to 10 and Comparative Examples 1 and 2. The measurement sensitivity of each immunoaggregation reaction reagent for measuring CRP concentration was evaluated under the following measurement conditions.

The amount of sample to be measured was 7 μl, serum having a known CRP concentration was used, and samples having various concentrations in the range of 0 mg / dl to 10 mg / dl were prepared and used.

The amount of the first reagent was 200 μl, and 10 mM Tris buffer (pH 8.0) prepared to have the same osmotic pressure as that of each Example and each Comparative Example was used.

The amount of the second reagent was 100 μl, and
To 10 and each CRP obtained in Comparative Examples 1 and 2
An immunoagglutination reaction reagent for concentration measurement was used. The measurement wavelength was 340 nm for the main wavelength and 700 nm for the sub wavelength, and the amount of change in absorbance for about 5 minutes was obtained by two-point analysis of 28 points and 50 points.

Using the absorbance at a CRP concentration of 4 mg / dl, the measurement sensitivity was calculated from the obtained amount of change in absorbance according to the following formula (2), and the results are shown in Table 1 below for each Example and each Comparative Example. The measurement sensitivity of the immune agglutination reagent for CRP concentration measurement was shown. As for the measurement sensitivity, the smaller the value is, the lower the concentration of the test substance can be measured and the higher the sensitivity is.

[0120]

[Equation 1]

[0121]

[Table 1]

From the above evaluation results, the CR according to the present invention
It was found that the measurement using the immunoagglutination reagent for P concentration measurement has a remarkably high measurement sensitivity as compared with the comparative example, which indicates that the low concentration measurement of CRP is possible.

[0123]

EFFECTS OF THE INVENTION According to the immunoaggregation reaction reagent of the present invention, in addition to the advantages that the liposome carrier has the characteristic that it can measure in the short wavelength range and the cell contamination of the measuring device is low, A general-purpose measuring device can be used for the measurement, and the substance to be measured can be measured with a higher sensitivity than before in a wide range from a low concentration range to a high concentration range, particularly in a low concentration range.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takahisa Ueno 1075-2 Kitanan Mororo, Yuki City, Ibaraki Prefecture Nissui Pharmaceutical Co., Ltd. Diagnostic Drug Research Laboratory (72) Seiji Tanaka 1075-2 Kitanan Mororo, Yuki City, Ibaraki Prefecture Nissui Pharmaceutical Co., Ltd. Diagnostic Research Laboratories (72) Inventor Makoto Umeda 1075-2 Kitanan Mororo, Yuki City, Ibaraki Prefecture Nissui Pharmaceutical Diagnostic Research Laboratories

Claims (5)

[Claims] 1. An immunoagglutination reagent for measuring the amount of agglutination caused by an immunological reaction with a substance to be measured in a test substance, which has an osmotic pressure of 120 mOsm or more higher than the osmotic pressure of a hydrated aqueous solution during liposome preparation. An immunoaggregation reaction reagent, wherein an osmotic pressure-loaded liposome obtained by substituting the outer aqueous phase of the liposome with an aqueous solution is used as a carrier. 2. The liposome carrier loaded with osmotic pressure comprises:
120mO from the osmotic pressure of the hydrated aqueous solution during liposome preparation
The external water phase of the liposome is first substituted with a first displacement aqueous solution having a higher osmotic pressure than sm, and then the first displacement external water phase is
The immunoagglutination reaction reagent according to claim 1, wherein the reagent is secondly substituted with a second substitution aqueous solution having an osmotic pressure lower than that of the first substitution aqueous solution. 3. The osmotic pressure of the hydrated aqueous solution is 0.5 to 30 mO.
The immunoagglutination reaction reagent according to claim 1 or 2, which is sm. 4. The osmotic pressure of the second displacement aqueous solution is 700 mOs.
The immunoagglutination reaction reagent according to claim 2, which is not more than m. 5. The immune agglutination reagent according to claim 1, 2, 3 or 4 is allowed to act on a substance to be measured of a subject, and the amount of agglutination generated by an antigen-antibody reaction is measured by transmitted light. Immunoassay method.