JPH0682449A - Immunological diagnostic agent - Google Patents

Abstract

PURPOSE:To provide diagnostic agent which enables easy, quick test at high sensitivity without using such a carrier as latex by including antibodies modified by an acyl group with a specific number of carbon atoms in the diagnostic agent as turbidity generating components. CONSTITUTION:The hydrophilic-hydrophobic balance of antibodies is optimized by the chemical hydrophobic modification of the antibodies themselves so as to prepare a material which at an antibody-antigen reaction is likely to cause turbidity suitable for detection; i.e., the antibodies modified with an acyl group of 2 to 24 carbon atoms are contained in diagnostic agent as turbidity, generating components. A fatty acid, especially one with 2 to 24 carbon atoms, is desirable for use as a compound for modifying the antibodies to provide hydrophobicity. The use of a fatty acid of 25 carbon atom or more is not desirable since, in that case, coagulation among the modified antibodies becomes too strong. To produce the hydrophobic modified antibodies by introducing a hydrophobic compound in the antibodies, a method in which the hydrophobic compound is directly reacted with the antibodies and that in which the hydrophobic compound is reacted with the antibodies after being temporarily activated, are employed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel immunological diagnostic agent based on an antigen-antibody reaction, which is highly sensitive and reproducible, and which is excellent in running cost and the like.

[0002]

In vitro diagnostic agents are classified into RI method, chemiluminescence method, enzyme method, latex method and the like according to their detection / quantification methods. Among these, the RI method uses an antibody (antigen) labeled with a radioactive element, and the chemiluminescence method is described in JP-A-63-57572 and JP-A-63-101368.
As disclosed in JP-A No. 63-141514 and JP-A No. 60-71958, an antibody (antigen) labeled with a chemiluminescent substance such as acridine is disclosed. As described above, the detection / quantification is performed by the light emission from the enzyme / substrate. By combining these with advanced immunological techniques such as the sandwich method, ultra-trace quantification of ng / ml to pg / ml is possible.

On the other hand, the latex method uses a latex having a particle size of submicron order on which an antibody (antigen) is immobilized, and an agglutination reaction of the latex produced as a result of an antigen-antibody reaction between the latex and the antigen (antibody) in a specimen. It is an assay, and generally has a detection sensitivity of mg / ml to μg / ml. In this method, an agglutination reaction occurs on a slide glass as an assay method, and the slide method is used to visually judge the agglutination reaction. Can be quantified as
It is a method that is easy and quick. In addition, as a means for suppressing the nonspecific agglutination reaction, which is a problem at this time,
57-29039 and Japanese Patent Publication No. 2-14661 disclose an attempt to modify an antibody with a 2,4-dinitrophenyl group or a hydrophobic group to enhance its physical adsorption ability to latex fine particles.

[0004]

[Problems to be Solved by the Invention] RI method, chemiluminescence method,
Although the enzyme method can provide high sensitivity, on the other hand, it requires large-scale equipment or devices, has a high running cost, and requires a long time from pretreatment to measurement. In addition, the latex method allows simple and quick diagnosis, but the detection sensitivity is mg /
There is a problem that it is inferior to the other, from ml to μg / ml.

An object of the present invention is to provide an immunological diagnostic agent in a system for assaying the occurrence of turbidity, which is highly sensitive and can be simply and quickly assayed without using a conventional carrier such as latex. It is to be.

[0006]

As a result of earnest studies, the present inventors have optimized the hydrophilic / hydrophobic balance of an antibody by chemically modifying the antibody itself without using a carrier such as latex. The inventors have found that a diagnostic agent with higher sensitivity and excellent reproducibility can be obtained by preparing a substance that easily causes turbidity that is convenient for detection during antibody-antigen reaction, and completed the present invention.

The present invention will be described in more detail below.

[0008] That is, the means for solving the problems of the present invention is an immunological diagnosis characterized by containing an antibody modified with an acyl group having 2 to 24 carbon atoms as a turbidity generating component. It is to provide medicine.

In the present invention, the compound that modifies the antibody to impart hydrophobicity (hereinafter abbreviated as "hydrophobic compound") is preferably a fatty acid, particularly a fatty acid having 2 to 24 carbon atoms. Thus, particularly preferred are fatty acids having 2 to 18 carbon atoms. The use of a fatty acid having 25 or more carbon atoms is not preferable because aggregation between modified antibodies remarkably increases.

Of the fatty acids, for example, saturated straight chain fatty acids (aliphatic monocarboxylic acids) are specifically exemplified by acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid,
Examples thereof include caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nondecanoic acid, and arachidic acid. By using these fatty acids, as the monovalent saturated linear acyl group, for example, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, etc. are modified in the antibody.

In order to produce a hydrophobically modified antibody by introducing the above hydrophobic compound into an antibody, a method of directly reacting the hydrophobic compound with the antibody and a method of activating the hydrophobic compound and then reacting with the antibody And the method of letting.

In the former case, as a dehydration condensation agent, BOP reagent (manufactured by Wako Pure Chemical Industries, Ltd.), 1,1'-carbonyldiimidazole,
Meso-p-toluene sulphonate (N-cyclohexyl-N '-(2-morpho-ethyl) carbodiimide meso-p-toluenesulfonate), dicyclohexylcarbodiimide, diethyl cyanophosphate, N-ethoxycarbonyl-2-ethoxy-1,2-dihydro Quinoline, 1-
Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the like are preferably used.

In the latter case, the carboxyl group in the hydrophobic group is converted to an acid chloride or an acid anhydride, or as an activating reagent, N, N'-disuccinimidyl carbonate, 1-hydroxybenzotriazole, N is used. -Hydroxy-5-norbornene-2,3-dicarboximide, N
Those activated with esterification using -hydroxysuccinimide, N-hydroxyphthalimide, isobutyl chloroformate, 4-hydroxyphenyldimethylsulfonium methylsulfate and the like are preferably used. Also,
A method in which an aliphatic aldehyde such as formaldehyde or acetaldehyde is allowed to act on an amino group in the antibody and further reduced with sodium borohydride or the like to carry out N-alkylation can also be used.

Any antibody may be used as the antibody used in the present invention, and examples thereof include an anti-human CRP antibody obtained by immunizing a rabbit and an anti-human CRP antibody obtained by immunizing a goat. be able to.

The molar ratio of the hydrophobic compound to 1 mol of the antibody at the time of reaction is not particularly limited, but it is 1: 0.01-.
It is 1: 10000, preferably 1: 1 to 1: 1000. In this case, if 0.01 or less, the expected effect is not obtained, and 10
When it is 000 or more, precipitation is generated only by the modified antibody itself, which is not preferable.

The introduction reaction of these hydrophobic compounds into the antibody is most preferably a reaction in water or a buffer solution in order to minimize the inactivation of the antibody, and is preferably 0 to 80 ° C., preferably 4 to The reaction is carried out at 40 ° C. for 10 minutes to 72 hours, preferably 1 hour to 24 hours.

As a solvent other than water or a buffer solution, an organic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, 1,4-dioxane, etc., or a mixed solvent of these with water or a buffer solution can be used.

The obtained hydrophobically modified antibody is as it is,
Alternatively, after further purification by dialysis, ultrafiltration, gel filtration, adsorbent treatment, extraction, etc., it is used as a component of the diagnostic agent of the present invention. At this time, the hydrophobically modified antibody has a pH of 4.
Dissolve or uniformly disperse it in an aqueous solution of 5 to 8.5 or a buffer solution, or an organic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, 1,4-dioxane or a mixed solvent of these and water or a buffer solution. Used. As additives, water-soluble polymers such as polyethylene glycol, polyvinyl alcohol and dextran, and anionic compounds such as sodium dodecylbenzene sulfonate and Triton X,
It is also possible to use various cationic and nonionic surfactants.

The concentration range of the modified antibody in the immunological diagnostic agent is not particularly limited because it depends on the antibody used, but the final concentration of the assay system is 10E-4 to 10E4.
mg / ml, particularly preferably 10E-1 to 10E2 mg / ml. At 10E-4 mg / ml or less, sufficient sensitivity cannot be obtained, and at 10E4 mg / ml or more, the modified antibodies are likely to aggregate with each other, which is not preferable.

The diagnostic agent of the present invention carries out an assay by generating turbidity caused by an antigen-antibody reaction that occurs when a sample is added to a solution in which the hydrophobic modified antibody is uniformly dissolved or dispersed. At this time, the degree of agglutination reaction can be quantified optically by agglutination turbidimetric method, integral turbidity method, particle size distribution method, nephelometric method and the like. As a light source, a xenon lamp, hydrogen / deuterium lamp, mercury lamp,
Tungsten lamp, tungsten-halogen lamp,
The measurement is performed using a helium lamp, a krypton lamp, various lasers, light emitting diodes, etc., using a wavelength of 340 nm or more, particularly preferably 340 to 700 nm or 800 to 2400 nm.

[0021]

INDUSTRIAL APPLICABILITY The present invention provides a novel diagnostic agent which has high sensitivity and excellent reproducibility and which enables rapid assay.

[0022]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples.

[Example 1] An anti-human CRP antibody obtained by immunizing a rabbit was diluted with 0.1 M phosphate buffer (pH = 8.0) to 50
Adjusted to μg / ml. To 1 ml of this, 1 g of sodium acetate was added and dissolved, then 2 μl of acetic anhydride was further added, and the mixture was reacted at 4 ° C. overnight. After the reaction was completed, dialysis was performed overnight at 4 ° C in 0.01M phosphate buffer (pH = 7.4) to remove acetic acid, and insoluble materials were removed by filtration with a filter (0.45 μm) to obtain an acetylated antibody. It was The titer of the obtained acetylated antibody was measured by the Becker method, and 0.05M NaCl-0.
It was adjusted to 0.5 mg Ag / ml with 01M phosphate buffer (pH = 8.2) and used for the following evaluation.

50 μl of the reagent obtained above and 1 to
20 μl of 100 mg / ml human CRP and 300 μ of 0.5% polyethylene glycol / 0.01M phosphate buffer (pH = 7.4)
and 1 were mixed. After 5 minutes, the absorbance (device used; Hitachi
Model 7150 automatic analyzer, main wavelength 340 nm, sub-wavelength 700 nm) was measured. Table 1 shows the measured values (× 10 ^-4 ).

[0025]

[Table 1]

[Example 2] 10 mg / ml of anti-human CRP antibody obtained by immunizing a goat with 0.1 M phosphate buffer (pH = 8.0)
Adjusted to. To 1 ml of this, 10 μ of acetaldehyde
1 was added and the mixture was reacted at 4 ° C. for 30 minutes, 1 mg of sodium borohydride was further added, and the mixture was reacted at 4 ° C. for 30 minutes. After completion of the reaction, dialysis was performed in 0.01 M phosphate buffer (pH = 7.4) at 4 ° C. overnight, and insoluble matter was removed by filtration with a filter (0.45 μm) to obtain an ethylated antibody. The titer of the obtained ethylated antibody was measured by the Becker method, and 0.05M Na
It adjusted to 0.5 mgAg / ml with Cl-0.01M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed. Table 1 also shows the measured values.

[Example 3] An anti-human CRP antibody obtained by immunizing a goat was added to 10 mg / 0.1% in 0.1 M phosphate buffer (pH = 7.4).
Adjusted to ml. To 1 ml of this was added 1 ml of a 500 mg / ml N-decanoyloxysuccinimide / dimethylformamide solution, and the mixture was reacted at 40 ° C. for 6 hours. After completion of the reaction, dialysis was performed overnight at 4 ° C. in 0.01 M phosphate buffer (pH = 7.4), and insoluble matters were removed by filtration through a filter (0.45 μm) to obtain a decanoylated antibody. The titer of the obtained decanoylated antibody was measured by the Becker method, and 0.05M Na
It adjusted to 0.5 mgAg / ml with Cl-0.01M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed. Table 1 also shows the measured values.

[Example 4] 10 mg of anti-human CRP antibody obtained by immunizing a goat with 0.1 M citrate buffer (pH = 4.5)
/ Ml. In 1 ml of this, 500 mg / ml of 4-
1 ml of a 0.1 M citrate buffer solution (pH = 4.5) containing (lauroyloxy) phenyldimethylsulfonium methyl sulfate was added, and the mixture was reacted at 40 ° C. for 4 hours. After the reaction,
Dialysis was carried out in 0.01 M phosphate buffer (pH = 7.4) at 4 ° C. overnight, and insoluble matters were removed by filtration through a filter (0.45 μm) to obtain lauroylated antibody. The titer of the obtained lauroylated antibody was measured by the Becker method, and 0.05M NaCl was used.
It was adjusted to 0.5 mgAg / ml with a -0.01 M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed below. Table 1 also shows the measured values.

[Example 5] 10 mg of anti-human CRP antibody obtained by immunizing a goat with 0.1 M citrate buffer (pH = 4.5)
/ Ml. In 1 ml of this, 50 mg / ml of 4-
1 ml of (myristoyloxy) phenyldimethylsulfonium methylsulfate / dimethylformamide solution was added and reacted at 40 ° C. for 4 hours. 0.01M after the reaction
Dialysis was performed overnight at 4 ° C. in a phosphate buffer (pH = 7.4), and insoluble materials were removed by filtration with a filter (0.45 μm) to obtain a myristoylated antibody. The titer of the obtained myristoylated antibody was measured by the Becker method, and 0.05M NaCl-
The concentration was adjusted to 0.5 mg Ag / ml with 0.01 M phosphate buffer (pH = 8.2), and the same evaluation as in Example 1 was performed. Table 1 also shows the measured values.

[Comparative Example] Unmodified goat-derived anti-human CRP
The titer of the antibody was measured by the Becker method, and 0.05M NaCl-0.01
The concentration was adjusted to 1.0 mgAg / ml with M phosphate buffer (pH = 8.2). 50 μl of this and 1-100 mg / ml human CRP
Was mixed with 300 μl of 0.01 M phosphate buffer (pH = 7.4) containing Absorbance after 5 minutes (used equipment: Hitachi 7150
Type automatic analyzer, main wavelength 340 nm, sub-wavelength 700 nm) was measured. Table 1 also shows the measured values.

From the above results, the hydrophobically modified antibody according to the present invention has a CRP concentration of 1 to 100 mg / ml as compared with the unmodified antibody.
It can be seen that it has a significantly high sensitivity over a wide range of.

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[Procedure amendment]

[Submission date] December 11, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction content]

[0004]

[Problems to be Solved by the Invention] RI method, chemiluminescence method,
Although the enzyme method can provide high sensitivity, on the other hand, it requires large-scale equipment or devices, has a high running cost, and requires a long time from pretreatment to measurement. The latex method allows simple and quick diagnosis, but latex is expensive.
Therefore, the running cost is high .

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction content]

[0012] In the former, as a dehydrating condensing agent, BOP reagent (manufactured by Wako Pure Chemical Industries, Ltd.), 1,1'-carbonyldiimidazole, N- cyclohexyl--N '- (2-Moruhorunoechiru) carbodiimide meso -p- toluenesulfonate , Dicyclohexylcarbodiimide, diethyl cyanophosphate, N-ethoxycarbonyl-2-ethoxy-1,
2-dihydroquinoline, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the like are preferably used.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

50 μl of the reagent obtained above,
20 μl of human CRP at 1-100 mg / dl ,
Mix with 300 μl of 5% polyethylene glycol / 0.01 M phosphate buffer (pH = 7.4). After 5 minutes, the absorbance (used equipment; Hitachi 7150 type automatic analyzer, main wavelength 340 nm, sub wavelength 700 nm) was measured. Table 1
^{Shows the} measured value (× 10 ⁻⁴ ).

[Procedure amendment 4]

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[Correction method] Change

[Correction content]

[Comparative Example] Unmodified goat-derived anti-human CRP
The titer of the antibody was measured by the Becker method, and 0.05MNaC
1-0.01 M phosphate buffer (pH = 8.2)
It was adjusted to 0 mg Ag / ml. 50 μl of this,
0.01 containing 1-100 mg / dl of human CRP
300 μl of M phosphate buffer (pH = 7.4) was mixed. After 5 minutes, the absorbance (apparatus used; Hitachi 7150 type automatic folding machine, main wavelength 340 nm, sub wavelength 700 nm) was measured. Table 1 also shows the measured values.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction content]

From the above results, the hydrophobically modified antibody according to the present invention has a CRP concentration of 1 to 100 m, as compared with the unmodified antibody.
It can be seen that it has significantly higher sensitivity over a wide range of g / dl .

[Procedure Amendment 5]

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[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

[0025]

[Table 1]

Continued Front Page (72) Inventor Shoichi Suto 1-18-18 Hinode Hinode, Saga City, Saga Prefecture

Claims (2)

[Claims] 1. An immunological diagnostic agent comprising an antibody modified with an acyl group having 2 to 24 carbon atoms as a turbidity generating component. 2. A method for measuring an antigen, which comprises mixing the immunological diagnostic agent according to claim 1 with a liquid containing an antigen and quantifying the generated turbidity.