JP3350762B2 - Immunological diagnostics - Google Patents

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 having high sensitivity and excellent reproducibility based on an antigen-antibody reaction, and also excellent in running costs and the like.

[0002]

2. Description of the Related Art In vitro diagnostic agents are classified into RI method, chemiluminescent method, enzymatic method, latex method and the like according to their detection and quantification methods. Among them, 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.
JP-A-63-141514 and JP-A-60-71958 disclose an antibody (antigen) labeled with a chemiluminescent substance such as acridine as disclosed in Japanese Unexamined Patent Publication No. As described above, detection and quantification are performed by luminescence from an enzyme / substrate. These can be combined with advanced immunological techniques such as a sandwich method to enable ultra-trace quantification of ng / ml to pg / ml.

[0003] On the other hand, the latex method uses a latex having a submicron order particle size on which an antibody (antigen) is immobilized, and a latex agglutination reaction resulting from an antigen-antibody reaction between the latex and an antigen (antibody) in a sample is performed. 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 means, and a slide method in which the agglutination reaction is visually determined, or the degree of the agglutination reaction is optically determined as transmitted light turbidity, scattered light turbidity, or integrated class turbidity. It is possible to quantify
This method is simple and quick. In addition, as a means for suppressing non-specific agglutination reactions,
JP-A-57-29039 and JP-B-2-14661 discloses an attempt to modify an antibody with a 2,4-dinitrophenyl group or a hydrophobic group to increase the physical adsorption ability to latex fine particles.

[0004]

SUMMARY OF THE INVENTION The RI method, the chemiluminescence method,
Although high sensitivity can be obtained with the enzymatic method, on the other hand, there are problems such as the necessity of large-scale equipment or equipment, high running cost, and a long time from pretreatment to measurement. The latex method allows simple and quick diagnosis, but latex is expensive.
And the running cost is high .

[0005] An object of the present invention is to provide an immunological diagnostic agent capable of performing a highly sensitive, simple and rapid assay without using a conventional carrier such as latex in a system for assaying the generation of turbidity. It is to be.

[0006]

Means for Solving the Problems As a result of intensive 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 present inventors have found that by preparing a substance which easily causes turbidity which is convenient for detection during an antibody-antigen reaction, a diagnostic agent having higher sensitivity and excellent reproducibility can be obtained, and the present invention has been completed.

Hereinafter, the present invention will be described in more detail.

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

In the present invention, as the compound that imparts hydrophobicity by modifying an antibody (hereinafter abbreviated as “hydrophobic compound”), a fatty acid, particularly a fatty acid having 2 to 24 carbon atoms is preferable. Thus, particularly preferred are fatty acids having 2 to 18 carbon atoms. It is not preferable to use a fatty acid having 25 or more carbon atoms, since the aggregation between the modified antibodies becomes remarkably strong.

Among the fatty acids, specific examples of the saturated linear fatty acid (aliphatic monocarboxylic acid) include acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, and the like.
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 arachiic acid. By using these fatty acids, for example, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl and the like are modified into monovalent saturated linear acyl groups.

In order to produce a hydrophobically modified antibody by introducing the above-mentioned hydrophobic compound into an antibody, a method of directly reacting the antibody with the hydrophobic compound or a method of reacting the antibody with the antibody after activating the hydrophobic compound once is used. And a method of causing it to be used.

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

In the latter, a carboxyl group in a hydrophobic group is converted to an acid chloride or an acid anhydride, or N, N'-disuccinimidyl carbonate, 1-hydroxybenzotriazole, -Hydroxy-5-norbornene-2,3-dicarboximide, N
Activated esterification using -hydroxysuccinimide, N-hydroxyphthalimide, isobutyl chloroformate, 4-hydroxyphenyldimethylsulfonium methyl sulfate or the like is 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 perform N-alkylation is also used.

As the antibody used in the present invention, any antibody may be used, 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 preparing the reaction is not particularly limited.
The ratio is 1: 10000, preferably 1: 1 to 1: 1000. In this case, if the value is less than 0.01, the expected effect cannot be obtained.
A molecular weight of 000 or more is not preferred because precipitation occurs only with the modified antibody itself.

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

Organic solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran and 1,4-dioxane, and mixed solvents of these with water or a buffer can also be used as solvents other than water or the buffer.

The resulting hydrophobically modified antibody 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 hydrophobic modified antibody has a pH of 4.
5 to 8.5 aqueous solution or buffer, or an organic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, 1,4-dioxane, or a mixed solvent of these and water or a buffer. Used. As additives, water-soluble polymers such as polyethylene glycol, polyvinyl alcohol and dextran and anionics such as sodium dodecylbenzenesulfonate and Triton X;
Various cationic and nonionic surfactants can also be used.

The concentration range of the modified antibody in the immunological diagnostic agent is not particularly limited because it varies depending on the antibody used, but the final concentration of the measurement system is 10 ^-4 to 10 ^4.
mg / ml, particularly preferably 10 ^-1 ~10 ² mg / m
l. If the concentration is less than 10 ⁻⁴ mg / ml, sufficient sensitivity cannot be obtained, and if the concentration is higher than 10 ⁴ mg / ml, the modified antibodies are likely to aggregate with each other, which is not preferable.

The diagnostic agent of the present invention performs 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 extent of the agglutination reaction can be optically quantified by an agglutination turbidimetric method, an integral class turbidity method, a particle size distribution method, a turbidimetric method, or the like. Xenon lamps, hydrogen / deuterium lamps, mercury lamps,
Tungsten lamp, tungsten-halogen lamp,
It is measured using a helium lamp, a krypton lamp, various lasers, a light emitting diode, or the like, and a wavelength of 340 nm or more, particularly preferably 340 to 700 nm or 800 to 2400 nm.

[0021]

According to the present invention, a novel diagnostic agent having high sensitivity and excellent reproducibility and capable of performing a rapid assay is provided.

[0022]

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to only these examples.

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

50 μl of the reagent obtained above,
20 μl of 1-100 mg / dl human CRP;
300 μl of 5% polyethylene glycol / 0.01 M phosphate buffer (pH = 7.4) was mixed. After 5 minutes, the absorbance (device used: Hitachi 7150 automatic analyzer, main wavelength 340 nm, auxiliary wavelength 700 nm) was measured. Table 1
^Shows measured values (× 10 ⁻⁴ ).

[0025]

[Table 1]

Example 2 An anti-human CRP antibody obtained by immunizing goats was immunized with a 0.1 M phosphate buffer (pH = 8.0) at 10 mg / ml.
Was adjusted. In 1 ml of this, acetaldehyde 10μ
1 was added and reacted at 4 ° C. for 30 minutes, and 1 mg of sodium borohydride was further added and reacted at 4 ° C. for 30 minutes. After completion of the reaction, dialysis was performed overnight at 4 ° C. in a 0.01 M phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain an ethylated antibody. The obtained ethylated antibody was measured for titer by the Becker method, and 0.05 M Na
It was adjusted to 0.5 mgAg / ml with a 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 treated with a 0.1 M phosphate buffer (pH = 7.4) at 10 mg / mg.
Adjusted to ml. To 1 ml of the solution, 1 ml of a 500 mg / ml N-decanoyloxysuccinimide / dimethylformamide solution was added and reacted at 40 ° C. for 6 hours. After completion of the reaction, dialysis was performed overnight at 4 ° C. in a 0.01 M phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain a decanoylated antibody. The obtained decanoylated antibody was measured for titer by the Becker method, and 0.05 M Na
It was adjusted to 0.5 mgAg / ml with a 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 an anti-human CRP antibody obtained by immunizing a goat in 0.1 M citrate buffer (pH = 4.5)
/ Ml. 1 ml of this contains 500 mg / ml 4-
1 ml of a 0.1 M citrate buffer (pH = 4.5) solution of (lauroyloxy) phenyldimethylsulfonium methyl sulfate was added, and the mixture was reacted at 40 ° C. for 4 hours. After the reaction,
Dialysis was performed overnight at 4 ° C. in a 0.01 M phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain a lauroylated antibody. The obtained lauroylated antibody was measured for titer by the Becker method, and 0.05 M NaCl
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. Table 1 also shows the measured values.

Example 5 10 mg of an anti-human CRP antibody obtained by immunizing a goat in 0.1 M citrate buffer (pH = 4.5)
/ Ml. 1 ml of this contains 50 mg / ml 4-
1 ml of a solution of (myristoyloxy) phenyldimethylsulfonium methyl sulfate / dimethylformamide was added and reacted at 40 ° C. for 4 hours. After the reaction is completed, 0.01M
Dialysis was performed overnight at 4 ° C. in a phosphate buffer (pH = 7.4), and insolubles were removed by filtration with a filter (0.45 μm) to obtain a myristoylated antibody. The obtained myristoylated antibody was measured for titer by the Becker method, and 0.05 M NaCl-
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. Table 1 also shows the measured values.

[Comparative Example] The titer of an unmodified goat-derived anti-human CRP antibody was measured by the Becker method, and 1.0 mg Ag in 0.05 M NaCl-0.01 M phosphate buffer (pH = 8.2). / Ml. Of these, 50 μl and 0.01 M phosphate buffer containing 1 to 100 mg / dl human CRP (pH =
7.4) 300 μl were mixed. Absorbance after 5 minutes (device used: Hitachi 7150 automatic analyzer, dominant wavelength 340
nm, subwavelength 700 nm). Table 1 also shows the measured values.

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

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shoichi Shuto 1-18-18 Hinode, Saga City, Saga Prefecture (56) References JP-A-61-100660 (JP, A) JP-A-59-224565 (JP, A) (58) Fields surveyed (Int. Cl. ⁷ , DB name) G01N 33/536

Claims (2)

(57) [Claims] 1. An immunological diagnostic agent comprising, as a turbidity-generating component, an antibody modified with an acyl group having 2 to 24 carbon atoms without using an insoluble carrier . 2. A method for measuring an antigen, comprising mixing the immunological diagnostic agent according to claim 1 and a liquid containing an antigen, and quantifying the generated turbidity.