JP3147992B2 - Labeling reagent for immunoassay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a labeling reagent used for the measurement of a physiologically active substance by an immunoassay, and uses an aminoglycan in which 20 to 80% of amino groups are acylated with a saturated fatty acid. The present invention relates to a labeling reagent capable of cryopreservation of the labeling reagent.

[0002]

2. Description of the Related Art Conventionally, as a means for increasing the sensitivity of a labeling reagent used in an immunoassay, a method having a plurality of reactive groups,
By using a compound in which most of the reactive groups are modified with a large number of labeling substances (enzymes, dyes, luminescent substances, etc.), the present inventor has specially attempted to increase the amount of labeling per molecule of immunological substance. No. 2,506,243 proposed an aminoglycan having a plurality of amino groups.

[0003]

As aminoglycan, chitosan is generally used because of its easy availability. Commercially available chitosan is produced by treating chitin extracted from crustaceans with an acid, and 10% of the amino group is obtained. Are less than acetylated. Since such chitosan has a low solubility in water, the labeling reagent using the chitosan is frozen or freeze-dried and stored, and when thawed and dissolved at the time of use, a part of the chitosan remains insoluble and remains undissolved during immunoassay. There is a problem that sensitivity and reproducibility are reduced.

[0004]

Means for Solving the Problems The present inventors acylate 20 to 80% of the amino groups of aminoglycans such as chitosan and the like, and the labeling reagent produced using the acylates can be stored frozen or lyophilized. I found The present invention is a labeling reagent for immunoassay, wherein the aminoglycan modified with a label and bound to an immunological substance is an aminoglycan in which 20 to 80% of amino groups are acylated with a saturated fatty acid.

Hereinafter, a method for producing the labeling reagent of the present invention will be described. The molecular weight is 10 ^{4 to} 5 × 10 ⁶ , preferably 10 ⁵ to 1
0 ⁶ chitosan is, the aminoglycan such as polygalactosamine, dissolved in an aqueous solution of saturated fatty acids having 1 to 6 carbon atoms, is reacted by addition of a condensing agent, 20 to 80 of the amino group
%, Which is purified by dialysis or gel filtration. The degree of acylation can be roughly determined by measuring fluorescence at 460 nm at an excitation wavelength of 225 nm with a fluorescence spectrophotometer, using aminoglycans of known molecular weight and concentration as standards. Further, instead of the combination of the fatty acid and the condensing agent, an anhydride of the fatty acid may be used.

[0006] By mixing a labeled substance such as a dye or an enzyme with the aminoglycan acylated as described above, adding a condensing agent and reacting the mixture, and reacting the labeled substance with the remaining amino group which is not acylated, An aminoglycan modified with a label is obtained. Fluorescein, rhodamines, coumarin-based dyes and cyanine dyes (for example, NK1160 manufactured by Japan Photographic Dye Laboratories) and the like are used as the dyes for the label. As the enzyme, an oxidoreductase such as peroxidase or luciferase, or a hydrolase such as phosphatase, glucosidase or esterase is used. As a substrate for the enzyme, other than a substance that emits color or fluorescence by the enzyme reaction, a substance that emits light by the enzyme reaction,
For example, luminol is used for peroxidase, luciferin for luciferase, and APPDM for phosphatase.

[0007] The aminoglycan modified with a label,
An immunological substance (antigen or antibody) is added together with a condensing agent and reacted to obtain a labeling reagent. The immunizing agent is attached to a non-labeled or non-acylated amino group. the above,,
As the condensing agent used in the above, carbodiimides, N-
Bromosuccinimide or the like can be used. In addition, if the aminoglycan modified with a label is modified with a plurality of labels via biotin and avidin,
It is possible to increase the amount of label per molecule of the immune substance,
preferable. Such a labeling reagent is obtained by reacting biotin instead of reacting the label in the above-mentioned step to bind biotin to the remaining amino group, and mixing avidin modified with the label after the above-mentioned step. Thus, for example, a labeling reagent of the formula (I) is obtained.

[0008]

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Furthermore, biotin + aminoglycan + immune substance and avidin modified with a label may not be bound. In such a case, biotin + aminoglycan + immune substance (antigen or the like) is immunoreacted with an immunological substance (antibody) on an optical fiber, and further, avidin modified with a label is reacted. Thus, by reacting the labeled avidin later, it is possible to prevent the labeled body from being oxidized or hydrolyzed during the immune reaction.

[0010]

The free amino group in the aminoglycan forms a hydrogen bond with the hydroxyl group and is tightly bound between the molecules. Therefore, since the amino group is not easily protonated, it becomes sparingly soluble in water. However, when a part of the amino group is acylated, hydrogen bonding at the site is hindered, so that the amino group is easily protonated and consequently easily dissolved in water. On the other hand, if the acylation proceeds too much, the number of free amino groups decreases, and the compound becomes sparingly soluble in water again. Therefore, by using an aminoglycan in which 20 to 80%, preferably 25 to 30% of amino groups are acylated, a water-soluble labeling reagent can be prepared even after freezing or freeze-drying.

[0011]

The labeling reagent produced by using aminoglycan in which 20 to 80% of the amino groups are acylated does not cause precipitation when the frozen or lyophilized one is redissolved. By using such a labeling reagent, the sensitivity and reliability of the labeling reagent were improved.

[0012]

【Example】

Example 1 Measurement of Calcitonin: Sandwich Method (1) Chitosan having a degree of acetylation of 5% and a molecular weight of about 10 ⁶ was dissolved in 10% acetic acid to a concentration of 3 mg / ml. Next, 60 mg / water-soluble carbodiimide was added to this chitosan solution.
ml, and reacted at room temperature for 6 hours. This reaction mixture was dialyzed against distilled water to obtain a chitosan stock solution having an acetylation degree of about 30%. (2) 4 mg of sodium carbonate and 10 of biotin in 100 ml of water
mg was dissolved. To this biotin solution, 2 ml of the chitosan stock solution of the above (1) was mixed, and then water-soluble carbodiimide 10
0 mg was added and reacted overnight. Then, add 0.
Biotinylated chitosan was precipitated by adding 4 ml of a mixture of 2 g / ml sodium carbonate and 0.1 g / ml sodium chloride. After collecting the precipitate by centrifugation, the precipitate was washed twice with a mixed solution of 0.1 g / ml sodium carbonate and 0.3 g / ml sodium chloride. This precipitate was suspended in 2 ml of 10 mM phosphate buffer (pH 7) and dialyzed in the same buffer at 4 ° C. overnight to give a dialyzed product as a biotinylated chitosan suspension.

(3) 100 μg of rabbit-derived human calcitonin antibody is added to the biotinylated chitosan suspension of (2), and 10 mg of water-soluble carbodiimide is further added.
The reaction was performed at 4 ° C. for 6 hours. The reaction mixture was dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.0), and then unreacted substances were removed using an anion exchange column to obtain biotinylated chitosan to which a human calcitonin antibody was bound. (4) Cyanine dye NK116 was added to a solution of 1 mg of avidin and 0.2 ml of triethylamine in 1 ml of ethanol.
0 mg was added and dissolved sufficiently. Further, 0.3 ml of dicyclohexylcarbodiimide was added and reacted at room temperature overnight. After recovering the precipitate by centrifugation, the precipitate was washed twice with ethanol, and the precipitate was recovered by centrifugation, and the ethanol remaining in the precipitate was removed under reduced pressure.
This residue was dissolved in 2 ml of 20 mM acetate buffer (pH 6.5) to obtain NK1160-modified avidin.

(5) 10 mg of nickel sulfate was dissolved in 0.5 ml of water, and 2.5 ml of ethanol was further added. The precipitate formed at this time was removed by centrifugation, and the collected supernatant was washed with Ni
-Ethanol solution. Next, Ni-ethanol solution was added to 0.4 ml of 20 mM potassium hydroxide in ethanol solution.
1 ml and 50 μl of 50% glutaraldehyde were added to prepare a reaction solution. (6) An optical fiber made of polymethyl methacrylate resin (manufactured by Mitsubishi Rayon) was cut into 3 cm, and both end faces were polished with a polishing film. After immersing one side of this optical fiber in the reaction solution of the above (5) at 50 ° C. for 10 minutes,
Washed with mM hydrochloric acid followed by phosphate buffered saline (PBS). Next, this optical fiber was immersed in a 2 mg / ml goat-derived calcitonin antibody solution and left at 4 ° C. overnight. Removed fiber from the solution, was dipped for 15 minutes in 1% NaBH ₄ solution, washed with PBS, and creates a goat human calcitonin antibody immobilized sensor was a detector this.

(7) The labeled reagent was obtained by mixing the biotinylated chitosan to which the rabbit-derived human calcitonin antibody of (3) was bound and the avidin modified with NK1160 of (4). This was freeze-dried and stored at −20 ° C. for 2 weeks. (8) 10 μl of each concentration of calcitonin solution was added to the above (6).
Was immersed in a solution obtained by suspending the freeze-dried powder of the above (7) in distilled water for 30 minutes. 1M KSCN containing 0.2% Tween 20
After washing with an aqueous solution and measuring the fluorescence intensity with the apparatus shown in FIG. 1, it was possible to measure up to 0.3 ng / ml. This was the same sensitivity as when the labeling reagent of (7) was used immediately after preparation without lyophilization and storage.

Comparative Example 1 (1) A labeling reagent was prepared in the same manner as in Example 1 except that chitosan (acetylation degree: 5%) not subjected to acetylation treatment was used, and this was lyophilized. At -20 ° C
Saved for a week. (2) Even if the freeze-dried sample of (1) was suspended in distilled water, it did not dissolve, so that measurement was impossible.

Example 2 Measurement of Calcitonin: Sandwich Method (1) Polygalactosamine having a degree of acetylation of 1% and a molecular weight of about 10 ⁵ was acetylated in the same manner as in Example 1 (1). The degree of acetylation of the product was about 40%. (2) A biotinylated polygalactosamine to which a human calcitonin antibody was bound was obtained in the same manner as in (2) of Example 1, except that the polygalactosamine of (1) was used instead of chitosan. (3) A labeling reagent modified with NK1160 and a goat-derived human calcitonin antibody-immobilized sensor were prepared using the same method as in (3) to (7) of Example 1. (4) When human calcitonin was measured in the same manner as in (8) of Example 1, it could be measured up to 0.4 ng / ml.

Comparative Example 2 (1) A labeling reagent was prepared in the same manner as in Example 2 except that polygalactosamine chitosan (acetylation degree: 1%) not subjected to acetylation treatment was used, and this was lyophilized. And stored at −20 ° C. for 2 weeks. (2) Even if the freeze-dried sample of (1) was suspended in distilled water, it did not dissolve, so that measurement was impossible.

Example 3 Measurement of Human Chorionic Gonadotropin (hCG): Competition Method (1) Chitosan having a degree of acetylation of 5% and a molecular weight of about 10 ⁶ was dissolved in 5% propionic acid to a concentration of 3 mg / ml. And
Next, water-soluble carbodiimide (CHMC) was added to the solution at a concentration of 50 mg / ml and reacted at room temperature for 6 hours. The degree of acylation of the product was about 25%. (2) A biotinylated chitosan suspension was obtained in the same manner as in Example 1, (2). (3) Instead of rabbit calcitonin antibody 100 μg, human chorionic gonadotropin (hCG) 100
Except for adding μg, a biotinylated chitosan to which hCG was bound was obtained in the same manner as in Example 1, (3). (4) 1.8 mg of fluorescein isothiocyanate (fluorescein) was dissolved in a solution of 1 mg of avidin and 0.2 ml of triethylamine in 1 ml of ethanol, and reacted at room temperature for 6 hours under light shielding. After recovering the precipitate by centrifugation, the precipitate was washed twice with ethanol, and the precipitate was recovered by centrifugation, and the ethanol in the precipitate was removed under reduced pressure. This residue was treated with 50 mM acetate buffer (pH 5.
5) The suspension was suspended in 1 ml to obtain avidin modified with fluorescein.

(5) hCG instead of calcitonin antibody
An hCG antibody-immobilized sensor was prepared in the same manner as in (5) and (6) of Example 1 except that an antibody was used, and this was used as a detection unit. (6) The biotinylated chitosan bound to hCG of (3) and avidin modified with fluorescein of (4) were mixed to obtain a labeling reagent. This was stored frozen at -20 ° C for one week. (7) The cryopreserved product of (6) was thawed and added in a fixed amount to 10 μl of the hCG solution at each concentration, and the detection section of (5) was immersed and left at room temperature for 30 minutes. The detection part was washed with PBS containing 0.2% Tween 20, and then washed with PBS.
% Sodium carbonate solution, and irradiated with a semiconductor laser of 780 nm and 1360 nm using the apparatus shown in FIG.
When the fluorescence at 8 nm was measured, it could be measured up to 0.4 ng / ml.

Comparative Example 3 (1) A labeling reagent was prepared in the same manner as in Example 3 except that chitosan (acetylation degree: 5%) not subjected to an acylation treatment was used. Stored frozen for one week. (2) When the cryopreserved sample of (1) was left at room temperature for 20 minutes and thawed, insoluble precipitates were partially formed, and the measured values fluctuated, and only up to 50 ng / ml could be detected.

Example 4 Measurement of Human Chorionic Gonadotropin (hCG): Competition Method (1) In the same manner as in (3) to (4) of Example 3, hC
G-linked biotinylated chitosan and avidin modified with fluorescein were obtained. (2) The biotinylated chitosan bound to hCG and the avidin modified with fluorescein of (1) above were separately lyophilized and cryopreserved at -20 ° C for one month. (3) An hCG antibody-immobilized sensor was prepared in the same manner as in (3) of Example 3 and used as a detection unit. (4) Add 10 μl of the hCG solution of each concentration to the hCG of (2).
A fixed amount of a solution obtained by dissolving a freeze-dried preparation of biotinylated chitosan to which CG is bound in distilled water is added, and then the above (3)
Was immersed and left at room temperature for 30 minutes. Next, the detection portion was washed with PBS containing 0.2% Tween 20, and immersed in a solution of the freeze-dried sample of avidin modified with fluorescein (2) in distilled water, and allowed to stand at room temperature for 10 minutes. I left it. The detection part was washed with PBS containing 0.2% Tween 20, then immersed in 1% sodium carbonate solution, and 780 nm and 136 nm were measured using the apparatus shown in FIG.
Irradiation with a semiconductor laser of 0 nm and measurement of fluorescence at 518 nm showed that the measurement was possible up to 0.4 ng / ml.

Comparative Example 4 (1) Chitosan having a degree of acetylation of 5% and a molecular weight of about 10 ⁶ was dissolved in 30% acetic acid so as to have a concentration of 3 mg / ml. Then, 20 mg / ml of a water-soluble carbodiimide was added to the chitosan solution.
, And 3 times 20 mg / ml every 2 hours to a final concentration of 80 mg / ml.
Allowed to react for hours. The reaction mixture was dialyzed against distilled water to obtain a chitosan stock solution having a degree of acetylation of about 85%. (2) In the same manner as in (2) to (7) of Example 1, NK
A labeling reagent modified with 1160 and a goat-derived human calcitonin antibody-immobilized sensor were prepared. (3) Add 10 μl of each concentration of calcitonin solution to (2)
Was immersed in a solution in which the freeze-dried powder of the above (2) was suspended in distilled water for 30 minutes. At this time, about 10 minutes after the start of immersion, a slight precipitation occurred. 0.2% Tween 20 detector
After washing with a 1M aqueous KSCN solution and measuring the fluorescence intensity with the apparatus shown in FIG. 1, only up to 10 mg / ml could be measured.

[Brief description of the drawings]

FIG. 1 Fluorescence measurement system using one laser

FIG. 2 Fluorescence measurement system using two lasers

[Description of References] 1 Optical fiber 2 Laser 3 Guide rail for optical axis alignment 4 Detector 5 Filter 6 Fluorescence detector 7 Half mirror

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-63-258579 (JP, A) WO 90/13029 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/48-33/98

Claims (3)

(57) [Claims] 1. An aminoglycan which binds to an immune substance and is modified with a label is an aminoglycan in which 20 to 80% of amino groups are acylated with a saturated fatty acid, and is stored frozen or freeze-dried. A labeling reagent for immunoassay, comprising: 2. The labeling reagent for immunoassay according to claim 1, wherein the aminoglycan modified with the label is modified with a plurality of labels via biotin and avidin. 3. An immunoassay labeling reagent comprising a combination of an aminoglycan modified with biotin, which binds to an immune substance, and avidin modified with a label, wherein the labeling reagent is 20 to 80% of the amino group of the aminoglycan. Is acylated with a saturated fatty acid, and is stored frozen or freeze-dried.