JPH02193070A - Measuring kit of morphine and measuring method thereof - Google Patents

Abstract

PURPOSE:To enable rapid, highly sensitive and easy measurement of morphine with high peculiarity by a measuring kit using a normorphine bearer and an antibody bearer. CONSTITUTION:A normorphine bearer is prepared by making a carrier bear a combination obtained by combining chemically normorphine with macromolecular protein having a molecular weight of ten thousands or above. As for a bearer (antibody bearer) bearing an antibody having a very high pecu liar immune reactivity to morphine and the normorphine, it is prepared by dissolving this antibody in a buffer solution adjusted to be neutral approximate ly, so as to prepare a solution of appropriate concentration, and by making a carrier bear this solution. Subsequently, a material for measurement and a standard morphine solution are prepared and added to the normorphine bearer and the antibody bearer respectively for reactions. By comparing and observing states of coehesion caused by the reactions, by quantity of the morphine in a sample of measurement can be measured rapidly with high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a kit and method for measuring morphine.

[Conventional technology]

In recent years, a method of alleviating pain by administering morphine has been adopted as one of the treatments for terminal cancer patients. In such a method, if the dose of morphine is too small, the analgesic effect will be poor, and if the dose is too high, the side effects will be too great.

Therefore, in order to determine the dosage to maintain the drug at an appropriate concentration, it is necessary to use a method that can easily and quickly monitor its blood concentration.

Conventionally, such methods include polyclonal antibodies [
Clinical Chemistry (C1ini(al Ch)
emistry) 20.243-248.1974]
or monoclonal antibodies (108th Annual Meeting of the Pharmaceutical Society of Japan;
A measurement method using enzyme immunoassay (Sawada et al., April 1988) is known.

However, when measuring a large number of specimens, these analysis methods require a considerable amount of time and the measurement operations are complicated.

Therefore, in monitoring blood concentration, the measurement method needs to be further improved so that it can be measured simply and quickly.

[Problems to be solved by the invention]

The object of the present invention is to provide a morphine drug that requires a carrier carrying an antibody having very high specific immunoreactivity to the analgesics morphine and normorphine, and a carrier carrying normorphine to the carrier. An object of the present invention is to provide a measurement kit for morphine and a method for measuring morphine using the measurement kit.

[Means for solving problems]

As a result of intensive research to solve the above problems, the present inventors have discovered that morphine can be measured more easily and quickly than conventional measurement methods by measuring morphine using a morphine measurement kit. This discovery led to the completion of the present invention.

That is, the first invention is used in the measurement of morphine, and comprises (a) a carrier carrying an antibody having very high specific immunoreactivity to morphine and normorphine; and (b) The present invention relates to a kit for measuring morphine, which is characterized in that it essentially includes a carrier in which normorphine is supported on the carrier.

A second invention is a morphine measurement kit characterized in that, using the morphine measurement kit, morphine in a measurement sample and normorphine supported on a carrier are competitively reacted with an antibody supported on a carrier. It concerns the measurement method.

The present invention will be explained in detail below.

The morphine measurement kit used in the morphine measurement method of the present invention is a carrier carrying an antibody having very high specific immunoreactivity for morphine and normorphine (hereinafter abbreviated as r-antibody carrier). and normorphine (
A carrier (hereinafter abbreviated as r-normorphine carrier j) supporting a morphine (similar compound of morphine) is essential.

In order to measure morphine, in addition to these reagents, you will also need a reaction plate as a place for these reactions, a known amount of morphine solution (hereinafter abbreviated as r standard morphine solution 1), and a stirring bar. Therefore, these may be incorporated into a morphine measurement kit in advance, or may be prepared before morphine measurement.

In the present invention, "antibody carrier 1" in a morphine measurement kit is used.
Antibodies to morphine and normorphine that can be used for the production of morphine are not particularly limited as long as they have very high specific immunoreactivity to morphine and normorphine, and antiserum or monoclonal antibodies can be used. However, it is preferable to use a monoclonal antibody.As such a monoclonal antibody having very high specificity for morphine and normorphine, normorphine as shown in Japanese Patent Application No. 63-234826 is used. Mouse hybridoma strains obtained by immunization [e.g., MO-2 strain (Feikoken Joyori No. 1910), MO-3 strain (Feikoken Joyori No. 1911), MO-4 strain, MO-5 strain (Feikoken Jokyo No. 1912), MO-6 strain, etc.] can be mentioned.

When the antibody carrier 1 used in the measurement of morphine in the present invention is a powder, it is dispersed in an appropriate solvent such as water or a buffer solution (e.g., phosphate buffer, Tris-HCl buffer, etc.). It is used as a suspension solution at an appropriate concentration.

r antibody carrier 1 in the morphine measurement kit of the present invention
In the preparation of r-normorphine carrier 1, materials for the carrier supporting antibody or normorphine include polyethylene, polystyrene, polypropylene, nitrocellulose, glass, nylon, PMMA (polymethyl methacrylate), styrene-divinylbenzene copolymer, Examples include styrene-butadiene copolymer, but polystyrene is preferred.

The shape of these carriers can be spherical, rod-like, needle-like, cubic, etc., but spherical is preferable.

The size of these carriers is 0.01 to 5 μm, especially 0.01 to 5 μm.
Although it can be used in a thickness of 1 to 3 μm, it is more preferably 0.2 to 2 μm. Further, it is preferable that the size and shape of the carriers used are uniform. These carriers may be appropriately colored depending on the color of the reaction plate used during measurement.

The r antibody carrier 1 in the present invention can be produced as follows.

An antibody that is highly specific for morphine is dissolved in a buffer adjusted to near neutrality (e.g., phosphate buffer, Tris/HCl buffer, etc.) to prepare a solution with an appropriate antibody concentration. The antibody can be supported on the carrier by contacting and reacting this solution with the above-mentioned carrier for a certain period of time by a method such as standing still or shaking. The heating temperature during this contact is preferably 10 to 50°C, preferably 20 to 40°C.
In addition, it is preferable that the heating time at this time be longer as the temperature in the heating temperature range is lower (
For example, at 30-50°C, 2-4 hours is sufficient;
At 0-29°C, 4-10 days are required. When heating for a long period of time, it is preferable to add a fluorescent preservative such as sodium azide, sodium ethylmercury thiosalicylate, etc.).

The r-antibody carrier 1 thus obtained can be stored at low temperature (for example, 2 to 10°C) in the state of suspension, but
Preferably, protein preservatives such as sodium azide, sodium ethylmercury thiosalicylate, etc., and protein stabilizing substances such as bovine serum albumin (BSA) may also be added in required amounts.

The r-normorphine carrier 1 in the present invention can be produced by supporting a bond obtained by chemically bonding normorphine and a polymeric fluorescent substance having a molecular weight of 10,000 or more on a carrier as described below. can.

Examples of high molecular weight proteins with a molecular weight of 10,000 or more used in the production of r-normorphine carrier j in the present invention include BSA (bovine serum albumin), 0VA (ovalbumin), and KLH.
(jinkasagai hemocyanin), biopolymers such as T-globulin, and polyamino acids such as poly-L-lysine (PLL).

In the preparation of r-normorphine carrier 1, the bonding between normorphine and the polymer protein was performed by converting normorphine into N-(4-bromobutyl) using N-(4-bromobutyl)phthalimide, and converting the N-(4-bromobutyl)-formed normorphine into 1-ethyl- 3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (hereinafter abbreviated as EDPC), 1-cyclohexyl-3-(2-
This can be carried out by binding to the above-mentioned high molecular weight protein using a carbodiimide such as morpholinoethyl)carbodiimidemeth-p-toluenesulfonate (hereinafter abbreviated as CMEC).

r-Normorphine carrier 1 is prepared by combining the thus obtained conjugate of normorphine with a high-molecular protein, as it is, or by purifying it by gel filtration using 5ephadex, 5ephacryl, etc., and then leaving it with a carrier or by a method such as shaking. This antibody can be supported on the carrier by contact reaction for a certain period of time. The heating temperature during this contact is preferably 10 to 50"C1, preferably 20 to 40C.
In addition, it is preferable that the heating time at this time be longer as the temperature in the heating temperature range is lower (
For example, at 30-50°C, 2-4 hours is sufficient;
At 10-29°C, 4-10 days are required. When heating for a long period of time, it is preferable to add a protein preservative such as sodium azide, sodium ethylmercury thiosalicylate, etc.).

The r-normorphine carrier 1 thus obtained can be stored as a suspended solution at a low temperature (for example, 2 to 10'C), but preferably sodium azide, sodium ethylmercury thiosalicylate, etc. Protein preservatives such as BSA, protein stabilizers such as BSA, etc. can also be added in required amounts.

As the measurement sample used in the measurement of morphine of the present invention, human body fluids such as human urine, blood, and serum can be used. These measurement samples can be used for measurement with or without dilution.

Examples of the reaction plate used as a reaction site in the measurement of morphine of the present invention include a glass plate, a plastic plate, and a plate coated with a waterproof material (wooden, paper, etc.). It is preferable to use a plate made of paper, and the colors of the reaction plates are r-normorphine carrier 1 and r-antibody carrier 1.
Although there is no particular limitation as long as the aggregated substance formed as a result of the reaction with the substance can be clearly confirmed, for example, if the aggregated substance is white, it is preferably black.

The stirring rod for the reaction solution used in the measurement of morphine in the present invention is not particularly limited as long as it is waterproof, but it is preferably a plastic or plastic-coated rod. The size of the rod is not particularly limited as long as it can stir to achieve the purpose of the present invention, but for example, one with a length of 3 to 10 cm and a diameter of 0.1 to 5 mm can be used.

Prepare the reaction plate, stirring rod for the reaction solution, measurement sample, and r standard morphine solution J as described above, and use the reagents of the morphine measurement kit, ``Antibody carrier J, r Normorphine carrier J,'' as follows. Morphine in the measurement sample can be measured by going through the steps (i) to (ii) (the measurement is the same as for morphine except that the normorphine measurement sample and a known amount of normorphine solution are used). By performing this operation, normorphine can be measured.)

rAntibody carrier J, rnormorphine carrier J, and measurement sample or rstandard morphine solution J are each kept constant! (e.g., 5 to 50 μ2), placed on the reaction plate as one set at a position interval that makes it easy to stir each other.
At this time, [r antibody carrier 1, r normorphine carrier 1
and measurement sample] and [r-antibody carrier],
r normorphine carrier 1 and r standard morphine solution 1]
Preferably, the set consisting of the following is placed on the same reaction plate at appropriate intervals.

Stir each solution quickly with a stirring bar, and react at a constant temperature (for example, room temperature) for a certain period of time (for example, 1 to 2 minutes) while moving the reaction plate back and forth and left and right. Observe the aggregation state of each set caused by the reaction with carrier 1. At this time, compare the aggregation state of the set using r standard morphine solution J and the aggregation state of the set using the measurement sample. The amount of morphine in the sample to be measured is determined by observation.

In the step of stirring and reacting each reagent on the reaction plate for each set of (it) in this morphine measurement operation procedure, the r-normorphine-carrying A competitive reaction occurs between normorphine in body 1 and morphine in the measurement sample or r standard morphine solution J, and an agglutination reaction occurs between antibody carrier 1 and r normorphine carrier 1, so that r standard morphine solution 1 By comparing and observing the aggregation state in the case of a set using a test sample and the aggregation state in a set using a measurement sample, the amount of morphine in the sample can be measured quickly and with high sensitivity.

〔Example〕

Hereinafter, the present invention will be specifically explained using reference examples, working examples, and comparative examples.

Note that these Examples are for illustrating the present invention, and do not limit the scope of the present invention.

Reference Example 1 [Production and purification of antibodies] Production of monoclonal antibodies with high specificity for morphine and normorphine produced by the hybridoma strain MO-3 (Feikoken Jokyo No. 1911) is as follows. I went there.

B A 10' cell line suspended in phosphate buffer
The test was administered intraperitoneally to LB/c mice (male, 8 weeks old, 0.5 m of Bristane was administered intraperitoneally 2 weeks earlier). A remarkable increase in mouse body weight was observed from around the first week, and ascites was removed as appropriate from the first to third weeks. The antibody titer of the monoclonal antibody thus obtained was 10&~101.

Purification of monoclonal antibodies from the obtained ascites was performed as follows.

The ascites was dialyzed against Tris-HCl buffer (pH 7.4) and applied to a DEAE-cellulose column equilibrated with the same buffer. The pass-through fraction was salted out with 50% saturated ammonium sulfate, and the resulting precipitate was dissolved in phosphate buffer (pH 7.4) and dialyzed against the same buffer.

The purity of the thus obtained monoclonal antibody having high specificity and reactivity with respect to morphine and normorphine was determined by slab gel electrophoresis using SDS polyacrylamide gel.

Example 1 [Preparation of r-normorphine carrier J] 271 mg of normorphine was dissolved in 20 mf of dioxane, and 340 mg of N- was dissolved in 5 ml of ethanol.
(4-bromobutyl)phthalimide dissolved and 6
00 mg of sodium carbonate was added thereto, and the mixture was subsequently heated under reflux for 20 hours. After filtering off inorganic substances from the reaction solution, the filtrate was dried under reduced pressure.

The obtained residue was dissolved in a very small amount of ethyl acetate and applied to a silica gel column (20 mm Φ x 300 mm L; Kiese
lgel 60 (7(1~200 mesh
ASTM)). This was mixed with 60 mf of ethyl acetate/
After elution with methanol (9:1), elution was continued with ethyl acetate/methanol (5:1), and the fraction was fractionated into 20 ml portions. TLC (Kieselgel 60; developing solvent:
Ethyl acetate/methanol/concentrated ammonia water - 85:10
: Analyze in step 5) and collect the fractions containing only the target substance.1. The solvent was removed under reduced pressure. The obtained residue was dissolved in 20 m, l of ethanol and then 100 μ! After adding 90% hydrazine hydrate and refluxing for 2 hours, ethanol was distilled off. The resulting residue is 15mj! After dissolving in 1% hydrochloric acid, the solution was washed twice with chloroform/isopropanol (5:1).

After adjusting the pH to 8 with concentrated ammonia water, chloroform/
After extraction with isopropanol (5:1), dehydration with anhydrous sodium sulfate and filtration, the filtrate was distilled off under reduced pressure to obtain 13
0 mg of N-(4-aminobutyl)normorphine (hereinafter abbreviated as ABNM) was obtained.

A combination of normorphine and BSA, a high-molecular protein (
It is abbreviated as normorphine-BSA. ) is the above ABN
It was prepared as follows using M.

After mixing 15 mg of ABNM dissolved in 0.3 mL of dimethylformamide and 10 mg of BSA, which is a polymer carrier, dissolved in 1.5 mL of pure water, 10% of 1- Add 0.5 ml of ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDPC) aqueous solution, adjust the pH to 5.5, and stir at room temperature to 1.
After stirring for 6 hours, it was dialyzed twice against pure water, the non-dialyzed fraction was lyophilized, and normorphine was used as an immunogen.
10 mg of BSA was obtained.

rNormorphine in the preparation of normorphine carrier j
The method for supporting normorphine on a carrier via BSA was carried out as follows.

100 μl of normorphine-BSA solution (400 μg/
mf) and 100μ! of polystyrene latex (solid component: 480% by weight, particle size: 0.8 μm, solvent: 0.1M phosphate buffer at pH 7.4),
The mixture was shaken at 37°C for 3 hours to physically support MA-BSA on the surface of the polystyrene latex, thereby obtaining an r-normorphine carrier J suspension.

Example 2 [Preparation of r-normorphine carrier] Half of the r-normorphine carrier 1 suspension of Example 1 was further heated at 37'C for 5 hours to obtain r-normorphine carrier J suspension. Ta.

Example 3 [Preparation of r-antibody carrier] r-antibody carrier 1 was prepared using 100 uit of a monoclonal antibody solution (1 mg/m!, solvent was p
0.1M phosphate buffer (H7,4) and 100μ2 of polystyrene latex (solid component: 4.0% by weight, particle size: 0.8μm, solvent: 0.1M (pH 1,4))
M phosphate buffer) and shaken at 37°C for 3 hours to obtain a monoclonal antibody that was physically supported on the polystyrene latex surface.

Example 4 [Measurement of morphine diluted with urine of healthy subjects]
Antibody carrier 1 is placed at two adjacent points on the reaction plate at a distance of 25 μ2 so that these reagents do not come into contact with each other.
The urine of a healthy person (HYLAND DI
A standard morphine solution 1 with a concentration of o, o, i, 0.2 or 0.3 ppm of morphine prepared by diluting it with AGNO3TIC was added dropwise in 5 μl portions and immediately stirred with a stirring bar. r-normorphine carrier 1 on the same reaction plate, ? The antibody carrier j and the measurement sample were mixed by stirring. If you keep moving the same plate slowly, it will take 16 seconds when the morphine concentration of standard morphine solution 1 is Oppm, 22 seconds when it is 0.1 ppm, and 0.2
It was observed with the naked eye that the polystyrene latex particles agglomerated in 35 seconds at ppm and over 180 seconds at 0.3 ppm.

Example 5 [Measurement of morphine diluted with urine of a healthy person] r Normorphine carrier 1 prepared in Example 2 and r prepared in Example 3
Antibody carrier 1 is placed at two adjacent points on the reaction plate at a distance of 25μ so that these reagents do not come into contact with each other.
The urine of a healthy person (HYLAND DI
The concentration of morphine prepared by diluting it with AGNO3TIC) is 0.0.1.0.2 or 0.3 p p m
The r-standard morphine solution 1 was added dropwise at 5 μi intervals, and immediately the r-normorphine support 1, the r-antibody support J, and the measurement sample on the same reaction plate were stirred and mixed using a stirring rod. If you keep moving the same plate slowly, when the morphine concentration of r standard morphine solution 1 is Oppm, 13
seconds, 0.1 p p m, 19 seconds, 0.2
0.3 p p m in 30 seconds when p p m
At this time, it was observed with the naked eye that the polystyrene latex particles aggregated in 150 seconds or more.

Comparative Example 1 [Measurement of Cocaine] All measurements were carried out in the same manner as in Example 4, except that a standard solution prepared with cocaine was used instead of morphine.

As a result, it was observed with the naked eye that polystyrene latex particles aggregated in 16 seconds when the cocaine concentration was Oppm, and in 16 seconds when the cocaine concentration was 0.3 ppm.

Comparative Example 2 [Measurement of Ethylmorphine] All measurements were carried out in the same manner as in Example 4, except that a standard solution prepared with ethylmorphine was used instead of morphine.

As a result, when the ethylmorphine concentration is Oppm, 1
It was observed with the naked eye that polystyrene latex particles agglomerated in 16 seconds even at 0.3 ppm in 6 seconds.

Comparative Example 3 [Measurement of Cocaine] All measurements were carried out in the same manner as in Example 5, except that a standard solution prepared with cocaine was used instead of morphine.

As a result, it was observed with the naked eye that polystyrene latex particles agglomerated in 13 seconds when the cocaine concentration was Oppm, and in 13 seconds when the cocaine concentration was 0.3 ppm.

Comparative Example 4 [Measurement of Ethylmorphine] All measurements were carried out in the same manner as in Example 5, except that a standard solution prepared with ethylmorphine was used instead of morphine.

As a result, when the ethylmorphine concentration is Oppm, 1
It was observed with the naked eye that polystyrene latex particles agglomerated in 13 seconds even at 0.3 ppm.

〔Effect of the invention〕

According to the present invention, morphine can be measured with high specificity (, rapid, high sensitivity, and easy .

Patent applicant: Ube Industries Co., Ltd.

Claims (2)

[Claims] (1) Used in the measurement of morphine, including (a) a carrier carrying an antibody with very high specific immunoreactivity to morphine and normorphine, and (b) a carrier carrying normorphine. A kit for measuring morphine, characterized in that it requires a carrier. (2) Using the morphine measurement kit according to claim 1, morphine in the measurement sample and normorphine supported on the carrier are competitively reacted with the antibody supported on the carrier. Method for measuring morphine.