JPH067190A - Anti-idiotype antibody of anti-morphine antibody, method for producing the same and assay method for morphine - Google Patents

Abstract

(57) [Summary] (Modified) [Object] To provide an anti-idiotype antibody of an anti-morphine antibody, a method for producing the same, and an immunological assay method for morphine using the anti-morphine antibody and the anti-idiotype antibody. [Structure] An animal is immunized with an anti-morphine antibody, and a lymphocyte obtained from the animal is fused with a myeloma cell to prepare a hybridoma strain, which is cultured in a general lymphocyte culture medium. Thus, an antibody having reactivity with the variable region of the antibody having immunological reactivity with morphine is obtained. Competitively reacting enzyme-labeled anti-morphine antibody or morphine with anti-morphine antibody anti-morphine antibody, or competitively reacting anti-morphine antibody with enzyme-labeled anti-morphine antibody anti-idiotype antibody or morphine By doing so, morphine can be measured easily and quickly.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a variable site of an antibody having immunological reactivity with morphine which is one of analgesics (hereinafter referred to as "anti-morphine antibody"). The present invention relates to a method for producing an antibody having the following (hereinafter referred to as "anti-idiotype antibody") and a method for measuring morphine using these antibodies.

[0002]

2. Description of the Related Art In recent years, a method for reducing pain by administering morphine has been adopted as one of the additional therapies for end-stage cancer patients. In such a method, when the dose of morphine is small, there is no analgesic effect, and when the amount is too large, the side effects become too large. Therefore, when deciding a dose for maintaining the appropriate concentration at all times, there is a need for a method capable of easily and rapidly monitoring the blood concentration.

As such an attempt, conventionally, a method using chromatography [Journal of Chromato] has been used.
graphy), 230, 427-432, 198.
2 years], an immunochemical assay method using a polyclonal antibody having immunological reactivity to morphine [European Journal of Pharmacology (Europe)
ean Journal of Pharmacolo
gy), 38, 149-156, 1976],
Immunochemical assay method using a monoclonal antibody having immunological reactivity to morphine [Molecular Immunology]
y), 25, 937-943, 1988] and the like.

However, there are the following problems with. However, the operation of separating morphine from serum is complicated, and since the separated morphine needs to be measured by liquid chromatography, a long time is required. In and, the antibody has low specificity (reactivity with codeine etc. is observed), and
There were problems such as the fact that the conditions for handling radioisotopes had to be in place.

[0005]

The object of the present invention is to provide an "anti-idiotype antibody", a method for producing the same, and an immunological assay for morphine using "anti-morphine antibody" and "anti-idiotype antibody". It is to be.

[0006]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have performed cell fusion of lymphocytes and myeloma cells obtained by immunizing with “anti-morphine antibody”. By culturing the hybridoma strain obtained as described above, an "anti-idiotype antibody" can be obtained,
The inventors have found that morphine can be measured by a competitive immunological assay using "anti-morphine antibody" and "anti-idiotype antibody", and have completed the present invention. That is, the present invention is as follows.

The first invention relates to "anti-idiotypic antibody" of "anti-morphine antibody". The second invention is the above-mentioned "anti-idiotypic antibody", which comprises culturing a hybridoma strain obtained by cell fusion of lymphocytes obtained by immunizing with "anti-morphine antibody" and myeloma cells. ] About the manufacturing method.
The third invention relates to the hybridoma strain described above. A fourth invention is a method for assaying morphine, which comprises reacting "anti-morphine antibody" immobilized on a solid support with morphine or "anti-idiotype antibody" labeled with a labeling substance in a competitive manner. It is about.
A fifth invention is a method for assaying morphine, characterized in that "anti-idiotype antibody" immobilized on a solid support is reacted competitively with morphine or "anti-morphine antibody" labeled with a labeling substance. It is about.

The present invention will be described in detail below. The "anti-idiotype antibody" of the present invention can form a complex of "anti-idiotype antibody" and "anti-morphine antibody" by an immunological reaction with the variable region of "anti-morphine antibody" (the former An antibody in an immunological reaction, and if the latter is replaced by the term antigen, both can form a complex by a so-called antigen-antibody reaction),
Moreover, it is an antibody having the property of blocking the immunological reaction between "anti-morphine antibody" and morphine. That is, the "anti-idiotype antibody" of the present invention forms a complex by an immunological reaction with the variable site of "anti-morphine antibody" (site having specific immunological reactivity with morphine), It has the property of being able to prevent the formation of a complex between "anti-morphine antibody" and morphine.

The "anti-idiotype antibody" having such characteristics is obtained by immunizing an animal with "anti-morphine antibody", and is obtained by cell fusion of lymphocytes and myeloma cells of the animal. Other examples include monoclonal antibodies produced by hybridoma strains; preferably monoclonal antibodies.

Examples of animals used for immunization of the present invention include experimental small animals (mouse, rat, guinea pig, rabbit, dog, etc.), livestock (goat, cow, horse, etc.); for producing monoclonal antibodies. For this, it is preferable to use an experimental small animal, and more preferable to use a mouse.

The "anti-morphine antibody", which is an antigen used as an immunogen for animals in the present invention, is not particularly limited as long as an "anti-idiotype antibody" that can be used in a competitive immunological assay can be prepared. , Serum having specific immunological reactivity with morphine obtained by immunizing the animal with morphine, monoclonal antibody produced by a hybridoma strain obtained by cell fusion of lymphocytes and myeloma cells of the animal, etc. Although it can be mentioned; preferably, a monoclonal antibody is preferable; more preferably, a monoclonal antibody that does not substantially cross-react with codeine (an antitussive agent) is preferable.

Monoclonal antibodies that do not substantially cross-react with codeine are, for example, lymphocytes obtained from immunized mice in which normorphine is bound to a biological macromolecule, immunoglobulin, and mice obtained from immunized mice. Which is a hybridoma strain obtained by fusing with myeloma cells of
-2 strains (Microtechnical Research Article No. 1910), MO-3 strains (Microtechnical Research Article 1911), MO-4 strains, MO-5 shares (Microtechnical Research Article 1912), MO -6 strain and the like can be obtained by statically culturing in a flask using a general lymphocyte culture medium or by culturing in mouse abdominal cavity, but codeine and M-6-G (morphine-6-glucuronide) can be obtained. In the case of requiring a monoclonal antibody which is not substantially reactive with MO), MO-3 strain, MO-4
By culturing the strain, MO-5 strain or MO-6 strain, a monoclonal antibody having higher specificity can be obtained.

The immunogen is not particularly limited as long as it has the variable region of such a monoclonal antibody, and for example, the monoclonal antibody may be used as it is, and Fab, Fab 'of the monoclonal antibody may be used.
Alternatively, F (ab ′) ₂ may be used, and / or those obtained by binding them to a polymer carrier having a molecular weight of 10,000 or more can be mentioned; preferably, the above-mentioned monoclonal antibody has a molecular weight of 10,000. What is bound to the above polymer carrier is preferable.

Examples of such a polymeric carrier having a molecular weight of 10,000 or more include biopolymers such as bovine serum albumin (BSA), ovalbumin (OVA), Jinkasai hemocyanin (KLH) and immunoglobulin, and poly L-. Lysine (P
Mention may be made of polyamino acids such as LL).

The preferred "anti-idiotype antibody" of the present invention is, for example, an immunogen in which an "anti-morphine antibody" having specific reactivity with morphine is bound to KLH which is a biopolymer. IMO-3-X1 strain, which is a hybridoma strain obtained by fusing lymphocytes obtained from immunized mice and mouse myeloma cells (Microtechnology Research Institute
1933), iMO-3-P1 strain, iMO-3-P2.
It can be obtained by statically culturing the strain or the like in a flask using a general lymphocyte culture medium, or by culturing the mouse intraperitoneally. An outline of a preferable method for producing such a hybridoma strain and a method for producing an antibody will be described.

[A] Preparation of Monoclonal Antibody-Producing Hybridoma Strain (1) Preparation of Immunogen and Labeled Antibody Immunogens used in mice include, for example, "anti-morphine antibody".
As a preferable example, a polymer carrier having a molecular weight of 10,000 or more (eg, biopolymer such as BSA, OVA, KLH, immunoglobulin, etc., and polyamino acid such as PLL, etc. can be mentioned using glutaraldehyde. Can be produced).

The labeling substance in the antibody labeled with the labeling substance is a radioisotope, a fluorescent substance, an enzyme [for example, oxidoreductase (peroxidase, catalase, glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, β-galactosidase, etc.). , Phosphate hydrolase (alkaline phosphatase, etc.)], but it is preferable to use an enzyme that is easy to handle. As the enzyme-labeled antibody, for example, "anti-morphine antibody" is prepared by the periodate oxidation method [Nakane, P. et al. K. and Kawai,
A. : J. Histochem. Cytochem. Two
2, pp. 1084 (1974)] by coupling with horseradish peroxidase (HRP).

(2) Preparation of immunized mouse lymphocytes The method for immunizing mice is PBS (phosphate buffered saline).
The immunogen (10 to 400 μg) dissolved in (1) above can be administered to mice once or several times at intervals of several weeks. The first immunization can be performed without administration of an adjuvant (immunity-promoting substance including alum, killed tuberculosis cells, nucleic acid, etc.), but it is preferable to administer an emulsion prepared using an adjuvant. Although lymphocytes can be obtained from blood, lymph nodes, spleen, etc. several days after the final immunization after confirming a sufficient antibody titer in the immunized mouse, lymphocytes are preferably obtained.

(3) Preparation of myeloma cells For cell fusion, MPC-11, P3-X6 derived from mouse are used.
3-Ag8 ・ 653 (653), P3-X63-Ag8
-U1 (P3U1), P3-NS-1 (NS-1), S
P2 / 0-Ag14 (SP2 / 0), etc., and rat-derived 210. RC Y3. Myeloma cells such as Ag1.2.3 (Y3) can be used;
It is preferable to use myeloma cells that do not secrete antibodies extracellularly such as 3U1, NS-1, SP2 / 0.

(4) Cell fusion In the cell fusion, the number of cells between the lymphocytes of the mouse immunized as described above and the myeloma cells is (5-20):
At a ratio of 1, a cell suspension solution that does not hinder cell fusion, for example, a commonly used lymphocyte culture medium component (medium component such as MEM, DMEM, McCoy, RPMI1640) solution, an isotonic buffer solution, etc. HVJ was added to the pellet (cell mass) after thorough mixing and centrifugation.
(Sendai virus) or PEG (polyethylene glycol) solution can be added, but it is preferable to use a PEG solution, more preferably 30 to 60 with an average molecular weight of 1000 to 8000.
A weight percent PEG solution is preferably used. At this time, in order to promote cell fusion, colchicine, dimethyl sulfoxide, poly-L-arginine and the like can be added in appropriate amounts. As the myeloma cells used for cell fusion, those derived from an animal different from the immunized mouse can be used, but considering the antibody production amount and stability of the resulting monoclonal antibody-producing hybridoma strain, It is preferable to use the same type of myeloma cell as the mouse, and it is more preferable to use the same type of mouse.

(5) Selection of hybridomas Hybridomas are selected by selecting H
AT medium [medium containing hypoxanthine, aminopterin, thymidine, and fetal calf serum (FCS). As the medium component, a commonly used medium component for lymphocyte culture can be used].
Hybridomas are cultivated by putting the number of cells suitable for searching antibody-producing wells into each well (culture well) of the culture plate. At this time, the hybridoma growth-promoting substance or cells producing it (eg, thymus, spleen) , Lymphocytes derived from lymph nodes) can be used as feeder cells as needed. Hybridomas selected by growing in HAT medium are HT medium (medium containing hypoxanthine, thymidine, FCS, and lymph nodes commonly used as components of this medium until the number of cells suitable for searching antibody-producing wells is reached). The medium components for sphere culture can be used) for several days, and further,
It is cultured in a commonly used FCS-containing lymphocyte culture medium.

(6) Selection of Antibody-Producing Hybridoma To test whether the hybridoma obtained in (5) above produces the desired antibody, for example, an enzyme-labeled secondary antibody against mouse antibody was used. ELISA (enzyme immunoassay), sandwich ELISA, plaque formation, agglutination, RIA (immunoassay using radioisotope), indirect fluorescent antibody (IFA), etc. It is preferable to use the ELISA method.

This sandwich ELISA method is performed as follows. EL with "anti-morphine antibody" immobilized
The hybridoma culture supernatant is added to each well (measurement well) of the ISA plate and left standing for a certain period of time. Then, the enzyme-labeled antibody prepared in (1) (the enzyme used for labeling may be, for example, peroxidase, alkaline phosphatase, β-galactosidase, etc.) is added to each of these washed measurement wells. In addition, leave it for a certain period of time. Next, these measurement wells are washed, and a substrate solution corresponding to the enzyme used is added to measure the enzyme activity. If the enzyme activity is observed, it can be seen that the hybridoma producing the desired antibody was present in the culture well from which the culture supernatant was collected. In this way, a hybridoma in which cell proliferation is recognized and which produces an antibody can be obtained.

(7) Hybridoma Straining (Cloning) Hybridomas in the culture wells in which antibody production was observed were the limiting dilution method and the single cell manipulation method (under an inverted microscope, one hybridoma per well). , A method of picking colonies using soft agar, FACS (Fluorescent Activat)
ed Cell Sorter) and the like. At this time, the antibody-producing hybridoma found in (6) was cultured by any of the above-mentioned cloning methods, and the supernatant of the culture well in which the growth was observed was used to select the antibody-producing hybridoma in (6). The antibody-producing wells are searched by the same method as the ELISA method, and the antibody-producing wells are further searched for the antibody-producing wells whose reactivity with the "anti-morphine antibody" is inhibited by the inhibition test using morphine. . In this way, a hybridoma strain that produces a monoclonal antibody that is an "anti-idiotype antibody" having high specificity for the variable region of "anti-morphine antibody" and a high antibody titer can be obtained.

[B] Method for Producing Monoclonal Antibody The above-mentioned monoclonal antibody can be produced by culturing the hybridoma strain obtained above in a flask or in the abdominal cavity of an animal. The production of the monoclonal antibody by in-flask culture of the hybridoma strain obtained in (7) above can be performed, for example, by using a commonly used lymphocyte culture medium containing 0 to 20% fetal bovine serum (for example, MEM, DMEM, McCoy, RPMI1
It can be carried out by culturing in a medium containing medium components such as 640) until the cell concentration reaches the upper limit. At this time, the monoclonal antibody is contained in the culture supernatant obtained by centrifugation.

On the other hand, the production of the monoclonal antibody by the intraperitoneal culture of the hybridoma strain obtained in (7) above
It is possible to use an animal of a different type from the animal from which the cells used for cell fusion are derived, but it is preferable to use an animal of the same species, and it is more preferable to use an animal of the same strain. .

"Anti-morphine antibody" by such a method
The production of a monoclonal antibody that is an "anti-idiotype antibody", which has a high specificity for the variable region of the mouse and a high antibody titer, makes the immunity of this animal intraperitoneal in an appropriate animal such as mouse, rat or hamster. Administer a substance that lowers, for example, mineral oil such as pristane,
It can be carried out by administering ⁶ to 10 ^{7 of} the hybridoma cell line obtained in the above (7) and proliferating the cell line into the abdominal cavity at high density for several weeks. At this time, the monoclonal antibody is contained in the ascites supernatant obtained by centrifugation. The antibody concentration is 10 to 1000 times the antibody concentration of the culture supernatant when the culture is performed in the flask.

The monoclonal antibody obtained by culturing the hybridoma strain in a flask or in the abdominal cavity of an animal is used in general protein purification methods such as salting out, dialysis, ion exchange chromatography, affinity chromatography, etc. By carrying out the procedure, a high-purity monoclonal antibody is obtained. The monoclonal antibody obtained as described above has a very high specific reactivity with the variable region of the "anti-morphine antibody".

[C] Measurement of morphine by competitive immunoassay using "anti-morphine antibody" and "anti-idiotype antibody" (1) Labeling of "anti-morphine antibody" or "anti-idiotype antibody" Labeling with a substance As a labeling substance of "anti-morphine antibody" or "anti-idiotype antibody", a radioisotope, a fluorescent substance, an enzyme [for example, a redox enzyme (peroxidase, catalase, glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, , Β-galactosidase, etc.), phosphate hydrolase (eg alkaline phosphatase)], etc., but it is preferable to use an enzyme that is easy to handle. The enzyme-labeled antibody is, for example, an “anti-morphine antibody” or an “anti-idiotype antibody” and an enzyme, which is subjected to a one-step method using glutaraldehyde [Immunochemistry (Immunoche
mistory), 6, 43, 1969] or 2
Stepwise method [Immunochemistry
(8), 1175, 1971], periodate oxidation method [Method in Enzymology (Met)
hods in Enzymology), Vol. 37, 1
33, 1975], Maleimide method [Journal of the Biochemisty (Journal of Bi
Chemistry), 78, 235, 1975.
Years, etc., but the periodate oxidation method and the maleimide method are preferable.

This can be directly used for the measurement of morphine, but in order to further enhance the measurement sensitivity of morphine, gel filtration using Sephadex, Shephacryl or the like is performed, or the enzyme used for labeling is such as peroxidase. In the case of a glycoprotein, those subjected to affinity chromatography using concanavalin A sepharose can be used. The enzyme-labeled fraction thus purified can be used as it is for the measurement of morphine, but it is dialyzed against a buffer solution near neutrality (for example, phosphate buffer solution, Tris-hydrochloric acid buffer solution, etc.) and lyophilized. Alternatively, it is stored after being filtered by a sterilizing filter, and as necessary, an enzyme-labeled "anti-morphine antibody" or an enzyme-labeled "anti-idiotype antibody" having a protein concentration of 0.01 to 100 μg / ml in a buffer solution near neutrality, Preferably 0.1-10
It is preferable to adjust the concentration to μg / ml before use.

(2) Measurement of morphine by competitive immunological reaction using "anti-morphine antibody", morphine, and "anti-idiotype antibody" labeled with a labeling substance. "Anti-morphine antibody" was used as a solid support. "Anti-idiotype antibody" immobilized and labeled with morphine or a labeling substance
Morphine can be measured by competitively reacting with "immobilized" anti-morphine antibody ". Examples of the shape of the solid phase support include those that can be used for immunoassays such as plates, tubes, beads, and membranes; examples of the material include polyethylene, polystyrene, polypropylene, nitrocellulose, Examples thereof include nylon, styrene-butadiene copolymer, polymethylmethacrylate, glass and the like. Morphine can be measured by going through the following major steps.

Immobilizing a fixed amount of "anti-morphine antibody" on the solid support A fixed amount of the "anti-morphine antibody" solution is fixed on the solid support for a certain period of time within a range that enables the measurement of the present invention. Contact. The temperature at the time of contact is not particularly limited, but preferably 2 to 4
0 ° C is good. "Anti-morphine antibody" not immobilized on a solid support
After removing the "anti-morphine antibody" solution, it is washed several times with a washing solution to remove the "anti-morphine antibody" that has not been immobilized on the solid support. As the washing solution, for example, water, a buffer solution used for immobilization, and a surfactant (Tween 20, Tw
Examples thereof include a solution containing 0 to 3% by volume of een60) and the like, but it is preferable to use a buffer solution containing 0.005 to 0.8% of a surfactant and having a pH adjusted during the reaction.

Step of preventing non-specific binding of the "anti-idiotype antibody" labeled with a labeling substance to the surface of the solid phase support The above-mentioned "anti-morphine antibody" -immobilized support is subjected to the assay of the present invention. Within a range that enables the above, a fixed amount of the blocking solution is contacted for a fixed time. The temperature at the time of contact is not particularly limited, but preferably 2 to 40 ° C. Examples of the blocking solution include high molecular proteins such as BSA (bovine serum albumin), OVA (ovalbumin), KLH (jinkamuskai hemocyanin), and γ-globulin, or serum of various animals, including saccharose and magnesium chloride. It can be prepared by dissolving in a washing solution. The concentration of these high molecular weight proteins is preferably 0.5 to 5, preferably 0.5 to 2 wt / vol%, and 1 to 50 wt / vol% in the case of serum of various animals. In addition, if necessary, such blocking solution may contain caisson CG,
A protein preservative such as sodium azide or sodium ethylmercury thiosalicylate may be added in a necessary amount.

A step of competitively reacting morphine or an "anti-idiotype antibody" labeled with a labeling substance with an "anti-morphine antibody" immobilized on a solid support A measurement sample (including morphine) and the above-mentioned label Equal volumes of "anti-idiotype antibody" solution are taken and reacted with "anti-morphine antibody" immobilized on the solid phase support. The temperature at the time of contact is not particularly limited, but preferably 2 to 4
0 ° C is good. Measurement sample and label "anti-idiotype antibody"
The solution may be contacted immediately after mixing, or may be an "anti-morphine antibody" immobilized on a solid support.
Alternatively, each of them may be sequentially added and immediately stirred to cause a catalytic reaction. Human body fluids such as human urine, blood, and serum can be used as the measurement sample.

Step of removing free labeled "anti-idiotype antibody" which has not been immobilized on the solid phase support through "anti-morphine antibody". The reaction solution is removed, and "anti-morphine antibody" is added. Free labeled "anti-idiotype antibody" and morphine that have not been immobilized on the solid support are removed by washing several times with a washing solution.

A step of reacting a labeled "anti-idiotype antibody" immobilized on a solid support through an "anti-morphine antibody" with a substrate solution of enzyme (hereinafter referred to as "substrate solution") When the substance used for labeling is an enzyme within a range that enables measurement, a certain amount of “substrate solution” is contacted for a certain period of time (preferably an acid such as H ₂ SO ₄ , an alkali such as NaOH, an enzyme activity It is better to stop the enzyme reaction with an inhibitor etc.), and measure the absorbance developed by the reaction.
The temperature at the time of contact is not particularly limited, but there is no particular problem as long as it is within the optimum temperature range of the enzyme used, but preferably 2 to
40 ° C is good.

As the "substrate solution", a substrate of H ₂ O ₂ which the enzyme used for labeling reacts with, and a coloring substance [o-phenylenediamine, 2,2'-aminobis] which is colored by a substance generated by the enzyme reaction. (3 ethylbenzothiazoline-6-sulfonic acid) and the like] in the optimum pH range can be used. Then, the absorbance of the reaction solution is measured at a wavelength at which the color of the reaction solution after the completion of the above-mentioned enzymatic reaction shows the maximum absorbance. When the substance used for labeling is a radioisotope, it is a scintillation counter, and when it is a fluorescent substance, it is a fluorometer, and a labeled "anti-idiotype antibody" is immobilized on a solid support via "anti-morphine antibody". To measure. As described above, a calibration curve was prepared from the measurement results using a known amount of morphine solution (0 to 40 ng / ml morphine solution; hereinafter referred to as “standard morphine solution”) instead of the measurement sample, The amount of morphine in the measurement sample can be known. In one of these morphine measurement steps, the measurement was performed by the competitive reaction between morphine and the labeled "anti-idiotype antibody" against "anti-morphine antibody" immobilized on the solid support. The amount of morphine in a sample can be measured quickly and with high sensitivity.

(3) Measurement of morphine by competitive enzyme immunoreaction using "anti-idiotype antibody", morphine, and "anti-morphine antibody" labeled with a labeling substance. "Anti-idiotype antibody" was used as a solid support. Morphine can be measured by competitively reacting with "anti-morphine antibody" immobilized and labeled with morphine or a labeling substance. In (2) above, "anti-idiotype antibody" is used instead of "anti-morphine antibody", and "anti-morphine antibody" labeled with a labeling substance is used instead of "anti-idiotype antibody" labeled with a labeling substance. By doing the same, the amount of morphine in the measurement sample can be measured rapidly and with high sensitivity.

[0039]

EXAMPLES The present invention will be specifically described below with reference to examples and examples. It should be noted that these examples do not limit the scope of the present invention. Reference Example 1 [Production and Purification of "Anti-Morphine Antibody"] BAL was used as a hybridoma strain [MO-3 strain (Mikori Kenjojo No. 1911)] of 2 × 10 ⁶ cells suspended in MEM.
B / c mice (♀, 8 weeks old, pristane 0.5 ml intraperitoneally administered 1-2 weeks before) were intraperitoneally administered.
Significant increase in mouse body weight was observed around the first week,
Ascites was appropriately removed from 1 to 3 weeks. The antibody titer of the ascites thus obtained was 10 ⁶ to 10 ⁸ . The "anti-morphine antibody", which is a monoclonal antibody, was purified from the obtained ascites fluid as follows. The ascites fluid was dialyzed against 0.1 M Tris-hydrochloric acid buffer solution (PH 7.4) and passed through a DEAE-cellulose column equilibrated with the same buffer solution. The pass-through fraction is salted out with 50% saturated ammonium sulfate,
The obtained precipitate was dissolved in phosphate buffered saline (PBS) and dialyzed against PBS. The "anti-morphine antibody" thus obtained was found to be highly pure by slab gel electrophoresis using SDS polyacrylamide gel.

Example 1 [Preparation of immunogen] The monoclonal antibody of Reference Example 1 (hereinafter referred to as "MO-3") and KLH (CALBIOCHEM # 3748).
Each of 1) and 1 was diluted to 1 mg / ml with PBS, mixed in equal amounts, and added with 10% glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd. for electron microscope) in an amount of 14 μl / ml, and allowed to stand at room temperature for 1 hour. It was stirred. The immunogen thus obtained was dialyzed against PBS and stored at 4 ° C.

Example 2 [Preparation of enzyme-labeled antibody] 7.32 mg of horseradish peroxidase (HRP)
Was dissolved in 1 ml of distilled water, 200 μl of 0.1 M sodium periodate was added, and the mixture was reacted at room temperature for 30 minutes. This enzyme solution was added to 1 mM acetate buffer (pH 4.5) at 4 ° C.
After overnight dialysis with 0.2M sodium carbonate buffer (pH
The pH was adjusted to 9.5 by adding 100 μl of 9.5).
On the other hand, 8 mg of monoclonal antibody (MO-3) dissolved in PBS was added to 10 mM sodium carbonate buffer (pH
It was dialyzed against 9.5) overnight at 4 ° C. The HRP solution thus obtained was mixed with the monoclonal antibody solution, and the mixture was stirred at room temperature for 2 hours and 30 minutes, sodium tetrahydridoborate was added to the reaction solution, and the mixture was stirred at 4 ° C. for 2 hours. The HRP-labeled monoclonal antibody thus obtained was dialyzed against PBS and stored at 4 ° C.

Example 3 [Preparation of "anti-idiotype antibody" -producing hybridoma strain] (1) Immunization of mouse and preparation of spleen lymphocytes 0.4 ml and 0.4 ml of the immunogen solution prepared in Example 1
0.2 ml of the emulsion obtained by thoroughly mixing with Freund's complete adjuvant was administered subcutaneously to the abdomen of BALB / c mice (♀, 8 weeks old). Two weeks after this initial immunization, 0.2 ml of the emulsion prepared as described above was subcutaneously administered to the abdomen of the mouse. Furthermore, after 7 weeks, 0.4 ml and 0.4 ml of the above immunogen solution was added.
0.2 ml of an emulsion obtained by thoroughly mixing ml of Freund's incomplete adjuvant was intraperitoneally administered to the mouse.

Furthermore, after 2 weeks and 4 weeks, 0.2 ml of the emulsion prepared in the same manner as above was subcutaneously administered to the abdomen of the mouse. Two weeks later, as the final immunization, 0.2 ml of the above immunogen solution diluted 2-fold with PBS was administered to the tail vein of the mouse. The spleen extracted from the mouse thus immunized on the third day after the final immunization was placed in a petri dish containing MEM (a lymphocyte culture medium powder dissolved in distilled water, containing 10 mM HEPES). It was washed, transferred to another Petri dish containing 10 ml of MEM, and disentangled using the frosted part of the sterilized slide glass. The floating lymphocytes thus obtained were suspended in MEM (37 ° C.), centrifuged (350 G, 6 minutes) and washed. After repeating this operation twice more, it was resuspended in MEM (37 ° C.) to obtain mouse spleen lymphocytes for cell fusion.

(2) Cell fusion 8-azaguanine-resistant mouse myeloma cells in the logarithmic growth phase [X63-Ag8 · 653 (653) or P3]
-X63-Ag8-U1 (P3U1)] was recovered from the culture flask and centrifuged (350 G, 6 minutes) to perform MEM.
It was washed twice at (37 ° C). This was resuspended in MEM (37 ° C.) to obtain mouse myeloma cells used for cell fusion. 1.8 × 10 ⁸ mouse spleen lymphocytes and 3.6 × 10 ⁷ 653 mouse myeloma cells or 1.8 × 10 ⁷ mouse spleen lymphocytes and 9 ×
50 with 10 ⁷ P3U1 mouse myeloma cells
Place in a conical centrifuge tube made of ml plastic, mix,
Then, after centrifuging the supernatant (350 G, 6 minutes), the same centrifuge tube was tapped to loosen the pellet.

While rotating the centrifuge tube, 1 ml of 40%
The PEG 1500 solution (37 ° C.) was added over 1 minute, and the reaction was continued for 1 minute while rotating. Next, while rotating, MEM (37 ° C) was gradually added, and finally 10
It was made up to ml and centrifuged (120 G, 6 minutes) to remove the supernatant by suction. The centrifuge tube is tapped to loosen the pellet, and 80 ml of HAT medium (1 × 10 ⁻⁴ M hypoxanthine, 4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 4
RPMI 1640 medium containing ⁵ M thymidine, 0.2 U / ml bovine insulin and 20% fetal calf serum,
Re-suspend at 37 ° C.) and dispense 100 μl of each to each culture well of 8 96-well culture plates.
Cultured with ₂ incubator (5% _CO 2, 9
5% air, 37 ° C, 100% humidity).

(3) Selection of Hybridoma From 2 to 4 weeks from the start of the culture in (2) above, in the culture supernatant of each well of the culture plate where cell growth was observed,
Whether or not an antibody against the "anti-morphine antibody" was contained was examined by the following ELISA method. First, the MO purified in Reference Example 1 was added to each analysis well of a 96-well U-bottom ELISA plate (hereinafter abbreviated as an ELISA plate).
-3 (“anti-morphine antibody”) solution (10 μg / ml, p
50 μl of H9.8 (dissolved in 0.05 M carbonate buffer) was dispensed into each well, and the mixture was allowed to stand at 4 ° C. overnight (by such treatment, MO-3 is adsorbed on the surface of each analysis well).

Then, each of the analysis wells was washed with a washing solution (0.
PBS with 05% Tween20. Below, PBS-
Abbreviated as T. ), 0.5% OVA solution (dissolved in PBS) was dispensed into each analysis well by 100 μl and left at room temperature for 30 minutes. Each of these analysis wells was washed with PBS-T, and 50 μl of the culture supernatant of each culture well of the culture plate was dispensed into each of the analysis wells and left at room temperature for 2 hours (for a negative control. , HAT medium was used as the positive control, and the serum of the mouse used for cell fusion in the present invention was diluted 100-fold with PBS-T).

Next, each of the above analysis wells was washed with PBS-T, and the enzyme-labeled antibody solution prepared in Example 2 was added to PBS.
50 μl of a solution appropriately diluted with −T was dispensed into each analysis well, and left still at room temperature for 1 hour. Then, after washing each analysis well with PBS-T, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (1.2 mg / ml) and 0.02% hydrogen peroxide were added. 100 μl of the containing solution was dispensed into each analysis well, and after reacting at room temperature for 30 minutes, the absorbance at 405 nm of each analysis well was measured using the absorbance measuring device for this analysis plate. The production of antibodies having immunological reactivity with "anti-morphine antibody" was observed.
One of 269 culture wells in the culture plate when using 653, and two of 553 culture wells in the culture plate when using P3U1. .

Next, these antibodies are "anti-morphine antibodies".
It was examined whether or not it had immunological reactivity with the variable region of. That is, for three culture wells that produced these antibodies, an inhibition test using morphine (instead of 50 μl of the supernatant of the culture well in the above sandwich ELISA,
The same operation was performed except that 25 μl of the supernatant of the culture well and 25 μl of PBS-T in which 250 ng of morphine were dissolved were simultaneously placed in the analysis well). As a result, all three culture wells containing the antibody inhibited by morphine were observed. That is, a hybridoma producing an antibody (“anti-idiotype antibody”) having reactivity with the variable region of “anti-morphine antibody” is present in these three culture wells (referred to as A, B, and C). It was confirmed.

(4) Hybridoma Cell Line (Cloning) Using the RPMI1640 medium containing 20% FCS, the antibody production shown in the above step (3) was confirmed 3
The hybridoma in each culture well was cloned by the limiting dilution method. The culture, using a 96-well culture plates, using the thymocyte suspension BALB / c mouse as feeder cells (10 ⁷ cells / ml), (1 to 5 cells hybridoma) / (thymus cell suspension 100 μl) / well, and 5 days after the start of culture, 100 μl of RPMI1640 medium containing 20% FCS was added to each culture well, and the culture was further continued. In cloning the hybridomas in the three culture wells A, B, and C, the supernatant of the culture well observed as a single colony on day 10 to 14 was collected, and the sandwich ELISA method [(3. The method similar to the step)] was performed to screen antibody producing wells. In this way, A, B,
At least one hybridoma strain was obtained from each C culture well. Then, among them, the hybridoma strains representative of the A, B, and C culture wells were selected and recloned one by one in consideration of cell growth and antibody production.

The strain thus obtained was used as iMO-3-
X1 strain (Microtechnology Research Institute, No. 11933), iMO-3-
P1 strain and iMO-3-P2 strain are referred to, and the monoclonal antibodies produced by these strains are respectively referred to as iMO-3-X.
1, iMO-3-P1, iMO-3-P2. The class / subclass of monoclonal antibody and the type of L chain contained in the culture supernatants of these three strains were determined by the following measurement test.

[Measurement test] The class / subclass of the "anti-idiotype antibody" was determined as follows. Mouse Monoclonal Antibody Isotyping Kit (Am
by the immunoblotting method using ERSHAM). iMO3-X1 strain, iMO3-P1 strain and iMO-
As a result of examining the class / subclass of the immunoglobulin produced by the 3-P2 strain, the iMO-3-X1 strain, iMO-3
-P1 strain and iMO-3-P2 strain produced the monoclonal antibody (iMO-3-X1, iMO-3-P1 and i
MO-3-P2) is an IgG having a κ type L chain.
It was found to be an antibody belonging to ₁ .

Example 4 [Production of "anti-idiotype antibody" by flask culture] Cultured cells of the iMO-3-X1 strain obtained by culturing in RPMI1640 medium containing 15% FCS were added with 10 ml of RPMI.
The cells were transferred to 1640 medium (without FCS) and cultured until just before almost all cells were killed. "Anti-idiotype antibody" against "anti-morphine antibody" (iMO-3
-X1) was contained in the supernatant obtained by centrifuging the culture (1,500 G, 5 minutes) at 50 μg / ml (measured by the one-dimensional plate immunodiffusion method).

Example 5 [Production of "anti-idiotype antibody" in mouse abdominal cavity] In order to obtain a large amount of "anti-idiotype antibody" against "anti-morphine antibody", iMO-3-X1 was intraperitoneally injected in a mouse.
The cells of the strain were cultured. 2 × cells of the iMO-3-X1 strain were intraperitoneally administered to BALB / c mice (♀, 8 weeks old, 0.5 ml of pristane was intraperitoneally administered 2 weeks before).
0.5 ml of PBS with 10 ⁶ suspended therein was administered. The weight of this mouse showed a remarkable increase from the first week,
Ascites was collected using a 19G injection needle on the 7th, 9th and 11th days after cell administration (total collected amount; 6 ml / animal). Then, the ascites was centrifuged (1,500 G, 5 minutes) to obtain an ascites supernatant.

"Anti-idiotype antibody" (iMO-3-X1) against "anti-morphine antibody" was added to this ascites supernatant at 10 mg / ml (measured by one-dimensional plate immunodiffusion method).
It was contained. By purifying the "anti-idiotype antibody" in the same manner as in the purification of "anti-morphine antibody" of Reference Example 1, it was found by slab gel electrophoresis using SDS polyacrylamide gel that the purity was high.

Example 6 [Measurement of morphine] (1) Preparation of "anti-morphine antibody" and "anti-idiotype antibody" labeled with a labeling substance The enzyme-labeled "anti-morphine antibody" was MO-5 strain (Microtechnology Research Institute). No. 1912), and the enzyme-labeled "anti-idiotype antibody" was used as iMO-3.
Using the purified antibody iMO-3-X1 produced by the -X1 strain (Microtechnology Research Institute No. 11933), it was prepared by the following maleimide method. First, this antibody (4 mg) was dissolved in 0.1 M citrate buffer (pH 3.5) (1 ml). Immobilized pepsin [manufactured by Sterogyne Co., Ltd.] was washed with this solution three times with the same buffer. 100 μl (0.3-0.4
mg of pepsin was added)], and the mixture was shaken at 37 ° C. for 6 hours for reaction. Then, a small amount of 1N sodium hydroxide was added to adjust the pH to 6 to 7 to stop the reaction.

The reaction solution was centrifuged (2,000 G, 1
The immobilized pepsin was removed by ultrafiltration with a 0.1 minute phosphate buffer (pH 6.0) and 500 μl of the supernatant was ultrafiltered with a molcut 30000 (Millipore).
And Next, 0.1M mercaptoethylamine solution [0.1M phosphate buffer solution (pH 6.0) containing 5 mM sodium ethylenediaminetetraacetate (EDTA)] (50 μl) was added to 470 μl of this solution, and the mixture was added at 37 ° C. for 90 minutes. The reaction was allowed to stand. Then, mercaptoethylamine was removed using a PD-10 column (Pharmacia), and the obtained target fraction was ultrafiltered with a molcut 10000 (Millipore) to obtain a 0.1 M phosphate buffer (5 m).
Includes M EDTA. The pH was adjusted to 900 μl at 6.0). In this way, a Fab ′ solution in which the Fc portion of the antibody used here was removed was obtained (this Fab ′ has a thiol group). Then, the concentration of Fab ′ was calculated from the absorbance of this solution at 280 nm (2% of 0.1% solution).
The absorbance at 80 nm was 1.48).

On the other hand, horseradish peroxidase (4 m
g) in 0.1 M phosphate buffer (pH 7.0) (1 ml)
Dissolved in 50 mM sulfosuccinimidyl-4
-(N-maleimidomethyl) cyclohexane-1-carboxylate (hereinafter abbreviated as sulfo-SMCC) solution [dissolved in 0.1 M phosphate buffer (pH 6.0)] (60 μl) was added and the mixture was added at 30 ° C. for 1 hour. It was left to stand for a reaction. Immediately after the reaction, perform ultrafiltration with a molcut of 10000, and 600 μ with 0.1M phosphate buffer (pH 6.0).
It was set to l. In this way, peroxidase- (su
lfo-SMCC) solution was obtained. And this 403n
From the absorbance at m, the peroxidase- (sulfo
-SMCC) concentration was calculated (403% of 0.1% solution)
The absorbance at m was 2.28). Fab ′ solution of antibody and peroxidase obtained as described above
(Sulfo-SMCC) solution was mixed so that the molar ratio of both was 1: 1 (final concentration of EDTA was 2.5 m
M) After reacting at 4 ° C. for 16 hours, 50 mM N-
Ethyl maleimide solution [0.1 containing 5 mM EDTA
The reaction was stopped by adding M phosphate buffer (pH 6.0)] (10 μl). This reaction solution was passed through a column packed with Concanavalin A Sepharose (Pharmacia) to remove unreacted Fab ′. This solution was dialyzed at 4 ° C. overnight using phosphate buffered saline (solution containing 0.14 M sodium chloride in 0.1 M phosphate buffer (pH 6.0), hereinafter abbreviated as PBS). Then, the target enzyme-labeled "anti-morphine antibody" or enzyme-labeled "anti-idiotype antibody" was obtained. When measuring morphine, these stock solutions were appropriately diluted with a "diluting solution" [0.1 M phosphate buffer (pH 6.0) containing 1% BSA] and used.

(2) Enzyme-labeled "anti-idiotype antibody"
Preparation of calibration curve for morphine using "Protein G column" (manufactured by Pharmacia) of Reference Example 1 [50 mM Tris-HCl buffer (pH 6.8) for the effluent and 0. 2M hydrochloric acid-glycine buffer (pH 2.5) was used. ] To obtain a highly pure "anti-morphine antibody". This "anti-morphine antibody" solution (10 μg / ml, P
10 times each of the BS solution) to each analysis well of the solid support (polystyrene microtiter plate manufactured by Nunc).
Aliquots of 0 μl were dispensed and the mixture was allowed to stand overnight at 4 ° C. to immobilize “anti-morphine antibody”. The plate is then washed with a washing solution (0.
After washing with PBS containing 01% Tween 20), blocking solution (0.5% BSA, 0.5% BSA, to prevent nonspecific adsorption of the enzyme-labeled "anti-idiotype antibody" on the plate
5% sucrose, 1 mM magnesium chloride and 0.0
PBS containing 1% caisson CG) 1 per assay well
It was dispensed in 00 μl aliquots and left at room temperature for 1 hour. Next, after washing with a washing solution, "standard morphine solution" (a 0-32 ng / ml morphine solution prepared using "diluting solution").
After adding (50 μl), the enzyme-labeled “anti-idiotype antibody” solution (50 μl) of (1) above was added and stirred,
It was allowed to stand at room temperature for 1 hour.

Next, after washing the same well with a washing solution, "substrate solution" (20 mg of o-phenylenediamine and 10 μl)
Of 35% H ₂ O ₂ in 25 ml of 0.1 M citrate buffer (pH 5.0) was added to each well in an amount of 100 μl, and the mixture was allowed to stand at room temperature for 15 minutes while protected from light, and then 2N.
Sulfuric acid was dispensed in 50 μl aliquots to stop the enzymatic reaction, and the reaction solution was analyzed at 492 nm using a microplate photometer.
The absorbance at was measured. The calibration curve of morphine thus obtained is shown in FIG.

(3) Preparation of calibration curve of morphine using enzyme-labeled "anti-morphine antibody" In the above (2), enzyme-labeled "anti-morphine antibody" was used instead of enzyme-labeled "anti-idiotype antibody".
0-1,000 ng / ml prepared by using "anti-idiotype antibody" of Example 5 instead of "anti-morphine antibody" and "diluting solution" as "standard morphine solution"
A morphine calibration curve was prepared using the morphine solution prepared in (1) by the same procedure as in (2) above. The result is shown in FIG.

(4) Morphine Addition Recovery Test In the above (2), instead of the “standard morphine solution”, a solution (10, 20, 50, or 100 ng) of morphine added to normal human serum (manufactured by Kaketsuken). / Ml) was diluted 10 times with a "diluting solution", and the amount of morphine was measured. As a result, the amount of morphine added (calculated value) and the amount of morphine actually measured (measured value) Was a very close value. The results are shown in Table 1.

[0063]

[Table 1]

[0064]

According to the immunological assay method for morphine using the anti-idiotype antibody of the anti-morphine antibody of the present invention, morphine can be easily and quickly performed.

[Brief description of drawings]

FIG. 1 is a calibration curve of morphine obtained by competitively reacting an enzyme-labeled “anti-idiotype antibody” with morphine against “anti-morphine antibody” in (6) of Example 6.

FIG. 2 is a calibration curve of morphine obtained by competitively reacting the enzyme-labeled “anti-morphine antibody” and morphine with “anti-idiotype antibody” in (6) of Example 6.

Front page continuation (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location G01N 33/53 G 8310-2J // C12N 15/06 G01N 33/577 B 9015-2J (C12P 21/08 C12R (71) (72) Inventor Takashi Usagawa 5 1978, Ogushi, Ube City, Yamaguchi Prefecture, Ube Research Co., Ltd. Ube Laboratory (72) Inventor Hiroyuki Setoguchi 5 1978, Obe, Kobu, Yamaguchi Prefecture Ube Industries Ube Co., Ltd. In the laboratory (72) Inventor Noriko Uzeki 5 1978, Kogushi, Ube, Yamaguchi Prefecture 5 Ube Kosan Co., Ltd. Ube Laboratory

Claims (5)

[Claims] 1. An anti-idiotype antibody of an anti-morphine antibody. 2. The anti-idiotype antibody according to claim 1, wherein the hybridoma strain obtained by cell fusion of lymphocytes obtained by immunizing with anti-morphine antibody and myeloma cells is cultured. Manufacturing method. 3. The hybridoma strain according to claim 2. 4. The anti-morphine antibody according to claim 1 immobilized on a solid support is allowed to competitively react with the anti-idiotype antibody according to claim 1 labeled with morphine or a labeling substance. Characteristic method of measuring morphine. 5. The anti-idiotype antibody according to claim 1 immobilized on a solid support is allowed to react competitively with morphine or the anti-morphine antibody according to claim 1 labeled with a labeling substance. Characteristic method of measuring morphine.