JP4509007B2 - Method for producing antibody against emamectin and its similar compounds, antibody, hybridoma, immunological assay method and assay kit - Google Patents

Description

The present invention relates to a method for producing an antibody against emamectin and its similar compounds, an antibody, a hybridoma, an immunological measurement method and a measurement kit thereof.
Here, emamectin refers to a mixture of emamectin B1a and emamectin B1b,
Emamectin B1a is a chemical name “(10E, 14E, 16E, 22Z)-(1R, 4S, 5 ′S, 6S, 6′R, 8R, 12S, 13S, 20R, 21R, 24S) -6 ′-[ (S)-sec-butyl] -21,24- hydroxy-5 ', 11,13,22- tetramethyl-2-oxo -3,7,19- trioxadodecanoic tetracyclo [15,6,1,1 ^{4 , 8, 0 20, 24]} Pentakosa -10,14,16,22- tetraene-6-spiro-2 '- (5', 6'-dihydro -2'H- pyran) -12-yl 2,6 Dideoxy-3-O-methyl-4-O- (2,4,6-trideoxy-3-O-methyl-4-methylamino-α-L-lyxo-hexopyranosyl) -α-L-arabino-hexopyranoside ” The substance represented,
Emamectin B1b is the chemical name “(10E, 14E, 16E, 22Z)-(1R, 4S, 5 ′S, 6S, 6′R, 8R, 12S, 13S, 20R, 21R, 24S) -21, 24-. hydroxy-6'-isopropyl-5 ', 11,13,22- tetramethyl-2-oxo -3,7,19- trioxadodecanoic tetracyclo ^{[15,6,1,1 4,8, 0 20, 24]} Pentacosa-10,14,16,22-tetraene-6-spiro-2 '-(5', 6'-dihydro-2'H-pyran) -12-yl 2,6-dideoxy-3-O-methyl- 4-O- (2,4,6-trideoxy-3-O-methyl-4-methylamino-α-L-lyxo-hexopyranosyl) -α-L-arabino-hexopyranoside ”.

（式中、Ｒ_２はメチル基およびエチル基を示す）
で表される構造を有する１６員環マクロライド系殺虫剤で、害虫の神経系に作用し速やかに殺虫効果を発揮して食害を抑制する。ミナミキイロアザミウマ，チャノキイロアザミウマ等のアザミウマ目害虫，シンクイムシ類，チャノホソガ，キンモンホソガ，ギンモンハモグリガ，モモハモグリガ，ミカンハモグリガ等の鱗翅目害虫，更にはマメハモグリバエ等の双翅目害虫やハダニ類などの幅広い害虫に低薬量で卓効を示し、既存薬剤に抵抗性を示す害虫に対しても優れた効果を示す作物に対して安全性の高い化合物である。 Emamectin has the following formula (2):

(Wherein R ₂ represents a methyl group and an ethyl group)
It is a 16-membered macrolide insecticide having a structure represented by the following formula: It acts on the nervous system of pests and quickly exerts an insecticidal effect to suppress food damage. Thripidae pests such as Thrips thrips and Thrips thrips, Thripidae, Chanohosuga, Kinmonhosoga, Gimmon's leafworm, Papilio's leafworm, and ticks such as legumes such as legumes It is a highly safe compound for crops that exhibits excellent effects at low doses against pests and also exhibits excellent effects against pests that are resistant to existing drugs.

  In recent years, there has been a great social interest in the residue of pesticide residues in the environment such as soil, water, and the atmosphere, and recently the increase in agricultural chemicals of imported agricultural products. For Emamectin, the residual standard values of food, additives, and other standards in the Food Sanitation Act are, for example, cabbage (0.1 ppm), roots of potatoes (0.1 ppm), leaves of potatoes (0.1 ppm), Japanese cabbage (0.1 ppm), broccoli (0.1 ppm), Brussels sprouts (0.1 ppm), cucumber (0.1 ppm), tomato (0.1 ppm), eggplant (0.1 ppm), tea (0.5 ppm), etc. It has been established. Therefore, in order to ensure safety regarding the environment and food, it is necessary to quickly and accurately measure the amount of emamectin contained in the above-mentioned crops (see Non-Patent Document 1).

Conventionally, for example, the content of emamectin in crops needs to be determined for metabolites at the same time, so it is extracted from wheat, fruit, vegetables, etc., and then processed in two ways to process fluorescence induction. Has been analyzed by high performance liquid chromatography (HPLC). That is, a sample is extracted with methanol, filtered, and then treated with a _C18 minicolumn and then with a sulfopropylsilica gel minicolumn. Next, the treatment liquid is divided into two, and after one is further treated with an NH ₂ silica gel minicolumn, fluorescence is induced, respectively, and a method of measuring by HPLC is employed (see Non-Patent Document 1).

  In recent years, simultaneous analysis methods by instrumental analysis that collectively analyze many types of pesticides have been recognized as official methods, and simultaneous analysis methods are becoming a major place in residual pesticide analysis. However, emamectin cannot be analyzed by the simultaneous analysis method because of its complicated pretreatment method, and it takes time even for a single analysis. Therefore, establishment of a simple analysis method is desired.

  On the other hand, an immunological measurement method measures an antigen using an antigen-antibody reaction, and is not only excellent in measurement accuracy but also a quick, simple and economical measurement method. Conventionally, immunological measurement methods have played a major role as one of patient pathological analysis methods in the field of clinical diagnosis, but their application to the measurement of environmentally hazardous chemical substances is progressing.

"Agricultural Chemical Registration Retention Standard Handbook (Revised 4th Edition)" [pp. 169-173] (Chemical Industry Daily)

  However, the method of measuring by the above-mentioned HPLC is a problem that sample preparation is complicated, requires a lot of procedures and time, requires skill in analysis, and requires high costs for measuring devices and equipment. There is a point. For the measurement of emamectin, it is necessary to obtain an analysis result of a huge number of samples in a short time, and there is a demand for a new measurement method that is not only accurate but also simple, quick, and economical.

  In addition, regarding immunological measurement methods, there has been no report on the production of antibodies against emamectin and measurement methods using the same, and there is an increasing demand for practical use.

（式中、Ｒ_１：−ＮＨ_２、−ＮＨＣＨＯおよび−Ｎ（ＣＨ_３）ＣＨＯを、Ｒ_２はメチル基およびエチル基を示す）
で表されるエマメクチンの代謝生成物（以後、Ｒ_１：−ＮＨ_２をアミノ体、−ＮＨＣＨＯをホルミルアミノ体、−Ｎ（ＣＨ_３）ＣＨＯをＮ−メチルホルミルアミノ体と称する）はその化学構造がエマメクチンに非常に類似していることから、エマメクチンの糖鎖の末端部分のアミノ基の部分にリンカーを導入したハプテン由来の抗体を作製した場合、その抗体はエマメクチンのみならずその代謝生成物に対しても高い反応性が期待でき、エマメクチンとその代謝生成物を同時に測定できる可能性が高い。 Immunoassays are often highly reactive to structurally similar compounds due to their nature. Conversely, the following general formula (3)

(In the formula, R ₁ : —NH ₂ , —NHCHO and —N (CH ₃ ) CHO, R ₂ represents a methyl group and an ethyl group)
A metabolite of emamectin represented by the formula (hereinafter R ₁ : —NH _{2 is referred} to as an amino form, —NHCHO as a formylamino form, and —N (CH ₃ ) CHO as an N-methylformylamino form). Is very similar to emamectin, so when an antibody derived from a hapten with a linker introduced into the amino group at the end of the sugar chain of emamectin is produced, the antibody is not only used for emamectin but also its metabolites. High reactivity can be expected, and it is highly possible to measure emamectin and its metabolites at the same time.

  An object of the present invention is to provide a method for producing an antibody highly sensitive and highly selective against emamectin and its similar compounds (mainly metabolites of emamectin), an antibody against emamectin and its similar compounds, and the antibody It is an object to provide an immunological measurement method and a measurement kit for emamectin and its similar compounds that are highly sensitive and excellent in quantification using the above.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have used the following emamectin derivatives as haptens to produce an emamectin and similar compound antibody production method, an antibody, a hybridoma producing the antibody, It has been found that the above object can be achieved by an immunological assay method and a measurement kit for emamectin and its similar compounds comprising the antibody, and the present invention has been completed.

（式中、Ｒ_１はＮＲ_３Ｒ_４またはＯＲ_３を、Ｒ_２はメチル基およびエチル基を、Ｒ_３は水素原子、−ＣＯ（ＣＨ_２）ｎＲ_５または−（ＣＨ_２）ｎＲ_５を、Ｒ_４は水素原子または炭素数１から１０のアルキル基を、Ｒ_５は水素原子、カルボキシル基、アミノ基、アルデヒド基またはヒドロキシル基を、ｎは０から１０の整数を表す。）
で表わされる構造を有する化合物をハプテンとし、当該ハプテンと高分子化合物との複合体を抗原として用いることによるエマメクチンに対する抗体の製造方法である（請求項１）。 That is, the present invention provides the following formula (1):

(Wherein R ₁ represents NR ₃ R ₄ or OR ₃ , R ₂ represents a methyl group and an ethyl group, R ₃ represents a hydrogen atom, —CO (CH ₂ ) nR ₅ or — (CH ₂ ) nR ₅ , R ₄ represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ₅ represents a hydrogen atom, a carboxyl group, an amino group, an aldehyde group or a hydroxyl group, and n represents an integer of 0 to 10.)
A method for producing an antibody against emamectin using a hapten as a compound having a structure represented by the following formula and using a complex of the hapten and a polymer compound as an antigen (claim 1).

  The present inventor has devised that it is possible to produce an antibody against emamectin and its similar compounds with high sensitivity and high selectivity by suitably using the above compound (1) as a hapten for emamectin and its similar compounds. Thus, it is possible to provide an immunological measurement method and measurement kit for emamectin and its similar compounds that are highly sensitive and excellent in quantification using the antibody.

In particular, in the compound represented by the formula (1), R ₁ preferably has a structure represented by —N (CH ₃ ) CO (CH ₂ ) ₃ COOH, and R ₂ represents methyl and ethyl. 2).

  By having such a configuration, a particularly excellent effect can be achieved as a hapten for producing antibodies against emamectin and its similar compounds.

  The present invention is an emamectin obtained by the above production method and an antibody against the similar compound or a fragment thereof capable of binding to an antigen (claim 3).

  In the present invention, by using an antibody obtained by using the above compound as a hapten and a complex of the above hapten and a high molecular compound as an antigen, an immune response to emamectin and its similar compounds can be preferably induced in animals. Specific and highly sensitive antibodies to emamectin and its analogs or fragments thereof can be obtained.

  The present invention is the above-mentioned antibody or a fragment thereof, wherein the antibody or fragment thereof to emamectin and its analogs is a polyclonal antibody or a fragment thereof or a monoclonal antibody or a fragment thereof (claim 4).

  In general, the antibody includes a polyclonal antibody or a monoclonal antibody, and any antibody can be used in the present invention. Furthermore, the antigen-antibody reaction according to the present invention includes not only such antibodies but also a part of antibodies having antigen binding properties such as Fab fragments and F (ab ') 2 fragments.

  The present invention is a hybridoma producing the above monoclonal antibody (Claim 5).

  The monoclonal antibody can be stably produced by the hybridoma according to the present invention, and a large amount of the monoclonal antibody is produced by administering the hybridoma into the abdominal cavity of an animal of culture or origin to produce ascites. Can do.

  Specifically, in the present invention, the hybridoma is preferably EMT2-17H7-2 (Claim 6).

  It has been found that such a hybridoma can stably produce a highly sensitive and highly selective antibody against emamectin and its similar compounds, and can be suitably used for immunological measurement of emamectin and its similar compounds. .

  The present invention is an immunoassay method for emamectin and its analogs comprising any of the above-mentioned antibodies or fragments thereof (Claim 7).

  The method for immunological measurement of emamectin and its similar compounds of the present invention exhibits excellent effects in sensitivity, specificity and ease of operation by using the antibody of the present invention or a fragment thereof.

  The present invention also provides a kit for measuring emamectin and a similar compound comprising any one of the above-described antibodies or fragments thereof (claim 8).

  By including the antibody of the present invention or a fragment thereof and the measurement method, the measurement kit of the present invention is suitably used for immunological measurement of emamectin and its similar compounds, and the emamectin and its similar compounds are specifically, highly sensitive and highly sensitive. It can be easily measured.

The production method of the present invention is suitably used for the production of antibodies to emamectin and its similar compounds. By using a complex of an emamectin hapten compound and a high molecular compound as an antigen, an immune response against emamectin and its similar compounds can be successfully induced in animals, and a specific and highly sensitive antibody against emamectin and its similar compounds Can be obtained. Further, in the compound represented by the formula (1), when R ₁ is —N (CH ₃ ) CO (CH ₂ ) ₃ COOH, it has a particularly excellent effect as a hapten for producing antibodies against emamectin and its similar compounds. .

The antibody of the present invention has sensitivity to emamectin and its similar compounds in either case of a polyclonal antibody or a monoclonal antibody, and can be used for the antigen-antibody reaction according to the present invention. In particular, in the case of a monoclonal antibody, it is highly sensitive to emamectin and its similar compounds, and cross-reactivity to other compounds is not observed, and emamectin and its similar compounds can be specifically detected. The hybridoma of the present invention can stably produce the monoclonal antibody. For example, a large amount of monoclonal antibody can be produced by administering the hybridoma into the abdominal cavity of a cultured or derived animal to produce ascites. Can do. In addition, in the present invention, by including any one of the above-described antibodies or fragments thereof, it can be suitably used in an immunological assay method for emamectin and its similar compounds. It is possible to provide a measurement kit that can be used for measurement.

（式中、Ｒ_１はＮＲ_３Ｒ_４またはＯＲ_３を、Ｒ_２はメチル基およびエチル基を、Ｒ_３は水素原子、−ＣＯ（ＣＨ_２）ｎＲ_５または−（ＣＨ_２）ｎＲ_５を、Ｒ_４は水素原子または炭素数１から１０のアルキル基を、Ｒ_５は水素原子、カルボキシル基、アミノ基、アルデヒド基またはヒドロキシル基を、ｎは０から１０の整数を表す。）
で表わされる構造を有する化合物をハプテンとし、当該ハプテンと高分子化合物との複合体を抗原として用いることによるエマメクチンに対する抗体の製造方法を提供する。該製造方法は、エマメクチンおよびその類似化合物の製造方法として好適に使用される。 The present invention provides the following formula (1):

(Wherein R ₁ represents NR ₃ R ₄ or OR ₃ , R ₂ represents a methyl group and an ethyl group, R ₃ represents a hydrogen atom, —CO (CH ₂ ) nR ₅ or — (CH ₂ ) nR ₅ , R ₄ represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ₅ represents a hydrogen atom, a carboxyl group, an amino group, an aldehyde group or a hydroxyl group, and n represents an integer of 0 to 10.)
A method for producing an antibody against emamectin by using a hapten as a compound having a structure represented by the above and using a complex of the hapten and a polymer as an antigen is provided. The production method is suitably used as a production method of emamectin and its similar compounds.

（式中Ｒ_２，ｎは前述と同じ意味を表す）
で示す方法は、高収率で化合物を得ることから好適に用いられる。 The production of the compound (1) used as the hapten compound can be carried out by a known synthesis method and is not particularly limited. For example, R _{1 of the} compound (1) is —N (CH ₃ ) CO (CH ₂ ) For ₃ COOH, the following reaction formula:

(Wherein R ₂ and n represent the same meaning as described above)
Is preferably used because it yields the compound in high yield.

  Specifically, in the above reaction formula, emamectin (2) as a starting material is reacted with ω-carboxyalkylcarboxylic acid in the presence of a base and DCC (dicyclohexylcarbodiimide) as a dehydrating agent in the presence of a solvent, and reacted. An N-ω-carboxyalkylcarbonyl derivative (4) is obtained.

Although it does not specifically limit as a base by this reaction, For example, alkali metal hydroxides, such as sodium hydroxide and potassium hydroxide, alkali metal hydrides, such as sodium hydride and potassium hydride, sodium methylate, sodium ethylate, etc. Alkali metal alcoholates, carbonates such as potassium carbonate and sodium carbonate, organic bases such as triethylamine, pyridine, 4-dimethylaminopyridine and DBU can be used. Examples of the solvent include alcohols such as methanol and ethanol, chlorinated solvents such as methylene chloride and chloroform, aromatic hydrocarbons such as benzene and toluene, ethers such as diethyl ether, dipropyl ether, and tetrahydrofuran, acetone, and methyl ethyl ketone. Ketones such as acetonitrile, nitriles such as acetonitrile and propionitrile, acid amides such as dimethylformamide and dimethylacetamide, sulfoxides such as dimethyl sulfoxide, water and a mixed solvent thereof. The reaction temperature is usually from room temperature to the boiling point of the solvent and is carried out for about 30 minutes to 20 hours.
Detailed synthesis methods are described in the examples.

  The thus-obtained emamectin derivatives are polymers such as bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), mussel hemocyanin (KLH), thyroglobulin (TG), and immunoglobulins. After forming a complex with a compound (protein), it is used as an antigen.

  The formation method of a composite_body | complex can be performed by a well-known method, and is not specifically limited. For example, the complex can be formed by reacting the carboxyl group of the emamectin derivative with the functional group of the polymer compound by a mixed acid anhydride method or an active ester method.

  The present invention provides an antibody against emamectin and its similar compounds obtained by using a complex of the above emamectin derivative and a polymer compound as an antigen. The antibody is an antibody having specificity for emamectin and its similar compounds.

  In general, antibodies are collected from blood from immunized rabbits or goats, and so-called polyclonal antibodies that separate and purify antibodies contained therein, and antibodies that are secreted by cloned hybridomas that have antibody-producing ability are separated and purified. There are monoclonal antibodies. In the present invention, a polyclonal antibody or a monoclonal antibody is included. Monoclonal antibodies are particularly preferred because of the high versatility of application, such as high sensitivity and high selectivity possessed by monoclonal antibodies, and further the reactivity that can be separated from a plurality of components by a combination of a plurality of monoclonal antibodies.

The monoclonal antibody preferably has the following IC ₅₀ value in order to clarify the specificity for emamectin and its similar compounds and the cross-reactivity for other substances. Here, the IC ₅₀ value refers to the concentration of a sample exhibiting 50% inhibition when a standard inhibition curve is obtained by indirect competitive ELISA or direct competitive ELISA. Here, ELISA refers to Enzyme-linked Immunosorbent Assay.

That is, when this antibody is to be measured for emamectin and its similar compounds, the IC ₅₀ for emamectin and its similar compounds by direct competitive ELISA is preferably ₅₀ ng / mL or less, more preferably 10 ng / mL or less.

  The antibody can be produced according to a usual production method (Current Protocol in Molecular Biology, chapters 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, a complex of an emamectin hapten and a polymer compound is formed according to a conventional method, and then the complex is immunized to a non-human animal such as a rabbit. It can be obtained from the sera of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, the complex is immunized to a non-human animal such as a mouse according to a conventional method, and a hybridoma cell prepared by cell fusion of the obtained spleen cell and myeloma cell is screened. It can be obtained by culturing an antibody-producing hybridoma (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 to 11.11).

  The antibody can be prepared by appropriately combining concentration and purification methods such as ultrafiltration, ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography, and affinity chromatography.

  The present invention also provides a hybridoma that produces the monoclonal antibody. Hereinafter, a method for producing a hybridoma using a mouse will be described in more detail.

As described above, the prepared antigen is dissolved in physiological phosphate buffer so as to be about 2 mg / mL, mixed with an adjuvant in an equal volume, and then administered intraperitoneally to Balb / c mice. Thereafter, booster immunization is carried out about every 2 weeks. The spleen of the mouse in which the antibody titer in the serum of blood collected from the tail blood vessel has been increased is removed, removed in a serum-free DMEM medium (Dulbecco's modified Eagle medium), and transferred to a new medium. The spleen cells are completely suspended in the medium. Centrifugation and supernatant removal are repeated several times to wash the cells, and then mixed with mouse myeloma cells (P3X63Ag8.653) at a cell number ratio of 5: 1 to 10: 1 (spleen cells: myeloma). After the cells are precipitated and the supernatant is removed, cell fusion is performed by slowly adding 50% polyethylene glycol (molecular weight 1500) while stirring. After cell fusion, the cells collected by centrifugation are suspended by adding HAT medium so that the number of cells is 5 × 10 ⁵ cells / mL, and the cell suspension is added to a 96-well plastic plate in an amount of 250 μL / well. Dispense and incubate in an incubator at 37 ° C., 5% carbon dioxide, humidified conditions. One week later, half of the medium in the well is replaced with HAT medium and cultured for 10 to 14 days. The activity of the antibody in the culture solution is examined by ELISA, and the hybridoma is cloned by limiting dilution for the cells of the well producing the target antibody. Cloning yields a stable hybridoma strain producing antibodies to emamectin and its analogs.

  In the present invention, a hybridoma was prepared by the above-described method, and EMT2-17H7-2 was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on September 27, 2005 (reception number FERM AP-20667).

  The hybridoma of the present invention can be cultured using a medium (for example, DMEM containing 10% fetal bovine serum), and the centrifuged supernatant of the culture solution can be used as a monoclonal antibody solution. In addition, ascites can be generated by injecting the hybridoma into the abdominal cavity of an animal from which the hybridoma is derived, and the obtained ascites can be used as a monoclonal antibody solution. These antibody solutions can be further purified and concentrated as described above.

  In addition, in the present invention, emamectin and its similar compounds can be easily measured by including an antibody that specifically binds to emamectin and its similar compounds, and immunological measurement of emamectin and its similar compounds described later. It can be suitably used for a method and a measurement kit as a measurement means.

  Furthermore, the present invention relates to an immunological assay method for emamectin and its similar compounds, characterized by using the antibody. The measuring method is not particularly limited as long as it uses a normal antigen-antibody reaction, and isotope immunoassay (RIA), enzyme immunoassay (EIA), fluorescence or luminescence measuring method, agglutination method, Examples include immunoblotting, immunochromatography, etc. (Meth. Enzymol., 92, 147-523 (1983), Antibodies Vol. II IRL Press Oxford (1989)). EIA is preferred from the standpoint of sensitivity and simplicity. From the viewpoint of versatility, ELISA is more preferable. Examples of the enzyme used for ELISA include peroxidase, alkaline phosphatase, β-galactosidase, luciferase and the like.

  Examples of the measurement method by ELISA include indirect competitive ELISA and direct competitive ELISA. For example, it can be performed by direct competition ELISA using the monoclonal antibody of the present invention as described below.

(1) The monoclonal antibody of the present invention is immobilized on a carrier.
The carrier to be used is preferably a microtiter plate having 96 holes, 48 holes, 192 holes or the like. For immobilization, for example, a buffer containing the immobilization antibody may be placed on a carrier and incubated. The concentration of the antibody in the buffer is usually about 0.01 μg / mL to 100 μg / mL. As the buffer solution, a known solution can be used according to the detection means.

(2) In order to prevent non-specific adsorption of the protein to the solid phase surface of the carrier, the solid phase surface portion where the antibody for immobilization is not adsorbed is blocked with a protein unrelated to the antibody.
As the blocking agent, BSA or skim milk solution, or commercially available Block Ace (Dainippon Pharmaceutical Co., Ltd.) can be used. Blocking is performed by adding the blocking agent to the carrier and, for example, incubating overnight at about 4 ° C. and then washing with a washing solution. Although there is no restriction | limiting in particular as a washing | cleaning liquid, The same buffer solution as said (1) can be used.

(3) A mixture is prepared by adding an enzyme-bound hapten obtained by binding an emamectin derivative and an enzyme to a sample containing various concentrations of emamectin and similar compounds.
The enzyme-linked hapten may be prepared by any method without particular limitation as long as it is a method for binding an emamectin derivative to an enzyme.

(4) The mixture of step (3) is reacted with the antibody-immobilized carrier obtained in step (2).
A complex between these and an immobilized carrier is formed by competitive inhibition reaction of emamectin and its similar compounds with enzyme-linked hapten. The reaction is carried out, for example, at about 25 ° C. for about 1 hour. After completion of the reaction, the carrier is washed with a buffer to remove the enzyme-bound hapten that did not bind to the immobilized antibody.
By measuring the amount of the immobilized antibody-enzyme-bound hapten complex, the amount of emamectin and its similar compounds in the sample is determined from a calibration curve prepared in advance.

(5) The amount of emamectin and its similar compounds can be calculated from the calibration curve by adding a chromogenic substrate solution that reacts with the labeling enzyme bound to the carrier and measuring the absorbance.
When peroxidase is used as the labeling enzyme, for example, a chromogenic substrate solution containing hydrogen peroxide and 3,3 ′, 5,5′-tetramethylbenzidine or o-phenylenediamine can be used. Usually, after adding a chromogenic substrate solution and reacting at room temperature for about 10 minutes, the enzyme reaction is stopped by adding sulfuric acid. When 3,3 ′, 5,5′-tetramethylbenzidine is used, the absorbance at 450 nm is measured. When o-phenylenediamine is used, the absorbance at 492 nm is measured. In order to correct the background value, it is desirable to simultaneously measure the absorbance at 630 nm.
When alkaline phosphatase is used as the labeling enzyme, for example, a method of coloring with p-nitrophenyl phosphate as a substrate, adding an NaOH solution to stop the enzyme reaction, and measuring the absorbance at 415 nm can be mentioned.
For the absorbance of the reaction solution to which no emamectin and its similar compounds are added, the rate of decrease in the absorbance of the solution reacted with the antibody by adding emamectin and its similar compounds is calculated as the inhibition rate. The concentration of emamectin and its similar compound in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which emamectin and its similar compound at known concentrations are added.

  In another embodiment, emamectin and its similar compounds can be measured by indirect competitive ELISA according to the following procedure.

(1) An immobilized antigen is immobilized on a carrier.
The carrier to be used is not particularly limited as long as it is a carrier used in ordinary ELISA, but a microtiter plate having 96 holes or the like is preferable. For immobilization, for example, a buffer containing the immobilization antigen may be placed on a carrier and incubated. The concentration of the antigen in the buffer is usually about 0.01 μg / mL to 100 μg / mL. As the buffer solution, a known solution can be used according to the detection means.

  (2) In order to prevent nonspecific adsorption of the protein to the solid phase surface of the carrier, the solid phase surface portion where the antigen for immobilization is not adsorbed is blocked with a protein unrelated to the antigen. As the blocking agent, BSA or skim milk solution, or commercially available Block Ace (Dainippon Pharmaceutical Co., Ltd.) can be used. Blocking is performed by adding the blocking agent to the carrier and, for example, incubating overnight at about 4 ° C. and then washing with a washing solution. Although there is no restriction | limiting in particular as a washing | cleaning liquid, The same buffer solution as said (1) can be used.

(3) Samples containing various concentrations of emamectin and its analogs and the monoclonal antibody solution of the present invention are added to the solid phase surface treated in (1) and (2) above, and the antibody is added to the immobilized antigen, and It reacts competitively with emamectin or a similar compound to produce an immobilized antigen-antibody complex and an antibody complex against emamectin or a similar compound.
The reaction can usually be performed at room temperature for about 1 to 2 hours.
Emamectin and its similar compounds are insoluble in water, and therefore the reaction solution can contain various organic solvents. What is necessary is just to select an organic solvent and its content as the said organic solvent in the range which dissolves emamectin and its analog, and does not inhibit an antigen-antibody reaction. Specific examples include methanol, dimethyl sulfoxide, N, N-dimethylformamide, and the content is about 5 to 50% by weight.

(4) By measuring the amount of the immobilized antigen-antibody complex, it is possible to determine the amount of emamectin and similar compounds in the sample from a calibration curve prepared in advance.
The amount of the immobilized antigen-antibody complex can be measured by adding an enzyme-labeled secondary antibody (an antibody that recognizes a mouse antibody). For example, when a mouse monoclonal antibody is used as an antibody against emamectin and its similar compounds, an enzyme-labeled anti-mouse-goat antibody (for example, peroxidase or alkaline phosphatase) is used to react with an antibody against emamectin and its similar compounds bound to a carrier. It is desirable to let them. The reaction may be performed under the same conditions as in (3) above. After the reaction, wash with buffer.

  (5) Add a chromogenic substrate solution that reacts with the labeling enzyme of the secondary antibody bound to the carrier, add a chromogenic substrate solution that reacts with the enzyme of the enzyme-bound hapten in the same manner as the direct competitive inhibition ELISA method described above, and measure the absorbance. Thus, the amount of emamectin and its similar compounds can be calculated from the calibration curve.

In the measurement method of the present invention, the sample can be pretreated according to the measurement object to obtain a sample, and then subjected to the step (3) of the direct competitive ELISA or the indirect competitive ELISA.
For example, when the measurement object is soil or food, all methods capable of extracting emamectin and its similar compounds can be used. The extract is dissolved in methanol, diluted with a buffer solution, and used as a measurement sample. As a simple method, a sample extracted with methanol and diluted with a buffer can be used as it is. Specifically, for example, 5 mL of methanol is added to 5 g of a sample and shaken for 20 minutes. After standing at room temperature for 30 minutes, the upper 2 mL of the extract is centrifuged at 2500 Rpm for 10 minutes. The supernatant is diluted 1: 4 with distilled water and subjected to step (3).
Further, when the measurement object is environmental water, an extraction step is not required, and it can be directly used as a measurement sample.

  The present invention also relates to a kit for measuring emamectin and its similar compounds, characterized by using the antibody and the immunological measurement method. Specifically, this measurement kit is prepared from an enzyme-labeled secondary antibody or enzyme-labeled emamectin derivative (antigen), a buffer solution, a detection reagent and / or a standard solution of emamectin and its similar compounds, depending on the measurement method. Composed. Further preferably, the present measurement kit can be used in an ELISA method, and when used in the direct competitive ELISA method, an antibody against a solid phased emamectin and its similar compounds, a carrier that holds the solid phase antibody. Enzyme-labeled antigen, standard solution, detection reagent, reaction stop solution and washing solution. The enzyme-labeled antigen and standard solution may be lyophilized. By using the measurement kit having such a configuration and using the measurement method by the direct competition ELISA and the indirect competition ELISA, it is possible to easily and rapidly measure emamectin and its similar compounds.

<Example>
EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1 (4 "-epi- [N- (4- carboxy-propionyl) -N- methylamino] -4" - Synthesis of deoxy avermectin B1a / B1b (emamectin hapten))
A 20 mL Schlenk tube was charged with 30 mg (0.034 mmol) of emamectin, 6.0 mg (0.051 mmol) of succinic acid and 4.0 mg (0.034 mmol) of 4-dimethylaminopyridine, and purged with argon. Further, 2 mL of methylene chloride was added under a nitrogen stream and cooled to 0 ° C. with an ice bath. Next, 14.0 mg (0.068 mmol) of dicyclohexylcarbodiimide was added and stirred at room temperature for 12 hours. The obtained reaction mixture was isolated and purified by silica gel chromatography (methylene chloride / methanol = 10/1) to obtain 8.0 mg of a colorless transparent viscous liquid product.

[Example 2] (Preparation of immunogen)
As an immunogen, a conjugate of scallop hemocyanin (KLH) and the emamectin hapten of the present invention was prepared using an active ester method.
4 prepared in Example 1 "-epi- [N- (4- carboxy-propionyl) -N- methylamino] -4" - deoxy-avermectin B1a / B1b (emamectin hapten) 1.5 mg, N-hydroxysuccinimide 1.2mg And 1.9 mg of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride are dissolved in 200 μL of N, N-dimethylformamide, and this solution is allowed to stand in the dark at 25 ° C. for 1.5 hours. A hapten solution was obtained.
Separately, 10 mg of KLH was added to 1 mL of 0.1 M borate buffer (pH 8.0) and stirred overnight to obtain a KLH solution.
To this KLH solution, the previously prepared emamectin hapten solution was gradually added dropwise and stirred for 1.5 hours at room temperature in the dark. After completion of the reaction, the mixture was dialyzed against physiological phosphate buffer (PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) at 4 ° C. for 2 days, and stored at −40 ° C. The conjugate of emamectin hapten and KLH thus obtained was used as an immunogen.

[Example 3] (Preparation of immobilized antigen)
A conjugate of bovine serum albumin (BSA) and emamectin hapten as a solid-phased antigen was prepared using the active ester method.
Emamectin hapten 1.5 mg, N-hydroxysuccinimide 1.2 mg and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride 1.9 mg were dissolved in 200 μL of N, N-dimethylformamide, and this solution was dissolved. It was left in a dark place at 25 ° C. for 1.5 hours to obtain an emamectin hapten solution.
Separately, 10 mg of BSA was added to 1 mL of 0.1 M borate buffer (pH 8.0) and stirred overnight to obtain a BSA solution.
To this BSA solution, the previously prepared emamectin hapten solution was gradually added dropwise and stirred at room temperature in the dark for 1.5 hours. After completion of the reaction, the mixture was dialyzed against PBS buffer (PBS, 10 mM phosphate buffer, 150 mM NaCl. PH 7.0) overnight at 4 ° C., and stored at 30 ° C. The conjugate of emamectin hapten and BSA thus obtained was used as the immobilized antigen.

[Example 4] (Immunity to mice)
The immunogen prepared in Example 2 was dissolved in PBS buffer (0.4% BSA, 10 mM sodium phosphate buffer (hereinafter referred to as “NaPB”), 150 mM NaCl, pH 7.0) to 2 mg / mL. This was mixed with an equal amount of complete adjuvant (trade name: Freund's complete adjuvant; FCA) to make an emulsion, and 100 μL thereof was intraperitoneally administered to 7-week-old female BALB / c mice. In the same procedure, an equal volume of incomplete adjuvant (trade name: Freund's incomplete adjuvant; FICA) was mixed and boosted 2 weeks later. Further, 0.25 mg / mL of the immunogen was mixed with an equal volume of incomplete adjuvant, and 100 μL thereof was boosted every 2 weeks. After four immunizations, blood was collected from the fundus and the antibody titer in the serum and inhibition against emamectin were confirmed by the indirect competition method.

[Example 5] (Indirect competition test of antiserum)
(1) One week after four immunizations, blood was collected from the fundus of the mouse, and the serum portion was diluted 100-fold with 10% Block Ace-PBS buffer to obtain an antiserum dilution.
(2) Prepare the solid-phased antigen prepared in Example 3 to 5.0 μg / mL with PBS buffer, dispense 100 μL / well into a 96-well microplate, and let stand at 4 ° C. overnight. Phased. Next, after removing the solution by suction, 25% Block Ace-PBS buffer solution was dispensed at 300 μg / well, and allowed to stand at 4 ° C. overnight to effect blocking to obtain a solid phase plate.
(3) The antiserum dilution was sequentially diluted with 10% Block Ace-PBS buffer, dispensed at 100 μL / well onto a solid-phased plate, allowed to stand at 20 ° C. for 1 hour, and then washed.
(4) A 10% PBS solution of horseradish peroxidase (HRP) -conjugated anti-mouse IgG goat antibody (second antibody) diluted 4000 times with Block Ace was added at 100 μL / well, and allowed to stand at room temperature for 1 hour, then the plate Was washed.
(5) A HRP substrate solution (0.1 M sodium acetate buffer (pH 5.5) supplemented with 100 μg / mL 3,3 ′, 5,5′-tetramethylbenzidine and 0.006% hydrogen peroxide) 100 μL / well was added, and the mixture was reacted and colored at 25 ° C. for 10 minutes. The enzyme reaction was stopped by adding 100 μL / well of 1N sulfuric acid, and the absorbance at 450 nm was measured. The antiserum of two mice (No1, No2) having an absorbance of about 1.0 at a final dilution factor of 800 was tested for inhibition of emamectin as follows.
(6) Antiserum solution diluted 400 times with 0.2% BSA-PBS buffer and emamectin standard solution of various concentrations diluted with 10% Block Ace BSA-PBS buffer were mixed in equal amounts, and the solid-phase plate Was dispensed at 100 μL / well and allowed to stand at 25 ° C. for 1 hour.
(7) Subsequent operations were performed in the same manner as (4) and (5). The results of the antiserum indirect inhibition test are shown in FIG.

[Example 6] (Production of monoclonal antibody-producing hybridoma)
After confirming that the titer was sufficiently high, an immunogen 10 μg / 100 μL / PBS was administered from the tail vein as a final immunization one week later. Three days later, the spleen was removed from the mouse and subjected to cell fusion.
Excess tissue fragments were excised from the excised spleen in serum-free DMEM medium (Dulbecco's modified Eagle medium), and then the cells were completely removed from the spleen and suspended in the medium. Allow to settle for 5 minutes to settle large floating tissue pieces. Collect the cell suspension in a centrifuge tube, centrifuge at 1500 rpm, aspirate and remove the supernatant, and add new serum-free DMEM to float the cells. I let you. This operation was repeated twice.
The myeloma cells (P3X63Ag8.653) that had been cultured in advance were collected, centrifuged, removed from the supernatant, and resuspended twice in serum-free DMEM medium.
Each cell number was counted, mixed so that the ratio of spleen cells to myeloma cells was 10: 1 to 7.5: 1, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed by suction.
While vigorously stirring the centrifuge tube, 2 mL of a 50% polyethylene glycol (molecular weight 1500) solution was added over about 60 seconds. About 10 mL of serum-free DMEM was then added over 3-4 minutes with stirring.
The centrifuge tube is centrifuged at 1000 rpm for 5 minutes to completely remove the supernatant, and HT medium (Hypoxanthine, thymidine, 10% fetal bovine serum-containing DMEM is added so that the number of splenocytes becomes 2.5 × 10 ⁶ cells / mL. The suspension was suspended in a medium), dispensed at 100 μL / well into a 96-well culture plate, and the culture was started under conditions of 37 ° C., 8% carbon dioxide and humidification.
On the next day, about 40 μL / well of HAT medium (DOX medium containing hypoxanthine, thymidine, aminopterin, 10% fetal bovine serum) was added, and observation was continued until myeloma cells were killed and hybridoma cell colonies were formed. Thereafter, HT medium was added while observing the state of the cells.
Ten days after the start of the culture, the culture solution was collected, and wells producing antibodies against emamectin were selected by the indirect competitive inhibition method, and the culture scale was sequentially increased to 96, 48, and 24 wells.
Cloning by the limiting dilution method was performed at the stage of 24 wells to obtain a monoclonal antibody-producing hybridoma strain [EMT2-17H7-2] against emamectin.

[Example 7] (Preparation of monoclonal antibody)
The hybridoma strain obtained in Example 6 was cultured in DMEM containing 10% fetal bovine serum, about 2 × 10 ⁷ cells were injected into the abdominal cavity of BALB / c female mice, and ascites fluid was collected.
The obtained ascites was subjected to IgG purification using a protein G column to obtain an anti-emamectin antibody MET2-17H7-2.

[Example 8] (Preparation of conjugate of emamectin hapten and HRP)
For use as a tracer in a direct competitive ELISA, a conjugate of emamectin hapten and HRP was prepared by the active ester method. Emamectin hapten 1.5 mg, N-hydroxysuccinimide 1.2 mg and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride 1.9 mg were dissolved in 200 μL of N, N-dimethylformamide, and this solution was dissolved. It was left in a dark place at 25 ° C. for 1.5 hours to obtain an emamectin hapten solution.
Separately, 10 mg of HRP was added to 1 mL of 0.1 M borate buffer (pH 8.0) and stirred overnight to obtain an HRP solution.
The previously prepared emamectin hapten solution was gradually added dropwise to this HRP solution and stirred at room temperature in the dark for 1.5 hours. After completion of the reaction, the mixture was dialyzed against physiological phosphate buffer (PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) overnight at 4 ° C., and stored at 30 ° C. Purification was performed by gel filtration, and the concentration was measured by a DC protein assay.

[Example 9] (Measurement of emamectin by direct competitive ELISA)
(1) Anti-mouse IgG goat antibody was diluted to 10 μg / mL with PBS buffer (10 mM NaPB, 150 mM NaCl), dispensed at 100 μg / well into a 96-well microplate, and allowed to stand overnight at 4 ° C. Phased. Next, after removing the solution by suction, blocking was performed by dispensing 300 μg / well of PBS buffer (0.4% BSA, 10 mM NaPB, 150 mM NaCl, pH 7.0) and allowing to stand at 4 ° C. overnight. Thereafter, the blocking solution was removed by suction.
(2) The monoclonal antibody EMT2-17H7-2 obtained in Example 7 was diluted to 0.05 μg / mL with PBS buffer (0.2% BSA, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0). The anti-mouse IgG goat antibody-immobilized plate prepared in (1) was dispensed at 100 μg / well, allowed to stand at 20 ° C. for 1 hour, and then washed with PBS to obtain an antibody-immobilized plate.
(3) A conjugate of HRP and emamectin hapten was diluted to 0.12 μg / mL with PBS buffer (0.4% BSA, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) to obtain an HRP diluted solution.
(4) Equal volume of emamectin standard solution (0.125, 0.25, 0.5, 1.0, 2.5, 5 and 10 μg / mL) dissolved in 10% methanol solution with HRP diluted solution Then, 100 μg / well of the mixture was added to the antibody-immobilized plate obtained in (2), allowed to stand at 20 ° C. for 1 hour, and then washed with PBS.
(5) HRP substrate solution (100 μg / mL of 3,3 ′, 5,5′-tetramethylbenzidine and 0.1 M sodium acetate buffer (pH 5.5) supplemented with 0.006% hydrogen peroxide) 100 μL Was added to the well and incubated at 25 ° C. for 10 minutes. Then, 100 μL of 1N sulfuric acid was added to the well to stop the enzyme reaction, and the absorbance at 450 nm was measured with a microplate reader.
The standard inhibition curve of emamectin by the direct competitive inhibition method for the monoclonal antibody EMT2-17H7-2 solution is shown in FIG. The memectin measurable range by direct competitive ELISA using the monoclonal antibody EMT2-17H7-2 solution is 0.25 ng / mL to 3 ng / mL, and the value indicating 50% inhibition (IC ₅₀ value) is 0.7 ng. / ML.

  From the above results, the direct competition ELISA method using the monoclonal antibody obtained by using the emamectin derivative of the present invention makes it possible to greatly simplify the emamectin analysis and shorten the measurement time, thereby allowing a large number of specimens to be rapidly, easily and easily obtained. It turns out that it can measure at low cost.

[Example 10] (Cross-reactivity of antibody to emamectin structural analog)
The cross-reactivity of antibody EMT2-17H7-2 was examined for pesticides and metabolites similar in chemical structure to emamectin and antibiotic pesticides. For cross reactivity, the IC ₅₀ value of the test compound was determined in the same manner as described in Example 9, and the cross reaction rate was calculated by the following formula.
Cross-reactivity rate (%) = _{(IC 50} values _{IC 50} values / test compound emamectin) × 100
As a result, it showed high cross-reactivity with the acaricide abamectin [Chemical 5] (unregistered in Japan) and the amino form, formylamino form and N-methylformylamino form of emamectin metabolites, whereas milbemectin [Chemical Formula 6], spinosad, and streptomycin all have a cross-reactivity of 0.1% or less, and the antibody EMT2-17H7-2 is emamectin, its analogs abamectin and metabolite amino, formylamino, It can be said to be an antibody specific for N-methylformylamino.
Table 1 shows the cross-reactivity with emamectin metabolites (amino, formylamino, N-methylformylamino) and abamectin in direct competition ELISA using antibody EMT2-17H7-2.

（式中Ｒは、Ｃ_２Ｈ_５（アバメクチンＢ１ａ）とＣＨ_３（アバメクチンＢ１ｂ）を表す） Here, abamectin is a 4: 1 mixture of abamectin B1a and B1b, and represents a compound represented by the following formula (5).

(Wherein R represents C ₂ H ₅ (abamectin B1a) and CH ₃ (abamectin B1b))

（式中Ｒは、Ｃ_２Ｈ_５（ミルベメクチンＡ４）とＣＨ_３（ミルベメクチンＡ３）を表す） Here, milbemectin is a 3: 7 mixture of milbemectin A3 and A4, and represents a compound represented by the following formula (6).

(Wherein R represents C ₂ H ₅ (milbemectin A4) and CH ₃ (milbemectin A3))

The inhibition curve (mouse No1, No2) with respect to emamectin in the indirect competition ELISA method using antiserum is shown.

The inhibition curve with respect to emamectin in the direct competition ELISA method using antibody EMT2-17H7-2 is shown.

Claims (9)

Following formula (1):

(Wherein R ₁ is —N (CH ₃ ) CO (CH ₂ ) ₂ COOH, R ₂ is a methyl group) and the above formula (1) (wherein R ₁ is —N ( _{CH 3) CO (CH 2)} 2 COOH, R 2 is a mixture of the compounds represented by ethyl groups) and haptens, emamectin by using the complex between said hapten and polymer compound as an antigen, the amino compound emamectin the method of manufacturing an antibody against the formylamino body emamectin, and N- methyl-formylamino emamectin. The production method according to claim 1, wherein the polymer compound is KLH. An emamectin, amino emamectin, formylamino emamectin, and N-methylformylamino emamectin antibody obtained by the production method according to claim 1 or 2, or a fragment thereof capable of binding to an antigen.   The antibody or fragment thereof according to claim 3, wherein the antibody or fragment thereof to emamectin, amino emamectin, formylamino emamectin, and N-methylformylamino emamectin is a polyclonal antibody or fragment thereof, or a monoclonal antibody or fragment thereof. A hybridoma producing the antibody according to claim 3 or 4. The hybridoma according to claim 5, wherein the hybridoma is EMT2-17H7-2 (FERM P-20667). A monoclonal antibody produced from a hybridoma represented by EMT2-17H7-2 (FERM P-20667). A method for immunological measurement of emamectin and an analog thereof, comprising the antibody or fragment thereof according to claim 3, 4 or 7. A kit for measuring emamectin and its similar compounds, comprising the antibody or fragment thereof according to claim 3, 4 or 7.

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