JPH08136540A - Immunological analytic method - Google Patents

Abstract

PURPOSE: To obtain a method for analyzing a pyrethroid based compound in the environmental water, soil, plant or food conveniently, economically and quickly. CONSTITUTION: The immunological analytic method for 2-(4-ethoxyphenyl)-2- methylpropyl 3-phenoxybenzyl ether and 2-(4-hydroxyphenyl)-2-methylpropyl 3-phenoxybenzyl ether employs a monoclonal antibody being bonded specifically to 2-(4-ethoxyphenyl)-2-methylpropyl 3-phenoxybenzyl ether and 2-(4- hydroxyphenyl)-2-methylpropyl 3-phenoxybenzyl ether.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a pyrethroid insecticide 2- (4-ethoxyphenyl) -2-methylpropyl.
The present invention relates to a monoclonal antibody that specifically reacts with 3-phenoxybenzyl ether (hereinafter referred to as etofenprox) and its derivative, a hybridoma producing the monoclonal antibody, and a method for analyzing etofenprox and its derivative using the monoclonal antibody.

[0002]

BACKGROUND OF THE INVENTION Etofenprox has insecticidal activity against many kinds of insects, including planthoppers and leafhoppers that have acquired drug resistance, and has good photostability and low fish toxicity. It is a pesticide used in paddy fields. In addition, since the compound has low toxicity to mammals and has an effect on internal or external parasites parasitic on animals and humans, it is expected to be used as an anthelmintic agent or a medicine. However, in consideration of such widespread use, it is necessary to first accurately measure the amount of etofenprox remaining in them in order to ensure the safety of the environment and food. Generally, residual pesticide analysis methods have been established for each crop at the time the pesticide was registered, and mainly after extracting the pesticide from the sample and purifying it, the content of the pesticide in the extract is determined by gas chromatography or liquid chromatography. It is measured by graphy. However, in the case of etofenprox, the sensitivity is poor if it is directly measured. Therefore, it is measured by gas chromatography equipped with an electron capture detector after induction with 3-phenoxybenzyl iodide. Although this measuring method has no problem in terms of accuracy, it has drawbacks in that cleaning up of a sample before measurement is complicated and it takes a long time, and that measuring devices and equipment require high cost. Especially in the analysis of residual pesticides, since the number of analyzes is large, simplicity and speed are important in addition to accuracy. Further, it is required that the analysis can be easily performed without using an expensive measuring device. In addition, a simple analysis method is desired similarly for the analysis of etofenprox in a biological sample. On the other hand, in recent years, immunological detection methods have been applied to medium and low molecular weight scientific substances, and various application studies have been conducted.Etofenprox and its derivatives are immunologically and selectively selected. It was not considered to measure.

[0003]

Therefore, the present invention provides a simple and rapid immunological analysis method for etofenprox and its derivatives without using an expensive measuring device for etofenprox and its derivatives. The task is to do. Further, the present invention avoids the use of an organic solvent to extract etofenprox and its derivatives from a test sample when immunologically analyzing etofenprox and its derivatives remaining in crops and soil. Therefore, it is an object of the present invention to provide an antibody that is resistant to an organic solvent and is capable of antigen-antibody reaction even in the presence of an organic solvent.

[0004]

[Means for Solving the Problems] The present inventors have conducted extensive studies for the purpose of immunologically measuring etofenprox and its derivatives. The present invention has been completed by successfully producing a monoclonal antibody that reacts and shows resistance even in the presence of an organic solvent.

That is, the present invention provides 2- (4-ethoxyphenyl) -2-methylpropyl-3-phenoxybenzyl ether and 2- (4-hydroxyphenyl) -2.
-Methylpropyl 3-phenoxybenzyl ether (hereinafter referred to as etofenprox and its derivatives).
The present invention also relates to a hybridoma producing the above monoclonal antibody, and a method for immunological analysis of etofenprox and its derivatives using the above monoclonal antibody.

The isolation of hybridomas producing the monoclonal antibody according to the present invention, preparation of the monoclonal antibody, and immunological analysis method will be described below in this order. The isolation of the hybridoma producing the monoclonal antibody according to the present invention can be carried out by a conventional method, for example, the method described in the Second Biochemistry Laboratory, Immunobiochemistry Research Method (edited by the Japanese Biochemical Society). The method for separating a hybridoma producing a monoclonal antibody will be specifically described below. First as immunogen, 2
-(4-hydroxycarbonylmethyloxyphenyl)
A hapten compound of 2-methylpropyl-3-phenoxybenzyl ether bound to a biopolymer (eg, serum albumin or immunoglobulin) is used.

Mammals and birds (for example, mice, rats, chickens, etc.) are immunized by the in vivo immunization method using these immunogen solutions. For example, an immunogen solution is emulsified and mixed with an equal amount of Freund's complete or incomplete adjuvant, and the mixture is subcutaneously administered to a mouse (primary immunization). Thereafter, the same operation is performed at intervals of 2 to 4 weeks, and immunization is performed several times. A few days after the final immunization, the spleen is aseptically removed, crushed with a stainless mesh or the like to prepare spleen cells, and used in the cell fusion step.

As the other parent cell of the cell fusion, myeloma cells (myeloma cells), various known cell lines such as p3.NS-1 / 1.Ag4.1 (Eu) can be used.
r. J. Immunol. , 5; 511-517 (19
75)), SP2 / 0-Ag14 (Nature, 27).
6; 269-270 (1978)), P3-X63-A.
g8.653 (J. Immunol., 123; 154)
8-1550 (1979)), NSO (Methods)
in Enzymology, 73, 3 (198
1)) and the like can be used.

[0009] Cell fusion can be carried out by an ordinary method, for example, using a known fusion promoter (polyethylene glycol etc.).
About 1/5 of myeloma cells to mouse spleen cells
The number used is about 1/10. As a cell fusion medium,
For example, a commercially available polyethylene glycol molecular weight for cell fusion 1500 (Boehringer Mannheim) can be used. The fusion is performed by thoroughly mixing the immune spleen cells and the myeloma cells in the above medium.

Subsequently, cells other than the hybridomas are removed using a selection medium (for example, HAT medium), and the presence or absence of antibody production in the hybridoma culture supernatant is determined by, for example, ELI.
It measures by SA method and isolate | separates the target hybridoma. A hybridoma producing an antibody can be confirmed to be a monoclonal antibody-producing cell by cloning it using a cell cloning method. When selecting a hybridoma that produces a monoclonal antibody resistant to an organic solvent, etofenprox is added to an aqueous solution containing an organic solvent at various concentrations, and the monoclonal antibody is added to By confirming that the reaction proceeds normally, the hybridoma producing the organic solvent-resistant monoclonal antibody is isolated. The isolated hybridoma can be subcultured in an ordinary medium, and can be easily stored for a long period of time in liquid nitrogen or the like.

The monoclonal antibody according to the present invention also comprises
It can be easily prepared by culturing a hybridoma that produces the desired monoclonal antibody. Hybridoma culture may be performed in vitro, for example, with 5
It can be performed using any medium suitable for culturing under conditions of% carbon dioxide and 37 ° C., but is preferably a medium containing 10% fetal bovine serum (hereinafter FBS) in Iscove's modified Dulbecco's medium (hereinafter, Iscove's medium) (Hereinafter Iskoff / 10
% FBS medium) is used. In the case of in vivo, it is preferable to culture in the abdominal cavity of a mouse, for example.

Further, the above culture solution or ascites fluid may be directly used as an antibody solution, or the desired monoclonal antibody may be separated and purified using these as starting materials. In order to separate and purify the monoclonal antibody from the culture solution or mouse ascites, general methods for protein isolation and purification can be used. Examples of such a method include salting out with ammonium sulfate, ion exchange chromatography, molecular sieve column chromatography using a molecular sieve gel, affinity chromatography using a protein A or protein G binding polymer, dialysis and the like.

Since the immunological analysis method according to the present invention is carried out using the above-mentioned monoclonal antibody, etofenprox and its derivatives can be accurately analyzed. In addition, since the analysis method of the present invention uses an organic solvent-resistant monoclonal antibody, an accurate antigen-antibody reaction proceeds even in an organic aqueous system containing an organic solvent capable of sufficiently dissolving etofenprox and its derivatives to be analyzed. Can be made.

The method of the present invention uses a monoclonal antibody that reacts with etofenprox and its derivatives,
And preferably using an organic solvent resistant antibody (thus carrying out the antigen-antibody reaction in the presence of an organic solvent)
Other than that, it can be applied as it is to a conventionally known immunological analysis method. The analysis method includes, for example, direct labeling of an antibody that reacts with etofenprox and its derivative and is resistant to the organic solvent to obtain the immobilized antigen and etofenprox and its derivative in the sample. There is a method called a direct competition method for causing a competitive reaction, or a method called an antigen labeling method for immobilizing the above antibody and competing the labeled antigen with etofenprox or a derivative thereof in a sample. Here, the method of the present invention will be described below with respect to a measurement method which is usually called an indirect competitive method among antibody labeling methods.

When measuring etofenprox and its derivatives according to the present invention, (1) a hapten and a biopolymer such as 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl-3-phenoxybenzyl ether (for example, Step of immobilizing a conjugate with serum albumin or immunoglobulin (hereinafter referred to as immobilizing antigen) (2) Etofenprox or its derivative dissolved in an organic solvent (particularly, a water-miscible organic solvent) , A step of mixing and reacting with an antibody that reacts to etofenprox and its derivative and is resistant to an organic solvent (hereinafter referred to as the first antibody) (3) Reaction of the first antibody with etofenprox and its derivative (4) First antibody bound to the solid-phased antigen and etofene in the sample A step of separating the first antibody bound Rocks and its derivatives (5) labeled anti-mouse antibody (hereinafter referred to as second antibody)
Is contacted with the first antibody bound to the immobilized antigen (6) The second antibody bound to the immobilized first antibody and the first antibody
A step of separating the second antibody that is not bound to the antibody (7) A step of measuring a signal from the label of the second antibody that is bound to one of the two separated in step (6), preferably the first antibody And a method for analyzing etofenprox or a derivative thereof in a sample.

The organic solvent used in the present invention is a solvent capable of dissolving etofenprox and its derivatives. The use of a water-miscible organic solvent is preferable because the antigen-antibody reaction can be carried out after appropriately diluting the organic solvent extract with water. As the organic solvent, for example, an alcohol compound (for example,
Mention may be made of lower alcohols having 1 to 3 carbon atoms, in particular methyl alcohol, ethyl alcohol,), ketone compounds (in particular acetone), acetonitrile, dimethyl sulphoxide or mixtures thereof.

When the method of the present invention is carried out, a known amount of labeled secondary antibody can be used to confirm or quantify etofenprox and its derivatives. For labeling an antibody, a known label, for example, a radioisotope (for example,
³² P, ³⁵ S, ³ H), enzymes (eg peroxidase, alkaline phosphatase), vitamins (eg
Biotin), a fluorescent substance (for example, FITC), a chemiluminescent substance (for example, acridinium) or the like can be used.

The labeled second antibody can be added to the reaction system after the step of contacting the first antibody with etofenprox or its derivative and the solid-phased antigen is completed. That is, after contacting the first antibody with etofenprox and its derivatives and the solid-phased antigen, the first antibody that did not bind to the solid-phased antigen is separated and removed, and then the labeled second antibody is added, Labeled second against the first antibody bound to the phased antigen
The antibody binds.

In the method of the present invention, the first antibody bound to the solid-phased antigen is separated after the competitive reaction between the first antibody, etofenprox or its derivative, and the solid-phased antigen is completed. Separation can be performed, for example, by filtration, centrifugation, or washing with a buffer solution. Further, after the reaction between the first antibody bound to the solid-phased antigen and the labeled second antibody is completed, the labeled second antibody that has not bound to the first antibody is separated, and subsequently, for example, the first antibody The signal from the label of the labeled second antibody bound to the antibody is measured. When measuring the signal, it is preferable to change the reaction system containing the labeled second antibody to conditions preferable for signal measurement. For example, when a fluorescent or chemiluminescent substance is used as the label, the signal is measured under the condition that quenching does not occur.

[0020]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1 Hapten compound 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3
-Preparation of phenoxybenzyl ether 3-phenoxybenzyl 2- (4-hydroxyphenyl) -2-methylpropyl ether 3.0 g, sodium hydride 0.36 g, acetonitrile 20 ml about 20
After heating under reflux for 1 minute, 2 ml of methyl bromoacetate at room temperature
Was added dropwise, and the mixture was stirred at 60 ° C. for 20 minutes. The reaction solution was concentrated, about 50 ml of ethyl acetate was added, washed with water, dried, concentrated, separated and purified by silica gel column chromatography, and 3-phenoxybenzyl 2- (4-methoxycarbonylmethyloxyphenyl) -2-methylpropyl ether. 3.0 g was obtained. NMR: δTMS (CDCl ₃ ) (ppm): 1.30 (6H, s), 3.40 (2H, s),
3.79 (3H, s), 4.43 (2H, s), 4.59 (2H, s), 6.81-7.35 (13H, m) IR: ν _MAX (neat) (cm ^-1 ): 1763,1585,1488,1252, 12
13,1080 Refractive index: 1.5641 (21.6 ° C.) 3-phenoxybenzyl 2- (4-methoxycarbonylmethyloxyphenyl) -2-methylpropyl ether 2.2 g, sodium hydroxide 2.5 g, water 7. 5g,
10 ml of ethylene glycol was heated under reflux for 2.5 hours. Dilute hydrochloric acid was added to the reaction solution to make it acidic, extracted with ethyl acetate, washed with water, dried, concentrated, separated and purified by silica gel column chromatography, and 0.9 g of 3-phenoxybenzyl 2- (4-hydroxycarbonylmethyloxyphenyl). 2-Methylpropyl ether was obtained. NMR: δTMS (CDCl ₃ ) (ppm): 1.30 (6H, s), 3.41 (2H, s),
4.43 (2H, s), 4.62 (2H, s), 6.81-7.35 (13H, m), 9.10-9.90 (1
H, br) IR: ν _MAX (neat) (cm ^-1 ): 3041,1733,1585,1488,12
50,1188,1076 Refractive index: 1.5680 (21.6 ℃)

Example 2 Preparation of monoclonal antibody-producing hybridoma and preparation of monoclonal antibody solution 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl
After dissolving 25 mg of ether in 2 ml of anhydrous dioxane, 50 μl of N-methylmorpholine was added, and the mixture was stirred at 10 ° C. for 10 minutes. Then, 20 μl of isobutyl chlorocarbonate was added little by little and stirred for 20 minutes. on the other hand,
80 mg of bovine serum albumin was dissolved in 1 ml of distilled water, adjusted to pH 9 with 1N NaOH, 2.6 ml of dioxane was added dropwise at 10 ° C., and p was added again with 1N NaOH.
Prepared to H9. To this bovine serum albumin solution, the previously prepared 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether solution was gradually added dropwise while keeping the pH at 9 with 1N NaOH, and the solution was added at 4 ° C at 4 ° C. After reacting for a time, it was dialyzed against distilled water. The conjugate of 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether thus prepared and bovine serum albumin was lyophilized and then used as an immunogen and an antigen for analysis.

Immunization of mice was carried out with Dalbeco PB using 100 μg of a conjugate of 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether and bovine serum albumin as an immunogen.
It was dissolved in 50 µl of S (-), mixed with an equal amount of Freund's complete adjuvant, and then subcutaneously inoculated into mice. One month later and two months later, 1/5 of the initial immunization amount was boosted.

Cell fusion was carried out using the spleen of a mouse 3 days after the final immunization. First, the excised spleen was washed three times with Iscove's medium, and the spleen cells in the tissue were taken out in Iscove's medium. The cell suspension was filtered through a stainless mesh to remove large solids, and the resulting spleen cells and mouse myeloma cells P3-X63-Ag8.653 were washed three times with Iscove's medium, and then the number of cells was determined by the number of cells. Mix 5: 1 (spleen cells: myeloma cells) and centrifuge (1,
(000 rpm, 5 minutes) to obtain cell sediment. 37 to this
1 ml of 50% polyethylene glycol (1,500 molecular weight) solution that had been warmed to 0 ° C. was slowly added over 60 seconds while rotating the centrifuge tube to fuse the cells. Cell fusion is performed by adding Iscove's medium 1 to the centrifuge tube in which the fusion is progressing.
After adding 0 ml over 30 seconds per ml, FBS
Was stopped by adding 1 ml. Centrifuge this,
The obtained cells were suspended in HAT medium (Hypoxanthine / aminobuterin / thymidine was added to Iscove / 10% FBS medium) so that the cell number was 2 × 10 ⁵ cells / ml. This cell suspension was dispensed into a 96-well polystyrene plate in an amount of 250 μl / well, and the cells were incubated at 37 ° C.
Culture was performed in the gas phase of% carbon dioxide-95% air for 10 to 14 days. After culturing, the activity of the antibody against the conjugate of 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether secreted into the liquid and bovine serum albumin was examined.

The antibody activity was measured by the ELISA method. First, the conjugate of 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether and bovine serum albumin was added to 0.5
Coating buffer (10 mM
phosphate buffer, pH 7.5,1
100 mM / well) in a 96-well polystyrene plate, and then added to a 96-well polystyrene plate at 4 ° C.
It was solidified by allowing it to stand overnight at. Then 250 μl
Buffer containing 1% skim milk (10 mM phosphate buffer, pH
7.2, 0.8% NaCl, 0.02% KCl) (hereinafter referred to as blocking buffer)
Blocked for hours. This well was washed 3 times with a washing solution (0.02% tween 20 added to phosphate buffer). 100 μl of hybridoma culture supernatant
/ Well, and reacted at room temperature for 1 hour. After washing again with the washing solution three times, an antibody dilution solution (phosphate buffer, 0.1.
1% skim milk, 0.05% EDTA) 10,000
Peroxidase-conjugated anti-mouse IgG antibody diluted 0-fold was added at 100 μl / well and reacted for 1 hour. After washing three times with a washing solution, a substrate solution of peroxidase (1
00mM phosphate-citrate bu
ffer, pH 5.0, 0.2% O-phenylen
edamine, 0.003% H ₂ O ₂ ) to develop color,
The absorbance at 492 nm was measured.

As a result, the cells in the wells producing the desired antibody were subjected to cell cloning by the limiting dilution method. As a result of cell cloning, 2- (4-
Eight strains of hybridoma producing an antibody that reacts with a conjugate of hydroxycarbonylmethyloxyphenyl) -2-methylpropyl-3-phenoxybenzyl ether and bovine serum albumin but not bovine serum albumin alone were obtained. Iskoff / 10 of these hybridomas
The culture supernatant cultivated in% FBS medium was used as the antibody solution in the following experiments.

Example 3 Reactivity of each monoclonal antibody with etofenprox by indirect competitive ELISA 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl
0.5 μg / m of conjugate of ether and bovine serum albumin
Dissolve in coating buffer to a concentration of 1,
100 μl / well was added to a 96-well polystyrene plate, and the mixture was left to stand at 4 ° C. overnight to be solid-phased. Then, 250 μl / well was replaced with the blocking buffer, and blocking was performed at room temperature for 1 hour. This well was washed with a washing solution three times. On the other hand, an etofenprox solution having a predetermined concentration was mixed with an antibody solution diluted to an appropriate concentration with an antibody diluent, and reacted at room temperature for 1 hour.
Add 100 μl of this to the previously blocked plate
It was added in a well and reacted at room temperature for 1 hour. 3 again with cleaning solution
After washing twice, 100 μl of peroxidase-conjugated mouse IgG antibody diluted 10,000 times with antibody diluent
/ Well, and reacted for 1 hour. After washing three times with a washing solution, color was developed with a substrate solution of peroxidase.
The absorbance at nm was measured. The inhibition rate was shown as% inhibition from the absorbance decreased by inhibition with etofenprox.

The results are shown in Table 1 (Table 1). First
The table revealed that all monoclonal antibodies react with etofenprox.

[0028]

[Table 1]

Example 4 Methyl Alcohol Resistance of Monoclonal Antibodies Among the eight monoclonal antibodies, the highly sensitive antibodies to etofenprox cl 8-2 and cl 205-
For 65 and cl 221-89, the resistance to methyl alcohol was examined using the ELISA method described in Example 2. The culture supernatant of each hybridoma was diluted with an antibody diluent such that cl 8-2 was 1: 100, cl 205-65 was 1: 100, and cl 221-89 was 1: 150. At the same time, methyl alcohol was added and used so that the final concentration of each was 0 to 90%. Thus, changes in reactivity of the monoclonal antibody with the conjugate of 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether and bovine serum albumin depending on the concentration of methyl alcohol were examined. The results are shown in Fig. 1. From FIG. 1, cl 8-2 and cl 205-65 are relatively stable up to 40% methyl alcohol concentration and rapidly decrease in reactivity at higher concentration, and cl 221-89 shows 30%.
It was revealed that it was relatively stable up to%, and that the reactivity rapidly decreased at higher concentrations.

Example 5 Reactivity of etofenprox Using the indirect competitive ELISA method described in Example 3, cl
8-2, cl 205-65, and cl 221-89 were examined for the measurement sensitivity of etofenprox. The dilution ratio of each monoclonal antibody was the same as in Example 4, and methyl alcohol was added at a final concentration of 10%. The results are shown in FIG. From FIG. 2, cl 8-2 and cl 205-
65 is cl 22 up to about 1 to 1,000 ng / ml
It became clear that 1-89 can quantitatively measure etofenprox up to about 1 to 100 ng / ml.

Example 6 Cross-Reactivity with Derivatives of Etofenprox and Related Pesticides Using the conditions shown in Example 5, the derivatives and related pesticides were reacted in place of etofenprox and cross-reactivity was investigated. . As a result, the absorbance in the case of reacting without adding these compounds was taken as 100%, the molar concentration of the compound inhibiting 50% was taken as the IC ₅₀ concentration, and the IC ₅₀ concentration of etofenprox was taken as 100%. The ratio of the IC ₅₀ concentration of each compound at that time was determined by the following formula (Equation 1), and the cross-reactivity value (%) was calculated.

[0032]

[Number 1] cross-reactivity value (%) = × 100 The results (IC ₅₀ of IC ₅₀ / compound of etofenprox), monoclonal antibodies cl 8-2, cl 2
05-65, cl 221-89 is 2- (4-hydroxyphenyl)-which is a derivative of etofenprox.
Although highly cross-reactive with 2-methylpropyl 3-phenoxybenzyl ether, 2- (4-ethoxyphenyl), a major metabolite of etofenprox
-2-Methylpropyl 3- (4-hydroxyphenoxy) benzyl ether, 2- (4-ethoxyphenyl) -2-methylpropyl 3-hydroxybenzyl
It did not show cross-reactivity with ether, 2- (4-methoxyphenyl) -2-methylpropyl 3-phenoxybenzoate.

[0033]

Etofenprox was conventionally measured by a method in which it was induced to 3-phenoxybenzyl iodide and then measured using gas chromatography with an electron capture detector. Took a considerable amount of time and effort. On the other hand, 2- (4-hydroxycarbonylmethyloxyphenyl) -2-methylpropyl 3-phenoxybenzyl of the present invention
By immunological analysis using a monoclonal antibody obtained by using ether as a hapten, 2- (4-ethoxyphenyl) -2-methylpropyl-3-phenoxybenzyl ether and 2- (4-hydroxyphenyl) -2 were obtained.
Each of -methylpropyl 3-phenoxybenzyl ether could be measured directly and specifically.
Furthermore, 2- (4-ethoxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether could be measured with high sensitivity with a measurement limit of about 1 ng / ml. In addition, since it was possible to measure etofenprox and its derivatives even when 10% of methyl alcohol was added, not only samples of paddy water and tap water, but also biological samples, samples extracted from crops, soil, etc. can be easily measured. . Therefore, by using the method of the present invention, it becomes possible to greatly simplify the measurement of etofenprox and its derivatives and shorten the measurement time, and it is possible to measure a large amount of sample quickly and inexpensively.

[Brief description of drawings]

FIG. 1 is a plot of the resistance of each monoclonal antibody to methyl alcohol using an ELISA method and plotting the absorbance at 492 nm against the concentration of methyl alcohol.

FIG. 2 shows the reactivity of each monoclonal antibody with respect to 2- (4-ethoxyphenyl) -2-methylpropyl-3-phenoxybenzyl ether (ethofenprox), measured by an indirect competitive ELISA method.

[Explanation of symbols]

The mark ■ represents the monoclonal antibody cl8-2, the mark □ represents the monoclonal antibody cl205-65, and the mark ● represents the monoclonal antibody c221-89.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasunori Kuro 1144 Togo, Mobara-shi, Chiba Mitsui Toatsu Chemical Co., Ltd. (72) Inventor Takako Kata 1144 Togo, Mobara-shi, Chiba Mitsui Toatsu Chemical Co., Ltd.

Claims (3)

[Claims] 1. A monoclonal antibody which specifically binds to 2- (4-ethoxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether and 2- (4-hydroxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether. . 2. A hybridoma which produces the monoclonal antibody according to claim 1. 3. 2- (4-Ethoxyphenyl), characterized in that the monoclonal antibody according to claim 1 is used.
Immunological analysis method of 2-methylpropyl 3-phenoxybenzyl ether and 2- (4-hydroxyphenyl) -2-methylpropyl 3-phenoxybenzyl ether.