JPH0854391A - Monochronal antibody specific to diphenylether compound, hybridoma, and method for detecting diphenylether compound - Google Patents

Abstract

PURPOSE:To establish a method for detecting a diphenylether compound using a monochronal antibody by using the monochronal antibody showing a reactivity with a diphenylether compound expressed by a specific formula. CONSTITUTION:The novel monochronal antibody showing a reactivity to a diphenylether compound expressed by the formula (in which R is a nitro group or an amino group) and a hybridoma secreting the monochronal antibody are obtained, thereby, to detect the diphenylether compound using the monochronal antibody. A compound represented by the formula is chloronitrophen if the R is a nitro group or 4-aminophenyl 2, 4, 6-trichlorophenyl ether if the R is an amino group. In preparing the monochronal antibody, it is desired to use the diphenylether compound represented by the formula or its salt, or its compound bonded with a biopolymer carrier as an immunity source.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel monoclonal antibody specific to a diphenyl ether derivative, a hybridoma producing the monoclonal antibody, and a method for detecting the diphenyl ether derivative using the monoclonal antibody. Said monoclonal antibody according to the invention is in particular an organic solvent resistant antibody.

[0002]

2. Description of the Related Art In recent years, pesticides remaining in the environment and food have been attracting attention as a major social problem in terms of their effects on the human body. In particular, the herbicide chloronitrophen (hereinafter also referred to as CNP) poses a problem because a causal relationship with gallbladder cancer is sought. Therefore, in order to ensure the safety of the environment and food, it is first necessary to accurately measure the amount of residual agricultural chemicals contained in the environment and food. The amount of residual pesticides is determined for each crop at the time the pesticides are registered, and conventional methods for analyzing residual pesticides mainly extract pesticides from a sample, then refine and then extract using gas chromatography. The content in the product was measured. These methods have the drawbacks that the preparation of the sample is complicated and requires a long time, and that the measuring device and the equipment are expensive. Since the number of analysis of pesticide residues 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.

[0003]

Conventionally, chloronitrophen (CN), which is a typical example of diphenyl ether herbicides, has been used.
P), that is, 4-nitrophenyl 2,4,6-trichlorophenyl ether (C ₁₂ H ₆ Cl ₃ NO ₃ ) was also analyzed by the method using gas chromatography. Took a considerable amount of time and effort. Also, for example, CN remaining in the soil
When measuring P, it was necessary to use an organic solvent at the stage of the extraction operation. In recent years, immunochemical detection methods have also been applied to the measurement of components other than biological samples, and their application has been studied in various ways. However, immunochemical detection of CNP has hitherto been known. Had not been considered.

[0004] Therefore, the present inventors have conducted intensive studies for the purpose of immunochemically detecting CNP, and as a result, succeeded in finding a novel monoclonal antibody specific to the diphenyl ether compound. Therefore, the object of the present invention is to
It is intended to provide the above novel monoclonal antibody, a hybridoma secreting the monoclonal antibody, and a method for detecting a diphenyl ether compound using the monoclonal antibody.

[0005]

Accordingly, the present invention provides a compound represented by the general formula (I):

Embedded image The present invention relates to a monoclonal antibody reactive with a diphenyl ether compound represented by the formula (wherein R is a nitro group or an amino group). In addition, the present invention relates to a monoclonal antibody which is resistant to an organic solvent among these monoclonal antibodies and therefore can carry out an immune reaction even in the presence of the organic solvent. Furthermore, the present invention also relates to a hybridoma producing the above-mentioned monoclonal antibody, and a method for immunochemically detecting the diphenyl ether compound represented by the general formula (I), characterized by using the above-mentioned monoclonal antibody.

In the general formula (I), when R is a nitro group (-NO ₂ ), the compound represented by the general formula (I) is chloronitrophene (CNP) and R is an amino group ( -NH ₂ ) (an analogue of a reduced CNP nitro group), the compound represented by the general formula (I) is 4-aminophenyl 2,4,6-trichlorophenyl ether (hereinafter, CNP). it is there) called -NH _2. The monoclonal antibody and the hybridoma according to the present invention can be prepared by a conventional method, for example, the method described in Zokusei Chemistry Laboratory, Immunobiochemistry Research Method (edited by the Japanese Biochemical Society). Specifically, as the immunogen, a diphenyl ether compound represented by the above general formula (I) or a salt thereof, and further, those compounds bound to a biopolymer (eg, bovine serum albumin or immunoglobulin) carrier It is preferable to use. Further, a diphenyl ether compound having a chemical structure similar to that of the diphenyl ether compound represented by the general formula (I), for example, 5
-[2-Chloro-4- (trifluoromethyl) phenoxy] -2-nitrobenzoic acid or salts thereof, even if those compounds are bound to a biopolymer carrier,
A hybridoma that secretes the desired monoclonal antibody can be obtained.

Mammals (eg, mice, rats, rabbits, goats or horses) are immunized by the in vivo immunization method using these immunogen solutions. For example, an immunogen solution is emulsified and mixed with an equal volume of Freund's complete or incomplete adjuvant, and the mixture is subcutaneously administered to mice (first 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 for cell fusion, myeloma cells (myeloma cells), various known cell lines such as p3 (p3 / * 63-Ag8) (Nature, 256, 495-497) are used.
(1975)), p3-U1 (Current Topics in Microbiology a
nd Immunology, 81; 1-7 (1978)), NS-1 (Eur.J.Immunol.,
6; 511-519 (1976)), MPC-11 (Cell, 8; 405-415 (1976)),
SP2 / 0 (Nature, 276; 269-270 (1978)), FO (J.Immunol.
Meth., 35; 1-21 (1980)), * 63.6.55.3 (J.Immunol., 123;
1548-1550 (1979)), S194 (J.Exp.Med., 148; 313-323 (197)
8)), or R210 (Nature, 277; 131-133 (1
979)) etc. can be used.

The cell fusion can be carried out by an ordinary method, for example, using a known fusion promoter (polyethylene glycol etc.), and the usage ratio is the same as in the conventional method, for example, about spleen cells to about myeloma cells. It is used in an amount of about 1 to 10 times. As the fusion medium, for example, 40% (W / V)
Dulbecco's modified Eagle medium containing polyethylene glycol 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.
The hybridoma of interest can be separated by detection and measurement by the SA method. In particular, when selecting a hybridoma producing a monoclonal antibody having resistance to an organic solvent, the diphenyl ether compound is added to an organic aqueous solution or an anhydrous organic solution containing an organic solvent at various concentrations, and the monoclonal antibody is added thereto. After the addition, by confirming that the antigen-antibody reaction proceeds normally, it is possible to select the hybridoma producing the organic solvent-resistant monoclonal antibody.

The thus obtained hybridoma of the present invention secreting the desired monoclonal antibody can be subcultured in an ordinary medium and can be easily stored in liquid nitrogen for a long period of time. As a medium for culturing the hybridoma, any medium suitable for the hybridoma can be used, and preferably Iscove's modified DMEM
(Hereinafter referred to as IMEM) with 10% fetal bovine serum (hereinafter referred to as F
A medium containing BS) is used. Hybridomas are preferably cultured in vitro, for example, in a medium at 5% carbon dioxide and 37 ° C., and in vivo, for example, in the peritoneal cavity of a mouse.

From the culture solution obtained by culturing the above hybridoma by a conventional method, or from the ascites of a suitable mammal (for example, mouse or rat) to which the hybridoma is administered,
The desired monoclonal antibody can be separated and purified. Isolation of protein when separating and purifying monoclonal antibody from culture medium or mouse ascites,
It is possible to use a method generally used for purification. Examples of such a method include salting out with ammonium sulfate, ion exchange chromatography, molecular sieve column chromatography using a molecular sieve gel, affinity column chromatography using a protein A or protein G binding polymer, dialysis, and lyophilization.

Since the method for detecting a diphenyl ether compound represented by the general formula (I) according to the present invention is carried out using the above monoclonal antibody, the diphenyl ether compound can be accurately detected. Also,
By using the organic solvent-resistant monoclonal antibody, an accurate antigen-antibody reaction can proceed even in an organic aqueous system containing an organic solvent capable of sufficiently dissolving the diphenyl ether compound to be tested. For example, when measuring CNP as a residual pesticide in soil, the organic extract used to extract CNP from a test sample such as soil can be used as it is as a sample, and a quick and simple operation is possible. It will be possible.

The method of the present invention uses a monoclonal antibody reactive with the diphenyl ether compound,
And preferably using an organic solvent resistant antibody (thus carrying out the antigen-antibody reaction in the presence of an organic solvent)
Otherwise, it can be applied as it is to the conventionally known immunochemical detection method. The detection method is
For example, an antibody having reactivity with the diphenyl ether compound and having resistance to an organic solvent is directly labeled to cause a competitive reaction between the immobilized antigen and the diphenyl ether compound in the sample, usually in the antibody labeling method, There is a method called a direct competition method, or a method called an antigen labeling method, in which the above antibody is immobilized and the labeled antigen and the diphenyl ether compound in the sample are allowed to compete with each other. The case where is applied to a detection method which is generally called an indirect competition method among antibody labeling methods will be described below.

In the method of the present invention, specifically, for detecting the diphenyl ether compound represented by the general formula (I) present in soil, for example, (1) antigen [that is, in the general formula (I) A diphenyl ether compound represented] and a biopolymer (for example, serum albumin or immunoglobulin) on a solid phase (hereinafter referred to as a solid phase antigen) (2) A test sample (for example, soil) Organic solvents (especially,
Water-miscible organic solvent) to prepare an organic solvent extract (3) An antibody that reacts with the diphenyl ether compound and is resistant to the organic solvent (hereinafter referred to as the first antibody); Contacting the organic solvent extract prepared in 2) and the solid-phased antigen prepared in the step (1) (4) The first antibody bound to the solid-phased antigen and the diphenyl ether compound in the sample (5) Labeled anti-mouse antibody (hereinafter referred to as second antibody)
Is contacted with the first antibody bound to the solid phase antigen (6) The second antibody bound to the first antibody bound to the solid phase antigen and the first antibody bound to the solid phase antigen A step of separating the antibody from the second antibody that is not bound (7) A step of detecting a signal from the label of the second antibody that is bound to either one of the two antibodies separated in the step (6), preferably the first antibody And a method for detecting a diphenyl ether compound in a sample.

When measuring the diphenyl ether compound in the aqueous sample, the step (2) can be omitted and the test sample can be directly contacted with the first antibody and the immobilized antigen. Otherwise, the procedure is the same as for detecting diphenyl ether compounds present in soil.

The organic solvent used in the present invention is an alcohol or a ketone, and is a solvent used when extracting the diphenyl ether compound represented by the general formula (I) from the sample to be inspected. The use of a water-miscible organic solvent is preferable because the extraction step can be performed with an organic aqueous solvent, or the organic-solvent extract can be appropriately diluted with water before the antigen-antibody reaction can be performed in the organic aqueous solvent. . Examples of the organic solvent include alcohol compounds (eg, lower alcohols having 1 to 3 carbon atoms, particularly methyl alcohol, ethyl alcohol, isopropyl alcohol), ketone compounds (eg, lower aliphatic compounds having 3 to 5 carbon atoms). Ketones, in particular methyl ethyl ketone, acetone or diethyl ketone,) or mixtures thereof. In the method of the present invention, any sample that may contain the diphenyl ether compound represented by the general formula (I) can be used, and examples thereof include soil, agricultural products, foods, paddy water, rivers, ponds, and swamps. Such as water can be mentioned.

When specifically carrying out the detection method of the present invention (particularly when soil is used as a sample), the sample is first extracted with the above-mentioned organic solvent. The obtained organic solvent extract is used as it is or diluted with water for the next step. For example, when the first antibody is contacted with the solid-phased antigen and the organic solvent extract, when the diphenyl ether compound (antigen) is present in the organic solvent extract, the antigen is competitively competed in the presence of the organic solvent. Antibody reaction occurs. This antigen-antibody reaction can be performed in the same manner as a usual antigen-antibody reaction except that it is performed in the presence of an organic solvent. Further, in the case of an aqueous sample, a competitive antigen-antibody reaction occurs by directly bringing it into contact with an antibody and a solid-phased antigen.

When carrying out the method of the present invention, a known amount of labeled secondary antibody can be used to confirm or quantify the diphenyl ether compound. Known labels such as radioisotopes (eg, ³² P,
³⁵ S, ³ H), enzymes (eg peroxidase, alkaline phosphatase), vitamins (eg biotin), fluorescent substances (eg FITC), chemiluminescent substances (eg acridinium) and 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 the organic solvent extract (or aqueous sample) and the solid-phased antigen is completed. When the first antibody is brought into contact with the organic solvent extract (or aqueous sample) and the solid-phased antigen, the first antibody that has not bound to the solid-phased antigen is washed off, and then the labeled second antibody is added. The labeled second antibody binds to the first antibody bound to the immobilized antigen.

In the method of the present invention, after the reaction between the first antibody, the diphenyl ether compound in the extract (or the aqueous sample), and the solid-phased antigen is completed, the first antibody bound to the solid-phased antigen is added. To separate. The separation can be performed, for example, by filtration, centrifugation, or washing with a buffer solution. Furthermore, the first antibody bound to the immobilized antigen and the labeled second antibody
After the reaction with the antibody is completed, the labeled second antibody that has not bound to the first antibody is removed, and subsequently, the signal from the label of the labeled second antibody that has bound to the first antibody is detected or measured. To do.

The signal derived from the label of the labeled second antibody bound to the thus separated first antibody is detected or measured.
When detecting or measuring a signal, it is preferable to change the reaction system containing the labeled second antibody to conditions preferable for signal detection or signal measurement. For example, when a fluorescent or chemiluminescent substance is used as the label, the signal is detected or measured under the condition that quenching does not occur.

[0023]

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: Preparation of monoclonal antibody-producing hybridoma
5- [2-chloro-4- (trifluoromethyl) phenoxy] -2-, which is one of the formed diphenyl ether herbicides
25 mg of nitrobenzoic acid (hereinafter referred to as AF) was dissolved in 2 ml of anhydrous dioxane, and then N-methylmorpholine 50 was added.
μl was added and stirred at 10 ° C. for 20 minutes. Then add 20 μl of isobutyl chlorocarbonate little by little and add 1
Stir for 5 minutes. On the other hand, after dissolving 80 mg of bovine serum albumin in 4.3 ml of distilled water, pH was adjusted with 1N NaOH.
It was adjusted to 9.5, and 2.6 ml of dioxane was added dropwise at 10 ° C. This bovine serum albumin solution was previously prepared
The AF solution was gradually added dropwise with 1N NaOH while keeping the pH at 9 and reacted at 4 ° C. for 4 hours, and then dialyzed against distilled water. The conjugate of AF and bovine serum albumin thus obtained was used as an immunogen and an antigen for analysis.

The immunogen was 10 in terms of bovine serum albumin.
0 μg was dissolved in 50 μl of Dulbecco's PBS (−) (hereinafter referred to as PBS), mixed with an equal amount of Freund's complete adjuvant, and then subcutaneously inoculated into Balb / c mice. 1/5 of the above amount after 1 month and 2 months respectively
The amount was boosted.

The spleen of the mouse in which the titer of anti-AF antibody in the serum was increased was excised, and the excised spleen was washed 3 times in a petri dish with IMEM medium, and then wound with an injection needle.
A single cell suspension was prepared by squeezing. The single cell suspension was filtered through a mesh to remove large solids. The obtained filtrate was added to mouse myeloma cells P3-X63-
Ag8.653 was mixed at a cell number ratio of 1: 5 (myeloma cells: spleen cells) and centrifuged (1000 rp).
m, 5 minutes) and the cells were collected. Next, the above-mentioned precipitated cells were resuspended in IMEM medium, centrifuged under the same conditions, the centrifuge tube was flipped with a finger to stir the precipitate, and then 50% polyethylene glycol (molecular weight 1500 ) 1 ml of solution was added slowly over 60 seconds while rotating the centrifuge tube. Stop the cell fusion by adding 10 ml of IMEM medium to the centrifuge tube in which the cell fusion is progressing at 30 ml per ml.
After adding for 2 seconds, 1 ml of FBS was added. After the addition was completed, the mixture was centrifuged, and the obtained cells were suspended in HAT medium so that the cell number was 5 × 10 ⁵ cells / ml. This cell suspension was dispensed into a 96-well polystyrene plate in an amount of 100 μl / well, and the mixture was incubated at 37 ° C.
Was cultured in a gas phase of 5% carbon dioxide-95% air for 10 to 14 days. The activity of the anti-AF antibody in the culture solution was examined, and the cells of the well producing the desired antibody were cloned into a 96-well polystyrene plate by the limiting dilution method using the HT medium in the hybridoma. As a result of cloning, CNP and CNP-
Antibodies reactive with NH ₂ (AF 75-14
4, IgG1) producing hybridoma (AF
75-144 strain) was obtained.

Example 2: Purification of AF 75-144 antibody Monoclonal antibody AF 75-144 was obtained by producing 10% FB of hybridoma strain AF 75-144.
Culture the cells in IMEM medium with S, and add A directly from the centrifuged supernatant.
Purified by affinity chromatography on a vid AL gel. The antibody was eluted with 0.1 M acetate buffer (p
H3.0) and dialyzed against PBS to give an antibody solution. As a result of electrophoresis of this antibody solution by SDS-PAGE, it was confirmed that the antibody solution was almost pure, and by the same operation as in Example 1, it was confirmed that the antibody solution specifically reacts with CNP and CNP-NH ₂ . .

Example 3: C by indirect competitive ELISA
Measurement of NP and CNP-NH ₂ The AF-bovine serum albumin conjugate was dissolved in PBS to a concentration of 0.4 μg / ml, added to a 96-well polystyrene plate at 100 μl / well, and then at 1 ° C. at 4 ° C.
It was solidified by allowing it to stand overnight. Then replace with 1% bovine serum albumin solution at 250 μl / well and
Blocked for hours. Wash the well (85 mM
Borate buffer pH 8.0; 60 mM NaCl)
After washing with a solution containing a predetermined concentration of CNP and CNP-NH ₂ (sample solution; CNP-NH ₂ is 0.5 mM
Hydrochloric acid was used as a buffer) at 50 μl / well, and 17.5 μg / ml of monoclonal antibody AF was added.
75-144 was added at 50 μl / well, stirred, and reacted at room temperature for 1 hour. After washing again with the washing solution three times,
A peroxidase-conjugated anti-mouse IgG antibody (manufactured by Capel) diluted 1000 times with a 0.3% bovine serum albumin solution was added at 100 μl / well and reacted for 1 hour. After washing three times with a washing solution, color was developed with a substrate solution of peroxidase, and the absorbance at 450 nm was measured. Using this indirect competitive ELISA method, methyl alcohol was added to the sample solution and the monoclonal antibody solution so that the final concentration was 0 to 60%, and the inhibition curves of CNP and CNP-NH ₂ were measured. The measurement results are shown in the CNP inhibition curve in FIG. 1 and the CNP-NH ₂ inhibition curve in FIG. 1 and 2, □ is 0% methyl alcohol, ⋄ is 20% methyl alcohol, ∘ is 40% methyl alcohol, and Δ is 60% methyl alcohol.

As is apparent from the results shown in FIGS. 1 and 2,
Monoclonal antibody AF 75-144 shows resistance up to 40% methyl alcohol concentration when CNP is measured, and at 0% methyl alcohol concentration, the concentration of CNP showing half the absorbance when CNP is not added (hereinafter referred to as IC ₅₀ ). ) Is 0.66 μg / ml, the IC ₅₀ is 1.2 μg / ml at 20% methyl alcohol concentration, and the IC ₅₀ is 7.0 μg / ml at 40% methyl alcohol concentration, and the higher the methyl alcohol concentration, the lower the measurement sensitivity. . Again 60
The reaction disappeared with% methyl alcohol. On the other hand, CNP
When -NH ₂ was measured, the IC ₅₀ was 5.8 μg / ml at a 0% methyl alcohol concentration, and the solubility was 100 μg / ml or more at a 20% methyl alcohol concentration, and it was not possible to measure, but at 100 μg / ml it was 43%. It showed inhibition. 400 μg / ml at 40% methyl alcohol concentration
Solubility was exceeded at the above concentrations, and inhibition was not exerted at concentrations of 400 μg / ml or less. Therefore it is possible to measure up to methyl alcohol concentration when 20% of CNP-NH _2.

Example 4: Addition recovery test Using a sample solution prepared by adding a predetermined concentration of CNP to tap water or paddy water (3 types), the absorbance was measured by the method shown in the above Example 3. The results are shown in Table 1 where the number of times that CNP could be detected / the total number of times of measurement was detected when the case where the inhibition of 33% or more of the absorbance without CNP was shown was detected and the case where it was less than that was not detected.

[0030]

[Table 1] Water for measuring CNP added to tap water or paddy water CNP addition amount (μg / ml) 2.0 1.0 0.5 0.25 Tap water 2/2 4/4 3/4 0/2 Paddy water (1) 2/2 4/4 3/4 0/2 Paddy water (2) 2/2 4/4 2/4 0/2 Paddy water (3) 2/2 3/4 2/4 0/2

As is clear from the above results, 2.0 μ
With the addition of g / ml, CNP could be detected in all, and with the addition of 1.0 μg / ml, it could be detected 15 times in 16 tests. With addition of 0.5 μg / ml,
It was possible to detect 10 times in 16 tests. Therefore, it became clear that in case of paddy water or tap water, CNP can be measured up to about 0.5 μg / ml. On the other hand, CNP-NH ₂
In the same manner, the addition recovery test was conducted, and CNP-NH ₂
Table 2 shows the results of the number of times that was detected / total number of measurements.

[0032]

TABLE 2 sample water CNP-NH ₂ addition amount of CNP-NH ₂ was added to tap water or paddy water (μg / ml) 8.0 4.0 2.0 1.0 Tap water 4/4 4/4 3/4 1/2 paddy water (1) 4/4 4/4 3/4 1/2 Paddy water (2) 4/4 4/4 2/4 0/2 Paddy water (3) 4/4 4/4 4/4 0/2

As is clear from the above results, 8.0 μ
CNP was added by addition of g / ml and 4.0 μg / ml.
-NH ₂ could be detected, and with the addition of 2.0 μg / ml, it could be detected 12 times in 16 tests. Therefore, in case of paddy water or tap water, CNP-NH ₂ is 2.0μ
It became clear that measurement can be performed up to about g / ml. Further, an addition recovery test was conducted for the case where a predetermined concentration of CNP was added to the paddy soil. First, a predetermined concentration of CNP dissolved in methyl alcohol was added to the soil. Next, add equal amount of methyl alcohol, stir at room temperature for 1 hour, and add CN
After P was extracted, the extract was diluted to a methyl alcohol concentration of 40%, and the absorbance was measured by the method described in Example 3. Evaluation of the measurement results is performed in the same manner as the paddy water,
The results in Table 3 were obtained.

[0034]

[Table 3] * Number of times CNP was detected / total number of measurements

As is clear from the above results, with the addition of 15.0 μg / g of CNP, CN was added at all times in 12 tests.
P can be detected, and at the addition of 10.0 μg / g,
I was able to detect CNP 11 times in 12 tests,
With 5.0 μg / g addition, no detection was possible during 12 tests. Therefore, the CNP in paddy soil is 1
It was revealed that the measurement can be performed up to about 0.0 μg / g.

[0036]

In the conventional detection of CNP, a method in which a sample is extracted and purified and then measured using gas chromatography has been adopted, but the analysis requires a considerable amount of time and labor. On the other hand, by using the immunochemical measurement method of the present invention, CNP in tap water or paddy water is 0.5 μm.
2.0 μg / ml of CNP-NH ₂ up to about g / ml
The degree can be measured directly. In addition, 10.0 μg / CNP of methyl alcohol extract in paddy soil
It can be directly measured up to g. Furthermore, it is considered that the measurement sensitivity can be increased by the concentration of the extract. Therefore, by using the method of the present invention, it is possible to greatly simplify the test operation and shorten the test time, and it is possible to rapidly measure a large amount of sample.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of methyl alcohol concentration on the CNP inhibition curve.

FIG. 2 is a graph showing the influence of methyl alcohol concentration on the inhibition curve of CNP-NH ₂ .

Claims (3)

[Claims] 1. A compound represented by the general formula (I): A monoclonal antibody having reactivity with a diphenyl ether compound represented by the formula (wherein R is a nitro group or an amino group). 2. A hybridoma which produces the monoclonal antibody according to claim 1. 3. A method for immunochemically detecting the diphenyl ether compound represented by the general formula (I) according to claim 1, characterized in that the monoclonal antibody according to claim 1 is used.