JPH06186229A - Residual agricultural chemical analysis method - Google Patents

Abstract

PURPOSE:To simply and rapidly measure the amount of residual asichrolfen(AF) in a natural environment sample by connecting a labeled enzyme to the connection carrier of a first antibody for reacting with a second antibody and adding a buffer liquid and measuring absorbance. CONSTITUTION:A liquid solution containing a specific amount of anti AF mammal antibody (first antibody) is allowed to react with the sample liquid solution containing AF and then a non-reaction first antibody included in a reaction liquid is connected to a carrier which is coated with a connected body of the serum albumin of the same type of mammal as the mammal producing the first antibody and AF. A labeled enzyme is connected to the carrier after washing and an antibody (second antibody) for the first antibody derived from the mammal which is different from that producing the first antibody is allowed to react with the carrier after washing. After washing the carrier, a buffer liquid containing a color developing reagent which develops color by the enzyme reaction with the substrate of the labeled enzyme is added and reacted for measuring absorbance. The AF concentration in a sample is calculated from the measurement value.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a diphenyl ether herbicide 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid [generic name: Acifluorfen] Write down. ] For measuring the residual amount in natural environment samples of

[0002]

2. Description of the Related Art Pesticide residues in the natural environment, and the problem of postharvest pesticides in imported foods, which has recently been increasing in recent years, has attracted great public interest. In order to ensure the safety of the natural environment and imported foods, it is necessary to accurately and quickly measure the amount of pesticide residues contained in them. Traditionally, pesticide residue analysis methods have been established for each crop at the time the pesticide is registered, and mainly after extracting and purifying the pesticide, the content in the extract is determined by gas chromatography or high-performance liquid chromatography. It was measured using chromatography and the like. These methods are sufficient in both sensitivity and accuracy, but they have the drawback of requiring considerable labor and time for sample preparation and of requiring high cost for measuring devices and equipment. .
Analysis of pesticide residues in imported foods, etc.
Moreover, since it is necessary to maintain freshness, simplicity and speed are important. Further, it is required that the analysis can be easily performed without an expensive measuring device. Therefore, it is desired to develop a simpler and faster method for analyzing residual pesticides as compared with the conventional analysis methods.

[0003]

Conventionally, in the analysis of AF, which is a diphenyl ether type herbicide, a method in which a carboxylic acid is mainly methylated and then measurement is carried out using gas chromatography has been adopted. The analysis required considerable effort and time. Therefore, it has been desired to establish a simple and rapid analysis method for AF by an immunological detection method using an AF antibody.

[0004]

Therefore, the present inventor has
As a result of sincere examination of the method for measuring the residual amount of AF in a natural environment sample, after immunizing a mammal against a conjugate of mammalian serum albumin and AF, blood was collected from the mammal, An antibody that recognizes AF obtained by separating and purifying blood is used as a first antibody, binds a labeling enzyme, and is an antibody against a first antibody derived from a mammal of a species different from the mammal producing the first antibody. The inventors have found that the residual amount of AF in a natural environment sample can be easily, rapidly and accurately analyzed by an indirect competitive inhibition method (ELISA method) in which is used as a second antibody, and completed the present invention. That is, the present invention provides a method for measuring 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid by an indirect competitive inhibition method (ELISA method) which is an immunological detection method. ) A solution containing a predetermined amount of anti-AF mammalian antibody (hereinafter referred to as the first antibody) is reacted with a sample solution containing AF, and (2) unreacted first antibody contained in the reaction solution is reacted. ,
The antibody is bound to a carrier whose surface is coated with a conjugate of serum albumin and AF of a mammal of the same species as the mammal producing the first antibody, (3) after washing the carrier, and (4) binding with a labeling enzyme. And, the antibody against the first antibody derived from a mammal of a species different from the mammal producing the first antibody (second antibody) is reacted with the carrier, (5) after washing the carrier, (6) A buffer solution containing a coloring reagent that develops a color by an enzymatic reaction with the substrate of the labeling enzyme is added, and after the reaction, the absorbance is measured, and (7) the AF concentration in the sample is calculated from the measured value. A method for measuring the amount of residual AF in a natural environment sample (hereinafter, referred to as the measurement method of the present invention) and a test kit for use in the measurement method, comprising:
{2-chloro-4- (trifluoromethyl) phenoxy} -2
-A carrier (solid support) whose surface is coated with a conjugate of AF and a serum albumin of a mammal of the same species as a mammal that produces a mammalian antibody against nitrobenzoic acid (hereinafter referred to as AF), (2) A reagent containing an anti-AF mammalian antibody (first antibody), (3) a first antibody that binds a labeling enzyme and is derived from a mammal of a species different from the mammal producing the first antibody (second The present invention provides a composition for immunological detection / analysis of AF, which comprises a reagent containing an antibody).

The first antibody used in the present invention is, for example,
Bovine serum albumin (BSA: molecular weight 66200), human serum albumin (HSA: molecular weight 58000), rabbit serum albumin (RS
A: molecular weight 68000), goat serum albumin (GSA: molecular weight 6800)
(0) such as mammalian serum albumin chemically bound to AF is used as an immunogen, for example, J.ASSOC.OFF.ANA.
WH New described in L.CHEM., 70 (6) 1025-1027 (1987), etc.
After immunizing a mammal such as rabbit, rat, dog, etc. according to a usual immunization method such as some, blood is collected from the mammal, and the blood is collected, for example, by centrifugation, ammonium sulfate or polyethylene glycol. It can be produced by separating and purifying by a conventional method such as precipitation by use, gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like. The immunogen used for the production of the first antibody is, for example, the acid anhydride method (J. Biol. Chem. Vol. 234, 1090-1094 (195
It can be prepared by chemically binding mammalian serum albumin to AF by a conventional method such as 9)). In addition, immunocompetent B from the above immunized mammal
The cells are isolated, the immunocompetent B cells are fused with tumor cells capable of serial cell division, the resulting fusion is isolated, and after selection, the hybridoma cells producing the desired antibody are cloned. Antibodies with high specificity and affinity can also be produced by culturing the hybridoma cells in vitro or in vivo to produce monoclonal antibodies.

As the carrier (solid carrier) used in the measuring method of the present invention, a carrier usually used in an indirect competitive inhibition method (ELISA method) can be used. For example, a plastic surface of a microtiter plate or a test tube can be used. ,
The surface of beads such as polystyrene, polypropylene, polyvinyl chloride, glass or plastic, the surface of filter paper, dextran, cellulose or nitrocellulose, or the surface of strips of other similar materials can be mentioned. The above carriers have a very wide range of designs,
It can have very different shapes, depending on the particular purpose intended for use. For example, a plate, a ball,
Examples include plates, small rods, cells, small bottles, small tubes, fibers and nets. As a specific example, a transparent plastic material such as a microtiter plate made of polyvinyl chloride or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod can be used. In particular, it is convenient to use a 96-well microtiter plate of polystyrene. In the measuring method of the present invention,
A coating buffer solution used for coating the carrier of the conjugate of serum albumin and AF of a mammal of the same species as the mammal producing the first antibody, for example, by a conventional method using glutaraldehyde or cyanogen bromide in advance. After being added to the activated carrier, the surface of the carrier is coated with a conjugate of serum albumin and AF of a mammal of the same species as the mammal producing the first antibody by incubating. As the coating buffer solution used here, for example, it is convenient to use a 10 mM phosphate buffer solution (pH 7.5) containing 100 mM of NaCl,
It is not limited only to this. A conjugate of mammalian serum albumin and AF can be produced in the same manner as in the method for preparing the immunogen used for producing the first antibody described above.

Regarding the above-mentioned coating conditions, the concentration of the coating buffer used for coating the carrier of the conjugate of serum albumin and AF of a mammal of the same species as the mammal producing the first antibody is about 1 μg / It is preferable that the amount is at least ml. Increasing the concentration to about 100 μg / ml did not change the final result, indicating that about 1 μg / ml equilibrates the adsorption of the antigen to the carrier. When using a 96-well microplate, the amount used is preferably about 0.1 ml / well. For the above incubation conditions, for example, about 4 ℃
Incubating for about 6 hours to about 24 hours, and preferably overnight. Serum albumin of a mammal of the same species as the mammal producing the first antibody
After coating the carrier of the conjugate of AF, in order to prevent non-specific binding of the first antibody, with a protein other than serum albumin of a mammal of the same species as the mammal producing the first antibody,
It is desirable to block a portion of the mammal of the same species as the mammal that produces the first antibody, to which serum albumin is not adsorbed. For this purpose, a simple method is, for example, a method of incubating a skim milk solution of about 3% and a carrier at about 20 ° C for about 2 hours. The carrier thus obtained may be washed buffer (e.g. NaCl
0.8% (W / V), KCl 0.02% (W / V) and Tween 20, 0.2% (V / V
V) -containing 10 mM phosphate buffer (pH 7.2) is preferred. )
Use after washing with.

According to the measuring method of the present invention, when a water system in a natural environment such as paddy water or tap water is used as a sample, the AF concentration in the sample should be measured in the range of about 100 to about 10,000 ng / ml. You can Therefore, when the AF concentration in the sample is higher than the above concentration, the sample is appropriately diluted before use. When the concentration is low, the sample is appropriately concentrated before use. When using non-aqueous compounds in the natural environment such as foods, grains, plants and soil as a sample, AF is extracted from the sample with a solvent such as methanol, and then the methanol extract is used, such as the above-mentioned washing buffer. Use after diluting with buffer etc. AF prepared in this way
And a buffer solution containing an excess amount of the first antibody are mixed and allowed to react by incubating overnight at about 20 ° C., for example. At this time, dilute the primary antibody about 3,000 to about 5,000 times in the sample.
It is desirable to react with AF, and a dilution of about 5,000 times can be more desirable. Ie about
It is desirable to mix the 2,500-fold diluted primary antibody with a sample solution containing the same volume of AF and allow to react overnight at about 20 ° C. As the diluent, for example, one having the same composition as the above-mentioned washing buffer can be used. In addition,
If necessary, it is desirable to add 3% skim milk or the like as a protective protein for stabilization when the primary antibody is excessively diluted.

The unreacted first antibody contained in the reaction solution of the sample prepared as described above and the first antibody is treated with serum albumin of a mammal of the same species as the mammal producing the first antibody.
A reaction of binding to a carrier whose surface is coated with a conjugate of AF is performed. Regarding this reaction condition, it is desirable to add a reaction solution of the sample and the first antibody to a carrier and react at about 20 ° C. for about 1 hour. After the reaction, after washing the carrier with the above-mentioned washing buffer, an antibody against the first antibody derived from a mammal of a species different from the mammal that binds the labeling enzyme and produces the first antibody (second antibody)
Subject to reaction with.

The second antibody used in the assay method of the present invention includes, for example, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, glucose-6-. An antibody to the first antibody bound with an enzyme such as phosphate dehydrogenase can be mentioned. As a specific example, when a rabbit antiserum is used as the first antibody, the second antibody is an anti-rabbit immunoglobulin (I) conjugated with peroxidase.
Preferred is gG) goat immunoglobulin (IgG). The rabbit IgG and goat IgG are commercially available and can be easily obtained. When labeled with peroxidase, it combines with hydrogen peroxide as a substrate and diaminobenzidine or O-phenylenediamine as a coloring reagent to give a brown or yellow color. When using the latter, the optimal color development time is about 10 minutes,
The final absorbance is about 1.3 to about 1.4. In addition, 4 for measurement
Absorbances at 90 nm and 655 nm are suitable. In the case of labeling with glucose oxidase, for example, 2,2′-acid-di- (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) or the like is used as a substrate.
The absorbance at 405 nm and 655 nm is suitable for the measurement. About 1,000 to about 2,000 times the carrier bound to the primary antibody,
After reacting the second antibody, which is preferably diluted about 1,000 times, the carrier is washed, and then a buffer solution containing a coloring reagent that develops color by the enzymatic reaction of the labeled enzyme bound to the second antibody and the substrate is added. After addition and reaction, the absorbance is measured using a device such as a multi-scanning spectrophotometer.

The present invention also comprises an anti-AF mammalian antibody as a reagent, and is suitable for use under field conditions for detection and analysis of AF that is convenient and can be processed rapidly and with high accuracy. It comprises means for immunological detection and analysis of AF in the form of a test kit that has been prepared. The above-mentioned test kit may contain the following components, for example: (1) A carrier whose surface is coated with a conjugate of serum albumin and AF of a mammal of the same species as a mammal producing a mammalian antibody against AF ( Solid support), (2) reagent containing anti-AF mammalian antibody (first antibody), (3) derived from a mammal of a species different from the mammal producing the first antibody, which binds the labeling enzyme Against the first antibody
(4) AF standardized solution (5) Buffer solution (6) Polypeptides that prevent non-specific adsorption and aggregate formation, additives such as surfactants, and (7) ) Pipettes, reaction vessels, calculation curves, etc.

[0012]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples, and ordinary modifications can be made in the technical field of the present invention. Example 1 (Preparation of a conjugate of mammalian serum albumin and AF) 25 mg of AF was dissolved in 2 ml of anhydrous dioxane, and 0.05 ml of N-
Methylmorpholine was added, and the mixture was stirred at 10 ° C for 20 minutes. Further, 0.02 ml of isobutyl chlorocarbamate was added little by little, and the mixture was stirred for 15 minutes. On the other hand, 80 mg of bovine serum albumin (BSA) was dissolved in 4.3 ml of distilled water, adjusted to pH 9.5 with a 1N NaOH solution, and 2.6 ml of dioxane was gradually added dropwise at 10 ° C. Then, while maintaining pH 9 with 1N NaOH solution, add the AF solution prepared above little by little and at 4 ° C.
The reaction was stirred for 4 hours. Then, purification by gel filtration using Sephadex G-25 was performed. The obtained protein fraction was dialyzed against distilled water for 24 hours and then freeze-dried to obtain a bovine serum albumin-AF conjugate.

Example 2 (Preparation of anti-AF mammalian antibody) The conjugate obtained in Example 1 was dissolved in physiological phosphate buffer to a concentration of 200 μg / ml. 0.5 ml of each of this aqueous solution and Freund's complete adjuvant (manufactured by DIFCO LABORATORIES) was added and mixed well. 1 ml of the obtained mixture was intramuscularly injected into a Japanese white rabbit (male, 4 weeks old, about 0.6 kg). Then, after 2, 4, and 6 weeks, a physiological phosphate buffer solution prepared so that the concentration of the conjugate obtained in Example 1 was 100 μg / ml was additionally administered as in the first administration. Whole blood was collected from the test rabbits immunized after the last administration, and the obtained blood was centrifuged (1000xg, 15 min, 4 min.
℃) and collected the supernatant. The obtained supernatant was salted out with 33% saturated ammonium sulfate, centrifuged (1000 xg, 15 min, 4 ° C), and the precipitate was dissolved in a small amount of physiological phosphate buffer. This was dialyzed against a physiological phosphate buffer solution at 4 ° C. overnight to obtain a first antibody.

Example 3 (Coating of Antibody Surface with a Conjugate of Serum Albumin and AF of a Mammal of the Same Type as the Primary Antibody-Producing Mammal) Rabbit serum albumin and AF obtained according to Example 1 Coating buffer containing the conjugate at a concentration of 1 μg / ml (10 mM phosphate buffer (pH 7.5) containing 100 mM NaCl)
To a polystyrene 96-well microtiter plate,
It was added at a ratio of 1 / well and incubated overnight at about 4 ° C. Then, add 300 μl of 3% skim milk solution, incubate at 20 ° C. for about 2 hours, and wash the microtiter plate with washing buffer (NaCl 0.8% (W / V), KC).
l Contains 0.02% (W / V) and Tween 200.2% (V / V) 1
The plate was washed 3 times with 0 mM phosphate buffer (pH 7.2).

Example 4 (Indirect competitive inhibition method (ELISA method)
Measurement of AF according to No. 1) The anti-AF rabbit antibody obtained in Example 2 was treated with a washing buffer (NaCl 0.8% (W / V), KCl 0.02% (W / V) and Tween 20).
10 mM phosphate buffer containing 0.2% (V / V) (pH 7.
2) Dilute 2,500 times with the buffer containing skim milk at 3% concentration in (), mix with the same volume of sample solution diluted with the above washing buffer, and react overnight at about 20 ° C. Let 0.1 ml / well of the obtained reaction solution was added to the carrier coated with the conjugate of rabbit serum albumin and AF obtained in Example 3 and reacted at 20 ° C. for 1 hour. Was bound 3 times with the above washing buffer. A solution of peroxidase-conjugated anti-rabbit IgG goat IgG fraction (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 1,000 times with the above buffer was added to the carrier at a rate of 0.1 ml / well, and the temperature was 20 ° C About 3
After incubation for a period of time, the carrier (reacted with the second antibody) was washed 3 times with the above washing buffer. o-
Phenylenediamine coloring solution (o-phenylenediamine 0.2% (W / V) 100 mM, phosphate / citrate buffer (pH 5) 10
0 mM, hydrogen peroxide 0.003% (V / V)) was added to the carrier after washing at a rate of 0.2 ml / well, incubated at 20 ° C. for 10 minutes, and then ₄ NH ₂ SO _4, 0.05 ml / The reaction was stopped at a well ratio. Then, the absorbance at 490 nm and 655 nm was measured. As a result of measuring the AF concentration in the natural environment sample as described above, AF of paddy water, tap water and washing buffer
The results of each measurement using the solution were in good agreement, and it was found that the substances contained in the paddy water and tap water had no effect on the measurement of AF concentration, and the AF in the sample was about 100 ng.
It was revealed that the residual amount of AF in the sample can be accurately measured in the concentration range of / ml to about 10,000 ng / ml.

Example 5 (Indirect competitive inhibition method (ELISA method)
Measurement of AF using No. 2) The anti-AF rabbit antibody obtained in Example 2 was treated with a washing buffer (NaCl 0.8% (W / V), KCl 0.02% (W / V) and Tween 20).
10 mM phosphate buffer containing 0.2% (V / V) (pH 7.
2) Dilute 2,500 times with the buffer containing skim milk at a concentration of 6% in (), mix with the same volume of sample solution diluted with the above washing buffer, and react overnight at about 20 ° C. Let 0.1 ml / well of the obtained reaction solution was added to the carrier coated with the conjugate of rabbit serum albumin and AF obtained in Example 3 and reacted at 20 ° C. for 1 hour. Was bound 3 times with the above washing buffer. A solution prepared by diluting the anti-rabbit IgG donkey IgG fraction bound with biotin as a labeling enzyme to 2,000 times with the above buffer was added to the carrier at a rate of 0.1 ml / well, and incubated at 20 ° C for about 1 hour. rear,
The carrier (reacted with the second antibody) was washed 3 times with the above washing buffer. A solution prepared by diluting avidin-conjugated alkaline phosphatase 1000 times was added at a rate of 0.1 ml / well, incubated for 1 hour, and then the carrier was washed 3 times with the above buffer solution. Alkaline phosphatase coloring reagent (1M diethanolamine containing 0.5M MgCl ₂ , 0.1% disodium nitrophenol phosphate-
Add hydrochloric acid buffer solution (pH 9.8)) to the carrier after washing at a rate of 0.2 ml / well, incubate at 25 ° C for 30 minutes, and add 1N NaOH at a rate of 0.05 ml / well to react. stopped. Then, the absorbance at 415 nm and 630 nm was measured. As a result of measuring the AF concentration in the natural environment sample as described above, each measurement result using AF solution of paddy water, tap water and washing buffer is almost the same, and the substances contained in paddy water and tap water Has no effect on the measurement of AF concentration, and the AF in the sample is about 100 ng / ml ~
It was revealed that the residual amount of AF in the sample can be accurately measured in the concentration range of about 10,000 ng / ml.

[0017]

EFFECTS OF THE INVENTION By using the present invention, it becomes possible to measure the residual amount of aciflorfen (diphenyl ether herbicide) in a natural environment sample that can be simply and rapidly processed with high accuracy. Especially, when measuring the residual amount of aciflorfen in natural environment samples such as paddy water, tap water, food, grains, plants and soil, a large number of measurements can be processed at one time.

Claims (3)

[Claims] 1. An indirect competitive inhibition method (EL) which is an immunological detection method for 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid (hereinafter referred to as AF).
ISA method) (1) reacting a solution containing a predetermined amount of anti-AF mammalian antibody (hereinafter referred to as the first antibody) with a sample solution containing AF, (2) The unreacted first antibody contained in the reaction solution is bound to a carrier whose surface is coated with a conjugate of serum albumin and AF of a mammal of the same species as the mammal producing the first antibody, (3) After washing the carrier, (4) reacting the carrier with an antibody (second antibody) that binds the labeling enzyme and is derived from a mammal of a species different from the mammal producing the first antibody ,(Five)
After washing the carrier, (6) a buffer solution containing a coloring reagent that develops color by an enzymatic reaction with the substrate of the labeling enzyme is added, and after reacting, the absorbance is measured, and (7) a sample is obtained from the measured value. A method for measuring the amount of residual AF in a natural environment sample, which comprises calculating the AF concentration in the sample. 2. An indirect competitive inhibition method (EL) which is an immunological detection method for 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid (hereinafter referred to as AF).
ISA method), (1) reacting a solution containing a predetermined amount of anti-AF rabbit antibody with a sample solution containing AF in the range of 100 ng / ml to 10000 ng / ml, (2) The unreacted anti-AF rabbit antibody contained in the reaction solution was bound to a carrier whose surface was coated with a conjugate of rabbit serum albumin and AF,
(3) After washing the carrier, (4) peroxidase-conjugated anti-rabbit IgG goat IgG is reacted with the carrier, (5) after washing the carrier, (6) an enzyme with a substrate for the labeling enzyme A buffer solution containing a coloring reagent that develops color by the reaction is added,
The absorbance is measured after the reaction, and (7) the AF concentration in the sample is calculated from the measured value.
Measuring method. 3. A test kit for use in the measuring method according to claim 1, wherein (1) 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid (hereinafter, A carrier (solid support) whose surface is coated with a conjugate of serum albumin and AF of a mammal of the same species as the mammal that produces a mammalian antibody against (AF), (2) anti-AF mammalian antibody
(Hereinafter, referred to as a first antibody), (3) a first antibody that binds a labeling enzyme and is derived from a mammal of a species different from the mammal producing the first antibody (second A composition for immunological detection / analysis of AF, which comprises a reagent containing an antibody).