JP3292937B2 - Pesticide residue analysis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND 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. ] The method for measuring the amount of residue in a natural environment sample of the above.

[0002]

BACKGROUND OF THE INVENTION There has been great public interest in the problem of pesticide residues in the natural environment, and in recent years, especially the problem of post-harvest pesticides on imported foods. 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 these. Conventionally, pesticide residue analysis methods are specified for each crop at the time the pesticide is registered. Mainly, after extracting and purifying pesticides from a sample, the content of the extract is determined by gas chromatography or high-performance liquid chromatography. It was measured using chromatography and the like. These methods are satisfactory in both their sensitivity and accuracy, but have the drawback that they require considerable labor and time for sample preparation, and that they require high costs for measuring devices and equipment. .
Analysis of pesticide residues in imported foods, etc. is very
Moreover, since it is necessary to maintain freshness, simplicity and quickness are important. In addition, it is required that analysis can be easily performed without an expensive measuring device. Therefore, there is a demand for a simpler and faster method for analyzing pesticide residues than conventional analysis methods.

[0003]

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

[0004]

Means for Solving the Problems For this reason, the present inventor has proposed:
As a result of sincerely examining the method of measuring the residual amount of AF in the natural environment sample, after immunizing the mammal against the conjugate of serum albumin and AF of the mammal, blood was collected from the mammal, An antibody that recognizes AF obtained by separating and purifying blood is used as a first antibody, a labeled enzyme is bound thereto, and an antibody against the first antibody derived from a mammal different from the mammal that produces the first antibody The present inventors have found that the residual amount of AF in a natural environment sample can be simply, quickly, and accurately analyzed by an indirect competitive inhibition method (ELISA method) using as a second antibody, and completed the present invention. That is, the present invention relates to a method for measuring 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid by an indirect competitive inhibition method (ELISA), which is an immunological detection method, A) reacting a solution containing a predetermined amount of an anti-AF mammalian antibody (hereinafter referred to as a first antibody) with a sample solution containing AF, and (2) removing unreacted first antibody contained in the reaction solution. ,
The first 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, (4) a labeling enzyme is bound. And reacting an antibody against the first antibody (second antibody) derived from a mammal of a different species from the mammal producing the first antibody (second antibody) with the carrier, (5) 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. (Hereinafter referred to as the measuring method of the present invention) and a test kit for use in the measuring method, wherein (1) 5-
{2-Chloro-4- (trifluoromethyl) phenoxy} -2
A carrier (solid support) having a surface coated with a conjugate of serum albumin and AF of a mammal of the same species as a mammal producing 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 which binds a labeling enzyme and is derived from a mammal of a different species from the mammal producing the first antibody (second antibody) And a reagent for immunological detection and analysis of AF.

[0005] 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) using a conjugate obtained by chemically binding mammalian serum albumin to AF as an immunogen, for example, J.ASSOC.OFF.ANA
L. CHEM., 70 (6) 1025-1027 (1987), etc.
Following immunization of mammals such as rabbits, rats, dogs, etc. according to the usual immunization method such as some, blood is collected from the mammal, and the blood is centrifuged, for example, by ammonium sulfate or polyethylene glycol. It can be produced by separation and purification by ordinary methods such as precipitation by use, gel filtration chromatography, ion exchange chromatography, chromatography such as affinity chromatography and the like. The immunogen used for the production of the first antibody is, for example, an acid anhydride method by BFErlanger et al. (J. Biol. Chem. Vol. 234, 1090-1094 (195
9)) and the like, and can be prepared by chemically binding mammalian serum albumin to AF. In addition, immunocompetent B
The cells are isolated, the immunocompetent B cells are fused with continuously mitogenic tumor cells, the resulting fusion is isolated, and after selection, the hybridoma cells producing the desired antibody are cloned. By culturing the hybridoma cells in vitro or in vivo to produce a monoclonal antibody, an antibody having a high degree of specificity and affinity can also be produced.

[0006] As the carrier (solid carrier) used in the measurement method of the present invention, a carrier usually used in an indirect competitive inhibition method (ELISA) can be used. For example, a plastic surface of a microtiter plate or a test tube can be used. ,
Surfaces of beads made of polystyrene, polypropylene, polyvinyl chloride, glass or plastic, etc., filter paper, strips of dextran, cellulose or nitrocellulose or other similar materials can be mentioned. The above carriers have a very wide range of designs,
And can have very different shapes depending on the particular purpose intended for use. For example, dishes, balls,
Plates, small rods, cells, small bottles, small tubes, fibers, nets and the like can be mentioned. As specific examples, a transparent plastic material, for example, a microtiter plate made of polyvinyl chloride or polystyrene, a small ball made of polystyrene and polystyrene latex, a tube or a rod, or the like can be used. In particular, it is convenient to use polystyrene 96-well microtiter plates. In the measurement method of the present invention,
A coating buffer solution used for coating the carrier with a serum albumin / AF conjugate of a mammal of the same species as the mammal producing the first antibody is prepared, 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 incubation. As the coating buffer solution used here, for example, it is convenient to use 10 mM phosphate buffer (pH 7.5) containing 100 mM NaCl, etc.
However, the present invention is not limited to this. Further, the 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.

[0007] Under the above-mentioned coating conditions, the concentration of the coating buffer used for coating the serum albumin-AF conjugate of the mammal of the same species as the mammal producing the first antibody on the carrier is about 1 μg / Desirably, it is not less than ml. Increasing the concentration to about 100 μg / ml did not change the final result, indicating that at about 1 μg / ml the adsorption of the antigen to the carrier was equilibrated. When a 96-well microplate is used, for example, about 0.1 ml / well can be preferably used. For the above incubation conditions, for example, about 4 ° C
For about 6 hours to about 24 hours, preferably overnight. A serum albumin of a mammal of the same species as the mammal producing the first antibody;
After coating the conjugate of the AF with the carrier, to prevent non-specific binding of the first antibody, 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 a mammal of the same species as the mammal producing the first antibody, on which serum albumin is not adsorbed. For this purpose, for example, a method of incubating a skim milk solution of about 3% and a carrier at about 20 ° C. for about 2 hours can be mentioned as a simple method. The carrier thus obtained can be used as a washing buffer (for example, NaCl
0.8% (W / V), KCl 0.02% (W / V) and Tween 20, 0.2% (V / V
10 mM phosphate buffer (pH 7.2) containing V) is preferred. )
Use after washing with.

In the measurement method of the present invention, when a water system in the natural environment such as paddy water or tap water is used as a sample, the measurement should be performed at an AF concentration in the sample of about 100 to about 10,000 ng / ml. Can be. Therefore, when the AF concentration in the sample is higher than the above, the sample is used after appropriately diluting it. When the concentration is low, the sample is used after being appropriately concentrated. When a non-aqueous system in a natural environment such as food, cereals, plants and soil is used as a sample, AF is extracted from the sample with a solvent such as methanol, and then the methanol extract is subjected to, for example, the aforementioned washing buffer or the like. Use after dilution with a buffer or the like. AF prepared in this way
Is mixed with a buffer solution containing an excess amount of the first antibody, and reacted by, for example, incubating at about 20 ° C. overnight. At this time, the primary antibody is diluted about 3,000 to 5,000 times to
It is desirable to react with AF, and a dilution of about 5,000-fold can be more desirable. That is, about
It is desirable to mix the 2,500-fold diluted primary antibody with a sample solution containing the same volume of AF and 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 first antibody is excessively diluted.

The unreacted first antibody contained in the reaction solution of the sample and the first antibody prepared as described above is combined with serum albumin of a mammal of the same species as the mammal producing the first antibody.
A reaction for binding to a carrier whose surface is coated with an AF conjugate is performed. Under these reaction conditions, it is desirable to add a reaction solution of the sample and the first antibody to the carrier, and to allow the reaction at, for example, about 20 ° C. for about 1 hour. After the reaction, after washing the carrier with the above-mentioned washing buffer, the labeling enzyme is bound, and the antibody against the first antibody (second antibody) derived from a mammal of a different species from the mammal producing the first antibody
For the reaction.

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

[0011] The present invention also relates to a method for use which comprises an anti-AF mammalian antibody as a reagent, and which is suitable for use under field conditions for high-precision detection / analysis which can be performed easily and rapidly with AF. Means for immunological detection and analysis of AF in the form of a test kit that has been prepared. The above-described test kit may contain, for example, the following components: (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 material), (2) reagent containing anti-AF mammalian antibody (first antibody), (3) derived from a mammal of a different species from the mammal that binds the labeled enzyme and produces the first antibody Against the first antibody
(2) a reagent containing (second antibody), (4) a standardized solution of AF, (5) a buffer solution, (6) an additive such as a polypeptide that prevents nonspecific adsorption and formation of aggregates, and an additive such as (7) ) Pipettes, reaction vessels, calculation curves, etc.

[0012]

EXAMPLES 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 changes in the technical field of the present invention can be made. Example 1 (Preparation of 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 then 2.6 ml of dioxane was added dropwise at 10 ° C. Next, while maintaining the pH at 9 with a 1N NaOH solution, the AF solution prepared above was added little by little, and the mixture was added at 4 ° C.
The reaction was performed by stirring for 4 hours. Thereafter, purification by gel filtration using Sephadex G-25 was performed. The obtained protein fraction was dialyzed against distilled water for 24 hours and lyophilized to obtain a conjugate of bovine serum albumin and AF.

Example 2 (Preparation of Anti-AF Mammalian Antibody) The conjugate obtained in Example 1 was dissolved in a physiological phosphate buffer to a concentration of 200 μg / ml. 0.5 ml each of this aqueous solution and Freund's complete adjuvant (manufactured by DIFCO LABORATORIES) were 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). Thereafter, 2, 4, and 6 weeks later, a physiological phosphate buffer solution prepared so that the concentration of the conjugate obtained in Example 1 became 100 μg / ml was additionally administered in the same manner as the first time. Whole blood was collected from test rabbits immunized after the last administration, and the obtained blood was centrifuged (1000 xg, 15 min, 4
C) and the supernatant was collected. The resulting supernatant was salted out with 33% saturated ammonium sulfate, centrifuged (1000 × g, 15 min, 4 ° C.), and the precipitate was dissolved in a small amount of a physiological phosphate buffer. This was dialyzed overnight at 4 ° C. using a physiological phosphate buffer 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 species as the mammal producing the first antibody) Rabbit serum albumin and AF obtained according to Example 1 Coating buffer containing conjugate at a concentration of 1 μg / ml (10 mM phosphate buffer (pH 7.5) containing 100 mM NaCl)
Into a polystyrene 96-well microtiter plate, 0.1m
1 / well, and incubated at about 4 ° C. overnight. Then, 300 μl of a 3% skim milk solution was added thereto, and the mixture was incubated at 20 ° C. for about 2 hours. Then, the microtiter plate was washed with a washing buffer (NaCl 0.8% (W / V), KC
l 1 containing 0.02% (W / V) and 200.2% (V / V) Tween
The plate was washed three times with 0 mM phosphate buffer (pH 7.2).

Example 4 (Indirect competitive inhibition method (ELISA method)
1) Anti-AF rabbit antibody obtained in Example 2 was washed with 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) In), dilute 2,500-fold using a buffer containing skim milk at a concentration of 3%, mix with the same volume of the sample solution diluted with the above washing buffer, and react overnight at about 20 ° C. I let it. 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 allowed to react at 20 ° C. for 1 hour. Was washed three times with the above washing buffer. A solution obtained by diluting an anti-rabbit IgG goat IgG fraction (manufactured by Wako Pure Chemical Industries, Ltd.) bound to peroxidase as a labeling enzyme by 1,000 times with the above buffer solution was added to the carrier at a rate of 0.1 ml / well at 20 ° C. About 3
After incubation for a period of time, the carrier (with which the second antibody was reacted) was washed three times using the above-mentioned washing buffer. o-
Phenylenediamine coloring solution (o-phenylenediamine-0.2% (W / V) 100 mM, phosphate / citrate buffer (pH5) 10
0 mM, hydrogen peroxide 0.003% (V / V)) was added to the carrier after washing at a rate of 0.2 ml / well, and after incubation at 20 ° C for 10 minutes, 4N H ₂ SO _4, 0.05 ml / The reaction was stopped at the rate of well. Thereafter, 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, the AF concentration of paddy water, tap water and washing buffer was measured.
The results of each measurement using the solution were almost the same, and it was found that the substances contained in the paddy water and tap water had no effect on the measurement of the AF concentration, and the AF in the sample was about 100 ng.
It has been found that the residual amount of AF in a sample can be accurately measured in a concentration range of / ng to about 10,000 ng / ml.

Example 5 (Indirect competitive inhibition method (ELISA method)
2) The anti-AF rabbit antibody obtained in Example 2 was washed 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-fold using a buffer containing skim milk at a concentration of 6%, mix with the same volume of sample solution diluted with the above washing buffer, and react at approximately 20 ° C overnight. I let it. 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 allowed to react at 20 ° C. for 1 hour. Was washed three times with the above washing buffer. A solution obtained by diluting the anti-rabbit IgG donkey IgG fraction conjugated with biotin as a labeling enzyme 2,000-fold with the above buffer solution 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 (with which the second antibody was reacted) was washed three times with the above-mentioned washing buffer. A solution obtained by diluting avidin-conjugated alkaline phosphatase 1000-fold was added at a rate of 0.1 ml / well, and incubated for 1 hour. Then, the carrier was washed three times with the above buffer solution. Alkaline phosphatase color reagent (1 M diethanolamine containing 0.5 M MgCl ₂ , 0.1% disodium p-nitrophenol phosphate)
Hydrochloric acid buffer (pH 9.8)) was added to the washed carrier at a rate of 0.2 ml / well, incubated at 25 ° C for 30 minutes, and 1N NaOH was added at a rate of 0.05 ml / well to perform the reaction. stopped. Thereafter, 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, the respective measurement results using the paddy water, the tap water and the AF solution of the washing buffer almost agree with each other, and the substances contained in the paddy water and the tap water. Has no effect on the measurement of the AF concentration, and the AF in the sample is about 100 ng / ml
It has been found that the residual amount of AF in a sample can be accurately measured in a concentration range of about 10,000 ng / ml.

[0017]

According to the present invention, it has become possible to measure the residual amount of aciflorfen (diphenyl ether herbicide) in a highly accurate natural environment sample which can be easily and quickly processed. In particular, in the measurement of the residual amount of aciflorfen in natural environment samples such as paddy water, tap water, food, cereals, plants and soil, a large number of measurements can be processed at one time.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/53

Claims (3)

(57) [Claims] 1. An indirect competitive inhibition method (EL) which is an immunological method for detecting 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid (hereinafter referred to as AF).
ISA method), wherein (1) reacting a solution containing a predetermined amount of an anti-AF mammalian antibody (hereinafter referred to as a first antibody) with a sample solution containing AF, and (2) 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) an antibody against the first antibody (second antibody) derived from a mammal of a different species from the mammal that produces the first antibody is bound to the labeled enzyme and reacted with the carrier. ,(Five)
After washing the carrier, (6) a buffer solution containing a coloring reagent that develops a color by enzymatic reaction with the substrate of the labeling enzyme is added, and after the reaction, 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, comprising calculating an AF concentration in the sample. 2. An indirect competitive inhibition method (EL), which is an immunological method for detecting 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid (hereinafter referred to as AF).
ISA method), comprising: (1) reacting a solution containing a predetermined amount of anti-AF rabbit antibody with a sample solution containing AF in a range of 100 ng / ml to 10,000 ng / ml; The unreacted anti-AF rabbit antibody contained in the reaction solution is bound to a carrier whose surface is coated with a conjugate of rabbit serum albumin and AF,
(3) After washing the carrier, (4) reacting a peroxidase-conjugated anti-rabbit IgG goat IgG with the carrier, (5) washing the carrier, and (6) reacting the enzyme with the substrate of the labeled enzyme. A buffer containing a coloring reagent that develops color by the reaction is added,
The method according to claim 1, wherein after the reaction, the absorbance is measured, and (7) the AF concentration in the sample is calculated from the measured value.
Measurement method. 3. A test kit for use in the method according to claim 1, comprising (1) 5- {2-chloro-4- (trifluoromethyl) phenoxy} -2-nitrobenzoic acid (hereinafter, referred to as AF)). 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 (a solid support); (2) an anti-AF mammalian antibody
(Hereinafter, referred to as a first antibody), and (3) a first antibody which binds a labeling enzyme and is derived from a mammal of a different species from the mammal producing the first antibody. A composition for immunological detection and analysis of AF, comprising a reagent containing an antibody.