JP2016223900A - Pretreatment method for inspecting residual pesticide using immunoassay method and method for inspecting residual pesticide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pretreatment method for inspecting residual pesticide in an agricultural product containing polyphenol and a method for inspecting the residual pesticide in the agricultural product.SOLUTION: Provided is a pretreatment method for inspecting residual pesticide using the immunoassay method. To inspect residual pesticide attached to or absorbed in an agricultural product containing polyphenol, an extraction liquid is extracted from the agricultural product and an antigen-antibody reaction in which the residual pesticide contained in the extraction liquid works as an antigen is used to quantify the residual pesticide. Prior to the quantification, the extraction liquid is pretreated with the pretreatment method using the immunoassay method with casein mixed in the extraction liquid.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a pretreatment method for a pesticide residue test for a crop containing polyphenol, and a method for testing a pesticide residue of the crop.

  In voluntary inspection of agricultural chemicals remaining on agricultural products, immunoassay inspection is attracting attention because it is a quick, simple and inexpensive measurement method compared to instrumental analysis. However, it has been pointed out that the measurement is disturbed by components contained in agricultural products.

  For example, when the chlorthalonil organochlorine fungicide in tea extract is measured by the Eliza method, it has been clarified that the reactivity of chlorothalonil with an antibody changes due to the influence of components derived from tea. As a means for removing such impurities, there is a pretreatment method using a minicolumn (Oasis HLB 60 mg, manufactured by Waters). Tests for fat-soluble pesticides such as chlorothalonil have been carried out using a pretreatment method using this mini-column.

Eriko Hatakeyama, 3 others, “Residual analysis of chlorothalonil in tea leaves by the Eliza method”, Journal of Pesticide Science Society of Japan, Japanese Pesticide Society, November 20, 2008, Vol. 33, No. 4, pp. 387-392

  However, for water-soluble pesticides, a pretreatment method using an immunoassay method including the Eliza method has not been established, and there has been a problem that the measurement of pesticides is hindered by the influence of contaminant components.

In order to solve such a problem, the present inventors have studied a technique for capturing a contaminating component contained in tea, and as a result, have found a simple method capable of measuring agricultural chemicals.
The technology described below has been completed based on the above knowledge, and one of its purposes is to provide a pretreatment method for the inspection of residual agricultural chemicals for crops containing polyphenols, and a method for inspection of residual agricultural chemicals of the crops. There is.

  In the pretreatment method described below, as a residual agricultural chemical test for residual agricultural chemicals attached to or penetrating into crops containing polyphenols, residual agricultural chemicals contained in the extract are extracted as antigens against the extracted liquid extracted from agricultural crops. In quantifying residual pesticides using the antigen-antibody reaction, a pretreatment method for residual pesticide inspection using an immunoassay method performed on the extract before performing the quantification, Casein is mixed with the liquid.

  In addition, the pesticide residue inspection method is a residue pesticide test for residue pesticides attached to or penetrated into crops containing polyphenols, with the residue pesticide contained in the extract as an antigen. A method for inspecting residual agricultural chemicals by a quantitative method using an antigen-antibody reaction, characterized in that casein is mixed with the extract before carrying out the quantitative determination.

FIG. 1A is a graph showing the measurement results of samples (samples 1 to 10) that were not treated with skim milk obtained in Example 1. FIG. FIG.1 (b) is a graph which shows the measurement result of the sample (samples 11-20) which performed the skim milk process obtained in Example 1. FIG. FIG. 2A is a graph showing the measurement results of the sample prepared from the concentration of acetamiprid 0 ppb in Example 2. FIG. 2B is a graph showing the measurement results of the sample prepared from the concentration of 50 ppb acetamiprid in Example 2. FIG. FIG. 3 is a graph showing the measurement results of the samples (samples 50 to 61) obtained in Example 3. FIG. 4 is a schematic view of the skim milk treatment in the immunoassay.

Hereinafter, an embodiment performed for verifying the operation and effect of the present invention will be described, but the present invention is not limited to this embodiment.
In the embodiments described below, the following materials are used unless otherwise specified. As acetamiprid, 52220 of Hayashi Junyaku Kogyo Co., Ltd. is used. As the catechin powder, Code No. of Wako Pure Chemical Industries, Ltd. 032-18231, catechin mixture, derived from green tea. As the casein, skim milk powder containing casein is used, and as the skim milk powder, code No. of Wako Pure Chemical Industries, Ltd. is used. 198-10605, skim milk powder is used.

[Example 1: Measurement of concentration of acetamiprid to tea exudate]
As a specimen sample, tea was used as an agricultural crop containing polyphenol, and commercially available green tea was prepared. In the tea extraction method, first, 50 mL of boiling water at 100 ° C. was added to 3 g of tea leaves and allowed to stand for 5 minutes while boiling at 100 ° C. Then, it removed from the hot water bath, left still at room temperature for 1 hour, and left still at 4 degreeC overnight. It is considered that the components of tea extracted into hot water by such an extraction method include polyphenols such as catechin and tannin. Centrifugation was performed at 5000 g for 5 minutes, and the supernatant was collected. The supernatant was used as tea infusion (hereinafter referred to as infusion A).

  In addition, the notification method in the Ministry of Health, Labor and Welfare Ministry of Health, Labor and Welfare, Food Safety Department Notification No. 012400 includes an extraction method in which 1 g of a specimen sample is leached in 50 mL of boiling water at 100 ° C. In the embodiment described below, tea infusion A having a higher concentration than this notification method was used. As a result, in the measurement of residual agricultural chemicals using the antigen-antibody reaction, it was clarified that the measurement was disturbed by the contaminated component and the effect of eliminating the influence of the contaminated component, and it was confirmed that it was applicable to various types of tea.

Perform the following procedure 1-1, procedure 1-2, procedure 1-3, procedure 1-4, procedure 1-5, procedure 1-6, procedure 1-7, procedure 1-8 in order, and target each sample. The absorbance was measured.
Procedure 1-1. Using distilled water, samples were prepared so that the concentration of acetamiprid in distilled water was 0.1, 1, 10, 100, and 1000 ppb.
Procedure 1-2. Using the above leachate A, samples were prepared such that the concentration of acetamiprid in leachate A was 0.1, 1, 10, 100, and 1000 ppb.
Procedure 1-3. Each sample of acetamiprid concentration 0.1, 1, 10, 100, 1000 ppb prepared in the above procedure 1-1 was mixed with an equal amount of distilled water and then shaken slowly for 5 minutes. 3, 4, and 5. Samples 1 to 5 were used as sample solutions for the Eliza measurement.
Procedure 1-4. Each sample of acetamiprid concentration 0.1, 1, 10, 100, 1000 ppb prepared in the above procedure 1-2 and distilled water were mixed in equal amounts, and then shaken slowly for 5 minutes. , 8, 9, and 10. Samples 6 to 10 were used as sample solutions for the Eliza measurement.
Procedure 1-5. Each sample of acetamiprid concentration 0.1, 1, 10, 100, 1000 ppb prepared in the above procedure 1-1 and 4% skim milk were mixed in equal amounts, respectively, and then gently shaken for 5 minutes. 12, 13, 14, and 15. Samples 11 to 15 were used as sample solutions for the Eliza measurement.
Procedure 1-6. Each sample of acetamiprid concentration 0.1, 1, 10, 100, 1000 ppb prepared in the above procedure 1-2 and 4% skim milk were mixed in equal amounts, and then shaken gently for 5 minutes. 17, 18, 19, and 20. Samples 16 to 20 were used as sample solutions for the Eliza measurement.
Procedure 1-7. A sample containing no acetamiprid was also prepared. Specifically, distilled water or 4% skim milk was mixed with distilled water in equal amounts, and then gently shaken for 5 minutes to obtain a sample solution for Eliza measurement. Furthermore, after mixing equal amounts of distilled water or 4% skim milk with the leachate A, the mixture was gently shaken for 5 minutes to obtain a sample solution for Eliza measurement.
Procedure 1-8. The Eliza method (indirect competition method) for the sample solution of the Eliza measurement obtained from the procedure 1-3, the procedure 1-4, the procedure 1-5, the procedure 1-6, and the procedure 1-7. Absorbance was measured by. The Eliza method (indirect competition method) will be described in detail later.

  The results are shown in FIG. 1 (a) and FIG. 1 (b) (however, since FIG. 1 (a) and FIG. 1 (b) are graphs with logarithmic scale on the horizontal axis, 0.1% is included in each graph. 1, 10, 100, and 1000 ppb for each sample). In addition, the measurement result in FIG. 1 was shown by the average +/- standard deviation (n = 4).

  As shown to Fig.1 (a), when skimmed milk was not processed (samples 1-10), the light absorbency of samples 6-10 was low compared with the light absorbency of samples 1-5. This decrease in absorbance means a decrease in the reactivity of the antigen-antibody reaction using acetamiprid as an antigen, and it was considered that components such as catechin and tannin in tea infusion A interfered with the antigen-antibody reaction. On the other hand, when the skim milk treatment shown in FIG. 1B was performed (samples 11 to 20), the absorbance of samples 16 to 20 almost coincided with the absorbance of samples 11 to 15. This means that by performing skim milk treatment, acetamiprid exhibits almost the same reactivity of antigen-antibody reaction as that when it exists in distilled water even if it exists in tea infusion A. That is, when skim milk was not treated, contamination of the antigen-antibody reaction was caused by contaminating components such as catechin and tannin in tea exudate A, but this interference was avoided by carrying out skim milk treatment.

  Furthermore, when a calibration curve was created based on the absorbance of samples 16 to 20, a calibration curve almost identical to the calibration curve created based on the absorbance of samples 11 to 15 was obtained. Therefore, it is considered that if skim milk treatment is performed, the influence of contaminating components in tea infusion liquid A can be eliminated, and accurate measurement of acetamiprid is possible. In other words, if a sample with a known acetamiprid concentration is subjected to skim milk treatment and the absorbance is measured, and a calibration curve is created based on the measurement result, a known amount of acetamiprid in the tea infusion is known. Absorbance is measured after applying skim milk treatment equivalent to the skim milk treatment applied to the sample, and the concentration of acetamiprid in the tea leachate is determined based on the measurement results and the calibration curve described above. can do.

[Example 2: Measurement of concentration of acetamiprid with respect to catechin solution]
The following procedure 2-1, procedure 2-2, procedure 2-3, and procedure 2-4 were performed in order, and the absorbance was measured for each sample.
Procedure 2-1 Samples were prepared using a 1.10% methanol solution so that the concentration of catechin in the 10% methanol solution was 0, 100, 300, 400, 500, 800, 1000, 3000 μg / mL.
Procedure 2-2 Using a 2.4% skim milk solution, a sample was prepared so that the concentration of acetamiprid in the 4% skim milk solution was 0, 50 ppb.
Procedure 2-3. Samples were prepared using distilled water so that the concentration of acetamiprid in distilled water was 0, 50 ppb.
Procedure 2-4. Each sample prepared in the above procedure 2-1 and each sample prepared in the above procedure 2-2 were mixed in equal amounts and then allowed to stand for 15 minutes to obtain a sample solution for Eliza measurement. Moreover, after mixing each sample prepared by the said procedure 2-1 and each sample prepared by the said procedure 2-3 by equal amounts, it was left still for 15 minutes and it was set as the sample liquid of an Eliza measurement. The absorbance of these sample solutions was measured by the Eliza method (indirect competition method). The Eliza method (indirect competition method) will be described in detail later.

The results are shown in FIGS. 2 (a) and 2 (b).
As shown in FIG. 2 (b), in the case of a sample containing acetamiprid, if the skim milk treatment is not performed (see the graph on the left side in FIG. 2 (b)), the catechin concentration is compared with the catechin concentration of 0 μg / mL. As the concentration increased, the absorbance decreased greatly. This decrease in absorbance means that the reactivity of the antigen-antibody reaction using acetamiprid as an antigen has changed. In the solution containing acetamiprid, the reactivity of the antigen-antibody reaction changed as the concentration of catechin increased, so it was considered that catechin hindered the antigen-antibody reaction. On the other hand, in the case of a sample containing acetamiprid, when the skim milk treatment is performed (see the graph on the right side in FIG. 2 (b)), the absorbance of the sample hardly decreases even when the concentration of catechin increases. The value was almost the same as the absorbance when the concentration was 0 μg / mL. This means that the skim milk treatment does not change the reactivity of the antigen-antibody reaction even when the concentration of the catechin solution is increased. That is, in the solution containing acetamiprid, when skim milk is not treated, the catechin, which is a contaminating component, interferes with the antigen-antibody reaction, but by performing the skim milk treatment, the interference with the antigen-antibody reaction was avoided.

  Further, as shown in FIG. 2 (a), in the case of the sample not containing acetamiprid, the same change in absorbance as in the case of the sample containing acetamiprid (FIG. 2 (b)) was shown. As described above, the same change in absorbance was shown regardless of whether or not acetamiprid was contained, and therefore, when skim milk treatment was not performed (left side in FIG. 2A and FIG. 2B). (Refer to the graph on the left side of the inside.) As the catechin concentration increased, it was shown that the cause of the decrease in the absorbance was the decrease in the antigen-antibody reaction.

  Furthermore, avoiding such interference with the antigen-antibody reaction makes it possible to accurately determine the concentration of acetamiprid. In other words, if a sample with a known acetamiprid concentration is subjected to skim milk treatment and the absorbance is measured, and a calibration curve is created based on the measurement result, a known sample is used to quantify the acetamiprid concentration in the catechin solution. The absorbance is measured after applying a skim milk treatment equivalent to the skim milk treatment applied to the catechin solution, and based on the measurement result and the calibration curve described above, the concentration of acetamiprid in the catechin solution can be quantified. it can.

[Example 3: Measurement of the concentration of acetamiprid on a sample subjected to ultrafiltration after skim milk treatment]
The following procedure 3-1, procedure 3-2, procedure 3-3, procedure 3-4, procedure 3-5, procedure 3-6, procedure 3-7, procedure 3-8, procedure 3-9, procedure 3- 10 was performed in order, and the absorbance was measured for each sample.
Procedure 3-1. First, 4 mL of a 1000 μg / mL catechin solution was added to a centrifugal filtration device (abbreviation: Microsep 10K, product name: Microsep Advance, Centrifugal Filtration Device Microsep 10K, manufacturer code: MCP010C41, manufactured by PALL Corporation), and then 7500 g to 5 Centrifuged for minutes. Furthermore, after adding 4 ml of distilled water, it was washed by centrifuging at 7500 g for 5 minutes, and a pre-treatment of the centrifugal filtration device was performed (the centrifugal filtration device subjected to the pre-treatment is hereinafter referred to as a centrifugal filtration device P). ). This pre-treatment was performed in order to prevent contaminant components such as catechin and tannin in the tea component from adsorbing to the centrifugal filtration device in the ultrafiltration treatment of the following procedure.
Procedure 3-2. Distilled water, tea infusion A extracted in Example 1, and 1000 μg / mL catechin solution were mixed in equal amounts with distilled water to prepare a mixed solution, and mixed solution B, mixed solution C, It was set as the liquid mixture D. Next, it divided into the case where the process of ultrafiltration is not performed with respect to this liquid mixture B, C, D using the centrifugal filtration device P, and the case where the process of ultrafiltration is not performed.
Procedure 3-3. Distilled water, tea infusion A extracted in Example 1, and 1000 μg / mL catechin solution were mixed with equal amounts of 4% skim milk solution to prepare mixed solutions, which were mixed solution E and mixed solution, respectively. F and liquid mixture G were used. Next, it divided into the case where the process of ultrafiltration is not performed with respect to this liquid mixture E, F, G using the centrifugal filtration device P, and the case where the process of ultrafiltration is not performed.
Procedure 3-4. In the case of performing ultrafiltration, 1 mL of the mixed solution B mixed in the above procedure 3-2 was added to the filtration device P, and then centrifuged at 7500 g for 50 minutes, and the filtrate H was taken out. Moreover, when performing ultrafiltration processing, after adding the liquid mixture C mixed by 1 mL said procedure 3-2 to the filtration device P, it centrifuged at 7500g for 50 minutes, and the filtrate I was taken out. Similarly, when performing ultrafiltration, 1 mL of the mixed solution D mixed in the above procedure 3-2 is added to the filtration device P, and then centrifuged at 7500 g for 50 minutes to obtain the filtrate J. I took it out.
Procedure 3-5. In the case of performing ultrafiltration, 1 mL of the mixed solution E mixed in the procedure 3-3 was added to the filtration device P, and then centrifuged at 7500 g for 50 minutes, and the filtrate K was taken out. Moreover, when performing ultrafiltration processing, after adding the liquid mixture F mixed by 1 mL said procedure 3-3 to the filtration device P, it centrifuged at 7500g for 50 minutes, and the filtrate L was taken out. Similarly, when performing ultrafiltration, 1 mL of the mixed solution G mixed in the above procedure 3-3 is added to the filtration device P, and then centrifuged at 7500 g for 50 minutes, and the filtrate M is obtained. I took it out.
Procedure 3-6. A sample was prepared by using the mixed solution B mixed in the above procedure 3-2 so that the concentration of acetamiprid in the mixed solution B was 50 ppb. In addition, a sample was prepared by using the mixed solution C mixed in the procedure 3-2 so that the concentration of acetamiprid in the mixed solution C was 50 ppb. Similarly, a sample was prepared by using the mixed solution D mixed in the above procedure 3-2 so that the concentration of acetamiprid in the mixed solution D was 50 ppb. These samples 50, 54, and 58 were used as sample solutions for the Eliza measurement.
Procedure 3-7. A sample was prepared by using the mixed solution E mixed in the above procedure 3-3 so that the concentration of acetamiprid in the mixed solution E was 50 ppb, and the sample 52 was obtained. In addition, a sample was prepared by using the mixed solution F mixed in the procedure 3-3 so that the concentration of acetamiprid in the mixed solution F was 50 ppb. Similarly, a sample was prepared by using the mixed solution G mixed in the procedure 3-3 so that the concentration of acetamiprid in the mixed solution G was 50 ppb. These samples 52, 56, and 60 were used as sample solutions for the Eliza measurement.
Procedure 3-8. A sample was prepared by using the filtrate H subjected to the ultrafiltration treatment in the above procedure 3-4 so that the concentration of acetamiprid in the filtrate H was 50 ppb. In addition, using the filtrate I subjected to the ultrafiltration treatment in the above procedure 3-4, a sample was prepared so that the concentration of acetamiprid in the filtrate I was 50 ppb. Similarly, a sample was prepared by using the filtrate J subjected to the ultrafiltration treatment in the above procedure 3-4 so that the concentration of acetamiprid in the filtrate J was 50 ppb. These samples 51, 55, and 59 were used as sample solutions for the Eliza measurement.
Procedure 3-9. Using the filtrate K subjected to the ultrafiltration treatment in the above procedure 3-5, a sample was prepared so that the concentration of acetamiprid in the filtrate K was 50 ppb, and the sample 53 was obtained. In addition, a sample was prepared by using the filtrate L subjected to the ultrafiltration treatment in the above procedure 3-5 so that the concentration of acetamiprid in the filtrate L was 50 ppb. Similarly, a sample was prepared by using the filtrate M subjected to the ultrafiltration treatment in the above procedure 3-5 so that the concentration of acetamiprid in the filtrate M was 50 ppb. These samples 53, 57, and 61 were used as sample solutions for the Eliza measurement.
Procedure 3-10. Absorbance was measured by the ELISA method (indirect competition method) for the sample solution of the ELISA measurement obtained from the procedure 3-6, the procedure 3-7, the procedure 3-8, and the procedure 3-9. . The Eliza method (indirect competition method) will be described in detail later.

The result is shown in FIG. In addition, the measurement result in FIG. 3 was shown by the average +/- standard deviation (n = 4).
About the result of FIG. 3, when neither the skim milk process and the ultrafiltration process were performed, when the ultrafiltration process was performed without performing the skim milk process, the ultrafiltration process was not performed after the skim milk process was performed. The case and the case where the ultrafiltration treatment is performed after the skim milk treatment are referred to as Condition 1, Condition 2, Condition 3, and Condition 4, respectively, and will be described below.

  As shown in FIG. 3, the absorbance of the sample 52 subjected to the skim milk treatment was increased by performing the ultrafiltration treatment after the skim milk treatment (condition 4) (sample 53). From this result, it was found that skim milk remained as a residue in the centrifugal filtration device, and the skim milk was removed from the sample.

  In addition, the absorbance of the sample 54 was lower than that of the sample 50, and when the sample 54 was subjected to ultrafiltration treatment (sample 55), the absorbance increased, but did not increase to the absorbance of the sample 51. The decrease in the absorbance of the sample 54 compared to the absorbance of the sample 50 is due to the fact that components such as catechin and tannin in the tea infusion liquid A interfered with the measurement. And, despite the pretreatment treatment of the centrifugal filtration device that prevents the adsorption of tea-derived catechins, when ultrafiltration treatment is performed (sample 55), tea-derived catechins are slightly adsorbed to the centrifugal filtration device. As a result, the absorbance slightly increased compared to the case where the ultrafiltration was not processed (sample 54), and the interference with the measurement was reduced. However, since the increase did not increase to the absorbance of the sample 51, it was found that the centrifugal filtration device did not completely adsorb the tea-derived contaminant components, and the contaminant components remained in the sample.

  On the other hand, the absorbance of the sample 56 showed almost the same value as the absorbance of the sample 52. This is considered to be due to the effect of the skim milk treatment, which was prevented from being disturbed by components such as catechin and tannin in the tea infusion A, and was affected in the same way as distilled water. When the ultrafiltration treatment was performed on the sample 56 (sample 57), the absorbance increased and increased to the absorbance of the sample 53. In other words, by performing ultrafiltration after skim milk treatment (condition 4), the interference with the measurement was avoided because the skim milk and contaminating components such as catechins and tannins in tea leachate A were added to the centrifugal filtration device. It means that it remained as a residue. From this point and the point that the skimmed milk described above remains as a residue in the centrifugal filtration device and that the contaminating component hardly adsorbs to the centrifugal filtration device, it was found that the contaminated component adsorbs to the skimmed milk. Therefore, contaminant components such as catechin and tannin can be removed from the sample 56, and the influence of the contaminant components on the measurement is omitted.

  Furthermore, the results for the catechins of the samples 58 to 61 showed the same tendency as the results for the tea of the samples 54 to 57. Therefore, it was found that catechin was adsorbed on skim milk and catechin was removed from the sample, thereby preventing interference with measurement by catechin. Moreover, it was estimated that the factor which the change of the light absorbency of samples 54-57 appears includes tannin which is a property similar to catechin which is a component in tea and catechin.

[Eliza method (indirect competition method)]
The procedure of the Eliza method (indirect competition method) used in this embodiment is described below. Procedure 4-1, procedure 4-2, procedure 4-3, procedure 4-4, procedure 4-5, procedure 4-6, and procedure 4-7 are performed in this order, and the sample solution is measured by the Eliza method (indirect competition method). Went.
Procedure 4-1. The acetamiprid-BSA solution was dissolved in 55 mM NaHCO 3 (pH 9) at a concentration of 0.01 μg / mL. This solution was designated as Solution 4-1. The method for preparing the acetamiprid-BSA solution will be described in detail later.
Procedure 4-2. 100 μL of the solution 4-1 was added to each well of a microplate (product name: F96 MaxiSorp Nunc-Immuno Plate, catalog number 442404, manufactured by Thermo Scientific). The mixture was allowed to stand at 4 ° C. overnight, and acetamiprid-BSA was immobilized on a microplate. The solution after standing overnight was designated as Solution 4-2.
Procedure 4-3. The solution 4-2 in each wheel was removed, and the plate was washed 5 times with a washing solution (abbreviation: PBS, product name: phosphate buffered saline) containing 0.01% Tween-20. Thereafter, 300 μL of blocking solution (abbreviation: SB, product name: Starting Block T20 (PBS) Blocking Buffer, catalog number 37539, manufactured by Thermo Scientific)) was added to each well and allowed to stand at room temperature for 30 minutes. The solution after standing was designated as Solution 4-3.
Procedure 4-4. Except for the solution 4-3 in each well, add 50 μL of the sample solution for ELISA measurement in Examples 1 to 3 above and 50 μL of anti-acetamipride monoclonal antibody diluted to 2 μg / mL with blocking solution, and cover. A film was attached and allowed to stand at room temperature for 2 hours. The solution after standing was designated as Solution 4-4.
Procedure 4-5. The secondary antibody (product name: ECL Anti-mouse IgG Horseradish linked whole antibody), catalog number NA931V was diluted 10,000 times with a blocking solution after removing the solution 4-4 in each well and washing 5 times with a washing solution. GE Healthcare Life Sciences) was added in an amount of 100 μL and allowed to stand at room temperature for 1 hour. The solution after standing was designated as Solution 4-5.
Procedure 4-6. After removing the solution 4-5 in each well and washing 5 times with a washing solution, 100 μL of a chromogenic substrate (abbreviation: TMB, product name: Sure Blue Reserve TMB Microwell Peroxidase Substrate, product code 53-00-01, manufactured by KPL) was added. And left to stand in the dark for 30 minutes. The solution after standing was designated as Solution 4-6.
Procedure 4-7. To the solution 4-6, 100 μL of a reaction stop solution (product name: TMB stop solution, product code 50-85-05, manufactured by KPL) was added, and the absorbance (450 nm) was measured using a plate reader.

[Eliser method (indirect competition method): Preparation method of acetamiprid-BSA solution]
A method for preparing an acetamiprid-BSA solution used in the Eliza method (indirect competition method) is described below. This acetamiprid-BSA solution was used for immobilizing a microplate used in the ELISA method (indirect competition method). Procedure 5-1, procedure 5-2, procedure 5-3, and procedure 5-4 were performed in this order to prepare an acetamiprid-BSA solution.
Procedure 5-1. A derivative in which a carboxyl group was introduced into acetamiprid was dissolved in dimethyl sulfoxide (abbreviation: DMSO) at a concentration of 20 mg / mL. This solution was designated as Solution 5-1.
Procedure 5-2. 200 μL of solution 5-1, 100 μL of 80 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (abbreviation: EDC) prepared with DMSO, and 80 mM N-hydroxysuccinimide (DMDC) Abbreviation: NHS) was added to 100 μL. Stir slowly at room temperature for 1.5 hours to give solution 5-2.
Procedure 5-3. Solution 5 in 0.5 mL of 10 mg / mL bovine serum albumin (abbreviation: BSA) dissolved in 0.1 M borate buffer (abbreviation: BB) (pH 8.0) containing 150 mM sodium chloride 2 was slowly added and stirred slowly at room temperature overnight. This solution is designated as Solution 5-3.
Procedure 5-4. Solution 5-3 was subjected to centrifugal filtration device (abbreviation: Microsep 10K, product name: Microsep Advance, Centrifugal filtration device Microsep 10K, manufacturer code: MCP010C41, manufactured by PALL Corporation) and 10 mM sodium phosphate buffer (pH 7.3). (Abbreviation: PB) was purified by ultrafiltration to obtain an acetamiprid-BSA solution.

[Schematic of skim milk treatment in immunoassay]
The above results will be described below with reference to the schematic diagram of FIG.
A plate of Eliza in which acetamiprid-protein, that is, pesticide-protein is solid-phased with an acetamiprid-BSA solution is used. The acetamiprid pesticide and anti-acetamiprid monoclonal antibody, which is an anti-pesticide antibody, or tea infusion A are added to the plate. When the tea infusion A is not added, the anti-acetamipride monoclonal antibody binds to the antigen, acetamiprid, and causes an antigen-antibody reaction. On the other hand, when the tea exudate A is added, contaminant components such as catechin in the tea exudate A interfere with the antigen-antibody reaction by adhering to the anti-acetamipride monoclonal antibody or the agricultural chemical-protein. However, when the process of adding skim milk is performed, the skim milk captures a contaminating component such as catechin, thereby preventing interference of the antigen-antibody reaction by the contaminating component. From this, if the skim milk treatment is not performed before quantifying the residual pesticide in the tea extract, it is not possible to accurately determine the residual pesticide due to the interference of antigen-antibody reaction with contaminant components, but by performing the skim milk treatment, Interference with the antigen-antibody reaction by contaminating components is avoided, and accurate quantification can be realized. In other words, when quantifying pesticides in extracts extracted from crops that contain polyphenols, if the absorbance is measured after applying skim milk treatment, a sample prepared after the same skim milk treatment is applied to samples with known pesticides. Pesticides can be quantified based on the line.

[Other Embodiments]
As mentioned above, although embodiment of this invention was described, this invention is not limited to said specific one Embodiment, In addition, it can implement with a various form.

  For example, in the above-described embodiment, an example of a method of leaching from a specimen sample as an extraction liquid of the specimen sample has been described, but the extraction method is not limited to this, and other extraction methods may be used. As other extraction methods, for example, the following methods can be used. Add double the amount of water to the sample tea and chop it with a food processor to make it even. The chopped and homogenized material is dispensed into a conical tube, 5 times the amount of methanol is added, and then extracted with shaking for 30 minutes. Centrifuge for 10 minutes at 1880 g and collect the supernatant. The supernatant was diluted so that the methanol concentration was 5%, and used as an extract of the specimen sample.

  Moreover, although the example of the green tea was shown as a specimen sample, a specimen sample is not limited to this, What is necessary is just a crop containing polyphenol. Examples of crops that contain polyphenols include, for example, tea, grapes, apples, blueberries, strawberries, bananas, strawberries, Soldam, raspberries, prunes, peaches, purple onions, onions, olives, spinach, broccoli, walnuts, oregano, sage, rose Examples include marie, almonds, cocoa powder, and chocolate. Examples of tea include, for example, Japanese tea (green tea), Chinese tea, and black tea.

Moreover, although the example of catechin and tannin was shown as polyphenol, polyphenol is not limited to this, Other polyphenol may be sufficient.
Moreover, although the example of acetamiprid was shown as an agrochemical, a residual agrochemical is not limited to this, What is necessary is just an agrochemical tested using an immunoassay method. Examples of pesticides used in the immunoassay include, for example, azoxystrobin, emamectin, fenitrothion, isoxathion, malathion, triflumizole, chlorophenapyl, iprodione, carbaryl, isoprothiolane, microbutanyl, flutolanil, vitertanol, boscalid, toluclophosmethyl , Fipronil, chlorantranilibrol, pyridalyl, mepanipyrim, cresoxime methyl, spinosad, flufenoxuron, buprofezin, dipermethrin, fenvalerate, procymidone, methidathion, chlorpyrifos, and phostiazate. Examples of residual agricultural chemicals include water-soluble agricultural chemicals and fat-soluble agricultural chemicals. Examples of water-soluble pesticides include neonicotinoid pesticides. Examples of neonicotinoid pesticides include acetamiprid, thiacloprid, imidacloprid, thiamethoxam, clothianidin, dinotefuran, and nitenpyram.

Moreover, although the example of the mixing process of skim milk was shown as a pre-processing method, the pre-processing method is not limited to this, What is necessary is just a mixing process of the solution containing casein.
In the above examples, 4% skimmed milk was used, but it may be adjusted to 1% -20%, in particular 1% -10%. Casein may be used at 0.3% -7%.

  Moreover, although the ELISA method (indirect competition method) was used as the immunoassay method, the immunoassay method is not limited to this, and other immunoassays may be used. As other immunoassay methods, for example, Eliza method (direct competition method), immunochromatography method, Western blotting method, surface plasmon resonance method, latex agglutination method, immunoturbidimetric method, and Luminex method can be used.

Claims (9)

As a residual pesticide test for residual pesticides attached to or permeating a crop containing polyphenol, an antigen-antibody reaction using the residual pesticide contained in the extract as an antigen is performed on the extract extracted from the crop. In quantifying the pesticide residue using, a pretreatment method for pesticide residue inspection using an immunoassay method performed on the extract before performing the quantification,
A pretreatment method in which casein is mixed with the extract. The pretreatment method according to claim 1, wherein the polyphenol is one or both of catechin and tannin. The pretreatment method according to claim 1, wherein the casein is casein derived from skim milk. The pretreatment method according to any one of claims 1 to 3, wherein the residual pesticide is a water-soluble pesticide. The pretreatment method according to claim 4, wherein the water-soluble pesticide is a neonicotinoid-based pesticide. The pretreatment method according to claim 5, wherein the neonicotinoid pesticide is acetamiprid, thiacloprid, imidacloprid, thiamethoxam, clothianidin, dinotefuran, or nitenpyram. The pretreatment method according to any one of claims 1 to 6, wherein the crop containing the polyphenol is tea. As a residual pesticide test for residual pesticides attached to or permeating a crop containing polyphenol, an antigen-antibody reaction using the residual pesticide contained in the extract as an antigen is performed on the extract extracted from the crop. It is a residual pesticide inspection method that quantifies the residual pesticide by a quantification method used,
A method for testing pesticide residues, wherein casein is mixed with the extract before carrying out the determination. The residual agricultural chemical test method according to claim 8, wherein the quantification method is any one of an Eliza method (indirect competition method), an Eliza method (direct competition method), an immunochromatography method, and a surface plasmon resonance method.