JP2001281250A - Method for detecting low-molecule compound using antigen/antibody reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting such low-molecule compound as mepronyl and iprodione more simply and rapidly as compared with the conventional ELISA method. SOLUTION: In the method for detecting a low-molecule compound that is a substance to be analyzed using the antigen/antibody reaction, a substance that specifically reacts with a substance to be analyzed on a porous material is subjected to solid phase, and a specimen containing the substance to be analyzed is filtered by the porous material, thus allowing the substance to be analyzed to react with the solid-phase substance on the porous material. Then, a labeled substance that reacts with the substance to be analyzed by completion to the solid-phase substance is filtered by the porous material, thus allowing the labeled substance to react with the solid-phase substance. By measuring the amount of substance being labeled while reacting with the solid-phase substance, the amount of substance to be analyzed is detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a low molecular weight compound using an antigen-antibody reaction.

[0002]

2. Description of the Related Art In recent years, there has been a great social concern about the effects of pesticide residues in the environment such as water, soil and air and in foods on human health. For example, with respect to mepronil and iprodione, reference values remaining in foods are respectively defined.

Conventionally, these compounds have been extracted and purified from samples such as cereals and fruits, and then purified by gas chromatography (GC) or high-performance liquid chromatography (HPLC).
And so on. These methods have problems that the preparation of the sample is complicated and requires a great deal of labor and time, that the analysis requires skill, and that a high cost is required for measuring devices and equipment. In the measurement of pesticides such as mepronil and iprodione, it is necessary to obtain an analysis result of an enormous number of samples in a short time, and there is a demand for a measurement method that is not only accurate but also simple, quick and economical. ing.

[0004] The immunochemical measurement method is a method for detecting an antigen based on an antigen-antibody reaction in which an antibody specifically recognizes the antigen. Due to its excellent accuracy, simplicity, rapidity, and economic efficiency, it has recently been used. Attracting attention. In immunochemical assays, a great variety of labels are used as detection methods, for example, enzymes,
Radiotracers, chemiluminescent and fluorescent labels, metal atoms and sols, stable free radicals, latex and bacteriophages have been applied.

[0005] Among immunochemical assay methods, enzyme immunoassay (adsorption) assay (ELISA) using an enzyme has been widely used as being particularly excellent in terms of economy and convenience.

In general, for a molecule having a large molecular weight,
By inoculating animals without further modification,
An appropriate immune response can be generated to produce an antibody that recognizes the antigen. However, low molecular weight compounds such as mepronil and iprodione do not usually elicit an immune response when inoculated into animals. For the first time, these molecules act as a panel of epitopes by binding to a macromolecule that has immunogenicity, and elicit an immune response in the presence of the T cell receptor, which results in a group of B lymphocytes that produce antibodies. Produced. Molecules that produce immunogenicity only when bound to a polymer compound in this way are collectively called “haptens”.

[0007] In the ELISA method, the above low-molecular compound can be detected immunochemically using an antibody prepared using such a hapten. In the conventional ELISA method, for example, a 96-well microtiter plate made of polystyrene is used as a carrier for immobilizing an antigen or an antibody. After immobilizing an antigen or antibody in each well, a labeled antigen or antibody is detected by an indirect competition method or a direct competition method, and the amount of a substance to be analyzed is detected.

[0008]

The conventional ELISA method is known as a simple and quick measurement method, but still requires about several hours for measurement. Therefore, there has been a demand for a simple and quick measurement method that can measure in a shorter time than the conventional ELISA method.

An object of the present invention is to provide a method capable of detecting low-molecular-weight compounds such as mepronil and iprodione more easily and quickly than conventional ELISA methods.

[0010]

SUMMARY OF THE INVENTION The present invention is a method for detecting a low molecular weight compound which is a substance to be analyzed using an antigen-antibody reaction, wherein a substance which specifically reacts with the substance to be analyzed is placed on a porous material. The solid phase is immobilized, and the analyte containing the analyte is filtered through the porous material, whereby the analyte is reacted with the immobilized material on the porous material. By filtering the labeled substance that reacts in competition with the analysis target substance through the porous material, the labeled substance is reacted with the solid-phased substance, and the solid-phased substance is reacted with the solid-phased substance. The method is characterized in that the amount of a substance to be analyzed is detected by measuring the amount of a labeled substance.

In the present invention, examples of the substance immobilized on the porous material include an antibody that specifically reacts with the substance to be analyzed. Such an antibody can be prepared using, for example, a hapten compound corresponding to the substance to be analyzed, and may be a polyclonal antibody,
It may be a monoclonal antibody.

The labeled substance which reacts with the analyte by reacting with the solid-phased substance such as an antibody includes a denaturant of the analyte, for example, a hapten or the like and a labeling agent directly. Or a substance bound via another compound. Examples of the labeling agent include an enzyme, a radioactive tracer, a colored marker, and the like. However, it is preferable to use an enzyme or a colored marker since semi-quantitative detection can be visually performed as a detection method.
As will be described later, when an enzyme is used, it takes time to develop a color. Therefore, from the viewpoint of enabling measurement in a short time, it is preferable to use a colored marker as a labeling agent. Examples of such a colored marker include colloidal gold, latex beads, and selenium.

The substance to be analyzed in the present invention is preferably a low molecular weight compound which cannot be a polyvalent antigen from the principle of measurement. In addition, the filtration by the porous material in the present invention is preferably performed using a plate having a plurality of wells each having a wall and a bottom. In this case, the porous material is preferably arranged at the bottom of the well. Such plates are available from Millipore as "Mul
This is commercially available as “tiScreen” (registered trademark), “IP” using hydrophobic PVDF (polyvinylidene fluoride) as a porous material, “HA” using mixed cellulose ester as a porous material, and 1 as a porous material.
“NC” using 00% nitrocellulose is known. These plates have 96 wells.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (Experiment 1) Mepronil having a structure represented by the following formula (Formula 1) was used as a substance to be analyzed, and MultiScreen IP (manufactured by Millipore) was used as a porous material.

[0015]

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MultiScreen IP: 0.4
96-well membrane plate with 5 μm hydrophobic PVDF membrane-Immobilon P- attached to the bottom of the well, hereinafter referred to as “IP plate”.

A monoclonal antibody obtained by synthesizing a mepronyl hapten represented by the following formula (Chem. 2) and immunizing a mouse with the hapten was used as a mepronil antibody.

[0018]

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The labeled substance which reacts in competition with mepronil is a substance obtained by binding mepronil hapten to horseradish peroxidase (hereinafter referred to as "mepronil hapten").
"Mepronil conjugate") was used.

Using the above IP plate, coating, blocking, competitive reaction, and color development were performed in the following procedures. (1) Coating: 50 μg / ml of mepronil antibody solution was added to each well of the IP plate at 10 / well.
Add to 0 μl and filter immediately after addition. Thereby, the mepronil antibody is immobilized on the membrane.

(2) Blocking: Block Ace is set to 2
5% sodium phosphate buffer (hereinafter referred to as “PB
S ") at a concentration of 300 µl per well, and reacted at 25 ° C for 2 hours, followed by filtration.

(3) Washing: PBS containing 0.05% Tween is added at 200 μl per well, and filtration is performed immediately after the addition. (4) Competition reaction: Addition is performed in the order and method shown in Table 1.

(5) Washing: PBS containing 0.05% Tween is added at 200 μl per well, and filtration is performed immediately after the addition. (6) Color development: OPD (o-phenylenediamine; 161)
-1971, manufactured by Wako Pure Chemical Industries, Ltd.)
After reacting for a minute, the mixture is filtered.

The above "Block Ace" is manufactured by Dainippon Pharmaceutical Co., Ltd., code No. UK-25B.

[0025]

[Table 1]

In the competitive reaction (4), the sample solution was a 10% methanol solution, and 5
0 μl was added. The mepronil conjugate solution was diluted 20000 times with PBS, and 50 μl was added per well.

As shown in Table 1, CF-SF is obtained by adding a mepronil conjugate first and filtering, and then adding a sample of a sample containing mepronil which is a substance to be measured and filtering. SF-CF is obtained by adding a sample containing mepronil according to the present invention, filtering the sample, adding a mepronil conjugate, and filtering. CF-SI
Is a sample obtained by adding a mepronil conjugate first and filtering, adding a sample containing mepronil, incubating at 37 ° C. for 1 hour, and filtering. SF-C
I was prepared by adding a mepronil-containing sample according to the present invention, filtering, and then adding a mepronil conjugate.
After incubating at 1 ° C for 1 hour, the mixture was filtered.
The SCI is obtained by adding a sample containing mepronil and a mepronil conjugate, incubating at 37 ° C. for 1 hour, and then filtering.

FIG. 1 is a graph showing the relationship between the concentration of mepronil in a sample and the detected absorbance when added by the method shown in Table 1 above. As shown in FIG.
For F and CF-SI, a sufficient change in absorbance corresponding to a change in the concentration of mepronil in the sample was not obtained, indicating that semi-quantitative detection of mepronil was not possible. In addition, the sample and the conjugate were mixed in a well,
Also in CI, a sufficient change in absorbance corresponding to a change in mepronil concentration was not obtained, indicating that semi-quantitative detection was not possible.

On the other hand, in SF-CF and SF-CI to which the sample was previously added according to the present invention, a sufficient absorbance change corresponding to the mepronil concentration change was obtained, and semi-quantitative detection was possible. You can see that there is.

FIG. 2 is a graph showing the relationship between the inhibition rate of mepronil on the reaction of mepronil conjugate with the antibody and the concentration of mepronil in the sample. The results shown in FIG. 2 also indicate that semi-quantitative detection is possible according to the method for adding SF-CF and SF-CI according to the present invention.

The following can be considered as the reason for the above result. That is, the methanol contained in the sample decreases the peroxidase activity of the mepronil conjugate bound to the antibody, so that when the mepronil conjugate is first filtered and then the sample is filtered, the methanol in the sample becomes Reduces the activity of For this reason, CF-SF and C
It seems that good sensitivity was not obtained with F-SI. In contrast, SF-CF and SF-C according to the present invention
It is considered that since the sample containing methanol is filtered first, the activity of the mepronil conjugate to be filtered later can be kept high, and good sensitivity can be obtained.

SF-CI is incubated for 1 hour after the addition of the mepronil conjugate, and requires a long measurement time. From the viewpoint of shortening the measurement time, SF-
Since a method of adding CF is preferable, hereinafter, this SF-CF
Is employed.

[Comparison of Membrane Materials] As a porous membrane material, nitrocellulose was compared with hydrophobic PVDF, which is the membrane material of the IP plate. The following nitrocellulose membrane plates were used.

MultiScreen HA: 0.4
96-well membrane plate with 5 μm mixed cellulose ester membrane attached to the bottom of the well, manufactured by Millipore,
Hereinafter, it is referred to as “HA plate”.

FIGS. 3 and 4 are diagrams showing the results obtained by the SF-CF method using an IP plate and an HA plate. FIG. 3 shows the relationship between the mepronil concentration and the absorbance, and FIG. 4 shows the relationship between the mepronil concentration and the inhibition rate.

As shown in FIG. 3, the absorbance of the IP plate is about 1/3 higher than that of the HA plate. Further, as shown in FIG. 4, there is no significant difference in the inhibition ratio, but the value of the inhibition ratio at a high concentration is high because the S / N of the HA plate is low. As described above, since the HA plate has a low S / N ratio, and thus the variation in measurement is large, it can be seen that the IP plate having a high S / N ratio is a more excellent membrane material.

[Effect of Sample Addition Amount on Competition Reaction] The effect of sample filtration amount on sensitivity was examined. The above SF-CF
The effect on the 50% inhibitory concentration (IC50) was examined by changing the amount of the mepronil sample at a concentration of 2 ng / ml from 25 μl to 500 μl by the method described above.

FIG. 5 shows the results. The horizontal axis indicates the amount (μl) of the filtered sample liquid. As is clear from FIG. 5, a stable IC50 is shown in the amount of the filtrate to which 50 μl has been added.

[Effect of Color Developing Agent] As the color developing agent, the same OPD and TMBE (tetramethylbenzidine (soluble), manufactured by Moss) as described above were used. Was set to 250 μl, and the others were detected in the same manner. FIG. 6 shows this result, and it can be seen that both OPD and TMBE have the same detection sensitivity.

[Dry Storage of Coated Plate] A coated plate having an antibody immobilized thereon was prepared in advance, and the possibility of dry storage of the coated plate was examined.

The IP plate after coating, blocking and washing according to the above procedure was freeze-dried and stored at 4 ° C. According to the above SF-CF method, the filtration amount of the mepronil sample was set to 250 μl, and detection was performed using OPD as a color former.

FIG. 7 shows the result. "Fr
"eeze Dry" indicates a freeze-dried product.
“No Freeze Dry” indicates a sample that has not been freeze-dried. As is clear from the results shown in FIG. 7, it is found that the freeze-drying does not particularly affect the measurement system.

From the above experiments, it can be seen that according to the present invention, a good detection sensitivity can be obtained by first filtering the sample solution and then filtering the conjugate solution.
In addition, it can be seen that detection can be performed in about 30 minutes.
Of the 30 minutes of this measurement time, 20 minutes is the time for coloring by the coloring agent. Therefore, it is possible to further reduce the measurement time by using a colored marker such as gold colloid.

The method of the present invention by filtration using a membrane has a sensitivity equivalent to that of a conventional ELISA plate.

(Experiment 2) A measurement method using colloidal gold as a coloring marker was examined below. [Preparation of Colloidal Gold Labeled Substance] Gold colloid having a particle size of 15 nm was bound as a coloring marker (labeling agent) to a binding substance of goat IgG and mepronil hapten shown in (Chemical Formula 2). The mepronil hapten and goat IgG were mixed and bound at a ratio of 1:20 by a mixed acid anhydride method. Thereafter, 0.1 mg of the binding substance and 15 nm of gold colloid (p
H9.5) was reacted.

[Measurement Procedure] Using the gold colloid-labeled substance obtained as described above, measurement was carried out according to the following procedure.

(1) Coating: A mepronil antibody solution having a concentration of 10 μg / ml is added to each well of the IP plate so as to be 50 μl per well, and then filtered. (2) Blocking: Block Ace is added at 50 μl per well and then filtered.

(3) Wetting: 10% glycerol is added at 50 μl per well, filtered and wetted. (4) Drying: Freeze-dry, seal and store at 4 ° C.

(5) Addition of sample: When necessary, take out the plate which has been freeze-dried and stored, and extract 50 μl of an extract sample containing 10% methanol per well.
And filtered.

(6) Addition of a gold colloid-labeled substance: 20 OD at an absorbance of 530 nm prepared as described above.
Gold colloid-labeled substance at a concentration of
Add to a volume of 1 and filter.

(7) Washing: PBS containing 0.05% Tween 20 is added at 50 μl per well, followed by filtration. This operation is repeated three times. As a sample, a sample extracted by the following addition / collection / extraction method was used.

Grain extraction method 2 g of mepronil methanol solution was added to 10 g of crushed grains (brown rice, buckwheat, barley, wheat, corn).
Dry at room temperature for hours. Thereafter, 10 ml of PBS was added, and the mixture was allowed to stand at 4 ° C. overnight to be hydrated. 30 ml of methanol was added, shaken for 2 hours, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used as an extract sample. Extract solution sample was PBS
And diluted 10-fold for the test.

Crop Extraction Method 1 ml of a mepronil methanol solution was added to 5 g of chopped crops (potato, pepper, spinach, apple, banana, strawberry), and 19 ml of methanol was added. Homogenizer (CELL MASTER CM-
100), shaken for 2 hours, 10,000 rpm
and the supernatant was used as an extract sample. Extract samples were diluted 10-fold with PBS and used for the test.

Juice, river water extraction method
5, 1, 0 mg / ml mepronil methanol solution
1 by 1 (0, 1, 5, 10 ppm). Then one
10 mM good buffer MES containing 50 mM NaCl
(PH 6.5) The sample was diluted 10-fold with a buffer to obtain a measurement sample.

FIG. 8 shows the measurement results of the various samples. As is clear from FIG. 8, it can be seen that the detection method according to the present invention makes it possible to detect mepronil from foods and agricultural products. In addition, about wheat, barley, and corn, it was measurable by centrifuging an extract and removing cloudiness. Grape juice (Concord) pH 7 could not be measured. The operations (5) to (7) and the determination in the present method were performed for 10 samples 10
Within minutes.

(Experiment 3) Next, iprodione was measured. Iprodione is a compound having a structure represented by the following formula (Formula 3).

[0057]

Embedded image

The iprodione hapten having the structure shown in the following formula (Formula 4) was used.

[0059]

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As an iprodione antibody, a mouse was immunized with an iprodione hapten, and the obtained monoclonal antibody was used. Using the above-mentioned IP plate as a membrane plate, measurement was performed in the following procedure.

(1) Coating: Add 10 μg / ml iprodione antibody solution to 50 μl per well and filter. (2) Blocking: 1% bovine serum albumin (BSA)
Is added to a volume of 50 μl per well, followed by filtration.

(3) Drying: The plate obtained as above is vacuum dried, sealed and stored at 4 ° C. (4) Addition of sample: When necessary, the plate is taken out, an extract sample containing 10% methanol is added to each well at 50 μl per well, and filtered.

(5) Addition of colloidal gold-labeled substance: A colloidal gold-labeled substance prepared in the same manner as in Experiment 2 using gold colloid having a particle diameter of 40 nm was adjusted to a concentration at which the absorbance at 530 nm was 2 OD, and this was used. 20 μl per well
And filtered.

(6) Washing: 50 μl of PBS containing 0.05% Tween 20 is added per well, followed by filtration.
This washing operation is repeated three times. As the extract sample, a sample prepared according to the extraction method of Experiment 2 was used.

FIG. 9 is a diagram showing the measurement results. In FIG. 9, the rightmost membrane in each sample shows the measured concentration when iprodione having the regulated value concentration of the target crop was added. 2 from right
The second position from the left indicates the amount of 1/5 of the regulation value, and the second position from the left indicates the amount of 1/10 of the regulation value. On the far left is a sample without iprodione.

As shown in FIG. 9, for a crop having a regulation value of 5 ppm, this regulation value can be detected. Also, 1
For crops in which 0 ppm is the regulation value, the regulation value and 1/5 of the regulation value can be detected. For crops having a regulation value of 20 ppm, it is possible to detect the regulation value and the concentration of 1/10 of the regulation value.

[0067]

By using the detection method of the present invention,
In about 10 minutes, low molecular compounds such as mepronil and iprodione can be detected at 1/5 to 1/10 level of the regulated value.

Therefore, according to the present invention, a low-molecular compound can be detected more easily and quickly than the conventional ELISA method.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between mepronil concentration and absorbance in a measurement method according to the present invention.

FIG. 2 is a graph showing the relationship between the mepronil concentration and the inhibition rate in the measurement method according to the present invention.

FIG. 3 is a diagram comparing an IP plate and an HA plate in terms of absorbance.

FIG. 4 is a diagram comparing an IP plate and an HA plate in terms of the inhibition rate.

FIG. 5 is a graph showing the relationship between the amount of sample to be filtered and IC50 (50% inhibitory concentration).

FIG. 6 is a graph comparing OPD and TMPE as color formers in terms of inhibition rate.

FIG. 7 is a diagram showing detection sensitivity when an IP plate is freeze-dried.

FIG. 8 is a graph showing the results of detecting mepronil concentration in various crops and foods.

FIG. 9 is a view showing the results of detecting the concentration of iprodione in various crops and foods.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yasuhiro Kagawa 868-98, Yamazaki, Kamakura City, Kanagawa Prefecture (72) Inventor Shozo Kanai 4-12-13, Imaizumidai, Kamakura City, Kanagawa Prefecture (72) Inventor Shigehisa Ito Shizuoka Prefecture 685-3 Nakajima, Fukuda-cho, Iwata-gun

Claims (7)

[Claims] 1. A method for detecting a low-molecular-weight compound as an analyte using an antigen-antibody reaction, comprising immobilizing a substance that specifically reacts with the analyte on a porous material, By filtering the specimen containing the, through the porous material, the analyte is reacted with the solid-phased substance on the porous material, and then competes with the analyte with the solid-phased substance. The labeled substance which reacts is filtered through the porous material, whereby the labeled substance is reacted with the solid-phased substance, and the amount of the labeled substance reacted with the solid-phased substance. A method for detecting a low molecular weight compound, wherein the amount of a substance to be analyzed is detected by measuring the amount of a low molecular weight compound. 2. The method for detecting a low molecular compound according to claim 1, wherein the substance to be analyzed is a low molecular compound that cannot be a polyvalent antigen. 3. The method according to claim 1, wherein the filtration is performed using a plate having a plurality of wells each having a wall and a bottom, and the porous material is disposed at the bottom of the well. 3. The method for detecting a low-molecular compound according to item 2. 4. The detection of a low molecular compound according to claim 1, wherein the porous material is formed of hydrophobic PVDF, mixed cellulose ester, or nitrocellulose. Method. 5. The labeling substance according to claim 1, wherein the labeling substance is a substance obtained by binding a denaturing substance of a substance to be analyzed and a labeling agent directly or via another compound.
5. The method for detecting a low-molecular compound according to any one of 4. 6. The method for detecting a low molecular compound according to claim 5, wherein the labeling agent is a colored marker. 7. The method for detecting a low-molecular compound according to claim 6, wherein the colored marker is colloidal gold, latex beads, or selenium.