JP2003098173A - Method for detecting or determining dioxins - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring dioxins at low costs by a simple and easy method. SOLUTION: In the method for detecting or determining dioxins, immunoassay using an anti-8 chlorinated dioxin antibody is performed on a sample, and the presence of dioxins in the sample is detected, or the amount of the dioxins is computed on the basis of the results of the immunoassay. An organic solvent is added to a soil or a sediment sample, and the dioxins are extracted by applying ultrasonic waves for 20 minutes or more. The extracted dioxins are analyzed by the immunoassay using the antibody to dioxins in the method for detecting or determining the dioxins in the soil or sediment sample. A kit or a detecting apparatus for detecting or determining the dioxins in the soil or sediment sample for implementing the method is provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting or quantifying dioxins and an antibody used therefor.

[0002]

2. Description of the Related Art G is a method for measuring dioxins.
Methods such as C-MS and simple ELISA are known.

[0003]

In the GC-MS method, as a pretreatment, it is necessary to manually concentrate and purify a sample before measurement.
Alternatively, it is necessary to perform solvent extraction under high temperature and high pressure conditions, which is this simple method. In addition, expensive equipment is required for measurement after pretreatment. Although the ELISA method is simpler than this, only an antibody against tetrachloride dioxine has been obtained so far, and with this antibody, dioxins having eight chlorine substitutions cannot be measured. Moreover, a method for measuring dioxin in soil and sediment samples by immunoassay has not been established. Dioxins are a general term for isomers of polychlorinated dibenzoparadioxins and polychlorinated dibenzofurans, and these include 75 types and 135 types of isomers, respectively. It may also include those having a coplanar structure. Of these, the compounds currently shown as toxic dioxins are the compounds shown in the table below.

[0004]

[Table 1]

In the table, TEF is a toxicity equivalent coefficient proposed by WHO in 1997, and is the most toxic.
It represents the relative strength of toxicity when the toxicity of 3,7,8-tetrachlorodibenzoparadioxin is 1. The value obtained by multiplying each of the dioxins by this coefficient and adding them together is called the toxic equivalent (TEQ). Therefore, when investigating the degree of environmental pollution due to dioxin, the TEQ value is obtained. Therefore, in order to obtain an accurate TEQ value, it is necessary to quantify each compound of dioxins and multiply this by the toxicity equivalent coefficient. Currently, the obtained dioxin antibody is dioxin octachloride (dibenzotrichloride). An accurate value could not be obtained because it has no cross-reactivity to paradioxin (hereinafter sometimes referred to as OCDD). Dioxin 7 chloride (dibenzoparadioxin 7 chloride, hereinafter sometimes referred to as HpCDD) and dioxin octachloride are contained in a large amount in soil and sediment, so it is important to measure these amounts in soil and sediment. It is especially important when knowing the toxicity of dioxin in the dioxin. In the present invention, production of an antibody against dioxin octachloride was successful. Further, the present inventors have found that this dioxin has cross-reactivity with dioxins having 7 or less chlorine substitutions and dibenzofuran having 8 or less chlorine substitutions, and completed the present invention. Further, it was found that dioxin in soil / sediment samples can be detected or quantified by performing a simple pretreatment with an organic solvent in the present invention.

[0006]

The present invention relates to the following method for detecting or quantifying dioxins. (1) Detection of dioxins by performing an immunoassay using an anti-8-dichlorodioxin antibody on the sample and detecting the presence of dioxins in the sample based on the result or calculating the amount of dioxins Or a quantification method. (2) After the sample is treated with an organic solvent, an immunoassay using an anti-8-chlorinated dioxin antibody is carried out, and based on the result, the presence of dioxins in the sample is detected or the amount of dioxins is calculated. A method for detecting or quantifying dioxins. (3) Divide the sample into a plurality of samples, treat each with a different organic solvent, and then perform an immunoassay using an anti-8-chlorinated dioxin antibody. From the results for each sample treated with each organic solvent, A method for detecting or quantifying dioxins, which comprises calculating the total amount or the amount of each dioxin. (4) The dioxins to be detected or quantified are one or more dioxins selected from the group consisting of dioxins 6 chloride, dioxins 7 chloride, dioxins 8 chloride, dibenzofuran 6 chloride, dibenzofuran 7 chloride and dibenzofuran 8 chloride. The method for detecting or quantifying dioxins according to any one of (1) to (3). (5) The method for detecting or quantifying dioxins according to any one of claims 1 to 4, wherein the anti-8-chloride dioxin antibody is a polyclonal antibody. (6) The method for detecting or quantifying dioxins according to any of (1) to (5), wherein the immunoassay is performed by a competitive method. (7) The method for detecting or quantifying dioxins according to any of (1) to (5), wherein the immunoassay is performed by a sandwich method.

The present invention also relates to the following antibodies. (8) A labeled or unlabeled anti-8-chlorinated dioxin antibody used in the method for detecting or quantifying dioxins according to any one of (1) to (7). (9) Reacts with dioxin octachloride and has at least 6
One or more dioxins selected from the group consisting of dioxins chloride, dioxins chloride, dibenzofuran hexachloride, dibenzofurans chloride 7 and dibenzofurans chloride and 0.01% or more, preferably 1% or more;
In particular, the antibody (8) having a cross-reactivity of 10% or more. The present invention also relates to the following kit or detection instrument.
A dioxin comprising a labeling agent and a means for binding the dioxin in the sample to the labeling agent by utilizing an immunospecific reaction with an anti-octachlorodioxin antibody and a means for measuring the activity of the labeling agent. A kit or detection instrument for detection or quantification. Furthermore, the present invention provides 199 from the values obtained by the above-mentioned method for detecting or quantifying dioxins.
Provide a method to obtain the toxicity equivalent amount specified by WHO in 7 years. Furthermore, the present invention divides the sample into a plurality of samples, treats each with a different organic solvent, and then performs an immunoassay using an antibody against dioxins, and from the results for each sample treated with each organic solvent, the dioxin in the sample is determined. The present invention also relates to a method for detecting or quantifying dioxins, which comprises calculating the total amount of dioxins or the amount of each dioxin. In the method, immunoassays using antibodies against different dioxins may be combined.

The present invention further relates to the following method for detecting or quantifying dioxins. (11) An organic solvent is added to a soil or sediment sample, dioxin is extracted by applying ultrasonic waves for 20 minutes or more, and the extracted dioxin is analyzed by an immunoassay using an antibody against dioxin. A method for detecting or quantifying dioxins in soil or sediment samples. (12) The method for detecting or quantifying dioxins according to (11), wherein the organic solvent is selected from the group consisting of alcohols having 1 to 5 carbon atoms, ketones having 3 to 8 carbon atoms, dimethyl sulfoxide, and dimethylformamide. (13) The dioxin according to (11) or (12), wherein the dioxins are extracted by applying ultrasonic waves in the organic solvent under normal pressure at a temperature of 10 to 80 ° C. for 20 to 180 minutes. Method for the detection or quantification of species. (14) Dioxins extracted with the organic solvent are
The method for detecting or quantifying dioxins according to (11) to (13), which comprises diluting with a buffer solution before performing the immunoassay. (15) Diluting with the buffer solution is performed so that the concentration of the organic solvent is less than 1.0% by volume, preferably less than 0.5% by volume, and the dioxin concentration is 0.7 ng / ml or more. (14) The method for detecting or quantifying dioxins according to (14). (16) The sample is 1 to 100 μm per 1 mg of the sample.
The method for detecting or quantifying dioxins according to (11) to (15), characterized in that the organic solvent is added in an amount of 1, preferably 5 to 25 μl. (17) The method for detecting or quantifying dioxins according to (11) to (16), wherein the antibody against dioxins is an anti-tetrachlorodioxin antibody and / or an anti-octachlorodioxin antibody. (18) The method for detecting or quantifying dioxins according to (11) to (16), wherein the antibody against dioxins is an anti-8-chloride dioxin antibody. (19) The method for detecting or quantifying dioxins according to (18), for detecting or quantifying dioxine octachloride or dibenzofuran octachloride. Further, the present invention comprises at least an anti-dioxin antibody, for example, an anti-tetrachloride dioxin antibody and / or an anti-8-chloride dioxin antibody, for carrying out the method according to any one of (11) to (19) above, The present invention relates to a kit or detection instrument for detecting or quantifying dioxins in soil or sediment samples. In the present specification, “dioxins” means polychlorinated dibenzoparadioxins (the following formulas)
I), polychlorinated dibenzofurans (formula II) and coplana PCB (formula III), which are known to be toxic. More specifically, it means the substances shown in Table 1 above.

[0009]

[Chemical 1]

(In the formula, each X independently represents a hydrogen atom or a chlorine atom.)

[0011]

[Chemical 2]

(In the formula, each X independently represents a hydrogen atom or a chlorine atom.)

[0013]

[Chemical 3]

(In the formula, each X independently represents a hydrogen atom or a chlorine atom.) In the present specification, "detection" means detecting dioxin as a whole and detecting each dioxin individually. That means both. In this specification,
"Quantitative" means not only determining the exact amount of each component, but also examining the approximate amount of each component.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a polyclonal antibody against dioxin octachloride is obtained, which is 6
Cross-reactivity with dioxins chloride, dibenzofuran hexachloride (hereinafter sometimes referred to as HeCDF), dioxines 7 chloride, dibenzofuran 7 chloride (hereinafter sometimes referred to as HpCDF), and dibenzofuran octachloride (hereinafter sometimes referred to as OCDF) It has been revealed that it is possible to measure these dioxins by an immunoassay using them. Furthermore, it was found that when the sample was previously treated with a specific organic solvent, the reactivity of each antigen with the antibody was different depending on the solvent used. This allows the sample to be divided into multiple parts, treated with different solvents, and then subjected to an immunoassay to identify the dioxins contained in the sample in a large amount, calculate the amount of each dioxin, and determine the total amount of dioxins. It has become possible to calculate. Such detection or quantification is based on the fact that the extraction rate in the immunoassay for dioxins, which was first found in the present invention, depends on the solvent used for the pretreatment. The extraction rate of dioxin tetrachloride by GC / MS is almost in agreement with about 70% for methanol and dimethyl sulfoxide (see Table 3). After considering the extraction rate (methanol, dimethylsulfoxide) when using the immunoassay kit, the measured value of the sample extracted with dimethylsulfoxide was extracted with methanol,
When it is higher than the measured value, dioxin hexachloride is often present. In an immunoassay using an octachlorodioxin antibody as well, since hexachlorodioxin responds, 6 when the value measured by extraction with dimethyl sulfoxide is higher than the result extracted by methanol.
Dioxin chloride is likely to be present. However, in this case, it is necessary to consider the extraction rate of GC / MS shown in Table 3.

The method of using the anti-octachlorodioxin antibody of the present invention can also be carried out in combination with an immunoassay in which the anti-tetrachlorodioxin antibody is used and, if desired, pretreatment with a different solvent is carried out. Almost no octachlorodioxin can be detected by the conventional tetrachlorodioxin antibodies, and thus it is highly possible that this method can separately detect tetrachlorodioxin and octachlorodioxin. In addition, dioxine octachloride and dibenzofuran octachloride can be distinguished by their solubility in dimethyl sulfoxide (hereinafter sometimes referred to as DMF).

Furthermore, the present invention also relates to a method for identifying the dioxins contained in a large amount in a sample and predicting the origin of the dioxins by the above method. For example, when a large amount of dioxin octachloride is contained, it is highly likely that the dioxins are derived from pesticides in the soil. When a large amount of hexachlorodioxin is contained, it is highly possible that the dioxin comes from the treated wastewater of a pulp mill or the like.

In the present specification, the terms "extraction rate in immunoassay" and "extraction rate in GC / MS" refer to the ratio of the amount quantified by the immunoassay and GC / MS to the amount actually contained in the sample. Is expressed as a percentage. This value is not necessarily related to the solubility of each compound in each solvent. The organic solvent is
It is an organic solvent that has high solubility of one or more kinds of dioxins and hardly affects the antigen-antibody reaction.
Preferably, solvents having different extraction rates by immunoassay depending on dioxins are used in combination. For example, dimethylformamide (DMF), dimethylsulfoxide (DMSO), methanol and the like can be used in combination.

[Sample] The sample to be the subject of the measuring method of the present invention may be soil, sediment, ash, agricultural products, food, drainage, blood, air, environmental water and the like. The measurement depends on the type and condition of the sample, but particularly in the case of soil, sediment and agricultural products, it is preferably carried out after treatments such as crushing, sieving and drying. Drying is performed up to a moisture content of 40% or less, for example. The treatment with an organic solvent is performed, for example, by adding a solvent to the sample and extracting while applying ultrasonic waves if desired. Preferably, 5 to 100 μl of a solvent is added per 1 mg of a sample, and the mixture is extracted with ultrasonic waves of 10 to 100 khz for 20 to 180 minutes without allowing the solvent to decrease by evaporation so as to affect the measurement. It is done by During extraction, if desired, 5 every 1 to 10 minutes
Shake for ~ 20 seconds.

[Anti-octaoctyl chloride dioxin antibody] The anti-octaoctyl chloride dioxin antibody used in the present invention is a monoclonal antibody or a polyclonal antibody, preferably a polyclonal antibody. For example, it may be a polyclonal antibody collected from an animal immunized with dioxine octachloride. More specifically, it reacts with octachlorodioxin,
And dibenzoparadioxin substituted with not more than 7 chlorines, particularly hexachlorodioxin and hexachlorodioxin, and dibenzofuran substituted with not more than 8 chlorines, especially one or more dioxins of 6-8 dibenzofuran. It is a cross-reactive antibody.
As an example, assuming that the reactivity with dioxine octachloride is 100%, the cross-reactivity with dibenzofuran chloride is 30 to 1
30%, the cross-reactivity with 1,2,3,4,6,7,8-heptachlorodibenzofuran is 0.01-70%, more specifically 1-50%, 1,2,3,7 Polyclonal antibodies having a cross-reactivity of 0.01 to 90%, more specifically 10 to 70% with 8,8,9-hexachlorodibenzoparadioxin. The polyclonal antibody against dioxin octachloride is, for example, produced by the following method.

[Preparation of Polyclonal Antibody] 8 Dioxin chloride bound to a protein or polypeptide was administered to a warm-blooded animal as an immunogen for a certain period of time, and ascites, blood, etc. were purified from the collected sample to obtain 8 Obtain a polyclonal antibody against dioxin chloride. The substance which binds to dioxine octachloride is preferably polylysine. The warm-blooded animals are, for example, mammals such as rabbits, goats, cows, sheep, rats, mice, guinea pigs, horses and pigs, and birds such as chickens, pigeons, ducks, geese and quails. The administration interval is, for example, 5 to 30 days.
The dose may be an amount effective for antibody production. For example, in the case of rabbit, about 0.01 to 1.0 mg at a time is emulsified with about 1 to 5 ml of physiological saline and Freund's complete adjuvant, and then subcutaneously administered. Administer. The administration frequency is, for example, 5 to 30 times. Antibodies formed in warm-blooded animals, eg, in rabbits, are usually harvested from the ear vein between 7 and 12 days after the final inoculation.

Purification of the octachlorodioxin antibody from serum can be carried out by a method for separating and purifying immunoglobulins, for example, salt folding method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, adsorption / desorption method using ion exchanger, and ultrafiltration method. It is carried out by a centrifugation method, a gel filtration method, a specific purification method in which only the antibody is collected by an antigen-antibody conjugate or an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody. More specifically, it can be separated by chromatography using recombinant Protein G lacking the albumin binding region as a ligand. In this case, the base carrier is preferably highly cross-linked spherical sepharose and the purification time is, for example, 5 to 2
The maximum flow rate for 0 minutes is, for example, 1 to 10 ml / minute.
The molecular weight of the anti-octachlorodioxin polyclonal antibody of the present invention is, for example, about 100 to 200 kilodaltons,
For example, an IgG antibody.

[Monoclonal Antibody] The antibody used in the measuring method of the present invention may be a monoclonal antibody. The monoclonal antibody is produced, for example, by the following method. The monoclonal antibody can be obtained, for example, by a method similar to the method described by Koehler and Milstein [Nature, 256 (1975), p. 495]. That is, a spleen or lymph node of an immunized warm-blooded animal is collected in the same manner as in the method for preparing the polyclonal antibody described above, and the antibody-producing cells contained therein are fused with myeloma cells to obtain a monoclonal antibody-producing hybridoma. be able to. Monoclonal antibody
It is preferably obtained using a rat or mouse. For example, when immunizing a mouse, it is preferable to inject subcutaneously, intraperitoneally, or intravenously. For example, a method is generally used in which immunization is carried out about 2 to 6 times at immunization intervals of 2 weeks, and spleen cells extracted about 1 to 5 days after the final immunization, and preferably about 2 to 4 days after, are used. The dose is, for example, about 0.1 μg or more per mouse, preferably about 10
It is μg to 300 μg. In addition, before extracting the spleen, partial blood sampling is performed to confirm an increase in the antibody titer in the blood, and the spleen of the individual having the increased antibody titer is excised.

The fusion operation can be carried out by a known method, for example, the above-mentioned Koehler and Milstein method or a method analogous thereto. Examples of myeloma cells include NS-
1, P3U1, SP2 / 0, etc. The preferable ratio of the number of antibody-producing cells (spleen cells) to the number of bone marrow cells is about 1: 1 to about 2
It is 0: 1. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. The degree of polymerization of PEG is usually about 1,
It is used at a concentration of 000 to 6,000 and a concentration of about 10% to 80%. The fusion time is about 0.5 to 30 minutes, the fusion temperature is 20 to 40 ° C.,
It is preferably 30 to 70 ° C. As an example of preferable conditions, PEG 6,000 can be efficiently fused by treating it with about 35 to 55% for about 4 to 10 minutes. The fused cells are hypoxanthine-aminopterin-thymidine medium [HAT medium; Nature, 256, 495 (1975)].
Etc. can be used to selectively proliferate.

The culture supernatant of the expanded cells can be screened for the production of the desired antibody, and the antibody titer can be screened as follows. That is, in this case, as a first step, the presence or absence of antibody production against the immunogen can be examined by a method such as a radioimmunoassay (RIA) method or an enzyme immunoassay (EIA) method, but various methods are also available. Can be modified. As one example of a preferable measurement method, one method using EIA will be described. For example, a rabbit anti-mouse immunoglobulin antibody is coupled to a carrier such as cellulose beads according to a conventional method, and a culture supernatant to be measured and mouse serum are added to the carrier, and the temperature is maintained at a constant temperature (about 4 to 40 ° C) for a certain period of time. React with. After that, the reaction product is thoroughly washed, an enzyme-labeled immunogen is added, and the reaction is allowed to proceed for a certain period of time at a constant temperature (about 4 to 40 ° C.). After thoroughly washing the reaction product, an enzyme substrate is added, and the reaction is carried out at a constant temperature (about 4 to 40 ° C.) for a predetermined time, and then the produced color product can be measured by absorbance or fluorescence. It is desirable that cells in the wells that show growth in the selective medium and that have antibody activity against the immunogen are cloned by the limiting dilution method or the like. A monoclonal antibody-producing hybridoma clone is obtained by similarly screening the supernatant of the cloned cells and increasing the number of cells in wells having high antibody titers.

The hybridoma thus cloned is grown in a liquid medium. Specifically, for example, about 0.1 to 4 in a liquid medium such as RPMI-1640 [Moore, GE et al., Journal of American Medical Association (J.Am.Med.Assoc.) 199,549 (1967)].
The monoclonal antibody can be obtained from the culture medium by culturing in a medium containing 0% bovine serum for about 2 to 10 days, preferably about 3 to 5 days. The antibody can be obtained by inoculating the mammal intraperitoneally, allowing the cells to grow, and collecting ascites. To do this, for example, in the case of mice, BA pre-inoculated with mineral oil etc.
LB / c mice are intraperitoneally inoculated with about 1 × 10 ⁴ to 1 × 10 ⁷ hybridomas, preferably about 5 × 10 ⁵ to 2 × 10 ⁶ hybridomas, about 7 to 20 days later, preferably about Ascites fluid is collected 10 to 14 days later. The antibody produced and accumulated in ascites can be easily isolated as a pure immunoglobulin from the monoclonal antibody by, for example, ammonium sulfate fractionation, DEAE-cellulose column chromatography and the like. [Immobilization of antibody] The antibody is immobilized on a carrier such as a 96-well plastic plate (for example, Nunc Co., Denmark immunoplate), glass beads, plastic beads, or the like. For plastics, 10 ~ 10 at about 4 ℃ for plastic
It is carried out by reacting for 20 hours or at room temperature to 40 ° C. for about 0.5 to 4 hours. In the case of glass, for example, Proceedings National of the Academy of Science (Proc.Natl.Acad.Sci.USA), Volume 80, 3513-35
Fix as described on page 16 (1983). In addition, various commercially available plates for immobilizing antibodies (described above, Nunc, etc.) can also be used.

[Immunoassay] The immunoassay is, for example, a radioimmunoassay, an enzyme immunoassay, or a fluorescent immunoassay. As a label for the immunoassay, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a gold colloid or the like is used, but it is preferable to use a so-called direct label such as an enzyme or a gold colloid. The antigen-antibody reaction can also be detected quantitatively or qualitatively by a surface plasmon resonance method (hereinafter referred to as SPR method) or a method using a crystal oscillator. The SPR method is a method in which an antibody-immobilized surface and an antibody-immobilized surface are formed on the same metal film, and the antigen-antibody reaction is measured by comparing the resonance wavelengths obtained on both surfaces. is there. The crystal oscillator method is a method of immobilizing an antigen or antibody on the crystal oscillator and detecting an antigen-antibody reaction by reducing vibration when the antibody or the antigen binds to the crystal oscillator. The ELISA method is particularly preferable as the enzyme immunoassay. As the enzyme, those which are stable and have a large specific activity are preferable, and peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase and the like can be used, and peroxidase is preferable. As the peroxidase, those of various origins can be used, and examples thereof include peroxidase obtained from horseradish, pineapple, fig, sweet potato, broad bean, corn, etc., and particularly extracted from horseradish. Horseradish peroxidase (HRP) is preferred. More specifically, a sample containing an unknown amount of dioxins is treated with a solvent if necessary, and the extract or untreated sample is mixed with a fixed amount of labeled octachlorodioxin. The mixture is contacted with a support on which an antibody of dioctyl octachloride is immobilized and washed. After that, the reaction is performed by contacting with a color-developing substrate such as TMB, the reaction is stopped, and the absorbance is measured to measure the amount of dioxins substituted with 8 or less chlorine. This reaction may be a fluorescent reaction. From the value obtained here, the total amount of dioxins substituted with 8 or less chlorines or the amount of individual dioxins can be calculated.

[Kit or Inspection Instrument] The present invention provides a means for binding a labeling agent and dioxins in a sample to the labeling agent by utilizing an immunospecific reaction with an anti-octachlorodioxin antibody, and the labeling agent. The present invention also relates to a kit or a detection instrument for detecting or quantifying dioxins, which comprises a means for measuring the activity of Dioxins. The labeling agent may be the one exemplified in the section "immunoassay". The means for binding with the labeling agent is appropriately selected from labeled or unlabeled anti-octaoctyl dioxin antibody and labeled or unlabeled octanoic dioxin depending on the type of immunoassay. The anti-octachlorodioxin antibody and the octaoctadiodioxin may be immobilized on a solid phase or unimmobilized depending on the type of immunoassay. That is, the means for binding to the labeling agent may include a sample and optionally a solid phase on which either octachlorodioxin or anti-octachlorodioxin antibody is mobile and which is immobilized. The means for measuring the activity of the labeling agent is, for example, the substrate and the reaction terminator when the labeling agent is an enzyme. The solid phase can be, for example, polystyrene beads, plates, tubes, etc., as well as nylon, nitrocellulose, cellulose acetate, glass fibers, or other porous polymers. The solid phase may be in the form of a dipstick. In the case of the kit, in addition to the above-mentioned means, for example, anti-8-dioxin, 8-octyldioxin, substrate, etc., a solvent, a detergent, a standard substance, etc. may be contained. The test device includes, for example, a solid phase composed of a porous material on which an anti-octachlorodioxin antibody is immobilized, and a labeled anti-octachlorodioxin antibody is movably carried when wet, and the solid phase
The detection instrument may be a casing including a casing in which a part of the solid phase can be brought into contact with a sample.

[Detection or Quantification of Dioxins in Soil Sample] The present invention is particularly directed to the method for detecting or quantifying dioxins in a soil sample described in (11) to (19) above, and to carry out the method. Of kits or detection instruments. In this case, the extraction of dioxins is 10
It is preferably carried out at 80 ° C, particularly 10 to 40 ° C. For example, it can be performed at room temperature. The organic solvent is a soil sample 1
1-100 μl, preferably 5-25 μl for mg
Is the amount of. Extraction time is 20 minutes or more, preferably 2
It is from 0 to 180 minutes, especially from 60 to 120 minutes. The dilution with the buffer solution is preferably performed so that the concentration of the organic solvent is less than 1.0% by volume, preferably less than 0.5% by volume, and the dioxin concentration is 0.7 ng / ml or more. In the measurement, the dioxin concentration is unknown, but it is preferable to adjust the amount of the buffer solution to be added so that the dioxin concentration falls within the above range based on the expected dioxin concentration. In addition, if the dioxin concentration is a normal environmental level, the extract extracted with the solvent can be directly subjected to the immunoassay without the need for operations such as dilution and concentration. The anti-dioxin antibody is a monoclonal antibody or a polyclonal antibody. The immunoassay method and the kit or detection instrument may be those described in the above items, but as the antibody, in addition to anti-octachlorodioxin antibody, anti-tetrachlorodioxin antibody and other anti-dioxin-type antibodies are used. can do. These antibodies may be monoclonal antibodies or polyclonal antibodies, and either of them may be commercially available or can be produced according to the method described above for the anti-8-dioxin dioxin antibody.

[0030]

EXAMPLES Example 1: Preparation of antibody 1-1 Reagent In this example, 1,2,3,4,6,7,8,9-octachlorobenzo-p-dioxin (OCDD) powder, HRP was used. (20,000 Unit
(For EIA) is manufactured by Wako Pure Chemical Industries, Peroxidase-conjugated Goat I
gG Fraction to Rabbit IgG (H + L) is ICN Pharmaceutical
s, Inc (CAPPEL), TMB is DAKO (substrate-chromoge
n, CA, USA), Block Ace used as a blocking agent was manufactured by Dainippon Pharmaceutical. All other reagents were special grade reagents manufactured by Wako Pure Chemical Industries.

1-2 Device Microplate reader manufactured by BIO-RAD (Model 550 (Japan Bio-Rad)) was used to measure at 450 nm. 96 holes
CORNING 9018 (Corning, NY, USA) was used as the EIA plate. As the antibody purification kit, MAbTrap ^™ G “kit (manufactured by Amersham Pharmacia Biotech Co., Ltd.) was used.

1-3 Preparation of 8-dioxin-labeled product OCDD was labeled with BSA or HRP by the following method. (1) Add HCl and NaNO ₂ to p-aminobenzoic acid to adjust pH to 6
The temperature was adjusted to -8 and the reaction was carried out at 0 ° C for 2 hours. (2) Powdery octachlorodioxin was solubilized with 60% ethanol, and an ammonium solution and sodium amide were added to the OCDD solution. As a result, it is considered that chlorine at the 1, 4, 6, and 9-positions is eliminated, and since it temporarily becomes benzene-like, an active group can be introduced and stabilized here. (3) The OCDD solution was concentrated under reduced pressure by an evaporator to a volume of about 1/10. The solution of (1) was added thereto and reacted for 2 hours. (4) A 10-fold amount of activated HRP / DMF solution or BSA / DMF solution was added to OCDD of (2), and the mixture was reacted for 2 hours.
At this time, the denaturation of the protein was prevented by using a DMF solution instead of ethanol. (5) Glycine was added to stop the reaction. 150 mM N
Dilute with 10 mM phosphate buffer pH 7.5 containing aCl, and add OCDD-HRP,
An OCDD-BSA concentration of 0.3 mg / mL was obtained. At this time, BSA, HR
The binding ratio of P to OCDD is about 1:10 in molar ratio (BSA or HRP: OCDD).

1-4 Preparation of 8-dioxin chloride antibody Polylysine was bound to OCDD and used as an antigen. This is referred to as OCDD-PLL (PLL: Poly-L-Lysine). OCDD-PLL
Is prepared by the same method as the preparation of the octachlorodioxin-labeled product shown in 1-3. The coupling ratio of PLL and OCDD is the molar ratio (P
It becomes about 1:20 as LL: OCDD). As the immunized animals, three healthy female rabbits (Japanese white breed) (weight 2.5 to 3.0 kg) (KAL-1, 2, 3) were used. Freund's complete adjuvant (FCA; Freund's complete adjuvant) at the time of the first immunization as an adjuvant (non-specific enhancer of the immune system), and the second and subsequent ones are Freund's incomplete adjuvant (FIA; incomple
te adjuvant) was used. In both cases, the immunization site is intradermal1
The dose of OCDD-PLL was 0.5 mg / bird and the emulsion concentration was 0.25 mg / mL. The administration frequency of OCDD-PLL was 15 times at 2-week intervals. Total dose concentration is 7.5
It was mg / feather. After the first administration, the second and third antigen administrations were performed at the second and fourth weeks. The first test blood sampling date is 3
One week after the first administration. Thereafter, the antigen was administered approximately every 2 weeks, and blood was collected 1 week after each administration. Whole blood collection was performed on the last blood collection day. Serum is stored for each individual,
NaN ₃ was added as a preservative so that the concentration was 0.05%.

1-5 Measurement of titer of antiserum against 8-dioxine chloride labeled product 50 mM carbonate buffer solution, pH 9.6 as coating buffer, 10 mM phosphate buffer solution (PBS), pH 7.2 as dilution buffer , 0.05% Tween 20-PBS pH 7.2 was used for washing. (1) 50 μg of 5 μg / ml OCDD-BSA on a 96-well plate
Add 1 / well, 37 ℃ for 2 hours or 4 ℃
The coating was performed by leaving it for 15 hours. After the completion of coating, OCDD-BSA was removed from each well, and the wells were washed 3 times. (2) Add 25% Block Ace in an amount of 100 µl / well, and add 3
After blocking by reacting at 7 ° C. for 2 hours or at 4 ° C. for 15 hours, the solution was removed and the plate was washed 3 times with a washing solution. (3) The antiserum was diluted 2n times with PBS (n = 0 to 10), and the antiserum diluted to each concentration was added in an amount of 50 μl / well. (4) After the reaction, remove the antiserum, wash three times with a washing solution,
Enzyme-labeled antibody Anti-HRP was added at 50 μl / well and incubated at 37 ° C. for 2 hours. (5) After the reaction, the enzyme-labeled antibody was removed, the plate was washed 3 times with a washing solution, and a coloring solution was added at 50 μl / well and reacted for 15 minutes. For the reaction, 100 μl / well of stop solution 2M H ₂ SO ₄ was used.
It was stopped by adding. After stopping, the absorbance was measured with a plate reader (450 nm).

1-6 Purification of anti-8-dichlorodioxin antibody Sepharose as a base carrier (highly cross-linked sphere as a matrix, average particle size 34 μm, pH stability in a short time 2
~ 9, long time 3 ~ 9) using HPMAbTrap G2 kit (Amersham Pharmacia Biotech Co., Ltd.),
Purified. This kit has the advantage that there are few reaction defects due to subclasses. In addition, recombinant Prptein G lacking the albumin binding region is used as a ligand, the amount of the ligand is 2 mg Protein G / ml carrier or less, and approximately 20 to 25 mg of IgG can be purified by one purification. . The amount of antiserum corresponds to approximately 1 ml, and the final amount of IgG recovered is 10 to 15 mg. Also, the coupling capacity is 25
mg human IgG / ml carrier. The purification time is 10 min and the maximum flow rate is 4 ml / min. Purification by such a kit enables high-purity IgG purification in one step without purification such as salting-out in IgG purification from antiserum.
In the case of a monoclonal antibody, it is necessary to consider denaturation of Fc and the like at the time of elution, but since this antibody is a polyclonal antibody, denaturation seems to be small. Specifically, it was purified by the following method. 3 ml of the binding buffer was sent by a syringe to equilibrate the column, and then the antiserum obtained in 1-4 was added. It was then washed with 5-10 ml binding buffer and eluted with 3-5 ml elution buffer. Further, 75 μl of the neutralization buffer solution per 1 ml was added to the elution fraction to stabilize it. The purified antibody was subjected to electrophoresis (SDS-PAGE). In other words, using 4-20% Gradient as the electrophoresis gel, the sample anti-
OCDD rb IgG purified product 2 μg / lane reduction treatment. The concentrations of KAL-1, 2 and 3 in the antiserum were 1.74 mg / ml and 1.36 m, respectively.
It was adjusted to g / ml and 3.14 mg / ml. Also, 0.15M Na
0.1M glycine buffer pH7.5 containing Cl and 0.1% NaN ₃
Was used as a buffer for electrophoresis.

Example 2: Confirmation of reaction of antibody with OCDD-HRP 2-1 Immobilization of anti-8-dioxin dioxin antibody or BSA The anti-OCDD antibody obtained in Example 1 was diluted with 0.05M carbonate buffer (p.
Dilute each concentration with H9.6) to 300 μl / well 9
Added to 6-well plate. BSA100 as blank
μg / ml was similarly added to the wells and incubated at 4 ° C. for 18 hours. After removing the liquid, washing with 0.01M PBS buffer (pH 7.2) containing 0.05% Tween 20 was performed twice for 3 minutes, and then only 0.01M PBS buffer was added,
Removed immediately. 2-2 Confirmation of reaction OCDD-HRP was adjusted to 0.29 with 0.01M PBS (pH 7.2).
Dilute to 4.69 (μg / ml) and add 3 at 50 μl / well
The reaction was carried out at 7 ° C for 4 hours. Remove the OCDD-HRP solution, wash, add TMB at 50 μl / well, and shield from light 3
The reaction was carried out at 7 ° C for 20 minutes. 1M H ₂ S as stop solution
50 μl / well of O ₄ was added and the absorbance was measured (450 nm) with a plate reader. The results are shown in the graph of FIG. From this, it was found that the antibody concentration of 2.72 μg / ml was optimal. In addition, OCDD-H at this antibody concentration
The graph of FIG. 2 shows the result of examining the optimum concentration of RP. From this, it can be seen that competition occurs when the OCDD-HRP concentration is 4.7 μg / ml or less.

Example 3 Measurement of OCDD by Competitive Method Instead of using OCDD-HRP diluted with 0.01M PBS (pH 7.2), it was diluted with PBS to a final concentration of 4.7 μg / ml. OCDD-HRP and final concentration 0-200ng / m
The OCDD was measured by substantially the same procedure as in Example 2 except that a mixed solution (volume ratio 1: 4) with OCDD diluted with PBS so as to be 1 was added at 50 μl / well. The results are shown in the graph of FIG.

Example 4 Measurement of HeCDD, HpCDD, OCDD and OCDF OCDD-HRP was diluted to 4.7 μg / ml with PBS and used in the same manner as above. Each dioxin is PBS (final concentration: 0.
Dilute to 25 ng / ml with 5% DMSO). 6
Dioxin chloride (HeCDD) and dibenzofuran chloride (H)
pCDF) was diluted to 25 ng / ml with a phosphate buffer containing 0.5% MeOH and 0.5% DMSO. Dioxin octachloride (OCDD) and dibenzofuran octachloride (OCDF) were diluted to 25 ng / ml with a phosphate buffer containing 0.5% DMF and 0.5% DMSO. Dioxins and OCDD
-HRP was mixed and reacted at a volume ratio of 1: 4. To eliminate the influence of the solvent, subtract the absorbance of the solvent from the absorbance,
OCDF when the reactivity of dioxin octachloride is 100,
The crossing rate of HeCDD and HpCDF was obtained. The results are shown in Table 2.

[0039]

[Table 2]

Example 5: Measurement of dioxin in a soil sample Soil was sampled, passed through a 2 mm sieve and dried at 70 ° C. for 24 hours (water content about 26%). 4 mg of obtained dry soil
In addition, 40 μl of 1 ppm dioxine octachloride (10 μg
/ G) was added, and the mixture was shaken thoroughly and then air-dried for 2 nights.
Using this as a sample, 100 μl of dimethyl sulfoxide was added as a solvent, and extraction was performed for 90 minutes while applying ultrasonic waves having a frequency of 43 kHz. In the meantime, it was shaken every 5 minutes for 10 seconds and diluted with PBS so that the concentration of dimethyl sulfoxide was 0.5 v / v% to prepare a sample solution. When dioxin was measured for this sample solution by the method of Example 4, a measured value corresponding to the actual weight was obtained.

Example 6 In order to examine the influence of the solvent used for pretreatment on the immunoassay,
Using the substances listed in Table 3, the extraction rate of tetrachloride dioxin was examined by an immunoassay using an anti-tetrachloride dioxin antibody. The results are shown in Table 3. For reference, the extraction rate by GC / MS is also shown. As shown in the table, the extraction rate is highest when dimethyl sulfoxide is used.

[0042]

[Table 3]

Example 7: For 3 to 6 dioxin chloride, the extraction ratio by the solvent used for the pretreatment was examined by GC / MS. The results are shown in Fig. 4. From the figure, the extraction rate when methanol was used was almost constant for 3 to 6 dioxin chloride, but the extraction rate when dimethyl sulfoxide was used was higher for dioxin hexachloride.

Example 8: In order to investigate the difference in extraction ratio between polychlorinated dibenzofuran and polychlorinated dioxin, a case where pretreatment was carried out with dimethylsulfoxide as a solvent and a case where pretreatment was carried out with methanol as a solvent The extraction rate was investigated. The results are shown in Tables 4 and 5.

[0045]

[Table 4]

[0046]

[Table 5]

Example 9: In order to examine the effect of the solvent on the anti-octachlorodioxin antibody, an immunoassay using the anti-octachlorodioxin antibody was carried out using methanol, acetone, dimethyl sulfoxide and dimethylformamide as the solvent. The results are shown in the graph of FIG.
As shown in the figure, when a solvent other than methanol was used, inhibition of the antibody was observed at 0.1% or more, and when methanol was used, inhibition of the antibody was observed at a concentration of 0.5% or more. .

Example 10: Soil was collected, passed through a 2 mm sieve and dried at 70 ° C. for 24 hours. To the obtained dry soil, octachlorodioxin was added so that the amount of octachlorodioxin was 10 μg per 1 g of dry soil, and the mixture was thoroughly shaken and air-dried for 2 nights. Using this as a sample, 100 μl of dimethylformamide was added as a solvent,
Extraction was performed while applying ultrasonic waves with a frequency of 43 kHz. FIG. 6 shows the relationship between the extraction time and the extraction rate.

Example 11: Soil was collected, passed through a 2 mm sieve and dried at 70 ° C. for 24 hours. To the obtained dry soil, 8 chloride dioxine, 8 chloride dibenzofuran, and a 1: 1 mixture of 8 chloride dioxine and 8 chloride dibenzofuran were added so that the amount per 1 g of dry soil was 10 μg, and the mixture was thoroughly shaken. Then, it was air dried for 2 nights. Using these as samples, dimethylformamide 1 as a solvent
00 μl was added, and the mixture was extracted for 90 minutes while applying ultrasonic waves having a frequency of 43 kHz. Add each extract to phosphate buffer (1
0 mM, pH 7.2), the dioxin content is 32.
Diluted to 5 ng / ml, 65 ng / ml, 130 ng / ml. Competitive immunoassays were carried out in the same manner as described in Example 3 by using the obtained diluted solution, with anti-octyl chloride dioxin antibody and HRP-labeled OCDD. The results are shown below. The absorbance shown below is a value obtained by subtracting the absorbance measured above from the absorbance measured in the same manner using a soil sample containing no dioxin.

[0050]

[Table 6]

From the above table, it is clear that the approximate amount of dioxane octachloride and dibenzofuran octachloride in a sample can be known by an immunoassay using an anti-octachlorodioxin antibody.

[0052]

[Effects of the Invention] Dioxins in a sample can be qualitatively or quantitatively analyzed by performing the method of the present invention alone or in combination with an ELISA method using an anti-tetrachlorodioxin antibody. This makes it possible to measure environmentally important dioxins with a simple method and at low cost. In particular, for dioxins in soil samples, the pretreatment does not require complicated treatment or expensive equipment required before,
It is possible to know the approximate amount of dioxins by performing an immunoassay after a simple pretreatment. In particular, dioxin octachloride is a dioxin contained in a large amount in soil and sediment, and by measuring this, it is possible to grasp the dioxin contamination status of the soil and sediment. Therefore, the method, kit, and detection instrument of the present invention are particularly suitable for the screening-like investigation of soil and sediment contamination status.

[Brief description of drawings]

FIG. 1 is a graph showing the reactivity of antioctachlorodioxin and octachlorodioxin of the present invention.

FIG. 2 is a graph showing the reactivity of antioctachlorodioxin and octachlorodioxin of the present invention.

FIG. 3 is a graph showing the relationship between the concentration of dioxine octachloride and the absorbance.

FIG. 4 is a graph showing the relationship between the solvent and the GC / MS extraction rate.

FIG. 5 is a graph showing the influence of a solvent on an antibody.

FIG. 6 is a graph showing the relationship between the extraction time of dioxins octachloride and the extraction rate.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/531 G01N 33/577 B // G01N 33/577 1/28 YZ F term (reference) 2G052 AA19 AB11 AB22 AD34 AD46 EA04 EB12 EB13 FB02 FB08 FB10 FC02 FC15 FD01 FD02 FD09 FD18 GA11 GA22 GA24 GA30 JA03 JA07 4H045 AA11 CA42 DA75 EA50 FA74 GA22

Claims (20)

[Claims] 1. A dioxin which comprises performing an immunoassay on a sample using an anti-8-chloride dioxin antibody and detecting the presence of dioxin in the sample based on the result or calculating the amount of dioxin. Method for detection or quantification of. 2. A sample is treated with an organic solvent, and then an immunoassay using an anti-8-dichlorodioxin antibody is carried out, and the presence of dioxins in the sample is detected or the amount of dioxins is calculated based on the result. A method for detecting or quantifying dioxins comprising. 3. A sample is divided into a plurality of samples, each of which is treated with a different organic solvent, and then an immunoassay using an anti-8-chlorinated dioxin antibody is carried out. From the results of each sample treated with each organic solvent, the dioxin in the sample is determined. A method for detecting or quantifying dioxins, which comprises calculating the total amount of dioxins or the amount of each dioxin. 4. Dioxins to be detected or quantified,
1. One or two or more dioxins selected from the group consisting of hexachlorodioxin, hexachlorodioxin, hexachlorodioxin, octachlorodioxin, hexachlorodibenzofuran, hexachlorodibenzofuran and octachlorodibenzofuran.
The method for detecting or quantifying dioxins according to any one of 3 above. 5. The method for detecting or quantifying dioxins according to any one of claims 1 to 4, wherein the anti-8-chloride dioxin antibody is a polyclonal antibody. 6. The method for detecting or quantifying dioxins according to any one of claims 1 to 5, wherein the immunoassay is performed by a competitive method. 7. The method for detecting or quantifying dioxins according to claim 1, wherein the immunoassay is performed by a sandwich method. 8. A labeled or unlabeled anti-octachlorodioxin antibody used in the method for detecting or quantifying dioxins according to any one of claims 1 to 7. 9. A dioxin that reacts with dioxine octachloride and is selected from at least one dioxin selected from the group consisting of dioxine hexachloride, dioxine hexachloride, dibenzofuran hexachloride, dibenzofuran chloride 7 and dibenzofuran octachloride and 0 or more. The antibody according to claim 8, which has a cross-reactivity of 0.01% or more. 10. A means for binding a labeling agent to a labeling agent by utilizing an immunospecific reaction of dioxins in a sample with an anti-octachlorodioxin antibody, and a means for measuring the activity of the labeling agent. A kit or detection instrument for the detection or quantification of dioxins, including. 11. An organic solvent is added to a soil or sediment sample,
A method for detecting or quantifying dioxins in soil or sediment samples, characterized by extracting dioxin by applying ultrasonic waves for 20 minutes or more and analyzing the extracted dioxin by an immunoassay using an antibody against dioxin. . 12. The detection of dioxins according to claim 11, wherein the organic solvent is selected from the group consisting of alcohol having 1 to 5 carbon atoms, ketone having 3 to 8 carbon atoms, dimethyl sulfoxide, and dimethylformamide. Or a quantification method. 13. The method according to claim 11, wherein the dioxins are extracted by applying ultrasonic waves in the organic solvent under atmospheric pressure at a temperature of 10 to 80 ° C. for 20 to 180 minutes. A method for detecting or quantifying dioxins. 14. The detection of dioxin according to any one of claims 11 to 13, wherein the dioxin extracted with the organic solvent is diluted with a buffer solution before performing the immunoassay. Quantitation method. 15. The method according to claim 14, wherein the buffer solution is diluted so that the concentration of the organic solvent is less than 1.0% by volume and the dioxin concentration is 0.7 ng / ml or more. A method for detecting or quantifying dioxins. 16. The sample is 1 to 1 per 1 mg of the sample.
The method for detecting or quantifying dioxins according to any one of claims 11 to 15, wherein the method is performed by adding the organic solvent in an amount of 00 µl. 17. The method for detecting or quantifying dioxins according to any one of claims 11 to 16, wherein the antibody against the dioxins is an anti-tetrachlorodioxin antibody and / or an anti-octachlorodioxin antibody. 18. The method for detecting or quantifying dioxins according to any one of claims 11 to 16, wherein the antibody against dioxins is an anti-octachlorodioxin antibody. 19. The method for detecting or quantifying dioxins according to claim 18, which is used for detecting or quantifying dioxine octachloride or dibenzofuran octachloride. 20. A kit or a kit for detecting or quantifying dioxins in a soil or sediment sample, which comprises at least an anti-dioxin antibody, for carrying out the method according to any one of claims 11 to 19. Equipment.