JP5199944B2 - Immunoassay for dioxins - Google Patents

Description

  The present invention relates to a method for measuring dioxin concentration (toxic equivalent amount) in a sample using an antibody that specifically reacts with dioxins. More specifically, the present invention relates to a method for calculating a toxic equivalent amount regardless of the isomer distribution of dioxins contained in a sample.

  Dioxins are a general term for polychlorodibenzo-para-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), and coplanar PCBs (Co-PCBs). Dioxins have many isomers and have different toxicities, and therefore, their emission regulations are controlled by the Toxicity Equivalency Quantity (TEQ). Toxicity equivalent is defined as 1 when the toxicity of 2,3,7,8-tetrachlorodibenzo-para-dioxin (2,3,7,8-TeCDD), which is the most toxic isomer of dioxins, is taken as 1. The sum of the product of the value of the toxicity of each isomer (toxic equivalent coefficient: TEF: Toxicity Equivalency Factor) and the concentration of each isomer.

  In the official method (High Resolution Gas Chromatography Mass Spectrometry (HRGC / HRMS) method), the isomer concentration of each of the 29 types of dioxins is measured, the TEF value is multiplied to each concentration, and the sum is calculated as the TEQ value. . Therefore, in order to calculate the TEQ value, it is necessary to quantify the isomers of individual dioxins, which requires not only a long time but also advanced separation / analysis techniques.

  Conventionally, when measuring the concentration of dioxins in a sample using an antibody, 2,3,7,8-TeCDD having the highest toxicity or 2,3,4,7,8-pentachlorodibenzofuran (2 , 3,4,7,8-PeCDF) and antibodies having high reactivity with pentachlorinated or hexachlorinated dibenzofurans have been used for the measurement.

  When an antibody having specific reactivity with 2,3,7,8-TeCDD, which is the most toxic isomer of dioxins, is used for measurement, the concentration of 2,3,7,8-TeCDD in the sample is determined. Although it is possible to know, the toxicity equivalent of the sample cannot be determined. This is because there is no significant correlation between 2,3,7,8-TeCDD concentration and toxicity equivalent in the same sample. In addition, environmental standards and emission standards for dioxins are regulated by the equivalent amount of toxicity for each scale of medium and discharge facility, so even if 2,3,7,8-TeCDD concentration is measured It is not possible to judge whether the sample exceeds the regulatory standard. On the other hand, from the analysis results of exhaust gas discharged from waste incineration facilities and dust by the HRGC / HRMS method, pentachlorinated and hexachlorinated dibenzofurans such as 2,3,4,7,8-PeCDF are toxic equivalent amounts. And these isomers are called TEQ index isomers (Non-patent Documents 1 and 2).

  In recent years, a method using an antibody exhibiting specific reactivity to 2,3,4,7,8-PeCDF using this relationship has been proposed (Patent Document 1). In this method, first, a sample (several tens of samples) with a known concentration is measured in advance, and a conversion coefficient is calculated from the obtained reaction amount and the toxic equivalent amount measured by the GC / MS method. Next, the sample having an unknown concentration is measured by an immunoassay, and the obtained measurement value (standard product equivalent concentration) is multiplied by a conversion factor to be converted into a toxic equivalent amount. If an antibody that reacts with an indicator isomer such as 2,3,4,7,8-PeCDF is used to measure exhaust gas and dust, the measurement results agree well with the toxicity equivalent measured by the GC / MS method. Can be obtained. For example, Non-Patent Documents 3 and 4 show that all types of hexachlorinated PCDFs and one type of hexachlorinated PCDD exhibit a cross-reactivity of about 35 to 46%, and also have a TEF value. An antibody having a wide range of cross-reactivity with dioxins having a reactivity of about 10% is also described in the dioxin isomers of the above, and as a result of measuring an environmental sample using the antibody, It was reported that there was a correlation.

  In addition, various anti-dioxin monoclonal antibodies have been developed for the purpose of measuring more types of dioxin isomers or for stability in the measurement system (Patent Documents 2 and 3). Furthermore, a low-toxic dioxin standard for quantification has also been developed (Patent Document 4).

  However, when the dioxin isomer distribution is almost constant regardless of the sampling location, such as exhaust gas and dust, good measurement results can be obtained, but sampling such as dioxin-contaminated soil and sediment is collected. There is a problem that when measuring a sample having a greatly different isomer distribution depending on the location, it is difficult to obtain a measurement result that is in good agreement with the GC / MS method. In particular, when a sample in which PCB accounts for 50% or more of the equivalent amount of toxicity, a measurement result significantly lower than that of the GC / MS method is calculated, and false negatives may be caused.

JP 2005-194238 A JP 2007-284392 A Japanese Patent No. 3969878 International Publication No. 2004/0209797 Pamphlet

Shibayama Moto et al., 11th Environmental Chemistry Symposium, 2002, pp. 136-137 Takahiko Matsueda et al., Proceedings of the 11th Environmental Chemistry Conference, 2002, pages 402-403 Hiroki Fujihira et al., Environmental Purification Technology, 2003, Vol. 2, pp. 63-66 Fujihira Hiroki et al., Resource Environment Countermeasures, 2003, 39, 62-68

  An object of the present invention is to provide a method for calculating a measurement result (toxic equivalent amount) consistent with the GC / MS method regardless of the contamination source.

The present invention provides an immunoassay method for toxic equivalents of dioxins, the method comprising:
Reacting a sample containing dioxins with an anti-dioxin antibody having affinity for 3,3 ′, 4,4 ′, 5,5′-hexachlorobiphenyl;
Detecting the amount of dioxins bound to the anti-dioxin antibodies or the amount of the anti-dioxin antibodies not bound to the dioxins; and calculating the toxicity equivalent in the sample from the detected amounts ;
Including
The anti-dioxin antibody is a monoclonal antibody.

  In one embodiment, the anti-dioxin antibody further has an affinity for 2,3,4,7,8-pentachlorodibenzofuran.

  In one embodiment, the detection step is performed based on a method selected from the group consisting of enzyme immunoassay, radioimmunoassay, and fluorescent immunoassay.

  According to the method of the present invention, it is possible to quickly calculate a measurement result consistent with TEQ (toxic equivalent amount) by the official method (GC / MS method) regardless of the pollution source. This method facilitates simple analysis and monitoring of dioxins in environmental samples.

It is a schematic diagram which shows the measurement principle and procedure of ELISA. It is a graph which shows the correlation of the ELISA measured value (antibody A) and GC / MS value in a soil sample. It is a graph which shows the correlation with the ELISA measured value (antibody A) and GC / MS value in a sediment sample. It is a graph which shows the correlation of the ELISA measured value (antibody B) and GC / MS value in a soil sample. It is a graph which shows the correlation of the ELISA measured value (antibody B) and GC / MS value in a sediment sample.

The immunoassay method for toxicity equivalents of dioxins of the present invention is:
Reacting a sample containing dioxins with an anti-dioxin antibody having affinity for 3,3 ′, 4,4 ′, 5,5′-hexachlorobiphenyl;
Detecting the amount of dioxins bound to the anti-dioxin antibodies or the amount of the anti-dioxin antibodies not bound to the dioxins; and calculating the toxicity equivalent in the sample from the detected amounts ;
including.

(sample)
The sample measured by the method of the present invention is not particularly limited. For example, it may be an environmental sample collected from the environment, a biological sample, a food, or a dioxin solution prepared for an experiment. The method of the present invention is particularly suitable when an environmental sample is used as a sample. Examples of environmental samples include air; exhaust gas from automobiles, equipment, factories, etc .; soil; water in rivers, lakes, harbors, etc .; combustion ash, fly ash, and the like. Examples of biological samples include breast milk, blood, urine and the like.

  The sample may be directly used for the immunoassay method of the present invention, or a fraction containing dioxins may be extracted from the sample by pretreatment (for example, cleanup). Preferably, pretreatment is performed according to the sample. For the extraction, a nonpolar organic solvent such as toluene, hexane, or dichloromethane is preferably used. The extracted fraction is preferably a water-miscible organic solvent such as dimethyl sulfoxide (DMSO), methanol or ethanol. Although the pre-processing method is illustrated below, the pre-processing method is not limited to these.

The pretreatment of the exhaust gas sample is performed according to the official method (JIS K0311). For example, first, an arbitrary amount (Nm ³ ) of gas is collected, a substance in the gas is extracted with a nonpolar organic solvent such as toluene, hexane, or dichloromethane, and quantified to 20 ml to obtain a crude extract. 10 ml of this crude extract is taken and treated with sulfuric acid until the sulfuric acid layer is no longer colored. This solution was transferred to hexane, and then cleaned up by passing 200 ml of hexane over a multilayer silica gel column laminated with 1 g of sodium sulfate, 1 g of 10% (w / v) silver nitrate silica gel, and 3 g of silica gel. The solution quantified in 1 ml DMSO is subjected to immunoassay.

  The pretreatment of soil and fly ash samples is as follows. First, an arbitrary amount (g) of soil or fly ash is collected, a substance in the sample is extracted with toluene, and quantified to 20 ml to obtain a crude extract. 1 ml of this crude extract is collected and cleaned up, and finally the solution quantified in 2 ml of DMSO is subjected to immunoassay.

(Anti-dioxin antibodies)
The anti-dioxin antibody used in the method of the present invention has an affinity for 3,3 ′, 4,4 ′, 5,5′-hexachlorobiphenyl (3,3 ′, 4,4 ′, 5,5′-HxCB) Have sex.

  The anti-dioxin antibody used in the method of the present invention is a monoclonal antibody. This is a fusion cell (hybridoma cell) of a spleen cell of a mammal (for example, a mouse) immunized with a conjugate in which a dioxin derivative is bound to a polymer compound as an antigen for immunization and a myeloma cell usually used for production of a monoclonal antibody. ) And purifying the monoclonal antibody produced by this hybridoma cell.

  In the present invention, a dioxin derivative used as an immunizing antigen can be prepared by a known method (Kun Chae et al., J. Agric. Food., 1977, 25, 1207-1209; Simona G. Merica Et al., Can. J. Chem., 1995, 73, 826-834). Dioxin derivatives are hapten compounds and are not themselves immunogenic, so they can be used as immunogens by forming a complex by binding an immunogenic polymer compound as a carrier compound. .

  Although the manufacturing method of the antigen for immunization of a dioxin derivative is not specifically limited, For example, it can manufacture with the following method. By refluxing 1,2-dichloro-4-fluoro-3,5-dinitrobenzene and 4,5-dichlorocatechol in acetone in the presence of potassium carbonate and crown ether for 6 hours, 1-nitro-2,3 , 7,8-tetrachlorodibenzo-p-dioxin. This compound is reduced in benzene with zinc powder and concentrated sulfuric acid to give 1-amino-2,3,7,8-tetrachlorodibenzodioxin. Next, acylation is carried out by reacting with N, N-dimethyl-4-aminopyridine (DMAP) as a base with ethyl glutaryl chloride under heating at 60 ° C. in anhydrous dimethylformamide (DMF), followed by further alkaline hydrolysis. As a result, a carboxylic-oxidized dioxin derivative is obtained. The resulting carboxylated dioxin derivative has the following formula:

(Wherein X represents chlorine or hydrogen, and n represents an integer of 1 to 10). Furthermore, this carboxylated dioxin derivative and N-hydroxysuccinimide are reacted in the presence of a coupling agent such as dicyclohexylcarbodiimide or water-soluble carbodiimide in an organic solvent to convert it to an imide ester. This imidoesterified dioxin derivative is used as an antigen for immunization. Moreover, you may refine | purify as needed by refinement | purification operations, such as silica gel column chromatography and recrystallization.

  The polymer compound used as a carrier compound for binding to the hapten compound preferably has a molecular weight of 10,000 or more. For example, proteins such as hemocyanin, ovalbumin, bovine serum albumin, rabbit serum albumin, polysaccharides such as agarose , Inactivated bacteria. Among these, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) are preferable in terms of operability and immunogenicity.

  The bond between the imide esterified hapten compound and the carrier compound is usually performed in a water-containing organic solvent. First, an imidoesterified dioxin derivative is dissolved in an organic solvent such as methanol or DMSO. Similarly, a carrier compound such as BSA or KLH is dissolved in a buffer solution made of a component not containing an amino group, such as a phosphate buffer solution. Next, the carrier solution dissolved in the buffer solution is gradually added to the imidoesterified dioxin derivative solution and reacted. The antigen for immunization thus obtained is purified by substituting with an appropriate buffer by gel filtration, dialysis or the like, if necessary.

  Using the immunizing antigen obtained as described above, a monoclonal antibody was prepared according to a known method, for example, the method of Kohler G. and Milstein C. (Nature, 1975, 256, pages 495-497). Can be produced. Although an example is shown below, it is not limited to this method.

  When immunizing a soluble substance as described above as an antigen for immunization, the antigen for immunization is usually administered to an animal in an emulsion state together with an adjuvant. Examples of adjuvants that can be used include Freund's adjuvant, alum adjuvant, and pertussis cells.

  As the animal to be immunized, BALB / c mice are usually used. Alternatively, F1 mice, hamsters, etc. may be used. The amount of antigen to immunize one mouse is usually about 5 to 20 μg / dose when the antigen for immunization is toxic. Specifically, for example, an immunizing antigen prepared with physiological saline to be 100 to 500 μg / ml and a complete Freund's adjuvant are mixed in equal amounts to prepare an emulsion, and 0.05 to 0.1 ml / Inject one by one. The administration of the emulsion is preferably intraperitoneal injection and subcutaneous injection. The antigen for immunization is usually administered several times every 1 to 2 weeks. The immunized mice are bled several days after each administration, and after confirming that the antibody titer has sufficiently increased, the same amount of antigen for immunization is injected intravenously or intraperitoneally, and the spleen 3 to 4 days later. And antibody-producing cells are collected.

  The myeloma cells used for cell fusion are not particularly limited, and known cells can be used. Examples of such myeloma cells include NS-1, P3U1, SP2, and X63.3.6.5.3 derived from BALB / c mice.

  Fusion of antibody-producing cells and myeloma cells may be performed by a method known to those skilled in the art, and is not particularly limited. The polyethylene glycol (PEG) method is preferred because the fusion operation is simple. Generally used PEG has an average molecular weight of 1000 to 6000, and is usually used at a concentration of 30 to 50% (v / v).

  Spleen cells collected from the removed spleen are mixed with myeloma cells in the logarithmic growth phase to perform cell fusion. Specifically, the mixed spleen cells and myeloma cells are centrifuged to obtain a pellet, and a PEG solution is added thereto, and they are fused by stirring and shaking.

  After cell fusion, the fused cells (hybridoma cells) are screened for the production of the desired antibody. For example, the hybridoma cells are washed by centrifugation, then transferred to a growth medium supplemented with HAT (hypoxanthine, aminopterin and thymidine), and cultured in a 96-well culture plate. A few days after the fusion operation, the culture supernatant of a well in which colony growth was observed was collected, and the reactivity with dioxins was measured by enzyme immunoassay (EIA method) using a plate on which dioxins were immobilized. Confirm with In the screening, for example, an antigen-antibody reaction is performed in an organic solvent (for example, DMSO) at a concentration of 20 to 50% (v / v) for a short time (10 to 30 minutes) to obtain a clone having an antigen binding activity. A clone is selected by evaluating the reactivity in the organic solvent. After screening, a well in which antibody production has been confirmed is selected and cloned by limiting dilution to establish a single antibody-producing clone.

  In order to prepare a monoclonal antibody from the established clone, hybridoma cells are cultured in a plate or flask, and the culture supernatant is obtained. Furthermore, the hybridoma cells were transplanted into the abdominal cavity of a mouse that had been previously administered pristane (2,6,10,14-tetramethylpentadecane), and the ascites collected in the abdominal cavity after 10 to 14 days was recovered by monoclonal antibody. Antibodies can be obtained.

  The culture solution or ascites obtained as described above is purified by ion exchange chromatography, hydrophobic chromatography, or affinity chromatography with protein A or protein G immobilized, if necessary, and then a monoclonal antibody preparation It can be. The monoclonal antibody thus obtained retains substantial antigen binding ability in a 30% (v / v) DMSO aqueous solution.

  The anti-dioxin antibody used in the method of the present invention may be any antibody that recognizes 3,3 ′, 4,4 ′, 5,5′-HxCB, and 2,3,4,7,8-PeCDF. Is also preferably recognized. The cross-reactivity for 2,3,7,8-TeCDD, which has the strongest toxicity, is less than 0.1%.

(Measurement of dioxins)
In the immunoassay method for toxicity equivalent amount of dioxins of the present invention, first, a sample containing dioxins is reacted with the anti-dioxin antibody, and then the amount of dioxins bound to the anti-dioxin antibody or the dioxin The amount of the anti-dioxin antibody that did not bind to the antibody is detected.

  Methods for immunologically detecting dioxins in a sample include radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), fluorescence immunoassay (FIA), immunochromatography, depending on the detection means. Chromatography, chemiluminescent enzyme immunoassay (CLEIA), immunoturbidimetry (TIA), latex immunoturbidimetry (LTIA) and the like. In the present invention, a method selected from the group consisting of enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescent immunoassay (FIA) is preferably employed. EIA is particularly preferred because of its simplicity of measurement.

  Each of the measurement methods includes a competitive measurement method, a non-competitive measurement method, a homogeneous method, and the like. Since dioxins are low molecular weight compounds, they can usually be performed by a competitive method. The competition method includes an indirect competition method in which an antigen is immobilized on a well or tube of a microplate, and a direct competition method in which an antibody is immobilized on a well or tube. Taking the case of EIA as an example, the indirect competition method and the direct competition method will be described.

  In the indirect competition method, dioxins or a complex of these and a carrier protein is used as a competitive antigen immobilized on a well. When using an untreated reaction vessel made of resin or glass, it is difficult to immobilize the dioxins alone in the vessel, and therefore it is preferable to use a complex with a carrier protein. In addition, when using a reaction vessel whose surface is activated with a highly reactive functional group such as an amino group or a carboxyl group, dioxins alone can be immobilized on the vessel via these functional groups. . Regardless of the presence or absence of a carrier protein, it is preferable to bond a linker to dioxins. This alleviates steric hindrance and improves the reactivity between the competitive antigen and the anti-dioxin antibody, so that the measurement sensitivity of dioxins in the sample is improved.

  The dioxins used as the competitive antigen are not particularly limited as long as they are recognized by the anti-dioxin antibodies used for the measurement. For example, the dioxins used for the immunizing antigen can be used. Alternatively, competitive antigens such as dioxins or dioxin-like compounds having low structural commonality with these dioxins can be used. By using such a competitive antigen, the reactivity between the competitive antigen and the anti-dioxin antibody is lower than the reactivity with the dioxins in the test sample, and the detection sensitivity of the dioxin concentration in the sample is improved. .

  The carrier protein is not particularly limited, and a known carrier protein can be used. Examples of such carrier protein include KLH and BSA. The linker is preferably one that does not sterically hinder the binding with the antibody or one that does not hinder the solubility in the reaction process, and includes, for example, a polymethylene chain. The linker is placed between dioxins or dioxin-like compounds or these and carrier proteins and / or containers.

  Furthermore, dioxins can be quantified with high sensitivity by using, for example, a chlorophenol derivative disclosed in Patent Document 4 as a competitive antigen. This chlorophenol derivative can also be used as a standard for preparing a calibration curve. In this case, an antigen for immobilization can be prepared by changing the amino acid or peptide moiety bound to the terminal of the chlorophenol derivative to a carrier protein such as BSA.

  The indirect competition method can be performed as follows, for example. FIG. 1 shows a schematic diagram of the measurement principle. First, the competitive antigen is solid-phased in the well of a reaction vessel such as a microplate as described above to obtain a solid-phased antigen. Next, the portion of the well surface where antigens are not bound is blocked with a commercially available blocking agent such as BSA or casein. A sample (preferably a pretreated sample) and an anti-dioxin antibody that is a primary antibody are added to the well, and the dioxins in the sample and the immobilized antigen are caused to compete with the anti-dioxin antibodies. After washing and removing the anti-dioxin antibody that did not bind to the immobilized antigen, the well is labeled with a secondary antibody such as goat anti-mouse immunoglobulin antibody with an enzyme such as peroxidase (PEO) or alkaline phosphatase (ALP). The enzyme-labeled antibody is added to bind to the primary antibody bound to the immobilized antigen. After washing several times with a buffer solution, a labeled enzyme substrate is added, and the absorbance of the colored enzyme reaction product is measured. When PEO such as horseradish peroxidase (HRP) is used as the enzyme, hydrogen peroxide as a substrate and o-phenylenediamine, tetramethylbenzidine or the like as a color former may be used. When ALP is used as an enzyme, p-nitrophenyl phosphate is usually used as a substrate.

  In the indirect competitive method described above, the ratio of the decrease in the absorbance of the reaction solution by adding the sample to the absorbance of the reaction solution to which no sample is added is measured as the inhibition rate by dioxins. In this case, for example, the competitive reaction is performed in the same manner except that the above chlorophenol derivative is used as a dioxin standard product and a solution having a plurality of known concentrations of this standard product is used instead of the sample. Create a calibration curve showing the relationship between the concentration of the standard product and the inhibition rate, and compare the obtained calibration curve with the inhibition rate due to dioxins in the sample to determine the concentration of dioxins in the sample as the standard product equivalent concentration. Can be calculated as

  When the concentration of dioxins in the sample is high, the amount of antibody bound to the immobilized antigen decreases because the amount of binding between the anti-dioxins antibody and the dioxins increases. Therefore, the amount of the enzyme-labeled secondary antibody that binds to the enzyme antigen is reduced, and the color intensity is weakened. In contrast, when the concentration of dioxins in the sample is low, the amount of antibody that binds to the immobilized antigen increases, so the amount of secondary antibody that binds to this antibody increases, resulting in strong color development. That is, the intensity of color development varies depending on the dioxin concentration in the test sample.

  In the direct competition method, the above-described anti-dioxin antibody is immobilized on a well of a container to obtain a solid-phased antibody. Next, the portion of the well where the antibody is not bound is blocked in the same manner as in the indirect competition method. Next, a competitive enzyme-labeled antigen and a specimen are added to the wells to cause the sample and the enzyme-labeled antigen to undergo a competitive reaction with the immobilized antibody. Next, the enzyme-labeled antigen that has not bound to the immobilized antibody (anti-dioxin antibody) is washed away, the enzyme substrate used for labeling is added, and the absorbance of the reaction product is measured.

  The enzyme-labeled antigen can be prepared by binding an enzyme such as PEO or ALP to the same dioxins or dioxin-like compounds as the competitive antigen used in the indirect competition method. Moreover, when using what changed the amino acid or peptide part which exists in the terminal of the chlorophenol derivative disclosed by patent document 4 to the enzyme like PEO or ALP, a sensitivity improves.

  The concentration of dioxins in the sample (concentration unknown sample) obtained by the above arbitrary measurement method is a standard product equivalent concentration. Converts the conversion factor, which is calculated in advance from the relationship between the toxic equivalent amount and the standard product equivalent concentration, to the toxic equivalent amount (TEQ) by multiplying the standard product equivalent concentration of the sample by measuring multiple samples with known toxic equivalents. To do.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

(Example 1: Measurement of dioxin isomer concentration and TEQ value in soil sample and exhaust gas sample)
About 25 to 50 g of 92 samples of soil were collected, and the substances in the sample were extracted with 500 ml of toluene, and quantified to 20 ml to obtain a crude extract. In addition, about 4 m ³ N was collected from 275 exhaust gas samples from the flue of the municipal waste incineration plant. The collected sample was extracted using toluene or dichloromethane for each form of filter paper, resin, absorbent, etc. according to the official method (JIS K0311). These extracts were combined and concentrated to 20 ml to obtain a crude extract.

  1 ml each was taken from the crude extract of soil and exhaust gas and purified by sulfuric acid treatment, multilayer silica gel chromatography and activated carbon column chromatography. About each refined sample, each dioxin isomer density | concentration was calculated | required with the gas chromatograph mass spectrometer (made by Micromass). Next, each dioxin isomer concentration was multiplied by its toxicity equivalent coefficient (TEF) to calculate the dioxin toxicity equivalent (TEQ) in the soil and exhaust gas samples. Table 1 shows the correlation coefficient between the dioxin isomer concentration and the toxicity equivalent (TEQ) in the soil sample and the exhaust gas sample.

  Table 1 shows the relationship between dioxin isomer concentration and toxicity equivalent (TEQ) calculated from the GC / MS measurement results of 92 soil samples derived from various pollutants and 275 exhaust gases derived from waste incinerators. Indicates the number of relationships. The exhaust gas sample has a high TEQ correlation with pentachlorinated and hexachlorinated dibenzofurans such as 2,3,4,7,8-PeCDF. On the other hand, in the soil sample, the concentration of 3,3 ′, 4,4 ′, 5,5′-HxCB (PCB # 169) has the highest correlation with TEQ. From this result, in order to obtain an ELISA measurement value highly correlated with GC / MS, an antibody having high reactivity with pentachlorinated and hexachlorinated dibenzofurans may be used for the exhaust gas sample. For soil samples with different sources of contamination, it has been found that antibodies that are highly reactive with PCB # 169 may be subjected to the measurement.

(Example 2: Preparation of anti-dioxin monoclonal antibody)
(1) Preparation of immunogen antigen

The antigen for immunity was produced by the active ester method using the hapten compound represented by these.

  First, the hapten compound was reacted with 1.2 equivalents of hydroxysuccinimide and 1.5 equivalents of 1-ethyl-3- (3-diethylaminopropyl) carbodiimide hydrochloride in DMF for 16 hours at room temperature. The reaction solution was extracted with methylene chloride, and then the solvent was distilled off under reduced pressure to obtain an active ester. Next, 15 mg of BSA was dissolved in 1 ml of 50 mM phosphate buffer (pH 8.0), and 4.98 mg (40 equivalents) of an active ester solution dissolved in 1 ml of DMSO was added dropwise and reacted at room temperature for 1 hour. It was. After the reaction, purification was performed with a gel filtration column (PD-10: manufactured by Amersham Biosciences) equilibrated with phosphate buffered saline (PBS) to obtain an antigen for immunization. The hapten introduction rate of the obtained antigen for immunization was 40 when the molecular weight of BSA was calculated as 66,000 and the number of hapten molecules bound per molecule of BSA by analyzing the absorption spectrum. In addition, this antigen can be used also as a solid-phased antigen at the time of selecting an antibody.

(2) Immunization of mouse 2 mg of the antigen for immunization prepared in (1) above was dissolved in 1 ml of PBS and mixed with an equal amount of complete Freund's adjuvant to prepare a 1 mg / ml emulsion. This emulsion was intraperitoneally administered to 5 BALB / c mice so as to obtain an antigen amount of 5 to 15 μg per mouse. In the same manner, booster immunization was performed 6 times every 2 weeks. One week after each immunization, blood was collected, and the blood was allowed to stand at room temperature for 1 hour, after which the clot was separated to obtain antiserum, and the antibody titer of the animal was determined.

(3) Measurement of antibody titer The antigen for immunization obtained in (1) above was diluted with PBS to 0.84 μg / ml. 50 μl of this diluted solution was dispensed into each well of a 96-well assay plate (manufactured by Costar: catalog number 3590), and the plate was sealed and allowed to stand at 37 ° C. for 1 hour. Each well was washed three times with PBS containing 0.005% (v / v) of Tween 20 and then 300 μl of PBS containing 1% (w / v) gelatin (BIO-RAD, catalog number 170-6537). The solution was dispensed into wells, and the plate was sealed and allowed to stand at 37 ° C. for 2 hours. Each well was washed three times with PBS containing 0.005% (v / v) Tween 20, and then 3,3 ′, 4,4 ′, 5,5′-HxCB at a final concentration of 10 ⁻⁶ to 10 ⁻⁸ M. 25 μl of 50% (v / v) DMSO aqueous solution containing (PCB # 169) and 2,3,4,7,8-PeCDF was added to each well. Immediately after the addition, 25 μl of antiserum (antiserum obtained in (2) above) diluted 1000-fold with PBS containing 1 mg / ml BSA was added to each well, and the plate was gently shaken and stirred. Left for 1 hour. Each well was washed three times with PBS containing 0.005% (v / v) of Tween 20, and then labeled with goat anti-mouse IgG (H + G) HRP diluted 2000 times with PBS containing 1% (w / v) gelatin. 50 μl of antibody (affinity purification) was dispensed. The plate was allowed to stand at room temperature for 1 hour, then washed 3 times with PBS containing 0.005% (v / v) of Tween 20, and 50 μl of TMB (manufactured by KPL) as a substrate for HRP was dispensed into each well. And allowed to stand at room temperature for 10 minutes. 50 μl of 1M phosphoric acid solution was added to each well, and the absorbance at a wavelength of 450 nm (control 630 nm) was measured with a microtiter spectrophotometer. Considering the background due to non-specific adsorption, the antibody titer was determined by subtracting the measured value of the well in which the antigen was not immobilized from the measured value of the well in which the antigen was immobilized.

(4) Preparation of anti-dioxin monoclonal antibody About the mouse confirmed to have a high antibody titer by the measurement of (3) above, an immunogen diluted with sterile PBS in the tail vein (compound of the above formula bound to BSA) (Hapten compound)) was administered as a booster at 50 μg (50 μl). Three days after the final administration, the spleen was aseptically removed from the mouse according to the method commonly used by those skilled in the art, and the spleen cells were separated and washed three times with RPMI 1640 medium to prepare a spleen cell suspension. Count the number of cells, mix so that SP2 / 0 myeloma cells (BALB / c mouse-derived myeloma cells) and spleen cells cultured in advance have a cell number ratio of 1:10, and centrifuge. The supernatant was removed. After removing the supernatant, PEG 1500 solution was added to stir the cells, and RPM1640 medium was further added to perform cell fusion. Next, the supernatant is removed by centrifugation, HAT medium is added to suspend the cells, 100 μl is seeded at a concentration of 1-2 × 10 ⁶ cells / ml in a 96-well culture plate, and then in a carbon dioxide incubator. Culturing was performed at 37 ° C. 100 μl of the supernatant was collected from the wells in which colonies appeared after 10 to 14 days, and the antibody titer was measured by the method (3) above without dilution. The clone in which the antibody titer was detected was transferred to a 24-well culture plate and the culture was continued. In order to confirm continued antibody production, the antibody titer was measured every few days. A clone that maintains a high antibody titer is selected, cloned by limiting dilution method 3 to 5 times using RPMI1640 medium containing 10% (w / v) FCS containing HT, and a plurality of antibody producing strains Got.

  Each of the obtained antibody-producing strains was transplanted into the peritoneal cavity of BALB / c mice pre-administered with pristane, and ascites collected in the peritoneal cavity was collected along with cell proliferation. The collected ascites was subjected to affinity chromatography using Protein G-Sepharose (manufactured by BIO-RAD) as a carrier, and the antibody-containing fractions were collected and dialyzed against PBS to obtain each purified monoclonal antibody.

(5) Specificity evaluation with respect to dioxin isomer of monoclonal antibody Specificity with respect to dioxin isomer was evaluated about each monoclonal antibody obtained by said (4). First, the antigen for immunization obtained in the above (1) was diluted with PBS to 1 μg / ml. 50 μl of this diluted solution was dispensed into each well of a 96-well assay plate (manufactured by Costar: catalog number 3590), and the plate was sealed and allowed to stand at 37 ° C. for 1 hour. Each well was washed 3 times with PBS containing 0.005% (v / v) of Tween 20, and then 300 μl of Block Ace (manufactured by Snow Brand) diluted to 25% (v / v) with distilled water was dispensed to each well. The plate was sealed and allowed to stand at 37 ° C. for 2 hours. Each well was washed 3 times with PBS containing 0.005% (v / v) of Tween 20, and then a final concentration of 5 × with 20% (v / v) DMSO containing 0.01% (v / v) of Triton X100. 25 μl of dioxin compound diluted to 10 ⁻⁷ M or the solvent used for dilution was added to each well. Immediately after the addition, 25 μl of an antibody solution diluted to 0.1 μg / ml with PBS containing 1 mg / ml BSA was added to each well, and the plate was gently shaken and stirred. I put it. Each well was washed three times with PBS containing 0.005% (v / v) of Tween 20, and then diluted 2000 times with 10% (v / v) Block Ace, and a goat anti-mouse IgG (H + L) HRP-labeled antibody ( (Affinity purification) 50 μl was dispensed. The plate was allowed to stand at room temperature for 1 hour, then washed 3 times with PBS containing 0.005% (v / v) of Tween 20, and 50 μl of TMB (manufactured by KPL) as a substrate for HRP was dispensed into each well. And allowed to stand at room temperature for 10 minutes. 50 μl of 1M phosphoric acid solution was added to each well, and the absorbance at a wavelength of 450 nm (control 630 nm) was measured with a microtiter spectrophotometer. In the reaction evaluation, the absorbance when no dioxin compound was added (dilution solvent) was taken as 100, and the absorbance when each dioxin was added was calculated as an inhibition rate (%). Table 2 shows the results of the specificity evaluation of the obtained monoclonal antibodies.

  As can be seen from Table 2, all have the same specificity, 1,2,3,6,7,8-HxCDD and 3,3 ', 4,4', 5,5'-HxCB. It was found that the reactivity was high. On the other hand, the reactivity with 2,3,7,8-TeCDD was low.

(6) Determination of subclass of monoclonal antibody About each monoclonal antibody obtained in (4) above, each culture supernatant is used, and as a secondary antibody, a typing antibody panel attached to a BIO-RAD typing kit is used. The subclass was determined. As a result of typing, all monoclonal antibodies were IgG1 (L chain, κ).

(Example 3: Examination of cross-reactivity)
The antibody disclosed in Non-Patent Documents 3 and 4 (referred to as antibody A; an antibody contained in a dioxin measurement kit, manufactured by Toyobo Co., Ltd.) and the monoclonal antibody prepared in Example 2 above (referred to as antibody B) ) Was examined by the ELISA described below (measurement methods disclosed in Patent Documents 3 and 4) with 29 types of dioxin isomers having TEF.

  For the measurement, 96 wells in which a predetermined amount of competitive antigen (2,4,5-trichlorophenol glycylglycine: hereinafter referred to as TCP-glycylglycine) was bound to BSA and immobilized (solid-phased antigen). A microplate was used. First, the sample was cleaned up and dissolved in DMSO. To 25 μl of the sample dissolved in DMSO, 25 μl of a phosphate buffer and 50 μl of an anti-dioxin antibody as a primary antibody were added and stirred, and then this mixture was placed in a well and left for 1 hour. At this time, the anti-dioxin antibody and the dioxin in the sample are preferentially bound, and an excessive amount of the anti-dioxin antibody is bound to the immobilized antigen.

  Next, after washing away the anti-dioxin antibodies that did not bind to the immobilized antigen, 100 μl of goat anti-mouse IgG (H + L) HRP-labeled antibody (affinity purified) as a secondary antibody was added to the wells and left at room temperature for 1 hour. Then, it was bound to the primary antibody bound to the immobilized antigen. After washing the well several times with a buffer solution, 100 μl of tetramethylbenzidine (TMB), which is a chromogenic substrate, was added and reacted at room temperature for 30 minutes in the dark. Subsequently, 100 μl of 0.5 mol / l sulfuric acid was added to the well to stop the enzyme reaction, and the color changed from blue to yellow. The absorbance at 450 nm, which is the color development wavelength, was measured with a microplate reader. Moreover, the light absorbency (blank light absorbency) of the reaction solution which does not add a sample was also measured, and the ratio of the amount of decrease in the light absorbency of the reaction solution due to the addition of the sample to the blank light absorbency was measured as the inhibition rate by the sample.

  On the other hand, TCP-glycylglycine was used as a standard product, and a plurality of known concentration solutions were subjected to a competitive reaction in the same manner to prepare a calibration curve showing the relationship between the standard product concentration and the inhibition rate. By comparing the obtained calibration curve and the inhibition rate by the sample, the concentration of dioxins in the sample was calculated as the standard product equivalent concentration. TCP-glycylglycine was measured simultaneously on the same plate as the sample. Finally, by measuring multiple samples with known toxic equivalents, a conversion factor that calculates the relationship between toxic equivalents and standard product equivalent concentrations in advance is obtained by measuring the test sample (concentration unknown sample). The product was converted to a toxic equivalent (TEQ) by multiplying the standard product equivalent concentration.

The IC ₅₀ value (antigen-antibody reaction) of the isomer to which each antibody reacts most (1,2,3,7,8-PeCDF for antibody A, 1,2,3,6,7,8-HxCDD for antibody B) 50% inhibition concentration: ng / ml) was defined as 100, and the cross-reaction rate (%) was calculated from the IC ₅₀ value of each isomer relative thereto. The results are shown in Table 3.

  Antibody A has the property of high reactivity with pentachlorinated and hexachlorinated dibenzofurans. On the other hand, antibody B was found to be highly reactive not only with pentachlorinated and hexachlorinated dibenzofurans but also with 3,3 ′, 4,4 ′, 5,5′-HxCB (PCB # 169). . Therefore, antibody A is effective for TEQ measurement in exhaust gas samples derived from waste incineration facilities, but has no reactivity with PCBs, so it can be used to measure soil samples with different isomer distribution depending on the sampling location. Is not suitable. On the other hand, antibody B is predicted to be suitable for TEQ measurement of soil samples because it has the property of having high reactivity with not only pentachlorinated and hexachlorinated dibenzofurans but also PCB # 169.

(Example 4: Examination of correlation with GC / MS method)
The concentration of dioxins in soil samples and sediment samples contaminated by various sources was measured by ELISA using antibody A and antibody B prepared in Example 2 above. The soil samples measured were 16 samples of 3 types: incineration contamination, PCB contamination and agricultural chemical contamination. In addition, the bottom sediment samples were 3 types and 12 samples derived from agricultural chemical contamination, PCB contamination, and industrial wastewater contamination.

  About 8 to 10 g of each of the soil sample and the bottom sediment sample were collected, the substance in the sample was extracted with 500 ml of toluene, and quantified to 20 ml to obtain a crude extract. 1 ml each was taken from the crude extract, and cleaned up by sulfuric acid treatment, multilayer silica gel chromatography and activated carbon column chromatography. About each clean-up sample, the dioxin density | concentration was measured according to the measurement procedure by said ELISA, and it converted into TEQ value. Further, the same sample was cleaned up, and then the TEQ value was measured with a gas chromatograph mass spectrometer (manufactured by Micromass). The results are shown in FIGS.

  In the graphs of FIGS. 2 to 5, the horizontal axis represents the GC / MS value (pg-TEQ / g) of each sample, and the vertical axis represents the ELISA value (pg-TEQ / g) measured using each antibody. The thick line shows a 1: 1 relationship between the GC / MS value and the ELISA value, and the range of 0.5 to 2 times is shown with dotted lines above and below it.

  2 and 3 show the results of measurement of soil samples and sediment samples in an ELISA measurement system using antibody A. FIG. Since antibody A has a characteristic of specifically reacting with pentachlorinated and hexachlorinated dibenzofurans, a result highly correlated with the GC / MS value was obtained in samples derived from incineration contamination and agricultural chemical contamination. However, since it does not react with PCBs, the measurement result of the soil sample derived from PCB contamination was calculated to be extremely lower than the GC / MS value.

  4 and 5 show the measurement results of soil samples and sediment samples in an ELISA measurement system using antibody B. FIG. Antibody B has high reactivity with not only pentachlorinated and hexachlorinated dibenzofurans but also with PCB # 169, so that a result highly correlated with the GC / MS value was obtained regardless of the contamination source. Therefore, if an antibody having a high affinity for 3,3 ′, 4,4 ′, 5,5′-HxCB (PCB # 169) is used, the GC / MS value is obtained regardless of the contamination source of soil or sediment. It was found that a highly correlated measurement value can be obtained. In particular, if there is a high affinity for both pentachlorinated and hexachlorinated dibenzofurans and PCB # 169, measurement is highly correlated with the GC / MS value regardless of the type of sample such as exhaust gas or soil. It turns out that the value can be obtained.

  According to the method of the present invention, it is possible to quickly calculate a measurement result consistent with TEQ (toxic equivalent amount) by the official method (GC / MS method) regardless of the pollution source. By this method, simple analysis, monitoring and the like of dioxins in environmental samples can be performed quickly and easily.

Claims (3)

An immunoassay method for toxic equivalents of dioxins in soil or sediment samples ,
Reacting with the soil or sediment sample, 3,3 ', 4,4', an anti-dioxin antibody having affinity for 5,5'hexachlorobiphenyl;
Detecting the amount of dioxins bound to the anti-dioxin antibodies or the amount of the anti-dioxin antibodies not bound to the dioxins; and calculating the toxicity equivalent in the sample from the detected amounts ;
Including
The anti-dioxin antibody is a monoclonal antibody;
Method.   The method according to claim 1, wherein the anti-dioxin antibody further has an affinity for 2,3,4,7,8-pentachlorodibenzofuran.   The method according to claim 1 or 2, wherein the detection step is performed based on a method selected from the group consisting of an enzyme immunoassay, a radioimmunoassay, and a fluorescence immunoassay.

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