JP2002119279A - Monoclonal antibody against dioxin and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish a simple and high-sensitivity enzyme immunoassay method for dioxins in a human biological sample. SOLUTION: Dioxins in a human biological sample are detected and assayed by an enzyme immunoassay method by a monoclonal antibody which is specific to one or more dioxins and has the maximum affinity for 2,3,7,8- tetrachlorodibenzo-p-dioxin among dioxins.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody against dioxins, in particular, dioxins,
The present invention relates to a monoclonal antibody having the highest affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin. The present invention also relates to a method for producing the monoclonal antibody, a method for detecting and measuring dioxins using the monoclonal antibody, and a kit therefor.

[0002]

2. Description of the Related Art In recent years, due to concerns about environmental pollution by chemical substances and their effects on human health, dioxins contained in air, incinerated ash, exhaust gas, wastewater, ordinary water, food, etc. are not only in Japan but also worldwide. It is a social concern in each country, and it is said that dioxins as environmental pollutants pose a persistent toxicity threat to living organisms and human beings. Dioxins are unintentional chemicals generated in the waste heat treatment process and the production process of organochlorine compounds, but their sources are diverse and their environmental pollution has become a major problem. It is urgent to investigate human exposures, ecosystems, and food pollution from the United States.
The developmental toxicity of dioxins is more sensitive than the toxicity of living organisms, and the effect is irreversible in many cases and has a serious problem of extending to the next generation. In particular, the effects on the offspring of pregnant and nursing mothers can be manifested by very low levels of dioxin. The establishment is eagerly awaited. Conventionally, high-resolution gas chromatography / mass spectrometry (HRGS / H) has been used to measure specific dioxins with high sensitivity and high accuracy in a sample containing various types of contaminants.
RMS) has been used. However, HRGS / HRM
To measure with S, any sample requires complicated clean-up operations at other stages, requires expensive equipment in well-maintained laboratories, and requires a skilled researcher to work for a long time. As a result, the cost is significantly higher. Since it is expected that the number and types of dioxins to be measured will increase in the future, there is a strong demand for the development of an inexpensive, simple, and highly sensitive measurement method. This H
One of the alternatives to RGS / HRMS is an immunoassay. Some measurements of dioxin by this method have been reported, but only on samples containing high concentrations of dioxin (environmental samples and standard solutions) (Kennel, SJ et al., Tox
icol.Appl.Pharmacol. 1986,82,256-263; Stanker, L.
H. et al., Toxicology. 1987, 45, 229-243; Vanderlaan, M.
Chem. 1988, 7, 859-870; Sugawara, Y. et al.
Anal.Chem. 1998,70,1092-1099; Sanborn, JR et al., JA
gric. Food Chem. 1988, 46, 2407-2416; Harrison, RO
Et al., Organohalogen Compounds 1999, 36, 129-132; Zenn
egg, M. et al., Organohalogen Compounds 1999, 36, 317-31
9) There are few reports on biological samples with low dioxin content, such as human breast milk and blood, for which measurement is required.

[0003] Dioxins can be further explained as follows.
Dioxins are dioxins represented by the following formula (a) [polychlorinated dibenzo-p-dioxins (PCDDs)]
And polychlorinated dibenzofuran (PCD) shown in formula (b)
Fs), which are tricyclic chlorinated compounds, which have many structural isomers depending on the number and position of substituted chlorines, and have similar biological actions and physicochemical properties, respectively. Some are quite different, and the toxicity varies significantly for each isomer. As described above, the toxicity differs greatly between isomers and homologues, so that it is difficult to evaluate the toxicity based on measured concentrations.

Embedded image

[0004] Structural isomers of dioxins include 75 kinds of polychlorinated dibenzo-p-dioxins (PCDDs) and 1
Although there are 35 kinds of polychlorinated dibenzofurans (PCDFs), the toxicity is significantly different between isomers, so the relative toxicity and toxicity when the toxicity for 2,3,7,8-TCDD, which is the most toxic, is defined as 1. Equivalent coefficient (Toxic Equ
ivalency Factor (TEF) has been proposed and seven highly toxic PCDDs and ten PCDs
Fs is a measurement target. In addition, polychlorinated biphenyls (P
Among CBs), four kinds of non-ortho PCBs and eight kinds of mono-ortho PCBs have been measured as compounds having the same biological action as dioxin. Therefore, for evaluation of dioxins, the sum of the values obtained by multiplying the measured concentration of each isomer by TEF is calculated as 2,3,7,8-TCDD toxic equivalent (Toxic Equivalent quat).
(teinity; TEQ), and the toxicity is evaluated using this value.

[0005] Most recently, the majority of dioxin TEQs in breast milk have been (1) 2,3,7,8-TCD
D, (2) 1,2,3,7,8-PeCDD and (1
0) It was revealed that the compounds were occupied by 3, 3, 4, 7, 8, 8-PeCDF, and that the toxicity of dioxins could be evaluated by enzyme immunoassay (ELISA) using an antibody having high affinity for these. Indicated (Nakazawa,
H. et al., Organohalogen Compounds 2000, 45, 86-89; Sait
o, K. et al., Organohalogen Compounds 2000, 45, 168-171;
Sugawara, Y. et al., Organohalogen Compounds 2000, 45, 172.
-175). However, since the antibody used here is a rabbit polyclonal antibody, it is impossible to supply a large amount of antibody without lot difference at the time of production or acquisition, and the measurement method and the stability and universality of the measurement result Had a problem. In addition, even if antisera from the same lot is used, a plurality of antibody species (chronotypes) involved in the reaction are mixed, so that the antibody species that reacts differs depending on the measurement sample, and the measured value may vary. In order to improve the above-mentioned drawbacks of the method using polyclonal antibodies, a monoclonal antibody against dioxins and a method for measuring dioxins using the same have been proposed [JP-A-63-14691 (US Pat.
P4,798,807) and JP-A-63-744494]. However, the specificity of these monoclonal antibodies for dioxins and the information on the anti-dioxin antibody titer are unclear, and the method described in JP-A-63-74494 discloses a method for cross-linking with other dioxin homologs. Since no reactivity has been shown, the measurement results obtained using this antibody cannot be evaluated from the viewpoint of toxicity, and JP-A-63-14691 has the highest toxicity 2,3, Because it has a higher affinity for other dioxin homologs than 7,8-TCDD, the measured values of actual samples with different homolog mixing ratios do not correlate with the toxic equivalents. However, the quantification limit of 8-TCDD was around 1 ng, and it could not be used for the quantification of real samples containing a small amount of dioxin such as biological samples.

[0006]

SUMMARY OF THE INVENTION The present invention establishes a simple and highly sensitive enzyme immunoassay (ELISA) for dioxins in a human biological sample, and is particularly useful for monitoring dioxin contamination and investigating the situation in humans. It is an object of the present invention to provide a method which can contribute to the above, and to provide a monoclonal antibody suitable for that purpose.

[0007]

In order to confirm the selection of an object to be measured by the ELISA method and to confirm its suitability, Nakazawa et al. Measured the results of each homologue of dioxin in human milk obtained by HRGC / MS. And toxic equivalent coefficient (T
The correlation with the toxicity equivalent amount (TEQ) calculated based on EF) was examined. As a result, in particular, (2) 1, 2, 3,
7,8-PeCDD and (10) 2,3,4,7,8-P
The two types of eCDF have particularly excellent correlation with TEQ,
Three kinds including 2,3,7,8-TCDD with high EF
It was confirmed that if it could be measured by LISA, it would be possible to evaluate dioxin exposure in humans as a toxic equivalent.
The present inventors have repeated studies to obtain monoclonal antibodies specific to the above three types, and as a result, 2,3 of dioxins
The present inventors have succeeded in obtaining a novel monoclonal antibody having the highest affinity for 7,8-TCDD, and have reached the present invention.

That is, the present invention relates to a monoclonal antibody specific to one or more dioxins, which has an affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin among the dioxins. This is for the largest monoclonal antibody. In the present invention, the dioxins refer to polyhalogenated dibenzo-p-dioxins or polyhalogenated dibenzofurans, and specific examples thereof include polyhalogenated-p-dioxins of the following (1) to (7) or ( 8) to (17) are polyhalogenated dibenzofurans. (1) 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378TCDD), (2) 1,2,3
7,8-pentachlorodibenzo-p-dioxin (1
2378 PeCDD), (3) 1, 2, 3, 4, 7, 8
-Hexachlorodibenzo-p-dioxin (1234)
78HxCDD), (4) 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (123678
HxCDD), (5) 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (123789Hx
CDD), (6) 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (12334678H
pCDD), (7) 1,2,3,4,6,7,8,9-
Octachlorodibenzo-p-dioxin (12346)
789OCDD), (8) 2,3,7,8-tetrachlorodibenzofuran (2378TCDF), (9) 1,
2,3,7,8-pentachlorodibenzofuran (123
78PeCDF), (10) 2,3,4,7,8-pentachlorodibenzofuran (23478PeCDF),
(11) 1,2,3,4,7,8-hexachlorodibenzofuran (123478HxCDF), (12) 1,
2,3,6,7,8-hexachlorodibenzofuran (1
23678HxCDF), (13) 1,2,3,7,
8,9-Hexachlorodibenzofuran (123789H
xCDF), (14) 2,3,4,6,7,8-hexachlorodibenzofuran (234678HxCDF),
(15) 1,2,3,4,6,7,8-heptachlorodibenzofuran (1234678HpCDF), (16)
1,2,3,4,7,8,9-heptachlorodibenzofuran (1234789HpCDF), (17) 1,2,2
3,4,6,7,8,9-octachlorodibenzofuran (123466789OCDF). The present invention relates to (1)
It has the highest affinity for 3,7,8-tetrachlorodibenzo-p-dioxin, and (2) of 1,2,3,7,
8-pentachlorodibenzo-p-dioxin, (8)
A monoclonal antibody having a high affinity for 2,3,7,8-tetrachlorodibenzofuran and / or 2,3,4,7,8-pentachlorodibenzofuran of (10). The present invention also provides hybridoma D
9-36 (FERM P-18057), Hybridoma D2-37 (FERM P-18056) or Hybridoma D35-42 (FERM P-18058)
And a monoclonal antibody produced from the same.

The present invention also provides a method of immunizing an animal with a conjugate of a hapten and a protein represented by the following formula, obtaining antibody-producing cells from the animal, and fusing the cells with tumor cells to produce a plurality of hybridomas. Selecting at least one hybridoma producing an antibody that reacts with dioxins from the plurality of hybridomas, and recovering the antibodies produced from the hybridomas. is there.

Embedded image In the formula, R1 is NHCO (CH ₂ ) ₂ COOH, NHCO
(CH ₂ ) ₃ COOH, NHCO (CH ₂ ) ₄ COOH, N
HCOCH = CHCOOH, CH = CHCOOH, CH
ＣC (CH ₃ ) COOH, (CH ＝ CH) ₂ COOH, H
R2 is H, Cl, OCH ₂
COOH, O (CH ₂ ) ₃ COOH, O (CH ₂ ) ₄ COO
H, CH = CHCOOH, a functional group selected from (CH = CH) ₂ COOH, R3 is Cl or H,
R4 is Cl, CH ₃ or C (CH _{3) 3.} Examples of the protein to be bound to the hapten include bovine serum albumin, ovalbumin, and limpet hemocyanin. The present invention also relates to a hybridoma producing the above-described monoclonal antibody, and particularly to D9-36 (FERM).
P-18057), D2-37 (FERM P-180)
56) or D35-42 (FERM P-18058)
This relates to a hybridoma represented by Furthermore, the present invention provides a method for detecting and measuring dioxins by an immunoenzyme assay using the monoclonal antibody of the present invention, a highly purified dioxin composition containing the monoclonal antibody of the present invention, and a dioxin containing the monoclonal antibody of the present invention. Compositions for use in biochemical, immunological, functional or other research assays. The present invention also relates to a kit for measuring the presence or concentration of dioxins in a sample containing the monoclonal antibody and the enzyme-labeled hapten of the present invention. The monoclonal antibodies of the present invention can be used for identifying dioxins contained in a sample, and can be used for measuring the concentration of dioxins in a sample. Examples of the target sample include environmental samples such as soil, water, air, and fly ash, and biological samples such as blood and breast milk.

When used as a reagent in various immunoassays to determine the presence or concentration of dioxins, the use of the monoclonal antibodies of the present invention improves the assay. Detection is more convenient, faster, more sensitive, and more specific. Examples of immunoassays that can use the monoclonal antibodies of the present invention include radioimmunoassay (RIA) (radioimmunoassay), competitive immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay. It is not limited to only these immunoassays. In the present invention, it is particularly preferable to use the method for the immunoenzyme assay. In this case, an indirect competition method in which the pseudoantigen is immobilized and the reaction between the dioxins in the sample and the monoclonal antibody are competed with each other, Direct competition method for observing the reaction rate between a class and an enzyme-labeled hapten. In the latter case, the monoclonal antibody of the present invention can be used as an antibody. It is excellent in that point and is preferable.
Enzymes for labeling haptens include horseradish peroxidase, β-galactosidase and the like.

The composition according to the present invention for assaying or measuring the presence or concentration of dioxins in a sample is effective for quantifying an antibody or a chemical substance at a concentration effective for detecting the presence of a chemical substance. Contains a concentration of antibody.
Latex particles or plastic microtiter
The antibody may be mixed with a suitable carrier such as a plate or the antibody may be adhered to these carriers. Depending on the immunological method used, enzymes or dyes can be added to the antibodies, and radioactive labels can be added. Therefore,
All analytical methods using monoclonal antibodies that react with dioxins including 2,3,7,8-TCDD are included in the technical scope of the present invention. The monoclonal antibodies of the present invention can be used to measure, isolate, purify, and / or remove dioxins from complex mixtures or solutions based on a selective immune response. The use of monoclonal antibodies that react with dioxins significantly improves the prior art. The properties that make these monoclonal antibodies useful in the reactions described above are distinguished by their remarkable specificity as compared to polyclonal antibodies, and their homogeneity on a large industrial or commercial scale. And the availability of virtually unlimited quantity.

For example, using the monoclonal antibody of the present invention, for example, 2,3,7,8-TCDD can be separated and purified from a mixture of other dioxins or similar organic compounds. When the mixture is contacted with the immobilized monoclonal antibody of the present invention, an immobilized complex of antibody-bound 2,3,7,8-TCDD is formed and 2,3,7,8-TCDD is removed from the mixture. After separation and removal of the mixture, the 2,3,7,8-TCDD is separated from the antibody, and the highly purified one is recovered by a known technique.
The composition according to the present invention used for purifying or recovering dioxins from a complex mixture may be immobilized on acceptable equipment or mixed with an acceptable carrier so as to be capable of reacting and binding with dioxins. It contains an effective amount of the monoclonal antibody of the invention. The monoclonal antibodies of the present invention are also useful reagents for studies relating to the structure and function of dioxins. Due to the sharp specificity of the antibodies of the present invention, they can be used for immunochemical analysis and structure-activity analysis of dioxins, and compared with the conventional polyclonal antibodies with poor specificity. It is more suitable for applications such as The composition according to the invention for use as a research reagent contains an amount of the antibody that is informative by mixing with dioxins and subsequent analysis. The amount can be appropriately determined.

The production of the novel monoclonal antibody of the present invention and the construction of the ELISA method are described in more detail below. 1) Synthesis of Hapten Antigen, Protein Conjugate, and Labeled Product A dioxin skeleton having a carboxyl group at the terminal C-1 or C-2 and a spacer having a different length via an acid amide, ether, or double bond. More than a dozen hapten antigens shown in Table 1 were synthesized.

[Table 1] Several haptens were selected from these and reacted with bovine serum albumin (BSA) by the active ester method, and reacted with western peroxidase (HRP) to obtain several enzyme-labeled products.

2) Monoclonal Antibodies Four hapten (I-2, I-3, I-5, II-2) BSA conjugates were repeatedly immunized to BALB / c or A / J mice, Individuals with good dioxin antibody titers were further examined for antibody affinity by the inhibitory effect of TMDD. Then, B obtained good results
Splenocytes obtained from two ALB / c mice and one A / J mouse were fused with P3 / NSI / 1-Ag4-1 myeloma cells using polyethylene glycol, and the fusion was obtained by HAT selective culture. ELISA of cell culture supernatant
Screened. As a result, it was shown that about 200 hybridomas secrete anti-dioxin antibodies. 3) selecting a hybridoma producing an antibody having a large inhibitory effect by TMDD and 2,3,7,8-TCDD;
As a result of cloning the obtained hybridoma to prepare a monoclonal antibody and examining its properties in detail,
A monoclonal antibody that is specific for one or more of the dioxins, wherein 2,3,
A novel monoclonal antibody having the highest affinity for 7,8-tetrachlorodibenzo-p-dioxin was successfully obtained. The monoclonal antibody of the present invention further comprises (2)
2,3,7,8-pentachlorodibenzo-p-dioxin, (8) 2,3,7,8-tetrachlorodibenzofuran, and / or (10) 2,3,4,7,8-pentane Some have great affinity for chlorodibenzofuran.

[0015]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Various conditions for conducting the experiment were as follows. 1. Experimental materials 1) Hapten-bovine serum albumin (BSA) conjugate and peroxidase (HRP) labeled hapten The four haptens shown in Table 1 above (I-2, I-3, I-5, II-
2) BSA conjugate and 8 haptens (I-2, I-3, I-5, I
-6, I-7, I-10, II-4, II-6). 2) Mouse BALB / c and A / J mice (both female, 8 weeks old)
Purchased from SLC. 3) Myeloma cell line The P3 / NS1 / 1-Ag4-1 myeloma cell line was provided by the Human Science Research Resource Bank.

2. Reagents and equipment 1) Immunology and ELISA Freund's complete and incomplete adjuvant: DIFCO 0638-60
-7 and 0639-60-6 affinity purified rabbit anti-mouse IgG + IgM antibody (second antibody): Jackson 315-005-044 1,2,7-trichloro-8-methyldioxin (TMDD): Wellingt
on Laboratories o-phenylenediamine dihydrochloride: Sigma P9029 30% hydrogen peroxide: Wako Pure Chemical Industries Microtiter plate for ELISA: Sumitomo Bakelite
MS-9596F 2) Cell fusion-related RPMI 1640 powder medium: GIBCO-BRL 31800-022 Fetal calf serum (FCS): GIBCO-BRL 26140-079 Hybridoma cloning factor (HCF): IGEN HAT Media Supplement: Sigma H0262 Polyethylene glycol 4000 ( PEG): Merck Art 9727 Dimethyl sulfoxide (DMSO): Sigma D2650 Culture flask (25 cm ² ): Iwaki Glass 3100-025 Culture flask (75 cm ² ): Becton Dickinson 3824 Cluster dish (96 wells): Costar 3598 Other salts -Special grade organic reagents were used. 3. Instruments ELISA plate reader (BL 312e): Bio-Tek Instrume
nt Inc. 4. Immunization and test blood sampling Each of the BSA conjugates of the above four haptens (I-2, I-3, I-5, II-2) was converted into BALB / c mice and A / J Each mouse
Five animals were repeatedly immunized. Hapten-BSA conjugate (50
μg) was dissolved in sterile physiological saline (0.1 mL), and as an emulsion with complete adjuvant (0.1 mL), the above mouse foot pad (one place per foot) and back (about 20 places by removing the hair with a hair clipper) ) Was administered subcutaneously. Thereafter, the same amount of immunogen was used as an emulsion with incomplete adjuvant, and boosted 6 to 8 times. Seven days after the fifth to seventh booster immunizations, test blood (20 to 40 μL) was collected from the ocular vein using a hematocrit tube. Serum was separated from the obtained blood by a conventional method, and its reactivity with HRP-labeled dioxin was determined.
It was examined by the following ELISA method.

5. ELISA 1) Buffer and substrate solution PBS: 0.05 mol / L NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer (pH 7.3) containing 0.9% NaCl. Substrate solution: 50 mmol / L citric acid-sodium acetate buffer (pH 5.0) containing 0.05% o-Phenylenediamine.HCl and 0.01% hydrogen peroxide. 2) Preparation of second antibody-immobilized plate In each well of a 96-well ELISA microtiter plate,
Dispense PBS solution (2μg / mL) of second antibody (100μL / well)
And left overnight at 4 ° C. After removing the antibody solution by suction,
Wash the wells three times with BS. BSA (0.5%) in PBS (150μL / wel
l) was dispensed into wells and left at room temperature for 2-3 hours. After the solution was removed by suction, the wells were washed three times with PBS to prepare a second antibody-immobilized plate. 3) ELISA method Add mouse serum, hybridoma culture solution or mouse ascites diluted with PBS containing 0.1% gelatin, and enzyme-labeled dioxin to the second antibody-immobilized plate (add a dioxin standard solution for the inhibition test) ( 100μL / well),
Leave at 4 ° C overnight. After the solution was removed by suction, the wells were washed three times with PBS, and a substrate solution was added (100 μL / well).
Leave at room temperature for 30 minutes to 1 hour. 3 mol / L sulfuric acid (50μL / wel
After l) was added to stop the enzyme reaction, the absorbance at 490 nm was measured with a plate reader.

6. Preparation of Monoclonal Antibody by Cell Fusion 1) Medium Basic medium: RPMI-1640 containing 10 mmol / L HEPES-NaOH buffer (pH 7.3) and kanamycin sulfate (0.06%). Culture medium: Basic medium containing 10% FCS, 0.05 mmol / L 2-mercaptoethanol, 2 mmol / L L-glutamine and 1 mmol / L sodium birubate. HAT medium: a culture medium containing 0.01 mmol / L hypoxanthine, 16 μmol / L thymidine and 0.4 μmol / L aminopterin. HT medium: a culture medium containing 0.01 mmol / L hypoxanthine and 16 μmol / L thymidine. 2) PEG solution Dissolve PEG (40 g) in Dulbecco's PBS (-) (50 mL) and add D
MSO (10 mL), 0.1% poly-L-arginine hydrochloride solution (1 m
After adding L), the pH was adjusted to about 7.5 using 1 mol / L NaOH. 3) Cell fusion Mice with increased antibody titer
(50 μg) of physiological saline solution (0.5 mL) was intraperitoneally administered. Three days later, the spleen was removed and splenic lymphocytes were loosened in a Petri dish containing a basic medium (10 mL) to prepare a cell suspension. After removing the tissue fragments using a stainless steel mesh, centrifugation was performed at room temperature (1600 rpm, 6 minutes) to remove the supernatant.
Add basal medium (10 mL) to the pellet, suspend the cells, centrifuge under the same conditions, and remove the supernatant. After this operation was performed once more, the cells were suspended by adding a basic medium (10 mL) to the pellet, and the number of cells was calculated using a part of the cells. Transfer the above splenocyte suspension (total amount) and about 1/5 of myeloma cells to a centrifuge tube and centrifuge at room temperature (1600 rpm, 5 minutes).
After sufficiently removing the supernatant, a PEG solution (1 mL) preliminarily heated to 37 ° C. was added dropwise to the pellet over 1 minute. After gently mixing the cell suspension for 1 minute, the basic medium was added in three portions (1 mL for 1 minute; 1 mL for 1 minute; 8 mL for 3 minutes),
Centrifuge under the same conditions. After removing the supernatant, the pellet was loosened well, and a HAT medium containing 10% HCF (50 mL per 1 × 10 ⁸ splenocytes used) was added to suspend the cells after the fusion operation.
This is dispensed into a 96-well cluster dish (100 μL
/ well) and cultured at 37 ° C., 5% CO ₂ . The next day, HAT medium
(100 μL / well), about half of the medium was removed by suction on the third and sixth days, and a HAT medium (100 μL / well) was newly added.

4) Screening of anti-dioxin antibody in culture supernatant A part of the culture supernatant of the hybridoma is collected 8 to 10 days after the start of the culture, mixed with PBS containing 0.1% BSA, and mixed with the above-mentioned second antibody. Added to immobilized ELISA plate. After 1 hour incubation at room temperature, the solution was aspirated off and the plate was washed three times with PBS. After adding HRP-labeled dioxin (0.1 μg; 100 μL) and incubating under the same conditions, the plate was washed similarly. A substrate solution (100 μL / well) was added to each well, and the HRP activity on the plate was measured by the method described above. Among the hybridomas producing antibodies against dioxin, TMDD
(50 pg / well), hybridomas in which the binding of the antibody-HRP-labeled dioxin was inhibited were selected. Further in it
Screening for hybridomas producing antibodies with high inhibitory effects on (1) 2,3,7,8-TCDD and (2) 1,2,3,7,8-PeCDD, and affinity for (1) Hybridomas producing strong monoclonal antibodies, D2-37, D9-36 and D35-
I got 42. 5) Cloning method Each hybridoma selected by screening was cloned by the limiting dilution method. The hybridoma was diluted to 10 cells / mL with HT medium containing 10% HCF,
Dispense 0.2 mL into each well of the well culture plate
Cultured for 10 days. For wells where hybridoma growth is observed, after confirming that they are single clones under a microscope, antibody-producing hybridomas are transplanted to 48-well culture plates and grown sequentially, and single clone hybridomas, D2-37, D9-36 and D35-42 were obtained.

6) Characteristics of Monoclonal Antibody 3) Obtained from the three hybridomas obtained in 5) above
The toxicity equivalency coefficients and crossover rates for the various monoclonal antibodies are shown in FIGS. The crossing rate is 2,3,7,
2,3 of each homolog calculated from the calibration curve of 8-TCDD
It was determined by dividing the equivalent amount of 7,8-TCDD by the amount of each homologue added. As can be seen from FIGS. 1 to 3, the above three types of monoclonal antibodies have the highest affinity for the most toxic (1) 2,3,7,8-TCDD among the dioxins, and similarly, Highly toxic (2) 1,2,3,7,8-
PeCDD, (10) 2,3,4,7,8-PeCDF
And / or (8) high affinity for 2,3,7,8-TCDF. The dioxin homolog content ratio in a biological sample such as breast milk has a small individual difference, and the three types (1), (2), (1)
Since the homolog of 0) occupies most of the total TEQ (toxic equivalent), it is possible to monitor TEQ in a biological sample by ELISA using the monoclonal antibody. In particular, the crossover rate of D9-36 with a dioxin homolog was determined by TEF (toxic equivalency).
factor), there is a possibility that the method can be applied to samples having different homolog content ratios. Also, D
Since 35-42 recognizes only TCDD and PeCDD, there is a possibility that the two homologs can be measured even in environmental samples containing significantly different homologs. The crossover rates of the monoclonal antibodies are as shown in Table 2.

[0021]

[Table 2] The characteristics of the three monoclonal antibodies are summarized below. D2-37: 2,3,7,8-TCDD, which has the highest affinity for 2,3,7,8-TCDD and the lowest crossover rate with other homologues, making it the most toxic 2,3,7,8-TCDD among dioxins Can be measured specifically and with high sensitivity. D9-36: In addition to 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 2,
Since it has a high affinity for 3,4,7,8-PeCDF, it is possible to monitor the equivalent amount of dioxin toxicity in biological samples such as breast milk. In addition, since the crossover rate with other homologs also correlates well with TEF, it can be used for toxicity evaluation of environmental samples and the like. D35-42: Reacts specifically with 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD, so the two dioxin homologues in any sample can be obtained by simple pretreatment. The body can be measured.

II. Standard Curve of 2,3,7,8-TCDD Mouse ascites diluted with PBS containing 0.1% gelatin on a second antibody-immobilized plate (D2-37: 200,000-fold, D9-36: 1) , 00
0,000-fold, D35-42: 400,000-fold) and I-5-HRP (0.4 μg /
90 μL of a mixed solution (mL) and 0.05% Triton X-100 solution of 2,3,7,8-TCDD (0.4, 2, 10, 50, 250, 1250 pg / 10 μL) 1
0 μL was added to each well and left at 4 ° C. overnight. Hereinafter, the absorbance was measured in the same manner as described above. As shown in FIG. 4, the measurement limit of 2,3,7,8-TCDD by this ELISA was about 1 pg, and the sensitivity was improved about 100 times compared to the known method.

Comparison with Known Monoclonal Antibodies The aforementioned JP-A-63-14691 (US Pat. No. 4,795)
No. 8,807), monoclonal antibodies DD-1, DD-3, DD-4, and DD against dioxins.
Table 3 shows comparisons of the crossover rate with dioxins and isotype with DD-5 and DD-6.

[Table 3] As can be seen from Table 3, (i) the antibody of the present invention is a novel monoclonal antibody having a different crossover rate with (2), (8) and (10) as compared with those of the known references. (Ii) Since the antibody of the present invention has a different crossover rate between the known compound and the similar compound, the correlation between the measured value and the toxic equivalent differs in the toxicity evaluation. (Iii) The three monoclonal antibodies of the present invention are the most toxic of dioxins (1) 2,3,7,8-
(2) 1,2,3,7,8-PeCDD, (10) 2, which has the greatest affinity for TCDD and is also highly toxic
3,4,7,8-PeCDF and / or (8) 2
It has a high affinity for 3,7,8-TCDF. The ratio of dioxin homologues contained in biological samples such as breast milk has a small individual difference.
(Toxicity equivalent), TEQ in biological samples was determined by ELISA using this monoclonal antibody.
Can be monitored. In particular, D9-3
6 with the dioxin homolog is TEF (toxi
Since it is close to C equivalency factor, it may be applicable to samples having different homolog content ratios. In addition, since D35-42 recognizes only TCDD and PeCDD, there is a possibility that the two homologs can be measured even in environmental samples containing significantly different homologs. As described above, the three monoclonal antibodies of the present invention have the following characteristics. D2-37: 2,3,7,8-TCDD, which has the highest affinity for 2,3,7,8-TCDD and the lowest crossover rate with other homologues, making it the most toxic 2,3,7,8-TCDD among dioxins Can be measured specifically and with high sensitivity. D9-36: In addition to 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD and 2,
Since it has a high affinity for 3,4,7,8-PeCDF, it is possible to monitor the equivalent amount of dioxin toxicity in biological samples such as breast milk. In addition, since the crossover rate with other homologs also correlates well with TEF, it can be used for toxicity evaluation of environmental samples and the like. D35-42: Reacts specifically with 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD, so the two dioxin homologues in any sample can be obtained by simple pretreatment. The body can be measured.

[0024]

The monoclonal antibodies of the present invention can be used for identifying dioxins contained in a sample, and can be used for measuring the concentration of dioxins in a sample. Examples of the target sample include soil, water, fly ash, air, tissues of animals and plants, and biological samples such as human blood and breast milk. Among the dioxins, the monoclonal antibodies of the present invention are particularly toxic, 2,3,7,8,
A so-called group-specific monoclonal antibody that shows almost the same affinity for both target homologues as -TCDD and 1,2,3,7,8-PeCDD. A highly sensitive primary simple screening method for dioxin contamination and toxicity It is useful as a monitoring method. Since the monoclonal antibody of the present invention can be supplied in a large quantity and semi-permanently with a certain quality, it is extremely useful not only for establishing an immunoassay, but also for developing an immunoaffinity extraction method as a simple cleanup method for an analysis sample. is there. In the present invention, an ELISA method was established using the above monoclonal antibody. Since the measurement range of the standard solution was 1 to 1000 pg, a very small amount of dioxin could be quantified, and the TEQ equivalent exposure level of dioxin in breast milk was It is sufficiently practical for evaluation.

[0025]

[Brief description of the drawings]

FIG. 1 is a graph showing the toxicity equivalency coefficient and the crossover rate of a monoclonal antibody obtained from hybridoma D2-37.

FIG. 2 is a graph showing the toxic equivalent coefficient and the crossover rate of the monoclonal antibody obtained from hybridoma D9-36.

FIG. 3 is a graph showing the toxic equivalent coefficient and the crossover rate of the monoclonal antibody obtained from hybridoma D35-42.

FIG. 4 is an ELISA using the monoclonal antibody of the present invention.
4 is a graph showing the measurement sensitivity and range of dioxin (2,3,7,8, -TCDD) by Method A.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/38 G01N 33/53 G C12P 21/08 33/543 545A G01N 33/53 33/577 B 33 / 543 545 C12R 1:91) 33/577 (C12P 21/08 // (C12N 5/10 C12R 1:91) C12R 1:91) C12N 15/00 C (C12P 21/08 5/00 B C12R 1:91 (C12R 1:91) (72) Inventor Masayuki Sato 52-1 Yata, Shizuoka City, Shizuoka Prefecture Inside the Faculty of Pharmacy (72) Inventor Mitsunobu Okuyama 729-5 Ochiai, Hadano-shi, Kanagawa Food and Drug Safety Center Hadano Research Laboratory F-term (reference) 4B024 AA11 AA17 BA08 BA11 BA53 DA02 FA10 GA05 HA15 4 B050 CC02 DD02 DD09 LL03 4B064 AG27 CA10 CA20 CC24 DA13 DA16 4B065 AA92X AB05 AC10 CA25 CA46 CA54 4H045 AA11 AA20 AA30 BA30 BA51 CA42 DA75 EA50 FA72

Claims (24)

[Claims] 1. A monoclonal antibody which is specific for one or more dioxins, and which has a maximum affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin among the dioxins. Monoclonal antibody. 2. The monoclonal antibody according to claim 1, wherein the dioxins are polyhalogenated dibenzo-p-dioxins or polyhalogenated dibenzofurans. 3. The dioxins are polyhalogenated-p-dioxins of the following (1) to (7) or (8) to
3. The monoclonal antibody according to claim 1, which is the polyhalogenated dibenzofuran of (17). (1) 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378TCDD), (2) 1,2,3
7,8-pentachlorodibenzo-p-dioxin (1
2378 PeCDD), (3) 1, 2, 3, 4, 7, 8
-Hexachlorodibenzo-p-dioxin (1234)
78HxCDD), (4) 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (123678
HxCDD), (5) 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (123789Hx
CDD), (6) 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (12334678H
pCDD), (7) 1,2,3,4,6,7,8,9-
Octachlorodibenzo-p-dioxin (12346)
789OCDD), (8) 2,3,7,8-tetrachlorodibenzofuran (2378TCDF), (9) 1,
2,3,7,8-pentachlorodibenzofuran (123
78PeCDF), (10) 2,3,4,7,8-pentachlorodibenzofuran (23478PeCDF),
(11) 1,2,3,4,7,8-hexachlorodibenzofuran (123478HxCDF), (12) 1,
2,3,6,7,8-hexachlorodibenzofuran (1
23678HxCDF), (13) 1,2,3,7,
8,9-Hexachlorodibenzofuran (123789H
xCDF), (14) 2,3,4,6,7,8-hexachlorodibenzofuran (234678HxCDF),
(15) 1,2,3,4,6,7,8-heptachlorodibenzofuran (1234678HpCDF), (16)
1,2,3,4,7,8,9-heptachlorodibenzofuran (1234789HpCDF), (17) 1,2,2
3,4,6,7,8,9-octachlorodibenzofuran (123466789OCDF). 4. The monoclonal antibody according to claim 3, which further has a high affinity for (2), (8) or (10). (5) When the affinity with (1) is set to 1.0,
5. The monoclonal antibody according to claim 4, wherein the crossover rate with (2) and (10) is around 0.5. 6. When the affinity with (1) is set to 1.0,
The monoclonal antibody according to claim 4, wherein the crossover rate with (2) is about 0.2 and the crossover rate with (8) and (10) is about 0.3. 7. When the affinity with (1) is set to 1.0,
The monoclonal antibody according to claim 4, wherein the crossover rate with (2) is about 1.0. 8. The hybridoma D9-36 (FERM P)
The monoclonal antibody according to claim 5, which is produced from -18057). 9. A hybridoma D2-37 (FERM P)
The monoclonal antibody according to claim 6, which is produced from -18056). 10. A hybridoma D35-42 (FERM).
P-18058).
The monoclonal antibody as described. 11. An animal is immunized with a conjugate of a hapten and a protein represented by the following formula, antibody-producing cells are obtained from the animal, and the cells are fused with tumor cells to produce a plurality of hybridomas (mixed species). 4. The method according to claim 1, wherein at least one hybridoma producing an antibody that reacts with dioxins is selected from the plurality of hybridomas, and the antibodies produced from the hybridoma are recovered. 8. The method for producing the monoclonal antibody according to 4, 5, 6, or 7. Embedded image In the formula, R1 is NHCO (CH ₂ ) ₂ COOH, NHCO
(CH ₂ ) ₃ COOH, NHCO (CH ₂ ) ₄ COOH, N
HCOCH = CHCOOH, CH = CHCOOH, CH
ＣC (CH ₃ ) COOH, (CH ＝ CH) ₂ COOH, H
R2 is H, Cl, OCH ₂
COOH, O (CH ₂ ) ₃ COOH, O (CH ₂ ) ₄ COO
H, CH = CHCOOH, a functional group selected from (CH = CH) ₂ COOH, R3 is Cl or H,
R4 is Cl, CH ₃ or C (CH _{3) 3.} 12. The method for producing a monoclonal antibody according to claim 11, wherein the protein in the hapten-protein conjugate used in claim 11 is bovine serum albumin, ovalbumin, or keyhole limpet hemocyanin. 13. The hybridoma is D9-36 (FERM).
P-18057), D2-37 (FERM P-18)
056) or D35-42 (FERM P-1805)
The method for producing a monoclonal antibody according to claim 11, which is 8). 14. A hybridoma which produces the monoclonal antibody according to claim 1, 2, 3, 4, 5, 6, or 7 obtained in claim 11. 15. D9-36 (FERM P-1805)
7), D2-37 (FERM P-18056) or D
The hybridoma according to claim 14, which is 35-42 (FERM P-18058). 16. The method of claim 1, 2, 3, 4, 5, 6, 7,
A method for detecting and measuring dioxins by an immunoenzymatic assay using the monoclonal antibody according to 8, 9, or 10. 17. An enzyme-labeled hapten for use in a dioxin-type assay system, wherein the hapten represented by the following formula is bound to an enzyme. Embedded image In the formula, R1 is NHCO (CH ₂ ) ₂ COOH, NHCO
(CH ₂ ) ₃ COOH, NHCO (CH ₂ ) ₄ COOH, N
HCOCH = CHCOOH, CH = CHCOOH, CH
ＣC (CH ₃ ) COOH, (CH ＝ CH) ₂ COOH, H
R2 is H, Cl, OCH ₂
COOH, O (CH ₂ ) ₃ COOH, O (CH ₂ ) ₄ COO
H, CH = CHCOOH, a functional group selected from (CH = CH) ₂ COOH, R3 is Cl or H,
R4 is Cl, CH ₃ or C (CH _{3) 3.} 18. The method according to claim 18, wherein the enzyme is horseradish peroxidase, β
The enzyme-labeled hapten according to claim 17, which is -galactosidase. (1) (1) Claims 1, 2, 3, 4, 5,
A solid phase on which an antibody against the monoclonal antibody according to 6, 7, 8, 9 or 10 is immobilized, (2) the solid phase.
The monoclonal antibody according to 3, 4, 5, 6, 7, 8, 9 or 10, (3) a sample containing dioxins, and (4)
18. The method according to claim 16, wherein the enzyme-labeled hapten according to claim 17 is reacted, and 2) the concentration of dioxins in the sample is measured by measuring the activity of the labeled enzyme immobilized on the solid phase. For the detection and measurement of dioxins. 20) 1) (1) Claims 1, 2, 3, 4, 5,
A solid phase on which the monoclonal antibody according to 6, 7, 8, 9 or 10 is immobilized, (2) a sample containing dioxins and (3) a reaction with the enzyme-labeled hapten according to claim 17, and 2) a solid phase 17. The method for detecting and measuring dioxins according to claim 16, wherein the concentration of dioxins in the sample is measured by measuring the activity of the immobilized labeled enzyme. 21. A composition for use in purifying dioxin, wherein the composition is used for purifying dioxin.
Or a composition comprising the monoclonal antibody according to 10. 22. A composition for use in dioxin biochemical, immunological, functional or other research assays, wherein the composition comprises an effective amount of dioxin. , 8,
A composition comprising the monoclonal antibody according to 9 or 10. 23. A kit for measuring the presence or concentration of dioxins in a sample, which is a kit according to claim 1, 2, 3, 4, 5, or
The monoclonal antibody according to 6, 7, 8, 9 or 10,
A kit comprising the enzyme-labeled hapten according to claim 17. 24. The method of claim 1, 2, 3, 4, 5, 6, 7,
A sample containing the antibody against the monoclonal antibody according to claim 8, 9, or 10, the monoclonal antibody according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the enzyme-labeled hapten according to claim 17. A kit for measuring the presence or concentration of dioxins in water.