KR20110100589A - Anti-pcb monoclonal antibody, method for producing the antibody, hybridoma producing the antibody, and method for measuring pcb using the antibody - Google Patents

Abstract

(단, 상기 구조식 (1) 중, X 및 Y는 각각 0∼3의 정수를 나타내고, X와 Y의 합은 1∼6의 정수를 나타낸다). It is a manufacturing method of the anti-PCB monoclonal antibody which uses the isomer mixture of the biphenyl derivative represented by following formula (1) as a hapten.

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6).

Description

Anti-PCB monoclonal antibody, method for producing the same, hybridoma producing the antibody, and method for measuring PCC using the antibody USING THE ANTIBODY}

The present invention provides an anti-PCB monoclonal antibody used for the measurement of PCBs (polychlorinated biphenyls) contained in environmental, industrial products, biological samples, and the like, a method for producing the same, a hybridoma producing the antibody, and the anti-antibody. The present invention relates to a PCB measurement method using a PCB monoclonal antibody, in particular, having a cross-reactivity with two or more PCB homologues with different chlorine numbers, and among all homologues of monochlorinated PCB-10 chlorinated PCB, An anti-PCB monoclonal antibody having a high affinity, a method for producing the same, a hybridoma producing the antibody, and a PCB measurement method using the anti-PCB monoclonal antibody.

The problem of environmental pollution by chemicals is a serious problem to be solved as soon as it causes great anxiety for health problems and social life and also affects industrial activities. In particular, PCBs (polychlorinated biphenyls, sometimes simply referred to as "PCB") whose harmfulness to humans and the environment have been confirmed are used on earth scales such as they are often used in insulating oils, heat medium, and mechanical oils of capacitors and transformers. This is expanding, and since it is hardly decomposable, its persistence and bioaccumulation are problematic.

Regarding PCBs, in 1974, bans on the manufacture, import, and new use of the chemicals are stipulated by the Act on the Examination and Manufacturing of Chemical Substances, and in 2001, appropriate disposal of polychlorinated biphenyl wastes. The Special Measures Act (PCB Special Act) on the Promotion of the National Responsibilities Act was implemented, and by 2016, the aptitude treatment of all PCB waste was legally mandated. Accordingly, monitoring in PCBs waste treatment and measurement of PCB concentrations after treatment are important issues.

In addition, even in the case of the replacement insulating oil in the transformer which is not considered to contain PCBs, the mixing of the trace amount of the remaining trace PCBs is found, and the rapid analysis of a large amount of the replacement insulating oil and the PCBs concentration of the transformer main body is also urgently required.

As a conventional PCB measurement, for example, high resolution gas chromatography / mass spectrometry (HRGC / HRMS) or gas chromatography (GC-ECD) equipped with an electron trap detector has been used as a process method. However, these methods are expensive due to equipment investment, analytical consumables, etc., and also require a lot of time for data acquisition and analysis due to complicated necessity of clean-up operation and acquisition of analytical skills. There was a limit as a method of processing a large amount of samples, such as being limited to an analysis engine.

In contrast, immunoassay using specific antibodies is known as a quick and easy measurement method and has recently been applied to the field of environmental analysis.

Receptor binding assays and immunoassays have also attracted attention as rapid assays for dioxin, a PCB-like substance, and in particular, immunoassays utilize antibodies that specifically bind to a single target. By measuring a specific homologue amount that correlates with the toxic equivalents of dioxins, a method for quickly and easily obtaining the total toxic equivalents (dioxine amount) of dioxins having a large number of homologues has been developed.

Furthermore, since the PCB is poorly soluble in water and it is necessary to dissolve the PCB in the presence of an amphipathic solvent in preparing a sample for analysis, the anti-PCB antibody used in the immunological assay is the parent present in the sample. Resistance to solvents is required. On the other hand, as an anti-PCB antibody used for the said immunoassay, the antimonoclonal antibody which does not substantially reduce antigen recognition characteristic even in 50 (v / v)% organic solvent aqueous solution (for example, Unexamined-Japanese-Patent No. 2000-). 191699).

As described above, in an immunological assay using an antibody, a low cross-reactivity has been focused on measuring a specific substance by using an inherent property of an antibody called high specificity to a measurement target.

Based on this idea, high specificity anti-PCB antibodies with controlled cross reactivity are required for PCBs in which a large number of isomers exist. For example, 3,3 ', 5, which is one of coplanar PCBs, Antibodies having specificity for 5'-4 chloride biphenyl (PCB80) (see, for example, Japanese Patent Laid-Open No. 2005-247822) and the like have been proposed.

On the other hand, in the PCB analysis, since there is no thinking of "toxic equivalence" in dioxin analysis, it is important to detect and measure as many PCB homologs as possible as well in the immunoassay method. However, in a measurement system using a single antibody having high specificity, it is difficult to cover homologues, and there is a risk of deviation from analytical values by a process method, false negative (determination or overlook of false negatives), In addition, there is a concern that the reproducibility is low.

Therefore, a method of analyzing PCB concentration in insulating oil using a specificity of an antibody or a measurement system having two different specificities and mixing the resulting antibody has been proposed (for example, 25th International Symposium on Halogenated Environmental Organic Pollutant and POPs). , 2005, ID2387). In addition, in a device for detecting dioxin and PCBs, it is disclosed that an isomer of a target substance is selected according to a case of estimating toxicity and a case of estimating total amount, and using an antibody of the target substance. (See, for example, Japanese Patent Laid-Open No. 2004-138550).

On the other hand, a method of detecting dioxins at the isomer level using plural kinds of antibodies in which cross-reactivity between the isomers is recognized has been proposed (see, for example, Japanese Patent Laid-Open No. 2002-228660).

However, the object of measurement in the immunological measurement method using the anti-PCB antibody described in JP-A-2004-138550 and JP-A-2002-228660 is a coplanar PCB, and the antibody used therein is another goat. It is not satisfactory as an antibody for quantitatively mixing a group of homologues (for example, a PCB derived from Kanechlor and KK Mitsubishi Chemical Co., Ltd., Arochlor, etc., manufactured by Mitsubishi Chemical).

In addition, anti-PCB monoclonal antibodies which have a cross-reactivity to a plurality of PCB homologs having different chlorine numbers, prepared using an isomeric mixture of biphenyl derivatives as hapten have been proposed (for example, Japanese Patent Laid-Open No. 2008-). See 137902). However, this proposed anti-PCB monoclonal antibody has a problem in that the suitability to the label by fluorescence is high, but the suitability to the label by gold colloid is not satisfactory enough.

The label by fluorescence is high in measurement cost, and in order to perform PCB analysis at low cost, the development of anti-PCB monoclonal antibody which is more suitable for the label by gold colloid which is cheaper than the label by fluorescence is highly demanded. It is the present situation.

This invention solves the above-mentioned conventional problem, and makes it a subject to achieve the following objectives. That is, the present invention relates to a plurality of PCB homologs having a high abundance in a mixture of a plurality of PCB homologues having different chlorine numbers such as trichloride biphenyl, tetrachloride biphenyl, pentachloride biphenyl, hexachloride biphenyl, and bichloride biphenyl. Anti-PCB monoclonal antibody having a cross-reactivity and high affinity and excellent compatibility with a label by gold colloid, its preparation method, hybridoma producing the antibody and anti-PCB monoclonal antibody It is an object to provide a PCB measurement method using.

MEANS TO SOLVE THE PROBLEM In order to solve the said subject, as a result of earnest examination by the present inventors, immunity mixture of the biphenyl derivative represented by following structural formula (1) is made into hapten, and the compound which couple | bonded this hapten with carrier protein as an immunogen is used for immunity. It was found that anti-PCB monoclonal antibodies having cross-reactivity with two or more PCB homologs having different chlorine numbers and high affinity and excellent compatibility with gold colloids can be obtained. The invention has been completed.

However, in said structural formula (1), X and Y respectively represent the integer of 0-3, and the sum of X and Y represents the integer of 1-6.

This invention is based on the said knowledge by this inventor, The means for solving the said subjects are as follows. In other words,

<1> A method for producing an anti-PCB monoclonal antibody, wherein an isomeric mixture of biphenyl derivatives represented by the following structural formula (1) is used as a hapten.

However, in said structural formula (1), X and Y respectively represent the integer of 0-3, and the sum of X and Y represents the integer of 1-6.

<2> a step of immunizing an animal using the compound represented by the following structural formula (2) obtained from the biphenyl derivative represented by the following structural formula (1) as an immunogen,

Recovering the antibody-producing cells of the immunized animal;

Fusing the antibody-producing cells and myeloma cells to obtain hybridomas,

Incubating the hybridoma

It is a manufacturing method of the anti-PCB monoclonal antibody as described in said <1> containing.

However, in said structural formula (1), X and Y respectively represent the integer of 0-3, and the sum of X and Y represents the integer of 1-6.

In the structural formula (2), X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

<3> In the following structural formula (2), Z is labeled with a labeling substance of any one of an enzyme, a fluorescent dye, a radioisotope, a magnetic substance, a pigment-encapsulated polymer, a rare earth, a coenzyme, a gold colloid, and a gold atom cluster. It is a manufacturing method of the anti-PCB monoclonal antibody as described in <2>.

In the structural formula (2), X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

<4> The anti-PCB according to any one of <1> to <3>, including the step of screening a hybridoma cell line using a compound represented by at least one of Structural Formula (1) and Structural Formula (2) It is a method for producing a monoclonal antibody.

However, in said structural formula (1), X and Y respectively represent the integer of 0-3, and the sum of X and Y represents the integer of 1-6.

In the structural formula (2), X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

<5> An anti-PCB monoclonal produced by the method for producing an anti-PCB monoclonal antibody according to any one of <1> to <4>, wherein the chlorine number has cross reactivity to two or more PCB homologues different from each other. Ronald antibody.

<6> Among the homologs of monochlorinated PCBs to 10-chlorinated PCBs, the affinity for trichlorinated PCBs to 7-chlorinated PCB homologs is higher than that of monochlorinated PCBs to 2-chlorinated PCB homologs and to 8-chlorinated PCBs to 10-chlorinated PCB homologs. It is a high anti-PCB monoclonal antibody as described in the above <5>.

<7> A hybridoma characterized by producing the anti-PCB monoclonal antibody according to the above <5> or <6>.

<8> The hybridoma according to the above <7>, wherein the accession number is FERM BP-11303.

To the sample to be measured, the anti-PCB monoclonal antibody according to the above <5> or <6> is added,

(1) a PCB in the sample under test bound to the anti-PCB monoclonal antibody, and

(2) A PCB measurement method comprising the step of detecting any of the anti-PCB monoclonal antibodies that are not bound to the PCB in the sample to be measured.

To the sample to be measured, an anti-PCB monoclonal antibody according to the above <5> or <6> and a compound represented by any one of the following structural formulas (1) and (2) as a PCB competition material are added, PCB detection method comprising the step of detecting the competitive substance bound to the anti-PCB monoclonal antibody.

However, in said structural formula (1), X and Y respectively represent the integer of 0-3, and the sum of X and Y represents the integer of 1-6.

In the structural formula (2), X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

<11> By using the carrier which added the anti-PCB monoclonal antibody as described in said <5> or <6> to the sample to be measured, and immobilized the compound represented by following structural formula (2) as a PCB competition material, And a step of competing a PCB competing material with a PCB in the sample to be measured to detect the anti-PCB monoclonal antibody that is not bound to the PCB in the sample to be measured.

In the structural formula (2), X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

<12> The anti-PCB monoclonal antibody is a PCB measurement method according to any one of <9> to <11>, wherein the antibody is labeled with a gold colloid.

According to the present invention, a plurality of PCB homologues having a high abundance in a mixture of a plurality of PCB homologues having different chlorine numbers such as trichloride biphenyl, tetrachloride biphenyl, pentachloride biphenyl, hexachloride biphenyl, and bichloride biphenyl are different. An anti-PCB monoclonal antibody which has reactivity and has high affinity and excellent compatibility with a label by gold colloid, a method for producing the same, a hybridoma producing the antibody and the anti-PCB monoclonal antibody The PCB measurement method used can be provided.

1 is a diagram showing the composition of PCB homologues of each Arochlor (Source: PCB treatment technology guidebook based on the new waste treatment method), edited by the Industrial Waste Disposal Business Promotion Foundation, Published by Kyosei Corporation, August 2005 1 Daily).
Fig. 2 is a graph showing the absorbance of each of the gold colloid-labeled antibodies in Test Example 2.
3 is a graph showing the sensitivity of each gold colloid-labeled antibody to PCB in Test Example 3. FIG.

(Anti-PCB monoclonal antibody)

<Method for producing anti-PCB monoclonal antibody>

Isomer mixtures of the biphenyl derivatives represented by formula (1) (hapten)-

The anti-PCB monoclonal antibody of the present invention is an anti-PCB monoclonal of the present invention using a hapten containing an isomeric mixture of the biphenyl derivative represented by the following structural formula (1) (compound represented by the following structural formula (1)) It is produced by a method for producing a ronal antibody.

However, in said structural formula (1), X and Y respectively represent the integer of 0-3, and the sum of X and Y represents the integer of 1-6.

On the other hand, there is no restriction | limiting in particular as an isomer in the isomeric mixture of the biphenyl derivative represented by the said Formula (1), According to the objective, it can select suitably, For example, trichlorinated biphenyl thru | or hexachlorinated biphenyl etc. mainly Etc. can be mentioned. Among these, bichlorinated biphenyl and pentachlorinated biphenyl are advantageous when the PCB in insulating oil is made into the measurement object.

There is no restriction | limiting in particular as a mixing ratio of these isomers in the isomer mixture of the said biphenyl derivative, According to the objective, it can select suitably, For example, when manufacturing the isomer mixture of the said biphenyl derivative as mentioned later, 5- From the point of view of the addition amount of the chlorinating agent to the phenyl ether and the orientation of the chlorine atom, the following compound 1A, compound 1B and compound 1C are considered to be the main isomers.

There is no restriction | limiting in particular as a manufacturing method of the biphenyl derivative represented by the said Formula (1), According to the objective, it can select suitably, For example, it can manufacture according to the following reaction formula.

In the following reaction formula, 3-phenylphenol is used as a starting material, and then first reacted with 6-bromohexanoic acid ethyl ester, followed by chlorination and hydrolysis, whereby the biphenyl derivative represented by the above structural formula (1) To manufacture.

In the above reaction formula, X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, Et represents an ethyl group, and Ph represents a phenyl group.

-A compound represented by the structural formula (2)-

Since the compound represented by the structural formula (1) alone does not have antigenicity, in order to produce an anti-PCB monoclonal antibody, the compound represented by the following structural formula (2) is represented by the following structural formula (2) It is preferable to prepare an anti-PCB monoclonal antibody by preparing an isomeric mixture of biphenyl derivatives (hereinafter sometimes referred to as "conjugate") and using this as an immunogen (antigen).

In the structural formula (2), X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

In the structural formula (2), the protein represented by Z (hereinafter sometimes referred to as "carrier protein") has immunogenicity, and the compound represented by the structural formula (1) and the spacer portion (-O- ( CH ₂₎ which can be connected via the ₅ -CONH-), it is not particularly limited, and may be selected according to the purpose, for example, key fissurellidae (keyhole-limpet) (KLH), ovalbumin-derived hemocyanin ( OVA), bovine serum albumin (BSA), human serum albumin, rabbit serum albumin, goat serum albumin, bovine gamma globulin, myoglobin, multiple antigen peptides (MAP), and the like. Among these, bovine serum albumin (BSA) and hemocyanin (KLH) derived from a keyhole hatch are preferable.

There is no restriction | limiting in particular as a preparation method of the compound (conjugate) represented by the said Formula (2), It can prepare by a well-known synthetic method, For example, as shown by following Reaction Formula, it is represented by the said Formula (1) Carboxyl groups of biphenyl derivatives are converted to active ester derivatives by condensation reactions such as N-hydroxysuccinimide, and then the amino esters of the active ester derivatives and carrier proteins are bonded in a usual manner. have.

In the above reaction formula, X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

The obtained compound (conjugate) represented by Structural Formula (2) is preferably purified by replacing the reaction solution with a neutral solution such as physiological phosphate buffer by a known method such as gel chromatography.

In the structural formula (2), the protein represented by Z may be labeled with a labeling substance. There is no restriction | limiting in particular as said labeling material, It can select suitably from a well-known labeling material, For example, an enzyme, a fluorescent dye, a radioisotope, a magnetic substance, a pigment encapsulation polymer, a rare earth, a coenzyme, a gold colloid, a gold atom cluster etc. Can be mentioned.

Preparation of Anti-PCB Monoclonal Antibodies

The anti-PCB monoclonal antibody is a step of immunizing an animal using, for example, a compound represented by the following structural formula (2) obtained from the biphenyl derivative represented by the structural formula (1) as an immunogen (hereinafter referred to as an "immune process"). Sometimes), a step of recovering the antibody-producing cells of the immunized animal (hereinafter may be referred to as an "antibody-producing cell recovery step"), and the hybrid of the antibody-producing cells and myeloma cells. By a production method including a step of obtaining a cutting board (hereinafter, referred to as a "hybridoma acquisition step") and a step of culturing the hybridoma (hereinafter, referred to as a "culture step"). Are manufactured.

The method for producing the anti-PCB monoclonal antibody further comprises the step of screening a hybridoma cell line using a compound represented by at least one of Structural Formula (1) and Structural Formula (2) (hereinafter, "screening process"). It may be referred to as))).

The method for producing a monoclonal antibody can be carried out according to, for example, Kohler G, Milstein C, Nature, 256, 495-7 (1975) and the like.

Immune Process

The said immune process is a process of immunizing an animal using the compound represented by the said structural formula (2) obtained from the biphenyl derivative represented by the said structural formula (1) as an immunogen.

There is no restriction | limiting in particular as said animal to immunize, Although it can select suitably according to the objective, Mammals, such as a mouse, a rat, a rabbit, a goat, are preferable.

There is no restriction | limiting in particular in the immunization method of the said animal, It can select suitably from a well-known method, For example, the emulsion obtained by emulsifying the said immunogen and an adjuvant is administered by subcutaneous, intravenous, intraperitoneal injection, etc. The method etc. are mentioned.

There is no restriction | limiting in particular in the administration method of the said immunogen, According to the objective, it can select suitably, For example, in the case of a mouse, 0.01-15 times / 0.5 g / horse three times at intervals of about two to three weeks. It is preferable to administer once.

There is no restriction | limiting in particular as said adjuvant, According to the objective, it can select suitably, For example, Freund's complete adjuvant, incomplete adjuvant, BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS) Alum adjuvant, silica adjuvant, etc. are mentioned, It is preferable to select and use the optimal thing from the relationship, such as antibody inducing ability and load to an animal.

Antibody-producing cell recovery process

The antibody producing cell recovery step is a step of recovering antibody producing cells of the immunized animal.

The antibody producing cell recovery process is achieved by recovering antibody producing cells from the immunized animal, eg, several days after the last administration of the immunogen.

There is no restriction | limiting in particular as said antibody producing cell, According to the objective, it can select suitably, For example, a spleen, B cell derived from a lymph node, etc. are mentioned.

Hybridoma Acquisition Process

The hybridoma acquisition step is a step of fusing the antibody-producing cells and myeloma cells to obtain hybridomas.

The hybridomas can be obtained by fusing the antibody producing cells with myeloma cells and then screening for fusion cells producing the desired antibody.

There is no restriction | limiting in particular as said myeloma cell, A well-known thing can be selected suitably. For example, a mouse, rat, human-derived cell line, etc. can be selected, and if it is a mouse-derived cell, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag41, Sp210-Ag14, FO, NSO / U, MPC- Examples of rat-derived cells such as 11, MPC11-X45-GTG1.7 and S194 / 5XXO.Bul include R210.RCY3, Y3-Ag.1.2.3, IR983F, and 4B210.

There is no restriction | limiting in particular in the fusion method of the said antibody producing cell and myeloma cell, A well-known method can be selected suitably, For example, a Sendai virus method, a polyethyleneglycol method, a protoplast method, etc. are mentioned. Among these, the polyethyleneglycol method is preferable at the point that the cytotoxicity is comparatively small, and fusion operation is also simple.

There is no restriction | limiting in particular and the method of screening the fusion cell which produces the said desired antibody can be suitably selected according to the objective, For example, radioimmunoassay (RIA), Enzyme immunoassay (EIA, ELISA) ), Fluoroimmunoassay (FIA), and the like.

In the said screening method, it is preferable to use the compound represented by at least any one of the said structural formula (1) and the said structural formula (2).

For example, the conjugate represented by the structural formula (2) is solidified, and the supernatant of the culture supernatant is measured by reacting the culture supernatant of the fusion cell (hybridoma). It can be selected as an antibody production well. In addition, the antibody coexists with a PCB replacement compound (e.g., a biphenyl derivative represented by the above structural formula (1)), PCBs, or the like at the time of measurement, to select wells that are highly reactive with these coexisting substances, and to target the target by limiting dilution. Monoclonalization of anti-PCB antibody producing hybridomas is possible.

In addition, in the conjugate represented by the above formula (2), the protein represented by Z is labeled with a labeling substance, which coexists during the measurement of the antibody titer of the anti-PCB antibody producing well to perform a direct competition reaction. It is also possible to screen anti-PCB antibody producing hybridomas.

Since the PCB to be measured for the anti-PCB antibody is poorly soluble in water, it is dissolved in an amphiphilic solvent and used for the measurement. For this reason, it is preferable that the said antiPCB antibody has tolerance to the said amphipathic solvent which exists in a sample. Therefore, screening of the anti-PCB antibody-producing hybridoma is also preferably performed in the presence of the amphiphilic solvent.

DMSO, methanol, ethanol, etc. are mentioned as said amphiphilic solution.

There is no restriction | limiting in particular and the density | concentration of the said amphiphilic solution used for the said screening can be suitably selected according to the objective, 50 mass% or less is preferable, 30 mass% or less is more preferable, 5 mass%-20 mass % Is particularly preferred.

There is no restriction | limiting in particular and the sensor used for the screening of the said anti-PCB antibody production hybridoma can be suitably selected according to the objective, For example, SPR (surface plasmon) sensor, QCM (crystal oscillator) sensor, optical sensor, Immobilized the biphenyl derivative represented by the structural formula (1) or the conjugate of the biphenyl derivative represented by the structural formula (2) and the carrier protein to the sensor functional part of a transducer such as an electrochemical sensor. Can be mentioned.

Moreover, what was made to react by previously adding PCB etc. to the culture supernatant of the said fusion cell can also be screened by contacting the said sensor functional part. In this case, anti-PCB antibody-producing hybridomas having high affinity which can be applied to PCB measurement by various transducers can be selected by evaluating the amount of antibody bound to the functional part.

<Hybridoma>

As the anti-PCB antibody-producing hybridoma, there is no particular limitation as long as the anti-PCB monoclonal antibody of the present invention which recognizes the PCB is produced and can be appropriately selected according to the purpose. The OD8B11 strains as shown in Examples described later are specifically mentioned.

The OD8B11 strain is a hybridoma produced by using the compound represented by the following structural formula (2), and was assigned to the Patent Biological Deposit Center of the Industrial Technology Research Institute, an independent administrative corporation, on August 6, 2009, under the accession number FERM P-21833 in Japan. It has been deposited internationally and thereafter has been deposited internationally as accession number FERM BP-11303.

In the structural formula (2), X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein.

-Cultivation Process-

The culture step is a step of culturing the hybridoma.

By the culture step, the anti-PCB monoclonal antibody can be obtained.

There is no restriction | limiting in particular in the method of the said culture process, According to the objective, it can select suitably, For example, the said anti-PCB antibody producing hybridoma cell line is previously made into pristane (2,6,10,14- tetramethylpentadecane). And culturing by implantation in the abdominal cavity of the mouse to which the administration.

After 10 days to 14 days after implantation into the abdominal cavity of the mouse, ascites containing anti-PCB monoclonal antibodies can be recovered to obtain the anti-PCB monoclonal antibodies from the ascites obtained.

In addition, the hybridomas can be cultured using a medium (for example, DMEM containing 10% fetal bovine serum), and the supernatant of the obtained culture can be used as an anti-PCB monoclonal antibody solution.

It is preferable that the said anti-PCB monoclonal antibody is refine | purified.

The method for purifying the anti-PCB monoclonal antibody from the anti-PCB antibody-producing hybridoma cell line culture supernatant obtained from the ascites or fetal bovine serum-containing medium is not particularly limited and may be appropriately selected from known methods according to the purpose. And may be purified by, for example, ultrafiltration, salting out, ion exchange chromatography, gel filtration chromatography, affinity chromatography, and the like.

<Evaluation of Anti-PCB Monoclonal Antibodies>

The anti-PCB monoclonal antibody of the present invention is produced by the method for producing the anti-PCB monoclonal antibody of the present invention, and has a cross-reactivity to two or more PCB homologs having different goat numbers. Moreover, it is preferable that the said anti-PCB monoclonal antibody has high affinity with respect to trichlorinated PCB-7 chlorinated PCB homolog among all the homologs of monochlorinated PCB-10 chlorinated PCB.

As an example of the high abundance ratio of the said trichlorinated PCB-7 chlorinated PCB homolog, Kanechlor (KC-300, KC-400, KC-500, KC-600: Kanega Fuchi Chemical Co., Ltd. make) contained in an oily sample, and Aroclor (Aroclor. 1242, Aroclor. 1248, Aroclor. 1254, Aroclor. 1260, Aroclor. 1232: Mitsubishi Monsanto (now Mitsubishi Chemical)).

In addition, since the anti-PCB monoclonal antibody of the present invention has high suitability for labeling by gold colloid (see Test Example 2 described later), the PCB can be measured at low cost.

Affinity to the PCB of the anti-PCB monoclonal antibody can be assessed, for example, by antigen concentration (IC ₅₀ value) that inhibits the binding of the anti-PCB monoclonal antibody to the PCB by 50%. In addition, cross-reactivity of the monoclonal antibody to the monoclonal PCB is, for example chlorine can not obtain the respective IC ₅₀ values for the PCB congeners or more of the other two species, can be evaluated by obtaining the ratio thereof.

Method of calculating the IC ₅₀ value is not particularly limited, and may be selected according to the purpose, for example, obtain and apply the approximate expression represented by the measurement data by a fluorescence spectrophotometer or the relative absorbances measuring the formula (1) This can be interpreted.

In Formula (1), y represents relative fluorescence intensity or absorbance when the value of fluorescence intensity or relative absorbance when no antigen is added is 100%, x represents antigen concentration, and P1 and P2 are approximations. Represents a parameter.

The measured data were converted into relative values using 100% of the fluorescence intensity or absorbance when no antigen was added, and then optimized P1 and P2 using graphing software (for example, Origin version 6.0 (manufactured by OriginLab)). Determine.

The approximation formula thus obtained is taken as the binding curve between the antibody and the antigen, and the value of x (= P1) when y = 50% is set as the IC ₅₀ value.

On the other hand, it is known that the IC ₅₀ value and the equilibrium dissociation constant (Kd) almost coincide with the IC ₅₀ value when the antibody concentration is sufficiently small (e.g., less than 1/10).

In addition, as the method of calculating the IC ₅₀ value, the measurement data by the fluorescence spectrophotometer or a relative absorbance measuring instrument, by applying a formula represented by the following formula (7) may be analyzed by determining the relative response (%). When the relative response is 50%, the value of IC ₅₀ is used.

However, in said Formula (7), R _G shows a relative response (%), I _CZ shows the measured value of the transmitted light of the measuring cell before the feeding, when no antigen is added, and I _CM shows the feeding liquid when the antigen is not added It represents an amount of transmitted light of the measuring cell after the measurement, _SZ I denotes a transmitted light amount measured in the measuring cell before the liquid-delivery of the antigen when added, I _SM represents the amount of transmitted light measurement of the measuring cell after the liquid feed when the antigen was added.

Specificity for PCBs with different goat numbers of the anti-PCB monoclonal antibody is, for example, canechlor (KC-300, KC-400, KC-500, KC-600) or arochlor (Ar-1242, Ar-1248, Ar -1254, Ar-1260, Ar-1232, Ar-1221) and the like can be evaluated as a sample to be measured.

Although said Canechlor and Arochlor contain PCB of different chlorine numbers in the ratio shown in following Table 1 and FIG. 1, respectively, For example, the anti-PCB monoclonal antibody of this invention is carried out by 2 or more types of PCB homologs with different chlorine numbers. It has cross-reactivity and has high affinity to trichlorinated to 7-chlorinated PCB homologues among all homochlorinated to 10-chlorinated PCB homologues, and among them, other canechlor and arochlor (KC-300) except Ar-1221. , KC-400, KC-500, KC-600, Ar-1242, Ar-1248, Ar-1254, Ar-1260 and Ar-1232).

(Source: Takasuga Takumi et al., `` Detailed Analysis Method for All Isomers of Polychlorinated Biphenyls (PCBs) Using Various Clean-up Methods and HRGC / HRMS, '' Environmental Chemistry, Vol. 5, No. 3, pp. 647-675, 1995)

(PCB measurement method)

The PCB measuring method of the present invention is not particularly limited as long as it is a method using the anti-PCB monoclonal antibody of the present invention, and can be appropriately selected according to the purpose. For example, the anti-PCB monoclonal antibody may be used in the sample. Methods of immunologically detecting and quantifying PCB, specifically, immunoassay (RIA), enzyme immunoassay (EIA, ELISA), fluorescence immunoassay (FIA), immunochromatography, chemiluminescent enzyme immunoassay (CLEIA) , Immunobinding (TIA), latex immunization (LTIA), binding equilibrium exclusion, and the like.

Examples of the PCB measurement method include a non-competition method, a contention method (indirect contention method or direct contention method), and a method of excluding bond equilibrium, and specifically, the following methods (I) to (III) may be mentioned. have.

(I) the anti-PCB monoclonal antibody of the present invention is added to the sample to be measured,

(1) a PCB in the sample under test bound to the anti-PCB monoclonal antibody, and

(2) A PCB measurement method comprising the step of detecting any of the anti-PCB monoclonal antibodies that are not bound to the PCB in the sample to be measured.

(II) The anti-PCB monoclonal antibody of the present invention and a compound represented by any one of the above formulas (1) and (2) are added as PCB competing substances to the sample to be measured, and the antiPCB monoclonal PCB measuring method comprising the step of detecting the competitive substance bound to the antibody.

(III) The PCB competition material and the blood sample are added to the sample to be measured by using a carrier obtained by adding the anti-PCB monoclonal antibody of the present invention and immobilizing the compound represented by the structural formula (2) as a PCB competition material. Competing PCB in a measurement sample and detecting the said anti-PCB monoclonal antibody which is not couple | bonded with PCB in the said sample to be measured.

Hereinafter, an example of the indirect contention method and the combined equilibrium exclusion method will be described in more detail.

In the indirect competition method and the binding equilibrium exclusion method, as an antigen (hereinafter sometimes referred to as "immobilized antigen") immobilized on a carrier, a compound containing a biphenyl derivative represented by the above formula (1), and The compound represented by the said structural formula (2), etc. are mentioned.

The indirect competition method and the binding equilibrium exclusion method immobilize the immobilized antigen on the carrier to block a portion of the carrier to which the immobilized antigen is not bound with a blocking agent. Subsequently, the sample to be measured and the anti-PCB monoclonal antibody are added to the carrier, and the immobilized antigen and the PCB in the sample to be tested are competitively reacted with the anti-PCB monoclonal antibody in the indirect competition method. In equilibrium exclusion, binding equilibrium exclusion is carried out.

After the anti-PCB monoclonal antibody which did not bind to the immobilization carrier was washed away, an antibody labeled as a secondary antibody was added to bind the anti-PCB monoclonal antibody to which the immobilized antigen was bound and washed. The color development or luminescence of is measured. In addition, the anti-PCB monoclonal antibody may be directly labeled and used without using a secondary antibody. With respect to the obtained absorbance and light intensity, the reduction rate for the value when no sample is added is obtained, and this is determined as the inhibition rate, using the calibration curve obtained in advance from the inhibition rate when the PCB of a known concentration is added. PCB concentration can be calculated.

As the carrier, a carrier having a structure identical or similar to a part of the immobilized antigen structure in a molecule may be used. There is no restriction | limiting in particular as a support | carrier which has the structure similar to or a part of the structure of the said immobilization antigen, According to the objective, it can select suitably, For example, the carrier described in Unexamined-Japanese-Patent No. 2007-206062, etc. are mentioned. have.

There is no restriction | limiting in particular as said label | substrate, According to the objective, it can select suitably, For example, using peroxide as an enzyme, hydrogen peroxide for a board | substrate, o-phenylenediamine or tetramethylbenzidine as a coloring agent, and as an enzyme When alkaline phosphatase is used and p-nitrophenyl phosphoric acid is used as a substrate, color development can be measured by absorbance. On the other hand, when the reaction product of the substrate is fluorescence or labeled using a fluorescent substance, fluorescence can be measured using a fluorophotometer or the like.

In the case of using a chemiluminescent material, light emission can be measured by a chemiluminescence meter or the like, and in the case of using a gold colloid, the transmitted light intensity or the like can be measured by a transmitted light quantity meter or the like.

The anti-PCB monoclonal antibody of the present invention is preferably used as the label in view of excellent suitability to the label by the gold colloid and the PCB can be measured at low cost.

There is no restriction | limiting in particular in the labeling method by the said gold colloid, A well-known method can be selected suitably, For example, the immunohistochemistry by the Yokota Sadaki "IMUNOGOLD method colloidal gold" It can carry out by the method of Kuwatanishoten (1992).

There is no restriction | limiting in particular as said transmitted light quantity measuring instrument, Although it can select suitably according to the objective, The relative absorbance measuring apparatus of Unexamined-Japanese-Patent No. 2006-215013 is preferable.

There is no restriction | limiting in particular as said sample to be measured, According to the objective, it can select suitably, For example, the sample which carries out liquid-liquid extraction of PCB in a test substance with polar solvents, such as dimethyl sulfoxide (DMSO) and an alcoholic solvent, etc. And liquid-liquid extraction of the PCB in the test substance with a polar solvent, solid phase extraction of the polar solvent including the extracted PCB with an adsorbent, and conversion of components adsorbed on the adsorbent to a desired solvent. The sample etc. which add the low polar solvent, such as normal hexane, cyclohexane, and toluene, to the sample and the said test substance and extract the PCB in the said test substance with a hydrophilic solvent etc. are mentioned.

In the liquid-liquid extraction, the mixing ratio (mass ratio) of the test substance and the polar solvent is not particularly limited and can be appropriately selected according to the purpose, (the test substance): (the polar solvent) = 1 1: 1: 1: 10 are preferable.

There is no restriction | limiting in particular if it is a substance which may contain a PCB (PCB contaminant) as said test substance, According to the objective, it can select suitably, For example, oil other than insulating oil, insulating oil (for example, vegetable oil, animal oil, Synthetic oil, mineral oil, etc.), factory drainage, soil, discharge gas, waste, such as a member used in the processing work of animal blood or body fluid, insulating oil, etc. are mentioned. Among these, insulating oil is mentioned suitably.

As said insulating oil, the insulating oil after carrying out PCB decomposition process, such as a dechlorination process, can also be mentioned suitably.

The concentration of the polar solvent in the sample to be measured is not particularly limited as long as the affinity to the PCB of the anti-PCB monoclonal antibody is not significantly degraded, and may be appropriately selected depending on the purpose. 2.0 mass% is preferable, and 10 mass%-20 mass% are more preferable.

Since the anti-PCB monoclonal antibody of the present invention has an affinity for PCBs in which a plurality of chlorine contained in canechlor or arochlor is substituted, and also has excellent suitability for labeling by gold colloid, an environmental sample As a result, PCBs contained in industrial products and biological samples can be analyzed quickly and reproducibly at low cost. The PCB measurement method using the anti-PCB monoclonal antibody is suitable as a rapid monitoring or analysis technique of PCBs.

Moreover, in the system using transducers, such as an SPR (surface plasmon) sensor, a QCM (crystal oscillator) sensor, an optical sensor, and an electrochemical sensor, the anti-PCB monoclonal antibody of this invention is provided in a sensor functional part. Alternatively, the biphenyl derivative and the like can be immobilized and used as an immune sensor.

Furthermore, the carrier on which the anti-PCB monoclonal antibody of the present invention is immobilized can be applied to efficient concentration or removal of PCB.

[Example]

Hereinafter, although the Example of this invention is described, this invention is not limited to these Examples at all, It is assumed that it contains a normal modification and a formula for these made in the art.

Example 1 Preparation of Anti-PCB Monoclonal Antibodies

(1) Preparation of Isomer Mixtures and Conjugates of Biphenyl Derivatives

According to the following reaction formula, a conjugate formed by preparing a biphenyl derivative (compound 3 in the following scheme) as a hapten in which a spacer molecule is introduced into 3-phenylphenol, and further binding to a carrier protein (compound 5 in the following scheme) Was synthesized.

In the above reaction formula, X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z is BSA (bovine serum albumin) or KLH (keyhole hatch-derived head). Mocyanine).

Dissolve 5.0 g (29.4 mmol) of 3-phenylphenol in 70 mL of acetone, add 8.11 g (58.8 mmol) of potassium carbonate, and then add 7.87 g (35.3 mmol) of 6-bromohexanoic acid ethyl ester to warm to 60 DEG C. Stirred for time. After the reaction was allowed to cool to room temperature, the insolubles were separated by filtration, and the solvent was distilled off from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (eluted with ethyl acetate: hexane = 1:20) to obtain 8.55 g of compound 1 in the above scheme.

To 1.00 g (5.88 mmol) of the compound 1, 10 mL of sulfuryl chloride and 50 mg of diphenyl sulfide (catalyst) were added, followed by stirring at room temperature for 20 hours, at 50 ° C for 20 hours, and at 70 ° C for 10 hours. It was. After cooling to room temperature, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluted with ethyl acetate: hexane = 1:20) to obtain 1.02 g of Compound 2 in the above reaction scheme as a chlorinated isomer mixture.

1.02 g of the compound 2 was dissolved in 4 mL of tetrahydrofuran, an aqueous solution of 0.5 g of potassium hydroxide dissolved in 4 mL of distilled water was added, and the mixture was stirred at room temperature for 50 hours. After the reaction, the solvent was distilled off under reduced pressure. The residue was dissolved in distilled water, the aqueous solution was made acidic with concentrated hydrochloric acid, and the precipitated solid was aggregated. This solid was purified by silica gel column chromatography (eluted with ethyl acetate: hexane = 1: 2) to obtain 0.72 g of Compound 3 in the above reaction scheme as a chlorinated isomer mixture.

0.72 g of the compound 3 and 0.291 g (2.53 mmol) of N-hydroxysuccinimide were dissolved in 12 mL of anhydrous dimethoxyethane. Dicyclohexylcarbodiimide 0.521g (2.53 mmol) was added to this reaction liquid under ice cooling, it heated up to room temperature and stirred for 24 hours. After filtering the reaction solution, the solvent was distilled off from the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (eluted with ethyl acetate: carbon tetrachloride = 1: 5) to obtain 0.60 g of Compound 4 in the above reaction scheme as a chlorinated isomer mixture.

0.60 g of Compound 4 was dissolved in dimethylsulfoxide (DMSO), added dropwise to BSA solution or KLH solution dissolved in 50 mM phosphate buffer (pH 7.4) under ice-cooled stirring, and stirred at room temperature overnight. The reaction solution was added to a Sephadex column equilibrated with PBS (-), developed with the same buffer, the carrier protein was separated, and the conjugate (Compound 5 in the above scheme) was purified.

(2) immunity of mice

Mice were immunized using the obtained conjugate as an immunogen.

Compound 5 and TiterMax Gold (manufactured by CytRx Corporation) as an adjuvant were mixed in a 1: 1 dose to sufficiently emulsify, and the obtained emulsion was administered 200 μL in the abdominal cavity of BALB / c mice (6 to 8 weeks old, female). The mice were then immunosensitized.

Additional immunization was carried out at intervals of about 2 to 3 weeks, blood was collected from the tail vein after one week from the additional immunization, and antibody titers in blood were measured by ELISA to observe the change in antibody titers.

(3) production of hybridomas

Final immunization was performed by administering the immunogen into the mouse tail vein where high antibody production to the immunogen was recognized in the blood. After 3 days from the final immunization, spleens were extracted from the immunized mice, splenocytes were prepared, myeloma cells (NS0) in the logarithmic growth phase and splenocytes were mixed 1: 5, and polyethylene glycol (PEG Cell fusion). The cells after fusion are suspended in 20% FCS-containing RPMI 1640 medium with HAT (hypoxanthine, aminopterin and thymidine) addition, and the spleen cell number is 1 × 10 ⁵ cells / well to 2 × 10 ⁵ cells / well. Seeds were sown in 96-well culture plates and incubated at 37 ° C. and 5% CO ₂ .

(4) Screening and Cloning of Antibody Production Hybridomas

After 7 days to 10 days after cell fusion, antibody titer was measured by the following method using the culture supernatant of the well in which the propagation of the clone was seen, and screening and cloning were performed.

-Preparation of Antigen Beads-

In agarose beads (manufactured by Pharmacia), a complex (antigen complex: the following compound X) of a dichlorophenol derivative and BSA (bovine serum albumin) was immobilized. Specifically, after immobilizing about 0.1 mg of the antigen complex to about 1 mL of agarose beads, the surface of the beads was blocked using 1 mL of 10 mg / mL BSA solution.

However, in the said compound X, "protein" represents BSA.

Screening and Cloning

Screening and cloning were performed as follows using KinExA3000 (manufactured by Sapidyne) as a fluorescence photometer.

First, 90 μL of the culture supernatant was recovered from each compartment of the 96 well plate, and one row (12 wells) was combined into one line of sampling tubes. Further, one plate (8 rows) was combined with one line of sampling tubes at 200 µL from the sampling tube.

The screening was first performed on the culture supernatant which was first combined for each plate, and the positive plate was subsequently screened every row.

Specific screening methods are as follows.

The culture supernatant was applied to the antigen beads for 120 seconds (0.5 mL total) at a flow rate of 0.25 mL / min. Subsequently, the antigen beads were washed with a Phosphate buffer saline (PBS) containing 0.1% BSA for 30 seconds at a flow rate of 0.25 mL / min, and then a secondary antibody (goat anti-mouse IgG antibody, manufactured by ImmunoResearch Laboratories) 5 nM was operated for 96 seconds (0.4 mL total) at a flow rate of 0.25 mL / min. Finally, washing of the antigen beads with PBS containing 0.1% BSA was performed for 30 seconds at a flow rate of 0.25 mL / min, followed by 90 seconds at a flow rate of 1.5 mL / min. A fluorescent substance (Cy5) binds to the secondary antibody, and the presence or absence of the target antibody in the culture supernatant is determined from the intensity of fluorescence remaining on the antigen beads after washing. No target antibody).

By judging the fluorescence intensity, the culture supernatant of the column determined to have the target antibody was divided into two lines of test tubes, DMSO was added to one line to a final concentration of 2% (control), and four types of PCB equivalent mixtures were added to the other line. (Kanechlor (KC-300, KC-400, KC-500, KC-600: manufactured by GL Science Corporation)) was added to a final concentration of 100 ppb. Then, the ratio (B / B0) of the fluorescence intensity (B) of the four PCB equivalent mixtures to the fluorescence intensity (BO) of the control is calculated to generate a heat for producing an antibody having high binding activity to the PCB. Selected.

The fluorescence intensity was measured as follows using KinExA3000 (manufactured by Sapidyne).

The antigen beads were applied to the control and the four PCB equivalent mixtures for 120 seconds (0.5 mL total) at a flow rate of 0.25 mL / min. Subsequently, the antigen beads were washed with PBS (Phosphate buffer saline) containing 0.1% BSA for 30 seconds at a flow rate of 0.25 mL / min, and then a secondary antibody (goat anti-mouse IgG antibody, manufactured by ImmunoResearch Laboratories). 5 nM was operated for 96 seconds (0.4 mL total) at a flow rate of 0.25 mL / min. Finally, washing of the antigen beads with PBS containing 0.1% BSA was performed for 30 seconds at a flow rate of 0.25 mL / min, followed by 90 seconds at a flow rate of 1.5 mL / min. Then, the intensity of fluorescence remaining on the antigen beads after washing was measured.

In addition, with respect to the heat producing the antibody having high binding activity to the PCB, the culture supernatant was recovered again in each compartment (well) on the day after the heat producing the antibody producing the high binding activity to the PCB was selected. ), And hybridomas growing in the positive compartments were cloned by colony formation with methylcellulose medium.

(5) Selection of antibody-producing clones in which chlorine water responds equally to other PCB homologues

Among the hybridomas selected in the above (4), hybridomas that were cloned one or more times were also selected using a fluorophotometer (KinExA3000 (manufactured by Sapidyne)) in the following manner.

The culture supernatant of the expanded culture clone was diluted appropriately with PBS containing 0.1% of BSA so that the signal (fluorescence intensity) was 2V, divided into 5 lines, and DMSO was added to the final line at 2% of final concentration. The remaining four lines were added with KC-300, KC-400, KC-500 and KC-600 to a final concentration of 10 ppb.

Then, the ratio (B / B0) of the fluorescence intensity (B) of each PCB-containing liquid to the fluorescence intensity (B0) of the control is calculated to obtain an antibody-producing clone showing a high affinity in common with each PCB. It was.

The fluorescence intensity was measured by the following formula using KinExA3000 (manufactured by Sapidyne).

For each of the controls and each PCB containing solution, the antigen beads were operated for 120 seconds (0.5 mL total) at a flow rate of 0.25 mL / min. Subsequently, the antigen beads were washed with PBS (Phosphate buffer saline) containing 0.1% BSA for 30 seconds at a flow rate of 0.25 mL / min, and then a secondary antibody (goat anti-mouse IgG antibody, manufactured by ImmunoResearch Laboratories). 5 nM was operated for 96 seconds (0.4 mL total) at a flow rate of 0.25 mL / min. Finally, washing of the antigen beads with PBS containing 0.1% BSA was performed for 30 seconds at a flow rate of 0.25 mL / min, followed by 90 seconds at a flow rate of 1.5 mL / min. Then, the intensity of fluorescence remaining on the antigen beads after washing was measured.

By the above method, the stable hybridoma which produces anti-PCB monoclonal antibody which has affinity for several PCB homologs with different goat water was obtained, and these were made into OD8B11 strain. The OD8B11 main cells formed colonies derived from a single cell using methyl cellulose medium, and the colonies were transferred to a liquid medium (20% FBS-added RPMI, GIBCO Co., Ltd.) to continue the culture. On the other hand, on August 6, 2009, the hybridoma OD8B11 stock was deposited in Japan with the Patent Biodeposit Center of the Industrial Technology Research Institute, an independent administrative corporation, and acquired FERM P-21833 as an accession number. It is transferred and FERM BP-11303 is acquired as an accession number.

(6) Preparation of anti-PCB monoclonal antibody

The colony of hybridoma OD8B11 strain established in (5) was transferred to HT (hypoxanthine, thymidine) medium based on 20% FBS-added RPMI medium on a 24-well plate, and cultured for 2 days, and then a part of the culture solution was Transfer to 40 mL square flask containing 10 mL of the above HT medium, incubate until just before confluent, and purify the antibody secreted by the OD8B11 strain by affinity chromatography using Protein A from the culture supernatant, An anti-PCB monoclonal antibody (hereinafter referred to as "OD8B11 antibody") was obtained.

(7) Mass preparation and purification of monoclonal antibodies

In the abdominal cavity of BALB / c mice over 8 weeks of age, 0.5 mL of pristane [2,6,10,14-tetramethylpentadecane] (manufactured by Wako Pure Chemical Industries, Ltd.) was administered and bred for two to three weeks.

The pre-logarithmic number of the monoclonal antibody producing cultures of hybridomas OD8B11 with anti-PCB monoclonal placed maintained in growth phase, after removal of the culture supernatant by centrifugation, FBS screening (沈査) as included RPMI 1640 1 × 10 ⁶ cells /0.5 mL Cell solution was prepared so as to be.

This cell solution was transplanted into the abdominal cavity of BALB / c mice pre-pristan administration, and ascites leaked into the abdominal cavity 10 to 10 days later was recovered from the abdomen with a syringe equipped with a 25 G syringe.

The collected ascites was filtered using a pore size 0.22 μm diameter filter, and the obtained filtrate was purified by affinity chromatography using a protein G-Sepharose column (manufactured by Amersham Bionsiens). Raw antibody (OD8B11 antibody) was obtained.

(8) Isotype Analysis

As the solidifying antigen, a compound represented by the following structural formula (2) was used, and the isotype of the anti-PCB monoclonal antibody was analyzed using a mouse typer kit (manufactured by BIO-RAD). .

As a result, the H chain of the anti-PCB monoclonal antibody produced by the anti-PCB monoclonal antibody-producing hybridoma OD8B11 was IgG1 and the L chain was κ.

(Comparative Example 1)

Established hybridoma WR3F6 strain disclosed in Japanese Patent Application Laid-Open No. 2008-137902 (October 26, 2006, was deposited with the Institute of Industrial and Biotechnology Research Institute of Patents and Biotechnology and acquired FERM P-21073 as an accession number). Anti-PCB monoclonal antibodies (hereinafter referred to as "WR3F6 antibodies") produced in the above were prepared and purified in large quantities in the same manner as the OD8B11 antibody.

(Test Example 1: Sensitivity characteristics for each PCB of the OD8B11 antibody)

Using OD8B11 antibody (anti-PCB monoclonal antibody) of Example 1 and WR3F6 antibody (anti-PCB monoclonal antibody) of Comparative Example 1, canechlor (KC-300, KC-400, KC-500, KC-600) and arochlor (Ar-1242, Ar-1248, Ar-1254, Ar-1260, Ar-1232, Ar-1221: manufactured by Wako Pure Chemical Industries, Ltd.) in the following formula Evaluated.

-Preparation of antibody binding carrier-

Agarose beads (manufactured by Amersham Bioscience) in which the N-hydroxysuccinimidyl ester group was introduced were covalently bound to the compound X (conjugate between the dichlorophenol derivative and the carrier protein) and blocked with BSA solution. It was set as the carrier for antibody binding.

Measurement of fluorescence intensity

For each of the OD8B11 antibody of Example 1 and the WR3F6 antibody of Comparative Example 1, an antibody concentration was set at a final concentration of 0.5 nM, and each was prepared to have a final concentration of 0.05 ng / mL to 20 ng / mL. 300, KC-400, KC-500, KC-600), and then reacted with a solution of arochlor (Ar-1242, Ar-1248, Ar-1254, Ar-1260, Ar-1232, Ar-1221), The carrier for antibody binding was operated at a flow rate of 0.25 mL / min.

Thereafter, a secondary antibody (goat anti-mouse IgG antibody, ImmunoResearch Laboratories) labeled with the fluorescent substance Cy5 (registered trademark) was allowed to act on the carrier for binding the antibody at a flow rate of 0.25 mL / min.

Subsequently, PBS (-) containing 5% DMSO, 0.1 w / v% BSA was passed through at a flow rate of 0.25 mL / min to exclude nonspecific binding to the carrier, and then the fluorescence intensity was measured.

Calculation of IC ₅₀ Values

The measured fluorescence intensity was applied to the approximation formula of the following formula (1) to calculate the IC ₅₀ value (value of x when y = 50%). The results are shown in Table 2.

In Formula (1), y represents relative fluorescence intensity when the value of fluorescence intensity when no antigen (PCB) is added to the sample is 100%, x represents antigen concentration, and P1 and P2 are approximations. Represents a parameter.

Cross Reaction Rate

The said cross reaction rate was computed by following formula (2)-formula (5). The results are shown in Table 2.

OD8B11 antibody

When the antigen is Canechlor

% Cross-reaction rate of OD8B11 antibody = {(IC ₅₀ value when antigen is KC-400) / (IC ₅₀ value of sample)} × 100 Formula (2)

% Cross-reaction rate of WR3F6 antibody = {(IC ₅₀ value when antigen is KC-400) / (IC ₅₀ value of sample)} × 100 Formula (3)

When the antigen is Arochlor

% Cross-reaction rate of OD8B11 antibody = {(IC ₅₀ value when antigen is Ar1254) / (IC ₅₀ value of sample)} × 100 Formula (4)

% Cross-reaction of WR3F6 antibody = {(IC ₅₀ value when the antigen is Ar1254) / (IC ₅₀ value of the sample)} × 100 Formula (5)

From the results of Table 2, the OD8B11 antibody is a 3-7 chlorinated PCB homolog (KC-300, KC-400, KC-500, KC-600, Ar-1242, Ar-1248, Ar-1254, Ar-1260, Ar -1232, Ar-1221), it has been found that the cross-reaction rate almost the same as the WR3F6 antibody.

In addition, the OD8B11 antibody is a 3-7 chlorinated PCB homolog (KC-300, KC-400, KC-500, KC-600, Ar-1242, Ar-1248, Ar-1254, Ar-1260, Ar-1232, Ar). -1221), it has a higher affinity than the WR3F6 antibody, and showed a high detection sensitivity.

(Test Example 2: Gold Colloidal Labeling Suitability of OD8B11 Antibody)

Using the OD8B11 antibody of Example 1 and the WR3F6 antibody of Comparative Example 1, gold colloid label suitability was evaluated in the following manner.

-Preparation of Gold Colloidal Solution-

A gold colloidal solution was prepared according to a general citric acid method (Immunohistochemistry with immunogold colloidal gold, Kuwatanishoten (1992) by Sadaki Yokota).

In detail, tetrachloro gold (III) acid tetrahydrate (made by Wako Pure Chemical Industries, Ltd.) was diluted with ultrapure water, and adjusted to the density | concentration of 0.01%. Then, the solution was boiled and 1.5 mL of sodium citrate diluted to 0.1% was added and the mixture was boiled for 10 minutes. Thereafter, the solution was cooled to obtain a gold colloidal solution.

-Labeling of antibodies-

See the immunohistochemistry with immunogold colloidal gold by Yokota Sadaki, Kuwatanishoten (1992). The OD8B11 antibody of Example 1 and the WR3F6 antibody of Comparative Example 1 were prepared. Labeled.

In detail, the prepared gold colloidal solution was maintained at 20 ° C, and the pH was adjusted to 9.15 using potassium carbonate (200 mM). Next, 0.6 mg of the antibody was added and allowed to react for 2 minutes. Subsequently, 10 mL of a 10% bovine serum albumin (BSA) solution was added to block. Finally, the mixture was centrifuged at 25 ° C. and 9,000 rpm for 25 minutes, and 10 mL of the precipitate was collected to obtain a gold colloid-labeled antibody.

- How to measure -

On the basis of the PCB biosensor method described in Naoya Omura et al., "Sensing using biological functions (7)", Electric Power Research Institute report V08053 (2009), the measurement was made in the following manner.

Preparation of Antibody Solution

The antibody solution containing the gold colloid labeled antibody was appropriately diluted with PBS containing 1% (w / w) bovine serum albumin (PBS-BSA).

To the DMSO solution, the antibody solution and PBS-BSA solution were finally added so that the DMSO concentration was 2%. On the other hand, the concentration of the gold colloid-labeled antibody in the added solution is prepared to be 500 pM, 400 pM, 300 pM, 200 pM, 100 pM in the OD8B11 antibody, and 700 pM, 560 pM, in the WR3F6 antibody. It was prepared to be 420 pM, 280 pM, 140 pM.

-Measuring device-

As the measuring device, the relative absorbance meter and the cell for measurement described in Experimental Example 2 of JP-A-2006-215013 were used.

As the diffuse reflection medium housed in the measurement cell, the cellulose (carrier for measuring immune response) described in paragraphs [0078] to [0079] of JP-A-2007-206062 was used.

-- Measure --

First, the measurement cell containing the diffuse reflection medium was fixed to the cell holder of the relative absorbance meter, and irradiated with light from the light emitting portion, and the amount of transmitted light L2 (transmitted light before liquid feeding) was measured.

Thereafter, 4 mL of the prepared antibody solution was transferred to the same detection cell at a flow rate of 8.5 mL / min. Subsequently, 1 mL of PBS-BSA was fed at a flow rate of 8.5 mL / min to wash the measuring cell. Thereafter, the measurement cell was centrifuged at 7,000 rpm for 5 minutes, dehydrated, and air dried for 30 minutes.

The airy measurement cell was again fixed to the cell holder of the relative absorbance meter and irradiated with light from the light emitting portion to measure the amount of transmitted light L1 (the amount of transmitted light after feeding).

Based on Lambert-Beer's law, absorbance A was calculated by applying L1 and L2 to the following formula (6). The results are shown in Fig.

A = -log (L1 / L2) equation (6)

In Figure 2, the axis of abscissas is antibody concentration (nM), and the axis of ordinates is absorbance. In addition, in FIG. 2, linear is a linear function calculated | required from the least square method, and R <^2> shows a correlation coefficient.

From the results in FIG. 2, it was found that at the same antibody concentration, the OD8B11 antibody had higher absorbance than the WR3F6 antibody. As a result, it was found that the OD8B11 antibody was more suitable for gold colloid label than the WR3F6 antibody.

Test Example 3: Sensitivity to PCB by Gold Colloid-labeled Antibody

In Test Example 2, the sensitivity of the gold colloid-labeled OD8B11 antibody and WR3F6 antibody to PCB was measured in the same manner as in Test Example 2 except that the concentration of the following gold colloid-labeled antibody and PCB were used. Was tested.

Preparation of Gold Colloid Labeled Antibody-Containing Solution-

The concentration of the gold colloid-labeled OD8B11 antibody in the solution at the time of antigen antibody reaction was set to 500 pM so that the gold colloid-labeled OD8B11 antibody and the WR3F6 antibody showed the same absorbance. 560 pM.

Preparation of PCB-containing solution

As said PCB containing solution, PCB (KC-300, KC-400, KC-500, KC-600 which mixed equal amount) was added to DMSO solution, and it was set as PCB containing solution.

On the other hand, PCB concentration in the solution at the time of antigen antibody reaction was 0.2 ppb, 1 ppb, 4 ppb, 20 ppb, 100 ppb.

-Antigen antibody response-

To the PCB containing solution, the antibody solution and the PBS-BSA solution were finally added so that the DMSO concentration was 2%.

The added solution was allowed to stand for 30 minutes or more in an air-conditioned room (25 ° C ± 5 ° C) to carry out antigen antibody reaction.

Using the prepared gold colloid-labeled antibody and PCB, the amount of transmitted light was measured by a relative absorbance meter in the same manner as in Test Example 2, and the relative response (%) was calculated by the following equation (7). It was. The results are shown in FIG.

In the above formula (7), R _G represents a relative response (%), I _CZ represents a measured amount of transmitted light of the measurement cell before the transfer when no antigen (PCB) is added, and I _CM represents an antigen (PCB). represents an amount of transmitted light measured in the measuring cell after the liquid feeding in the case that are not subject, I _SZ denotes the amount of transmitted light measured in the measurement cell before the liquid-delivery when added to the antigen (PCB), I _SM is a liquid feed when added to the antigen (PCB) The measured amount of transmitted light of the subsequent measuring cell is shown.

In Figure 3, the axis of abscissas is PCB concentration (ppb), and the axis of ordinates is relative response (%).

From the results in FIG. 3, it was confirmed that the OD8B11 antibody responded to a PCB at a lower concentration than the WR3F6 antibody. In addition, the IC ₅₀ value at this time was 4.89 ppb of the WR3F6 antigen, whereas the OD8B11 antibody was 2.96 ppb, and the sensitivity improvement of about 1.7-fold was recognized.

The anti-PCB monoclonal antibody (OD8B11 antibody) of the present invention is a mixture of a plurality of PCB homologues having different chlorine numbers, specifically, canechlore contained in an oily sample without a decrease in binding to PCBs in the presence of a polar solvent. KC-300, KC-400, KC-500, KC-600: Kanega Fuchigagaku Kogyo Co., Ltd. and Aroclor (Aroclor. 1242, Aroclor. 1248, Aroclor. 1254, Aroclor. 1260, Aroclor. 1232: Mitsubishi It is cross-reactive with a plurality of PCB homologues with high abundance in Monsanto (now Mitsubishi Chemical), and has a high affinity for trichlorinated PCB-7 chlorinated PCB homolog with high abundance among all homologues of 1-10 chlorinated PCB. Inspection objects that may contain PCBs, such as insulating oils whose composition is not yet known, can be detected and quantified with high sensitivity with minimal error.

In addition, the anti-PCB monoclonal antibody of the present invention has excellent suitability for labeling by gold colloids, and the PCB measurement method using the anti-PCB monoclonal antibody has a low cost of PCB residual concentration after waste treatment and PCB in environment. The concentration can be checked.

Patent Biodeposit Center, Korea Institute of Industrial Technology FERMBP11303 20090806

Claims (11)

A method for producing an anti-PCB monoclonal antibody, characterized in that an isomeric mixture of biphenyl derivatives represented by the following structural formula (1) is used as a hapten:

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6). The method of claim 1,
Immunizing an animal using a compound represented by the following structural formula (2) obtained from a biphenyl derivative represented by the following structural formula (1) as an immunogen, and
Recovering the antibody-producing cells of the immunized animal;
Fusing the antibody-producing cells and myeloma cells to obtain hybridomas,
Incubating the hybridoma
Method for producing an anti-PCB monoclonal antibody comprising:

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6).

(Wherein X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein). The method for producing an anti-PCB monoclonal antibody according to claim 1, comprising the step of screening a hybridoma cell line using a compound represented by at least one of the following structural formulas (1) and (2):

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6).

(Wherein X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein). An anti-PCB monoclonal antibody prepared by using an isomeric mixture of biphenyl derivatives represented by the following structural formula (1) as a hapten, and characterized by having cross-reactivity to two or more PCB homologs having different chlorine numbers. PCB Monoclonal Antibodies:

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6). The method according to claim 4, wherein the affinity for the trichlorinated PCB to the 7-chlorinated PCB homologue is higher than the affinity for the monochlorinated PCB to the 2-chlorinated PCB homolog and the 8-chlorinated PCB to the 10-chlorinated PCB homologue. High affinity antiPCB monoclonal antibody. An anti-PCB monoclonal that is prepared by a method for producing an anti-PCB monoclonal antibody using an isomeric mixture of biphenyl derivatives represented by the following structural formula (1) as a hapten, and having cross-reactivity to two or more PCB homologs having different chlorine numbers. Hybridomas that produce antibodies:

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6). The hybridoma according to claim 6, wherein the accession number is FERM BP-11303. The sample to be measured is prepared by a method for producing an anti-PCB monoclonal antibody using an isomeric mixture of a biphenyl derivative represented by the following structural formula (1) as a hapten, and having cross-reactivity with two or more PCB homologs having different chlorine numbers. By adding anti-PCB monoclonal antibody,
(1) a PCB in the sample under test bound to the anti-PCB monoclonal antibody, and
(2) the anti-PCB monoclonal antibody that is not bound to the PCB in the sample under test
PCB measuring method comprising the step of detecting any of:

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6). The sample to be measured is prepared by a method for producing an anti-PCB monoclonal antibody using an isomeric mixture of a biphenyl derivative represented by the following structural formula (1) as a hapten, and having cross-reactivity with two or more PCB homologs having different chlorine numbers. Adding an anti-PCB monoclonal antibody and a compound represented by any one of the following structural formulas (1) and (2) as a PCB competitive material to detect the competitive substance bound to the anti-PCB monoclonal antibody; PCB measurement method characterized in that:

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6).

(Wherein X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein). The sample to be measured is prepared by a method for producing an anti-PCB monoclonal antibody using an isomeric mixture of a biphenyl derivative represented by the following structural formula (1) as a hapten, and having cross-reactivity with two or more PCB homologs having different chlorine numbers. By adding an anti-PCB monoclonal antibody and using a carrier immobilized with the compound represented by the following structural formula (2) as a PCB competing material, the PCB competing material and the PCB in the sample to be measured are subjected to competition. PCB measuring method comprising the step of detecting the anti-PCB monoclonal antibody that is not bound to the PCB in:

(However, in the structural formula (1), X and Y each represent an integer of 0 to 3, and the sum of X and Y represents an integer of 1 to 6).

(Wherein X and Y each represent an integer of 0 to 3, the sum of X and Y represents an integer of 1 to 6, and Z represents a protein). The method of claim 8, wherein the anti-PCB monoclonal antibody is labeled with a gold colloid.

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