JP5096023B2 - Anti-PCB monoclonal antibody capable of measuring a plurality of PCB homologues having different chlorine numbers, hybridoma producing said antibody, and PCB measuring method using said antibody - Google Patents

Description

  The present invention relates to an anti-PCB monoclonal antibody used for monitoring a PCB decomposition treatment process of PCB waste, a hybridoma producing the antibody, and a PCB measurement method using the anti-PCB monoclonal antibody, and in particular, two kinds having different chlorine numbers. An anti-PCB monoclonal antibody having cross-reactivity with the above PCB homologues and having a high affinity for a PCB homologue having 1 to 2 chlorine atoms, a hybridoma producing the antibody, and the anti-PCB monoclonal antibody It relates to the PCB measurement method used.

The problem of environmental pollution caused by chemical substances is a serious problem that should be solved as soon as possible because it causes great concerns about health problems and social life, and also affects industrial activities. In particular, PCBs that have been confirmed to be harmful to the human body and the environment (polychlorinated biphenyls, hereinafter may be simply referred to as “PCB”) have been widely used in the past as insulation oil, heat medium, and machine oil for capacitors and transformers. Environmental pollution is expanding on a global scale, and its persistence and bioconcentration are problematic because it is difficult to decompose.
As for PCBs, in 1974 the “Law on the Examination and Production of Chemical Substances” was prohibited in principle for production, import, and new use. In 2001, “Appropriate disposal of polychlorinated biphenyl waste” The Special Measures Law on the Promotion of Disposal (PCB Special Measures Law) was enforced, and by 2016, all PCB wastes were legally mandated. Accordingly, monitoring in the process of processing PCBs waste and measurement of PCB concentration after processing are important issues.
In addition, in the alternative insulating oil in the transformer that was supposed not to contain PCBs, it was revealed that the trace amount of PCBs remaining remained, so that a rapid analysis of the PCBs concentration in a large amount of alternative insulating oil and transformer body Is also an urgent need.

For the measurement of conventional PCBs, for example, high resolution gas chromatography / mass spectrometry (HRGC / HRMS) and gas chromatography with electron capture detector (GC-ECD) have been used as official methods. However, these methods are costly for capital investment, analysis consumables, etc., and require a lot of time for data acquisition and analysis due to the need for complicated clean-up operations and analysts' technical skill. There is a problem, and there is a limit as a method for processing a large amount of samples, such as analysis work being limited to a specific analysis organization.
On the other hand, an immunological measurement method using a specific antibody is known as a rapid and simple measurement method, and has recently been applied to the field of environmental analysis.

  Receptor binding assays and immunoassays are also attracting attention as rapid analysis methods for dioxins that are PCB-analogous substances. In particular, immunoassays have the property that antibodies specifically bind to a single analyte. A method to quickly and easily determine the sum of the toxic equivalents (dioxin content) of dioxins containing a large number of congeners by measuring the amount of a specific homologue that correlates with the toxic equivalents of dioxins. Has been developed.

  Furthermore, since PCB is poorly soluble in water, and it is necessary to dissolve PCB in the presence of an amphiphilic solvent when preparing a sample for analysis, as an anti-PCB antibody used in the immunoassay method, Requires resistance to the amphiphilic solvent present in the sample. On the other hand, as an anti-PCB antibody used in the immunological assay, for example, an anti-monoclonal antibody whose antigen recognition property does not substantially decrease even in a 50 (v / v)% organic solvent aqueous solution (see Patent Document 1). Etc. have been proposed.

In this way, immunoassays using antibodies focus on measuring specific substances by utilizing the intrinsic nature of antibodies, which have low cross-reactivity and high specificity for the analyte. I came.
Based on such an idea, a PCB having a large number of isomers is required to have a highly specific anti-PCB antibody with controlled cross-reactivity, for example, 3,3 which is one of coplanar PCBs. An antibody having specificity for ', 5,5'-bichlorinated biphenyl (PCB80) (see Patent Document 2) has been proposed.

  On the other hand, in PCB analysis, since there is no concept of “toxic equivalent” in dioxin analysis, it is important that many PCB congeners can be detected and measured as widely as possible even in immunoassays. However, in a measurement system using a single antibody with high specificity, it is difficult to cover homologues, deviation from analysis values by official methods, risk of false negative (false negative judgment or oversight), Furthermore, there are concerns about low reproducibility.

Therefore, a method has been proposed in which two types of antibodies having different specificities or measurement systems are mixed and the PCB concentration in the insulating oil is analyzed using the specificity of the resulting antibody (Non-patent Document 1). reference). Further, in a low molecular weight substance detection device for detecting dioxins and PCBs, it is possible to select an isomer of a target substance according to a case of estimating toxicity and a case of estimating a total amount, and use an antibody of the target substance It is disclosed (see Patent Document 3).
On the other hand, a method of detecting dioxins at the isomer level using a plurality of types of antibodies in which cross-reactivity is recognized between isomers has been proposed (see Patent Document 4).

  However, the measurement target in the immunoassay method using the anti-PCB antibody described in Patent Documents 3 and 4 is a coplanar PCB, and the antibody used therein is a homologous mixture of different chlorine groups (for example, bell It is not satisfactory as an antibody for quantitatively quantifying Kanechlor manufactured by Sakai Chemical Industry and PCB derived from Arrochlor manufactured by Mitsubishi Monsanto (currently Mitsubishi Chemical) with high sensitivity.

  Therefore, it can be used for monitoring in the process of processing PCBs waste that removes chlorine from PCBs with chlorine added to the biphenyl skeleton and decomposes into harmless biphenyl, and for measurement of PCB concentration after treatment. There is a need for antibodies that can quantify different PCB congeners with high sensitivity. In particular, in the PCB decomposition treatment process, chlorine is removed from the PCB one by one, and finally decomposed into biphenyl via a PCB homologue having 1 to 2 chlorine atoms. There is a need for an antibody that can quantitate urine with high sensitivity. However, a PCB having a high affinity for a PCB homologue having 1 to 2 chlorine atoms and containing a large amount of biphenyl monochloride and biphenyl dichloride without reducing the binding property to the PCB in the presence of a polar solvent (for example, As for the anti-PCB monoclonal antibody capable of quantifying with high sensitivity PCB (derived from Arrochlor 1221 (Ar1221) manufactured by Mitsubishi Monsanto (currently Mitsubishi Chemical), etc.), a satisfactory one has not been provided, and improvement is required. Is the current situation.

JP 2000-191699 A JP 2005-247822 A JP 2004-138550 A JP 2002-228660 A 25th International Symposium on Halogenated Environmental Organic Pollutant and POPs, 2005, ID2387

  An object of the present invention is to solve the above-described problems and achieve the following objects. That is, the present invention is a mixture of a plurality of PCB homologues having different chlorine numbers, specifically, Kanechlor (KC300, KC400, KC500, KC600: produced by Kaneka Chemical Co., Ltd.) contained in an oil sample, and Aroclor ( Ar1221: An anti-PCB monoclonal antibody having cross-reactivity with a plurality of PCB homologues having a high abundance ratio in Mitsubishi Monsanto (currently Mitsubishi Chemical), and having a high affinity for a PCB homologue having 1 to 2 chlorine atoms An object of the present invention is to provide a hybridoma producing the antibody and a PCB measurement method using the anti-PCB monoclonal antibody.

Means for solving the problems are as follows. That is,
<1> The relationship between the equilibrium dissociation constant K1 for a PCB homologue having 1 to 4 chlorine atoms and the equilibrium dissociation constant K2 for a PCB homologue having 5 to 7 chlorine atoms satisfies the following equation: K1 <K2. Anti-PCB monoclonal antibody.
<2> The anti-PCB monoclonal antibody according to <1>, wherein the equilibrium dissociation constant K2 is 4.0 ppb or more.
<3> The anti-PCB monoclonal antibody according to <2>, wherein the equilibrium dissociation constant K1 is 3.0 ppb or less.
<4> The anti-PCB monoclonal antibody according to any one of <1> to <3>, wherein the equilibrium dissociation constant K3 for the PCB homologue having 1 to 2 chlorine atoms is 2.0 to 3.0 ppb.
<5> An animal is immunized with a compound represented by the following general formula (II) obtained from a biphenyl derivative represented by the following general formula (I) as an immunogen, and antibody-producing cells of the immunized animal are collected. The anti-PCB monoclonal antibody according to any one of <1> to <4>, obtained by culturing a hybridoma obtained by fusing the antibody-producing cells and myeloma cells.
However, in said general formula (I) and (II), n represents the integer of 1, X and Y represent the integer of 0-3, respectively, and the sum of X and Y represents the integer of 1-6.

However, in said general formula (II), n represents the integer of 1, X and Y represent the integer of 0-3, respectively, the sum of X and Y is 1-6, Z represents protein.
<6> The anti-PCB monoclonal antibody according to <5>, obtained by culturing a screened hybridoma cell line using the compound represented by any one of the general formula (I) and the general formula (II) It is.
<7> A hybridoma characterized by producing the anti-PCB monoclonal antibody according to any one of <1> to <6>.
<8> The hybridoma according to <7>, which is a hybridoma having an accession number of FERM P-21148.
<9> To the sample to be measured, add the anti-PCB monoclonal antibody according to any one of <1> to <6>,
(1) detecting any of PCB in the sample to be measured bound to the anti-PCB monoclonal antibody, and (2) the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured. Is a PCB measurement method characterized by
<10> An anti-PCB monoclonal antibody according to any one of <1> to <6>, and a compound represented by any one of the general formula (I) and the general formula (II) Is added as a PCB competitor, and the competitive substance bound to the anti-PCB monoclonal antibody is detected.
<11> A carrier obtained by adding the anti-PCB monoclonal antibody according to any one of <1> to <6> to a sample to be measured and immobilizing the compound represented by the general formula (II) as a PCB competitor And measuring the anti-PCB monoclonal antibody not bound to the PCB in the sample to be measured by competing the PCB competitor with the PCB in the sample to be measured. Is the method.
<12> The PCB measurement method according to any one of <9> to <11>, wherein the PCB homologue having 1 to 2 chlorine atoms is quantified.

  According to the present invention, a mixture of a plurality of PCB congeners having different chlorine numbers, specifically, Kanechlor (KC300, KC400, KC500, KC600: manufactured by Kaneka Chemical Co., Ltd.) contained in an oil sample, and Aroclor (Ar1221). : An anti-PCB monoclonal antibody having cross-reactivity to a plurality of PCB congeners having a high abundance ratio in Mitsubishi Monsanto (currently Mitsubishi Chemical), and having a high affinity for a PCB congener having 1 to 2 chlorine atoms, It is possible to provide a hybridoma producing the antibody and a PCB measurement method using the anti-PCB monoclonal antibody.

(Anti-PCB monoclonal antibody)
<Method for producing anti-PCB monoclonal antibody>
The anti-PCB monoclonal antibody of the present invention is obtained from a biphenyl derivative using a hapten composed of an isomer mixture of biphenyl derivatives represented by the following general formula (I) (compound represented by the following general formula (I)). In addition, an animal is immunized with a compound represented by the following general formula (II) as an immunogen, antibody-producing cells of the immunized animal are collected, and a hybridoma obtained by fusing the antibody-producing cells and myeloma cells is cultured. Can be obtained.

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

However, in said general formula (II), n represents the integer of 1, X and Y represent the integer of 0-3, respectively, the sum of X and Y is 1-6, Z represents protein.

  The biphenyl derivative represented by the general formula (I) can be produced, for example, according to the following reaction formula.

However, in said reaction formula, R represents a linear or branched lower alkyl group having 1 to 4 carbon atoms, provided that in the general formula (I), n represents an integer of 1, X And Y each represents an integer of 0 to 3, and the sum of X and Y is 1 to 6, preferably 4 to 6.

  Since the compound represented by the general formula (I) alone has no antigenicity, in order to produce an anti-PCB monoclonal antibody, a compound represented by the following general formula (II) (the following general formula (II) It is preferable to prepare the above-mentioned anti-PCB monoclonal antibody by preparing a biphenyl derivative isomer mixture represented by the following formula (hereinafter sometimes referred to as “conjugate”) and using it as an immunogen (antigen).

In the general formula (II), the protein represented by Z (sometimes referred to as “carrier protein”) has an immunogenicity and a compound represented by the general formula (I) and a spacer moiety ( As long as it can be linked via —O— (CH ₂ ) _n —CONH—, there is no particular limitation, and it can be appropriately selected according to the purpose. For example, mussel derived hemocyanin (KLH), ovalbumin (OVA) Bovine serum albumin (BSA), human serum albumin, rabbit serum albumin, goat serum albumin, bovine gamma globulin, myoglobin, multiple antigen peptide (MAP), and the like. Among these, mussel-derived hemocyanin (KLH) is preferable.

  The method for preparing the compound (conjugate) represented by the general formula (II) is not particularly limited and can be prepared by a known synthesis method. For example, as shown in the following reaction formula, the general formula A method in which the carboxyl group of the biphenyl derivative represented by (I) is converted into an active ester derivative by a condensation reaction such as N-hydroxysuccinimide, and then the active ester derivative and the amino group of the carrier protein are bonded according to a conventional method. Etc.

  The obtained compound (conjugate) represented by the general formula (II) is purified by substituting the reaction solution with a neutral solution such as a physiological phosphate buffer by a known method such as gel chromatography. Is preferred.

  In the general formula (II), the protein represented by Z may be labeled with a labeling substance. The labeling substance can be appropriately selected from known labeling substances such as enzymes, fluorescent dyes, radioisotopes, magnetic substances, dye-encapsulated polymers, rare earths, coenzymes, gold colloids, and gold atom clusters.

The anti-PCB monoclonal antibody, for example, immunized an animal using an emulsion obtained by emulsifying the immunogen and an adjuvant using the compound represented by the general formula (II) as an immunogen, It is produced by collecting spleen cells of an animal and culturing a hybridoma obtained by fusing the spleen cells and myeloma cells.
The monoclonal antibody can be produced according to, for example, the method of Kohler and Milstein (Kohler G, Milstein C, Nature, 256, 495-7 (1975)).

As the animal to be immunized, mammals such as mice, rats, rabbits and goats are preferable.
The animal immunization method is not particularly limited and can be appropriately selected from known methods. For example, an emulsion obtained by emulsifying the immunogen and an adjuvant is subcutaneously, intravenously or intraperitoneally. And a method of administration by injection or the like.
For example, in the case of mice, the immunogen is preferably administered at 0.01 to 0.5 g / animal 3 to 15 times at intervals of about 2 to 3 weeks.
The adjuvant is not particularly limited and may be appropriately selected depending on the intended purpose. For example, Freund's complete adjuvant, incomplete adjuvant, BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS), alum An adjuvant, a silica adjuvant, etc. are mentioned, It is preferable to select and use the optimal thing from relations, such as the induction | guidance | derivation ability of an antibody and the load to an animal.

Several days after the last administration of the immunogen, antibody-producing cells are collected from the immunized animal. Examples of the antibody-producing cells include spleen and lymph node-derived B cells.
The hybridoma can be obtained by fusing the antibody-producing cells with myeloma cells and then screening for fused cells that produce the desired antibody.

The myeloma cells are not particularly limited, and known ones can be used. For example, it can be selected from mouse, rat, human-derived cell lines, etc., and if it is a mouse-derived cell, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.1. If it is a rat-derived cell such as Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, S194 / 5XXO · Bul, R210. RCY3, Y3-Ag · 1.2.3, IR983F, 4B210 and the like.
Methods for fusing the antibody-producing cells and myeloma cells may be performed by methods known to those skilled in the art, such as Sendai virus method, polyethylene glycol method, protoplast method, and the like. It is preferable because of its relatively low cytotoxicity and easy fusion operation.

The method for screening the fused cells producing the desired antibody is not particularly limited and may be appropriately selected according to the purpose. For example, radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), fluoroimmunoassay (FIA).
For example, by immobilizing the conjugate represented by the general formula (II) and reacting the culture supernatant of the fusion cell (hybridoma), the antibody titer of the culture supernatant is measured, and the antibody titer is high. Wells can be selected as anti-PCB antibody producing wells. Furthermore, by coexisting PCB substitute compounds (for example, biphenyl derivatives represented by the above general formula (I)) and PCBs at the time of antibody titer measurement, wells that are highly reactive with these coexisting substances are selected, and limiting dilution is performed. The desired anti-PCB antibody-producing hybridoma can be monocloned by the method.
Further, in the conjugate represented by the general formula (II), the protein represented by Z is labeled with a labeling substance, and this is allowed to coexist when measuring the antibody titer of the anti-PCB antibody production well, and a direct competitive reaction is performed. An anti-PCB antibody-producing hybridoma can also be screened by the method.

Since PCB which is a measurement target of the anti-PCB antibody is hardly soluble in water, it is dissolved in an amphiphilic solvent and used for measurement. For this reason, it is preferable that the said anti- PCB antibody has tolerance with respect to the said amphiphilic solvent which exists in a sample. Therefore, it is preferable that the anti-PCB antibody-producing hybridoma is also screened in the presence of the amphiphilic solvent.
Examples of the amphiphilic solution include DMSO, methanol, ethanol and the like.
The concentration of the amphiphilic solution used for the screening is preferably 50% by mass or less, more preferably 30% by mass or less, and particularly preferably 5 to 20% by mass.

Examples of the sensor used for screening the anti-PCB antibody-producing hybridoma include the sensor functional part of a transducer such as an SPR (surface plasmon) sensor, a QCM (quartz crystal) sensor, an optical sensor, and an electrochemical sensor. Examples include a biphenyl derivative represented by the formula (I) or a conjugate of a biphenyl derivative represented by the general formula (II) and a carrier protein.
Screening can also be carried out by bringing PCBs or the like added to the culture supernatant of the fusion cells and reacting them in advance with the sensor functional part. In this case, an anti-PCB antibody-producing hybridoma having high affinity that can be applied to PCB measurement using various transducers can be selected by evaluating the amount of the antibody bound to the functional part.

The anti-PCB antibody-producing hybridoma is not particularly limited as long as it produces the anti-PCB monoclonal antibody of the present invention that recognizes PCB, but is preferably prepared and established by the above method. Examples include ES2G6 strains as shown in Examples.
The ES2G6 strain is a hybridoma prepared by using a compound represented by the following general formula (II), which was registered with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as of December 28, 2006, under the accession number FERM P. -Deposited as 21148.

However, in said general formula (II), n represents the integer of 1, X and Y represent the integer of 0-3, respectively, the sum of X and Y is 1-6, Z represents protein.

― Production and purification of anti-PCB monoclonal antibody ―
The anti-PCB monoclonal antibody is cultured, for example, by transplanting the anti-PCB antibody-producing hybridoma cell line into the abdominal cavity of a mouse previously administered with pristane (2,6,10,14-tetramethylpentadecane), After 14 days, ascites containing the anti-PCB monoclonal antibody can be collected and obtained from the obtained ascites.
In addition, the hybridoma can be cultured using a medium (for example, DMEM containing 10% fetal bovine serum), and the supernatant of the obtained culture solution can be used as an anti-PCB monoclonal antibody solution.
A method for purifying the anti-PCB monoclonal antibody from the culture supernatant of the anti-PCB antibody-producing hybridoma cell line obtained from the ascites or fetal bovine serum-containing medium is not particularly limited and may be a known method depending on the purpose. For example, it can be purified by ultrafiltration, salting out, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like.

<Evaluation of anti-PCB monoclonal antibody>
The anti-PCB monoclonal antibody of the present invention is produced by the above-described method for producing an anti-PCB monoclonal antibody of the present invention, and has cross-reactivity with two or more kinds of PCB homologs having different chlorine numbers. The anti-PCB monoclonal antibody preferably has a high affinity for 1 to 10 chlorinated PCB homologs among 1 to 10 chlorinated PCB homologs.

Specifically, in the anti-PCB monoclonal antibody, the relationship between the equilibrium dissociation constant K1 for a PCB homologue having 1 to 4 chlorine atoms and the equilibrium dissociation constant K2 for a homologue having 5 to 7 chlorine atoms is such that K1 <K2. Fulfill. The equilibrium dissociation constant K2 is 4.0 ppb or more, and the equilibrium dissociation constant K1 is 3.0 ppb or less, preferably 0.5 to 3.0 ppb.
In particular, the equilibrium dissociation constant K3 in the PCB homologue having 1 to 2 chlorine atoms is 2.0 to 3.0 ppb.

  Further, the anti-PCB monoclonal antibody preferably has a very low affinity for biphenyl, and the equilibrium dissociation coefficient K4 for biphenyl is preferably 500 ppb or more.

The equilibrium dissociation constant (affinity) of the anti-PCB monoclonal antibody is determined, for example, by the antigen concentration (IC ₅₀ value) that inhibits the binding of the anti-PCB monoclonal antibody to PCB by 50%. In addition, the cross-reactivity of the PCB monoclonal antibody can be evaluated by, for example, obtaining respective IC ₅₀ values for two or more kinds of PCB homologues having different chlorine numbers and obtaining their ratio.

The IC ₅₀ value calculation method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the measurement data obtained by a fluorometer is applied to the approximate expression represented by the following expression (1). It is possible to analyze by obtaining.

In the formula (1), y represents the relative fluorescence intensity when the fluorescence intensity value when no antigen is added is 100%, x represents the antigen concentration, and P1 and P2 are approximate. Represents the parameters.

The measurement data is converted into a relative value with the fluorescence intensity value when no antigen is added as 100%, and then the optimum P1 and P1 are obtained using graphing software (for example, Origin version 6.0 (manufactured by OriginLab)). P2 is determined.
The approximate expression obtained in this way is the binding curve between the antibody and the antigen, and the value of x (= P1) when y = 50% is the IC ₅₀ value.
It is known that when the antibody concentration is sufficiently small with respect to the IC ₅₀ value (for example, 1/10 or less), the IC ₅₀ value and the equilibrium dissociation constant (Kd) are almost the same.

The equilibrium dissociation constant, that is, the affinity of the anti-PCB monoclonal antibody for PCBs having different chlorine numbers, for example, Kanechlor (KC300, KC400, KC500, KC600: Kaneka Chemical Co., Ltd.) or Aroclor (Ar1242, Ar1248, Ar1254, Ar1260, Ar1016, Ar1262, Ar1232, Ar1221: Mitsubishi Monsanto (currently manufactured by Mitsubishi Chemical Corporation))) and the like can be used and evaluated.
The Kanechlor and Aroclor contain PCBs having different chlorine numbers in the ratios shown in Table 1 and FIG. 1, respectively. For example, the anti-PCB monoclonal antibody of the present invention has two or more PCBs having different chlorine numbers. Kanechlor (K300, K400: manufactured by Kaneka Chemical Co., Ltd.) and Aroclor (Ar1221: Mitsubishi Monsanto (current Mitsubishi Chemical)), which have cross-reactivity with homologs and a high abundance ratio of PCB homologues having 1 to 4 chlorine atoms And the equilibrium dissociation constant K2 for Kanechlor (K500, K600: manufactured by Kaneka Chemical Co., Ltd.) having a high abundance ratio of PCB homologues having 5 to 7 chlorine atoms. Equilibrium solution for Aroclor (Ar1221: manufactured by Mitsubishi Monsanto (now Mitsubishi Chemical)), which is K2 and has a high abundance ratio of PCB congeners having 1 to 2 chlorine atoms Constant K3 is a 2.0～3.0Ppb, a high affinity to the PCB congeners chlorine number 1-2.

(Source: Takuzo Takatsuki et al., “Detailed analysis method of all isomers of polychlorinated biphenyls (PCBs) using various cleanup methods and HRGC / HRMS”, Environmental Chemistry, Vol. 5, No. 3, pp. 647- 675, 1995)

(PCB measurement method)
The PCB measurement 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, using the anti-PCB monoclonal antibody, Of immunologically detecting and quantifying PCBs, specifically, radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), fluoroimmunoassay (FIA), immunochromatography, chemiluminescent enzyme immunoassay (CLEIA) ), Immunoturbidimetric method (TIA), latex immunoturbidimetric method (LTIA), binding equilibrium exclusion method, and the like.

Examples of the PCB measurement method include a non-competitive method, a competitive method (indirect competitive method or direct competitive method), a binding equilibrium exclusion method, and the like. Specifically, the following methods (I) to (III) are exemplified. It is done.
(I) The anti-PCB monoclonal antibody of the present invention is added to the sample to be measured,
(1) PCB including a method for detecting any of PCB in the sample to be measured bound to the anti-PCB monoclonal antibody and (2) the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured Measuring method.
(II) The anti-PCB monoclonal antibody of the present invention and the compound represented by any one of the general formula (I) and the general formula (II) are added as a PCB competitor to the sample to be measured, A method for measuring PCB comprising detecting the competitor substance bound to a PCB monoclonal antibody.
(III) An anti-PCB monoclonal antibody of the present invention is added to the sample to be measured, and a carrier in which the compound represented by the general formula (II) is immobilized as a PCB competitor is used. A method for measuring PCB, comprising: competing with PCB in a measurement sample and detecting the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured.

Hereinafter, an example of the indirect competition method and the binding equilibrium exclusion method will be described more specifically.
In the indirect competition method and the binding equilibrium exclusion method, as an antigen to be immobilized on a carrier (hereinafter referred to as “immobilized antigen”), for example, a compound containing a biphenyl derivative represented by the general formula (I), and The compound etc. which are represented with the said general formula (II) are mentioned.
In the indirect competition method and the binding equilibrium exclusion method, the immobilized antigen is immobilized on the carrier, and a portion of the carrier to which the immobilized antigen is not bound is blocked with a blocking agent. Next, the sample to be measured and the anti-PCB monoclonal antibody are added to the carrier, and the immobilized antigen and PCB in the sample to be measured are subjected to a competitive reaction with the anti-PCB monoclonal antibody in the indirect competition method, In the binding equilibrium exclusion method, a binding equilibrium exclusion reaction is performed.
After washing and removing the anti-PCB monoclonal antibody that did not bind to the immobilized carrier, an antibody labeled as a secondary antibody was added, and the anti-PCB monoclonal antibody bound to the immobilized antigen was bound and washed. The color development or luminescence of the label is measured. Further, the anti-PCB monoclonal antibody may be directly labeled without using a secondary antibody. For the absorbance and light intensity obtained, the rate of decrease relative to the value when no sample was added was determined, and this was used as the inhibition rate, using a calibration curve obtained in advance from the inhibition rate when PCB of a known concentration was added. The PCB concentration in the sample can be calculated.

  The label is not particularly limited and may be appropriately selected according to the purpose. For example, when peroxidase is used as an enzyme, hydrogen peroxide is used as a substrate, and o-phenylenediamine or tetramethylbenzidine is used as a color former. When alkaline phosphatase is used as the enzyme and p-nitrophenyl phosphate is used as the substrate, color development can be measured by absorbance. On the other hand, when the reaction product of the substrate is fluorescent or is labeled with a fluorescent substance, the fluorescence can be measured using a fluorometer or the like. When a chemiluminescent substance is used, the luminescence can be measured with a chemiluminescence measuring instrument or the like, and when using a gold colloid, the transmitted light intensity or the like can be measured with a transmitted light quantity measuring instrument or the like.

Examples of the sample to be measured include a sample obtained by liquid-liquid extraction of PCB in a test substance with a polar solvent such as dimethyl sulfoxide (DMSO) or an alcohol solvent, and PCB in the test substance with a polar solvent. Liquid-liquid extraction, the sample obtained by solid-phase extraction of the polar solvent containing the extracted PCB with an adsorbent, and the components adsorbed on the adsorbent transferred to a desired solvent, and the test substance in normal Examples include a sample obtained by adding a low-polarity solvent such as hexane, cyclohexane, and toluene and extracting PCB in the test substance with a hydrophilic solvent.
In the liquid-liquid extraction, the mixing ratio (mass ratio) between the test substance and the polar solvent is preferably, for example, (the test substance) :( the polar solvent) = 1: 1 to 1:10.

The test substance is not particularly limited as long as it is a substance (PCB contaminant) that may contain at least PCB, and can be appropriately selected according to the purpose. For example, insulating oil, oil other than insulating oil (For example, vegetable oils, animal oils, synthetic oils, mineral oils, etc.), factory wastewater, soil, exhaust gas, animal blood and body fluids, wastes such as members used in the treatment of insulating oils, and the like. Among these, insulating oil is preferably exemplified.
In particular, as the test substance, PCBs waste after PCB decomposition treatment such as dechlorination treatment is preferably exemplified.

  The concentration of the polar solvent in the sample to be measured is not particularly limited as long as the affinity of the anti-PCB monoclonal antibody for PCB is not significantly deteriorated. For example, it is preferably 1 to 20% by mass, More preferably, it is 20 mass%.

The anti-PCB monoclonal antibody of the present invention has affinity for PCBs substituted with multiple (3 to 7) chlorines contained in Kanechlor and Aroclor, and is a PCB homologue having 1 to 2 chlorine atoms. Because of its high affinity, it enables rapid and reproducible analysis of PCBs contained in environmental samples, industrial products, and biological tests. The PCB measurement method using the anti-PCB monoclonal antibody is a PCB degradation process. It is suitable as a rapid monitoring and analysis technique for PCBs in the process.
In a system using a transducer such as an SPR (surface plasmon) sensor, a QCM (quartz crystal) sensor, an optical sensor, or an electrochemical sensor, the anti-PCB monoclonal antibody of the present invention or A biphenyl derivative or the like can be immobilized and used as an immunosensor.
Furthermore, the carrier on which the anti-PCB monoclonal antibody of the present invention is immobilized can be applied to monitoring in the PCB decomposition treatment step.

  EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples, and includes ordinary alterations and modifications to these performed in the technical field.

Example 1
(1) Preparation of isomer mixture of biphenyl derivative and conjugate According to the following reaction formula, a biphenyl derivative (compound 3 in the following reaction formula) as a hapten obtained by introducing a spacer molecule into 4-phenylphenol was prepared. A conjugate (compound 5 in the following reaction formula) formed by binding with a carrier protein was synthesized.

However, in Chemical formulas 1-5, n represents the integer of 1, X and Y represent the integer of 0-3, respectively, the sum of X and Y is 4-6, and Z is KLH.

  To 511 mg (3.0 mmol) of 4-phenylphenol, 4 mL of trifluoroacetic acid was added, and 1.20 g (9.90 mmol) of N-chloromorpholine prepared for use under ice cooling was added dropwise. After stirring at the same temperature for 30 minutes, the temperature was raised to room temperature and further stirred for 1 hour. Thereafter, distilled water was added, and the reaction solution was neutralized with an aqueous sodium hydroxide solution. The organic layer was extracted with dichloromethane, and the organic layer was washed with saturated brine, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluted with dichloromethane: hexane = 1: 10) to obtain 619 mg of Compound 1 in the above reaction formula as a chlorinated isomer mixture.

  515 mg of the above compound 1 was dissolved in anhydrous N, N-dimethylformamide, 390 mg (2.82 mmol) of potassium carbonate and then 630 mg (2.82 mmol) of 6-bromohexanoic acid ethyl ester were added, and the mixture was heated to 60 ° C. Stir for 17 hours. After the reaction, the mixture was allowed to cool to room temperature, distilled water was added, the organic layer was extracted with dichloromethane, washed with saturated brine, and the solvent was distilled off under reduced pressure to give a chlorinated isomer mixture in the above reaction formula. Compound 2 was obtained. Compound 2 was used in the next reaction without further purification.

  The compound 2 was dissolved in 10 mL of dioxane and 12.5 mL of methanol, 2.5 mL of 10N aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 2 hours. After the reaction, the reaction solution was made weakly acidic with 6N hydrochloric acid, and the organic layer was extracted with dichloromethane. After washing with saturated brine, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluted with dichloromethane) to obtain 484 mg of compound 3 in the above reaction formula as a chlorinated isomer mixture.

  Next, 384 mg of the compound 3 and 137 mg (1.19 mmol) of N-hydroxysuccinimide were dissolved in 2 mL of anhydrous N, N-dimethylformamide. To this reaction solution, 207 μL (1.49 mmol) of triethylamine and 285 mg (1.49 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were added and stirred at room temperature for 14 hours. After the reaction, distilled water was added and the organic layer was extracted with dichloromethane. The organic layer was washed with saturated brine, and then the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluted with dichloromethane) to obtain 361 mg of Compound 4 in the reaction formula as a chlorinated isomer mixture.

  The compound 4 was dissolved in dimethyl sulfoxide (DMSO) and added dropwise to a BSA solution or KLH solution dissolved in 50 mM phosphate buffer (pH 8.0) under ice-cooling and stirring at room temperature overnight. The reaction solution was added to a Sephadex column equilibrated with PBS (−) and developed with the same buffer to separate the carrier protein, and the conjugate (compound 5 in the above reaction formula) was purified.

(2) Immunization of mice Mice were immunized using the obtained conjugate as an immunogen.
The compound 5 and adjuvant RAS R-700 (RIBI) were mixed in a 1: 1 volume and sufficiently emulsified, and the resulting emulsion was used as the abdominal cavity of BALB / c mice (6-8 weeks old, female). The mice were immunized by administration of 200 μL.
Booster immunization was performed at intervals of about 2 to 3 weeks, and blood was collected from the tail vein 1 week after booster immunization, and the antibody titer in blood was measured by ELISA to observe the change in antibody titer.

(3) Preparation of hybridoma Final immunization was carried out by further administering the immunogen into the tail vein of mice in which high antibody production against the immunogen was observed in the blood. Three days after the final immunization, the spleen is removed from the immunized mouse, spleen cells are prepared, and myeloma cells (P3-X63-Ag8.653 or Sp2 / O) in the logarithmic growth phase and spleen cells are 1: 5. And cell fusion was performed by the polyethylene glycol (PEG) method. The cells after the fusion are suspended in RPMI1640 medium containing 10% FCS supplemented with HAT (hypoxanthine, aminopterin and thymidine), and cultured in 96 wells so that the number of spleen cells is 1-2 × 10 ⁵ cells / well. The cells were seeded and cultured at 37 ° C. under 5% CO ₂ .

(4) Screening and cloning of antibody-producing hybridomas Seven to ten days after cell fusion, antibody titers were measured by ELISA using the culture supernatant of wells in which clone growth was observed.

(5) Screening of antibody-producing hybridoma (measurement of antibody titer)
To each well of a microtiter plate (CORNING), 0.25 to 1 μg / mL of each immunogen in PBS (−) diluted solution (50 μL) was added and allowed to stand at room temperature for 1 hour to be immobilized. In addition, in order to exclude antibody-producing clones against the carrier protein in the conjugate, wells immobilized with BSA or KLH were used as controls.
Each well was washed three times with 300 μL of PBS (−) (washing solution) containing 0.05% Tween 20 and 4 g of Block Ace powder (Yuki-Ink, UK-B80) was dissolved in 100 mL of purified water. A solution prepared by diluting 4 times with purified water (300 μL) was added to the wells and allowed to stand at room temperature for 2 hours for blocking. After the wells were washed three times with a washing solution (300 μL), 25 μL of 20% DMSO, 0.01% Titon-X100, and a culture supernatant (25 μL) containing the primary antibody were added to each well, mixed, and then mixed at room temperature. The antigen-antibody reaction was allowed to stand for 10 to 30 minutes under the conditions.
Subsequently, each well was washed three times with a washing solution (300 μL), and then 3,000 with a Block Ace solution diluted 10-fold with purified water in order to detect the primary antibody bound to each immobilized immunogen. Horseradish peroxidase-labeled anti-mouse IgG (γ chain recognition) prepared by doubling dilution (manufactured by KPL) was added as a secondary antibody solution (50 μL) and reacted at room temperature for 1 hour. After the wells were washed three times with 300 μL of the washing solution in the same manner, 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution (manufactured by KPL) was added to each well, and a color reaction was performed at room temperature. It was.
After 5 to 15 minutes, 50 μL of 1M phosphoric acid was added to stop the reaction, and the absorbance at 540 nm was immediately measured with a microplate reader (Multiscan MS, manufactured by Labsystems).
The difference in absorbance between the well in which each immunogen was immobilized and the control well was defined as the binding activity of the antibody to the immunogen.

(6) Selection of specific antibody-producing clones by indirect competition method According to (5) above, each immunogen is solid-phased in each well of a microtiter plate, and after washing, 300 μL of a block ace solution diluted 4-fold with purified water is added to the well. And allowed to stand at room temperature for 2 hours for blocking. The wells were washed 3 times with 300 μL of washing solution.
Subsequently, all were prepared so that the final concentration of the solvent was 20% DMSO containing 0.01% Titon-X100. (I) Each immunogen, (ii) Individual PCB congeners, (iii) 4 types of Kanechlor ( KC300, KC400, KC500, KC600) and one type of Aroclor (Ar1221), (iv) an equal mixture of four types of Kanechlor and one type of Aroclor (KC-Mix), and (v) a control (20% DMSO and 0.01% Titon-X100) was prepared, and 25 μL each was added to each well of the immunogen-immobilized plate.
Next, after adding and mixing in a hybridoma culture supernatant (25 μL) well, the mixture was allowed to stand at room temperature for 10 to 30 minutes to carry out an antigen-antibody reaction.
Each well was washed 3 times with 300 μL of washing solution, and then horseradish peroxidase prepared by diluting 3000 times with Block Ace diluted 10 times with purified water to detect the primary antibody bound to the immobilized immunogen. 50 μL of labeled anti-mouse IgG (γ chain recognition) was added as a secondary antibody solution and reacted for 1 hour at room temperature.
After the wells were washed three times with 300 μL of the washing solution in the same manner, a TMB solution was added to each well and a color reaction was performed at room temperature. After 5 to 15 minutes, 50 μL of 1M phosphoric acid was added to stop the reaction, and the absorbance at 450 nm was immediately measured with a microplate reader (Multiscan MS, manufactured by Labsystems). The ratio (B / B0) of the well absorbance value (B) in the coexistence of PCB, kanechlor, etc. to the absorbance value of the control well (control OD; B0) was calculated, and the reactivity of the antibody to the coexisting substance was judged. Hybridomas in wells that had high binding activity to PCB and were confirmed to have high antibody production continuously were selected and subjected to cloning by limiting dilution.

(7) Selection of clones having adaptability to transducers Among the hybridomas selected in (6) above, hybridomas that have been cloned one or more times are further analyzed by immunosensors (KinExA 3000, Selection was performed using Sapidyne.
The expanded culture supernatant of the clone was reacted in advance with a Cy5-labeled anti-mouse IgG (Jackson) for 2 hours or more in a solvent of PBS (−) containing 5% DMSO and 0.1 w / v% BSA at room temperature. After that, it was diluted to a range of 4 to 800 times and used for a transducer.
About 0.5 mL of the hybridoma culture supernatant containing the antibody labeled with Cy5 was passed through the compound 5 immobilized on the sensor functional part at a flow rate of 0.75 mL / min.
The amount of antibody in which the fluorescence intensity derived from Cy5 was bound to the sensor functional part after washing nonspecifically bound antibodies and contaminants with PBS (-) solution containing 5% DMSO and 0.1 w / v% BSA Detected as.
Furthermore, after reacting the clonal culture supernatant with Cy5-labeled anti-mouse IgG in the presence of PCB, the high affinity for PCB is obtained based on the fluorescence intensity detected when the solution is similarly sent to the sensor functional part. The indicated antibody producing clones were obtained.

  As described above, a stable hybridoma producing an anti-PCB monoclonal antibody having affinity for a plurality of PCB congeners having different chlorine numbers and having high affinity for PCB congeners having 1 to 2 chlorine atoms is obtained. Was designated as ES2G6 strain. The ES2G6 strain cells formed a colony derived from a single cell using a methylcellulose medium, and then transferred the colony to a liquid medium and continued the culture. This hybridoma ES2G6 was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on December 28, 2006, and has acquired FERM P-21148 as a deposit number.

(8) Preparation of anti-PCB monoclonal antibody The colony of the hybridoma ES2G6 strain established in (7) above was transferred to a HAT medium based on RPMI1640 medium on a 24-well plate and cultured for 4 days. It is transferred to a 50 ml square flask containing 10 ml of the HAT medium, cultured until just reaching confluence, and the antibody secreted by the ES2G6 strain is purified from the culture supernatant by affinity chromatography using protein A. An antibody (hereinafter referred to as “ES2G6 antibody”) was obtained.

(8) Large-scale preparation and purification of monoclonal antibody Pristan [2,6,10,14-tetramethylpentadecane (Wako Pure Chemicals)] (0.5 mL) was administered into the peritoneal cavity of BALB / c mice over 8 weeks of age. And raised for 2-3 weeks. The culture solution of the anti-PCB monoclonal antibody-producing hybridoma WR3F6 previously maintained in the logarithmic growth phase was collected, the culture supernatant was removed by centrifugation, and then the precipitate was examined with RPMI1640 without FCS at 1 × 10 ^6. Cell fluid was prepared to be cells / 0.5 mL.
This cell solution was transplanted into the abdominal cavity of BALB / c mice pre-administered with pristane, and ascites leaked into the abdominal cavity after 10 to 14 days was collected from the abdomen with a syringe equipped with a 25G syringe.
The collected ascites was filtered using a filter having a pore size of 0.22 μm, and the obtained filtrate was purified by affinity chromatography using a protein G-Sepharose column (manufactured by Amersham Biosciences), and the anti-PCB monoclonal antibody was purified. Obtained.

(Comparative Example 1)
Anti-PCB monoclonal antibody produced from the established hybridoma WR3F6 strain (deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on October 26, 2006, and obtained FERM P-21073 as the accession number) (Hereinafter referred to as “WR3F6 antibody”), a large amount of monoclonal antibody was prepared and purified in the same manner as the ES2G6 antibody.

(Evaluation of anti-PCB monoclonal antibody)
The anti-PCB monoclonal antibody (ES2G6 antibody) obtained in Example 1 and the anti-PCB monoclonal antibody (WR3F6 antibody) obtained in Comparative Example 1 were evaluated for affinity and cross-reactivity to PCB homologues and biphenyls having different chlorine numbers. did.

<Experimental Example 1: Measurement of PCB congeners using ES2G6 antibody (preparation of calibration curve)>
Using the ES2G6 antibody, abundance ratio is high in Kanechlor (KC300, KC400, KC500, KC600: Kaneka Chemical Co., Ltd.) and Aroclor (Ar1221: Mitsubishi Monsanto (current Mitsubishi Chemical)) contained in oil samples. PCB homologues were measured and a calibration curve was created. Further, biphenyl contained in the oil sample was measured using the ES2G6 antibody, and a calibration curve was prepared. As a measuring device, an immunosensor (KinExA 3000) based on fluorescence analysis was used.

― Adjustment of measurement sample ―
The ES2G6 antibody is applied to each oil sample containing different concentrations of Kanechlor (KC300, KC400, KC500 and KC600), each oil sample containing different concentrations of Aroclor (Ar1221), and each oil sample containing different concentrations of biphenyl. After mixing, each measurement sample was adjusted so that the final concentrations of canechlor, arochlor and biphenyl were 100-fold diluted, and the concentration of ES2G6 antibody was 0.2 nM.
The ES2G6 antibody was mixed and then allowed to stand at room temperature for 10 minutes, whereby the ES2G6 antibody was reacted with the antigens (canechlor, arochlor and biphenyl) contained in each measurement sample, and the antigen and antibody were bound. The reaction was allowed to reach equilibrium.

― Measurement ―
The compound 5 (conjugate of biphenyl derivative and carrier protein) is covalently bound to agarose beads (manufactured by Amersham Bioscience) into which an N-hydroxysuccinimidyl ester group has been introduced, and blocking is performed with a BSA solution. An antibody-binding carrier was obtained.
The measurement sample reacted with the ES2G6 antibody was allowed to act on the antibody-binding carrier at a flow rate of 0.25 mL / min. Thereafter, a secondary antibody (goat anti-mouse IgG antibody, ImmunoResearch Laboratories) labeled with a fluorescent substance Cy5 (registered trademark) was allowed to act on the antibody-binding carrier at a flow rate of 0.25 mL / min.
Next, PBS (−) containing 5% DMSO and 0.1 w / v% BSA was passed at a flow rate of 0.25 mL / min to eliminate non-specific binding to the carrier, and then a fluorometer (KinExA, manufactured by Sapidyne). 3000) was used to measure the fluorescence intensity of the antibody-bound carrier, and the amount of ES2G6 antibody bound to the carrier was measured. The measurement (reaction) of each measurement sample was performed at room temperature.
In the measurement by the immunosensor (KinExA 3000) based on the fluorescence analysis described above, the amount of fluorescence shown when measuring an antibody solution to which none of Kanechlor, Aroclor, and Biphenyl is measured is taken as a control (B0) value, and Kanechlor against the B0 value. The ratio (B / B0) of the fluorescence amount (B) decreased by the addition of Aroclor and biphenyl is output as the signal intensity (%). From this obtained value, the affinity and cross-reactivity for the antigens (K300, K400, K500, K600, Ar1221 and biphenyl) in the measurement sample in the ES2G6 antibody are obtained, and the antigen concentration in the measurement sample is determined. It can be quantified.
The affinity and cross-reactivity can be determined from the obtained values and quantified by measuring the signal intensity derived from a label other than the fluorescent label.
Specifically, when the label is a colloidal gold, the amount of light transmitted through the carrier is used as the signal intensity, and measurement can be performed using a transmitted light amount measurement device (for example, Imny, manufactured by Shibata Kagaku Co., Ltd.).

― Creation of calibration curve ―
Values (signal intensity (%)) measured by an immunosensor (KinExA 3000) based on fluorescence analysis were plotted with respect to the concentrations of kanechlor, arochlor and biphenyl in each measurement sample, and a calibration curve was created. The results are shown in FIG.
In FIG. 2, the horizontal axis represents the concentrations of Kanechlor, Aroclor and Biphenyl in the measurement sample (concentration obtained by diluting the oil sample 100 times), and the vertical axis represents the signal intensity (%) measured with KinExA 3000.

As shown in the calibration curve of FIG. 2, the ES2G6 antibody is not only for PCB homologues having 3 to 7 chlorine atoms (K300, KC400, KC500, KC600), but also for PCB homologues having 1 to 2 chlorine atoms. It is an extremely sensitive antibody, and by using the ES2G6 antibody, 0.05 ppm (the concentration in the measurement sample is 0.5 ppb) of Ar1221 contained in the oil sample can be quantified (detection lower limit (IC ₈₅ ) ) Was confirmed.

<Experimental Example 2: Measurement of PCB homologue using WR3F6 antibody>
Using the WR3F6 antibody, measurement of PCB congeners with high abundance ratio in Aroclor (Ar1242, Ar1016, Ar1248, Ar1262, Ar1254, Ar1232, Ar1260 and Ar1221: made by Mitsubishi Monsanto (current Mitsubishi Chemical)) contained in oil samples A calibration curve was created. As in Experimental Example 1, an immunosensor (KinExA 3000) based on fluorescence analysis was used as a measuring device.

― Adjustment of measurement sample ―
The WR3F6 antibody is mixed with each oil sample containing Aroclor having different concentrations (Ar1242, Ar1016, Ar1248, Ar1262, Ar1254, Ar1232, Ar1260 and Ar1221), and each concentration of Aroclor is finally diluted 100 times. And each measurement sample was adjusted so that the concentration of the WR3F6 antibody was 0.2 nM.
The WR3F6 antibody is mixed and then allowed to stand at room temperature for 10 minutes, whereby the WR3F6 antibody reacts with the antigen (arochlor) contained in each measurement sample, and the antigen-antibody binding reaction reaches an equilibrium state. I did it.

― Preparation of measurement and calibration curve ―
A measurement and a calibration curve were prepared in the same manner as in Experimental Example 1 except that the measurement sample was used. The results are shown in FIG.
In FIG. 3, the horizontal axis represents the concentration of Aroclor in the measurement sample (concentration obtained by diluting the oil sample 100 times), and the vertical axis represents the signal intensity (%) measured with KinExA 3000.

  As shown in FIG. 3, the WR3F6 antibody can be quantified with high sensitivity to PCB homologues having 3 to 7 chlorine atoms (Ar1242, Ar1016, Ar1248, Ar1262, Ar1254, Ar1232, Ar1260), It was confirmed that the quantitative property was low with respect to 2 PCB congeners (Ar1221).

<Experimental Example 3: Evaluation of affinity and cross-reactivity>
The ES2G6 antibody and the WR3F6 antibody were evaluated for affinity and cross-reactivity with respect to PCB homologues having 1 to 7 chlorine atoms (KC300, KC400, KC500, KC600 and Ar1221). In addition, the affinity and cross-reactivity for biphenyl were evaluated for the ES2G6 antibody.
In addition, each measurement sample was prepared and measured in the same manner as in Experimental Example 1.

(I) Measurement of equilibrium dissociation constant For the ES2G6 antibody and WR3F6 antibody, the average dissociation constant (Kd) for canechlor (KC300, KC400, KC500 and KC600), arochlor (Ar1221) and biphenyl was determined, and the affinity was evaluated.
Measurement data obtained by the fluorometer (KinExA 3000) was applied to an approximate expression to obtain an IC ₅₀ value. As the approximate expression, the following expression (1) was used.

In the formula (1), y represents the relative fluorescence intensity when the fluorescence intensity value when the antigen (PCB) is not added to the sample is 100%, x represents the antigen concentration, P1 and P2 represent approximate parameters.

It is known that when the antibody concentration is sufficiently small with respect to the IC ₅₀ value (for example, 1/10 or less), the IC ₅₀ value and the equilibrium dissociation constant (Kd) are almost the same. For the evaluation of affinity, the equilibrium dissociation constant (Kd) was used. The results are shown in Table 2 below.

(Ii) Cross-reactivity Cross-reactivity was calculated by the following formulas (2) and (3), respectively. It is a value (%) calculated based on the IC ₅₀ value of Ar1221 in the ES2G6 antibody. The results are shown in Table 2 below.
ES2G6 antibody cross-reactive (%) = (Ar1221 _{IC 50} values / _{an IC 50} value of the sample)
... Formula (2)
WR3F6 antibody cross-reactive (%) = (Ar1221 _{IC 50} values / _{an IC 50} value of the sample)
... Formula (3)

As shown in Table 2, the WR3F6 antibody of Comparative Example 1 has an equilibrium dissociation constant with respect to PCB homologues having 3 to 7 chlorine atoms (KC300, KC400, KC500 and KC600), but is less than 3.0 ppb. The equilibrium dissociation constant for the PCB homologue of number 1 to 2 (Ar1221) is 14.0 ppb or more, and the affinity for the homologue of 1 to 2 chlorine atoms is very low.
In contrast, the ES2G6 antibody of Example 1 has an equilibrium dissociation constant K1 for PCB homologues having 1 to 4 chlorine atoms (Ar1221, KC300 and KC400) and a PCB homologue having 5 to 7 chlorine atoms (KC500 and KC600). The relationship with the equilibrium dissociation constant K2 is K1 <K2, the equilibrium dissociation constant K2 is 4.0 ppb or more, and the equilibrium dissociation constant K1 is 3.0 ppb or less. In particular, the equilibrium dissociation constant K3 for a PCB homologue having 1 to 2 chlorine atoms (Ar1221) is 2.0 to 3.0 ppb, and has a high affinity for homologous pairs having 1 to 2 chlorine atoms.

  Regarding the cross-reactivity, the WR3F6 antibody of Comparative Example 1 has a low cross-reactivity with respect to PCB homologues having 1 to 2 chlorine atoms (Ar1221), whereas the ES2G6 antibody of Example 1 has 1 to 2 chlorine atoms. It had high cross-reactivity with respect to the PCB homologue (Ar1221).

  From these results, the anti-PCB monoclonal antibody (ES2G6 antibody) of the present invention can quantitate the PCB homologue PCB having 1 to 2 chlorine atoms, which was difficult to detect with conventional anti-PCB antibodies, with high sensitivity. It has been found useful in monitoring the PCB decomposition process of PCBs waste.

(Monitoring of PCB degradation process using ES2G6 antibody)
The monitoring of the PCB decomposition treatment step using the ES2G6 antibody of Example 1 will be described based on the calibration curve of the ES2G6 antibody against PCB homologues having different chlorine numbers, prepared in Experimental Example 1 (see FIG. 4).
As shown in the calibration curve of FIG. 4, the ES2G6 antibody is capable of detecting a PCB homologue having 1 to 2 chlorine atoms (Ar1221), as well as the detection sensitivity for PCB homologues having 3 to 4 chlorine atoms (KC300, KC400). Sensitivity is very high.
Table 3 below shows the concentrations of PCB homologues having 4 chlorine atoms, PCB homologues having 3 chlorine atoms, homologues having 1 to 2 chlorine atoms and biphenyl contained in the oil sample in the measurement using the ES2G6 antibody. It shows the intensity of each signal when the concentration is 0.05 ppm and 0.10 ppm (the concentrations in the measurement sample are 0.5 ppb and 1.0 ppb). The higher the signal intensity, the lower the affinity between the PCB homologue in the oil sample and the antibody, and the signal intensity of 85% was defined as the lower limit of detection of PCB quantification (IC ₈₅ value).

  As shown in FIG. 4 and Table 3, for example, when a dechlorination treatment is performed on an oil sample containing 0.05 ppm concentration of tetrachlorobiphenyl (for example, Kr400), the ES2G6 antibody is contained in the oil sample. For PCB congeners with 4 to 3 chlorine atoms, concentrations of 0.05 ppm and 0.10 ppm can be quantified, respectively, and for PCB congeners with 1 to 2 chlorine atoms contained in oil samples, concentrations of 0.05 ppm and 0 ppm are possible. .10 ppm can be quantitatively determined. Therefore, by using the ES2G6 antibody, the PCB decomposition treatment process can be monitored until all the chlorine is removed from the PCB and decomposed into biphenyl (harmless).

(Monitoring of PCB degradation process using WR3F6 antibody)
The monitoring of the PCB decomposition treatment step using the WR3F6 antibody of Comparative Example 1 will be described based on the calibration curve of the WR3F6 antibody against PCB congeners having different chlorine numbers, which was prepared in Experimental Example 2 (see FIG. 5).
As shown in the calibration curve of FIG. 5, the WR3F6 antibody has a very low detection sensitivity for the PCB homologue having 1 to 2 chlorine atoms (Ar1221) compared to the detection sensitivity for the PCB homologue having 3 to 7 chlorine atoms.
Table 4 below shows the measurement using the WR3F6 antibody, in which the concentrations of PCB homologues with 4 chlorine atoms, PCB homologues with 3 chlorine atoms, homologues with 1 to 2 chlorine atoms and biphenyl contained in the oil sample are as follows: It shows the intensity of each signal when it is 0.05 ppm and 0.10 ppm (when the concentration in the measurement sample is 0.5 ppb and 1.0 ppb). The higher the signal intensity, the lower the affinity between the PCB homologue in the oil sample and the antibody, and the signal intensity of 85% was defined as the lower limit of detection of PCB quantification (IC ₈₅ value).

  As shown in FIG. 5 and Table 4, for example, when dechlorination treatment was performed on an oil sample containing tetraphenyl biphenyl (Ar1248) at a concentration of 0.05 ppm and a concentration of 0.10 ppm, the WR3F6 antibody Concentrations of 0.05 ppm and 0.10 ppm can be determined for PCB homologues containing 4 to 3 chlorine atoms, respectively, but for PCB homologues containing 1 to 2 chlorine atoms contained in oil samples, concentrations of 0.05 ppm and None of 0.10 ppm can be quantified. That is, the WR3F6 antibody has low detection sensitivity of PCB homologues having 1 to 2 chlorine atoms, and the PCB decomposition treatment process is monitored until the final process in which all chlorine is removed from the PCB and decomposed into biphenyl (harmless). I can't.

The anti-PCB monoclonal antibody of the present invention has a high affinity for a PCB homologue having 1 to 2 chlorine atoms, and does not reduce the binding property to PCB in the presence of a polar solvent, and thus biphenyl monochloride and biphenyl dichloride. PCBs containing a large amount (for example, PCB derived from Arrochlor 1221 (Ar1221) manufactured by Mitsubishi Monsanto (now Mitsubishi Chemical)) with high sensitivity and quantification, such as insulating oil whose composition is unknown, especially PCBs with a low number of chlorine It is possible to detect and quantify a test object that may contain a large amount of homologues with high sensitivity with minimal error, and is suitable for inspection of PCB residual concentration after disposal of waste, PCB concentration in the environment, etc. Can be used for

FIG. 1 is a diagram showing the PCB homolog composition of Aroclor (Source: PCB treatment technology guidebook based on the new standard for waste treatment law, edited by Industrial Waste Treatment Business Foundation, published by Gyosei Co., Ltd.) (Published August 1, 2005). FIG. 2 is a calibration curve of PCBs (Ar1221, KC300, KC400, KC500 and KC600) and biphenyl having different chlorine numbers in the ES2G antibody of Example 1. In FIG. 3, “■” indicates Ar1221, “●” indicates KC300, “▲” indicates KC400, “▼” indicates KC500, “♦” indicates KC600, and “△” indicates biphenyl. FIG. 3 is a calibration curve of PCB (Ar1242, Ar1016, Ar1221, Ar1248, Ar1262, Ar1254, Ar1232, Ar1260 and Ar1221) and biphenyl having different chlorine numbers in the WR3F6 antibody of Comparative Example 1. In FIG. 3, “●” indicates Ar1242, “◯” indicates Ar1016, “▲” indicates Ar1248, “Δ” indicates Ar1261, “■” indicates Ar1254, “□” indicates Ar1232, “♦” indicates Ar1260, “◇” indicates The results of Ar1221 are shown respectively. FIG. 4 is a calibration curve of PCBs (Ar1221, KC300, KC400, KC500 and KC600) and biphenyl having different chlorine numbers in the ES2G6 antibody of Example 1, and is a diagram showing monitoring of the PCB decomposition treatment process. In FIG. 3, “■” indicates the result of Ar1221, “●” indicates the result of KC300, “▲” indicates the result of KC400, “▼” indicates the result of KC500, “♦” indicates the result of KC600, and “Δ” indicates the result of biphenyl. FIG. 5 is a calibration curve of PCBs having different chlorine numbers (Ar1242, Ar1016, Ar1221, Ar1248, Ar1262, Ar1254, Ar1232, Ar1260 and Ar1221) and biphenyl in the WR3F6 antibody of Comparative Example 1, and monitoring the PCB decomposition treatment process FIG. In FIG. 3, “●” indicates Ar1242, “◯” indicates Ar1016, “▲” indicates Ar1248, “Δ” indicates Ar1261, “■” indicates Ar1254, “□” indicates Ar1232, “♦” indicates Ar1260, “◇” indicates The results of Ar1221 are shown respectively.

Claims (12)

An anti-PCB monoclonal antibody, which is an anti-PCB monoclonal antibody produced by a hybridoma having the accession number FERM P-21148. The relation between the equilibrium dissociation constant K1 for a PCB homologue having 1 to 4 chlorine atoms and the equilibrium dissociation constant K2 for a PCB homologue having 5 to 7 chlorine atoms satisfies the following formula: K1 <K2. Anti-PCB monoclonal antibody. The anti-PCB monoclonal antibody according to any one of claims 1 to 2, which has an equilibrium dissociation constant K2 of 4.0 ppb or more. The anti-PCB monoclonal antibody according to any one of claims 1 to 3, wherein the equilibrium dissociation constant K1 is 3.0 ppb or less. The anti-PCB monoclonal antibody according to any one of claims 1 to 4, wherein an equilibrium dissociation constant K3 for a PCB homologue having 1 to 2 chlorine atoms is 2.0 to 3.0 ppb. A hybridoma characterized by being a hybridoma having a deposit number of FERM P-21148 . An animal is immunized with a compound represented by the following general formula (II) obtained from a biphenyl derivative represented by the following general formula (I) as an immunogen, and antibody-producing cells of the immunized animal are collected, and the antibody The hybridoma according to claim 6, which is obtained by fusing a production cell and a myeloma cell.
However, in said general formula (I), n represents the integer of 1, X and Y represent the integer of 0-3, respectively, and the sum of X and Y represents the integer of 1-6.
However, in said general formula (II), n represents the integer of 1, X and Y represent the integer of 0-3, respectively, the sum of X and Y is 1-6, Z represents protein. The hybridoma of Claim 7 obtained by screening using the compound represented by either general formula (I) or general formula (II). An anti-PCB monoclonal antibody according to any one of claims 1 to 5 is added to a sample to be measured,
(1) detecting any of PCB in the sample to be measured bound to the anti-PCB monoclonal antibody, and (2) the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured. PCB measuring method characterized by this. The anti-PCB monoclonal antibody according to any one of claims 1 to 5 and a compound represented by any one of the general formula (I) and the general formula (II) are added as a PCB competitor to a sample to be measured. And detecting the competing substance bound to the anti-PCB monoclonal antibody. Using the carrier in which the anti-PCB monoclonal antibody according to any one of claims 1 to 5 is added to a sample to be measured and the compound represented by the general formula (II) is immobilized as a PCB competitor, the PCB competition A method for measuring PCB, comprising: competing a substance with PCB in the sample to be measured, and detecting the anti-PCB monoclonal antibody not bound to PCB in the sample to be measured.   The PCB measuring method according to claim 9, wherein the PCB homologue having 1 to 2 chlorine atoms is quantified.