JP2007010583A - Immunoassay of pcb - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple high-sensitivity immunoassay of PCB. <P>SOLUTION: In the immunoassay of PCB, an antibody mixture containing at least two kinds of antibodies having different specificities with respect to PCB is brought into contact with a specimen and the formed PCB-antibody composite is detected. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an immunological measurement method for PCB and the like.

Polychlorinated biphenyl (PCB) is non-flammable, has excellent insulation and electrical properties, and is chemically stable, so it can be used as an insulation oil for transformers, capacitors, and wires, and as a heat medium and lubricant in the food, paper, and chemical industries. It has been used in a wide range of industrial fields as a solvent for plasticizers, flame retardants, marine paints, inks and non-carbon paper.
However, since then, it has been found that PCB acts as an endocrine disrupting substance on living organisms and exhibits various toxicities. In Japan, its production and use was prohibited in 1972, and it has reached the present day.
Since PCB is a very stable substance and generates dioxins by incineration, it accurately grasps the amount of PCB contained in discarded insulating oil, heat medium, lubricating oil, plasticizer, contaminated soil, etc. Although it is necessary to manage strictly, since the quantity is huge, it is urgent to establish a simple and highly sensitive PCB measurement method.
However, PCB is a general term for compounds in which hydrogen (H) of a biphenyl structure having two benzene rings is substituted with chlorine (Cl), and there are 209 isomers depending on the number and position of substituted chlorine, PCBs commercially available as industrial products (for example, trade names, Kanechlor: KC and Aroclor: Ar) are also mixtures of PCB isomers of various chlorine numbers (see Tables 1 and 2). . Furthermore, the composition of PCB contained in insulating oil or the like is not constant for each sample. Thus, there has been a demand for a method for easily and highly sensitively measuring commercially available PCBs having complicated compositions containing various isomers having different chlorine numbers.
Gas chromatograph mass spectrometry (GC-MS method), gas chromatograph electron capture detection method (GC-ECD method), etc. are known as high-sensitivity measurement methods for PCBs, but expensive equipment is required. Therefore, there is a problem as a method for measuring a huge amount of samples. In order to avoid this problem, an immunological measurement method using an antibody recognizing PCB has been developed (Patent Document 1). Patent Document 2 discloses a low molecular substance detection instrument such as PCB using immunochromatography. However, since PCB has 209 isomers, if a highly specific antibody is used, only a small part of the isomer of PCB is detected as a result, and a wide variety of isomers are detected at a time. I can't.
In addition, samples such as insulating oil contain a large amount of substances that can interfere with the immunoassay of PCBs, and remove the interfering substances by pre-treatment with an appropriate method prior to measurement. It is necessary to extract the contained PCB into a suitable solvent that does not inactivate the antibody. As a sample pretreatment method, a method of removing monoorthohalopolychlorobiphenyl (moPCB) from a sample by column treatment using a support containing activated carbon or alumina has been reported (Patent Document 3).
JP 2000-191699 A JP 2004-138550 A JP 2003-287539 A

  In view of the above circumstances, an object of the present invention is to provide a PCB measurement method capable of detecting or quantifying PCBs having complicated and diverse compositions with high sensitivity and ease.

As a result of diligent research to solve the above-mentioned problems, the present inventors have used a mixture of two or more antibodies having different specificities for PCBs in an immunological measurement method, thereby making it complicated and diverse. As a result, it was found that PCBs having various compositions can be detected or quantified with high sensitivity at a time, and the present invention has been completed. That is, the present invention relates to the following.
[1] A method for measuring PCB, comprising contacting a sample with an antibody mixture containing two or more antibodies having different specificities for PCB, and detecting or quantifying a PCB-antibody complex that can be formed.
[2] The method according to [1] above, wherein the antibody mixture contains at least the following two antibodies:
(a) an antibody having specificity for PCBs mainly composed of PCBs having 4 or less chlorine atoms;
(b) An antibody having specificity for PCBs mainly composed of PCBs having 5 or more chlorine atoms.
[3] A kit for measuring PCB, comprising a reagent for providing an antibody mixture containing two or more antibodies having different specificities for PCB.

  By using the measurement method of the present invention, PCBs having complicated and diverse compositions can be measured at once and simply and with high sensitivity.

(1. PCB measurement method and kit therefor)
The present invention relates to a method for measuring PCB (cocktail) comprising contacting a sample with an antibody mixture comprising two or more antibodies having different specificities for PCB, and measuring the amount of PCB-antibody complex that can be formed. (Sometimes referred to as an antibody method).

  PCB is a general term for compounds in which hydrogen (H) having a biphenyl structure having two benzene rings is substituted with chlorine (Cl), and has the following formula (I);

(In the formula, n represents an integer of 1 to 5, and m represents 0 or an integer of 1 to 5.)
Or a mixture thereof. The number of chlorine in PCB (the sum of n and m) is an integer of 1 to 10. The position of chlorine substitution on the benzene ring is not particularly limited.

  PCBs commercially available as industrial products (for example, trade names, Kanechlor (KC) and Aroclor (Ar)) are a mixture of PCB isomers of various chlorine numbers. Table 1 shows the composition of the commercially available PCB (component weight ratio for each number of chlorine), and Table 2 shows the chlorine content and main components.

  The antibody mixture used in the measurement method of the present invention contains two or more kinds of antibodies having different specificities for PCB, and preferably two or more kinds of specificities for PCB mainly composed of PCBs having different chlorine numbers. Contains antibodies. By using such an antibody mixture, it becomes possible to detect or quantify PCBs having complicated and various compositions such as commercially available PCBs and PCBs in insulating oil with high sensitivity at a time.

Examples of antibodies include polyclonal antibodies, natural antibodies such as monoclonal antibodies (mAbs), recombinant antibodies produced using gene recombination techniques (for example, single-chain Fv fragments (scFv), bispecific-chimeric scFV (χ- scFv), tandem scFV (scFv) ₂ , bispecific- (scFv) ₂ , disulfide-linked scFv, disulfide-stabilized Fv fragments (dsFv), diabody, single-chain diabody (scDb), bivalent diabody, bispecific diabody, knob-into -hole stabilized diabody, disulfide-stabilized diabody, triabody, tetrabody, trispecific triabody, CL-dimerized scFv, CH1-CL-dimerized scFv, CH3-dimerized scFv, knob-into-hole CH3-dimerized scFv, CH3-dimerized bivalent diabody, Fc-dimerized scFv, Fab-scFv fusions, Ig-scFv fusions, leucine-zipper stabilized scFv dimers, helix-stabilized scFv dimers, 4 helix-bundle stabilized scFv tetramers, streptavidin-scFv, intrabody, Fab 'fragments, F (ab' ) fragments, Fv fragments (Fv), chimeric antibodies, humanized antibodies, This chain antibodies, etc.), human antibodies, and the like that can be produced using human antibody-producing transgenic animal or the like, but is not limited thereto. As the antibody, a polyclonal antibody or a monoclonal antibody is preferably used from the viewpoint of ease of production. Further, it is more preferable to use a polyclonal antibody in order to detect PCBs having complicated and diverse compositions at a time. This is because when a monoclonal antibody is used, due to its high specificity, only a small part of the isomer of PCB may be detected as a result.

Further, in the present invention, “antibody” is a concept including a binding fragment thereof. The antibody-binding fragment means a partial region of the above-mentioned antibody, specifically, for example, F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody), scFv, dsFv (disulfide stabilized) Fv), dAb (single domain antibody), etc. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996), antibody fragments produced by the Fab expression library, etc. Illustrated.

  "An antibody is a specific PCB (PCB having a specific chlorine number, PCB having a specific chlorine number as a main component (for example, 40 wt% or more, preferably 50 wt% or more, more preferably 60 wt% or more as a component weight ratio) ) "Specificity for a commercially available PCB, etc.") means that the affinity of the antibody for a specific PCB (PCB with a specific chlorine number, commercially available PCB containing a PCB with a specific chlorine number as a main component) Is higher than the affinity for other PCBs (PCBs having other chlorine numbers, commercially available PCBs containing PCBs having other chlorine numbers as main components). Specificity for a PCB having a specific chlorine number is determined as specificity for a commercially available PCB containing the PCB having the chlorine number as a main component (for example, trade name: Kanechlor: KC or Aroclor: Ar). obtain. The affinity of an antibody can be determined using, for example, a binding constant or cross-reactivity in ELISA as an index. Cross-reactivity is determined by the antibody binding to a reference PCB (PCB with a reference chlorine number, commercially available PCB containing a PCB with a reference chlorine number as a main component (for example, KC-400), etc.) by a competitive ELISA method, etc. This value is proportional to the reciprocal of the concentration of a specific PCB (PCB with a specific chlorine number, commercially available PCB containing a PCB with a specific chlorine number as a main component) required to inhibit 50%, and is described in the Examples, etc. It can be calculated by the method. For example, when cross-reactivity in ELISA is adopted as an antibody affinity index, the antibody against a specific PCB (PCB having a specific chlorine number, commercially available PCB containing a PCB having a specific chlorine number as a main component, etc.) Cross-reactivity is, for example, 20%, preferably 50% or more compared to cross-reactivity with other PCBs (PCBs with other chlorine numbers, commercially available PCBs containing PCBs with other chlorine numbers as main components). When large or small, it can be defined as “the antibody has specificity for the particular PCB”.

  An antibody having specificity for a specific PCB is produced by immunizing a mammal with a conjugate of PCB and a carrier protein, and isolating an antibody that specifically recognizes the specific PCB Can do.

  Although it does not specifically limit as said carrier protein, For example, bovine serum albumin (BSA), mussel hemocyanin (KLH), ovalbumin (OVA), rabbit serum albumin (RSA), bovine thyroglobulin (BTG) etc. are mentioned.

  The conjugate between the PCB and the carrier protein is not particularly limited as long as the PCB is a compound in which PCB is bound directly or indirectly to the carrier protein, but preferably the conjugate is bound to the carrier protein via a spacer. It is a bound compound. Examples of the spacer include a divalent group containing alkylene and a divalent group derived from polyethylene glycol.

Next, the mammal is sensitized with the conjugate.
In the case of producing a monoclonal antibody, the above-mentioned conjugate is administered to a mammal itself or together with a carrier, a diluent, etc., and the mammal is immunized with the conjugate. In administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be used in order to enhance antibody production ability. The administration is usually performed once every 2 to 6 weeks, and about 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice, rats or chickens are preferably used.

  Selecting an individual with an antibody titer from an immunized mammal, collecting spleen or lymph nodes 2 to 5 days after the final immunization, and fusing the antibody-producing cells contained therein with myeloma cells Thus, a monoclonal antibody-producing hybridoma can be produced. The fusion operation can be performed according to known methods, for example, the method of Kohler and Milstein (Nature, 256, 495 (1975)) and known variations thereof. The monoclonal antibody-producing hybridoma is cultured according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).

  Subsequently, a hybridoma that produces a monoclonal antibody that specifically recognizes a specific PCB is selected from the monoclonal antibody-producing hybridomas. Various methods can be used for the selection. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a specific PCB carrier protein conjugate is adsorbed, and then a radioactive substance or enzyme (horseradish) is used. Anti-immunoglobulin antibody labeled with peroxidase, alkaline phosphatase, etc. (anti-mouse immunoglobulin antibody is used when spleen cells used for cell fusion are derived from mouse) or protein A, and monoclonal antibody bound to a solid phase For example, a method for detecting The hybridoma which produces the antibody which recognizes specific PCB specifically by the above is obtained.

  Subsequently, a monoclonal antibody that specifically recognizes a specific PCB is isolated from the culture supernatant of the hybridoma or ascites obtained by transferring the hybridoma to a pristane-treated nude mouse. Monoclonal antibodies are isolated by methods known per se, such as immunoglobulin separation and purification methods (eg, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Absorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which only the antibody is collected with an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the binding is dissociated to obtain the antibody. Can do.

  When producing a polyclonal antibody that specifically recognizes a specific PCB, like a monoclonal antibody, a conjugate of the specific PCB and a carrier protein is administered to a mammal with itself or a carrier, diluent, etc. A mammal is immunized with the conjugate. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and rabbits, sheep, goats and the like are preferably used.

  Polyclonal antibodies can be isolated from the serum, preferably ascites of mammals immunized by the above method, preferably from serum. Polyclonal antibodies can be isolated according to the same immunoglobulin separation and purification method as the above monoclonal antibody isolation. Preferably, the polyclonal antibody is purified by separating an antibody fraction that specifically recognizes the specific PCB by affinity chromatography using a column to which the specific PCB is bound.

  Furthermore, by treating the antibody fraction with a column to which a PCB of an unintended type (PCB having an undesired chlorine number, a commercially available PCB containing an undesired chlorine number as a main component, etc.) is bound, it is not intended. By removing the antibody fraction having cross-reactivity with the PCB having the chlorine number, an antibody having high specificity for the desired PCB can be obtained.

The type of PCB that is specifically recognized by the antibody contained in the antibody mixture is not particularly limited. However, in order to detect a wider range of PCB isomers with high sensitivity at one time, or the major types contained in insulating oil or the like. In order to reliably measure PCB isomers, the antibody mixture preferably comprises at least the following two antibodies:
(a) an antibody having specificity for PCBs mainly composed of PCBs having 4 or less chlorine atoms;
(b) An antibody having specificity for PCBs mainly composed of PCBs having 5 or more chlorine atoms.

  A PCB mainly composed of a PCB having a chlorine number of 4 or less (for example, 1 to 4, preferably 2 to 4) is 40% by weight or more, preferably 50% by weight, based on a PCB having a chlorine number of 4 or less. As mentioned above, it refers to a PCB composition containing 60% by weight or more. A PCB mainly composed of a PCB having a chlorine number of 5 or more (for example, 5 to 10, preferably 5 to 8) is 40% by weight or more, preferably 50% by weight, based on a PCB having a chlorine number of 5 or more. As mentioned above, it refers to a PCB composition containing 60% by weight or more.

  Here, in the antibody of (a) above, specificity to PCBs mainly composed of PCBs with 3 chlorine atoms (KC-300, etc.) and PCBs mainly composed of PCBs with 4 chlorine atoms (KC-400). It is preferred that the specificity for the “Equivalent specificity” means that the difference in antibody specificity (cross-reactivity etc.) is 10% or less, preferably 6% or less, more preferably 3% or less. In the antibody (b), the specificity for a PCB mainly composed of PCB having 5 chlorine atoms and the specificity for a PCB mainly composed of PCB having 6 chlorine atoms are preferably equivalent. . By using an antibody having such specificity, it is possible to achieve a highly sensitive measurement that gives substantially equivalent sensitivity to PCBs having various compositions.

The content ratio of each antibody contained in the antibody mixture can be appropriately set so as to detect a wider range of PCB isomers with higher sensitivity according to the type and specificity of the antibody.
For example, in the measurement method of the present invention, when an antibody mixture containing the antibodies described in the above (a) and (b) is used, the mixing ratio is the molar ratio [(PCB mainly composed of PCB having 4 or less chlorine atoms). For example, 5: 1 to 1: 5, preferably 3: 1 to 1: 3. For example, 5: 1 to 1: 5, preferably 3: 1 to 1: 3. More preferably, it is 2: 1 to 1: 2.

  In the present specification, “antibody mixture” means a state in which multiple types of antibodies are present in the same phase (solid phase, liquid phase) (for example, multiple types of antibodies are immobilized on the same solid phase). A plurality of types of antibodies are immobilized on a plurality of different solid phases (for example, beads). The state which exists in the same liquid phase is also included.

The contact between the antibody mixture and the sample is usually performed by bringing the sample into contact with a solid phase carrier on which the antibody mixture is immobilized so that the PCB contained in the sample can bind to the antibody and form a PCB-antibody complex. Is done.
The antibody mixture is described, for example, in “Metabolism”, Vol. 8, 696 (1971), bromocyan method, glutaraldehyde method, carbodiimide method, epoxy activation method, “Enzyme Immunoassay”, pages 268-296. Can be immobilized on a solid phase carrier by a known method such as the method described in “Affinity Chromatography Handbook” (Amersham Pharmacia Biotech Co., Ltd. (issued on Dec. 20, 1998)). Examples of the immobilization carrier include microplates (eg, 96-well microplates, 24-well microplates, 192-well microplates, 384-well microplates), test tubes (eg, glass test tubes, plastic test tubes), Glass particles, polystyrene particles, modified polystyrene particles, polyvinyl particles, latex (eg, polystyrene latex), nitrocellulose membrane, cyanogen bromide activated filter paper, DBM activated filter paper, granular solid phase (eg, sepharose, sephadex, agarose) , Cellulose, Sephacryl, etc.), iron-containing polycarbonate films, magnetic beads, crystal resonators, gold colloids and the like.

  Examples of the sample used in the measurement method of the present invention include, but are not limited to, insulating oil, lubricating oil, plasticizer, flame retardant, marine paint, solvent such as ink and non-carbon paper, soil, and the like. . Samples such as insulating oil contain substances that can interfere with the measurement method of the present invention. Pretreatment with an appropriate method removes the interfering substances and inactivates the PCB contained in the sample with antibodies. It is preferable to use for the method of this invention, after extracting to the suitable solvent which does not have it. Examples of the pre-treatment method include polychlorinated biphenyl (PCB) in JEAC1201 insulating oil established by the Electricity Research Committee of the Japan Electric Association, for example, No. 1 of Appendix 192 of the Ministry of Health and Welfare Notification No. 1992. Examples include the method described in the analysis method definition (1991) and the method described below.

  PCB-antibody complexes that can be formed include radioisotope immunoassay (RIA method), ELISA method (Engvall, E., Methods in Enzymol., 70, 419-439 (1980)), fluorescent antibody method, plaque method. Various methods commonly used for antigen detection, such as spot method, agglutination method, and Ouchterlony ("Hybridoma method and monoclonal antibody", R & D Planning, Inc., pages 30-53, Showa 57 March 5, 2011), a method using a crystal resonator (Japanese Patent Laid-Open No. 2002-310874), or the like. The ELISA method is widely used from the viewpoints of sensitivity and convenience.

In the method for measuring PCB of the present invention, a so-called competition method in which a sample and a known amount of labeled PCB are quantified by quantitative competition reaction of binding to an antibody is preferably used. Non-competitive methods such as “High-sensitivity non-competitive measurement of small molecules by the open sandwich method” (Abstracts of the 9th (2004) Scientific Symposium of Immunochemical Assays, 9-10 (2004)) It is. In the competition method, a sample solution containing an unknown amount of PCB and a known amount of labeled PCB are mixed, and the mixture is brought into contact with the antibody mixture immobilized on a carrier. Then, the labeled PCB-antibody complex held on the carrier is detected.
Examples of labeling agents for labeling PCB include labeling enzymes (horseradish peroxidase, alkaline phosphatase, etc.), radioisotopes ( ^125I, etc.), enzyme substrates, fluorescent substances (FITC, PE, APC, GFP, etc.), biotin, etc. Is mentioned. For binding of PCB and these labeling agents, maleimide method [Journal of Biochemistry (J. Biochem.), 79, 233 (1976)], activated biotin method [Journal of American Chemical Society ( J. Am. Chem. Soc.), 100, 3585 (1978)].

  More specifically, for example, a sample solution containing an unknown amount of PCB and a known amount of labeled PCB are mixed, and the mixture is brought into contact with the antibody mixture immobilized on a carrier. Next, the solid phase is thoroughly washed, and the activity of the labeling agent bound on the solid phase is measured. When the labeling agent is a radioisotope, it is measured with a well counter or a liquid scintillation counter. When the labeling agent is a labeling enzyme, the substrate is added and allowed to stand, and the enzyme activity is measured by a colorimetric method or a fluorescence method. Even if the labeling agent is a fluorescent substance or the like, it can be measured according to a known method.

When the competition method is used in the method for measuring PCBs of the present invention, it is preferable to mix and use labeled PCBs corresponding to PCBs that are specifically recognized by the respective antibodies contained in the antibody mixture.
The mixing ratio of the labeled PCB can be determined as follows.
First, for each antibody contained in the antibody mixture, the PCB concentration in the sample is reduced to 0 by the competition method using only one type of each antibody, the PCB that each antibody specifically recognizes, and the corresponding labeled PCB. In this case, the label signal intensity (absorbance, fluorescence intensity, etc.) is measured. Next, for each antibody, when the PCB concentration is 0, the concentration of the labeled PCB is adjusted so as to give the same label signal intensity when the same antibody amount is used. Then, the corresponding labeled PCB is mixed at the same ratio as the mixing ratio (molar ratio) of each antibody in the antibody mixture.
As described above, by mixing different labeled PCBs, it is possible to carry out a highly quantitative measurement having substantially the same sensitivity even for PCBs having different compositions.

  The present invention also provides a kit for measuring PCB, comprising a reagent for providing an antibody mixture containing two or more antibodies having different specificities for PCB.

Examples of the reagent include an antibody mixture itself containing two or more kinds of antibodies having different specificities for the above-mentioned PCB, or each antibody in an unmixed state. In addition, the kit may contain various reagents that can be used in the above method, such as labeled PCB, washing solution, chromogenic substrate, chromogenic stop solution, standard PCB, instructions describing the test protocol, and the like. As the washing solution, distilled water, PBS (phosphate buffered saline), T-PBS (Tween-20-containing phosphate buffered saline, etc.) can be used. 3,3 ′, 4,4′-tetramethylbenzidine (TMBZ), etc. As the color stop solution, 0.5N sulfuric acid, 0.5M phosphoric acid, etc. can be used. In addition, a combination of PCB (or a derivative thereof) and an enzyme (for example, peroxidase) can be used.
If the kit for PCB measurement of the present invention is used, PCB measurement can be easily performed by the above method.

(2. Preparation method of sample for immunological measurement of PCB)
This specification describes a method for preparing a sample for immunological measurement of PCB, comprising the following steps.
(a) mixing the sample with hexane and DMSO and collecting the DMSO fraction;
(b) diluting the DMSO fraction with an aqueous mineral salt solution, mixing the dilution with hexane, and collecting the first hexane fraction;
(c) mixing the first hexane fraction with the sulfonating agent and collecting the second hexane fraction;
(d) DMSO is added to the second hexane fraction, hexane is evaporated, and the residue is obtained as a sample for immunoassay of PCB.

  Samples that can be subjected to the preparation method described herein include insulating oils, lubricating oils, plasticizers, flame retardants, marine paints, solvents such as ink and non-carbon paper, soil, etc. It is not limited to these.

The amount of hexane used in the step (a) can be appropriately selected depending on the type of the sample and the like. For example, when the sample is insulating oil, it is usually about 0.05 to 2 mL per 50 mg of the sample. . The amount of DMSO used is not particularly limited as long as PCB extraction into the DMSO fraction is achieved, but it is usually about 0.25 to 5 mL with respect to 1 mL of hexane. From the viewpoint of suppressing the generation of emulsion during mixing with hexane and improving operability, it is preferable to use about 0.5 to 3 mL (for example, about 2 mL) of DMSO with respect to 1 mL of hexane. The DMSO is preferably saturated with hexane.
The sample is mixed with hexane and DMSO using a multimixer or the like so that PCB in the sample is efficiently extracted into DMSO.

  From the viewpoint of more reliably removing the interfering substance, step (a) may be repeated. The number of repetitions of step (a) is not particularly limited, but is usually about 2 to 5 times.

In step (b), first, the DMSO fraction collected in step (a) is diluted with an aqueous inorganic salt solution. Examples of inorganic salts include NaCl. The concentration of the inorganic salt in the aqueous inorganic salt solution is not particularly limited as long as the preparation method of the present invention is not inhibited. For example, when NaCl is used as the inorganic salt, it is usually 2 to 10 (w / v)% (for example, about 5 (W / v)%). The addition amount of the inorganic salt aqueous solution is not particularly limited, but is usually about 2.5 to 6 mL (for example, about 4 mL) per 1 mL of the DMSO fraction.
The diluted DMSO fraction is then mixed with hexane. The amount of hexane to be added is not particularly limited as long as it does not inhibit the preparation method of the present invention, but is usually about 0.5 to 2.5 mL (for example, about 1.5 mL) with respect to 10 mL of diluted DMSO fraction. is there. By vigorously stirring the mixture with a vortex mixer or the like, the PCB is back-extracted into the hexane fraction.

  In step (c), the hexane fraction collected in step (b) is mixed with a sulfonating agent. By the sulfonating agent treatment, the interfering substances contained in the hexane fraction are sulfonated and removed to the sulfonating agent layer. The sulfonating agent is not limited as long as the preparation method of the present invention can be achieved, but fuming sulfuric acid diluted in sulfuric acid is preferable. The concentration of fuming sulfuric acid in sulfuric acid is not limited as long as the preparation method of the present invention can be achieved, but is usually about 5 to 30%, preferably about 7.5 to 25% (for example, about 10%). By using fuming sulfuric acid having a concentration of about 5 to 30%, interfering substances can be removed with high efficiency. The amount of fuming sulfuric acid diluted in sulfuric acid is not particularly limited, but is usually about 0.5 to 2 mL (for example, about 1 mL) per 0.7 mL of hexane fraction.

  Prior to the sulfonating agent treatment, the hexane fraction collected in step (b) may be dehydrated. Dehydration can be performed by a method known per se, for example, by mixing a hexane fraction with anhydrous sodium sulfate.

  Moreover, you may remove the acid mixed in the hexane fraction by mixing the hexane fraction treated with the sulfonating agent with an inorganic salt aqueous solution. The inorganic salt is not particularly limited as long as the preparation method described in the present specification is not inhibited, and for example, NaCl can be mentioned. The concentration of the inorganic salt in the aqueous inorganic salt solution is not particularly limited as long as it does not inhibit the preparation method described herein. For example, when NaCl is used as the inorganic salt, the concentration is usually 2 to 10 (w / v)% ( For example, about 5 (w / v)%). Although the addition amount of inorganic salt aqueous solution is not specifically limited, It is about 0.5-2 mL (for example, about 1 mL) normally about 1 mL of hexane fractions.

In step (d), DMSO is first added to the hexane fraction treated with the sulfonating agent. DMSO is preferably saturated with hexane. The amount of DMSO added is not particularly limited, but is usually about 0.5 to 2 mL (for example, about 1 mL) per 1 mL of hexane fraction.
Next, hexane in the hexane fraction is evaporated. The evaporation is usually performed at room temperature using an extractor concentrator or the like. By evaporating hexane, PCB in the hexane fraction is extracted into a DMSO fraction (residue), and the residue can be obtained as a sample for immunoassay of PCB.

  The residue may be used directly for PCB immunoassay, but in order to avoid the possibility that contaminated hexane will affect the immunoassay, the residue is diluted appropriately with DMSO, and then immunologically analyzed. It may be used for measurement.

  According to the preparation method described in the present specification, it is possible to easily prepare a sample for PCB immunological measurement from which interference substances are removed at a high rate, on a small scale. By measuring PCB by the measurement method of the present invention using a sample prepared by using the method, it is possible to measure PCB in a large number of samples with extremely high sensitivity, simplicity, and low cost. Become.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

Reference Example 1 Pretreatment of insulating oil sample About 50 mg of insulating oil sample was accurately weighed into a test tube. 1 mL of hexane was added to the sample and stirred for 1 minute with a multimixer. Further, 2 mL of hexane-saturated DMSO was added, and the mixture was stirred for 1 minute with a multimixer. When an emulsion was formed, the layers were separated cleanly by centrifugation (2000 rpm / min) (the same applies to the following stirring operation).
Next, the hexane layer was removed, 1 mL of hexane was added to the DMSO layer, and the mixture was stirred with a multimixer for 1 minute. The hexane layer was removed again, 1 mL of hexane was added to the DMSO layer, and the mixture was stirred for 1 minute with a multimixer.
The hexane layer was removed and the DMSO layer was transferred to a test tube. The DMSO layer was diluted by adding 8 mL of 5% NaCl aqueous solution to the DMSO layer, 1.5 mL of hexane was further added, and the mixture was stirred with a vortex mixer for 1 minute.
1 mL of the hexane layer was transferred to a test tube in which about 10-30 mg of anhydrous sodium sulfate had been put in advance, and the mixture was stirred for 20-30 seconds by a vortex mixer. To a test tube in which 1 mL of 10% fuming sulfuric acid had been dispensed in advance, 0.7 mL of the hexane layer was transferred and stirred with a multimixer for 1 minute.
The hexane layer (0.4 mL) was transferred to a test tube in which 0.4 mL of 5% NaCl aqueous solution had been dispensed in advance, and the mixture was stirred with a vortex mixer for 20-30 seconds.
Into a test tube in which 0.2 mL of hexane-saturated DMSO was dispensed in advance as a keeper, 0.2 mL of the hexane layer was transferred, and the hexane layer was evaporated at room temperature (about 20-28 ° C.) using an extractor concentrator. .
The remaining DMSO layer (0.2 mL) was diluted by adding DMSO (0.8 mL) and used for ELISA measurement.

Example 1 Measurement of PCB concentration in insulating oil sample by ELISA method
Reagents and methods 1) ELISA kits For the measurement of PCB concentration in insulating oil samples, two types of ELISA kits manufactured by Abraxis (medium to highly chlorinated PCB (HC-PCB) measurement kit: product number PN530001), and (low Chlorinated PCB (LC-PCB) measurement kit: product number PN530021)
Were used alone or in appropriate combination. The composition of main reagents included in each kit is as follows.
1. Anti-PCB antibody-bound magnetic beads (composition: magnetic beads bound with anti-PCB antibodies)
2. PCB-enzyme complex (AgE) (Composition: PCB (or its derivative) and enzyme (eg, peroxidase) combined)
3. Coloring solution (composition: enzyme substrate (for example, 3,3 ', 4,4'-tetramethylbenzidine (TMBZ) in the case of peroxidase))
4). Stop solution (Composition: 0.5N sulfuric acid, 0.5M phosphoric acid, etc.)
5. Wash solution (composition: PBS (phosphate buffered saline) or T-PBS (phosphate buffered saline containing Tween-20))

  The anti-PCB antibody contained in the anti-PCB antibody-bound magnetic beads in the HC-PCB measurement kit is a polyclonal antibody (anti-HC-PCB antibody) having specificity for medium to high chlorinated PCB, and is a product number PN530001 from Abraxis. Is commercially available.

  The anti-PCB antibody contained in the anti-PCB antibody-bound magnetic beads in the LC-PCB measurement kit is a polyclonal antibody (anti-LC-PCB antibody) having specificity for low-chlorinated PCB, and is commercially available from Abraxis as part number PN530021 Has been.

In some tests, anti-HC-PCB antibody-bound magnetic beads and anti-LC-PCB antibody-bound magnetic beads were mixed at an appropriate ratio (volume ratio) and used (cocktail antibody method).
In some tests, commercially available HC-AgE and commercially available LC-AgE were mixed at an appropriate ratio (volume ratio).

2) Preparation of standard sample
Kanechlor KC-400 (GL-Science: Cat No. 1021-19104) was used as the PCB reference material unless otherwise specified. Kanechlor KC-400 was dissolved in 100% DMSO, and a 100% DMSO solution containing 0.5, 1, 2, 5, 10, 30, 100, 300, 1000 ppb PCB was prepared as a standard sample.
When testing cross-reactivity for PCBs with different chlorine numbers,
KC-300 (GL-Science: Cat No. 1021-19103)
KC-500 (GL Sciences: Cat No. 1021-19105)
KC-600 (GL Science: Cat No. 1021-19106)
A standard sample was prepared in the same manner as KC-400.

3) Measurement of sample In a polystyrene test tube, 80 μL of the sample pretreated by the above method or a PCB standard sample was placed. Add distilled water (125 μL), PCB-enzyme complex (250 μL), and PCB antibody-bound magnetic beads (500 μL), stir for 1-2 seconds with a vortex mixer, and let the mixture stand at room temperature for 30 minutes. Was reacted. The test tube was attached to a magnetic separator and allowed to stand for 2 minutes to separate the magnetic beads. The solution in the test tube was discarded while being attached to the magnetic separator. After the test tube was removed from the separator, a washing solution (1 mL) was added, stirred for 1-2 seconds with a vortex mixer, attached to the magnetic separator, and allowed to stand for 2 minutes to separate the magnetic beads. The solution in the test tube was discarded while being attached to the magnetic separator. Furthermore, after removing the test tube from the separator, a washing solution (1 mL) was added, stirred for 1-2 seconds with a vortex mixer, attached to the magnetic separator, and allowed to stand for 2 minutes to separate the magnetic beads. The solution in the test tube was discarded while being attached to the magnetic separator. After removing the test tube from the separator, a coloring solution (500 μL) was added, stirred for 1-2 seconds with a vortex mixer, and the mixture was allowed to stand at room temperature for 40 minutes for reaction. Stop solution (500 μL) was added, and absorbance measurement (450 nm) was measured using a visible spectrophotometer and a microcell. The PCB concentration (ppb) in the sample was calculated from the standard curve, and the value was corrected as necessary based on the amount of sample collected, the pretreatment recovery rate, and the like.

4) Preparation of standard curve Using the prepared standard sample for ELISA measurement, the absorbance at each concentration (0, 0.5, 1, 2, 5, 10, 30, 100, 300, 1000 ppb) was measured according to the above measurement flow. Next, a measured value was obtained from the obtained absorbance by a 4-parameter equation using Deltasoft.
4-parameter formula: Y = ((AD) / (1+ (X / C) ^B ) + D
A: absorbance at 0 concentration, B: slope at 50% inhibitory concentration (IC ₅₀ ), C: IC ₅₀ ,
D: Minimum absorbance (background), Y: Absorbance (Abs. 450 nm),
X: Concentration (ppb) in sample (100% DMSO solution)

5) Conversion of PCB concentration in the sample PCB concentration (ppm) in the sample is obtained by obtaining the ELISA concentration conversion value (ppb) from the ELISA signal value (absorbance) using the standard curve, and then the sample dilution multiple according to the following formula And corrected by the recovery rate in the pretreatment.
[PCB concentration in sample (ppm)] = [ELISA concentration conversion value (ppb)] X [Sample dilution multiple ^* ] / [PCB recovery rate in pretreatment] / 1000
* Sample dilution factor: When the sample weighed about 50 mg was diluted 150 times and measured by ELISA, the value obtained by dividing 50 X 150 = 7500 by the actual sample amount (mg) was used.

6) Measurement of PCB concentration by gas chromatography electron capture detector (GC-ECD) method In some tests, the GC-ECD method is applied according to the analysis method specification (1991) for polychlorinated biphenyls (PCB) in JEAC1201 insulating oil. Was used to measure the PCB concentration.

Result 1) Reactivity of PCB-enzyme complex to anti-PCB antibody Binding to each anti-PCB antibody of each PCB-enzyme complex (AgE) contained in the medium to high chlorinated PCB measurement kit or low chlorination PCB measurement kit The reactivity to magnetic beads was confirmed. As a result, the reactivity of HC-AgE to the anti-LC-PCB antibody was OD: 0.475, which was about 2/3 of the reactivity to the anti-HC-PCB antibody (OD: 0.715) (Table 3). On the other hand, LC-AgE showed almost no reactivity with anti-HC-PCB antibody (OD: 0.156). Therefore, when mixing anti-HC-PCB antibody-bound magnetic beads and anti-LC-PCB antibody-bound magnetic beads, the effects of anti-HC-PCB antibody and anti-LC-PCB antibody can be exhibited. In addition, it was considered necessary to dilute each AgE so that the absorbance would be about the same, and then mix and use.
Anti-HC-PCB antibody-bound magnetic beads for both AgEs: The reactivity of the anti-LC-PCB antibody-bound magnetic beads (1: 1) with the mixed beads was confirmed. For HC-AgE, the kit stock solution and LC- For AgE, it was found that the same OD (0.64) was obtained by diluting the kit stock solution with PBS 1.7 times (Table 3, FIG. 1). From this, when using 1: 1 mixed beads, HC-AgE (stock solution) and LC-AgE (1.7-fold dilution) were mixed and used in an equal volume.

2) AgE adjustment concentration at various antibody bead mixing ratios Anti-HC-PCB antibody-binding magnetic beads: Anti-LC-PCB antibody-binding magnetic beads = 1 to 4: 1, and the AgE adjustment concentration at each bead mixing ratio is the same as described above. Determined (Table 4).

3) KC cross-reactivity at various bead mixing ratios KC cross-reactivity at various antibody bead mixing ratios compared to those when anti-HC-PCB antibody-bound magnetic beads or anti-LC-PCB antibody-bound magnetic beads are used alone (FIG. 2). By using a mixture of two types of antibody beads, the distribution of cross-reactivity with KC converged, suggesting the effectiveness of the cocktail antibody method.
Therefore, anti-HC-PCB antibody-coupled magnetic beads that have an antibody bead mixing ratio that can be maintained at a relatively high OD when the PCB concentration is 0: anti-LC-PCB antibody-coupled magnetic beads = 1: 1 and 2: 1 Therefore, it was decided to obtain comparison data with instrumental analysis of actual samples.

4) Evaluation of cocktail antibody method using real sample Using insulation oil sample, cocktail antibody method (anti-HC-PCB antibody-coupled magnetic beads: anti-LC-PCB antibody-coupled magnetic beads = 1: 1 or 2: 1) FIG. 3 shows the result of comparison between the measured value of ELISA and the measured value by GC-ECD method.
In the cocktail antibody method, the correlation coefficient was improved, and in particular, R = 0.991 for anti-HC-PCB antibody-bound magnetic beads: anti-LC-PCB antibody-bound magnetic beads (2: 1). From the above results, it was considered that “anti-HC-PCB antibody-bound magnetic beads: anti-LC-PCB antibody-bound magnetic beads = 2: 1” is preferable as the bead mixing ratio in the cocktail antibody method.

5) Cross-reactivity with cocktail antibody method KC cross-reactivity with cocktail antibody method (bead mixing ratio / anti-HC-PCB antibody-coupled magnetic beads: anti-LC-PCB antibody-coupled magnetic beads = 2: 1) (ELISA measurement) Was compared with the case of using only anti-HC-PCB antibody-bound magnetic beads or anti-LC-PCB antibody-bound magnetic beads.
In addition, the cross-reactivity was calculated by the following equation from the concentration (IC ₅₀ ) when the inhibition rate = 50% for each KC.
Cross reactivity (%) = (IC _{50 of} KC-400) / (IC _{50 of} each KC) × 100
As a result, cross-reactivity increased from 49 to 101% with KC-300, 217 to 118% with KC-500, and 217 to 55% with KC-600 compared to using only anti-HC-PCB antibody-bound magnetic beads. It was confirmed that each was improved (Table 5, FIG. 4).

6) Evaluation of cocktail antibody method using insulating oil sample ELISA measurement using cocktail antibody method (anti-HC-PCB antibody-bound magnetic beads: anti-LC-PCB antibody-bound magnetic beads = 2: 1) using insulating oil samples The values were compared with those measured by the gas chromatography electron capture detector (GC-ECD) method. Tables 6 and 7 show the measurement results for the standard curve and the measurement results for the insulating oil sample.

(Calculation of conversion factor (PCB pretreatment recovery rate in cocktail antibody method))
The correlation between the GC-ECD method measurement values and the cocktail antibody ELISA measurement values in Table 7 was examined (FIG. 5). From this result, the concentration conversion factor in the insulating oil sample was determined to be 0.468 from the ELISA method measurement value.

(Effect of cocktail antibody method)
The quantitative values of the anti-HC-PCB antibody-coupled magnetic bead method (described as General) and the cocktail antibody method (described as Cocktail) were compared with the quantitative values of the GC-ECD method (FIG. 6). By using the cocktail antibody method, excess and under-quantification due to cross-reactivity are greatly reduced, and the ratio between the ELISA quantitative value and the GC-ECD instrumental analysis value is 47 to 50 for the anti-HC-PCB antibody-coupled magnetic bead method. While it was 197%, the cocktail antibody method was 61 to 119%, which was as accurate as the instrumental analysis method.

(Correlation between cocktail antibody conversion value and GC-ECD measurement value)
The correlation between the cocktail antibody method conversion value and the GC-ECD method measurement value was examined (FIG. 7). By using the cocktail antibody method, excess and under quantification due to cross-reactivity is reduced, and the slope is 1.117 → 1.002 compared to the measurement by ELISA method using only anti-HC-PCB antibody-conjugated magnetic beads. The correlation coefficient (R) was also improved from 0.93 to 0.99.

(Creation of standard curve and setting of detection lower limit and quantitative range)
A typical calibration curve when using a standard sample (KC-400 in 100% DMSO) for cocktail antibody ELISA is shown in FIG. Concentrations indicating inhibition rates of 5-10% (B / B0 = 90-95%) and 15-20% (B / B0 = 80-85%) are the lower detection limit (3ppb) and lower limit of quantitation (7ppb), respectively. The upper limit concentration (1000 ppb) of the standard substance that provides linearity between the standard substance concentration and B / B0 was defined as the upper limit of quantification.
Furthermore, from the dilution rate (150 times) and the recovery rate (46.8%) in the insulating oil pretreatment, the detection and quantification range at the time of measuring the insulating oil sample was set as follows.
Detection lower limit: 3ppb (kit detection lower limit) X 150 (dilution factor) /0.468 (recovery rate) / 1000 = 0.96ppm
Lower limit of quantification: 7 ppb (lower limit of kit quantification) X 150 (dilution ratio) /0.468 (recovery rate) / 1000 = 2.2 ppm
Upper limit of quantification: 1000ppb (upper limit of kit quantification) X 150 (dilution ratio) /0.468 (recovery rate) / 1000 = 320ppm

  By using the measurement method of the present invention, it is possible to measure a wide range of PCB isomers at once, simply and with high sensitivity.

It is the figure which showed the correlation with the dilution rate of LC-AgE, and a light absorbency when 1: 1 mixed beads are used in the cocktail antibody method. It is the figure which showed the cross-reactivity with canechlor in various antibody bead mixing ratio. It is the figure which showed the correlation with the measured value of ELISA, and the measured value by GC-ECD method. It is the figure which showed the cross-reactivity with canechlor in the cocktail antibody method. It is the figure which showed the correlation with the measured value of ELISA by the cocktail antibody method, and the measured value by GC-ECD method. It is the figure which showed the comparison of the quantitative value of ELISA method and GC-ECD method. It is the figure which showed the correlation with the conversion value by a cocktail antibody method, and the measured value by GC-ECD method. It is the figure which showed the typical calibration curve in the cocktail antibody method.

Claims (3)

  A method for measuring PCB, comprising contacting a sample with an antibody mixture comprising two or more antibodies having different specificities for PCB, and detecting or quantifying a PCB-antibody complex that can be formed. 2. The method of claim 1, wherein the antibody mixture comprises at least the following two antibodies:
(a) an antibody having specificity for PCBs mainly composed of PCBs having 4 or less chlorine atoms;
(b) An antibody having specificity for PCBs mainly composed of PCBs having 5 or more chlorine atoms.   A kit for measuring PCB, comprising a reagent for providing an antibody mixture comprising two or more antibodies having different specificities for PCB.

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