KR100893030B1 - Immune reaction measurement carrier, immune reaction measuring apparatus, and immune reaction measuring method - Google Patents

Abstract

SUMMARY OF THE INVENTION An object of the present invention is to measure an immune response comprising a carrier for measuring an immune response that can capture antibodies with high uniformity and high density and can detect a measurement with high sensitivity and can be efficiently prepared at low cost, and the carrier for measuring the immune response. An apparatus and an immune response measuring method using the carrier for measuring an immune response are provided.

In other words, the present invention, the test material X having no immunogenicity, the polymer material Z having immunogenicity, the test material X and the test material produced by using a complex represented by XYZ consisting of a linker Y combining the polymer material Z The present invention relates to a carrier for measuring immune response, which is used to capture antibody X 'against substance X, and has any one of at least part of a structure of linker Y, and an analog thereof. The present invention also relates to an immune response measuring apparatus including the immune response measuring carrier, and an immune response measuring method using the carrier for measuring the immune response.

Immune response carrier, device for measuring immune response, method for measuring immune response, immunogenicity

Description

Immune reaction measurement carrier, immune reaction measuring apparatus, and immune reaction measuring method}

The present invention relates to a carrier for measuring an immune response used for measuring the amount of an antibody, an apparatus for measuring an immune response provided with the carrier for measuring an immune response, and a method for measuring an immune response using the carrier for measuring an immune response.

PCBs (polychlorinated biphenyls) have been widely used as electrical insulation materials such as transformers and capacitors, and heat carriers because of their high stability and insulation properties. Manufacture or use is prohibited. PCB is regulated as a representative chemical of persistent organic pollutants that are difficult to decompose in the environment, accumulate in living organisms through plant chains, and affect human health and ecosystem. It is compulsory to store the waste containing it until it is disposed of properly so as not to interfere with the preservation of the living environment.

However, PCB wastes that have been stored without proper treatment are likely to be lost or leaked due to prolonged storage, and in 2001, "Promoting Proper Treatment of Polychlorinated Biphenyl Wastes." Act on Special Measures for the Act (Special Act on PCBs) was implemented. According to this PCB special law, proper disposal of all PCB waste within 15 years is required.

For example, in the case of insulating oil, in addition to the insulating oil stored, it is known that the replacement insulating oil substituted in the transformer which used the PCB as insulating oil in the past is also contaminated with the PCB remaining in a small amount in the transformer. In addition, it is necessary to inspect the insulating oil used for a myriad of electric devices, including replacement insulating oils among such transformers, and to quickly clarify the necessity of processing. In addition, it is also very important to inspect the PCB residual concentration after treatment and the PCB concentration in the environment for the PCB waste treated.

Conventionally, as a method for analyzing environmental pollutants such as PCB, for example, as a process method, high resolution gas chromatograph-high resolution mass spectrometry (HRGC-HRMS) or gas trapping with electron trapping detector ( The GC-ECD Act is used (see 「Regulations of Methods for Analyzing Polychlorinated Biphenyls (PCB) in Insulating Oils” (Edited by the Electrotechnical Standards Investigation Committee, published by the Japan Electric Association, September 30, 1991). Although these methods have high resolution and low quantitative lower limit, the time required for analysis is long, and the operation of a sample for removing a contaminant that obstructs the analysis is complicated, and the cost is high. There is a problem that is large. For this reason, as a simple and quick analysis method, the method which can be measured easily and easily in a measurement site has been calculated | required.

On the other hand, as a simple and rapid analysis method, an immunological assay (immunoassay) using an antigen antibody reaction has been proposed. Among the immunoassays, the most common measurement method is enzyme immunoassay (ELISA). As the ELISA method, radioimmunoassay (RIA), fluorescent immunoassay (FIA), enzyme immunoassay (EIA, ELISA) and the like are known.

However, the ELISA method has a problem of requiring a special device for detecting a test substance, or requiring a long time for pretreatment or measurement of a sample. For this reason, the immunochromato method which is easy to operate is proposed.

However, in the ELISA method and immunochromatography method, there is a fundamental problem in detection. In both methods, a so-called commonly known sandwich method is used in which a test substance is sandwiched with two molecules of antibodies on a carrier in order to obtain the most sensitive detection sensitivity. However, when the test substance is a low molecular compound like the PCBs described above. The sandwich method does not hold. This is because, since the antigen as the test substance is a low molecule, when the antibody as the biopolymer is bound to the test substance, the test substance is buried at the binding site of the antibody, and other antibodies cannot bind.

 For this reason, in most cases of the immunoassays represented by the ELISA method and the immunochromatography method, the competition method is employed instead of the sandwich method.

A competition method is a method of competing the said low molecular weight compound which is a test substance, and the said low molecular weight compound or the analogous compound with respect to the said low molecular weight compound in binding to an antibody, when detecting the said test material. However, this competition method has a principle disadvantage in that detection sensitivity up to the binding ability of the original antibody is difficult to be obtained as a result of the competitive binding of the test substance in the sample and the similar compound of the test substance to the antibody.

As a method other than the ELISA method, a flow-through immunoassay is known.

As the flow-through immunoassay, for example, an antibody to the test substance is reacted in a sample containing the test substance, and the free antibody which does not bind the test substance in the sample is detected. As a result, a method of detecting a test substance in the sample has been proposed (for example, see Japanese Patent Laid-Open No. 2004-138550). In addition, a capture reagent for capturing the subject (for example, when the subject is an antibody, the antibody is an antigen or pseudo antigen that is specifically recognized, and when the subject is an antigen, A sample containing the measurement object is supplied onto a carrier on which an antibody that specifically recognizes an antigen is immobilized, and when the sample passes the carrier, the measurement object captured by the capture reagent is randomly selected. The method of detection by the method of the following is proposed (for example, refer Unexamined-Japanese-Patent No. 2005-214670).

However, in the flow-through immunoassay, however, similarly to the ELISA method, since competitive binding occurs between the test object and the capture reagent in the sample solution, detection sensitivity up to the binding ability of the antibody is not obtained. .

In the flow-through immunoassay, the contact time between the carrier and the sample is made very short, so that the antibody is specifically fixed when the measurement object or capture reagent (e.g., the measurement object is an antibody) immobilized on the carrier. When the antigen to be recognized or the pseudo antigen is high and the object to be measured is an antigen, it is difficult for a competitive binding to occur between the antibody to specifically recognize the antigen) and the object or capture reagent in the sample solution. As a result, it is considered that good detection sensitivity is obtained.

 In the above method, since the carrier and the sample are only in contact with each other at an instant, a carrier capable of capturing a large number of objects or capture reagents in a limited time is required to ensure detection sensitivity and quantitative quality. . For this reason, the said carrier which immobilized the said to-be-measured object or capture reagent with high density is calculated | required. There is a need for a carrier for measuring immune response that is uniformly and at high density and efficiently capable of immobilizing the measurement target or capture reagent, capable of detecting the measurement with high sensitivity, and efficiently preparing at low cost, but such a carrier is important. As it is not recognized, it is not developed yet and does not exist.

On the other hand, although the improvement of the fixation method of a to-be-measured object or a capture reagent is proposed with respect to a support | carrier, there is a limitation to the improvement of a fixation method fundamentally, and as long as an operation called a fixation is performed, a to-be-measured object or a capture reagent is fixed. There is a problem that the cost and time of fixation itself and the fixing itself are taken. This is a common problem in immunoassays.

This invention solves the said problem in the past, and makes it a subject to achieve the following objectives. In other words, the present invention provides an immune response carrier having a uniform and high density of an antibody, capable of detecting an object to be detected with high sensitivity, and capable of efficiently preparing at low cost, and a carrier for measuring the immune response. An object of the present invention is to provide a measuring device and a method for measuring an immune response using the carrier for measuring an immune response.

In order to solve the above-mentioned problems, the inventors of the present invention have intensively studied, and as an antibody against a low molecular weight substance, the above-described low molecular weight substance, a linker, and an antibody preparation compound comprising the high molecular weight substance having immunogenicity are used. The produced antibody was found to be captured by a carrier having any one of the structure of a part of the linker and an analog thereof in the molecule even when the antibody did not fix the pseudo antigen to the carrier, thus completing the present invention. .

This invention is based on the said fact by this inventor, As means for solving the said subject, it is as follows. In other words,

<1> antibody X to test substance X prepared using a complex represented by test substance X, a polymer substance Z having immunogenicity, and XYZ, which is a linker Y that combines the test substance X and the polymer substance Z Used to capture ´,

It is a carrier for measuring immune response, which has at least part of a structure of linker Y and any one of its analogs in a molecule.

In <1>, the antibody X 'to test substance X is a carrier for measuring immune response, which is any one of a monoclonal antibody, a polyclonal antibody, and a mixture thereof.

<3> The linker Y according to any one of <1> to <2>, is a carrier for measuring immune response having a structure represented by any one of the following structural formulas I and II.

[Formula 1]

[Structure Formula II]

However, in said structural formula (I) and (II), n represents the integer of 1-20.

<4> The carrier for measuring immune response according to <3>, which is at least one of cellulose, humic acid, nitrocellulose, lignin sulphonic acid, alkylbenzene, alkylphenol, and derivatives thereof. .

<5> The carrier for measuring immune response according to any one of <1> to <4>, which is a fibrous body.

<6> The material of any one of <1> to <5>, wherein n is a test substance X ₁ to X _n , a polymer substance Z having immunogenicity, the test substance X, and the It is a carrier for measuring immune response that captures n kinds of antibodies against the test substance X ₁ to X _n produced by using a complex of n kinds of linker Y that binds the polymer substance Z.

<7> An apparatus for measuring immune response, comprising the carrier for measuring immune response in any one of <1> to <6>.

<8> The measuring cell according to <7>, which contains a carrier for measuring an immune response and is formed with a through hole through which light can pass;

A light emitting unit for irradiating light to the carrier for measuring immune response contained in the measuring cell;

Of the light irradiated to the carrier for measuring the immune response from the light emitting unit, the transmitted light passing through the carrier for measuring the immune response received in the measuring cell is received and the amount of light of the transmitted light received ( A light-receiving unit for measuring light

It is an apparatus for measuring immune response comprising a transmission amount measurement device having a.

<9> In <8>, the amount of transmitted light measuring means is provided between the light emitting part and the light receiving part so as to penetrate in the irradiation direction of the irradiation light, and the inner surface reflects the light reflected by the carrier for measuring the immune response. It is an apparatus for measuring immune response provided with a reflex barrel.

<10> The device for measuring immune response according to any one of <8> to <9>, further comprising a relative absorbance calculator that calculates a relative absorbance from the amount of light used as a reference to the amount of transmitted light measured by the light receiver.

The test sample to which the antibody X 'to <11> test substance X was added is supplied to the carrier for measuring the immune response in any one of <1>-<6>,

The antibody X ', which is not bound to the test substance X in the test sample, is captured on the carrier for measuring the immune response,

The capture amount of the antibody X 'is measured, and the concentration of the test substance X in the test sample is determined from the capture amount.

<12> The carrier for measuring immune response according to <11>, wherein the antibody capture amount of the carrier for measuring immune response supplied with the test sample containing test substance X and the reference sample containing no test substance X are supplied. The relative antibody capture amount is calculated from the antibody capture amount, and the concentration of the test substance X in the test sample is obtained from the relative antibody capture amount.

<13> The antibody X 'to test substance X has a label, and the method for measuring an immune response according to any one of <11> to <12> for measuring color development derived from the label. Here, "measuring color development" includes detecting the color emitted by the labeling substance, and also includes an aspect for detecting a change in color such as discoloration or coloration of the labeling substance.

<14> The method for measuring immune response according to any one of <11> to <13>, wherein the antibody X 'captured on the carrier for measuring immune response is reacted with a secondary antibody having a labeling substance.

<15> The method for measuring immune response according to any one of <11> to <14>, wherein the amount of antibody captured in the carrier for measuring immune response is optically detectable.

<16> The method according to any one of <11> to <15>, wherein the labeling substance is any one of an enzyme, a radioisotope, a fluorescent substance, and colored fine particles.

<17> The method according to any one of <12> to <16>, wherein the labeling substance is a gold colloid.

<18> The method according to any one of <11> to <17>, wherein the test substance is any one of PCB, dioxin, hormones, vitamins, pesticides, and heavy metals.

<19> The method of measuring immune response according to any one of <11> to <18>, wherein the immune response measuring device according to any one of <7> to <10> is used.

<20> The light is transmitted to the carrier for measuring the immune response contained in the measuring cell, and the transmitted light transmitted through the carrier for measuring the immune response received in the measuring cell is received from the irradiated light. It is an immune response measurement method for measuring the amount of received light.

<21> The transmitted light of the carrier for measuring immune response according to any one of <19> to <20>, wherein the carrier for immune response is irradiated with monochromatic light to supply a test sample containing test substance X. And an immune response measurement method for calculating the relative absorbance from the transmitted light of the carrier for measuring the immune response supplied with the reference sample containing no test substance X.

The antibody for measuring the immune response according to the present invention can be suitably used as a carrier for various immunoassays because the antibody captures the antibody at a uniform and high density and can detect the measured object with high sensitivity and can be efficiently prepared at low cost. In particular, since the antibody against the low molecular weight substance is useful for the capture of an antibody prepared using the low molecular weight substance, the linker, or an antibody preparation compound comprising the high molecular weight substance having immunogenicity, for example, It is suitable for the measurement of low molecular weight harmful substances such as pollutants and low molecular weight such as vitamin-like substances.

In addition, since the immune response measuring apparatus of the present invention includes the carrier for measuring the immune response of the present invention, it is suitable for various immunoassays, and for example, low molecular weight harmful substances such as environmental pollutants or vitamin-like substances. It is suitable for the measurement of low molecular weight such as substances.

Furthermore, the immune response measuring method of the present invention is suitable for various immunoassays because it uses the carrier for measuring the immune response of the present invention and the immune response measuring apparatus of the present invention, and is suitable for, for example, low environmental pollutants. It is suitable for the measurement of low molecular weight such as harmful substances of molecular weight and vitamin-like substances.

According to the present invention, an immune response measurement comprising a carrier for measuring an immune response capable of capturing an antibody at a uniform and high density and detecting a measured object with high sensitivity and efficiently preparing at low cost, and the carrier for measuring the immune response It is possible to provide an apparatus for measuring immune response using the carrier, and the carrier for measuring the immune response.

(Carrier for measuring immune response)

The carrier for measuring the immune response according to the present invention is a complex represented by XYZ consisting of a test substance X, a polymer substance Z having immunogenicity, a test substance X, and a linker Y which binds the test substance X (hereinafter, referred to as "antibody preparation Compound ”, which is used to capture the antibody X 'to the test substance X produced by using the compound), and has at least one structure of the linker Y and any one of its analogs in the molecule.

<Compound for Antibody Preparation>

The compound for preparing the antibody is a complex used to produce antibody X 'against test substance X, and antibody X' produced by the antibody preparation compound is a monoclonal antibody to the test substance X. It is preferred that it is either a clonal antibody, or a mixture thereof.

There is no restriction | limiting in particular as said test substance X, According to the objective, it can select suitably, For example, it may be a low molecular weight hazardous substance, such as an environmental pollutant, or a low molecular weight useful substance, such as a vitamin-like substance, Examples thereof include PCBs, dioxins, natural or environmental hormones, pesticides, heavy metals (chelate complexes), and the like.

The polymer Z is not particularly limited as long as it can induce an immune response and can be appropriately selected according to the purpose. For example, polysaccharides, proteins, nucleic acids, microorganisms derived from organisms or the environment are preferable, and among these, proteins This is more preferable.

Examples of the protein include hemocyanin (KLH), egg white albumin (OVA), bovine serum albumin (BSA), human serum albumin, rabbit serum albumin, goat serum albumin and bovine gamma globulin Etc. can be mentioned.

As the linker Y, if the test substance X and the polymer Z can be connected, there is no particular limitation, and the linker Y can be appropriately selected according to the purpose. For example, the linker Y can form a covalent bond with the polymer Z. The compound which contains at least 1 type of functional group, and has 1-20 carbon molecular chains is preferable, For example, the compound which has a structure represented by the following structural formulas I-III is mentioned.

Among these, compounds having structures represented by the following structural formulas (I) and (II) are preferable, and those carriers for measuring the immune response also have at least some of the structures represented by the following structural formulas (I) and (II) and any one of analogues thereof. desirable.

[Formula I]

However, in said structural formula I, n shows the integer of 1-20.

[Structure Formula II]

However, in the structural formula II, n represents the integer of 1-20.

[Structure Formula III]

In addition, as an analog of the structure represented by the said structural formula (I) and (II), for example,

O (CH ₂ ) _L ONH ₂ (wherein L represents an integer of 1 or more)

OCO (CH ₂ ) _L ONH ₂ (wherein L represents an integer of 1 or more)

CH ₃ (CH ₂ ) _n CH (CH ₂ ) _m CH ₃ (wherein n and m each represent an integer satisfying n + m ≧ 2).

Etc. can be mentioned.

There is no restriction | limiting in particular as a preparation method of the said compound for antibody preparation, It can prepare by a well-known synthetic | combination method, For example, the compound which consists of the said linker Y and the said test substance X (Hereinafter, a "hapten compound." And a method of preparing a compound by adding the same to a solution containing the polymer Z, and binding the reactive functional group reactor of the linker Y to the polymer Z.

It is preferable to refine | purify the obtained said compound for antibody preparation into the neutral solution suitable for using a reaction liquid as an immunogen as needed, and to refine | purify by well-known methods, such as filtration and dialysis.

In addition, the compound for the preparation of the antibody, n (where n is a natural number of 2 or more) of the test substance X ₁ ~ X _n , the polymer substance Z having immunogenicity, the test substance X and the above-mentioned polymer substance Z If the composite of n species to the linker Y, the _n-1 antibody X'~ _X'n of n kinds for said test substance X ₁ ~ X _n manufactured using antibodies for the preparation of compound species, the linker Y As long as the compound is the same compound, at least a part of the structure of the linker Y can be captured by the carrier for measuring the immune response.

Examples of the n kinds of test substances X ₁ to X _n include, for example, isomers of n kinds of PCBs, and the like are represented by the following structural formulas. The complex which becomes.

<Carrier for measuring immune response>

In the carrier for measuring the immune response of the present invention, neither the test substance X nor an analog thereof is added as an antigen substance (pseudo antigen), and neither the test substance X nor an analog thereof is fixed.

The carrier for measuring the immune response is not particularly limited as long as it has at least part of a structure of the linker Y and any one of its analogs in the molecule, and for example, at least one of the structures represented by the structural formulas I and II above. As a compound which has some structure and its analog, For example, the polysaccharide (for example, cellulose, nitrocellulose) represented by the following structural formulas A and B, the benzene skeleton represented by the following structural formulas C-E The compound which has (for example, alkylbenzene, alkylphenol, etc.), lignin, lignin sulfonic acid, humic acid, and derivatives thereof are mentioned.

[Formula A]

However, in the structural formula A, M represents O (CH ₂ ) _L ONH ₂ , and L represents an integer of 1 or more.

[Formula B]

However, in the structural formula B, N represents OCO (CH ₂ ) _L ONH ₂ , and L represents an integer of 1 or more.

[Formula C]

However, in the structural formula C, M represents O (CH ₂ ) _L ONH ₂ , L represents an integer of 1 or more, and Q represents any one of OH and SO ₃ Na.

[Formula D]

In Formula (D), N represents OCO (CH ₂ ) _L ONH ₂ , L represents an integer of 1 or more, and Q represents any one of OH and SO ₃ Na.

[Formula E]

However, in the formula (E), R represents CH ₃ (CH ₂ ) _n CH (CH ₂ ) _m CH ₃ , n and m each represent an integer satisfying n + m ≧ 2, and Q represents OH and SO. Any one of ₃ Na is shown.

Moreover, as a compound which has any of the structure of at least one part of the structure represented by the said Formula (III), and its analog, polybenzene, polybenzenevinyl, aminobenzene sulfonate lignin, polyphenol, etc. are mentioned, for example.

At least a part of the structure of the linker Y and a molecule which does not have any of its analogs may be appropriately added to at least part of the structure of the linker Y and its analog, and the addition may be performed by the linker Y. Any one of the compounds, proteins, nucleic acids, polymers, and complexes thereof, which are any one of the structures and analogs thereof, are physical (atomic force, minority), electrical, It can be performed by a well-known method by chemical (hydrogen bond, coordination bond, covalent bond).

As the carrier for measuring the immune response, as long as uniform contact with the sample is possible, the shape and the material are not particularly limited and can be appropriately selected according to the purpose. For example, the carrier is preferably made of a fiber, and a large number of fibers are made of high density. It is more preferable to have. As such a fiber body, a filter paper, a moisture absorbing paper, a woven fabric, a knit fabric, a nonwoven fabric etc. are mentioned, for example.

The carrier for measuring the immune response may be prepared by molding the fibers into a target shape or by cutting or punching a sheet-like fiber body in a target size and shape by a known method.

In addition, there is no restriction | limiting in particular as a shape of the said carrier for immune response measurement, Although it can select suitably according to the objective, For example, a thin film shape, a disk shape, a fine particle shape, a needle shape, a flat plate shape Shapes, such as (平板 狀), are preferable.

In addition, as the air permeability of the carrier for measuring the immune response, the sample to be passed through the physical means such as a pump or centrifugal, electrochemical means such as electrophoresis, etc. passes at a uniform flow rate. As long as it does not have a restriction | limiting in particular, Although it can select suitably according to the objective, For example, 1-100 cm <3> / cm <2> / min is preferable.

The carrier for measuring the immune response is passed through at a constant flow rate, so that the amount of the antibody X 'captured is cumulatively increased. As the quantity of the said antibody X 'captured, it is preferable that it is 0.01 to 10% of the total amount of the added antibody in a flat state.

(Immune Response Measurement Device)

The device for measuring the immune response of the present invention is not particularly limited as long as it includes the carrier for measuring the immune response of the present invention. For example, a flow-through detection device, an immunochromatographic device, a spectrophotometer, a microplate Although a microplate reader device, a fluorescence photometer, an electrochemical measuring device, a magnetic / magnetic measuring device, a refraction measuring device, etc. are mentioned, it is preferable that it is a flow-through detection device among these.

The apparatus for measuring immune response of the present invention can be suitably used, for example, in the immune response measuring method of the present invention described later.

As the apparatus for measuring the immune response, for example,

A measurement cell accommodating a carrier for measuring an immune response, and through-holes through which light passes are formed;

A light emitting unit irradiating light to the carrier for measuring immune response contained in the measuring cell;

Of the light irradiated to the carrier for measuring the immune response from the light emitting unit, the transmitted light passing through the immune response measuring carrier accommodated in the measuring cell is received, and the amount of transmitted light which becomes a light receiving unit for measuring the light amount of the received transmitted light. A device having a measuring device is suitably used.

As the method for measuring the immune response using the device, for example, a test sample containing the antibody X 'to the test substance X is transferred into the measuring cell containing the carrier for measuring the immune response. Thereby capturing the antibody X 'which is not bound to the test substance X in the test sample on the carrier for measuring immune response, and indirectly determining the concentration of the test substance X in the test sample from the amount of the test sample. And an antibody trapped amount of the carrier for measuring the immune response supplied with a test sample containing the test substance X and the antibody X 'to the test substance X, and the immunity supplied with the reference sample containing only the antibody X'. A method of calculating the relative capture amount from the antibody capture amount of the reaction measuring carrier and determining the concentration of the test substance X in the test sample from the relative capture amount. The can.

There is no restriction | limiting in particular as a raw material of the said measuring cell, According to the objective, it can select suitably, For example, a translucent material may be sufficient and a non-transmissive material may be sufficient. Since the measuring cell is provided with a through hole through which light can pass, it is not necessary to configure the measuring cell with a light-transmissive material. Therefore, even if the measuring cell is scratched or contaminated, it is necessary to pass through the light ( It does not affect the transit, and is advantageous in that no measurement error occurs. Especially, since the non-translucent material is light-shielding as a raw material of the said measuring cell, since it does not need to shield by a lid | cover etc., it is preferable. There is no restriction | limiting in particular as said non-translucent material, According to the objective, it can select suitably, For example, ABS resin (acrylonitrile butadiene-styrene resin), PP (polypropylene), PE (polyethylene), PS (polystyrene) ), PMMA (polymethyl methacrylate), PET (polyethylene terephthalate), PPE (polyphenylene ether), nylon, PA (polyamide), PC (polycarbonate), POM (polyacetal), PBT (polybutyl Lenterepthalate), PPS (polyphenylene sulfide), PEEK (polyether ether ketone), LCP (liquid crystal polymer), fluorine resin, urethane resin, elastomer, PF (phenol resin), MF ( Melamine resin), FRP (glass fiber reinforced plastic), etc. are mentioned.

In the measurement cell, the through hole is not particularly limited as long as it is a hole through which light can pass, and can be appropriately selected according to the purpose.

In the measuring cell, the carrier for measuring the immune response is not particularly limited as long as it is accommodated in the measuring cell, so that light passing through the through-hole is irradiated to at least part of the carrier for measuring the immune response. It arrange | positions accordingly. In particular, it is preferable that the carrier for measuring the immune response is fixed in the through hole so as not to fall off from the through hole. There is no restriction | limiting in particular as said fixing method, According to the objective, it can select suitably, For example, the said through-hole is carried out by the force of the said carrier for measuring immune response which applies a force to the inner wall of the said through-hole. The groove may be fixed to the inner wall of the through hole, and may be fixed by fitting the outer circumference of the carrier for measuring the immune response to the groove.

The measuring cell may be an aspect of an integrated disposable cell together with the carrier for measuring immune response, and can be used repeatedly as a structure in which only the carrier for measuring immune response can be exchanged. You may also

Since the measuring cell has a through hole as described above, only by transferring the test sample to the through hole, the test sample can be easily passed through the carrier for measuring the immune response. The test sample may be passed through the carrier for measuring the immune response by the liquid passing means detachably connected to the cell for the reaction. Examples of the liquid passing means include means for pressurizing the test sample so that the test sample is uniformly passed to the carrier for measuring the immune response, and feeding the test sample to the carrier for measuring the immune response. Can be. Specifically, a means for connecting a syringe, a pump, a centrifugal separator, or the like to the measurement cell, for pressurizing the test sample and for feeding the liquid, etc. may be mentioned. Moreover, the said liquid passing means may be equipped with the control part which controls a flow volume, and can control the operation | movement of pressurization.

There is no restriction | limiting in particular if the said light emitting part is equipped with the light source at least, and a light can be irradiated to the said carrier for immune reaction measurement in the said measuring cell, According to the objective, it can select suitably. As said light source, a laser, a light emitting diode, a halogen lamp, a tungsten lamp, etc. are mentioned, for example. The light emitting portion may have two or more light sources.

As the light emitted from the light emitting portion, one monochromatic light may be used, the monochromatic light may be used, or two or more monochromatic light may be used. As the light irradiated to the carrier for measuring immune response, one light is used. It is preferable that it is monochromatic light. Therefore, when the light emitted from the light emitting portion is monochromatic light or two or more monochromatic light, the light emitting portion can select only one monochromatic light among the monochromatic light or two or more monochromatic light, It is preferable to provide a monochromatic light selection unit capable of changing monochromatic light.

 According to the monochromatic light selection unit, the monochromatic light or two or more monochromatic light emitted from the light emitting unit can be irradiated to the carrier for measuring the immune response as one monochromatic light, and the monochromatic light is changed to another monochromatic light, for example, The amount of transmitted light of two or more coloring materials attached to the carrier for measuring the immune response may be measured.

As the light-receiving unit, if the light transmitted from the light-emitting unit is irradiated to the carrier for measuring the immune response, the transmitted light transmitted through the carrier for measuring the immune response (the part through which the test sample is passed) can be received and the amount of received light can be measured. There is no restriction | limiting in particular and it can select suitably according to the objective.

When the transmitted light from the carrier for measuring the immune response is monochromatic light or two or more monochromatic light, the light receiving unit includes a monochromatic light selection unit capable of selecting only one monochromatic light and capable of changing the selected monochromatic light. desirable.

According to the said monochromatic light selection part, the monochromatic light or two or more monochromatic light which permeate | transmitted the said carrier for measuring immune response can be received as one monochromatic light, and the monochromatic light is changed into another monochromatic light, for example, the said immune response The amount of transmitted light of two or more chromophores attached to the carrier for measurement can be measured.

It is preferable that the said apparatus for measuring immune response is provided with the measuring means which can measure the quantity of light received by the said light receiving part as the signal intensity of an electrical signal. For example, the measuring means can be connected to the light receiving unit to convert the amount of light received by the light receiving unit into a signal intensity of an electrical signal and output the signal.

 Moreover, it is preferable that the said apparatus for measuring immune response is provided with a reflecting tube between the said light emitting part and the said light receiving part. The reflecting tube is configured to penetrate in the direction of irradiation of light emitted from the light emitting portion so as to contain the carrier for measuring the immune response, and an inner surface thereof reflects the reflected light from the carrier for measuring the immune response. By providing the reflecting cylinder, the light reflected by the carrier for measuring the immune response can be converged, so that the measurement sensitivity can be improved.

The apparatus for measuring immune response preferably includes a relative absorbance calculator that calculates a relative absorbance from the amount of light used as a reference to the amount of transmitted light measured by the light receiver. Specifically, light is irradiated to the carrier for measuring the immune response, thereby supplying the transmitted light of the carrier for measuring the immune response, which is supplied with the test sample containing the test substance X, and a reference sample not containing the test substance X. It is more preferable to provide a relative absorbance calculator which calculates a relative absorbance from the transmitted light of the carrier for measuring the immune response.

In the apparatus for measuring an immune response, the contact time between the test sample (liquid phase) and the carrier for measuring the immune response (solid phase) is short, and the antibody in the test sample is flown. In order to capture continuously, the measurement can be carried out without breaking the bonding equilibrium state in the liquid phase. Thus, the lowering of the sensitivity and the competing reaction of the liquid phase and the solid phase which complicates the measurement can be avoided.

In addition, since the antibody can be continuously captured, specifically, the equilibrium dissociation constant of the antibody itself until the binding of the antigen to the antibody in the liquid phase becomes the affinity domination of the antibody. I) The concentration of the antibody can be lowered to below, and the signal value can be amplified until the sensitivity necessary for detection is further obtained.

(Measurement of immune response)

In the method for measuring immune response of the present invention, a test sample to which the antibody X 'to the test substance X is added is supplied to a carrier for measuring the immune response of the present invention, and the test sample X is bound to the test substance X in the test sample. The antibody X ', which is not present, is captured on the carrier for measuring the immune response and the amount of the antibody is measured.

The immunoassay method of the present invention can be suitably carried out using the apparatus for measuring the immune response of the present invention as a method of using the carrier for measuring the immune response of the present invention.

As a method for measuring the amount of antibody captured by the carrier for measuring an immune response, a method for measuring a signal emitted from a label of the antibody X when the antibody X 'to the test substance X is an antibody having a label, When using the secondary antibody which has a label | marker for antibody X ', the method of measuring the signal emitted from the label | marker of the said secondary antibody is mentioned.

There is no restriction | limiting in particular as a method of measuring the said signal, According to the kind of said label | substrate, it can select suitably. For example, when the said label | substrate is an enzyme, it reacts after making the said enzyme react with a substrate. And a method for measuring the reflected absorbance, the fluorescence absorbance, and the luminescence intensity of the product, and the method of measuring radiation dose as a signal when the labeling substance is a radioisotope, or as a signal when the labeling substance is a fluorescent substance. A method for measuring fluorescence intensity, a method for measuring luminescence intensity as a signal when the labeling substance is a luminescent material, a method for measuring a quantity of transmitted light changed by coloring as a signal when the labeling substance is colored fine particles, the label When the substance is a substance exhibiting an electrochemical reaction, a method for measuring a change in current or voltage and potential as a signal, the label If the quality of the substance showing an electron spin and a method for measuring a spectrum change in ESR (electro spin resonance) as a signal.

These measured signals are, for example, measured every second, recorded on a computer, or the like, and a method of displaying the difference between the finally obtained residual signal value and the baseline value as a measured value in volts units is preferable.

There is no restriction | limiting in particular as said labeling material, Although it can select suitably according to the objective, For example, peroxidase, an alkaline phosphatase, tyrosinase, galactosidase ), Luciferase, enzymes such as glucose oxidase, radioactive isotopes such as I ¹²⁵ , I ¹³¹ and H ³ , tetramethylrhodamine, europium chelate, fluorescein, etc. Fluorescent materials such as isocyanate derivatives, acridinium esters, acridinium sulfonic acids, luciferin, NAD oxidoreductase, lucigenin, lucigenin, lophine, gold Colored fine particles such as colloid, selenium colloid, colored latex, magnetic fine particles, piperidine-N-oxide derivative, pyrrolidine-N-oxide derivative, oxazoli The substance which shows free radicals, such as a dean-N-oxide derivative, etc. are mentioned.

Among these, the aspect in which the said label | substrate is colored fine particles is preferable, and the method of measuring the amount of transmitted light which changes by coloring as a signal as said immune reaction measuring method is preferable.

Here, n ₁ antibody X'~ _X'n of n kinds for said test substance X ₁ ~ X _n of the species, by the addition in the same sample, wherein the immune response to the carrier at the same time antibodies _X'1 ~ X for measurements _N may be captured and the amount of the test substance X ' ₁ to X' _n may be quantified as the total amount from the captured amount. As a result, when quantifying a test substance containing a large number of homologues or isomers as a total amount, accurate quantification, which was difficult with a single antibody, can be performed in one measurement.

As described above, the immune response measuring method of the present invention avoids a competitive reaction between the liquid phase and the solid phase, and by the accumulation effect, the antibody concentration at the time of measurement can be set to be equal to or less than the equilibrium dissociation constant of the antibody itself. Multiple antigen-antibody reactions in the same solution occur simultaneously and independently, depending on each equilibrium dissociation constant. Therefore, even if there are n types of test substances, it can be grasped | ascertained as the sum total of the antigen antibody reaction of n types of antibodies.

In addition, by changing the binding affinity for each antigen of the n kinds of antibodies, the composition ratio (content ratio) of the homologues and isomers of the n kinds of antigens can be clarified.

In this case, for example, in the case where the test substance in the sample of X ₁ and X _2, coupled to both the antibody _X'2, X _1, and X ₂ which binds to the antibody _X'1, X ₂ binding to X ₁ The antibody is mixed. When the test substance X ₁ and X ₂ have the same concentration, the signal derived from the antibody which binds to X ₁ and the signal derived from the antibody which binds to X ₂ can be made identical by the design of measurement conditions. In addition, although the sum total becomes a signal originating in the antibody couple | bonded with the said test substance X <_1> and X <_2> , when the density | concentration of X <_1> and X <_2> is respectively different, the signal originating in the antibody couple | bonded with X <_1> , and X _The signals derived from the antibody binding to ₂ are different. Because even if the signal originating from the antibody binding to the signal originating from the antibody, X ₂ binding to X ₁ different from each other, the sum of the concentrations being determined from the signal derived from the antibody binding to X ₁ and X _2, as described above By selecting the binding specificity of the antibody to be added, the composition ratio (content ratio) of n kinds of test substances X ₁ to X _n can be estimated.

Further, n kinds of samples in which the antibodies X ' ₁ to X' _n are added alone may be prepared, the amount of antibody captured in each sample may be quantified, and the amount of the test substance may be quantified from the amount of the test. As a result, when evaluating the composition of a test substance containing a large number of homologues or isomers, it is possible to quantify the amount of antibody captured or the amount of test substance using the same carrier for measuring immune response. In this case, the measurement can be performed simultaneously and in parallel, and it is also possible to estimate the composition ratio (content ratio) of the test substance X ₁ to X _n as described above from the results of the parallel measurement.

The carrier for measuring immune response of the present invention is considered to be applicable as a carrier for conventional immunoassays such as ELISA method and immunochromatography method, and thus has a very wide application range.

The method for measuring the immune response according to the present invention is preferably a method for determining the amount of the test substance in the test sample from the amount of the antibody captured on the carrier for measuring the immune response, and the example is shown below.

In the measurement of the signal generated from the labeling substance, the signal intensity (a blank, for example, "F0") when the antibody X 'at a certain concentration is bound to the carrier for measuring the immune response is measured. Subsequently, the signal strength when the mixed solution to which the antibody X 'of the same density | concentration as the blank measurement was added to the test sample was supplied to the said carrier for measuring immune response, and the said unbound antibody X' was bound (for example, For example, "F1" is measured.

Here, when the test substance X is present in the test sample, since the antibody X 'binds to the test substance X in the test sample, the amount of the unbound antibody X' that binds to the carrier for measuring the immune response is small. Lose. Therefore, when the signal intensity becomes F1 <F0, it can be determined that the test substance X exists in the test sample.

Since the rate of decrease of F1 has a correlation with the amount of the test substance X contained in the test sample, a calibration curve is prepared in advance by using a test substance X of a known concentration, and thus the test rate is determined in accordance with the rate of decrease of F1. Quantification of substance X can be performed.

Hereinafter, although the Example of this invention is described, this invention is not limited to this Example at all.

(Example)

(1) Preparation of Antibody

In order to prepare an anti-PCB monoclonal antibody against a test substance PCB, a trichloride PCB (trichlorinated biphenyl) was used as a carrier protein (hemocyanine derived from a scavenger clam) (hereinafter referred to as "KLH") via a linker. Complex) was synthesized.

The complex was immunized with mice (Bulb / c, female, 5 weeks old) as antigen.

First time immunization was performed subcutaneously after mixing the said antibody preparation compound (about 0.3 mg as a protein mass) with a complete adjuvant. Two weeks after and four weeks after the first immunization, the same amount of the antibody preparation was mixed in an incomplete adjuvant and subcutaneously injected. Then, after one week or more, the same amount of the compound for antibody preparation is injected into the abdominal cavity or tail vein, and the spleen is extracted after 4 to 5 days (摘 出).

The splenocytes prepared from the spleens removed were mixed with myeloma cells in a polyethylene glycol solution for 2 minutes to carry out cell fusion.

The fusion cells after the fusion reaction were cultured and the presence or absence of the desired anti-PCB monoclonal antibody was screened using a hybridoma culture supernatant 2 weeks or more after cell fusion to produce an anti-PCB monoclonal antibody. A stable hybridomer was obtained. The hybridomer was incubated and the culture supernatant was purified to obtain an anti-PCB monoclonal antibody.

(2) Carrier for measuring immune response

In the complex used for preparation of the anti-PCB monoclonal antibody, the linker had a structure represented by the following structural formula (IV).

[Structure IV]

Therefore, cotton (cotton) and cellulose are selected as the fibrous bodies having a structure similar to that of Structural Formula IV, and the filters made of these fibrous bodies are cut out in a circular shape having a diameter of about 5 mm, and the carrier (immune reaction of the present invention) is selected. Carrier for measurement) was prepared.

As a comparison object, polyolefins and polyesters were selected as the fibrous bodies having no structure similar to that of the above structural formula IV, and the filters made of these fibrous bodies were cut out in the same manner to prepare a carrier.

Evaluation of antibody capture

The anti-PCB monoclonal antibody was labeled with gold colloid by a conventional method, and dissolved in physiological saline containing 1 g / L bovine serum albumin to obtain an antibody solution of about 1 nM.

Next, the carrier was placed in a holder (measurement cell) through which the antibody solution can be passed. The holder (cell for measurement) is provided with a hole having a diameter of 3 mm, and the antibody solution can be passed through the carrier from the connected syringe.

Evaluation of the antibody trapping ability of the carrier was carried out by putting 2 mL of the antibody solution into a syringe and passing the liquid at a constant flow rate of 0.85 mL / min with a connected pump. After passing through the antibody solution, 1 mL was passed through physiological saline containing 1 g / L bovine serum albumin not containing the antibody at the same flow rate, and then the amount of transmitted light of the holder (cell for measurement) was measured. .

In addition, 1 mL of physiological saline containing 1 g / L of bovine serum albumin is passed through the same volume before passing through the antibody solution, and the amount of transmitted light is measured with a measuring device to determine the change in the amount of transmitted light before and after the passage of the antibody solution. It was used as an index of antibody trapping ability. The amount of transmitted light was measured using a device having a light source and a photodiode at the position where the holder (measurement cell) was inserted, and capable of measuring the transmission of light passing through the carrier as an electric signal. Therefore, the greater the amount of antibody captured, the darker the red color on the film derived from the labeled gold colloid, and the smaller the signal value.

The results are shown in FIGS. 1A-F and FIGS. 2A-D.

(3) Quantification of Test Substance (PCB)

PCB containing insulating oil was processed by the following method, and the test sample was prepared. The PCB-containing insulating oil was added to insulating oil not containing PCB in various concentrations of Canekrol-300 (manufactured by GL Science Co., Ltd.) and prepared as insulating oil containing a PCB having a known concentration.

A diatomaceous earth column (Extrelut NT3, manufactured by Merk) and a silica gel column (manufactured by SepPak Silica Plus, Waters) were connected, and 1 mL of 25% fuming sulfuric acid and 2 mL of concentrated sulfuric acid were added to the diatomaceous earth column. It was added sequentially and it left to stand for 15 minutes.

Subsequently, after layering 1.5 g of anhydrous sodium sulfate on the diatomaceous earth column, 0.5 g of the said test sample (PCB containing insulating oil) was added, and 1 mL of n-hexane was further added to penetrate into a column. It was left to stand for 5 minutes, and the development was performed after promoting the decomposition of the insulating oil component in the test sample.

After adding 5 mL of n-hexane and confirming that the liquid surface was lowered to the anhydrous sodium sulfate layer, 10 mL of n-hexane was further added, and the whole amount of the eluate was branched. Recovered to the flask. 0.5 mL of dimethyl sulfoxide (hereinafter referred to as "DMSO") was added to the eluate, and n-hexane was removed by a rotary evaporator in a 30 ° C hot water bath. The residue was transferred to a microtube, centrifuged (2000 rpm, 5 minutes), and the DMSO layer was aliquoted to obtain a test sample (pretreatment liquid).

 In the antibody solution, the anti-PCB monoclonal antibody was labeled with gold colloid by a conventional method, dissolved in physiological saline containing 1 g / L bovine serum albumin, and an antibody solution of about 1 nM was prepared.

The test sample (pretreatment solution) was added to the antibody solution so as to have a 100-fold dilution to prepare a solution for measurement.

The carrier for measuring immune response, prepared by cutting a filter made of cellulose fibers into a circular shape having a diameter of about 5 mm, was placed in a holder (measurement cell) capable of passing the antibody solution. As the measuring device, the same device as used in the evaluation of the antibody trapping property was used.

2 mL of the measurement solution was placed in a syringe and passed through a connected pump at a constant flow rate of 0.85 mL / min. After passing through the antibody solution, 1 mL of physiological saline containing 1 g / L bovine serum albumin not containing the antibody was passed at the same flow rate, and then the amount of transmitted light from the holder (cell for measurement) was measured as a signal. Measured.

As a control, the amount of transmitted light when the physiological saline containing 1 g / L bovine serum albumin was passed without a PCB was measured, and the signal ratio (%) of the amount of transmitted light with the amount of transmitted light as 100% was measured. It was set as an index of the PCB concentration in the test sample. The results are shown in FIG.

From the results in Fig. 3, anti-PCB monoclonal antibodies can be captured by the carrier for measuring immune response without performing a process of immobilizing a pseudo antigen for capturing anti-PCB monoclonal antibodies to the antibody. It was found that the concentration of the test substance can be accurately determined.

Moreover, compared with the case where the conventional pseudo antigen is immobilized to a carrier, it turns out that the cost and time of preparation of a carrier can be reduced, and an antibody can be captured with high density.

FIG. 1A is a photograph showing the results of evaluating an immunoreaction measurement carrier made of cotton (cotton) in Example 1. FIG.

FIG. 1B is a photograph showing the results of evaluating the carrier for measuring an immune response of cellulose in Example 1. FIG.

1C is a photograph showing the results of evaluating a carrier for measuring an immune response of cellulose in Example 1. FIG.

1D is a photograph showing the results of evaluating a carrier for measuring an immune response of cellulose in Example 1. FIG.

FIG. 1E is a photograph showing the results of evaluating a carrier for measuring an immune response of cellulose in Example 1. FIG.

1F is a photograph showing the results of evaluating a carrier for measuring an immune response of cellulose in Example 1. FIG.

FIG. 2A is a photograph showing the results of evaluating a carrier for measuring an immune response of polyolefin in Example 1. FIG.

FIG. 2B is a photograph showing the results of evaluating the carrier for measuring immune response of polyolefin in Example 1. FIG.

FIG. 2C is a photograph showing the results of evaluating the carrier for measuring immune response of polyester in Example 1. FIG.

FIG. 2D is a photograph showing the results of evaluating the carrier for measuring immune response of polyester in Example 1. FIG.

It is a graph which shows the calibration curve of the PCB (Canekrol 300) created in the Example, and the photograph of the carrier for measuring the immune response at each concentration.

Claims (13)

The test material manufactured by using a complex represented by test material X, a polymer material Z having immunogenicity, the test material X and a linker Y represented by any one of the following structural formulas I and II combining the polymer material Z Used to capture antibody X 'against substance X, A carrier for measuring an immune response, characterized by having the structure of the linker Y in a molecule. [Formula I]

[Structure Formula II]

However, in said structural formula (I) and (II), n represents the integer of 1-20. The carrier for measuring immune response according to claim 1, wherein the antibody X 'to test substance X is any one of a monoclonal antibody, a polyclonal antibody, and a mixture thereof. The carrier for measuring immune response according to claim 1, wherein the carrier having the structure of the linker Y is in at least one of cellulose, humic acid, nitrocellulose, lignin sulfonic acid, alkylbenzene, alkylphenol, and derivatives thereof. The carrier for measuring immune response according to claim 1, wherein the carrier having the structure of the linker Y in a molecule is a fibrous body. According to claim 1, n kinds of test material X ₁ ~ X _n , a polymer material Z having an immunogenicity, the test material X and the linker Y to combine the polymer material Z is produced using a complex of n types the test substance X ₁ ~ X _n of the n kinds of the antibodies _X'1 ~ capture _X'n, and the immune response substrate for measuring the n is a natural number of 2 or more. An apparatus for measuring immune response, comprising the carrier for measuring immune response according to claim 1. The measuring cell according to claim 6, further comprising a measuring cell for receiving a carrier for measuring an immune response, and having a through hole through which light passes; A light emitting unit for irradiating light to the carrier for measuring immune response contained in the measuring cell; A light receiving unit for receiving the transmitted light transmitted through the carrier for the immune response measurement received in the cell for the measurement of the light irradiated to the carrier for measuring the immune response from the light emitting unit, the light receiving unit for measuring the amount of transmitted light received; Apparatus for measuring immune response comprising a transmission amount measurement device having a. The test material manufactured by using a complex represented by test material X, a polymer material Z having immunogenicity, the test material X and a linker Y represented by any one of the following structural formulas I and II combining the polymer material Z The test sample to which the antibody X 'with respect to the substance X was added is supplied to the carrier for measuring the immune response of Claim 1, The antibody X ', which is not bound to the test substance X in the test sample, is captured on the carrier for measuring the immune response, The concentration of the test substance X in the test sample is determined from the capture amount of the antibody X '. [Formula I]

[Structure Formula II]

However, in said structural formula (I) and (II), n represents the integer of 1-20. The antibody of the carrier for measuring immune response according to claim 8, wherein the antibody capture amount of the carrier for measuring the immune response supplied with the test sample containing test substance X and the reference sample containing no test substance X are supplied. A method for measuring immune response, wherein the relative antibody capture amount is calculated from the capture amount, and the concentration of the test substance X in the test sample is obtained from the relative antibody capture amount. The method according to claim 8, wherein the antibody X 'to test substance X has a label and measures the color development derived from the label. The method for measuring an immune response according to claim 8, wherein the antibody captured on the carrier for measuring an immune response is reacted with a secondary antibody having a label. The method of claim 8, wherein the labeling substance is any one of an enzyme, a radioisotope, a fluorescent substance, and colored fine particles. The method of claim 8, wherein the test substance is any one of polychlorinated biphenyl (PCB), dioxin, hormones, vitamins, pesticides, and heavy metals.

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