JP2013181889A - Immunoassay of ck-mb (creatine kinase isozyme mb) using spfs (surface plasmon excitation enhanced fluorescence spectrometry) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for quantifying or detecting CK-MB in a sample with high sensitivity.SOLUTION: An immunoassay targets CK-MB in a sample. The immunoassay includes the steps of: forming a sandwich type immune complex containing a sold phased anti-CK-MB antibody, an anti-CK-MM antibody or anti-CK-BB, CK-MB, a fluorescent labelled anti-CK-MB antibody, and an anti-CK-MM or anti-CK-BB antibody; and measuring fluorescence intensity emitted from a fluorescent body contained in the formed sandwich type immune complex with SPFS (surface plasmon excitation enhanced fluorescence spectrometry).

Description

  The present invention relates to SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) and CK-MB known as biomarkers such as myocardial infarction, which are used in fields such as medicine and biotechnology.

  Acute myocardial infarction is a disease in which the myocardium falls into necrosis due to occlusion of the coronary arteries, and chest pain, electrocardiogram and myocardial infarction markers have been used as examination methods for this disease. This myocardial infarction marker is roughly divided into a biochemical index and an immunological index.

  As biochemical indicators, the enzyme activity of creatine kinase (CK) or its specific myocardial isozyme, creatine kinase isozyme MB (CK-MB), is used. The quantity is measured. For example, when targeting CK-MB, after adding a CK-M inhibitor to a sample that may contain three types of isozymes (details will be described later), the enzyme activity of CK-B is measured. A method (immunoinhibition method) is used in which the measured value is doubled to obtain a measured value of CK-MB. However, although such a measurement method can be easily performed, it is assumed that CK-BB is negligibly small and macro CK (such as CK bound to immunoglobulin) is also detected. There is a point.

  On the other hand, CK-MB, myosin light chain, troponin, and the like have been put to practical use as immunological indices, and the content in a sample is measured through formation of an immune complex by an antigen-antibody reaction. For example, the following method for CK-MB is known. (1) Chemiluminescent enzyme immunization using a magnetic latex reagent (anti-CK-MB mouse monoclonal antibody-sensitized magnetic latex), a labeled antibody reagent (alkaline phosphatase-labeled anti-CK-BB mouse monoclonal antibody) and a luminescent substrate corresponding to the alkaline phosphatase Measurement method. (2) A chemiluminescent enzyme immunoassay method using solid-phased streptavidin, biotinylated anti-CK-BB mouse monoclonal antibody, HRP-labeled anti-CK-MB mouse monoclonal antibody, and the above-mentioned HRP luminescent substrate. (3) An immunochromatography method performed on a test slip provided with a biotinylated anti-CK-MB mouse monoclonal antibody, a colloidal gold-labeled anti-CK-MB mouse monoclonal antibody and streptavidin. However, these chemiluminescent enzyme immunoassays have room for improvement in terms of increasing sensitivity, that is, enabling quantification or detection at as low a concentration as possible.

  CK is a protein having a molecular weight of about 82,000, which is constituted as a dimer composed of two subunits, and is an enzyme that catalyzes a reaction in which the phosphate at the γ position of ATP is transferred to creatine. Since there are two types of subunits, M (muscle) and B (brain), there are three isozymes of CK-MM, CK-MB, and CK-BB depending on their binding mode. In general, CK-MM is considered to have high diagnostic significance for skeletal muscle disease, CK-MB for myocardial infarction, and CK-BB for cerebrovascular disorders and malignant tumors.

  In addition, Patent Document 1 includes a surface plasmon resonance biosensor comprising a transparent substrate, a metal film disposed on the transparent substrate, and an antibody against a myocardial infarction marker fixed on the metal film. (SPR) measurement chips and methods for measuring myocardial infarction markers using such measurement chips are described. As the myocardial infarction marker in this method, troponin T and myoglobin are specifically described.

  On the other hand, SPFS is known as one of measurement systems for detecting or quantifying various biological substances (see, for example, Patent Document 2). In SPFS, when the metal thin film formed on the dielectric member is irradiated with excitation light at an angle at which total reflection attenuation (ATR) occurs, the evanescent wave transmitted through the metal thin film is several tens of times due to resonance with the surface plasmon. It is a method of efficiently exciting a phosphor for labeling an analyte (analyte) captured in the vicinity of a metal thin film and measuring the fluorescence signal by utilizing the fact that it is enhanced several hundred times. Since such SPFS is extremely sensitive compared to a general fluorescent labeling method, it can be quantified even when only a very small amount of analyte is present in the sample.

Japanese Patent Laid-Open No. 11-023575 JP 2010-145272 A

  It is desirable that CK-MB in a blood sample serving as a biomarker such as myocardial infarction can be quantified or detected at as low a concentration as possible. The problem to be solved by the present invention is to provide means for quantitatively detecting CK-MB in a sample with high sensitivity.

  The present inventor forms a sandwich-type immune complex containing anti-CK-MB antibody and the like immobilized on SPFS, CK-MB, and anti-CK-MB antibody fluorescently labeled, and the like. By measuring the fluorescence intensity emitted from the phosphor contained in the immune complex, it was found that the sample can be quantified even when the sample contains only a very small amount of CK-MB, and the present invention is completed. It came to.

That is, the present invention includes the following inventions.
[1] An immunological assay for CK-MB (creatine kinase isozyme MB) in a sample, which is an immobilized anti-CK-MB antibody and anti-CK-MM (creatine kinase isozyme MM) antibody Alternatively, anti-CK-BB (creatine kinase isozyme BB) antibody (hereinafter referred to as “solid-phase antibody”), CK-MB, and fluorescently labeled anti-CK-MB antibody, anti-CK-MM antibody or anti-CK A step of forming a sandwich-type immune complex containing a -BB antibody (hereinafter referred to as “fluorescence-labeled antibody”), and the fluorescence intensity emitted from the phosphor contained in the formed sandwich-type immune complex is expressed by SPFS ( An immunological measurement method comprising a step of measuring by surface plasmon excitation enhanced fluorescence spectroscopy.

  [2] The step of forming the sandwich-type immune complex includes the step of reacting the immobilized antibody with CK-MB in a sample to form an immune complex, and the formed immune complex and the fluorescence The immunoassay method according to [1], comprising a step of reacting with a labeled antibody to form the sandwich-type immune complex.

  [3] The immobilized antibody is one or more anti-CK-MB monoclonal antibody, anti-CK-MM monoclonal antibody or anti-CK-BB monoclonal antibody, and the fluorescently labeled antibody is immobilized on the solid phase. One or two or more anti-CK-MB monoclonal antibodies, anti-CK-MM monoclonal antibodies or anti-CK-BB monoclonal antibodies different from one or more monoclonal antibodies as an antibody [1] or The immunoassay method according to [2].

  [4] A solid-phased anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody used in the immunoassay method according to any one of [1] to [3], and CK A sensor chip for SPFS, in which a sandwich-type immune complex including MB and a fluorescently labeled anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody is formed in the measurement region.

  By the immunological measurement method using SPFS of the present invention, the quantitative performance or detection performance of CK-MB can be remarkably enhanced as compared with the conventional method of forming a sandwich type immune complex such as ELIZA. Therefore, according to the present invention, it is possible to quantify even when CK-MB is contained in a blood sample in an extremely small amount, and the usefulness of CK-MB as a biomarker such as myocardial infarction is improved and diagnosed. It is possible to obtain highly reliable data that is useful for.

FIG. 1 is a plot of CK-MB quantitation limit results in Examples 1-1, 1-2 and 1-3.

In the present specification, the expression “X to Y” indicating a numerical range means X or more and Y or less.
Hereinafter, although this invention is demonstrated according to the aspect based on SPFS alone, this invention is applicable also in the aspect which combines SPFS and another measurement system thru | or systems, such as a SPFS-LPFS measurement system, for example. It goes without saying that known or commonly used means can be appropriately combined with the immunological measurement method based on SPFS of the present invention.

-Measuring member for SPFS-
The measurement member for SPFS is generally used for forming a sandwich-type immune complex, a sensor chip on which a field (measurement region) for performing fluorescence measurement by SPFS is formed and a sandwich-type immune complex is formed. A structure in which various solutions used (analyte-containing samples, labeled ligand solutions, other reaction reagents, etc.) can be held on the measurement area and laminated with members for constructing channels or wells. Take.

  That is, in one aspect, the present invention provides a solid-phased anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody used in the immunological assay of the present invention, and CK-MB. An SPFS sensor chip is provided in which a sandwich-type immune complex containing a fluorescently labeled anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody is formed in a measurement region.

  The sensor chip basically includes a transparent support for introducing excitation light into the back surface of the metal thin film, a metal thin film for generating surface plasmon resonance formed on the transparent support, and the metal thin film. Including a reaction layer formed on the sensor surface for capturing the analyte on the sensor surface, and if necessary, the phosphor formed between the metal thin film and the reaction layer is too close to the metal thin film. A spacer layer may be included to prevent the metal quenching of fluorescence caused by.

In the present specification, for convenience, the side of the sensor chip on which the metal thin film, reaction layer, etc. are formed may be referred to as “upper” or “front”, and the opposite side as “lower” or “back”.
The site where the reaction layer is formed corresponds to the measurement region. A reaction layer may be formed over the entire bottom surface of the flow path or the well to be a measurement region, or a reaction layer may be formed only on a part of the bottom surface (with a desired patterning if necessary) to be a measurement region. The area of the measurement region can be adjusted in consideration of the irradiation area of excitation light generally irradiated as laser light. For example, if the spot diameter of excitation light is about 1 mmφ, the assay area is usually designed to have an area of at least several mm square.

  When the SPFS system is a “channel type” that sends various solutions through a sealed channel, a “flow cell” with holes to form the channel is loaded on the sensor chip. Correspondingly, a measurement member is constructed by loading a “top plate” with a liquid feed inlet and a liquid feed outlet at a position corresponding to the hole of the flow cell and fixing them in close contact with each other. To do. The sensor chip surface at a position corresponding to the hole of the flow cell forms the bottom surface of the flow path, and a measurement region is formed there. In the case of the flow channel type, for example, various liquids can be fed into the flow channel from the liquid feed inlet and discharged from the liquid feed outlet using a liquid feeding means including a pump and a tube. Thus, reciprocating and circulating liquid feeding can also be performed. Conditions such as the feeding speed and feeding (circulation) time are as follows: the amount of the sample, the concentration of the analyte in the sample, the size of the flow channel or well, the mode of the reaction layer (the density of the immobilized ligand, etc.), the pump It can be adjusted as appropriate in consideration of performance and the like.

  On the other hand, when the SPFS system is a “well type” in which various solutions are stored in a space wider than the flow path, a “well member” having a through hole for forming a well is formed on the sensor chip. A measuring member is constructed by loading and fixing. In the case of the well type, various liquids can be added to the well and removed using, for example, a pipette-shaped member.

  The flow cell can be made of, for example, a sheet-like polydimethylsiloxane (PDMS). The top plate is made of a material having transparency so that fluorescence emitted from the measurement region can be measured, and can be made of, for example, plate-shaped polymethyl methacrylate (PMMA). Alternatively, the flow cell and the top plate can be made of plastic having a desired shape by molding or photolithography.

  The means for fixing the flow cell or well member in close contact with the sensor chip is not particularly limited. In general, the pressure may be physically applied from above and below, and if necessary, a transparent support. Adhesives having the same optical refractive index, matching oil, transparent adhesive sheets, and the like may be used.

(Transparent support)
The transparent support can take the form of a prism or a planar substrate. The prism-shaped transparent support is used after being mounted on the SPFS measuring device as it is after the sensor chip is manufactured. The flat substrate-shaped transparent support is mounted on the horizontal surface of the prism included in the SPFS measuring device after the sensor chip is manufactured. To use. The size of the transparent support is not particularly limited, and can be adjusted according to the SPFS measuring apparatus. When the transparent support takes the shape of a flat substrate, the thickness is usually in the range of 0.01 to 10 mm.

Examples of the transparent support material include transparent dielectric materials such as glass, plastics such as polycarbonate (PC) and cycloolefin polymer (COP), and the refractive index [n _d ] at d-line (588 nm) is Substances in the range of 1.40 to 2.20 are preferred. The surface of the transparent support is preferably cleaned with an acid or plasma before forming the metal thin film.

(Metal thin film)
The metal thin film is composed of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, which is stable against oxidation and has a large electric field enhancement effect by surface plasmons (that is, any one of the above) It may be composed of such a metal alone or may be composed of an alloy of a plurality of metals.), And particularly preferably composed of gold.

  When a glass member is used as the dielectric member, in order to bond the glass and the metal thin film more firmly, an adhesive thin film made of, for example, chromium, nickel chromium alloy or titanium is interposed, and the glass dielectric member is used. It is preferable to laminate in the order of / adhesive thin film / metal thin film.

  Examples of the method for forming the metal thin film and the adhesive thin film include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. A sputtering method or a vapor deposition method is preferable because adjustment is easy.

  It is appropriate to adjust the thickness of the metal thin film and the adhesive thin film so that surface plasmons are easily generated according to the material of the metal thin film. In general, the thickness of the metal thin film is preferably 5 to 500 nm, and the thickness of the adhesive thin film is preferably 1 to 20 nm. From the viewpoint of the electric field enhancement effect, the thickness of the metal thin film is as follows: gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20-70 nm, and alloys thereof: 10-70 nm is more preferable. The thickness of the adhesive thin film is more preferably 1 to 3 nm, for example, in the case of chromium.

(Reaction layer)
The reaction layer is generally composed of a ligand and a molecule (linker) for immobilizing the ligand on a metal thin film or a spacer layer provided as necessary.

  The linker is not particularly limited as long as it can immobilize the ligand on the sensor surface, capture the analyte, and appropriately perform the analysis by SPFS. It may be a thing or a thing (complex) consisting of two or more types of molecules.

  For example, an embodiment using SAM (Self-Assembled Monolayer) as a linker can be mentioned. In this embodiment, a SAM-ligand conjugate formed on the surface of a metal thin film or the like forms a reaction layer.

  Moreover, the aspect which uses SAM and a high molecular compound together as a linker is also mentioned, In this aspect, the SAM-high molecular compound-ligand conjugate | bonded_body formed in the surfaces, such as a metal thin film, makes a reaction layer. When a ligand is immobilized on the sensor surface via such a polymer compound, in other words, in a mode in which the ligand is supported on the polymer compound, the layer made of the polymer compound is referred to as a “solid phase layer”. There is also. In such an embodiment, the ligand is three-dimensionally arranged in the solid-phased layer, so that the ligand is arranged on the sensor surface at a higher density than the embodiment in which the ligand is two-dimensionally (planarly) arranged on the upper layer of the SAM. This can be said to be a more preferable embodiment.

・ SAM
SAM can bind a functional group (silanol group, thiol group, etc.) that can bind to a metal thin film (or a spacer layer provided if necessary) at one end of the molecule to a molecule that immobilizes at the other end. It can be formed by a compound having a reactive functional group (amino group, carboxyl group, glycidyl group, etc.). Such a compound can be easily obtained as a SAM-forming reagent. For example, a carboxyalkanethiol having about 4 to 20 carbon atoms (such as 10-carboxy-1-decanethiol) has little optical influence, that is, a SAM having a high transparency, a low refractive index, and a thin film thickness. It is preferable because it can be formed.

・ Polymer compound The solid phase layer used as needed includes other molecules (typically SAM) constituting the reaction layer or layers (metal thin film, spacer layer, etc.) formed under the reaction layer. It is comprised by the high molecular compound which has the functional group for couple | bonding, and the functional group for couple | bonding with a ligand.

  In addition, since various aqueous solutions are usually used for various solutions used in SPFS, the polymer compound constituting the solid phase layer has a high affinity with hydrophilic groups such as hydroxyl groups and carboxyl groups. It is preferably a rich hydrophilic polymer (these hydrophilic groups may function as the predetermined functional group). Since the hydrophilic group includes water molecules by hydrogen bonding, it has an advantage that nonspecific adsorption hardly occurs. On the other hand, polymers with few hydrophilic groups (many hydrophobic groups) and hydrophobic polymers as a whole tend to localize on the sensor surface, resulting in a decrease in reaction efficiency and non-specificity due to hydrophobic interactions. There is a risk that it will become more likely to occur.

  Examples of the polymer compound constituting the solid phase layer include glucose or a derivative thereof (for example, carboxymethylated glucose), vinyl ester, acrylic acid ester, methacrylic acid ester, olefin, styrene, crotonic acid ester, itaconic acid diester, A homopolymer or copolymer (random, block, graft) containing units derived from monomers such as maleic acid diester, fumaric acid diester, allyl compound, vinyl ether, and vinyl ketone can be used.

  For example, carboxymethyl dextran or carboxymethyl cellulose in which a carboxymethyl group is introduced into dextran or cellulose (via a hydroxyl group in these glucose units), or alginic acid that originally has a carboxyl group in the glucose unit. Saccharides are suitable as a polymer compound constituting the solid phase layer because they have the above-mentioned predetermined functional groups and have a small branched structure and high solubility in cold water. In addition, polymer compounds such as polyaspartic acid, polyglutamic acid, polyacrylic acid, and polymethacrylic acid can also be used to form the solid phase layer.

  The thickness of the solid phase layer in the wet state is such that the phosphor that labels the analyte trapped by the ligand is efficiently excited by the enhanced evanescent wave, and the quenching by the metal thin film does not occur. In view of the above, about 3 to 80 nm is preferable. Therefore, a polymer compound having a molecular weight capable of generating such a thickness is preferable. For example, in the case of carboxymethyl dextran, those having a molecular weight in the range of about 10 to 1,000,000 are preferable.

  The density of the ligand to be bound to the polymer compound (that is, supported on the solid phase layer) depends on the polymer compound used and the nature of the ligand (number of functional groups in one molecule related to the reaction, etc.) It can be adjusted according to the reaction conditions (addition amount, pH, WSC concentration, etc.) between the ligand and the ligand.

-Solid-phased ligand The ligand in the present invention is a substance capable of forming an immune complex with CK-MB in a sample which is an analyte, that is, an anti-CK-MB antibody, an anti-CK-MM antibody or an anti-CK-BB antibody. is there. Since CK-MB is a dimer composed of CK-M subunit and CK-B subunit, anti-CK-MM antibody can also bind to CK-MB via an epitope present on CK-M subunit. Since anti-CK-BB antibody can also bind to CK-MB via an epitope present on CK-B subunit, it can be used in the same manner as anti-CK-MB antibody.

The “antibody” in the present invention includes not only a complete immunoglobulin (antibody in a narrow sense) but also Fab, Fab ′ ₂ , CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv), etc. Antibody fragments or antibody derivatives known in the art are included.

  In the present invention, anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody is bound to a ligand immobilized on the sensor surface in order to capture the analyte on the sensor surface (ie, “solid-phased ligand” to “ Used as “solid-phase antibody”, the primary antibody of a sandwich immunoassay).

  On the other hand, in the present invention, a ligand complexed with a fluorophore (that is, a “fluorescent labeled ligand” or a “fluorescent labeled antibody”, a sandwich immunoassay, for fluorescently labeling an analyte captured on the sensor surface. As the secondary antibody, anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody is used.

  As the anti-CK-MB antibody, anti-CK-MM antibody and anti-CK-BB antibody, each polyclonal antibody can be used. Since a combination of antibodies can be constructed, it is preferable to use each monoclonal antibody. As anti-CK-MB monoclonal antibody, anti-CK-MM monoclonal antibody and anti-CK-BB monoclonal antibody, those having various sites of CK-MB, CK-MM and CK-BB as epitopes are known, for example, Those shown in Table 1 are commercially available and can be easily obtained.

In the present invention, one kind or two or more kinds of anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody can be used as the immobilized antibody, and one or two fluorescently labeled antibodies can be used. More than one species of anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody can be used. However, in order to avoid competitive binding of the immobilized antibody and the fluorescently labeled antibody to the same epitope of the analyte, the immobilized antibody is one or more anti-CK-MB monoclonal antibodies. An anti-CK-MM monoclonal antibody or an anti-CK-BB monoclonal antibody, wherein the fluorescently labeled antibody is different from one or more monoclonal antibodies as the solid-phased antibody, one or more The anti-CK-MB monoclonal antibody, anti-CK-MM monoclonal antibody or anti-CK-BB monoclonal antibody is preferably used.

  Depending on the combination of anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody used for each of the solid-phased antibody and the fluorescence-labeled antibody, the detection (quantitative) sensitivity of CK-MB by SPFS may differ. Thus, it is appropriate to use a combination of antibodies that can achieve the desired sensitivity. For example, it is desirable to use a combination of a highly sensitive anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody with a detection (quantitative) limit concentration (1 pg / mL or more) and less than 10 pg / mL or less. .

-Formation method of reaction layer The reaction layer is a layer formed under the reaction layer (metal thin film, spacer layer, etc.), SAM constituting the reaction layer, polymer compound constituting the solid phase layer, ligand, etc. Can be formed by sequentially bonding them using a known method.

  In order to form a SAM on an upper layer of a metal thin film or the like, generally, a solution of a SAM constituent molecule is brought into contact with the metal thin film or the like, and a functional group (for example, a thiol group) at one end of the molecule and the metal thin film or the like ( For example, gold).

  Subsequently, in order to immobilize the ligand on the upper layer of the SAM, generally, the ligand solution is brought into contact with the SAM, and the functional group of the ligand and the functional group at the other end of the SAM constituent molecule are combined. Let it react. For example, a ligand having an amino group (anti-CK-MB antibody in the present invention) and a SAM component having a carboxyl group include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and the like. A solution of said ligand with addition of water-soluble carbodiimide (WSC) and optionally N-hydroxysuccinimide (NHS) for activating (esterifying) the carboxyl group by contacting with SAM And a carboxyl group through a reaction.

  On the other hand, in order to form a solid phased layer by binding a polymer compound rather than directly immobilizing a ligand on the upper layer of the SAM (after that, the ligand is supported on the solid phased layer) The solution of the polymer compound is brought into contact with the SAM, and the functional group of the polymer compound is reacted with the functional group at the other end of the SAM constituent molecule. For example, when carboxymethyl dextran is used as the polymer compound, the aldehyde group (reducing terminal) possessed by carboxymethyl dextran and the amino group possessed by SAM can be reacted to form a Schiff base to bind them. it can. By adjusting the reaction conditions, the number of polymer compounds linked to SAM or the like can be increased or decreased.

  Alternatively, by using a polymer compound having a functional group capable of adsorbing to a metal thin film or the like (for example, a polysaccharide modified with thiosemicarbazide), the polymer compound is directly bonded to the metal thin film or the like without using SAM. It is also possible to carry a ligand on this.

  The polymer compound and the ligand can be bound according to a known method such as an amine coupling method, a thiol coupling method, or an indirect capture method (capture method). For example, when the amine coupling method is used, a ligand having an amino group (anti-cTn antibody in the present invention) and a polymer compound having a carboxyl group (solid phase layer) are 1-ethyl-3- ( Said water-soluble carbodiimide (WSC) such as 3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and optionally N-hydroxysuccinimide (NHS) for activating (esterifying) the carboxyl group By bringing the ligand solution into contact with the polymer compound, the ligand can be bound via a reaction between an amino group and a carboxyl group. It is possible to increase or decrease the number of ligands bound to the linear polymer by adjusting the reaction conditions.

  After immobilizing the ligand, in order to prevent nonspecific adsorption of the analyte, the sensor surface (including the channel or the side wall of the well / top plate) is blocked with bovine serum albumin (BSA), casein, etc. It is desirable to keep it.

(Spacer layer)
The spacer layer provided as necessary can be formed of a dielectric. As the dielectric for the spacer layer, various optically transparent inorganic substances and natural or synthetic polymers can be used. Among these, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) is preferable because of excellent chemical stability, production stability, and optical transparency.

  The thickness of the spacer layer is usually 10 nm to 1 mm, and preferably 30 nm or less, more preferably 10 to 20 nm from the viewpoint of resonance angle stability. On the other hand, from the viewpoint of the electric field enhancement effect, the thickness is preferably 200 nm to 1 mm, and more preferably 400 nm to 1,600 nm from the stability of the electric field enhancement effect.

  Such a spacer layer can be formed by sputtering, electron beam evaporation, thermal evaporation, a method using a chemical reaction using a material such as polysilazane, or application by a spin coater.

-Measurement equipment for SPFS-
The immunological measurement method of the present invention can be carried out using a general measurement apparatus for SPFS. The SPFS measurement device basically has an SFPFS measurement member that can be attached and detached, and a light source and excitation for irradiating excitation light (preferably laser light) with an appropriate wavelength according to the phosphor used. Prism for making light incident on the back of the metal thin film of the sensor chip at a predetermined angle (when the transparent support uses a sensor chip with a flat substrate), receiving the light reflected by the metal thin film and measuring its intensity Light receiver, lens for condensing fluorescence emitted from the phosphor, detector for measuring the intensity of the fluorescence, excitation light and only light having a predetermined wavelength is transmitted from the fluorescence and cut the other light It is equipped with various filters for doing so. For more specific modes, various documents such as JP 2010-145272 A (Patent Document 2) can be referred to.

− Immunological assay −
The immunological measurement method of the present invention is a method capable of quantifying the concentration of CK-MB in a sample or detecting the presence thereof, and includes performing the following steps 1 and 2:
(Step 1) Solid-phased anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody (solid-phase antibody), CK-MB, and fluorescently labeled anti-CK-MB antibody, A step of forming a sandwich-type immune complex comprising an anti-CK-MM antibody or an anti-CK-BB antibody (fluorescently labeled antibody), and (step 2) emitted from a phosphor contained in the formed sandwich-type immune complex A step of measuring the fluorescence intensity by SPFS (surface plasmon excitation enhanced fluorescence spectroscopy).

The step of forming the sandwich type immune complex shown as step 1 is not particularly limited as long as the sandwich type immune complex can be formed before moving to step 2, but for example, the following step 1a And embodiments comprising 1b are common:
(Step 1a) reacting the immobilized antibody with CK-MB in the sample to form a complex, and (Step 1b) reacting the formed immune complex with the fluorescently labeled antibody. Forming the sandwich-type immune complex.

  It is to be noted that a cleaning for cleaning the flow path or well with a cleaning liquid (for example, a surfactant solution) between the steps 1a and 1b or between the steps 1 (step 1b) and 2 as necessary. A process may be provided.

  On the other hand, in step 2, in the same manner as in general SPFS, the metal thin film is irradiated with excitation light, and an evanescent wave enhanced by surface plasmon resonance occurring in the metal thin film is generated, whereby a sandwich-type immune complex is formed. What is necessary is just to measure the intensity | strength (equivalent to a "signal") of the fluorescence emitted from the fluorescent substance contained (the fluorescent labeled antibody had). In addition, before the step 2 or in a region different from the measurement region, the fluorescence intensity generated by irradiating the metal thin film with excitation light in the same manner as in the step 2 in the state where the sandwich immune complex is not formed ( It is preferable that the signal value is corrected (subtracted or removed) by the noise value.

  When measuring the fluorescence intensity emitted from the phosphor in step 2, the flow path or well is usually filled with an aqueous solvent (for example, phosphate buffer) containing neither analyte nor fluorescently labeled antibody. However, it may be in a state filled with a solvent other than the aqueous solvent or air.

  The concentration of the analyte in the analyzed sample based on the signal obtained as described above (preferably corrected using noise) and a calibration curve prepared separately using a sample of known concentration Can be quantified.

  The concentration of CK-MB in the sample (particularly blood sample) measured in this way can be used as reference data when diagnosing various diseases or symptoms in which the CK-MB functions as a biomarker. .

・ Analytes, samples (blood samples, etc.)
The analyte (substance to be quantified or detected by SPFS) in the present invention is CK-MB that is contained in a blood sample and can be used as a biomarker such as myocardial infarction.

  In the present invention, a blood sample that may contain CK-MB is used for analysis as a sample to be put into the SPFS analyzer and brought into contact with the reaction layer provided on the sensor chip. The blood sample can be the same as that used to analyze various biomarkers and other biological substances, and may be whole blood that has been anti-coagulated if necessary, or may be precipitated by coagulating whole blood. Serum obtained by removing substances (blood clots) may be used, or these may be processed by centrifugation, dilution, mixing with reagents, etc. as necessary (in the present invention, all of them are “blood samples”. Generically).

  In addition, CK-MB prepared in advance by purchasing CK-MB in a phosphate buffer or the like instead of the blood sample as described above was used for experimental measurement or calibration curve preparation. -MB solution may be used as a sample in the present invention. That is, the immunological measurement method of the present invention may target CK-MB in a sample other than the blood sample as described above, similarly to CK-MB in the blood sample.

-Fluorescently labeled ligand In the present invention, as a fluorescently labeled ligand for fluorescently labeling the analyte captured on the sensor surface, a phosphor, an anti-CK-MB antibody, an anti-CK-MM antibody or an anti-CK-BB A fluorescently labeled antibody that is a complex with the antibody is used. As described above, the detection sensitivity of CK-MB by SPFS may differ depending on the combination of anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody used for each of the immobilized antibody and the fluorescently labeled antibody. Therefore, a suitable anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody is selected depending on the anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody used as the immobilized ligand. It is used for the production of fluorescently labeled antibodies.

  The fluorescence-labeled antibody can be prepared in the same manner as a complex (conjugate) of a fluorophore and an antibody that are also used in general immunoassay methods, and the mode is not particularly limited. For example, using a commercially available phosphor kit (for example, Alexa Fluor protein labeling kit, Invitrogen), the functional group introduced into the phosphor and the functional group possessed by the anti-CK-MB antibody are determined according to the attached protocol. By reacting in the presence of a predetermined reagent, a phosphor-anti-CK-MB antibody complex can be prepared. In order to bind the fluorophore and the ligand, reactions in various combinations of functional groups can be used, as well as a biotin-avidin bond between a biotinylated fluorophore and an avidinylated anti-CK-MB antibody. It is also possible to use it. The same applies when an anti-CK-MM antibody or an anti-CK-BB antibody is used instead of the anti-CK-MB antibody.

-Phosphor In the present invention, various known phosphors used in general SPFS and conventional fluorescence measurement methods can be used. In addition, “phosphor” is a general term for substances that emit fluorescence by irradiating with predetermined excitation light or by using the electric field effect, and “fluorescence” includes not only fluorescence in a narrow sense, It also includes phosphorescence and other luminescence.

A fluorescent dye is typically used as a phosphor used in SPFS, but other known phosphors such as semiconductor nanoparticles may be used.
Specific examples of fluorescent dyes include fluorescein family fluorescent dyes (Integrated DNA Technologies), polyhalofluorescein family fluorescent dyes (Applied Biosystems Japan), hexachlorofluorescein family fluorescent dyes (Applied Biosystems) Japan Co., Ltd.), Coumarin Family Fluorescent Dye (Invitrogen Corp.), Rhodamine Family Fluorescent Dye (GE Healthcare Biosciences), Cyanine Family Fluorescent Dye, Indian Carbocyanine Family Fluorescence Dyes, fluorescent dyes of the oxazine family, thiazine family fluorescent dyes, squaraine family fluorescent dyes, chelated lanthanide family fluorescent dyes, BODIPY® family fluorescent dyes Organic fluorescent dyes such as Vitrogen Corp., naphthalene sulfonic acid family fluorescent dyes, pyrene family fluorescent dyes, triphenylmethane family fluorescent dyes, Alexa Fluor® dye series (Invitrogen Corp.) Is mentioned.

Further, rare earth complex fluorescent dyes such as Eu and Tb (for example, ABTTA-Eu ³⁺ ), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), Examples of fluorescent dyes include fluorescent proteins typified by red fluorescent protein (DsRed) or Allophycocyanin (APC; LyoFlogen (registered trademark)), and fluorescent fine particles such as latex and silica.

  When a sample derived from a blood sample is used for analysis, in order to minimize the influence of light absorption by iron derived from blood cell components in blood, the maximum fluorescence wavelength is set in the near infrared region such as Cy5 or Alexa Fluor 647. It is desirable to use a fluorescent dye having the same. Further, it is preferable to consider the influence of light absorption by the metal contained in the metal thin film of the sensor chip. For example, when gold is used for the metal thin film, a fluorescent dye having a maximum fluorescence wavelength of 600 nm or more is preferable, and when silver is used for the metal thin film, a fluorescent dye having a maximum fluorescence wavelength of 400 nm or more is used. preferable.

The monoclonal antibodies used in the following examples are the products listed in Table 1.
[Example 1-1] 10R-CR3013M2 (solid phase) / 10R-C146A (label)
(1) Production of sensor chip After plasma cleaning of a glass transparent support “S-LAL 10” (Ohara Co., Ltd., refractive index (n _d ): 1.72) having a thickness of 1 mm, A chromium thin film was formed on one surface by sputtering, and a gold thin film was formed on the surface by sputtering. The thickness of the chromium thin film was 1 to 3 nm or less, and the thickness of the gold thin film was 42 to 47 nm.

  The transparent support provided with the metal thin film obtained by the above process was immersed in 10 mL of 11-amino-1-undecanethiol ethanol solution adjusted to 1 mM for 24 hours to form SAM on the surface of the gold thin film. The transparent support was taken out from the ethanol solution, washed with ethanol and isopropanol, and then dried using an air gun.

Subsequently, the transparent support provided with the SAM obtained by the above process was prepared by adding 1 mg / mL of carboxymethyldextran (CMD) having a molecular weight of 500,000 to 1,000,000 and 0.5 mM of N-hydroxysuccinimide (NHS). MES buffered saline (MES) having a pH of 7.4, and 1 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as water-soluble carbodiimide (WSC) (ionic strength: (10 mM) for 1 hour to immobilize CMD as a solid phase layer on the surface of the SAM, and then immersed in a 1 M NaOH aqueous solution for 30 minutes to hydrolyze unreacted succinate. The average film thickness of the solid phase layer made of CMD was 70 nm, and the density was 1.9 ng / mm ² .

  Subsequently, the transparent support provided with the solid phase layer obtained by the above step was immersed in MES containing 50 mM NHS and 100 mM WSC for 1 hour, and then anti-CK-MB monoclonal antibody “10R- The monoclonal antibody was immobilized on CMD by immersing in CR3013M2 ”solution (FGD, diluted to 2.5 μg / mL) for 30 minutes.

Further, non-specific adsorption prevention treatment was performed by circulating and feeding PBS containing 1% by mass of bovine serum albumin (BSA) and 1M aminoethanol for 30 minutes.
A PDMS sheet having a thickness of 10 mm and a hole having a width of 5 mm and having a thickness of 0.5 mm was placed on the sensor chip obtained as described above, and a silicone rubber spacer was arranged around the PDMS sheet. On the PDMS sheet and the silicone rubber spacer, a PMMA top plate in which a hole for introducing a liquid supply and a hole for discharging a liquid were formed at a position corresponding to the hole of the PDMS sheet was placed. . A laminate of these sensor chip, PDMS sheet and silicone rubber spacer, and PMMA top plate was pressure-bonded at the outer periphery and fixed with screws to produce a measurement member for SPFS.

(2) Preparation of fluorescently labeled antibody Using an anti-CK-MB monoclonal antibody “10R-C146a” solution (FGD, diluted to 2.5 μg / mL) and AlexaFluor647 labeling kit (Invitrogen), AlexaFluor647 labeled 10R-C146a was produced according to a predetermined procedure. Thereafter, unreacted substances were removed using a molecular weight cut filter (Nippon Millipore Corporation), and AlexaFluor647-labeled 10R-C146a was purified and stored at 4 ° C. until the following assay was performed.

(3) Implementation of assay 0.1 mL of PBS solution containing 1000 pg / mL (= 1 ng / mL) of CK-MB was sent to the flow path of the measurement member prepared in the above step (1) and circulated for 25 minutes. .

  Subsequently, Tris-buffered saline (TBS) containing 0.05% by weight of Tween 20 was fed, circulated for 10 minutes to wash the flow path, and then AlexaFluor 647-labeled 10R-C146a prepared in the above step (2). In PBS containing 2 μg / mL was circulated for 5 minutes.

  Again, Tris-buffered saline (TBS) containing 0.05% by weight of Tween 20 was fed, circulated for 10 minutes to wash the flow path, and the flow path was filled with PBS buffer (pH 7.4). Then, laser light (640 nm, 40 μW) was irradiated from the back surface of the metal thin film in the measurement region, and the amount of fluorescence was measured with a photomultiplier tube (PMT) installed at the top of the measurement region. This measured value was defined as “signal” (S) when the CK-MB concentration was 1000 pg / mL.

  On the other hand, instead of a PBS solution containing 1000 pg / mL of CK-MB, a PBS solution containing no CK-MB (0 pg / mL) was fed, and the amount of fluorescence was measured by the same procedure as above except that. This measured value was defined as “noise” (N).

  In addition, the concentration of PBS solution of CK-MB to be fed is set to 100000 pg / mL, 31600 pg / mL, 10000 pg / mL, 3160 pg / mL, 316 pg / mL, 100 pg / mL, 31.6 pg / mL, 10 pg / mL, The amount of fluorescence was measured by the same procedure as above except that it was changed to 16 pg / mL, 10 pg / mL, 3.16 pg / mL, 1 pg / mL, and 0.316 pg / mL. “Signal” (S).

  The result of each concentration was plotted with the vertical axis representing “signal”-“noise” (SN) value (unit: au) and the horizontal axis representing cTnI concentration (unit: pg / mL) ( FIG. 1, rhombus). Based on this plot, it was determined that the quantification limit of CK-MB by the measurement system was 2 pg / mL.

[Example 1-2] 1011820 (solid phase) / 10-C40A (label)
In the sensor chip production step (1), the anti-CK-BB monoclonal antibody “1011820” (COSMO BIO / COR) is immobilized on CMD, and in the production step (2) of the fluorescent labeled antibody, the anti-CK-MB monoclonal antibody is immobilized. Except that the antibody “10-C40A” (FGD) was fluorescently labeled and the obtained AlexaFluor647-labeled 10-C40A was changed to be used in the step (3) of the assay, the same as in Example 1-1 The operation was performed and “signal” and “noise” were measured. The result of each concentration was plotted (FIG. 1, square), and it was determined that the quantification limit of CK-MB by the above measurement system was 20 pg / mL.

[Example 1-3] 10-C40A (solid phase) / 10-C40B (label)
The anti-CK-MB monoclonal antibody “10-C40A” is immobilized on CMD in the sensor chip production step (1), and the anti-CK-MB monoclonal antibody “10-C40B” is produced in the fluorescent-labeled antibody production step (2). (FGD Co., Ltd.) and the same procedure as in Example 1-1 was performed except that the obtained AlexaFluor647-labeled 10-C40B was used so as to be used in the execution step (3) of the assay. And "noise" was measured. The result of each concentration was plotted (FIG. 1, triangle), and it was determined that the quantification limit of CK-MB by the above measurement system was 180 pg / mL.

  In addition to the above, various combinations of the immobilized anti-CK-MB monoclonal antibody (solid phase antibody) and the anti-CK-MB monoclonal antibody (labeled antibody) used for the fluorescently labeled ligand are the same as in Example 1-1. The limit of detection or the limit of quantification was obtained by the procedure described above. The results (including Examples 1-1 to 1-3) are shown in the following table.

[Example 2]
In the step (3) of the assay, Examples were changed except that 0.1 mL of human serum containing 1000 pg / mL of CK-MB was used instead of 0.1 mL of PBS solution containing 1000 pg / mL of CK-MB. The same operation as in 1-1 was performed, and “signal” and “noise” were measured. When the result of each density | concentration was plotted, it was determined that the same sigmoid curve as Example 1-1 was obtained, and it had the same quantitative limit.

Claims (4)

  An immunological assay for CK-MB (creatine kinase isozyme MB) in a sample, which is an immobilized anti-CK-MB antibody, anti-CK-MM (creatine kinase isozyme MM) antibody or anti-CK -BB (creatine kinase isozyme BB) antibody (hereinafter referred to as "solid-phase antibody"), CK-MB, fluorescently labeled anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody (Hereinafter referred to as “fluorescence-labeled antibody”), and the fluorescence intensity emitted from the phosphor contained in the formed sandwich-type immune complex is expressed by SPFS (surface plasmon excitation). An immunoassay comprising the step of measuring by enhanced fluorescence spectroscopy.   The step of forming the sandwich-type immune complex includes the step of reacting the solid-phased antibody with CK-MB in a sample to form an immune complex, and the formed immune complex and the fluorescently labeled antibody. The immunoassay method according to claim 1, further comprising a step of forming a sandwich-type immune complex by reacting with an immunological compound.   The immobilized antibody is one or more anti-CK-MB monoclonal antibody, anti-CK-MM monoclonal antibody or anti-CK-BB monoclonal antibody, and the fluorescently labeled antibody is used as the immobilized antibody. The anti-CK-MB monoclonal antibody, anti-CK-MM monoclonal antibody or anti-CK-BB monoclonal antibody different from one or more monoclonal antibodies is one or more types. Immunological assay for   A solid-phased anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody, CK-MB, and fluorescence used in the immunological assay according to any one of claims 1 to 3 A sensor chip for SPFS, in which a sandwich-type immune complex containing a labeled anti-CK-MB antibody, anti-CK-MM antibody or anti-CK-BB antibody is formed in a measurement region.