JP4436110B2 - Optical measuring apparatus and optical measuring method for specific binding substance using the same - Google Patents

Description

The present invention relates to an optical measuring apparatus and a method for optically measuring a specific binding substance using the same, and more specifically, a second specific binding member to which a fluorescent label or a light scattering label is bound and a substance to be measured. The specific binding substance bound to the target substance and the first specific binding member is fixed to the surface of the waveguide, the light incident side edge surface of the waveguide is irradiated with light, and the excited excitation Optical that measures the specific binding substance by measuring the fluorescence or scattered light converted or scattered by the fluorescent label or light scattering label by the evanescent wave component generated by the light beam, and thus the measurement substance in the fluid sample The present invention relates to a measuring apparatus and an optical measuring method.

Conventionally, the light from the light source is propagated while totally reflecting through the optical waveguide, and the fluorescent substance constrained near the surface of the optical waveguide by the antigen-antibody reaction is excited by the evanescent wave component, and the intensity of the resulting fluorescence is measured. Therefore, studies on fluorescent immunoassay devices that indirectly measure the degree of immune reaction have been conducted.

As a fluorescence immunoassay device with excellent measurement sensitivity and measurement accuracy, the antigen-antibody reaction is carried out on the surface of the optical waveguide that propagates while totally reflecting the introduced excitation light, and further a substance labeled with a fluorescent substance is reacted. Fluorescence immunoassay that measures the degree of immune reaction based on the fluorescence emitted from the optical waveguide by introducing the fluorescence emitted by the fluorescent material excited by the evanescent wave component of the excitation light into the optical waveguide and propagating it while totally reflecting it. A fluorescence immunoassay device characterized in that a fluorescent material having an absorption peak near 650 nm is used as the device and light having a wavelength near 635 nm is used as excitation light has been proposed (see, for example, Patent Document 1). ).
JP-A-7-63756

However, in the above-described fluorescence immunoassay apparatus, the light to be irradiated has to be irradiated with a laser beam having a specific wavelength at a specific angle (an angle at which the light is totally reflected in the waveguide), and an ordinary incandescent lamp, xenon lamp, etc. Since the light source cannot be used, the apparatus becomes large, the cost is high, and the measurement is difficult.

In the past, B / F separation was required to perform high-sensitivity and high-precision analysis.
Since the / F separation process requires complicated processing, analysis could not be performed at high speed in a short time.

In view of the above-described drawbacks, the object of the present invention is to use an arbitrary light source to irradiate a light incident side edge surface of a waveguide with light rays, to attenuate and extinguish excitation light rays that are not totally reflected in the waveguide, and to totally reflect light. Only the light is propagated through the waveguide to the reaction part, and the fluorescence or scattered light converted or scattered by the fluorescent label or the light scattering label by the evanescent wave component generated in the reaction part by the excitation light is measured by the light receiving element. Accordingly, an object of the present invention is to provide an optical measuring apparatus capable of measuring a specific binding substance at high speed and with high sensitivity and accuracy without performing B / F separation, and an optical measurement method for a specific binding substance using the same.

The optical measurement apparatus according to claim 1 is an apparatus for optically measuring a specific binding substance in a fluid sample, which is bound to a second specific binding member to which a fluorescent label or a light scattering label is bound. The first specific binding member that can specifically bind to the substance to be measured to form the specific binding substance has a reaction part fixed on one surface and a transparent guide having a light incident side edge surface. A transparent intermediate layer (B) and a light absorption layer (C) are sequentially laminated on the other surface of the waveguide (A). The refractive index of the waveguide (A) is n _w and the refractive index of the intermediate layer (B) is Where n _m and the refractive index of the fluid sample are n _s , each refractive index satisfies the following formula (1).
n _w > n _m ≧ n _s (1)

In the waveguide (A), one side surface is a light incident side edge surface, and the excitation light incident on the light incident side edge surface from the light source propagates to the reaction part formed on one surface while being totally reflected. Therefore, it is a film, sheet or plate made of an optically transparent material.

Examples of the optically transparent material include glass, quartz, acrylic resin, fluorine resin, polycarbonate resin, aromatic polyester resin, polyarylate resin, epoxy resin, silicon resin, polystyrene resin, polyimide resin, and fluorine thereof. In particular, glass, quartz, acrylic resin, polycarbonate resin, and polystyrene resin are preferable.

Examples of the acrylic resin include polymethyl methacrylate (refractive index = 1.4
90), polybenzyl methacrylate (refractive index = 1.568), polyphenyl methacrylate (refractive index = 1.571), poly 1-phenylethyl methacrylate (refractive index = 1.5).
49), polycyclohexyl methacrylate (refractive index = 1.507), methyl methacrylate-benzyl methacrylate copolymer, methyl methacrylate-phenyl methacrylate copolymer, methyl methacrylate-1,1,2-trifluoroethyl methacrylate Examples thereof include a polymer and a methyl methacrylate-styrene copolymer.

The refractive index n _w of the waveguide (A) must be greater than the refractive index n _{s of} the fluid sample, and when the fluid sample is an aqueous solution, the refractive index of water is 1.33, so n _w > 1.33. And preferably n _w > 1.49.

The thickness of the waveguide (A) is generally preferably 5 μm to 2 mm, more preferably 0.1 to 1.
mm. The size of the light source is preferably about the same as the thickness of the waveguide. When the light source is larger than the waveguide, the light source may be condensed to the same thickness as the waveguide by a lens or the like.

The reaction part has a first specific substance that can specifically bind to a substance to be measured bound to a second specific binding member to which a fluorescent label or a light scattering label is bound to form the specific bound substance. Static binding member is fixed.

The first specific binding member varies depending on the substance to be measured. For example, an enzyme, a microorganism, an antigen, an antibody, an antibody fragment, a lectin, a receptor, an ionophore, a proton pump, a biological membrane, an artificial biological element, DNA , One molecule selected from the group consisting of RNA molecules, proteins, sugar chains, glycoproteins, metalloproteins, or a mixture thereof.

As a method for fixing the first specific binding member to the reaction part, any conventionally known method may be employed. For example, a reactive group capable of covalently binding to the first specific binding member on the reaction part surface. Covering the reaction part surface with a compound layer that can be adsorbed by the first specific binding member such as protein, and the like. Examples include a method of adsorbing the first specific binding member.

The fluorescent label is a substance that generates fluorescence by converting light when irradiated with light. For example, a compound that generates fluorescence when irradiated with light, a compound that generates fluorescence. Examples thereof include synthetic resin particles.

Examples of the compound generating fluorescence include fluorin isothiocyanate, fluorescein, fluorescein N-hydroxysuccinimide ester, 6-(((4- (
4,4-difluoro-5- (2-thienyl) -4--4-bora-3a, 4a-diaza-5-indacene-3-yl) phenoxy) acetyl) amino) hexanoic acid, succinimidyl ester, 4-acetamide -4'-isocyanatostilbene-2,2'-disulfonic acid,
7-amino-4-methylcoumarin, 7-amino-4-trimethylcoumarin, N- (4-anilino-1-naphthyl) maleimide, dansyl chloride, 4′6-diamidino-2-phenylindole, 5- (4 6-dichlorotriazin-2-yl) aminofluorescein, 4
, 4'-Diisothiocyanatostilbene-2,2'-disulfonic acid, eosin isothiocyanate, erythrosin B, fluoresamine, fluorescein-5 (6) -carboxyamidocaproic acid N-hydroxysuccinimide ester, 5-isothiocyanate (is
othioscanante) diacetate, 4-methylumbelliferone, o-phthaldialdehyde, QFITC, rhodamine B isothiocyanate, rhodamine 101 acid chloride, tetramethylrhodamine isothiocyanate, 2 ′, 7′-difluorofluorescein, cyanine dye, Examples thereof include rhodamine and rare earth metal complexes, and rare earth metal complexes having a long emission lifetime are preferably used.

Examples of the rare earth metal constituting the rare earth metal complex include scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, and the like. And samarium, europium, terbium and dysprosium are preferably used.

The ligand constituting the rare earth metal complex together with the rare earth metal is not particularly limited as long as it can constitute a rare earth metal complex that reacts with the rare earth metal to generate fluorescence. For example, ethylenediaminetetraacetic acid, 4, 7-bis- (chlorosulfophenyl) -1,10-
Phenanthroline-2,9-dicarboxylic acid, 4,4′-bis-1 ″, 1 ″
, 1 ", 2", 2 ", 3", 3 "-heptafluoro-4", 6 "-hexanedione-6"
-Yl) -chlorosulfo-o-terphenyl, hexafluoroacetylacetone, triphenylphosphine oxide, diphenyl sulfoxide, 1,10-phenanthroline and the like.

Synthetic resin particles containing a compound that generates fluorescence are less likely to be reduced by coexisting substances in the fluid sample compared to when the compound that generates fluorescence is dissolved in the fluid sample. preferable.

Any conventional method may be employed as a method for causing the synthetic resin particles to contain the compound that generates fluorescence. For example, emulsion polymerization of a polymerizable monomer in the presence of a compound that generates fluorescence,
Examples thereof include a polymerization method by a polymerization method such as suspension polymerization.

Examples of the polymerizable monomer include aromatic vinyl compounds such as styrene, α-methylstyrene, o-vinyltoluene, m-vinyltoluene, and p-vinyltoluene; methyl (meth) acrylate, ethyl (meth) acrylate, n -Propyl (meth) acrylate,
acrylates such as i-propyl (meth) acrylate, n-butyl (meth) acrylate, t-butyl (meth) acrylate, n-hexyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, and cyclohexyl (meth) acrylate; Vinyl cyanide compounds such as (meth) acrylonitrile and vinylidene cyanide; vinyl halide compounds such as vinyl chloride, vinylidene chloride, vinyl fluoride, vinylidene fluoride and tetrafluoroethylene; vinyl esters such as vinyl acetate and vinyl propionate Is mentioned.

The polymerizable monomer may be a copolymer of a monofunctional monomer or a polyfunctional monomer that is generally copolymerized when these polymerizable monomers are polymerized. Examples of the monofunctional monomer include (meth) Acrylic acid, maleic anhydride, glycidyl acrylate and the like can be mentioned. Examples of the polyfunctional monomer include divinylbenzene, (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and trimethylol. Examples thereof include propane tri (meth) acrylate.

Since the content of the compound that generates fluorescence in the synthetic resin particles decreases, the amount of generated fluorescence decreases and the measurement sensitivity decreases. 5-80 weight% is preferable in a synthetic resin particle, More preferably, it is 15-60 weight%.

The synthetic resin particles are preferably as small as possible, preferably 10 μm or less, more preferably 20 nm to 500 nm, and even more preferably 30 nm to 200 nm.
nm.

In order to synthesize such synthetic resin particles having a small particle diameter, it is only necessary to prepare and polymerize a fine droplet of a polymerizable monomer. For example, the polymerizable monomer and necessary components such as an emulsifier, a dispersant, and a polymerization initiator The mixed solution may be supplied to a homogenizer, a micromixer, a microchannel or the like to produce fine droplets and polymerized according to a conventional method.

The light scattering label is a compound or fine particles that can scatter light when irradiated with light, for example, colloidal fine particles of metal such as gold and silver, chalcogenite fine particles such as CdS and CdSe, polystyrene resin, polycarbonate Examples thereof include polymer fine particles such as resin and poly (meth) acrylic resin, inorganic oxide fine particles such as silica gel, alumina and titanium oxide, and core-shell fine particles formed by a combination of two or more of these. The polymer fine particles and inorganic oxide fine particles may be dyed, or fluorescent molecules or metal nanoparticles may be dispersed.

The size of the light scattering label particles is not particularly limited, but is generally 10 nm to 1 nm.
0 μm, preferably 50 nm to 500 nm, more preferably 70 to 200 n.
m.

The second specific binding member is capable of specifically binding to the analyte to be measured, and the analyte is sandwiched between the first specific binding member and the second specific binding member. Is something that can be done.

Accordingly, the second specific binding member differs depending on the substance to be measured and the first specific binding member, but for example, an enzyme, a microorganism, an antigen, an antibody, an antibody fragment, a lectin, a receptor, an ionophore, a proton Pumps, biological membranes, artificial biological elements, DNA molecules, RNA
1 selected from the group consisting of molecules, proteins, sugar chains, glycoproteins, and metalloproteins
Seeds or a mixture thereof.

Therefore, in the case of an immunoassay method, the substance to be measured is an antibody or an antigen, and an antigen or an antibody that reacts specifically with the first specific binding member and the second specific binding member is used. The

While the excitation light beam incident on the light incident side edge surface from the light source propagates to the reaction part formed on one surface while totally reflecting inside the waveguide (A), the excitation light beam that is not totally reflected attenuates and disappears. Therefore, it is preferable that the excitation light is reflected as much as possible in the waveguide (A) before propagating to the reaction part.

Therefore, the distance from the reaction part to the light incident side edge surface is preferably at least twice the distance of one round trip in which the excitation light propagates through the waveguide (A) with total reflection, and more preferably 20 It is more than double.

Since the transparent intermediate layer (B) is a layer that is laminated on the other surface of the waveguide (A) and receives excitation light that does not totally reflect among excitation light propagating through the waveguide (A), Transparent and refractive index _nm
Needs to satisfy the following formula (1).
n _w > n _m ≧ n _s (1)

That is, when the refractive index n _m of the intermediate layer (B) is larger than the refractive index n _{w of the} waveguide (A), all of the excitation light rays introduced into the waveguide (A) pass through the waveguide (A). When the refractive index n _s of the fluid sample becomes larger than the refractive index n _w of the waveguide (A) without being reflected, the excitation light beam leaks into the fluid sample. This is because the floating fluorescent label or the light scattering label in the fluid sample is converted or scattered by the emitted and leaked excitation light, and fluorescence or scattered light is generated.

Furthermore, if the refractive index n _s of the fluid sample is greater than the refractive index n _m of the intermediate layer (B), a waveguide (
Although it is totally reflected between A) and the intermediate layer (B), it is not totally reflected between the waveguide (A) and the fluid sample, and excitation light leaking from the waveguide (A) to the fluid sample is not reflected. Will exist,
This is because the floating fluorescent label or light scattering label in the fluid sample is converted or scattered by the leaked excitation light, and fluorescence or scattered light is generated.

Examples of the material constituting the transparent intermediate layer (B) include a fluorinated epoxy resin, a fluorinated acrylic resin, and water. The refractive index n _s of the fluid sample and the refractive index n of the intermediate layer (B) are included. _{When m} is the same, in the reaction part to which the fluid sample is supplied, all light rays that are not totally reflected in the waveguide (A) can be absorbed by the intermediate layer (B), so that the refractive index n _{s of the} fluid sample is almost the same. preferably a substance having a refractive index, a substance having a refractive index of within 10% of the refractive index n _s of the fluid sample is preferred.

The thickness of the intermediate layer (B) is not particularly limited. However, when the thickness is reduced, it becomes difficult to introduce a liquid such as water, and when the thickness is increased, it is difficult to stack the waveguide (A) and the light absorption layer (C). So 0.
6-1000 micrometers is preferable.

The light absorption layer (C) is a layer that absorbs the excitation light that has entered the intermediate layer (B). By absorbing the light, the non-total reflection excitation light in the waveguide (A) is attenuated and extinguished. For example, a layer of metal or synthetic resin colored with a black pigment such as carbon, which is colored on the entire surface or inside, is preferable.

Examples of the synthetic resin include olefin resin, vinyl chloride resin, polystyrene resin, acrylic resin, ABS resin, fluorine resin, polycarbonate resin, polyester resin, polyarylate resin, epoxy resin, silicon Thermoplastic resins such as epoxy resins and polyimide resins, thermosetting resins, and photocurable resins.

For coloring, a colorant may be kneaded into the synthetic resin, or a colorant layer may be laminated on the surface of the synthetic resin layer. Examples of the colorant include azo, phthalocyanine, selenium, and dyes. Organic colorants such as lakes; inorganic colorants such as carbon black, oxides, molybdenum chromates, sulfides / selenides, ferrocyanides, etc., and carbon black is preferred.

The method for optically measuring a specific binding substance according to claim 6, wherein the fluid sample containing the substance to be measured and the second specific binding member to which the fluorescent label or the light scattering label is bound, and the substance to be measured are used. The second specific binding member to which a fluorescent label or a light scattering label is bound, and the second specific binding member, which is supplied to the reaction part of the optical measurement device according to any one of the above, and is immobilized on the reaction part via the substance to be measured After binding the first specific binding member to form a specific binding substance, the light incident side edge surface is irradiated with light, and the incident excitation light is totally reflected in the waveguide (A) and reacted. And the fluorescence or scattered light converted or scattered by the fluorescent label or the light scattering label by the evanescent wave component generated in the reaction section by the excitation light is measured by the light receiving element.

Next, a method for optically measuring the specific binding substance according to claim 6 will be described with reference to the drawings. FIG.
FIG. 2 is a schematic diagram showing an example of an optical measurement method of a specific binding substance using an optical measurement device, and FIG. 2 shows a second specific binding member to which a fluorescent label or a light scattering label is bound; FIG. 3 is a schematic diagram showing a state in which a substance to be measured and a specific binding substance to which a substance to be measured and a first specific binding member are bound are immobilized on the reaction surface.

In the figure, reference numeral 1 denotes an optical measuring device, which is formed by laminating a transparent waveguide (A) having a light incident side edge surface 11, a transparent intermediate layer (B), and a light absorption layer (C). A reaction portion 13 in which the first specific binding member 5 is fixed is formed on the light incident side edge surface 11 of the waveguide (A) and the one surface 12 near the opposing surface. Reference numeral 15 denotes a spacer, which bonds the end of the other surface 14 of the waveguide (A) and the end of the light absorption layer (C), and injects water 16 to form an intermediate layer (B).

In the method for optically measuring a specific binding substance, the first step is to provide a fluid sample containing a substance to be measured and a second specific binding member to which a fluorescent label or a light scattering label is bound.
The second specific binding member to which a fluorescent label or a light scattering label is bound is supplied to the reaction part of the optical measurement device according to any one of the items, and is fixed to the reaction part via the substance to be measured. And binding a first specific binding member to form a specific binding product.

That is, a larger amount of the second specific binding member 7 to which the fluorescent label or the light scattering label 8 is bound than the substance to be measured 6 contained in the fluid sample 2 and the fluid sample containing the substance to be measured 6 are reacted with the reaction unit 13. , The substance 6 to be measured specifically binds to the second specific binding member 7 to which the fluorescent label or the light scattering label 8 is bound, and the first specific binding immobilized on the reaction unit 13 The second specific binding member 7 to which the member 5 is specifically bound and the fluorescent label or the light scattering label 8 is bound, the specific substance 9 to which the analyte 6 and the first specific binding member 5 are bound. Is formed.

Since the first specific binding member 5 is fixed to the reaction part 13, the specific binding substance 9 is fixed to the reaction part 13. In addition, the second specific binding member 7 to which the excess fluorescent label or light scattering label 8 is bound is suspended in the fluid sample 2.

Next, the light incident side edge surface 11 is irradiated with excitation light from the light source 3, propagated while being totally reflected in the waveguide (A), and formed on the surface of the reaction portion 13 by the excitation light reaching the reaction portion 13. The light receiving element 4 measures fluorescence or scattered light converted or scattered by the fluorescent label or light scattering label 8 in the specific binding substance 9 fixed to the reaction unit 13 by the evanescent wave component. In addition, 31 is a slit and is installed so that the excitation light from the light source 3 may be irradiated only to the light incident side edge surface 11.

That is, when the light incident side edge surface 11 is irradiated with light from the light source 3 as indicated by the arrow D, the excitation light having a shallow angle propagates while totally reflecting the waveguide (A) as indicated by the light E. The reaction unit 13 is reached. The excitation light that has reached the reaction unit 13 forms an evanescent field on the surface of the reaction unit 13, and the fluorescent label in the specific binding substance 9 fixed to the reaction unit 13 by the evanescent wave component formed on the surface of the reaction unit 13. Alternatively, fluorescent light or scattered light converted or scattered by the light scattering label 8 is generated.

In this case, the floating fluorescent label or light scattering label 8 in the fluid sample 2 is away from the surface of the reaction portion 13 and the evanescent wave component does not reach, so that no fluorescence or scattered light is generated.

Therefore, the specific binding substance 9 can be measured by receiving the fluorescence or scattered light G generated in the reaction unit 13 with the light receiving element 4 and measuring it by electrical processing. The emitted light may be received through a filter that selectively transmits fluorescence or scattered light.

On the other hand, the excitation light having a deep angle passes through the waveguide (A) and enters the intermediate layer (B) from the other surface 14 of the waveguide (A) as shown by the light F, and the light absorption layer (C ) Is partially absorbed and partially reflected. The reflected light beam F enters the waveguide (A) again, is reflected by one surface 12 of the waveguide (A), enters the intermediate layer (B) from the other surface 14 of the waveguide (A), and absorbs light. layer(
C) is partially absorbed and partially reflected. The light F is repeatedly reflected and absorbed, and attenuates and disappears.

As the light source, any known light source can be used, and examples thereof include lasers, light emitting diodes, flash lamps, arc lamps, incandescent lamps, fluorescent lamps, xenon lamps, mercury lamps, and the like. Usable, for example, visible light, ultraviolet light, infrared light, and the like.

As the light receiving element, any measuring device capable of sensing light can be used. In general, a photodiode, a CMOS camera, and a CCD camera are preferably used.

Since the configuration of the optical measurement apparatus of the present invention and the optical measurement method of a specific binding substance using the same is as described above, it can be easily irradiated using an arbitrary light source, and the inside of the waveguide can be irradiated. The non-totally reflected excitation light is attenuated and extinguished when propagating through the waveguide, and does not reach the reaction part. Only the excitation light that totally reflects inside the waveguide reaches the reaction part. Only fluorescent or dispersed light that is converted or scattered by fluorescent or light-dispersive labels present in the object is generated,
Specific binding substances can be measured at high speed, with high sensitivity and accuracy, without B / F separation.

Further, it is preferable that the incident angle of the excitation light incident on the light incident side edge surface of the waveguide (A) is shallower,
The waveguide can be thinned, the optical measuring device can be thinned, and the light beam to be measured becomes bright and the measurement sensitivity is excellent. Therefore, the substance to be measured in the fluid sample can be measured with high sensitivity and accuracy.

  Examples of the present invention will be described below, but the present invention is not limited to the following examples.

Example 1
Monoclonal antibody against human hemoglobin and human glycohemoglobin A1c
Preparation of Monoclonal Antibody Balb / c mice were immunized 4 times every 2 weeks with 100 μl of a solution (20 μg / ml) in which human hemoglobin and human glycohemoglobin A1c were sufficiently dispersed in Freund's complete adjuvant.

Two weeks after the end of immunization, the spleen of the immunized Balb / c mouse was excised and 106 splenocytes were obtained. The obtained splenocytes were fused and cultured in the presence of myeloma cells and PEG (polyethylene glycol). The supernatant of the proliferated cells was collected and examined for the presence or absence of each antibody by ELISA.

Cells positive for each antibody were tested by the limiting dilution method, and cells producing each antibody were confirmed. These cells are cultured in large quantities, injected into the peritoneal cavity of Balb / c mice, and ascites is collected every 3 days from 2 weeks, and a monoclonal antibody against human hemoglobin and human glycohemoglobin A are collected.
Monoclonal antibody against 1c was obtained.

Preparation of fluorescent synthetic resin particles 80 parts by weight of water and 1.5 parts by weight of sodium dodecyl sulfonate are supplied to a glass polymerizer and stirred to dissolve sodium dodecyl sulfonate, and then 20 parts by weight of styrene, rare earth metal complex (Hexafluoroacetylacetone / trioctylphosphine oxide ligand complex coordinated to europium) 4 parts by weight and azobisisobutyronitrile 0,15 parts by weight are supplied and dispersed at high speed with a homogenizer while cooling to obtain an emulsion. Got.

The obtained emulsion was purged with nitrogen while being dispersed at high speed with a homogenizer, heated to 80 ° C. with warm water, and polymerized at 80 ° C. for 8 hours to obtain fluorescent synthetic resin particles having a volume average particle diameter of 120 nm.

Preparation of anti-human glycohemoglobin A1c antibody-adsorbing fluorescent synthetic resin particles Anti-human glycohemoglobin A1c antibody was dissolved in 0.05 M glycine buffer (pH 8.6) to obtain an antibody solution having a concentration of 1 mg / ml.

The obtained fluorescent synthetic resin particles were washed three times with 0.05 M glycine buffer (pH 8.6), and then 0.05 M glycine buffer (so that the fluorescent synthetic resin particle concentration was 1.0% by weight ( 0.5 ml of the obtained antibody solution was added to 1 ml of the resulting dispersion, and the mixture was stirred and mixed at 35 ° C. for 1 hour.

Next, 1 ml of bovine serum albumin 1.0 wt% phosphate buffer solution (pH 7.2) was added, stirred and mixed at room temperature for 1 hour, centrifuged (15000 rpm, 1 hour), and phosphate buffer solution. Washed with liquid (pH 7.2). After performing this centrifugation and washing three times, 2 ml of a phosphate buffer solution (into the obtained anti-human glycohemoglobin A1c antibody-adsorbed fluorescent synthetic resin particles (
pH 7.2) was added, and the mixture was stirred with an ultrasonic homogenizer, and anti-human glycohemoglobin A1c was added.
A dispersed phosphate buffer solution of antibody-adsorbed fluorescent synthetic resin particles was obtained.

Production of reaction part As a waveguide (A), a transparent polymethyl methacrylate sheet having a length of 10 mm, a width of 1 mm and a thickness of 100 μm is used, and the position of the reaction part 13 of the polymethyl methacrylate sheet (waveguide (A)) is distilled. After washing with water, a buffer solution in which a monoclonal antibody against human hemoglobin and a monoclonal antibody against human glycohemoglobin A1c were dissolved in 0.1 M Tris-HCl buffer (pH 9.0) to a concentration of 1 mg / ml was prepared. The mixture was supplied to the surface of the reaction part 13 with an injection needle and allowed to stand at 30 ° C. for 1 hour.

Next, after washing with 0.1 M Tris-HCl buffer (pH 9.0), 1 of bovine serum albumin
. A 0 wt% phosphate buffer solution (pH 7.2) was supplied and allowed to stand at 30 ° C. for 1 hour to coat the surface of the reaction part 13 that was not covered with the above monoclonal antibody.

Next, after washing again with 0.1 M Tris-HCl buffer (pH 9.0), it was covered with 0.1 M Tris-HCl buffer (pH 9.0) and coated with a polymethyl methacrylate sheet (waveguide (A)
) The reaction part 13 was formed on the top.

A transparent polymethyl methacrylate sheet on which the reaction part 13 is produced is used as a waveguide (A) for producing an optical measuring device, and a light absorption layer (C) is made of ABS resin and carbon black and has a length of 10 m.
A black ABS resin sheet having a width of 3 mm, a width of 3 mm, and a thickness of 100 μm was used.

As shown in FIG. 1, a spacer 15 made of a black ABS resin sheet having a thickness of 0.5 mm is attached around one surface of a black ABS resin sheet having a thickness of 0.3 mm (light absorption layer (C)). Then, a polymethylmethacrylate sheet (waveguide (A)) was pasted on it, and water 16 was injected into the space formed by both with a syringe to form an intermediate layer (B) to produce an optical measuring device. .

Preparation of blood sample for measurement Immediately after collecting the blood of a subject, sodium fluoride (blood anticoagulant) 10 per ml of whole blood
mg was added, and then 3 μl of blood was diluted 150-fold by adding a hemolysis reagent to obtain a diluted blood sample.

The hemolysis reagent was polyoxyethylene (10) octylphenyl ether (Pharm).
0.1% by weight of manufactured by Inc., Inc., trade name "TritonX-100")
A 0.05 M phosphate buffer (pH 6.0) obtained by dissolving 0.1% by weight of tetrapolyphosphate was used as a removal reagent.

The obtained blood sample was measured using an A1c dedicated measuring device (liquid chromatography, manufactured by Kyoto Daiichi Kagakusha,
Supplied to the trade name “Hi-AutoA1cHA-8121” and human glycohemoglobin A1
c concentration was measured, and based on the result, the hemolysis reagent was further added to the blood sample to prepare blood samples for measurement having human glycohemoglobin A1c concentrations of 3%, 5%, 10% and 12%.

Measurement To the obtained blood sample for measurement, an excessive anti-human glycohemoglobin A1c antibody-adsorbed fluorescent synthetic resin particle-dispersed phosphate buffer solution was mixed, and then allowed to stand for 5 minutes.
0 ml was supplied to the reaction unit 13 of the optical measuring device.

Using a xenon lamp with a filament diameter of 1 mm as the light source 3, 0.1 J / Flas
The light of h was irradiated 1000 times at an interval of 2000 times / second, a photodiode was used as the light receiving element 4, and the intensity of light having a wavelength of about 650 nm emitted from the reaction unit 2 was measured.

When the obtained light intensity and the human glycohemoglobin A1c concentration were plotted on a graph, a linear graph was obtained, and it was found that the human glycohemoglobin A1c concentration can be measured with the above optical measuring device.

It is a schematic diagram which shows an example of the optical measuring method of a specific binding material using an optical measuring device. A state in which the second specific binding member to which the fluorescent label or the light scattering label is bound, the substance to be measured, and the specific binding substance to which the first specific binding member is bound are immobilized on the reaction surface. It is a schematic diagram.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Optical measuring apparatus A Waveguide B Intermediate | middle layer C Light absorption layer 2 Fluid sample 3 Light source 4 Light receiving element 5 1st specific binding member 6 Measured substance 7 2nd specific binding member 8 Fluorescence label or light-scattering label 9 Specific binding substance 11 Light incident side edge surface 13 Reaction part

Claims (9)

An apparatus for optically measuring a specific binding substance in a fluid sample, which specifically binds to a substance to be measured bound to a second specific binding member to which a fluorescent label or a light scattering label is bound. having a reaction zone in which the first specific binding member capable of constituting the specific binding substance is fixed on the one surface, and a transparent waveguide having a light incident side edge face on one side (a) A transparent intermediate layer (B) and a light absorption layer (C) are sequentially laminated on the other surface of the waveguide (A), the refractive index of the waveguide (A) is n _w , and the refractive index of the intermediate layer (B) An optical measurement apparatus characterized in that each refractive index satisfies the following formula (1), where n _{m is} the refractive index and n _s is the refractive index of the fluid sample.
n _w > n _m ≧ n _s (1) The optical measuring device according to claim 1, wherein the waveguide (A) is made of a material selected from the group consisting of glass, quartz, acrylic resin, polycarbonate resin, and polystyrene resin. Intermediate layer (B) is an optical measuring apparatus according to claim 1 or 2, wherein the refractive index of the fluid sample is a substance having substantially the same refractive index as n _s. 4. The optical measuring device according to claim 1, wherein the light absorbing layer (C) is a black layer. The distance from the light incident side edge surface of the waveguide (A) to the reaction part is at least twice the one-way distance in which the incident excitation light beam is totally reflected and propagates in the waveguide (A). Claim 1 characterized
The optical measuring device of any one of -4. 6. A fluid sample containing a second specific binding member to which a fluorescent label or a light scattering label is bound and a substance to be measured is supplied to the reaction part of the optical measuring device according to any one of claims 1 to 5. A second specific binding member to which a fluorescent label or a light scattering label is bound via a substance to be measured;
The first specific binding member fixed to the reaction part is combined with the first specific binding member to form a specific binding substance, and then the light incident side edge surface is irradiated with light, and the incident excitation light is guided into the waveguide (A). The fluorescent light or the scattered light converted or scattered by the fluorescent label or the light scattering label by the evanescent wave component generated in the reaction part by the excitation light is measured by the light receiving element. And a method for optically measuring a specific binding substance. 7. The specific binding according to claim 6, wherein the substance to be measured is an antibody or an antigen, and the first specific binding member and the second specific binding member are an antigen or an antibody that specifically binds thereto. Optical measurement method for objects. The method for optically measuring a specific binding substance according to claim 6 or 7, wherein the fluorescent label is a rare earth metal complex. 9. The method for optically measuring a specific binding product according to claim 6, 7 or 8, wherein the fluorescent label is a synthetic resin particle containing a compound that generates fluorescence.

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