JP2006119011A - Method for detecting substance by luminescent reaction on porous carrier and reaction vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To reduce a background noise from a luminescent reaction, and improve a capability for detecting a measured substance even if a porous carrier as a reaction field is not colored. <P>SOLUTION: In a method, a complex is formed from a first substance for specifically combining the measured substance in a sample and the measured substance having a labeled low-molecular compound, and a second substance for specifically combining the measured substance in the sample and the measured substance having a labeled luminescent substance or a labeled luminescent reaction inducing substance, the complex is captured by the colorless porous carrier 3 having a previously fixed physiologically active substance for capturing the low-molecular substance labeled in the first substance, and the measured substance is detected by measuring an intensity of luminescence finally generated from the luminescent substance or the luminescent reaction inducing substance labeled in the second substance. The method for detecting the measured substance employs a reaction vessel 1 having a color other than white on a face of a layer 4 located below and contacting the carrier. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for detecting a substance to be measured such as nucleic acid, antigen, antibody or hormone in a sample by using a luminescent reaction on a porous carrier and its reaction container, and more particularly to directly receiving light from a luminescent label. The present invention relates to a simple and highly sensitive detection method and its reaction container.

  In recent years, a technique has been used in which various reactions are performed on a porous carrier to detect a substance to be measured in a sample. In this measurement method, measurement can be performed simply by sequentially dropping a sample or reagent onto a porous carrier housed in a reaction vessel, and the measurement operation is simple. The reaction rate is increased due to the large specific surface area of the carrier. There are advantages such as shortening the measurement time and keeping the waste liquid inside the container and preventing leakage.

  On the other hand, it is common to use a radioisotope, a dye, colored fine particles, etc. as a detection system for measuring a measurement target substance. However, in recent years, in order to detect a trace amount of a measurement target substance, it has become more sensitive. Increasingly, a fluorescence method or a luminescence method as a detection system is used. In addition, when measuring a measurement target substance in a sample using a fluorescence method or a luminescence method, it is well known to use a black microplate for the purpose of reducing the background noise of fluorescence or luminescence, Black microplates are also available from various companies.

  In Patent Document 1, in the luminescence immunoassay method using a fiber matrix, in order to reduce the luminescence background, “an element bonded to the water absorption pad to reduce chemiluminescence emitted from the absorption pad” is used. Although disclosed, it is mentioned that the reaction product is captured on the fiber matrix using the fine particle capture separation technique and the ion capture separation technique, but the binding bioactive substance is immobilized on the fiber matrix in advance. No mention is made of the case where the reaction product is trapped on the fiber matrix via the activated and immobilized bioactive substance.

Patent Document 2 discloses a measurement method using a “filter composed of glass fibers having an average diameter of 0.5 to 2.0 μm and an average fiber length of 0.5 to 2 mm,” among them. Examples of applying the luminescence method are described, but in the method of this document, the “first substance that specifically reacts with the substance to be measured” is directly immobilized on the “filter composed of glass fibers”. Therefore, since all the reactions are carried out on a solid phase, it is difficult to obtain sufficient sensitivity especially when it is necessary to detect a substance with a low concentration of 10 ng / mL or less, and different reaction containers are used for each measurement item. I have to prepare.
No. 2880801 patent Japanese Patent No. 3134231

  Examples of the porous carrier include membrane filters made of nitrocellulose, nylons, cellulose acetates, polyvinylidene fluorides, 4-fluoroethylenes, so-called filter papers made of cellulose fibers, glass fibers, and the like. These commercially available products are often white, and it has been difficult to obtain a colored porous carrier. In addition, when directly coloring these porous carriers, manufacturing know-how such as immobilization cultivated for the carrier cannot be applied because of changes in physical and chemical properties of the carrier, or dyes and pigments used for coloring And the residual solvent adversely affects the storage stability of various reaction systems such as a luminescent reaction performed on the porous carrier, or the physiologically active substance immobilized on the porous carrier.

  As a result of intensive studies to solve the above problems, the present inventors have finally completed the present invention. That is, the present invention captures a complex of a substance to be measured in a sample on a porous carrier, and measures the intensity of luminescence finally generated from the luminescent substance contained in the complex or a substance that induces a luminescence reaction. In the method of detecting a substance to be measured by using a reaction container, a reaction container in which at least a surface in contact with the carrier of a layer located immediately below the carrier is colored in a color other than white is used. The present invention relates to a method for detecting a substance and a reaction container thereof.

  In the present invention, the measurement target substance in the sample is labeled with a low molecular weight compound, a first substance that can specifically bind to the measurement target substance, and a luminescent substance or a substance that induces a luminescent reaction. A physiologically active substance capable of forming a complex in a liquid phase with a second substance that can specifically bind to the substance to be measured, and capturing the low-molecular compound labeled with the first substance. Measured by measuring the intensity of luminescence finally captured from a luminescent substance labeled with a second substance or a substance that induces a luminescent reaction, captured on an uncolored porous carrier that has been immobilized in advance. In the method for detecting a target substance, a measurement target substance is detected, characterized in that a reaction vessel in which at least a surface in contact with the support of a layer located immediately below the support is colored in a color other than white is used. Method, and On the reaction vessel.

  Furthermore, the present invention comprises forming a complex in a liquid phase with a first substance labeled with a low molecular compound and capable of specifically binding to the substance to be measured, in a liquid phase. The bioactive substance capable of capturing the low-molecular compound labeled with the first substance is captured on a non-colored porous carrier that has been immobilized in advance, and the complex on the multi-modal carrier is luminescent. A substance or a substance that induces a luminescence reaction is labeled, and a second substance that can specifically bind to the substance to be measured is bound, and the second substance is labeled with a luminescent substance or a substance that induces a luminescence reaction. In the method of detecting a substance to be measured by measuring the intensity of light emission finally generated, a reaction vessel in which at least the surface in contact with the carrier of the layer located immediately below the carrier is colored in a color other than white is used. It is characterized by using Method for detecting a constant target substance, and to the reaction vessel.

  According to the present invention, the background noise of the luminescence reaction can be reduced without coloring the porous support itself as a reaction field, and the measurement target substance and the substance that can specifically bind to the measurement target substance By combining means for forming the complex in the liquid phase, the detection power of the substance to be measured can be improved. In particular, it is optimal for quantification of low concentration substances of 10 ng / mL or less. Furthermore, a low concentration region of 0.5 pg / mL to 2000 pg / mL can be accurately measured.

  Hereinafter, the present invention will be described in detail. The porous carrier used in the present invention is not particularly limited as long as it can pass a liquid, can immobilize a physiologically active substance in advance, and can cause a luminescence reaction on the porous carrier. As materials, for example, membrane filters made of nitrocellulose, nylons, cellulose acetates, polyvinylidene fluoride (PVDF), 4-fluoroethylenes, or so-called filter paper made of cellulose fibers, glass fibers, etc. are used. obtain. Among them, a glass fiber filter is preferable, and it is more preferable to use a glass fiber filter having a relatively large and relatively thick weight, specifically, a weight of 100 to 200 g per cubic meter and a thickness of 0.3 to 1.0 mm. preferable.

  The reaction vessel used in the present invention is provided in a state in which a binding physiologically active substance that can be captured via a substance that forms a complex with a substance to be measured is immobilized on a porous carrier in advance. Examples of the binding physiologically active substance to be immobilized are not particularly limited as long as they can capture a low molecular weight compound labeled with the first substance that specifically binds to the measurement target substance. Examples include antibodies or antibody fragments against low molecular weight compounds such as antibodies, anti-dinitrophenol antibodies or anti-digoxigenin antibodies, avidin that binds to biotin, streptavidin, and the like.

  In order to immobilize the physiologically active substance on the porous carrier, a generally known method such as a physical adsorption method or a chemical bonding method may be used. At that time, the physiologically active substance may be directly immobilized on the porous carrier, or a substance such as an antibody or a receptor that specifically binds to the physiologically active substance may be immobilized on the porous carrier, The physiologically active substance may be immobilized. Alternatively, the immobilized porous carrier may be cut out and attached to the reaction vessel, or the immobilization may be performed after the non-solid phase porous carrier is attached to the reaction vessel. In consideration of stability during storage and transportation, the reaction vessel is preferably provided in a dry state after immobilizing the physiologically active substance. As drying methods, natural drying, air drying, vacuum drying, reduced pressure are preferable. Examples thereof include drying, freeze vacuum drying, drying using infrared rays or far infrared rays, and drying using high frequency such as microwaves.

  The layer located immediately below the porous carrier of the present invention is a layer that is in direct contact with the bottom surface of the porous carrier and is housed in a liquid-impermeable container together with the porous carrier, and at least the surface that is in contact with the porous carrier. There is no particular limitation except that is colored in a color other than white, but as shown in FIG. 2, a colored water-absorbing layer for holding a dropped sample or reagent is preferable. Further, as shown in FIG. 4, a colored liquid permeable layer may be provided immediately below the porous carrier. The liquid permeable layer as used herein refers to a layer existing without interfering with the transition of the sample or reagent dropped onto the porous carrier from the opening to the water absorption layer. The carrier material for forming these layers is, for example, fibers or filters made of nitrocellulose, nylons, cellulose acetates, polyvinylidene fluoride (PVDF), 4-fluoroethylenes, cellulose fibers, glass fibers, or the like. And so-called filter papers manufactured from the above.

  If the color of the layer located immediately below the porous carrier of the present invention is at least the surface in contact with the carrier, other than white, and any color that reduces background noise of fluorescence or light emission, black, red, brown, Although not limited to blue, yellow and the like, black is particularly preferable. Further, the coloring method is not particularly limited, but when a layer located immediately below the porous carrier is manufactured, the material for forming the layer is first dyed with a dye in advance, or a pigment or the like is kneaded into the raw material. The colored product may be molded by mixing, or the layer may be formed without coloring, and the finished product may be colored. Although what is used for coloring is not specifically limited, Dyes and pigments, commercially available inks, paints, etc. are mentioned. Moreover, when the liquid permeability of a reagent or a sample falls by coloring, ie, water is repelled by surface tension, you may implement a hydrophilic process as needed. The hydrophilic treatment mentioned here is a treatment applied to improve the liquid permeability of a reagent or a sample or to reduce the surface tension, and is not particularly limited as long as it is a method for improving the liquid permeability. Examples thereof include a method of adding a liquid containing various surfactants and a commercially available cleaning liquid, or drying after immersion. In addition, it is preferable that the color does not ooze from the colored layer to the surface of the porous carrier during the measurement.

  The opening of the reaction container in the present invention is provided for dropping a sample or reagent containing a substance to be measured on the porous carrier and for detecting luminescence generated on the porous carrier. It is desirable to be provided.

  The liquid impervious container in the present invention is not particularly limited as long as it does not allow liquid to permeate, but is preferably made from plastics such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate (PET), and ABS resin. It is desirable that the material is opaque and does not transmit light.

  The substance to be measured in the present invention is not particularly limited, but is a low molecular weight compound such as nucleic acids such as DNA and RNA, proteins and peptides having antigenicity, antibodies that recognize antigens and haptens, hormones and endocrine disrupting chemicals, etc. Etc. Examples of substances that can specifically bind to the measurement target substance include nucleic acids that are complementary to the nucleic acid to be measured, antibodies that specifically bind to the measurement target substance, antibody fragments or receptors, and antibodies that are the measurement target substance. And lectins that bind to sugar chains.

  Examples of the low molecular weight compound labeled with the first substance that can specifically bind to the measurement target substance include biotin, dinitrophenol, digoxigenin, and the like. Examples of a luminescent substance labeled with a second substance that can specifically bind to a measurement target substance or a substance that induces a luminescent reaction include acridinium ester or a derivative thereof, acridinium sulfonamide or a derivative thereof, Examples thereof include luminol, isoluminol and derivatives or anthracene derivatives thereof, enzymes, luminescent substrates or fluorescent substances. Examples of the enzyme include peroxidase, alkaline phosphatase, and β-galactosidase. Examples of the luminescent substrate include 1,2-dioxetanes (for example, AMPPD). Examples of the fluorescent substance include luciferin and derivatives thereof, oxalate ester and derivatives thereof, and the like.

1) Preparation of colored water absorbing layer Hobby spray black (manufactured by Nippon Paint Co., Ltd.) is sprayed on the surface of the cylindrical reagent solution absorbing filter to color it, and then left to stand in a well-ventilated place overnight to dry. It was immersed in a 1% polyoxyethylene monolaurate solution and freeze-dried.

2) Preparation of reaction vessel A glass fiber filter paper (manufactured by Advantech Toyo Co., Ltd.) is rounded and placed on the colored water-absorbing layer prepared in 1) above, and a polyethylene liquid having an opening at the top as shown in FIG. Housed in an impermeable container.

3) Preparation of antibody-immobilized carrier From the opening of the reaction container prepared in 2) above, (A) 100 mM citrate buffer: 100 μL of pH 3.0, (B) 10 μg / mL of rabbit anti-biotin polyclonal antibody (100 mM citrate) 100 μL of buffer solution (pH 3.0), (C) 100 μL of 5% glycerol / 1% bovine serum albumin (BSA) solution (10 mM phosphate-saline buffer solution: pH 7.4) were supplied immediately before After waiting for the liquid to be sucked in, it was sequentially supplied and then lyophilized.

4) Measurement of CEA 1% bovine serum albumin (BSA) solution (10 mM phosphate-saline buffer: pH 7.4), and CEA (Carcinoembryonic Antigen) in the solution at 5 ng / mL The sample added as described above was mixed with a biotin-labeled mouse anti-CEA monoclonal antibody solution (1% BSA / 10 mM phosphate-saline buffer solution: pH 7.4) at a volume ratio of 1: 1, The reaction was allowed to proceed at room temperature for 5 minutes (primary reaction solution). 50 μL of 25% Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.), 50 μL of the above primary reaction solution, and horseradish peroxidase-labeled mouse anti-CEA monoclonal antibody solution (1% BSA · 10 mM phosphorus) from the opening of the reaction vessel prepared in 3) above 20 μL of acid-saline buffer solution: pH 7.4) and 160 μL of 0.05% polyoxyethylene monolaurate solution (10 mM phosphate-saline buffer solution: pH 7.0) were supplied immediately before. After the liquid was aspirated, the solution was dropped in succession, and after 30 μL of a luminescent substrate containing luminol / enhancer and hydrogen peroxide was dropped, the intensity of light generated from the opening of the reaction vessel was changed to a photoelectron installed in a dark room. Measured with a multiplier.

[Comparative example]
A reaction vessel was manufactured in the same manner as in the example except that step 1) of the example was not performed, and CEA was measured.

  In Examples and Comparative Examples, CEA samples (0 ng / mL, 5 ng / mL) were measured five times, and the average value (mean), standard deviation (SD), and coefficient of variation (CV) of luminescence intensity (RLU) were calculated. The results are shown in Table 1.

  As shown in Table 1, the emission intensity (ie, background noise) of CEA 0 ng / mL is significantly lower and the variation (CV) is reduced in the examples as compared with the comparative example, thereby reducing the CEA 5 ng / mL against the background noise. The emission intensity (ie, Signal / Noise (S / N) ratio) was 21 times in the comparative example, but improved to 31 times in the example.

  As described above, by using the reaction container of the present invention, the background noise in the luminescence reaction on the non-colored porous carrier on which the physiologically active substance is immobilized is reduced, thereby the detection power of the measurement target substance. Improved.

  The present invention can reduce the background noise of the luminescence reaction even if the porous carrier itself that is the reaction site is not colored, and as a result, the detection power is improved and a trace amount of the measurement target substance can be detected. It can be used for various analytical purposes such as in-vitro diagnostic drugs and contributes greatly to the industry.

The perspective view in one Example of the reaction container of this invention Sectional view of the reaction vessel shown in FIG. The perspective view in one Example of the reaction container of this invention Sectional view of the reaction vessel shown in FIG.

Explanation of symbols

1: Reaction vessel 2: Opening portion 3: Porous carrier 4: Colored water absorbing layer 5: Liquid impermeable vessel 6: Colored liquid permeable layer 7: Uncolored water absorbing layer

Claims (20)

  A measurement target substance is captured by capturing a complex of a measurement target substance in a sample on a porous carrier and measuring the intensity of luminescence finally generated from the luminescent substance or the substance that induces a luminescent reaction contained in the complex. In a method for detecting a substance, a method for detecting a substance to be measured, comprising using a reaction vessel in which at least a surface in contact with the carrier of a layer located immediately below the carrier is colored in a color other than white .   The measurement target substance in the sample is labeled with a low molecular weight compound, a first substance that can specifically bind to the measurement target substance, and a luminescent substance or a substance that induces a luminescent reaction. A biologically active substance that can form a complex in a liquid phase with a second substance that can specifically bind to the substance and capture the low-molecular compound labeled with the first substance is immobilized in advance. The target substance is detected by measuring the intensity of luminescence that is finally generated from the luminescent substance labeled with the second substance or the substance that induces the luminescent reaction. And a method of detecting a substance to be measured, characterized in that a reaction vessel in which at least a surface in contact with the carrier of the layer located immediately below the carrier is colored in a color other than white is used.   A substance to be measured in a sample is formed into a complex in a liquid phase with a first substance that can specifically bind to the substance to be measured, labeled with a low molecular compound, and the complex is converted into the first substance. A physiologically active substance capable of capturing a labeled low molecular weight compound is captured on a non-colored porous carrier on which a physiologically active substance has been immobilized in advance, and a luminescent substance or a luminescent reaction is applied to the complex on the multinomial carrier. Luminescence finally produced from the luminescent substance labeled with the second substance, which is labeled with the substance to be induced and capable of specifically binding to the substance to be measured, or the substance that induces the luminescence reaction In the method for detecting the substance to be measured by measuring the intensity of the above, a reaction vessel in which at least the surface in contact with the carrier of the layer located immediately below the carrier is colored in a color other than white is used. The substance to be measured How to leave.   The method according to claims 1 to 3, wherein the porous carrier is a glass fiber filter.   The method according to claim 4, wherein the weight of the glass fiber filter is 100 to 200 g per cubic meter and the thickness is 0.3 to 1.0 mm.   The method according to claim 1, wherein a substance to be measured having a concentration in the sample of 10 ng / mL or less is detected.   The method according to claim 2, wherein the low molecular compound is biotin.   The method according to claim 2, wherein the physiologically active substance capable of capturing the low molecular compound is at least one of anti-biotin antibody, avidin or streptavidin.   The method according to claim 2, wherein the substance that induces a luminescent reaction is an enzyme.   The method according to claim 9, wherein the enzyme is at least one of peroxidase, alkaline phosphatase or β-galactosidase.   A measurement target substance is captured by capturing a complex of a measurement target substance in a sample on a porous carrier and measuring the intensity of luminescence finally generated from the luminescent substance or the substance that induces a luminescent reaction contained in the complex. A reaction container for detecting a substance, wherein at least a surface in contact with the carrier of a layer located immediately below the porous carrier housed in a liquid-impermeable container having an opening is colored in a color other than white. A reaction vessel characterized by comprising:   The measurement target substance in the sample is labeled with a low molecular weight compound, a first substance that can specifically bind to the measurement target substance, and a luminescent substance or a substance that induces a luminescent reaction. A biologically active substance that can form a complex in a liquid phase with a second substance that can specifically bind to the substance and capture the low-molecular compound labeled with the first substance is immobilized in advance. The target substance is detected by measuring the intensity of luminescence that is finally generated from the luminescent substance labeled with the second substance or the substance that induces the luminescent reaction. A layer located immediately below the porous carrier housed in the liquid-impermeable container having an opening, and at least the surface in contact with the carrier is colored in a color other than white Reaction volume characterized by .   A substance to be measured in a sample is formed into a complex in a liquid phase with a first substance that can specifically bind to the substance to be measured, labeled with a low molecular compound, and the complex is converted into the first substance. A physiologically active substance capable of capturing a labeled low molecular weight compound is captured on a non-colored porous carrier on which a physiologically active substance has been immobilized in advance, and a luminescent substance or a luminescent reaction is applied to the complex on the multinomial carrier. Luminescence finally produced from the luminescent substance labeled with the second substance, which is labeled with the substance to be induced and capable of specifically binding to the substance to be measured, or the substance that induces the luminescence reaction A reaction vessel for detecting a substance to be measured by measuring the strength of the liquid, and is in contact with at least the carrier in a layer located immediately below the porous carrier housed in a liquid-impermeable vessel having an opening Colored surface other than white Reaction vessel, wherein it is.   The reaction container according to claim 11, wherein the porous carrier is a glass fiber filter.   The method according to claim 14, wherein the weight of the glass fiber filter is 100 to 200 g per cubic meter and the thickness is 0.3 to 1.0 mm.   The reaction container according to claim 11, wherein a measurement target substance having a concentration in the sample of 10 ng / mL or less is detected.   The reaction container according to claim 12, wherein the low molecular compound is biotin.   The reaction container according to claim 12 to 13, wherein the physiologically active substance capable of capturing the low molecular weight compound is at least one of anti-biotin antibody, avidin or streptavidin.   The reaction container according to claim 12, wherein the substance that induces a luminescence reaction is an enzyme.   The reaction container according to claim 19, wherein the enzyme is at least one of peroxidase, alkaline phosphatase or β-galactosidase.

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