JP2007178316A - Reduction method of matrix effect - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To reduce a matrix effect regardless of a measuring item to improve measurement precision in a measuring container equipped with an insoluble carrier. <P>SOLUTION: The measuring container, used in a method for detecting a substance to be measured in a biosample, is equipped with: an opening part; the absorbing element positioned in close vicinity to the opening part; and the liquid impermeable insoluble carrier arranged between the opening part and the absorbing element, wherein a bondable physiologically active substance for directly or indirectly capturing the substance to be measured is fixed in the insoluble carrier in advance, the simultaneous measurement of many items of the substance to be measured can be performed, and the insoluble carrier is provided in a dried state. The reduction method of the matrix effect includes a process for dripping an aqueous solution with a proton concentration of 1×10<SP>-6</SP>M or above on the insoluble carrier before the fixation of the physiologically active substance or at the same time in the measuring container for dripping a definite amount or above of moisture on the insoluble carrier before measurement. <P>COPYRIGHT: (C)2007,JPO&INPIT

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 biological sample on an insoluble carrier and an improved measurement container used therefor.

  Techniques for performing immunoassays are generally known in the art. For example, in a conventional enzyme immunoassay (EIA) procedure, a series of steps is used in which a substance to be measured in a biological sample is first combined with a corresponding antigen reagent or antibody reagent. Next, a second antigen labeled with an enzyme or other substance that can be detected directly or after adding an appropriate reagent such as a chromogenic substance, a fluorescent source substance, or a trigger solution that activates chemiluminescence, or The antibody is introduced into the sample. The generated signal is then read to indicate whether an antigen or antibody is present in the biological sample.

In recent years, in immunoassay, a method of detecting a substance to be measured in a sample by causing a sample and a reagent to drop sequentially on an insoluble carrier housed in a measurement container to cause a reaction is used. One of these methods is a measurement container that can perform B / F separation on an insoluble carrier and contain used sample and reagent waste liquid by arranging an absorbing element in the vicinity of the insoluble carrier. . In the measurement using such a measurement container, a decrease in measurement accuracy often becomes a problem due to a phenomenon caused by using an insoluble carrier. This is the “matrix effect”. The matrix effect is defined in the diagnostic drug field as a substance (Matrix) other than the object to be measured in the sample to be measured interferes with and influences the measurement method at the time of measurement. This matrix effect can also occur in a measurement method using a solution-like reagent, but it is known that the method using an insoluble carrier is frequently generated and has a great influence on the measurement result.
Japanese Patent No. 2880801

 At present, the importance of immunoassay in the analysis of biological samples is increasing, and there are many voices that desire a higher level of measurement accuracy under such circumstances. The present invention provides a means for improving the measurement accuracy for a method for detecting a substance to be measured in a biological sample, regardless of measurement items.

The inventor diligently studied a method for improving measurement accuracy in a method for detecting a measurement target substance in a biological sample.
For example, Patent Document 1 discloses a luminescence immunoassay method using a fiber matrix in order to reduce non-specific reactions occurring on an insoluble carrier, for example, luminescence background, “absorption to reduce chemiluminescence emitted from an absorption pad”. The use of “elements coupled to pads” is disclosed, and the capture of reaction products onto a fiber matrix using particulate capture and ion capture separation techniques is described.
However, in this method, the “element bonded to the absorbent pad” dissociates from the fiber and floats on the insoluble carrier due to capillary action, which may cause a nonspecific reaction.
As a result of further intensive studies by the inventor, a measurement container used in a method for detecting a substance to be measured in a biological sample, having an opening, an absorption element positioned in the vicinity of the opening, and the opening An insoluble carrier having a structure capable of penetrating a liquid and disposed between the absorbent element and the absorbent element, and the insoluble carrier captures the substance to be measured directly or via a substance forming a binding pair complex. Bindable physiologically active substances that can be immobilized are preliminarily immobilized, and it is possible to measure a variety of measurement target substances without modifying the measurement container. The insoluble carrier is provided in a dried state. In a measurement container that can be stored for a long time if it is in a dried state, and can be immediately returned to a measurable state by dripping a certain amount or more of water into the insoluble carrier before measurement. Physiological activity Before immobilizing the substance, or immobilized at the same time, by dropping the proton concentration 1 × 10 ^-6 M or more aqueous insoluble carrier, it found that matrix effects in the insoluble carrier can be reduced, completion of the present invention did.

That is, the gist of the present invention is as follows.
[Item 1] A measurement container used in a method for detecting a substance to be measured in a biological sample,
(1) having an opening,
(2) an absorbent element located proximate to the opening;
(3) provided with an insoluble carrier that is disposed between the opening and the absorbent element and has a structure capable of transmitting liquid;
(4) A binding physiologically active substance capable of capturing the substance to be measured directly or via a substance forming a binding pair complex is immobilized on the insoluble carrier in advance.
(5) It is possible to measure multiple measurement target substances without modifying the measurement container,
(6) the insoluble carrier is provided in a dry state;
(7) Before measurement, a predetermined amount or more of water is dropped on the insoluble carrier.
In the measurement container,
(8) including a step of dropping an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more into the insoluble carrier before or simultaneously with the immobilization of the physiologically active substance.
A method for reducing a matrix effect in the insoluble carrier in a method for detecting a substance to be measured in a biological sample.
[Item 2] The measuring method according to item 1, wherein the absorbent element is cellulose or a cellulose derivative.
[Item 3] The measuring method according to item 1, wherein the insoluble carrier is a fiber matrix.
[Item 4] The measuring method according to item 3, wherein the fiber matrix is glass fiber.
[Item 5] The measuring method according to Item 1, wherein the physiologically active substance is IgG.
[Item 6] A measurement container used in a method for detecting a substance to be measured in a biological sample,
(1) having an opening,
(2) an absorbent element located proximate to the opening;
(3) provided with an insoluble carrier that is disposed between the opening and the absorbent element and has a structure capable of transmitting liquid;
(4) A binding physiologically active substance capable of capturing the substance to be measured directly or via a substance forming a binding pair complex is immobilized on the insoluble carrier in advance.
(5) It is possible to measure multiple measurement target substances without modifying the measurement container,
(6) the insoluble carrier is provided in a dry state;
(7) Before measurement, a predetermined amount or more of water is dropped on the insoluble carrier.
In the measurement container,
(8) including a step of dropping an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more into the insoluble carrier before or simultaneously with the immobilization of the physiologically active substance.
A method for producing a measurement container having a reduced matrix effect in the insoluble carrier in a method for detecting a substance to be measured in a biological sample.
[Item 7] A measurement container used in a method for detecting a substance to be measured in a biological sample,
(1) having an opening,
(2) an absorbent element located proximate to the opening;
(3) provided with an insoluble carrier that is disposed between the opening and the absorbent element and has a structure capable of transmitting liquid;
(4) A binding physiologically active substance capable of capturing the substance to be measured directly or via a substance forming a binding pair complex is immobilized on the insoluble carrier in advance.
(5) It is possible to measure multiple measurement target substances without modifying the measurement container,
(6) the insoluble carrier is provided in a dry state;
(7) Before measurement, a certain amount or more of water is dropped on the insoluble carrier,
In the measurement container,
(8) Obtained by a method comprising a step of dropping an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more onto the insoluble carrier before or simultaneously with immobilization of the physiologically active substance.
A measurement container having a reduced matrix effect in the insoluble carrier in a method for detecting a substance to be measured in a biological sample.
A measurement container having a reduced matrix effect in the insoluble carrier.
[Item 8] A measurement system for detecting a measurement target substance in a biological sample, wherein the measurement is performed using the measurement container according to Item 7.
[Item 9] A kit for use in measurement for detecting a substance to be measured in a biological sample, comprising the measurement container according to item 7.

  The invention of the present application is a measurement container comprising an insoluble carrier, and reduces the matrix effect and improves the measurement accuracy universally regardless of the measurement item.

  The present invention relates to a measurement container improved so that non-specific reaction in a method for detecting a substance to be measured such as nucleic acid, antigen, antibody or hormone in a biological sample on an insoluble carrier 3 is suppressed. Examples of the measurement target substance in the present invention include DNA, RNA, plasmid, albumin, alkaline phosphatase, cholinesterase, γ-GTP, LDH, acid phosphatase, cholesterol, amylase, LAP, CPK, aldolase, HDL cholesterol, GOT, GPT, LDL Cholesterol, RLP cholesterol, chylomicron, monoamine oxidase, sialic acid, lipase, fructosamine, G-6-Pase, G-6-PDH, adenosine deaminase, lipoprotein, guanase, catalase, ferritin, H-FABP, ACE, hemoglobin A1C , Antitrypsin, DD dimer, macroglobulin, antiplasmin, von Willebrand factor, PIVKA-II, blood coagulation factor, t A, protein C, glycoalbumin, 1,5-AG glycocholic acid, ceruloplasmin, troponin I, troponin T, myoglobin, microglobulin, type I collagen cross-linked N-telopeptide (NTx), type I collagen cross-linked C-telo Peptide (CTx), osteocalcin, deoxypyridinoline, IGFBP, cystatin C, vitamin B12, trypsin, hyaluronic acid, vitamin B2, remnant-like lipoprotein cholesterol, fibronectin, HGF, vitamin B1, vitamin C, 1,25-dihydrovitamin D3, HCG, HVA, VMA, prolactin, T3, FT3, TSH, T4, FT4, gastrin, ANP, BNP, CK-MB, insulin, GH, LH, FSH, PTH, hydroxycorticosteroid , Estrogen, ACTH, secretin, anti-GAD antibody, cortisol, aldosterone, testosterone, thyroglobulin, lactogen, glucagon, calcitonin, progesterone, c-AMP, pregnanediol, erythropoietin, arginine vasopressin, somatomedin, AFP, CEACC, DUPAN-2 -ST-439, CA15-3, elastase 1, PSA, BFP, CA19-9, CA72-4, CA-50, SPan-1 antigen, sialyl Tn antigen, enolase, sialyl Lex-i antigen, sialyl Lex antigen, CA125 , BCA225, CA602, CA130, cytokeratin, ProGRP, CA54 / 61, GAT, POA, γ-seminoprotein, HER, anti-streptridine O, toxo Razma antibody, anti-streptokinase, mycoplasma antibody, anti-streptococcal polysaccharide antibody, syphilis antibody, tsutsugamushi antibody, exogenous allergens such as house dust and pollen, Helicobacter pylori antibody, chlamydia antibody, pertussis antibody, HTLV-I antibody, Anti-acid-fast bacteria antibody, Shigella amoeba antibody, anti-DNase B, anti-streptococcal esterase antibody, HIV, Escherichia coli O157 LPS antigen, RS virus antigen, Vibrio parahaemolyticus heat-resistant hemolysin, influenza antigen, neuraminidase, Candida antigen, Chlamydia antigen, Aspergillus antigen Legionella antigen, E. coli antigen, anti-Anisakis antibody, cryptocox neoformans antigen, varicella virus antigen, leptospira antibody, glucan, cytomegalovirus antibody, Mycobacterium tuberculosis group antigen, anti-acetylcholine receptor antibody, Bs antigen, HBs antibody, HBe antigen, HBe antibody, HCV antibody, HBc antibody, hepatitis delta virus antibody, rheumatoid factor, antigalactose deficient IgG antibody, thyroid autoantibody, anti-DNA antibody, antinuclear antibody, insulin antibody, MMP-3 Anti-SS-A / Ro antibody, anti-SS-B / La antibody, anti-Scl-70 antibody, anti-Jo-1 antibody, anti-thyroid peroxidase antibody, anti-RNP antibody, anti-Sm antibody, anti-centromere antibody, anti-mitochondrial antibody, Anti-cardiolipin β2 glycoprotein I complex antibody, anti-LKM-1 antibody, anti-cardiolipin antibody, TSH receptor antibody, anti-desmoglein antibody, anti-neutrophil cytoplasmic antibody, anti-neutrophil cytoplasmic myeloperoxidase, anti-glomerular basement membrane antibody , TSH stimulating receptor antibody, CRP, immunoglobulin, serum amyloid A, Transferrin, C3, C4, apolipoprotein, total IgE, specific IgE, prealbumin, retinol binding protein, factor VIII-like antigen, free histamine, etc. can be mentioned, and the measurement target substance is not limited at all .

  The biological sample in the present invention includes biological fluids such as whole blood, spinal fluid, prostate fluid, urine, ascites, joint fluid, tear fluid, saliva, serum, and plasma. It is also possible to analyze other fluid samples of non-biological nature using this improved measuring vessel.

  One embodiment of the measurement container of the present invention is shown in FIGS. In the present specification, for example, “opening 2” indicates that it corresponds to the number in the drawing.

The present invention is preferably applied to the following measuring container used in a method for detecting a measurement target substance in a biological sample.
(1) It has an opening 2, (2) an absorbing element 4 located close to the opening 2, and (3) a liquid disposed between the opening 2 and the absorbing element 4 can pass therethrough. An insoluble carrier 3 having a structure; and (4) a binding physiologically active substance capable of capturing the substance to be measured directly or via a substance that forms a binding pair complex. Immobilized in advance, (5) Without changing the measurement container, it is possible to measure a number of measurement target substances,
(6) The insoluble carrier 3 is provided in a dry state,
(7) A measurement container in which a predetermined amount or more of water is dropped onto the insoluble carrier before measurement.

One preferred embodiment of the present invention is that in the measurement container, (8) an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more is dropped onto the insoluble carrier before or simultaneously with the immobilization of the physiologically active substance. This is a measurement container with a reduced matrix effect in the insoluble carrier, and a method for reducing the matrix effect.

  The outer shell 1 can have a combined structure of a cap (lid) 7 and a cup 5 (container) so that the insoluble carrier 3 and the absorbent element 4 can be inserted therein.

  The measurement container of the present invention and the method for detecting a measurement target substance in a biological sample using the measurement container are used for diagnostic assays using insoluble carriers of various detection systems such as chemiluminescence, fluorescence, color development, and electrical signals. The detection method is not limited.

 The measurement container of the present invention has an opening 2 for adding at least one sample and detecting a substance to be measured, and by sequentially introducing a reagent from the opening 2 as necessary, the substance to be measured can be directly or A binding physiologically active substance that can be captured is made to reach the insoluble carrier 3 that has been immobilized in advance through a substance that forms a binding pair complex, and the insoluble carrier 3 is passed through a specific binding pair complex. Retained. The measurement is performed by detecting the specific binding pair complex retained and immobilized.

  In a preferred embodiment of the present invention, an antigen or antibody is used as the binding physiologically active substance in the measurement container. The antibody is not limited to either a polyclonal antibody or a monoclonal antibody, and may be any of full-length, Fab, Fab ', F (ab)' 2, and Fab2. Preferably, one kind is used, and more preferably, the monoclonal antibody is selected from one of an anti-biotin antibody, an anti-dinitrophenol antibody, and an anti-digoxigenin antibody, and more preferably an IgG class antibody.

The aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more is an aqueous solution having protons of 1 × 10 ⁻⁶ M or more in the aqueous solution, and the presence or absence of the buffer capacity is not limited. Preferably, it is an aqueous solution having a proton concentration of 1 × 10 ⁻⁴ M or more and 1 × 10 ⁻¹ M or less, more preferably an aqueous solution in which protons are supplied from a weak acid according to the Bronsted definition, and more preferably a Bronsted definition to be used. This is an aqueous solution having a proton concentration in the vicinity of the first acid dissociation constant (pKa1) of the weak acid. The proton concentration generally adjusts the supply from an acidic substance by adding a basic substance, and this basic substance is preferably a strongly basic aqueous solution according to the Bronsted definition, an aqueous sodium hydroxide solution, Examples include potassium hydroxide aqueous solution and barium hydroxide aqueous solution. The type and concentration of the coexisting salt are not limited, but the type of salt is preferably selected from citric acid, acetic acid, glycine, succinic acid, and phthalic acid, and the salt concentration is preferably 5 to 5 It is 500 mM, More preferably, it is 5-100 mM.

The proton concentration is measured, for example, when a substance called AH reaches ionization equilibrium in an aqueous solution, and protons are supplied as shown in the following formula (1):
AH → A ⁻ + H ⁺ (1)
It is obtained by the following equation (2).
[H ⁺ ] = Ka · [AH] / [A ⁻ ] (2)
(At this time, Ka is a value inherent to the substance supplying protons with an acid dissociation constant, [AH] is the weak acid concentration in the aqueous solution in an ionization equilibrium state, and [A ⁻ ] is the concentration of the ionized weak acid.)
A general and simple method for measuring the proton concentration of an aqueous solution in which protons are supplied from a weak acid according to the Bronsted definition adjusted by a strong base according to the Bronsted definition is that [AH] is added to the aqueous solution before the ionization equilibrium is reached. Since [A ⁻ ] is approximated to the concentration of the added strong base, it is approximately calculated by the following equation (3).
[H ⁺ ] = Ka · [weak acid concentration] / [strong base concentration] (3)
There is also a method of measuring the proton concentration using the glass electrode method. The glass electrode method uses two electrodes, a glass electrode with a glass thin film (electrode film) and a standard electrode, and knows the voltage (potential difference) generated between the two electrodes, thereby determining the proton concentration of the solution. It is a method of measuring. This principle uses the fact that if there are solutions with different proton concentrations inside and outside the glass thin film, an electromotive force is generated in the thin film portion in proportion to the proton concentration difference. Thus, the proton concentration is relatively measured from the electromotive force generated in the electrode film.

The fiber matrix used as the insoluble carrier is often a glass fiber filter that is less likely to be clogged. However, since the glass has a negative charge, the glass fiber is produced or the reaction vessel is produced. In addition, substances existing in the air or substances that come into contact with glass fibers are likely to bind non-specifically by ionic bonds. Therefore, in the step of immobilizing the physiologically active substance, such a non-specific substance that has already adhered is immobilized, and it is considered that the measurement system is disturbed, that is, the matrix effect is caused.
Therefore, before or simultaneously with the step of immobilizing the physiologically active substance, an aqueous solution having a high proton concentration of 1 × 10 ⁻⁶ M or more is dropped to allow the non-specific substance to be adsorbed via ionic bonds. On the carrier, a large amount of protons come into contact with the negative charge of the insoluble carrier, and the non-specific substance is replaced with the proton by an ion exchange phenomenon and can be removed by liberation. Since immobilization can be performed in the absence of non-specific substances on an insoluble carrier, immobilization of measurement values due to non-specific substances that have the greatest effect on the measurement is reduced. It is thought to bring about improvement. Moreover, this reduction effect is effective even at the same time as the step of immobilizing the physiologically active substance, but a further effect can be obtained by carrying out it before the step of immobilizing the physiologically active substance. If this is performed only at the same time as the step of immobilizing the physiologically active substance, ion exchange between the target physiologically active substance and the nonspecific substance to be immobilized is performed in the same manner as ion exchange between the nonspecific substance and proton occurs. First, it is because the non-specific substance removal effect is greater when the non-specific substance is exchanged for protons and then the proton and the target physiologically active substance are exchanged.

In the present invention, it is important to add an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more before or simultaneously with the step of immobilizing the physiologically active substance. it can.

 For example, the insoluble carrier 3 is preferably a sheet having a uniform thickness. The thickness is not particularly limited, but since the change resulting from the reaction is detected from the top surface, it is preferably as thin as possible within the range in which the strength as a carrier can be maintained. Preferably it is 0.3-2.0 mm. The area of the sheet may be smaller or larger than the cross section of the absorbent element 4, but it must have a size and shape that covers the entire range of the opening 2. The size is preferably 100% to 400% compared to the area of the opening 2.

 The absorbent element 4 is arranged as a fluid removal means below the insoluble carrier 3 in order to enhance the passage of the biological sample and the measurement reaction mixture through the insoluble carrier 3. Its function is that the liquid that has passed through the insoluble carrier 3 is absorbed by the porous material by capillary action. For this reason, the surface in contact with the insoluble carrier 3 is preferably flat, and a columnar shape having a sufficient capacity to absorb the reaction solution is preferable. The length of the column is usually larger than the thickness of the insoluble carrier 3, and preferably the length of the column is 5 to 30 times the thickness of the insoluble carrier 3. The cross section of the column may have any shape, but is preferably a cylinder. The surface in contact with the insoluble carrier 3 is preferably perpendicular to the cylinder. The opposite surface of the cylinder may be any shape, but a flat surface that is perpendicular and flat is preferable so that the pressure in contact with the outer shell 1 is uniformly received and can be uniformly transmitted to the surface in contact with the insoluble carrier 3. In addition, it is preferable that the absorbent element 4 is shaped so as to fit tightly between the inner walls of the outer shell part 1, but the distance between the outer wall portion of the absorbent element 4 and the inner wall of the outer shell part 1 is the same in all parts. Need not be. Although there may be some play in the fit, it is preferable that the movement width does not exceed 10% of the maximum diameter of the absorbent element 4. In the case of a cylinder, the outer diameter is preferably 90% or more of the inner diameter of the outer shell 1.

  Further, if necessary, another structure may be inserted between the opening 2 and the insoluble carrier 3 or between the insoluble carrier 3 and the absorbing element 4 within a range not impeding the effect of the present invention.

 If the outer portion 1 has a structure in which the reaction solution or reagent added to the upper portion of the cap 7 does not leak and the inside can appropriately hold the insoluble carrier 3 and the absorbing element 4, the outer shape thereof is particularly good. It is not limited. The shape seen from above may be a circle, a regular polygon, or a shape close to them, or a collar such as 6 in FIG. In addition, a container for performing a reaction in a liquid phase, a container for storing a reagent necessary for the reaction, and the like may be integrated. In addition, in order to increase the fluid absorption rate, the outer portion 1 has one vent hole so that the air contained in the measurement container is replaced by the added reaction mixture and reagent before the measurement. More preferably.

 The cap 7 has an opening 2, is firmly fixed to the outer shell 1 at the time of mounting, and comes into contact with the insoluble carrier 3 and a part of the cap 7 to press the insoluble carrier 3 so that it does not move. Is not particularly limited. The shape of the opening 2 viewed from above may be a circle, a regular polygon, or a shape close to them. Preferably, it is a funnel-shaped structure with the opening as the central axis so that the added reaction solution and reagent do not leak to other than the opening.

  In the present invention, the size, shape, material and the like are not particularly limited. Preferably, all the reagents may be absorbed to the extent that they can be absorbed in the range visible from the opening 2 of the insoluble carrier 3.

  Specifically, the outer shell 1 is not limited as long as it can absorb a biological sample and a reagent necessary for measurement without leaking, but preferably has a size suitable for handling in an automatic analyzer. As a matter of fact, it is desired to be 10 to 25 mm. Moreover, it is preferable that a shape is cylindrical shape, and it is more preferable that an internal diameter is 5.0-20 mm. If the material is molded from polyethylene, polypropylene, polystyrene, acrylonitrile-butadiene-styrene terpolymer (ABS), polycarbonate, polyacrylate, HIPS, or any other moldable material that is inert to the measurement component, limited Not.

 The size of the cap 7 is not limited as long as it has the opening 2, but is preferably 10 to 25 mm that can cover the entire outer portion 1. If the material is molded from polyethylene, polypropylene, polystyrene, acrylonitrile-butadiene-styrene terpolymer (ABS), polycarbonate, polyacrylate, HIPS, or any other moldable material that is inert to the measurement component, limited Not. It is preferable that it is resistant to natural drying, air drying, vacuum drying, vacuum drying, freeze vacuum drying, drying using infrared rays or far infrared rays, drying using high frequency waves such as microwaves, etc. without passing water.

 The opening 2 is not limited as long as the insoluble carrier 3 having a size capable of detecting a signal necessary for measurement is exposed and the added reagent or sample can be absorbed within 10 seconds. It is a circle of 2.0 to 10 mm, more preferably 2.0 to 6.0 mm.

 The insoluble carrier 3 is not particularly limited as long as it is a structure that can permeate fluid. The material can be selected from various porous structures, but is preferably a fiber matrix such as a filter made of paper, glass, cellulose, nylon, other natural or synthetic fiber materials, and the like. More preferably, it is a binderless fiber. More preferably, it is a glass fiber binderless fiber filter known to be inexpensive and have less non-specific protein adsorption. Further, a glass fiber filter cut out into a circle having a diameter of 4 to 10 mm and a weight of relatively large and relatively thick, specifically, a weight of 80 to 220 g per square meter and a thickness of 0.3 to 2.0 mm. More preferably, it is used. A commercial item etc. can be used for these.

The absorbent element 4 is not particularly limited as long as it can hold a certain amount of fluid. Preferred are paper, glass, cellulose, cellulose derivatives, nylon, other natural fiber materials or synthetic fiber materials, and more preferred are cellulose acetate derivatives which are less susceptible to chemical changes due to fluid properties of fluids. The structure of the absorbent element 4 is preferably a porous structure in order to increase the surface area of the fiber that can hold the fluid. A commercially available tobacco filter can also be used for this. The size of the outer element 1 is 5.0 to 20 mm and the height is 10 to 25 mm, and the absorbing element 4 is 5.0 to 20 mm in inner diameter and 10 to 25 mm in height and has no gap. More preferably.

  The measurement system for detecting a measurement target substance in a biological sample of the present invention is, for example, conventionally known various physicochemical changes caused by a reaction using the measurement container of the present invention depending on the detection system. It can implement by using the detection method (measuring instrument).

  The kit of the present invention for use in the measurement for detecting a substance to be measured in a biological sample includes, for example, the reaction container (whole) of the present invention and, if necessary, the substance to be measured and a binding pair complex. Reagents containing substances to be formed, reagents containing substrates that produce a detectable signal, blocking solutions, washing solutions, and the like.

 In the measurement container of the present invention, a physiologically active substance binding to the insoluble carrier 3 that can capture the substance to be measured “directly” or “through a substance that forms a binding pair complex” is immobilized in advance. Supplied in the finished state. More preferably, a binding physiologically active substance that can be captured via a substance that forms a “binding pair complex that can be used as a highly versatile measurement container independent of the substance to be measured” It is in a fixed state.

  In this specification, the binding pair complex refers to a low molecular weight compound bound to a “first substance that specifically binds to a measurement target substance by a chemical or physical method” and the compound specifically. It means a complex with a molecule to be recognized. Representative combinations include, but are not limited to, hapten-antihapten complexes, such as biotin-antibiotin, avidin-biotin, carbohydrate-lectin, complementary Examples include nucleotide sequence, effector-receptor, enzyme cofactor-enzyme, enzyme inhibitor-enzyme.

  It is also possible to use this measurement container for measurement without using a biologically active substance immobilized in advance. For example, after using immunoassay such as latex beads in the liquid phase, it can be added to the measurement container of the present invention, and B / F separation can be performed on the insoluble carrier 3. Cross.

  In order to immobilize the physiologically active substance on the insoluble carrier 3, for example, a physically adsorbing method or a chemical bonding method may be used. At that time, the physiologically active substance may be directly immobilized on an insoluble carrier, or a substance such as an antibody or a receptor that specifically binds to the physiologically active substance may be immobilized on the insoluble carrier, and the physiologically active substance is interposed via the substance. The active substance may be immobilized. Alternatively, the immobilized insoluble carrier may be cut out and attached to the measurement container, or immobilization may be performed after the non-solid phase insoluble carrier is attached to the measurement container.

“(5) It is possible to measure a large number of measurement target substances without modifying the measurement container,” but this is dedicated to specifically detecting and measuring each of the various measurement items. This means that this measurement container is not necessary, and this measurement container can be used in common regardless of the measurement item.

 “(6) The insoluble carrier is provided in a dried state,” but this takes into account stability during storage and transportation, and the measurement container is dried after immobilizing the physiologically active substance. It is meant to be provided in the state. In addition, if the measurement container is stored in an airtight container that blocks moisture, it can be stored for a long period of about one year. Examples of the drying method include natural drying, air drying, vacuum drying, reduced pressure drying, freeze vacuum drying, drying using infrared rays or far infrared rays, and drying using high frequency such as microwaves.

“(7) Before measurement, a certain amount or more of water is dripped onto the insoluble carrier,” but by performing this step, it is possible to immediately return to a measurable state. The amount of water at this time is preferably such that at least 30 seconds later, an amount of water that is sufficiently wetted by the insoluble carrier 3 that is exposed from the opening 2 and is large enough to detect a signal necessary for measurement is dropped. More preferably, 50 to 100 μl of water is dropped, and after 1 to 5 minutes, a biological sample containing the measurement target substance is dropped and measurement is started.

“(8) Before immobilizing the physiologically active substance”, this is not limited to the above, as long as the insoluble carrier 3 can be moistened in the step of immobilizing the antibody. May be. Preferably, the antibody immobilization step is performed within 30 minutes.

[Example 1] (Measurement of PSA)
(1) Production of absorbent element A commercial tobacco filter was cut 11.5 mm at a time.
(2) Production of measurement container As shown in FIG. 1, glass fiber filter paper (manufactured by Advantech Toyo Co., Ltd.) having a diameter of 9.0 mm and a thickness of 1.0 mm is rounded and laminated on the absorbent element produced in (1) above. It was stored in a liquid impermeable container made of polyethylene. The liquid impervious container used this time had an inner diameter of 9.0 mm and a measurement container having a length of 15 mm.
(3) Immobilization of antibody From the opening of the measurement container prepared in (2) above, (a) 100 mM citrate buffer solution (containing [H ⁺ ] = 1.0 × 10 ⁻⁴ M) (b) anti-mouse 50 μl of biotin monoclonal antibody 47 μg / mL (100 mM citrate buffer (containing [H ⁺ ] = 1.0 × 10 ⁻⁴ M)), (c) 5% glycerol / 1% casein solution (10 mM phosphate) -Saline buffer solution (pH 7.4) was supplied in an amount of 100 μl, the solution supplied immediately before being aspirated, and then lyophilized.
(4) Preparation of HRP-labeled anti-PSA antibody solution Using 10 mg of anti-PSA antibody and horseradish-derived peroxidase, they were combined according to the Nakane method to prepare 2.0 μg / mL, and used as the first antibody solution.
(5) Preparation of biotin-labeled anti-PSA antibody solution 10 mg of anti-PSA antibody and biotinamide caproic acid-N-hydroxysuccinimide ester were reacted at 25 ° C. for 4 hours, and fractionated using NAP-5 (Amersham Biosciences). The second antibody solution was prepared to 8.0 μg / mL.
(6) Implementation of measurement A biological sample (40 μl) was mixed with 20 μl of the first antibody solution and 20 μl of the second antibody solution to prepare a reaction solution, which was then incubated at 40 ° C. for 5 minutes. 50 μl of physiological saline is added to the reaction container prepared in (3), and after 5 minutes, 50 μl of the reaction solution is added and incubated at 40 ° C. Two minutes later, physiological saline containing 0.05% Tween 20 was added twice by 80 μl, and after another 2 minutes, 30 μl of 3,3 ′, 5,5′-tetramethylbenzidine was added as a chromogenic substrate. One minute after the addition of the chromogenic substrate, the reflected absorbance at 670 nm was read.

[Example 2] (Measurement of PSA)
(1) Production of absorbent element A commercial tobacco filter was cut 11.5 mm at a time.
(2) Preparation of measurement container As shown in FIG. 1, the measurement container is circularly cut on a glass fiber filter paper (manufactured by Advantech Toyo Co., Ltd.) having a diameter of 9.0 mm and a thickness of 1.0 mm, and laminated on the absorbent element produced in (1) above. It was stored in a liquid impermeable container made of polyethylene. The liquid impervious container used this time had an inner diameter of 9.0 mm and a measurement container having a length of 15 mm.
(3) Immobilization of antibody From the opening of the measurement container prepared in (2) above, (a) mouse anti-biotin monoclonal antibody 47 μg / mL (100 mM citrate buffer ([H ⁺ ] = 1.0 × 10 ^{− 4} M-containing)) to 50μl, (b) 5% glycerol 1% casein solution (10 mM phosphate - saline buffer: a pH7.4) from 100 [mu] l, the liquid supplied immediately before is sucked After waiting and supplying sequentially, it was freeze-dried.
(4) Preparation of HRP-labeled anti-PSA antibody solution Using 10 mg of anti-PSA antibody and horseradish-derived peroxidase, they were combined in accordance with the Nakane method to prepare 2.0 μg / mL, which was designated as the first antibody solution.
(5) Preparation of biotin-labeled anti-PSA antibody solution 10 mg of anti-PSA antibody and biotinamide caproic acid-N-hydroxysuccinimide ester were reacted at 25 ° C. for 4 hours, and fractionated using NAP-5 (Amersham Biosciences). The second antibody solution was prepared to 8.0 μg / mL.
(6) Implementation of measurement A biological sample (40 μl) was mixed with 20 μl of the first antibody solution and 20 μl of the second antibody solution to prepare a reaction solution, which was then incubated at 40 ° C. for 5 minutes. 50 μl of physiological saline was added to the reaction container prepared in (3), and after 5 minutes, 50 μl of the reaction solution was added and incubated at 40 ° C. Two minutes later, physiological saline containing 0.05% Tween 20 was added twice by 80 μl, and after another 2 minutes, 30 μl of 3,3 ′, 5,5′-tetramethylbenzidine was added as a chromogenic substrate. One minute after the addition of the chromogenic substrate, the reflected absorbance at 670 nm was read.
[Comparative example]
In the step (3) of Example 2, a 100 mM citrate buffer solution (containing [H ⁺ ] = 1.0 × 10 ⁻⁴ M) was added to a 100 mM phosphate buffer solution ([H ⁺ ] = 1.0 × 10 ^{−). A} measurement container was manufactured in the same manner as in Example except that the content was ⁷ M), and PSA was measured.

  In Example 1, Example 2 and Comparative Example, the standard PSA reagent (2.5 ng / mL) was measured five times, and the average value (Mean) and CV% of the reflectance change amount (K / S) are shown in Table 1. .

  In the comparative example, the K / S value is small and the CV% is also large. This is because the non-specific substance adhering to the insoluble carrier physically hinders the immobilization of the physiologically active substance, resulting in a decrease in sensitivity and the non-specific substance to be measured by the non-specific substance. It is thought that CV% became large because adsorption occurred and interference with the measured value occurred.

  In Example 2, an aqueous solution having a high proton concentration was dropped simultaneously with the immobilization of the physiologically active substance, so that a large amount of protons were absorbed on the insoluble carrier on the non-specific substance adsorbed via ionic bonds. Contact with electric charge, non-specific substance is replaced with proton by ion exchange phenomenon, it can be removed by liberation, and non-specific substance is reduced on insoluble carrier when immobilizing target bioactive substance As a result, it is possible to immobilize the measured values, reducing fluctuations in measured values due to non-specific substances that have the greatest effect on the measurement, and improving sensitivity and CV%, resulting in improved measurement accuracy. Conceivable.

  In Example 1, a further effect is obtained by carrying out it before the step of immobilizing the physiologically active substance. If this is performed only at the same time as the step of immobilizing the physiologically active substance, ion exchange between the target physiologically active substance and the nonspecific substance to be immobilized is performed in the same manner as ion exchange between the nonspecific substance and proton occurs. First, it is because the non-specific substance removal effect is greater when the non-specific substance is exchanged for protons and then the proton and the target physiologically active substance are exchanged. Therefore, the improvement in sensitivity and CV% is more remarkable than in Example 2.

  As described above, by using the measurement container of the present invention, it is possible to reduce the matrix effect and improve the measurement accuracy in a general manner regardless of the measurement item without changing the setting of the measurement device. For this reason, manufacturing know-how cultivated so far can be used for various analysis applications such as in-vitro diagnostic drugs and contribute 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.

Explanation of symbols

1: Outer part 2: Opening part 3: Insoluble carrier 4: Absorbing element 5: Cup 6: Brim 7: Cap

Claims (9)

A measurement container used in a method for detecting a measurement target substance in a biological sample,
(1) having an opening,
(2) an absorbent element located proximate to the opening;
(3) provided with an insoluble carrier that is disposed between the opening and the absorbent element and has a structure capable of transmitting liquid;
(4) A binding physiologically active substance capable of capturing the substance to be measured directly or via a substance forming a binding pair complex is immobilized on the insoluble carrier in advance.
(5) It is possible to measure multiple measurement target substances without modifying the measurement container,
(6) the insoluble carrier is provided in a dry state;
(7) Before measurement, a predetermined amount or more of water is dropped on the insoluble carrier.
In the measurement container,
(8) including a step of dropping an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more into the insoluble carrier before or simultaneously with the immobilization of the physiologically active substance.
A method for reducing a matrix effect in the insoluble carrier in a method for detecting a substance to be measured in a biological sample. The measurement method according to claim 1, wherein the absorbent element is cellulose or a cellulose derivative. The measurement method according to claim 1, wherein the insoluble carrier is a fiber matrix. The measurement method according to claim 3, wherein the fiber matrix is glass fiber. The measurement method according to claim 1, wherein the physiologically active substance is IgG. A measurement container used in a method for detecting a measurement target substance in a biological sample,
(1) having an opening,
(2) an absorbent element located proximate to the opening;
(3) provided with an insoluble carrier that is disposed between the opening and the absorbent element and has a structure capable of transmitting liquid;
(4) A binding physiologically active substance capable of capturing the substance to be measured directly or via a substance forming a binding pair complex is immobilized on the insoluble carrier in advance.
(5) It is possible to measure multiple measurement target substances without modifying the measurement container,
(6) the insoluble carrier is provided in a dry state;
(7) Before measurement, a predetermined amount or more of water is dropped on the insoluble carrier.
In the measurement container,
(8) including a step of dropping an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more into the insoluble carrier before or simultaneously with the immobilization of the physiologically active substance.
A method for producing a measurement container having a reduced matrix effect in the insoluble carrier in a method for detecting a substance to be measured in a biological sample. A measurement container used in a method for detecting a measurement target substance in a biological sample,
(1) having an opening,
(2) an absorbent element located proximate to the opening;
(3) provided with an insoluble carrier that is disposed between the opening and the absorbent element and has a structure capable of transmitting liquid;
(4) A binding physiologically active substance capable of capturing the substance to be measured directly or via a substance forming a binding pair complex is immobilized on the insoluble carrier in advance.
(5) It is possible to measure multiple measurement target substances without modifying the measurement container,
(6) the insoluble carrier is provided in a dry state;
(7) Before measurement, a certain amount or more of water is dropped on the insoluble carrier,
In the measurement container,
(8) Obtained by a method comprising a step of dropping an aqueous solution having a proton concentration of 1 × 10 ⁻⁶ M or more onto the insoluble carrier before or simultaneously with immobilization of the physiologically active substance.
A measurement container having a reduced matrix effect in the insoluble carrier in a method for detecting a substance to be measured in a biological sample. A measurement system for detecting a measurement target substance in a biological sample, which performs measurement using the measurement container according to claim 7. A kit for use in measurement for detecting a substance to be measured in a biological sample, comprising the measurement container according to claim 7.

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