JP2010048754A - Method for reducing measuring error - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for equalizing the time required for passing a specimen or a reagent through a reaction chamber by increasing a rotation speed in the second half of liquid feed, and improving measuring precision to reduce a measuring error. <P>SOLUTION: The method for reducing a measuring error includes the steps of liquid-feeding a specimen or a regent to the reaction chamber by rotating an analysis chip having the reaction chamber capable of receiving a carrier bound with an antigen or antibody to the rotation axis of the outside of the analysis chip, and switching to a two or more times rotation speed before the whole of a specimen or a reagent solution is passed through the reaction chamber in the immunological analysis for measuring the quantity of a substance to be tested in the reaction chamber. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for reducing measurement errors in the use of an analysis chip for immunological analysis in which reagents and specimens are fed using centrifugal force due to rotation.

  Conventionally, most of the analysis of trace molecules related to clinical diagnosis, food hygiene, and environmental analysis requires expensive equipment and is skilled in operation, and thus has been performed by clinical testing companies and analysis companies. In recent years, as a trend of the world, simple and quick diagnosis at the bedside, analysis and measurement at each site of food processing and import, to prevent accidents and analyze harmful substances in rivers and waste The importance of conducting on-site such as rivers and waste disposal sites has attracted attention, and the development of detection methods and analysis chips that can be measured easily, quickly, inexpensively, with high accuracy and high sensitivity is emphasized. .

  In an immunological analysis for analyzing a trace amount of a test substance in a specimen, use of a small analysis chip (apparatus) called Lab-on-chip (μTAS) has been proposed. In recent years, a technique related to an analysis chip for feeding liquid using centrifugal force caused by such rotation has been developed, and application to immunological measurement is being studied.

  In immunological analysis using a small analysis chip, generally, a specimen and a reagent are fed into a reaction chamber filled with a carrier on which an antigen or antibody is solid-phased using centrifugal force of rotation, and pass through the reaction chamber. In some cases, an antigen-antibody reaction is performed. However, the time required for the specimen and reagent to pass through the reaction chamber varies depending on the difference in viscosity between the specimen and the reagent. For example, when the specimen is a sample derived from a living body such as blood, the viscosity of the living body derived sample such as blood is different among individuals, and thus it has been difficult to align the passage time in the reaction chamber. Variation in the time required for the sample to pass through the reaction chamber caused by individual differences in the sample results in variation in the time during which the carrier is in contact with the sample, resulting in an error in measurement or an error between measurements.

  The reaction chamber passage time also varies depending on the rotational speed and centrifugal force applied to the analysis chip. Furthermore, it varies depending on differences in the reaction chamber sectional area, channel sectional area, channel surface hydrophobicity, carrier amount, carrier packing density, carrier damming section sectional area, etc. of the apparatus. In the case of an analysis chip having minute channels, it has been difficult to control properties such as the shape, area, and hydrophobicity of these chips with high accuracy. Due to such chip variations, the time required for the specimen and reagent to pass through the reaction chamber varies. Variation in the time required for the specimen and reagent to pass through the reaction chamber results in variation in the time during which the carrier is in contact with the specimen and the reagent, resulting in an error within measurement or an error between measurements.

  For example, Patent Document 1 discloses a CD-like (disk-shaped) microfluidic device (analytical chip) having a plurality of measurement channels, and a method and operating profile for performing an immunoassay by rotating the microfluidic device. It is disclosed. Specifically, in each of the cleaning process, the sample transfer process, the sample cleaning process, the bonded body transfer process, and the bonded body cleaning process, the detailed effect is unknown, but the rotation increases the rotation speed step by step. A profile is disclosed. Further, it is disclosed that in the moving process, the liquid amount is defined in the capacity measuring unit during the first two processes accompanied by an increase in rotational speed.

  Patent Document 2 also discloses a CD-like (disk-shaped) microfluidic device (analytical chip) having a plurality of measurement channels and a method and operating profile for performing an immunoassay by rotating the microfluidic device. It is disclosed. Specifically, in each of the washing process, the sample moving process, the sample washing process, the conjugate moving process, and the conjugate washing process, the detailed effect is unknown, but the rotation increases the rotation speed step by step. A profile is disclosed. It is also disclosed that the volume is metered and defined during the first two steps of spin flow with increased rotational speed in sample and conjugate transfer. Further, it is disclosed that a pulse having an increased rotation speed for breaking through a hydrophobic break is employed.

However, a specific method for achieving a low measurement error between samples having various properties and achieving a low measurement error between chips having different properties is not disclosed. Further, there is no disclosed technique for solving the problem of increasing the measurement accuracy and reducing the measurement error by controlling the time taken for the specimen or reagent to pass through the reaction chamber.
JP-T-2006-524816 JP-T-2004-529336

  In view of such a state, the present invention controls the time required for the sample or reagent to pass through the reaction chamber by increasing the rotation speed of the analysis chip at an appropriate timing when the sample or reagent is delivered by rotation. It is an object of the present invention to provide a technique capable of increasing measurement accuracy and reducing measurement errors.

As a result of intensive studies, the inventors have solved the above problems with the configuration described in any one of (1) to (5) below.
(1) Rotating an analysis chip having a reaction chamber capable of accommodating a carrier to which an antigen and / or an antibody are bound with respect to a rotating shaft outside the analysis chip, thereby feeding a specimen and a reagent to the reaction chamber, In an immunological analysis for measuring the amount of a test substance in the reaction chamber,
A method for reducing measurement errors in immunological analysis by switching the rotational speed of an analysis chip to twice or more before the entire amount of a specimen or reagent solution passes through a reaction chamber.
(2) The measurement error reduction method according to (1), wherein the time point at which the rotation speed of the analysis chip is switched to twice or more is the time point when 50% or more of the amount of the sample or reagent solution has passed through the reaction chamber.
(3) The method for reducing measurement error according to (1) or (2), in which the rotational speed of the analysis chip is reduced to 90% or less before feeding the liquid before switching the rotational speed of the analysis chip to more than twice.
(4) The method for reducing measurement error according to (3), wherein the time point when the rotational speed of the analysis chip is reduced to 90% or less is a time point before 50% of the amount of the sample or reagent solution passes through the reaction chamber. .
(5) The measurement error reduction method according to any one of (1) to (4), wherein the immunological analysis is a two-step method.

  According to the present invention, by increasing the rotation speed of the analysis chip in the later stage of liquid feeding, it becomes possible to align the time taken for the specimen or reagent to pass through the reaction chamber between the analysis chips, and as a result, the measurement It is possible to improve the accuracy of the measurement and reduce the measurement error. In addition, by reducing the rotation speed of the analysis chip at the initial stage of liquid feeding, the reaction time of the sample or reagent is extended, and by increasing the rotational speed at the later stage of liquid feeding, the measurement error is reduced while increasing the sensitivity. Is possible.

  The method for reducing a measurement error of the present invention is used for an analysis chip for immunological analysis in which a specimen or reagent is fed using a centrifugal force due to rotation.

  The immunological analysis in the present invention means a technique for analyzing a test substance in a specimen using an antigen-antibody reaction, and a representative example thereof is an ELISA (Enzyme-Linked Immunosorbent Assay solid phase enzyme immunoassay). Method), RIA (Radioimmunoassay radioimmunoassay), FIA (Fluorescence immunoassay fluorescent immunoassay), FLISA (Fluorescence-Linked Immunosorbent Assay solid phase fluorescent immunoassay).

As an analysis method,
1) A direct method for directly recognizing and detecting a target substance by means of a labeled antibody,
2) An indirect method of recognizing a target substance with an antibody and recognizing and detecting an antibody bound to the target substance with a labeled antibody,
3) Competition method,
4) A two-antibody sandwich method in which a target substance is captured by an immobilized antibody (primary antibody) and further detected by another labeled antibody (secondary antibody),
5) A three-antibody sandwich method in which the target substance is captured by an immobilized antibody, the target substance is recognized by another antibody, and the target substance is detected by an antibody labeled with the recognized antibody.
Etc.

  The analysis method in the present invention includes a one-step method in which a complex of an antigen and an antibody labeled with at least one is reacted with an antigen or an antibody on a solid phase, After reacting with the antigen, the labeled antibody or the two-step method of reacting with the antigen, and further, after reacting the unlabeled antigen with the antibody or antigen on the solid phase, reacting with the unlabeled antibody and further labeling There is a three-step method in which an antibody is reacted. In particular, the measurement error reducing method of the present invention is a two-step method or 3 in which the measurement error due to the difference in passage time between the specimen and the reagent in the reaction chamber increases in order to pass the antigen solution, antibody solution and labeled antibody solution through the reaction chamber a plurality of times. In the step method, the measurement error can be pushed low, and therefore it is preferably employed.

  The test substance in the immunological analysis may be any substance that specifically binds to an antigen or antibody, such as a protein, sugar, lipid, nucleic acid, glycoprotein, glycolipid, or cell. Examples include cytokines, chemokines, interleukins, allergens, DNA, RNA, antibodies, lipids, enzymes, and other chemical substances. In particular, IL-6, IL-8, and TNF are preferable. The organism from which the test substance is derived does not matter. The test substance may be one type or two or more types.

  Further, the purpose of immunological analysis is not particularly limited, and there is no particular limitation such as detection of the presence or absence of a test substance in a sample, quantification of a test substance, and the like. The immunological epidemiological analysis in the present invention can be used for analysis in clinical tests, food tests, environmental tests and the like.

  The specimen in the present invention refers to a sample that may contain the test substance, and is preferably a liquid. For example, liquids collected from living bodies including body fluids such as blood, urine, spinal fluid, saliva, sputum, and cell suspension can be exemplified.

  The reagent in the present invention means a reagent for detecting a specimen, specifically, blocking solution, diluent, denaturant, labeled antibody, labeled antigen, unlabeled antibody, unlabeled antigen, labeled substance, luminescence. Substrate, fluorescent substrate, chromogenic substrate, hydrogen peroxide solution, washing solution and the like can be mentioned. The reagent for which the measurement error reducing method of the present invention is adopted preferably includes standard substances, labeled antibodies, and labeled antigens.

  In the immunological analysis method of the present invention, the specimen and the reagent are removed by rotating an analysis chip having a reaction chamber containing a carrier for binding an antigen and / or an antibody with respect to a rotation axis outside the analysis chip. The sample is sent to the reaction chamber and the test substance in the sample is analyzed.

  In the present invention, an analysis chip having a reaction chamber containing a carrier that binds an antigen and / or an antibody is used. The shape and size of the reaction chamber need only accommodate the carrier that binds the antigen and / or antibody. The shape is preferably tubular, and the cross section of the tube is not particularly limited, such as a circle or a polygon. As for the size of the reaction chamber, it is desirable that the test substance in the sample is narrow to some extent to the antigen and / or antibody of the carrier, and the minor axis of the cross section is usually 0.1 to 1 mm, preferably 0.2 to 0. 0.5 mm, and the length is usually 0.5 to 10 mm, preferably 0.5 to 5 mm.

  The reaction chamber contains a carrier to which an antigen and / or an antibody is bound. The number of carriers may be one or more, and it is preferable to accommodate a plurality of carriers from the viewpoint of increasing the efficiency of immunological analysis. At this time, it is preferable that a carrier damming portion having a channel depth or channel width smaller than the minor axis of the carrier is provided. By providing the support damming portion, it is possible to suppress the carrier from flowing out of the reaction chamber even when a centrifugal force is applied.

  The shape of the carrier may be a so-called microrod such as a cylinder or a polygonal column, or a plate-like microplate, in addition to spherical or elliptical microbeads.

  The size of the carrier depends on the size of the reaction chamber, but the minor axis is preferably in the range of 1 to 1000 μm, preferably 10 to 200 μm, regardless of the shape of the carrier.

  The material of the carrier is not particularly limited, and glass, ceramic (eg, yttrium partially stabilized zirconia), metal (eg, gold, platinum, stainless steel), resin (eg, nylon, polystyrene, polyvinyl alcohol, polymethyl methacrylate, polyacrylamide), agarose Of these, resins, particularly polystyrene, are preferred.

  When a plurality of carriers are stored in the reaction chamber, the shape, size, and material of each carrier may be uniform or varied. In addition, it is not necessary that antigens and / or antibodies are bound to all the carriers stored in the reaction chamber, and a part of the carriers that do not bind anything may be included.

   The antigen and / or antibody to be bound to the carrier may be selected from among various antibodies, antigen-binding fragments of antibodies such as Fab fragments and F (ab ′) 2 fragments, and various antigens. An antigen or an antibody that specifically binds to the test substance in the medium can be selected as appropriate, and may be one kind or plural. There are no particular restrictions on the binding density, number of bindings, binding mode, etc. of the antigen or antibody to the carrier.

  The method of binding and solidifying the antigen and / or antibody to the carrier can be performed by, for example, mixing the carrier and the antigen or antibody in a solution such as a buffer and bringing them into contact with each other. Bonding by contact can be carried out usually under conditions of 1 to 24 hours and 4 to 37 ° C. with stirring as necessary. The obtained carrier may be washed with a buffer solution, a washing solution or the like before use. In addition, the method of binding and solid-phase formation is not limited to this, for example, using a crosslinking agent containing a hydrophilic polymer (polyethyleneimine, polyethylene glycol, polyvinyl alcohol, sodium polysulfonate, etc.) for an antigen or antibody and a carrier chemically. It is also possible to use a method of binding to the.

  In addition to the above-described reaction chamber, the analysis chip in the present invention may be provided with a reservoir for holding a sample and a reagent, and a waste tank for storing the sample and the reagent after passing through the reaction chamber.

  In the immunological analysis method of the present invention, a specimen and a reagent are fed into a reaction chamber filled with a carrier using centrifugal force due to rotation, and pass through the reaction chamber. The time required for the total amount of the specimen and reagent to pass through the reaction chamber (reaction chamber passage time) varies depending on the difference in viscosity between the specimen and the reagent. When the sample is a sample derived from a living body such as blood, the viscosity of the sample derived from a living body such as blood is different between individuals, so it has been difficult to align the passage time in the reaction chamber. The reaction chamber passage time also varies depending on the rotational speed and centrifugal force applied to the analysis chip (device). Further, it varies depending on differences in the reaction chamber cross-sectional area, flow path cross-sectional area, flow path surface hydrophobicity, carrier amount, carrier packing density, carrier damming section cross-sectional area, etc. In the case of analysis chips having minute flow paths, it has been difficult to control the shape, area, and hydrophobicity of these analysis chips with high accuracy. The measurement error reduction method of the present invention can solve the plurality of measurement error factors simply by switching the rotation speed of the analysis chip to 2 times or more at an appropriate timing, that is, increasing the rotation speed to 2 times or more. it can.

  The rotation speed of the analysis chip in the present invention means the rotation speed of the analysis chip per unit time, and can be controlled, for example, by setting the rotation speed of a centrifuge used for rotation of the analysis chip.

  Variation in time required for the total amount of the specimen and reagent to pass through the reaction chamber results in variation in the time during which the carrier is in contact with the specimen or reagent, resulting in an error within measurement or an error between measurements. The method for reducing measurement errors in the present invention reduces the difference in time required for the entire amount of the specimen and reagent to pass through the reaction chamber. A method is adopted in which the rotational speed of the analysis chip is switched to twice or more before the entire amount of the sample or reagent solution passes through the reaction chamber. The timing for increasing or switching the rotational speed may be any time before the total amount (100%) of the sample or reagent passes through the reaction chamber, that is, when less than 100% of the amount of the sample or reagent solution has passed. It is preferable that 95% of the amount of the sample or reagent solution is at a point before passing through the reaction chamber. More preferably, 90% is the point before passing through the reaction chamber.

  In the present invention, the time point before the total amount of the sample or reagent passes through the reaction chamber means a time point when the amount of the sample or reagent that has passed through the reaction chamber is less than 100% of the total amount of the sample or reagent. It means a state where there is a specimen or reagent solution that has not passed through the reaction chamber.

  In addition, since most of the sample or reagent is not wasted, the sensitivity can be maintained as a result, and each reaction step can be shortened as much as possible, and the measurement time can be shortened. It is preferable to switch the rotation speed of the chip to twice or more. More preferably, at the time when 70% or more, most preferably 80% or more has elapsed, the rotational speed of the analysis chip is switched to twice or more.

  In addition, it is possible to effectively use a sample or reagent involved in an antigen-antibody reaction, and as a result, it is possible to maintain sensitivity. As a result, when 50% or more of the amount of the sample or reagent solution passes through the reaction chamber. It is preferable to switch the rotational speed of the analysis chip to twice or more. More preferably, when 70% or more, most preferably 80% or more has passed through the reaction chamber, the rotational speed of the analysis chip is switched to twice or more.

  In the present invention, an increase in the rotation speed and switching to a rotation speed higher than 2 times are performed by the method of switching the rotation speed of the analysis chip to more than 2 times in a process in which an antigen-antibody reaction that is a main cause of measurement error is performed. Therefore, it is preferably employed in the step of feeding a solution containing a specimen or the step of feeding a reagent solution containing any one of a labeled antibody, a labeled antigen and a standard substance. Since the problem that a difference in specimen viscosity due to individual differences causes a measurement error can be solved, it is more preferably employed in a step of feeding a solution containing a specimen.

  Since the increase in the rotation speed in the present invention can effectively reduce the measurement error, the rotation speed of the analysis chip is switched to twice or more. Preferably, the rotational speed of the analysis chip is switched to 3 times or more. More preferably, the rotational speed of the analysis chip is switched to 4 times or more. The effect of reducing the measurement error increases as the rotation speed increase factor increases.

  In addition, the increase in the rotational speed in the present invention has the effect of shortening the time required for measurement and enabling quick inspection and analysis.

  In the present invention, the rotation speed is preferably switched rapidly in order to shorten the measurement time. Specifically, the rotation speed is preferably increased at an acceleration of 100 rpm / second or more, that is, a rotation speed change speed. More preferably, the rotational speed is increased at an acceleration of 300 rpm / second or more.

  In the present invention, before the rotation speed of the analysis chip is switched to more than twice in order to suppress a decrease in the efficiency of the antigen-antibody reaction and a decrease in detection sensitivity caused by feeding a part of the specimen and reagent at a high rotation speed. In this case, it is preferable to reduce the rotational speed of the analysis chip to at least 90% or less. By reducing the rotation speed, the liquid feeding speed of the specimen or reagent can be reduced. In addition, since the time during which the carrier contacts the specimen or reagent can be increased, the efficiency of the antigen-antibody reaction can be increased, and as a result, the sensitivity can be improved. Further, since the rotational speed is increased with respect to the rotational speed once lowered, even if the rotational speed is switched to the same rotational speed, the rotational speed increase magnification is increased by once decreasing the rotational speed. As a result, the variation suppressing effect is increased and the measurement error can be further reduced.

  The timing at which the rotational speed is reduced is preferably less than 50% of the sample or reagent feeding step because the sample or reagent involved in the antigen-antibody reaction can be used more effectively. More preferably, the rotational speed is reduced to a time point of less than 30%, most preferably a time point of less than 10%.

  In addition, since it is possible to use a sample or reagent involved in the antigen-antibody reaction more effectively, it is preferable to reduce the rotation speed before 50% of the amount of the sample or reagent solution passes through the reaction chamber. . More preferably, the rotational speed is reduced at a time before 30% passes through the reaction chamber, most preferably at a time before 10% passes through the reaction chamber.

  In the present invention, the time point before 50% of the sample or reagent liquid amount passes through the reaction chamber is the time point when less than 50% of the sample or reagent solution amount passes through the reaction chamber. It means a time point at which the amount of the sample or reagent is less than 50% of the total amount of the sample or reagent.

  It is preferable to reduce the rotation speed of the analysis chip to at least 90% or less. Further, it is more preferable to reduce it to 80% or less because the liquid feeding speed of the specimen or reagent can be lowered and the efficiency of the antigen-antibody reaction can be increased.

  The amount and ratio of the specimen and reagent that have passed through the reaction chamber in the present invention are directly or indirectly calculated by measuring the amount of liquid upstream or downstream of the reaction chamber by stopping rotation over time. Alternatively, it may be calculated directly or indirectly by stopping the rotation at the time of liquid feeding by the rotation of the analysis chip and measuring the amount of liquid upstream or downstream of the reaction chamber.

  In addition, the time required for the specimen and reagent to pass through the reaction chamber by using a plurality of specimens and a plurality of chips in advance and examining the reaction chamber passage time and the reaction chamber passage amount over time at a specific rotational speed. Can be determined. Based on the time required for passing through the reaction chamber, the protocol and program can be set so that the rotational speed of the analysis chip is switched at the timing before the total amount of the specimen and reagent passes through the reaction chamber.

Reference example 1
An analysis chip was manufactured as follows. FIG. 1 shows a top view and a longitudinal sectional view of the analysis chip A. The analysis chip includes a tubular reaction chamber 1 and a sample / reagent reservoir 2 that is cylindrical and has a throttle shape on the reaction chamber side. The opening 4 of the specimen / reagent reservoir is provided with an ear 3 that protrudes to the outside in order to suspend the analysis chip above the centrifuge tube. Since the reaction chamber is filled with a bead carrier to which a primary antibody is bound, a hole (5) for press-fitting a wire mesh (not shown) and a filter is provided for blocking the carrier.

  First, an analysis chip body was manufactured by cutting a resin made of polymethylmethacrylate (manufactured by Kuraray Co., Ltd.). A stainless steel wire mesh having an opening diameter of about 20 μm was cut into a diameter of about 2 mm and subjected to hydrophobic treatment with Fluorosurf (manufactured by Fluoro Technology). A metal mesh subjected to hydrophobic treatment was inserted from the filter press-fitting hole 5 and placed downstream of the reaction chamber, and then the filter was press-fitted to form a carrier capping portion, thereby producing an analysis chip. A carrier damming portion is formed by press-fitting a metal mesh filter subjected to hydrophobic treatment from the filter press-fitting hole 5.

  Subsequently, the reaction chamber of the analysis chip was filled with a bead carrier.

  Polystyrene beads (Polyscience, particle size: 25 μm) were washed with a phosphate buffer, and the same amount of 30 μg / ml anti-hIL-8 antibody (manufactured by Kamakura Technoscience) / phosphate buffer was added to the polystyrene beads. Shake overnight at 4 ° C. After shaking, 2 μl of polystyrene beads were suspended in 150 μl of a phosphate buffer containing 0.05% Tween 20. After pouring this suspension into the reservoir of the analysis chip, the analysis chip is placed in a centrifuge tube (trade name: Eppendorf tube, manufacturer: Eppendorf, size: 1.5 mL), and centrifuged (trade name: trace amount). High-speed cooling centrifuge, manufacturer name: Tommy Seiko Co., Ltd., model name: MX-300) Centrifugation was performed at 2000 G for 30 seconds, and the support was filled in the reaction chamber.

Reference example 2
To the reservoir of the analysis chip prepared in Reference Example 1, 100 μl of 0.05% Tween 20-containing phosphate buffer was poured, and centrifuged at 4700 rpm (2000 G) for 1 minute to wash the carrier.

  Thereafter, 80 μl of 0 pg / ml and 100 pg / ml hIL-8 (human interleukin-8, Kamakura Technoscience) dissolved in a phosphate buffer containing 1% bovine serum albumin (BSA) was added as a sample solution to the reservoir. The solution was fed at 1800 rpm (300 G) for 10 minutes.

  150 μl of 0.05% Tween 20-containing phosphate buffer was added to the reservoir, and the solution was fed at 4700 rpm (2000 G) for 1 minute for washing.

  80 μl of 0.6 μg / ml HRP (horseradish peroxidase) -labeled anti-hIL-8 antibody (Kamakura Technoscience) dissolved in 1% BSA-containing phosphate buffer is added to the reservoir as a labeled antibody solution, and 1800 rpm (300 G). For 10 minutes.

  150 μl of 0.05% Tween 20-containing phosphate buffer was added to the reservoir, and the solution was fed at 4700 rpm (2000 G) for 1 minute for washing.

  After washing, 100 μl of 0.01M phosphate buffer (pH 7.5) containing 20 μM hydrogen peroxide solution and 13 μg / ml Amplex Red (Molecular Probes) was poured into the reservoir and centrifuged at 4700 rpm (2000 G) for 10 seconds to send the solution. .

  After standing at room temperature for 5 minutes, the analysis chip was taken out from the centrifuge tube, and the amount of resorufin produced in the reaction chamber was measured using a fluorescence microscope IX-71 (Olympus). The measurement results of the fluorescence intensity of hIL-8 at each concentration of 0 pg / ml and 100 pg / ml are shown in FIG. Measurement conditions were an excitation wavelength of 510 to 560 nm, an emission wavelength of 575 to 650 nm, and an exposure time of 10 milliseconds. The analysis protocol (profile) is shown in Table 1.

Comparative Example 1
Using the 12 analysis chips prepared in Reference Example 1, a specimen with an antigen (hIL-8) concentration of 100 pg / ml was measured according to the analysis protocol shown in Table 1. Table 2 shows the average value, standard deviation, and CV value of the fluorescence intensity. The CV value indicating measurement error and variation was 12.7%.

  Each of the 12 analysis chips after the measurement was washed with 150 μl of 0.05% Tween 20-containing phosphate buffer. 80 μl of 1% BSA-containing phosphate buffer was added to the reservoir, and the amount of liquid passing through the reaction chamber every 2 minutes at 1800 rpm (300 G) was measured (FIG. 3). After 2 minutes, it was confirmed that 50% or more of the sample liquid amount passed through the reaction chamber in all 12 analysis chips, and after 3 minutes, all of the 12 analysis chips had 100% of the sample liquid amount. It was read from the results shown in FIG. 3 that less than% passed through the reaction chamber and that most of the chips passed through the reaction chamber less than 90%.

  The correlation between the fluorescence intensity at the time of antigen measurement in each analysis chip and the amount of liquid passing through the reaction chamber after 2 minutes at 1800 rpm (300 G) was confirmed (FIG. 4). The results shown in FIG. 4 indicate that the difference in the reaction chamber passage speed has an influence on the fluorescence intensity, and that the difference in the reaction chamber passage speed causes the measurement error.

Example 1
Using the 12 analysis chips prepared in Reference Example 1, 80 μl of a sample having an antigen (hIL-8) concentration of 100 pg / ml was measured. At this time, when 50% or more of the sample and the amount of the labeled antibody have passed through the reaction chamber, a protocol that increases the rotation speed by about 2.6 times, that is, a protocol that switches the analysis chip rotation speed to 2.6 times is adopted. Then, the reduction of measurement error was examined. Measurements were performed according to the analytical protocol shown in Table 3. When 75% of the sample and labeled antibody solution feeding process has elapsed, the rotational speed of the analysis chip is switched to 2.6 times. Table 4 shows the average value, standard deviation, and CV value of the fluorescence intensity. The CV value indicating measurement error and variation could be reduced to 8.5%.

  Each of the 12 analysis chips after the measurement was washed with 150 μl of 0.05% Tween 20-containing phosphate buffer. 80 μl of the sample solution was added to the reservoir, and the amount of liquid passing through the reaction chamber every 2 minutes at 1800 rpm (300 G) was measured (FIG. 5). After 2 minutes, it was confirmed that 50% or more of the sample liquid volume passed through the reaction chamber in all 12 analysis chips, and after 3 minutes, less than 90% of the sample liquid volume in all 12 analysis chips. 5 was read from the results shown in FIG. 5 that the sample had passed through the reaction chamber, that is, 90% of the amount of the sample liquid was before the passage through the reaction chamber.

  The correlation between the fluorescence intensity at the time of antigen measurement in each analysis chip and the amount of liquid passing through the reaction chamber after 2 minutes at 1800 rpm (300 G) was confirmed (FIG. 6). From the results shown in FIG. 6, it became clear that the difference in the reaction chamber passage speed did not affect the fluorescence intensity, and the measurement error due to the difference in the reaction chamber passage speed could be greatly reduced.

Example 2
In this example, studies were made to reduce measurement errors while suppressing a decrease in fluorescence intensity that affects detection sensitivity.

  Using the 12 analysis chips prepared in Reference Example 1, 80 μl of a sample having an antigen (hIL-8) concentration of 100 pg / ml was measured. At this time, measurement was performed using 6 analysis chips according to the protocol shown in Table 3, and the remaining 6 analysis chips were measured using the protocol shown in Table 5.

  In the protocol shown in Table 5, at the initial stage of feeding by rotation of the specimen and labeled antibody solution, that is, when the specimen and labeled antibody pass through the reaction chamber at 50% or less, that is, 50% of the specimen or labeled antibody solution passes through the reaction chamber. Before this, a method of reducing the rotation speed to about 83%, that is, a protocol for reducing the rotation speed of the analysis chip to about 83% is adopted. Furthermore, when 50% or more of the amount of the sample and the labeled antibody solution has passed through the reaction chamber, a protocol that increases the rotation speed by about 3.1 times, that is, a protocol that switches the rotation speed of the analysis chip to 3.1 times is adopted. ing.

  From the viewpoint of time, a technique is adopted in which the rotational speed is reduced to 83% at the beginning of the liquid feeding by the rotation of the specimen and the labeled antibody solution, that is, at about 9% of the specimen and reagent feeding process. Further, a protocol is adopted that increases the rotational speed by about 3.1 times when about 82% of the liquid feeding process by the rotation of the specimen and the labeled antibody has elapsed.

  Measurements were performed according to the analysis protocol shown in Table 3 and the analysis protocol shown in Table 5. Table 6 shows the average value, standard deviation, and CV value of the fluorescence intensity. Almost the same as in Example 1, the measurement error in the protocol shown in Table 3 was 8.7%. On the other hand, the measurement results using the protocol shown in Table 5 were able to further reduce the CV value indicating the measurement error and variation to 4.3%. This is considered to be an effect of further increasing the increase rate of the rotational speed. Furthermore, the fluorescence intensity was increased by about 1.4 times from 311 to 431. This is considered to be the effect of reducing the rotation speed in the initial stage of feeding the specimen and the labeled antibody.

  Each of the 12 analysis chips after the measurement was washed with 150 μl of 0.05% Tween 20-containing phosphate buffer. 80 μl of the sample solution was added to the reservoir, and the amount of liquid passing through the reaction chamber every 2 minutes at 1500 rpm (200 G) was measured (FIG. 7). After 1500 rpm (200 G) for 4 minutes, 50% of the amount of the sample and the labeled antibody solution passed through the reaction chamber, and 90% was confirmed to be in the state before passing through the reaction chamber.

  The protocol shown in Table 3 and the protocol A which is considered to show the effect of reducing the measurement error are summarized as a graph showing the change over time in the rotational speed when the sample or labeled antibody is fed (FIG. 8A).

  The protocol shown in Table 5 and the protocol B and protocol C which are considered to show the effect of reducing measurement error and improving the signal intensity are summarized as a graph showing the change over time in the rotational speed when the sample or labeled antibody is fed (Fig. 8B).

FIG. 1 is a top view and a cross-sectional view schematically showing an example of an analysis chip used in the present invention. FIG. 2 is a diagram showing the measurement results of Reference Example 2. FIG. 3 is a diagram showing the result of measuring the liquid feeding amount in Comparative Example 1. FIG. 4 is a diagram showing the correlation of Comparative Example 1. FIG. 5 is a diagram showing the results of measuring the liquid feeding amount in Example 1. FIG. 6 is a diagram illustrating the correlation of the first embodiment. FIG. 7 is a diagram showing the results of measuring the amount of liquid fed in Example 2. FIG. 8 is a diagram showing a protocol adopting the measurement error reduction method of the present invention.

Explanation of symbols

1 Reaction Chamber 2 Reservoir 3 Ear 4 Reservoir Opening 5 Filter Press-In Hole A Analysis Chip

Claims (5)

By rotating an analysis chip having a reaction chamber capable of accommodating a carrier bound with an antigen and / or an antibody with respect to a rotating shaft outside the analysis chip, a specimen and a reagent are fed to the reaction chamber, and the reaction chamber In immunological analysis to measure the amount of test substance in
A method for reducing measurement errors in immunological analysis by switching the rotational speed of an analysis chip to twice or more before the entire amount of a specimen or reagent solution passes through a reaction chamber.   The method for reducing a measurement error according to claim 1, wherein the time point at which the rotation speed of the analysis chip is switched to twice or more is a time point when 50% or more of the amount of the sample or reagent solution has passed through the reaction chamber.   The method for reducing measurement error according to claim 1 or 2, wherein the liquid is fed by reducing the rotation speed of the analysis chip to 90% or less before switching the rotation speed of the analysis chip to twice or more.   The method for reducing measurement error according to claim 3, wherein the time point when the rotational speed of the analysis chip is reduced to 90% or less is a time point before 50% of the amount of the sample or reagent solution passes through the reaction chamber. The method for reducing measurement error according to claim 1, wherein the immunological analysis is a two-step method.

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