JP4359416B2 - Measuring method of substance immobilized on microparticle solid phase - Google Patents

Description

【００１９】
【表２】

[0001]
[Technical field to which the invention belongs]
The present invention relates to techniques used in many fields such as human clinical examinations, veterinary examinations, and food hygiene examinations. More specifically, a method for measuring a test substance by mixing a microparticle solid phase on which a test substance containing an enzyme labeling substance for measurement is fixed and a reagent solution for measuring the test substance, and a reagent used for the measurement About the kit.
[0002]
[Prior art]
Conventionally, in many fields such as human clinical examinations, veterinary examinations, food examinations, measurement of various substances such as antigens, antibodies, DNA, physiologically active substances, pathogen-related substances, tumor markers, etc. A complex with a binding substance has been formed, immobilized or trapped on a solid phase, and the complex on the solid phase has been measured. In order to measure the complex on the solid phase, an enzyme, fluorescent substance, luminescent substance, radio, etc. immobilized on the complex through various bonds such as antigen-antibody bond, DNA hybrid bond, biotin-avidin bond, ionic bond, etc. Labeling substances for measurement such as isotopes are widely used. As a solid phase for forming, fixing, or trapping a complex, a microparticle solid phase such as a latex plate or a magnetic bead is used in addition to the inner surface of a microplate, a polyethylene sphere, a glass sphere, or a polystyrene tube. Measurement of the test substance containing the complex on the solid phase and the labeling substance for measurement is performed by contacting the reagent solution for measurement with the solid phase and then measuring the signal corresponding to the type of the labeling substance for measurement. Has been. For example, when the measurement labeling substance is an enzyme, the absorbance, luminescence intensity, fluorescence intensity, magnetic properties of the measurement reagent solution are brought into contact with the measurement reagent solution containing the enzyme substrate and the solid phase on which the enzyme is immobilized. such as resonance degree of Ru is measured. During this, it is necessary for high sensitivity measurements contact that is uniform instantaneously accurate and reproducible with the solid phase analyte is fixed to the measuring reagent solution containing a measuring enzyme-labeled substance. In particular, when the solid phase is a microparticle solid phase, uniform mixing with the measurement reagent solution may be hindered by aggregation of the microparticle solid phase, reducing the accuracy, reproducibility and / or sensitivity of the measurement. Is the problem.
[0003]
[Problems to be solved by the invention]
It is an object to enable measurement with higher accuracy, reproducibility, and / or sensitivity of a test substance containing a measurement enzyme labeling substance immobilized on a fine particle solid phase.
[0004]
[Means for Solving the Problems]
The present invention relates to 1. A measurement method for measuring a signal generated by bringing a solid particle solid phase on which a test substance containing a measurement enzyme labeling substance is immobilized into contact with an enzyme substrate, and comprising a test substance containing the measurement enzyme labeling substance A step of immobilizing on a microparticle solid phase, a step of dispersing a microparticle solid phase in which a test substance containing an enzyme labeling substance for measurement is immobilized, in a dispersion, and a dispersion in which the microparticle solid phase is dispersed And an enhancer-containing solution containing an enhancer, wherein either the dispersion liquid or the enhancer-containing solution contains an enzyme substrate, and the dispersion liquid does not contain an enhancer, or immobilization of a test substance A measurement method characterized in that it does not contain an enhancer at a concentration that interferes with the uniform dispersion of the solid phase of the fine particles .
2. A test substance consists of a complex formed, fixed or trapped on a solid phase, and an enzyme labeling substance for measurement for measuring this complex. The complex consists of an antigen, an antibody, DNA, and a physiologically active substance. 2. The method according to 1 above, comprising any one of a pathogen-related substance and a tumor marker, and a specific binding substance for the substance.
3. 3. The measuring method according to 1 or 2 above, wherein the fine particle solid phase is a magnetic bead having a diameter of 0.1 to 10 μm.
4). 3. The measuring method according to 1 or 2 above, wherein the fine particle solid phase is latex particles having a diameter of 0.1 to 10 μm.
5. 5. The measuring method according to any one of 1 to 4, wherein the enzyme of the enzyme labeling substance for measurement is alkaline phosphatase, peroxidase or β-D-galactosidase.
6). 5. The method according to any one of 1 to 4, wherein the enzyme of the enzyme labeling substance for measurement is alkaline phosphatase or β-D-galactosidase.
7). 7. The measuring method according to any one of 1 to 6, wherein the labeling substance for measurement is an enzyme, and the enhancer-containing solution contains an enzyme substrate.
8). 7. The measuring method according to any one of 1 to 6, wherein the labeling substance for measurement is an enzyme, and the dispersion liquid contains an enzyme substrate.
9. The method according to claim 1, wherein the enzyme substrate is a dioxetane derivative.
10. 10. The measuring method according to any one of 1 to 9, wherein the enhancer is a surfactant.
11. Measurement method according to any one of the 1 to 10 dispersion and wherein the go, such contain enhancer.
12 A dispersion for dispersing a microparticle solid phase on which a test substance containing an enzyme labeling substance for measurement is immobilized, and an enhancer-containing solution that includes an enhancer and is mixed with the dispersion in which the microparticle solid phase is dispersed A reagent kit containing either a dispersion liquid or an enhancer-containing liquid containing an enzyme substrate, and the dispersion liquid does not contain an enhancer, or uniform dispersion of a microparticle solid phase on which a test substance is immobilized A reagent kit for measuring a test substance, which does not contain an enhancer at a concentration that interferes with
Consists of.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
The present invention is applied to a method for measuring a test substance by measuring a signal after contacting a microparticulate solid phase to which a test substance containing an enzyme labeling substance for measurement is fixed and a reagent solution for measurement. Can do.
The solid phase used in the present invention is preferably a fine particulate solid phase having a diameter of 0.1 to 10 μm, and is not limited by the material, shape, manufacturing method, and the like. Examples thereof include commercially available latex particles, preferably magnetic beads, but are not limited thereto.
[0006]
Enzyme-labeled substance for measurement in the present invention is not limited by examples, A alkaline phosphatase, peroxidase, beta-D-galactosidase of earthen others are exemplified their derivatives etc., respectively.
[0007]
How measuring the enzyme-labeled substance is immobilized to the complex of the above fine particulate solid phase, since the fixed state is no limit, measuring the enzyme-labeled substance is fixed to the various conditions in various ways. Although not limited by exemplification, for example, the enzyme labeling substance for measurement is immobilized on a complex on a microparticulate solid phase via various substances and various bonds. That is, the enzyme labeling substance for measurement is antigen, antibody, hormone, pathogen-related substance, tumor marker, avidin, DNA, streptavidin, biotin fragments thereof, derivatives thereof, antigen-antibody bond, ion bond, DNA hybrid bond, It is immobilized on a complex on a microparticulate solid phase via a biotin-avidin bond, a biotin-streptavidin bond, a hapten-anti-hapten antibody bond, or the like. These are substances and bonds used in immunoassays of various substances, DNA measurements and the like.
[0008]
As described above, in order to accurately and reproducibly measure the test substance immobilized on the microparticulate solid phase by various methods, the microparticulate solid phase is contained in the reagent solution for measuring the test substance. It is necessary to be uniformly dispersed without agglomeration. However, the reagent solution for measuring the test substance contains various substances for the test substance measurement. For example, a β-D-galactosidase or alkaline phosphatase measurement reagent solution that uses a dioxetane derivative as a substrate contains a surfactant or a surfactant-like substance as an enhancer. This prevents uniform dispersion of the microparticulate solid phase once accumulated in the measuring reagent solution, thereby hindering measurement of a test substance with high accuracy, reproducibility and / or sensitivity.
[0009]
In the present invention, a fine particulate solid phase in which a test substance containing a measurement enzyme labeling substance is immobilized in a dispersion is uniformly dispersed, and then mixed with a measurement reagent solution for the test substance. The measurement of the test substance with high accuracy, reproducibility and / or sensitivity is performed by attenuating the effect of the substance that prevents the uniform dispersion of the microparticulate solid phase contained in the measurement reagent solution.
The dispersion is not particularly limited as long as the solid phase is uniformly dispersed without agglomeration and there is no hindrance to measurement of a test substance with high accuracy, reproducibility, and / or sensitivity. Known or later-developed solutions, solutions or buffers containing or not containing various substances or substance groups, such as enzyme substrates for measuring test substances, various proteins, lipids, carbohydrates, various sera, etc. Although illustrated, it is not limited to an illustration. There are no restrictions on pH, ionic strength, etc., and the dispersion may be simple water. However, an enhancer that prevents uniform dispersion of the microparticle solid phase to which the test substance is immobilized and interferes with measurement of a test substance with high accuracy, reproducibility, and / or sensitivity, At concentrations that interfere, it is important that no dispersion be present. The method of using the dispersion is not particularly limited as long as the solid phase is dispersed without aggregation, for example, and the test substance can be measured accurately, with good reproducibility and with high sensitivity. The solid phase may be dispersed in the dispersion by any method such as stirring and shaking. The volume of the dispersion is preferably smaller than the volume of the reagent solution for measurement, but may be larger. The temperature at the time of dispersion is usually 20 to 40 ° C., but is not particularly limited.
The measurement reagent solution in the present invention is mixed with the microparticle solid phase dispersed in the above dispersion liquid and measuring the signal for measurement of a test substance with high accuracy, reproducibility and / or sensitivity, The solution to be contacted and thus contains the substance that allows the measurement in a concentration that allows the measurement. Although not limited by illustration, the case where the reagent solution for measurement contains an enzyme substrate and an enhancer is illustrated. However, when an enzyme substrate is already added to the dispersion, the enzyme substrate does not necessarily need to be contained in the measurement reagent solution. That is, the enzyme substrate may be added once or twice or more at any stage. The same applies to the addition of an enhancer, and the concentration of the enzyme substrate and enhancer is not limited, and the number of the reagent for measurement containing the enzyme substrate or / and enhancer and the number of additions are not limited. The fine particle solid phase dispersed in the above dispersion and the measurement reagent solution containing the enhancer may be mixed and further mixed with the measurement reagent solution containing the enzyme substrate. Preferably, the enzyme substrate is added at the same time as the enhancer or before the enhancer.
Signal measurement after dispersing a microparticle solid phase in which a test substance containing an enzyme labeling substance for measurement is immobilized in a dispersion and mixing it with a reagent solution for measurement is performed by a known method or a method developed in the future. Can be implemented. If the concentration of the microparticle solid phase is low and there is no hindrance in the signal measurement, the signal can be measured as it is, but the concentration of the microparticle solid phase is high and it is difficult to measure the signal due to quenching or the like. In this case, the signal can also be measured after accumulating the microparticle solid phase (Japanese Patent Application No. 2001-189419).
[0010]
The present invention includes solid phases, reagents, and the like for carrying out the measurement methods described above, and also exemplified by the solid phases and buffers, blocking solutions, washing solutions, substrate solutions, antibodies, haptens, etc. It also extends to a measurement kit comprising at least one solid phase and / or reagent used in the above. Furthermore, the present invention extends to a measurement system including a solid phase, a reagent, and automation software for carrying out the measurement method described above. Although the above was demonstrated by the illustration, this invention is not limited by these illustrations.
[0011]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.
[0012]
[Example 1]
In this example, a method for measuring ALP luminescence on a magnetic bead on which a test substance containing at least a complex containing human hepatitis B virus surface antigen (HBsAg) and an anti-HBsAg antibody and alkaline phosphatase (ALP) is immobilized . There are, it shows the effect of dispersing the said magnetic beads previously ALP activity measuring reagent solution other than the buffer.
[0013]
(Materials and methods)
・ Magnetic beads MG210 (diameter 1.7μm, specific gravity 1.3)
JSR Corporation, Tokyo, Japan
・ Alkaline phosphatase (hereinafter referred to as ALP)
Oriental Yeat Co. , Ltd., Ltd. , Tokyo, Japan
Monoclonal antibodies Anti-human hepatitis B virus surface antigen (hereinafter referred to as HBsAg) IgG-649 and 85 and anti-2,4-dinitrophenyl group (hereinafter referred to as DNP) IgG-1753
International Reagents Corporation, Kobe, Japan
DNP biotin-anti-HBsAg Fab ′ and ALP-anti-HBsAg Fab ′
DNP biotinylated bovine serum albumin-anti-HBsAg Fab′-649 (DNP-biotin-anti-HBsAg Fab ′) and ALP-anti-HBsAg Fab′-85 (ALP-anti-HBsAg Fab ′) were reacted with maleimide group and thiol group. Known methods to be used (E. Ishikawa, Ultrasensitive and Rapid Enzyme Immunoassay, Laboratory Techniques in Biochemistry, P., p. I., P., et al., Bio. 1999).
Blocking solution 10 mM sodium phosphate buffer, pH 7.0 containing 0.15 M NaCl, 2.5 mM EDTA, 2.5 g / L bovine serum albumin, 10 g / L sucrose, 1 g / L NaN3
Anti-DNPIgG Insolubilized Magnetic Beads Monoclonal anti-DNPIgG-1753 was insolubilized in magnetic beads according to the instructions of JSR Corporation, washed with a blocking solution, and stored in the same solution at 4 ° C.
DNP-biotin-anti-HBsAg Fab ′, ALP-anti-HBsAg Fab ′, HBsAg, anti-DNPIgG insolubilized magnetic beads and streptavidin insolubilized magnetic beads dilution TEA buffer TEA buffer 1 mM MgCl2, 0.1 mM ZnCl2, 1 g / L Bovine serum albumin, 0.1 M triethanolamine / HCl buffer solution containing 0.5 g / L NaN3, pH 7.6
Washing solution for ALP 20 mM Tris-HCl buffer solution containing 0.15 M NaCl, 0.1% Tween 20, 1 g / L NaN3, pH 7.4
Incubation temperature The incubation temperature in all steps of the non-competitive transfer solid phase measurement method was 37 ° C.
Binding of DNP-biotin-anti-HBsAg Fab ′ to anti-DNPIgG insolubilized magnetic beads 100 μmol / mL DNP-biotin-anti-HBsAg Fab ′, 25 μL and 10 mg of anti-DNPIgG insoluble magnetic beads washed with TEA buffer After incubating for a minute, the magnetic beads were separated by magnetic separation for 10 seconds, and the supernatant was removed by aspiration, then washed once by 200 μL of ALP washing solution for 10 seconds and suspended in 80 μL of TEA buffer.
Binding of HBsAg to DNP-biotin-anti-HBsAg Fab′-conjugated anti-DNPIgG insolubilized magnetic beads Incubate 20 μL of 2.5 IU / mL HBsAg and the washed DNP-biotin-anti-HBsAg Fab′-conjugated anti-DNPIgG insoluble magnetic beads for 2 minutes. After that, the magnetic beads were washed once with 200 μL of ALP washing solution by magnetic separation for 10 seconds.
Binding of ALP-anti-HBsAg Fab ′ to HBsAg-bound DNP-biotin-anti-HBsAg Fab′-bound anti-DNP IgG insolubilized magnetic beads 4.8 pmol / mL ALP-anti-HBsAg Fab ′, 100 μL and washed HBsAg-bound DNP- The biotin-anti-HBsAg Fab′-conjugated anti-DNPIgG insolubilized magnetic beads were incubated for 1 minute and then washed once with 200 μL of ALP washing solution for 10 seconds.
Phosphorus buffer solution 0.1 M sodium phosphate buffer solution containing 0.5 g / L NaN3, pH 7.5
Streptavidin Type II, Wako Pure Chemical Industries, Ltd, Osaka, Japan
-TEA buffer solution containing 3 mM εN-2.4-ditrophenyl-L-lysine-DNP-Lys solution-Streptavidin-insolubilized magnetic beads Biotinylated bovine serum albumin was prepared by a known method (E. Ishikawa, Ultrasensitive N Rapid Immunoassay, Immuno Immunoassay). in Biochemistry and Molecular Biology Vol. 27, S. Pillai, P. C van der Vliet eds., Elsevier, Amsterdam, pp. 141-176, 1999), which is insolubilized by JSR Corporation beads. And 30 μg / mL streptavidin in phosphate buffer The reaction was dissolved in the solution, washed once with a phosphate buffer, and stored at 4 ° C. in a blocking solution.
-Elution of immunocomplex from anti-DNPIgG-insolubilized magnetic beads Anti-DNPIgG-insolubilized magnetic beads to which the above-mentioned immune complex consisting of DNP-biotin-anti-HBsAg Fab ′, HBsAg, ALP-anti-HBsAg Fab ′ is bound After incubating 100 μL of the DNP-Lys solution for 0.5 minutes, the supernatant obtained by accumulating the magnetic beads with a magnet for 15 seconds was used as an eluate.
Binding of immune complex to streptavidin-insolubilized magnetic beads 0.5 mg of the above eluate and streptavidin-insolubilized magnetic beads were incubated for 1 minute, and then the magnetic beads were accumulated with a magnet for 10 seconds. After removing the supernatant, ALP The magnetically-poured beads were dispersed in 200 μL of the cleaning solution for washing, and once again collected by accumulating with a magnet for 10 seconds and immediately removing the supernatant.
ALP luminescent substrate solution CDP-star ready to use With Sapphire II, Tropix, Inc. , Bedford, MA
ALP dispersion 1M Dietanolamine / HCl buffer, pH 10.0
-Luminescence measuring device Lumicounter 2500, Microtec Co. Ltd .. , Chiba, Japan
Measurement of ALP activity of ALP-bound magnetic beads Measurement 1: The washed ALP-bound magnetic beads were dispersed in 30 μL of dispersion, mixed with 100 μL of substrate solution, incubated at 37 ° C. for 2.75 minutes, Five cases of the amount of luminescence per 0.1 second of the supernatant obtained by collecting the magnetic beads with a magnet were measured simultaneously.
Measurement 2: The washed ALP-coupled magnetic beads were dispersed in 130 μL of a mixed solution of 30 μL of dispersion and 100 μL of substrate solution, incubated at 37 ° C. for 2.75 minutes, and then the magnetic beads were collected with a magnet. The amount of light emission for 0.1 seconds was measured simultaneously for 5 cases.
[0014]
(result)
The results of Example 1 are shown in Table 1.
The luminescence intensity is 92,892 and the reproducibility is CV2 when ALP-bound magnetic beads are dispersed in a dispersion liquid not containing a substrate and an enhancer and then mixed with a substrate solution containing a substrate and an enhancer to measure ALP activity. 8% (5 cases). When the ALP activity was measured by directly dispersing in a mixed solution of the substrate solution and the dispersion, the luminescence intensity was 63,228 and the reproducibility CV was very high at 19.4%. In other words, the ALP-bound magnetic beads are dispersed in advance in a dispersion containing no enhancer, and then mixed with a measuring reagent solution containing a substrate and an enhancer to measure the luminescence intensity. Both reproducibility was significantly improved as compared with the case where the magnetic beads were directly mixed with a measuring reagent solution containing an enzyme substrate and an enhancer without using a dispersion.
[0015]
[Example 2]
In this embodiment, a method for measuring the emission of ALP for similar magnetic beads as in Example 1, shows the effect of adding an enzyme substrate before the enhancer.
[0016]
(Materials and methods)
The materials and methods described below are the same as those in Example 1.
-Substrate solution A solution containing only CDP-star as a substrate, Tropics.
Enhancer solution A solution containing only Sapphire II as an enhancer, Tropics.
Measurement of ALP activity of ALP-bound magnetic beads Measurement 1: Washed ALP-bound magnetic beads were dispersed in a mixture of 30 μL of dispersion and 90 μL of substrate solution, and 15 seconds later, the ALP luminescent substrate solution added in Example 1 Instead of the substrate CDP-star and the enhancer Sapphire II, 10 μL of an enhancer solution was added, incubated in the same manner as in Example 1, and the luminescence intensity was measured.
Measurement 2: Measured in the same manner as Measurement 1 except that 10 μL of enhancer solution was added with a delay of 2 minutes and 15 seconds from the addition in Measurement 1.
[0017]
(result)
The results of Example 2 are shown in Table 2.
The luminescence intensity was reduced from 121,500 to 79,090 by adding the enhancer later than the addition of the substrate and shortening the time from the addition of the enhancer to the luminescence measurement. This result shows that adding an enhancer as early as possible gives a high signal, that is, high sensitivity, and at the same time indicates that the enzymatic reaction proceeds even in the absence of an enhancer. It has also been shown that a higher signal can be obtained by adding the substrate at an early stage where it is not possible, for example, at the stage of dispersion addition.
[0018]
[Table 1]

[0019]
[Table 2]

Claims (12)

A measurement method for measuring a signal generated by bringing a solid particle solid phase on which a test substance containing a measurement enzyme labeling substance is immobilized into contact with an enzyme substrate, and comprising a test substance containing the measurement enzyme labeling substance A step of immobilizing on a microparticle solid phase, a step of dispersing a microparticle solid phase in which a test substance containing an enzyme labeling substance for measurement is immobilized, in a dispersion, and a dispersion in which the microparticle solid phase is dispersed And an enhancer-containing solution containing an enhancer, wherein either the dispersion liquid or the enhancer-containing solution contains an enzyme substrate, and the dispersion liquid does not contain an enhancer, or immobilization of a test substance A measurement method characterized in that it does not contain an enhancer at a concentration that interferes with the uniform dispersion of the solid phase of the fine particles .   A test substance consists of a complex formed, fixed or trapped on a solid phase, and an enzyme labeling substance for measurement for measuring this complex. The complex consists of antigen, antibody, DNA, physiologically active substance. The method according to claim 1, comprising any one of a pathogen-related substance and a tumor marker, and a specific binding substance for the substance.   The measuring method according to claim 1 or 2, wherein the fine particle solid phase is a magnetic bead having a diameter of 0.1 to 10 µm.   The measuring method according to claim 1 or 2, wherein the fine particle solid phase is latex particles having a diameter of 0.1 to 10 µm.   The method according to any one of claims 1 to 4, wherein the enzyme of the enzyme labeling substance for measurement is alkaline phosphatase, peroxidase or β-D-galactosidase.   The measurement method according to any one of claims 1 to 4, wherein the enzyme of the enzyme labeling substance for measurement is alkaline phosphatase or β-D-galactosidase.   The measurement method according to claim 1, wherein the enhancer-containing solution contains an enzyme substrate.   The measurement method according to claim 1, wherein the dispersion contains an enzyme substrate.   The method according to claim 1, wherein the enzyme substrate is a dioxetane derivative.   The measuring method according to any one of claims 1 to 9, wherein the enhancer is a surfactant. Measurement method according to any one of claims 1 to 10 dispersion and wherein the go, such contain enhancer. A dispersion for dispersing a microparticle solid phase on which a test substance containing an enzyme labeling substance for measurement is immobilized, and an enhancer-containing solution that includes an enhancer and is mixed with the dispersion in which the microparticle solid phase is dispersed A reagent kit containing either a dispersion liquid or an enhancer-containing liquid containing an enzyme substrate, and the dispersion liquid does not contain an enhancer, or uniform dispersion of a microparticle solid phase on which a test substance is immobilized A reagent kit for measuring a test substance, which does not contain an enhancer at a concentration that interferes with