JP2002238544A - Method for measuring substance immobilized on solid phase by light emission - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the measurement of a specimen immobilized on a solid phase by a light-emission method without being limited by the kind, shape, size, quantity and immobilization method of the solid phase. SOLUTION: This method for measuring the specimen comprises bringing a light-emitting reagent solution into contact with a solid phase on which the specimen is immobilized, separating the product from the solid phase and then measuring the emission intensity of the separated product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a test substance by luminescence measurement, and more particularly, to measurement of luminescence intensity of a luminescent substance produced in the presence of a test substance immobilized on a solid phase. Including the new measurement method of the test substance when performing the measurement, the solid phase for performing the measurement method, the reagent, and the measurement system including the automation software, and the reagent and / or the solid phase used for performing the measurement method The present invention relates to a reagent kit for measurement.

[0002]

2. Description of the Related Art Conventionally, when a test substance immobilized on a solid phase is measured by luminescence, the luminescence intensity has been measured as it is by bringing the solid phase into contact with a luminescent reagent solution. That is, the solid phase on which the test substance was immobilized had to be suitable for luminescence measurement. For example, when an antigen, an antibody, a hapten, a receptor, DNA, etc. are measured by a solid phase method and a luminescence method, a solid phase suitable for luminescence measurement has to be selected in advance. But the antigen,
Solid phase suitable for solid phase immunoassay (detection) for measuring or detecting antibodies, haptens, etc., solid phase DNA measurement (detection) for measuring or detecting DNA, etc. and solid phase suitable for luminescence measurement , Material, shape, size, transparency, and the like. If the shape, size, and the like are different, it cannot be mounted on the luminescence measuring device, and thus cannot be measured. In addition, it is often extremely difficult to modify the luminescence measuring device.

[0003] One of the typical examples of the solid phase, which is suitable and widely used for the solid-phase immunoassay (detection) method but is not suitable for the luminescence immunoassay, is a fine-particle solid phase such as magnetic beads and latex. . The particulate solid phase is suitable for performing an immune reaction quickly, but is not suitable for luminescence measurement. That is, the light is scattered and / or absorbed by the fine particles and quenching occurs, and thus is not suitable for measuring the emission intensity. Therefore, when the measurement is performed by the solid-phase immunoassay (detection) method and the luminescence measurement method, the amount of fine particles used is significantly limited.

[0004] In order to solve such a disadvantage, the following technique can be used. The first is a technique for separating a substance immobilized on a solid phase through antigen-antibody binding from the solid phase using an acid (E. Ishikawa, Ultrasensitive and
Rapid Enzyme Immunoassay (Laboratory Techniques in
Biochemistry and Molecular Biology Vol. 27), S. Pil
lai, PC van der Vliet eds., pp. 193-210, Elsevie
r, Amsterdam 1999) Secondly, there is a technique for separating a substance immobilized on a solid phase from a solid phase by an excess of hapten through binding of a hapten and an anti-hapten antibody (E. Ishikawa, Ultrasensitive an).
d Rapid Enzyme Immunoassay, p. 79-210) Third, there is a technique for separating a substance immobilized on a solid phase by physical adsorption from the solid phase using a surfactant (S. Ishikaw).
a et al., Anal.Lett., Vol. 33, No. 11 p.2183-219
6, 2000). Fourth, there is a technique for separating a substance immobilized on a solid phase through an ionic bond from the solid phase by ion conversion (K. Nakamura et al., Anal, Chem., Vol. 70, p.
954-957, 1998). Fifth, there is a technique for separating a substance immobilized on a solid phase through a -SS- bond from the solid phase using a reducing agent (see E. Ishikawa, Ultrasensitive and Ra.
pid Enzyme Immunoassay P. 79-210). Sixth, -CH
This is a technique for separating a substance immobilized on a solid phase through an OH-CHOH-bond from the solid phase using periodic acid (E. Ishikawa, Ultrasensitive and Rapid Enzyme Immu).
noassay p. 223-248). Seventh, there is a technique for separating a substance immobilized on a solid phase by antigen, antibody binding or / and DNA hybridization from the solid phase by ultrasonic waves (Title of Invention: Sample measurement method; Japanese Patent Application No. 2000-2000). 22765
3). Eighth, there is a technique for separating a substance immobilized on a solid phase by DNA hybridization from the solid phase at a high temperature.

While these techniques are useful for the above purposes, each has its own drawbacks. In the first and eighth techniques, the test substance may be deactivated. In the third technique, the efficiency of separation is not always high. The seventh technique requires a separate ultrasonic generator. In other techniques, it is necessary to use a specific technique for immobilization on a solid phase in advance.

[0006]

An object of the present invention is to provide a method for measuring a test substance based on the amount of a luminescent substance produced in the presence of the test substance immobilized on a solid phase. Provided is a method that allows a test substance immobilized on a solid phase to be measured by luminescence without being limited by the type, shape, size, amount, immobilization method, etc. of the immobilized solid phase. The purpose is to:

[0007]

Means for Solving the Problems The present inventors contact a solid phase on which a test substance is immobilized and a luminescent reagent solution that produces a luminescent substance in the presence of the test substance, and then separate the two. As a result, they have found that the latter emission intensity can be measured, and have completed the present invention.

That is, the present invention provides: In a method for measuring a test substance based on the amount of a luminescent substance produced in the presence of a test substance immobilized on a solid phase, the method comprises the steps of: 1. A method for measuring a test substance, which comprises contacting a luminescent reagent solution that produces a substance, and then measuring the luminescence intensity of the luminescent reagent solution separated from the solid phase. 2. The measurement method according to the above item 1, wherein the test substance is a hydrolase; 3. The measuring method according to the above 1, wherein the test substance is alkaline phosphatase; 4. The method according to the above 1, wherein the test substance is β-D-galactosidase; 5. The measuring method according to any one of the above items 1 to 4, wherein the luminescent substrate that produces a luminescent substance is a dioxetane derivative; 6. a measurement system including a solid phase, a reagent, and automation software for performing the measurement method according to any one of the above items 1 to 5, It comprises a reagent kit for measurement containing at least one or more reagents and / or a solid phase used for carrying out the measurement method according to any one of the preceding items 1 to 5.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be described below. The method provided by the present invention can be applied to the case where a test substance is measured based on the amount of a luminescent substance produced in the presence of the test substance immobilized on a solid phase.

The test substance in the present invention refers to a substance that produces a luminescent substance or a substance that controls the production of a luminescent substance in a luminescent reagent solution, and the test substance can be immobilized on a solid phase. There is no particular limitation on the type of solid phase, immobilization method, purpose of immobilization, and the like. As the test substance, for example, substances contained in biological components such as saliva, pancreatic juice, blood, serum, and urine, substances contained in cell culture, cell lysate, culture medium, and the like are measured by a solid phase immunoassay. In this case, an antigen, an antibody, avidin, streptavidin, a labeling substance indirectly immobilized on a solid phase via biotin and the like, and a hydrolase, more specifically, alkaline phosphatase, β-D-galactosidase and the like Enzymes, inhibitors of these enzymes, activators and the like.

The method of immobilizing a test substance on a solid phase can be carried out, for example, by various methods using conventional techniques for immunoassay (E. Ishikawa, Ultrasensitive and
Rapid Enzyme Immunoassay (Laboratory Techniques in
Biochemistry and Molecular Biology Vol. 27), S. P
illai, PC van der Vliet eds., pp. 17-221, Elsev
ier, Amsterdam 1999).

In addition, for detection of nucleic acids, especially DNA, DN
A labeling substance immobilized on a solid phase by A hybridization, a substance immobilized on a solid phase directly by physical adsorption or covalent bonding, and the like can also be measured in the present invention.
It goes without saying that a substance immobilized by one or a combination of two or more of immunological reaction, biotin, avidin binding, DNA hybridization, receptor, ligand binding and the like can also be measured.

The solid phase used in the measurement method of the present invention comprises:
It also includes solid phases conventionally used in immunoassays, DNA assays, and other solid phases, and is not limited by the type, shape, size, amount, etc. of the solid phase. For example,
Any solid phase can be used, such as polystyrene balls, nylon balls, polystyrene tubes, magnetic beads, latex, agarose, glass balls, glass powder, ferrite, cellulose powder, cellulose sheets, polystyrene sticks, and polystyrene microplates. Regardless of whether or not it is suitable for luminescence measurement,
It is a feature of the present invention that it can be used.

According to the technique of the present invention, a luminescent reagent solution is brought into contact with a solid phase on which a test substance is immobilized, then separated from the solid phase, and the luminescence intensity is measured. It is a feature of the present invention that a method of dissociating the test substance from the solid phase is not used, but the test substance may be dissociated from the solid phase by using a luminescent reagent solution.

The luminescent reagent solution according to the present invention is used to produce a luminescent substance in the solution or to control the production of the luminescent substance by contact with a test substance immobilized on a solid phase. This refers to a solution that allows measurement of a test substance based on the amount of luminescent substance produced. When the test substance is an enzyme, a solution containing a luminescent substrate from which a luminescent substance is produced by the action of an enzyme, that is, a luminescent substrate solution is a luminescent reagent solution. For example, if the test substance is alkaline phosphatase (AL
In the case of P), a dioxetane derivative (for example, 2-chloro-5- (4-methoxyspiro {1,2-dioxetane)
-3,2 '-(5'-chloro) -tricyclo [3,3,1,1,3,7,] decan} -4
A luminescent substrate solution containing (-yl) -1-phenylphosphate) becomes a luminescent reagent solution. A working solution of this compound is Tropix
It is commercially available as CDP-Star solution from the company. A practical solution of this analogous compound is also commercially available from Tropix as a reagent solution for luminescence of ALP and β-D-galactosidase (I. Bronstein, et.al., Journal of Bioluminescenc.
e and Chemiluminescence, Vol. 4, 99-111, (1989)). From these compounds, a luminescent substance is produced by hydrolysis activity such as ALP and β-D-galactosidase.
The amount of the test substance can be known from the amount of the produced luminescent substance. When the test substance is a control substance for these enzymes, the production of the luminescent substance is controlled by adding a solution of the above compound or a similar compound as a luminescence reagent solution to the mixture of the test substance and the enzyme. The amount of the test substance can be known from the amount of the produced luminescent substance.

The measurement of luminescence in the present invention can be performed by various commercially available luminescence measuring devices. As a measuring device, for example, MLX microtiter luminescence measuring system (Dynex Technologies, Inc.), Lumi counter 25
00 (Microtech Nithion) and the like. There is no limitation on the stage of measuring the emission intensity, the measurement time, the number of measurements, the measurement method, and the like.

The amount of the luminescent reagent solution to be used is not limited. However, the use of a sufficient amount to cover all or almost all of the solid phase to which the test substance is fixed is facilitated. The temperature at which the luminescence reagent solution is brought into contact with the solid phase is not limited,
A temperature optimal for luminescence measurement is preferred.

It is desirable that the luminescent reagent solution is brought into uniform contact with the solid phase, and shaking and stirring may be performed, but the method is not limited.

There is no limitation on the time from contact of the luminescence reagent solution with the solid phase until separation from the solid phase. However, if the lapse of time during which the luminescence intensity changes after the contact is confirmed in advance, Based on that, it is easy to select and implement the time from contact to separation.

The method for separating the luminescent reagent solution from the solid phase is not limited, and various methods can be used depending on the type, shape, size, etc. of the solid phase. When the solid phase is a tube type, a ball type, or the like, the luminescent reagent solution can be simply sucked by a pipette or the like and separated from the solid phase. When the solid phase is magnetic beads, it can be separated by adsorption with a magnet, centrifugation, filtration, or the like. In the case of latex, it can be separated by centrifugation, filtration and the like. In order to efficiently separate the luminescent substance from the solid phase, an appropriate solvent or solution may be added, and the separation operation may be performed after stirring. When the test substance is a substance that controls the production of a luminescent substance, it is necessary to stop the production of the luminescent substance before separating the luminescence reagent solution from the solid phase. For example, if the test substance is an alkaline phosphatase inhibitor, the ED
It is advisable to stop production of the luminescent substance by adding TA or the like, and then to separate the luminescent reagent solution from the solid phase.

The luminescence reagent solution separated from the solid phase is transferred to a tube, cell, or the like that is compatible with the luminescence measurement device.
Perform luminescence measurements. When the solid phase is a magnetic bead or a fine particle solid phase such as latex, if the solid phase and the reagent solution for luminescence are separated in a tube, cell, or the like adapted to the luminescence measuring device,
The measurement operation is simplified. Since the luminescence intensity of the luminescence reagent solution separated from the solid phase to which the test substance is fixed decreases with the passage of time, it is preferable to quickly perform the operation from separation to measurement of the luminescence intensity. In addition, it is useful to check in advance how much time the luminescence intensity of the luminescence reagent solution separated from the solid phase is reduced. The amount of the luminescence reagent solution separated from the solid phase used for measuring the luminescence intensity is not limited, and may be a part or all of the amount.

When the amount of the luminescence reagent solution to be brought into contact with the solid phase is reduced, the nonspecific luminescence of the luminescence reagent solution is reduced, so that the measurement sensitivity can be increased. In this case, in order to sufficiently increase the luminescence intensity after the contact, it is necessary to perform an operation such that a sufficient amount of the luminescent substrate or the like is secured on the entire surface of the solid phase to which the test substance is fixed. For example, when the solid phase is a polystyrene ball, a small amount of a luminescence reagent solution is placed in a test tube, and the polystyrene ball is randomly rotated in the test tube to constantly supply a luminescent substrate or the like to the entire surface of the polystyrene ball. be able to. (The above
E. Ishikawa, Ultrasensitive and Rapid Enzyme Immun
oassay, p. 141-176, Elsevier, Amsterdam 1999).

Further, after the solid phase is brought into contact with a sufficient amount of the reagent solution for luminescence, the minimum amount of the reagent solution for luminescence necessary for the measurement of a trace amount of the test substance is left, and the residue is removed to produce the product. The luminescent substance can be collected in a small amount of a solvent or a solution with a small amount of nonspecific emission (Title of Invention: Method for measuring substance fixed on solid phase; Japanese Patent Application No. 2000-345803). Then,
The emission intensity can be measured by the technique of the present invention. That is, a solution or a solvent in which non-specific luminescence is reduced and the luminescent substance is collected can be separated from the solid phase, and the luminescence intensity can be measured. This further improves the measurement sensitivity.

The present invention provides a solid phase used in the above-mentioned measuring method,
It contains a measurement reagent. Specific examples include a solid phase for immobilizing a test substance, various buffers, a labeling compound, and a luminescence measurement reagent. In addition, among these measurement reagents,
A measurement reagent kit including one or more measurement reagents is also included in the present invention. Further, the method of the present invention can be applied to a measurement system including automation software.

[0025]

EXAMPLES Examples of the present invention will be described below, but the present invention is not limited by these contents.

[0026]

Example 1 In this example, after the alkaline phosphatase (ALP) immobilized on the microplate well was brought into contact with the reagent solution for luminescence, the reagent solution for luminescence was transferred to the untreated microplate well and the luminescence intensity was changed. This indicates that the measurement can be performed.

(Materials) Phosphate buffer 0.1 M sodium phosphate buffer containing 0.1% NaN ₃ , pH 7.5 Microplate Fluoro Nunc module plate, C8 Maxisorp white,
Nunc Co., Ltd.-Eldia F washing solution-Kokusai Reagent Co., Ltd.-Streptavidin Type II, manufactured by Wako Pure Chemical Industries, Ltd.-BSA blocking solution 0.15 M NaCl, 2.5 mM EDTA, 2.5 g / L bovine serum albumin
(BSA), 10 mM sodium phosphate buffer pH 7.0 containing 10 g / L sucroseTEA buffer 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 1 g / L bovine serum albumin (BS
A), 0.1 M triethanolamine HCl buffer containing 0.5 g / L NaN ₃ pH 7.6 • Biotinyl • ALP Biotinyl • Alkaline phosphatase, manufactured by BIEARS • Washing solution for ALP 20 mM Tris • HCl buffer containing 0.1% Tween 20, 0.15 M NaCl Solution pH 7.4 ・ Reagent solution for luminescence CDP-star with Sapphire II, manufactured by Tropics ・ Luminescence measuring device Luminometer MLX for microplate, manufactured by Dynex Technologies

(Preparation of Biotinyl Bovine Serum Albumin) A known method utilizing the reaction between a maleimide group and a thiol group (E. Ishikawa, Ultrasensitive and Rapid Enzyme)
Immunoassay, Laboratory Techniques in Biochemistr
y and Molecular Biology Vol. 27, S. Pillai, PC v
an der Vliet eds., pp. 177-191, Elsevier, Amsterdam
1999).

(Preparation of streptavidin-insoluble microplate well) 100 μL of 30 μg / mL biotinyl / bovine serum albumin dissolved in phosphate buffer was dispensed into microplate wells, and the mixture was allowed to stand at 4 ° C. overnight. After washing three times with an Eldia F washing solution, 100 μL of 30 μg / mL streptavidin dissolved in a phosphate buffer was dispensed.
After standing at 4 ° C overnight, wash 3 times with Elgia F washing solution and BSA
300 μL of the blocking solution was dispensed and stored at 4 ° C.

(Binding of biotinyl-ALP to streptavidin-insoluble microplate well) 1.9 fmol / mL biotinyl-A dissolved in TEA buffer or the same solution
100 μL of LP was dispensed into the streptavidin-insolubilized microplate wells and shaken at room temperature for 40 minutes. Thereafter, the wells were washed three times with 300 μL of a washing solution for ALP.

(Measurement of Luminescence) 200 μL of a luminescence reagent solution was added to the washed microplate well to which the biotinyl-ALP was bound, and the plate was allowed to stand at 37 ° C. for 10 minutes. The luminescence was measured by transferring to a well.

(Results) The results of Example 1 are shown in Table 1.
As a result, even when the reagent solution for luminescence was transferred, the luminescence intensity of 67% of the case where the transfer was not performed could be measured, and the luminescence intensity did not decrease rapidly.

[0033] The numbers in parentheses indicate%.

[0034]

Example 2 In this example, the effect of the present invention is shown on the luminescence measurement of the activity of alkaline phosphatase (ALP) bound to magnetic beads.

Except for the following materials and methods, it is the same as Example 1.・ Magnetic beads manufactured by JSR, MG205, diameter 880nm, specific gravity 1.3 ・ Luminometer L2500, Microtechnion, Inc. Thereafter, 30 μg / mL of streptavidin was dissolved in a phosphate buffer and reacted, washed with the phosphate buffer, and stored at 4 ° C. in a BSA blocking solution.・ Binding of biotinyl ・ ALP to magnetic beads insolubilized with streptavidin 9.5 × 10 ^-17 mol / mL biotinil dissolved in TEA buffer
ALP 100μL and streptavidin-insolubilized magnetic beads 0.
5 mg were mixed and stirred at room temperature for 30 minutes. Thereafter, the magnetic beads that were magnetically separated were washed three times with 400 μL of the ALP washing solution.・ Measurement of ALP 200 μL of a luminescence reagent solution was added to the biotinyl / ALP-bound streptavidin-insoluble magnetic beads, and the mixture was incubated at room temperature for 5 minutes.
After incubation for 5 minutes, the magnetic beads were magnetically separated and removed for 30 seconds, and the luminescence intensity of the supernatant was measured. The emission intensity before removing the magnetic beads was also measured.

(Results) The results of Example 2 are shown in Table 2.
As a result, when ALP bound to the magnetic beads and the reagent solution for luminescence were mixed and the luminescence intensity was measured, an extremely low measured value was observed. However, when the luminescence intensity was measured with the magnetic beads separated, a value of 5.7 times was obtained. was gotten. In other words, the interference of the emission intensity measurement due to the quenching of the magnetic beads could be eliminated by removing the magnetic beads, and the effect of the present invention was shown.

[0037]

[0038]

According to the method for measuring a test substance based on the amount of a luminescent substance produced in the presence of the test substance immobilized on a solid phase, the method comprises the steps of: After contacting a luminescent reagent solution that produces a luminescent substance in the presence, by measuring the luminescence intensity of the luminescent reagent solution separated from the solid phase, the type, shape, size, amount, Luminescence measurement became possible without being limited by the immobilization method and the like, and a highly sensitive test substance measurement became possible.

Claims (7)

[Claims] 1. A method for measuring a test substance based on the amount of a luminescent substance produced in the presence of the test substance immobilized on a solid phase, the method comprising the steps of: A method for measuring a test substance, comprising: contacting a luminescent reagent solution that produces a luminescent substance in the presence of, and measuring the luminescence intensity of the luminescent reagent solution separated from the solid phase. 2. The method according to claim 1, wherein the test substance is a hydrolase. 3. The method according to claim 1, wherein the test substance is alkaline phosphatase. 4. The method according to claim 1, wherein the test substance is β-D-galactosidase. 5. The method according to claim 1, wherein the luminescent substrate producing the luminescent substance is a dioxetane derivative. 6. A measurement system comprising a solid phase, a reagent, and automation software for performing the measurement method according to claim 1. 7. A measuring reagent kit containing at least one or more reagents and / or a solid phase used for carrying out the measuring method according to any one of claims 1 to 5.