JP3365517B2 - Method for detecting an analyte by chemiluminescence and composition therefor - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting an analyte such as a nucleic acid or protein in a sample by chemiluminescence, and a composition therefor.

[0002]

2. Description of the Related Art In recent years, a technique for detecting a nucleic acid fragment having a specific sequence using a probe, for example, Southern blotting, Northern blotting, colony hybridization, plaque hybridization, etc. It has come to be widely used as an effective means for diagnosing cancer, infectious diseases and the like. Further, the Western blotting method using an antibody, ELISA, immunoassay and the like can similarly detect the antigen (protein). In these detection methods, ³² P and ³⁵ S are conventionally used.
^{Alternatively} , radioactive substances such as ¹²⁵ I have been used as labels. Since these radioactive substances can be integrated over a long period of time, detection with extremely high sensitivity is possible. However, considering the danger to the human body and environment, a labeling method that does not use radioactive isotopes is desirable. Currently, the method of labeling enzymes such as peroxidase and alkaline phosphatase is non-isotop
In addition to being ic, it has come to be used in many laboratories with the development of various analyzers. The colorimetric measurement method is generally the mainstream for measuring these enzyme activities, but there is a problem that the sensitivity is lower than that of the conventional method using a radioisotope. So, in recent years, from the colorimetric measurement method to the fluorescence measurement method
Alternatively, luminescence measurement methods are becoming popular. As one of the most sensitive and convenient methods, stable 1,2
-A method of using a dioxetane compound and measuring the luminescence caused by its decomposition (Japanese Patent Laid-Open No. 63-937).
77 publication). This method is considered to be a very excellent method because 1,2-dioxetane is very stable, the detection sensitivity is high, the reaction time is short, and the emission time is long.

On the other hand, when the target nucleic acid or protein is detected by the labeled probe or the labeled antibody using the solid phase, the non-specific reaction of the substance other than the target nucleic acid or protein with the solid phase is noisy. Is a serious problem that increases measurement sensitivity and decreases measurement sensitivity. In order to avoid this, it was essential to add a surfactant to the reaction system. In the method using a nucleic acid probe as the surfactant, SDS (sodium lauryl sulfate) (WO90 / 10718, etc.),
SLS (N-lauroyl sarcosine sodium) (Clin
ical Chemistry, 35, 6, 1989) and the like are common, and Tween-20, Triton-X100 and the like are common in the immunoreaction. However, when the present inventors have proceeded with the study of high-sensitivity detection using 1,2-dioxetane, it was revealed that some of these surfactants inhibit luminescence due to the decomposition of 1,2-dioxetane. Became. SDS
Is a typical example. SDS was originally known as an enzyme inhibitor, and when an enzyme was used as a label, the decrease in luminescence was thought to be due to a decrease in enzyme activity. At the same time, when the enzyme activity was measured by a coloring method using a substrate, the activity was It was clear that SDS inhibited chemiluminescence because no decrease was observed. A surfactant that prevents non-specific reactions is added to the reaction solution during the step of fixing the sample to the solid phase, the pretreatment of the solid phase in the previous step, or the step of reacting the labeled probe or labeled antibody with the sample. It is effective to do. However, at that time, it is considered that the surfactant was adsorbed on the solid phase and inhibited the luminescence reaction, resulting in a decrease in detection sensitivity.

[0004]

SUMMARY OF THE INVENTION The object of the present invention is to use a labeled ligand to detect an analyte in a sample with high sensitivity, instead of a surfactant that causes chemiluminescence inhibition, instead of a non-specific It enables highly sensitive detection using a composition that prevents a reaction.

[0005]

[Means for Solving the Problems] As a result of intensive studies to solve these problems, the inventors of the present invention prevent nonspecific reactions as well as general surfactants and do not inhibit luminescence reactions. It was found that diiodosalicylic acid or a salt thereof is useful as the substance.

That is, according to the present invention, an analyte in a sample immobilized on a solid phase is reacted with a labeled ligand that specifically binds to the analyte, and the label of the reacted ligand is measured by chemiluminescence. In the method for detecting an analyte contained in 1., a reagent containing diiodosalicylic acid or a salt thereof is used, which is a method for detecting an analyte by chemiluminescence.

In the present invention, the diluted solution of the labeled ligand and / or the reaction solution of the ligand and the analyte, and / or the reaction solution for treating the solid phase to prevent nonspecific reaction are diiodosalicylic acid or its A composition for detecting an analyte by chemiluminescence, which is a reagent containing a salt.

In the present invention, the analyte is an antigen or an antibody in the immunoassay utilizing the antigen-antibody reaction, and in the nucleic acid probe measurement utilizing the complementarity of nucleic acids,
It is DNA or RNA. The solid phase refers to a substance that binds an analyte, and is selected from microtiter plates, latex particles, magnetic particles, nylon membranes, nitrocellulose membranes and the like. The bonding method is physical adsorption, covalent bonding via an amino group or a carboxyl group,
A method of immobilizing the analyte directly on the solid phase such as binding by cross-linking, or a method of immobilizing a ligand that specifically binds to the analyte on the solid phase and immobilizing the ligand via the ligand can be considered. Then, the immobilization of the sample is direct binding to the solid phase by physical adsorption, binding through a functional group, binding by cross-linking or the like, or specifically binds to the analyte immobilized on the solid phase. It is the binding that takes place via the ligand.

As the label, an enzyme that catalyzes a reaction such that a decomposition product of the 1,2-dioxetane compound can be used as a substrate, such as alkaline phosphatase, β-galactosidase, and allyl esterase (T
etrahedron Letters; 28,935,1987). It is also possible to directly label the 1,2-dioxetane compound.

The 1,2-dioxetane of the present invention is a compound represented by the following formula.

[0011]

[Chemical 1] [In the formula, R ₂ is an aryl group substituted with an OX group, X is a group that easily reacts with an enzyme, and R _1, R _{3 and} R ₄ are each a passive organic group that allows the generation of light. It is a sexual group. Further, in the formula, the groups R ₁ and R ₂ may be bonded to each other by a spiro bond, and the groups R ₃ and R ₄ may be bonded to each other to form a cyclic group. ]

A specific compound is a compound represented by the following formula.

[Chemical 2] [In the formula, R is a lower alkyl group having 1 to 8 carbon atoms, and X is a group easily reactive with an enzyme. ] As a more specific compound, for example, Lumiphos 480 (manufactured by Wako Pure Chemical Industries, Ltd.) (4-Methoxy-4 (3-phosphate phenyl) spiro [1,
2-dioxetane-3,2'-adamantane] disodium salt and the like.

These compounds are disclosed in JP-A-63-93777.
It is manufactured by the method described in JP-A No. 2-72424. The chemiluminescence can be measured using a general luminometer (Dynatech, Microlite 1000, etc.). It is also possible to print on a polaroid film, an X-ray film or the like.

The method for detecting an analyte of the present invention is performed by any of the following methods. a) The analyte is directly bound to the solid phase, the analyte and the labeled ligand are reacted, and the unreacted labeled ligand or the nonspecifically reacted labeled ligand is removed by washing, and the solid phase The label that binds to is emitted and the amount of emitted light is measured. b) A ligand that specifically binds to the analyte is bound to the solid phase to capture the labeled analyte, and the labeled analyte that has not reacted with the ligand or the labeled analyte that has reacted nonspecifically. The analyte is removed by washing, the label bound to the solid phase is allowed to emit light, and the amount of emitted light is measured. c) A ligand that specifically binds to the analyte is bound to the solid phase, the analyte reacts with the ligand, and the labeled ligand reacts with the analyte, and the unreacted labeled ligand or non-specific The labeled ligand that has been chemically reacted is removed by washing, and the label bound to the solid phase is made to emit light,
The amount of light emission is measured.

Alternatively, the analyte may be reacted with the labeled ligand and then with the ligand bound to the solid phase. Furthermore, the reaction between the ligand bound to the solid phase and the analyte, and the analyte and the enzyme-labeled ligand can be performed simultaneously. The ligand bound to the solid phase specifically binds to a site that specifically binds to the analyte and a site different from the site that specifically binds to the labeled ligand.

In the present invention, in order to prevent non-specific binding of the labeled ligand and the analyte, in a), the analyte is bound to the solid phase and then treated with an inactivating agent. b),
In c), the ligand is bound to the solid phase and then the solid phase is treated with an inactivating agent. Further, in a), in the reaction solution used when reacting the labeled ligand, in b) and c) when the analyte is reacted and / or when the labeled ligand is reacted, and / or the labeled ligand At the time of reacting with, a deactivator for preventing non-specific reaction can be added to the reaction solution used. Here, the inactivating agent is diiodosalicylic acid or a salt thereof, and examples of the salt include sodium salt, potassium salt, magnesium salt, calcium salt and the like, and the concentration thereof is 0.1 to 10%, preferably 1 to 10. 2%.

The reagent containing diiodosalicylic acid or a salt thereof is used as a diluent for the labeled ligand and / or a reaction solution between the ligand and the solid phase and / or a reaction solution for treating the solid phase to prevent nonspecific reaction. use.

[0018]

Industrial Applicability According to the present invention, non-specific adsorption can be prevented without inhibiting luminescence due to the decomposition of 1,2-dioxetane, and an analyte, nucleic acid or protein in a sample can be accurately detected. You can

[0019]

EXAMPLES The present invention will be further clarified by exemplifying Examples of the present invention and Comparative Examples below. still,
In the examples, “n × SSC” represents a (20 / n) -fold diluted solution of a 0.3M sodium citrate and 3.0M NaCl mixture.

Reference Example 1 [Preparation of probe for detecting Vibrio parahaemolyticus] A labeled probe was a DNA synthesizer 391 (Applied Biosystems).
Was synthesized by the phosphoamidide method. The base sequence is 5-CCCCGGTTCTGAXAGATATTGTT-3. X in the array
Represents a uridine having a linker arm at the 5-position. This probe is described in JP-A-4-20299.

Reference Example 2 [Enzyme Labeling of Probe] The binding of the probe obtained in Reference Example 1 and its alkaline phosphatase via the linker arm was analyzed by literature (Nucleic Acids Research). , 14,6155,1
968). Linker oligonucleotide (labeled probe) 1.0 A ₂₆₀ was dissolved in 60 μl of 0.2 MH ₃ BO ₃ and then disuccinimidyl suberate (DDS) 1.25 was added.
After adding mg, the mixture was reacted at room temperature for 2 minutes. Add 1 mM CH ₃ C to the reaction mixture.
The excess DSS was removed by gel filtration through a Sephadex G-25 (Pharmacia) column (1 cm φ × 30 cm) equilibrated with OONa (pH 5.0). An alkaline phosphatase-labeled nucleic acid probe was obtained by further reacting the linker oligonucleotide in which the terminal amino group was activated with a two-fold equimolar amount of alkaline phosphatase (Boehringer Mannheim) at room temperature for 16 hours.

Reference Example 3 [Biotinylation of probe] The probe obtained in Reference Example 1 was biotinylated by the following method. Probe (500 μg / m
l) 112.5 μl, 1M Tris-HCl buffer (pH 7.6
) 37.5 μl, 15 mM NHS-biotin (Pierce) 150 μm
l was put in an Eppendorf tube, reacted overnight at room temperature, and then purified by gel filtration with Sephadex G-25 (Pharmacia).

Reference Example 4 [Preparation of Vibrio parahaemolyticus gene] A TDH-producing Vibrio parahaemolyticus strain was inoculated into Brain Heart Infusion Agar containing 3% NaCl and cultured at 37 ° C overnight. Each grown colony is scraped into a 1.5 ml Eppendorf tube and diluted with a dilution buffer (0.1 MN).
aH ₂ PO ₄ , pH 7.0) was suspended in 300 μl. Further to this, Proteinase K (Nacalai Tesque) 0.6 mg, lysate (8M
Urea, 0.25% sodium dodecyl sulfate, 0.25% sodium lauryl sarcosine, 50 mM EDTA, pH 7.6) 600 μl
Was added, stirred, and incubated at 60 ° C. for 30 minutes. The lysate thus obtained was extracted twice with phenol and once with chloroform, followed by ethanol precipitation to obtain a nucleic acid.

Example 1 (Binding of Vibrio parahaemolyticus Gene to Solid Phase) A nylon membrane (High Bond N +; Amersham) was used as the solid phase. The nucleic acid obtained in Reference Example 2 was diluted with sterilized water to obtain an equal volume of 0.3N NaO.
H 2 was added and the mixture was treated at room temperature for 15 minutes to denature it. As a negative sample, human placenta-derived DNA (Pharmacia) 1 μg / 1 μl
Was denatured in a similar manner. After denaturation, 100 μl of each sample was added on the membrane using a dot blotter (BRL) and slowly sucked. 5 x SSC after all solution is aspirated
100 μl of each of the above was suctioned, the membrane was removed from the dot blotter, rinsed lightly with 5 × SSC, and then dried.

(Hybridization) A nylon membrane on which the Vibrio parahaemolyticus gene has been immobilized is used as a hybrid bag (BRL).
Hybrid buffer [5 × SSC, 1.0% lithium diiodosalicylate (Nacalai Tesque), 0.5)
% Bovine serum albumin] and seal while shaking
Prehybridization was performed for 15 minutes. The buffer was removed from the hybrid bag, an alkaline phosphatase-labeled probe solution was added, and hybridization was carried out at 50 ° C. for 15 minutes while shaking. The membrane was taken out from the hybrid bag, transferred to a vat containing Wash Solution-1 (2 × SSC, 1% SDS), and washed at 50 ° C. for 15 minutes while shaking.
Next, the solution was transferred to a vat of Wash Solution-2 (1 × SSC, 0.5% Triton X-100) and washed at room temperature for 10 minutes while shaking.

(Detection of labeled probe) Hybridase-
After the treatment, the washed membrane was put in a cuvette and the chemiluminescent substrate, Lumiphos 480 (Wako Pure Chemical Industries) (4-Methoxy-4 (3-p
hosphatephenyl) spiro [1,2-dioxentane-3,2'-adamantan
e] disodium salt (Lumigen PPD)) in 100 μl,
After enzymatic reaction at 37 ° C. for 15 minutes, the amount of luminescence was measured with a TD4000 Lumiphotometer. The results are shown in Table 1. The amount of luminescence of the negative sample was low and non-specific adsorption was suppressed. It seems that there is no inhibition on luminescence.

Comparative Example 1 The same Vibrio parahaemolyticus fixed membrane as in Example 1 was prepared by using the surfactant SDS (sodium lauryl sulfate) (WO90 / 10).
718), SLS (N-lauroyl sarcosine sodium), and Tween-20 were used for hybridization, light was emitted in the same manner as in Example 1, and the amount of light emission was measured. The results are shown in Table 1. It can be seen that, with any of the reagents, the amount of luminescence of the positive sample was lower than that of the result of Example 1, and luminescence was inhibited.

[0028]

[Table 1]

Example 2 The nucleic acid-immobilized membrane obtained in Example 1 was prehybridized with the hybridization buffer of Example 1. The buffer was removed from the hybrid bag, the biotin probe solution obtained by the method of Reference Example 3 was added, and hybridization was carried out at 50 ° C. for 15 minutes while shaking. Remove the membrane from the hybrid bag and wash solution-1 (2 x SS
C, 1% SDS) while transferring to a vat with shaking
It was washed at 50 ° C for 15 minutes. Next, wash solution-2 (1 x SSC, 0.5
% Triton X-100), and washed at room temperature for 10 minutes while shaking. Next, the membrane was placed in a hybrid bag, an alkaline phosphatase-streptavidin-conjugate (Clontech) solution was added, and the mixture was reacted at room temperature for 1 hour. The membrane was then washed with distilled water at room temperature for 10 minutes.

(Detection of labeled probe) Hybridase-
After the reaction, the washed membrane was put in a cuvette, 100 μl of chemiluminescent substrate, Lumiphos 480 (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the enzyme reaction was carried out at 37 ° C for 15 minutes, and then the luminescence amount was measured by TD4000 Lumiphotometer . The results are shown in Table 2.

COMPARATIVE EXAMPLE 2 The same Vibrio parahaemolyticus membrane as in Example 2 was prepared by using the surfactant SDS (sodium lauryl sulfate) (WO90 / 10).
718), SLS (N-lauroyl sarcosine sodium), Tween-20, and pre-hybridization using a hybridization buffer,
Hybridization and luminescence were carried out by the method of Example 2,
The amount of luminescence was measured. The results are shown in Table 2. It can be seen that the amount of luminescence of the positive sample is lower than that in the case of performing prehybridization with diiodosalicylic acid in any of the reagents, indicating that luminescence inhibition occurs.

[0032]

[Table 2]

Reference Example 5 [Inhibition of chemiluminescence in the presence of diiodosalicylate] In addition to alkaline phosphatase, diiodosalicylate, SDS,
The SLS was made to coexist and the amount of luminescence was measured using 1,2-dioxetane. Alkaline phosphatase (Boehringer Mannheim) was diluted with 50 mM Tris-HCl buffer containing 0.1% lithium diiodosalicylate, or 0.1% SDS, or 0.1% SLS. Dispense 10 μl of diluted alkaline phosphatase into a cuvette,
Next, add 100 μl of chemiluminescent substrate, Lumiphos 480, and incubate at 37 ℃.
After 15 minutes of enzymatic reaction, the amount of luminescence was measured with a TD4000 lumiphotometer. As a control, alkaline phosphatase diluted with 50 mM Tris-HCl buffer was measured by the same method. The results are shown in Fig. 1. A large decrease in the amount of luminescence was observed in the presence of SDS and SLS, compared with the presence of lithium diiodosalicylate, suggesting that luminescence inhibition occurred.

[0034]

[Sequence list]

SEQ ID NO: 1 Sequence length: 24 Sequence type: Nucleic acid topology-: Single-stranded sequence type: Other nucleic acid synthetic DNA Sequence features Location: 1..24 Method of determining features: S Other features : Vibrio parahaemolyticus TDH (Thermostable Direct
Haemolysin) has a sequence homologous to the 102nd to 125th nucleotide sequences. Array CCCCGGTTCT GAATAGATAT TGTT 24

[Brief description of drawings]

FIG. 1 shows chemiluminescence inhibition in the presence of an inactivating agent.

[Explanation of symbols] In the figure, ● indicates coexistence of lithium diiodosalicylate and ○ indicates SD.
S coexistence, Δ indicates SLS coexistence, and □ indicates control.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-1-311272 (JP, A) JP-A-2-273200 (JP, A) JP-A-3-53897 (JP, A) JP-A-3- 139298 (JP, A) JP-A-5-103696 (JP, A) Special table 5-501203 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 21/78 G01N 33 / 543 4B063 BIOSIS / WPI (DIALOG)

Claims (4)

(57) [Claims] 1. An analyte in a sample immobilized on a solid phase,
A reagent containing diiodosalicylic acid or a salt thereof, which comprises reacting with a labeled ligand that specifically binds to it, and detecting the analyte contained in the sample by measuring the label of the reacted ligand by chemiluminescence. A method for detecting an analyte by chemiluminescence, characterized by using. 2. The method for detecting an analyte by chemiluminescence according to claim 1, wherein the label is an enzyme, and the substrate specifically decomposed by the enzyme is a 1,2-dixetane compound. 3. A reaction in which a reagent containing diiodosalicylic acid or a salt thereof is diluted with a labeled ligand and / or a reaction liquid between a ligand and a solid phase and / or a solid phase is treated to prevent a nonspecific reaction. The method for detecting an analyte by chemiluminescence according to claim 1, which is a liquid. 4. A diluted solution of a labeled ligand and / or
Alternatively, the reaction solution of the ligand and the solid phase and / or the reaction solution for treating the solid phase for preventing nonspecific reaction is a reagent containing diiodosalicylic acid or a salt thereof, and the analyte by chemiluminescence is characterized. A composition for detection.