JPH05103696A - Chemically luminescing sensitizing method - Google Patents

Abstract

PURPOSE:To eliminate troubles caused by the employment of an organic solvent and highly sensitively detect a chemiluminescence by adding a specific sensitizer having rich water solubility to a system in which a chemically luminescing substance is treated with an oxidant and a peroxidase to emit chemilumi nescence. CONSTITUTION:A compound of formula I (X is halogen; M is metal atom; R<1>, R<2> are halogen, etc.; n is 1-3) is added as a sensitizer to a system in which a chemically luminescing substance (preferably aluminol, lophine, etc.) is treated with an oxidant (preferably hydrogen peroxide or peroxyboric acid) and a peroxidase (preferably originated from horse radish) or peroxidase-like substance (preferably hemoglobin) to emit chemiluminescence. The compound of formula I includes sodium p-iodophenoxide and is obtained by reacting 1 mole of a compound of formula II with 0.9-1.1 mole of a compound of the formula M(OH)n.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescence sensitization method in an analytical method utilizing a chemiluminescence reaction.

[0002]

2. Description of the Related Art A method for analyzing a minor component utilizing chemiluminescence, for example, a method for quantifying a target minor component using an antigen or antibody labeled with a chemiluminescent substance such as luminol or isoluminol. Alternatively, for example, a method for quantifying a trace amount of a target substance by measuring the enzyme activity by utilizing chemiluminescence in EIA using an antigen or antibody labeled with an enzyme such as peroxidase, or an oxidizing agent or peroxidase (Or peroxidase-like substance)
And the like using chemiluminescence for the detection and quantification of (these are collectively referred to as chemiluminescence analysis methods).
Because of its extremely high measurement sensitivity, it is one of the analytical methods that has recently been attracting attention as a method for detecting and quantifying trace components. However, this method has a problem in terms of measurement accuracy because the light emission duration is short. Therefore, as a method for solving this problem, a method has been proposed in which a chemiluminescent sensitizer is allowed to coexist during a chemiluminescent reaction to significantly increase the amount of light emission.

The sensitizers that have been reported so far as the sensitizers that can be used in this method include phenol derivatives such as p-iodophenol and p-bromophenol.
59-171839, JP2-291299), aromatic amines (JP61-54453), indophenol derivatives (JP2-174694), indoaniline derivatives (left), Examples thereof include phenathiazine derivatives (same as left), and among them, p-iodophenol is known as a particularly effective sensitizer.

However, since all of these existing sensitizers are poor in water solubility, in order to coexist in a reagent solution for chemiluminescence analysis, the sensitizer is once dimethyl sulfoxide (hereinafter abbreviated as DMSO). There is no choice but to use the method of using the one dissolved in an organic solvent such as. However, since the reagent solution thus prepared naturally contains an organic solvent, there is a problem in terms of compatibility with an automatic analyzer and waste liquid treatment. This is very inconvenient in practical use because it may cause a problem such as deactivating the enzyme activity in the reagent solution and lowering the storage stability of the reagent solution itself.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above situation, and chemiluminescence is obtained by the action of an oxidizing agent and peroxidase (hereinafter abbreviated as POD) or a POD-like substance on a chemiluminescent substance. As a chemiluminescent sensitizer (hereinafter simply referred to as a sensitizer) in a reaction that causes a reaction, a compound that is highly water-soluble and can be used without being dissolved in an organic solvent is used. It is an object of the present invention to provide a highly efficient chemiluminescence analysis method.

[0006]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a system for generating chemiluminescence by reacting a chemiluminescent substance with an oxidizing agent and a POD or POD-like substance.

[Chemical 2] [Wherein, X represents a halogen atom, M represents a metal atom, R ¹ and R ² are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, an alkenyl group, a hydroxy group or Represents a nitro group. Also, R
¹ and R ² may combine with each other to form a ring. n represents an integer of 1 to 3. ] It is a chemiluminescent sensitization method, characterized in that one or more compounds represented by In the compound represented by the general formula (1), X is a halogen atom such as a fluorine atom, chlorine atom, bromine atom or iodine atom, and M is an alkali metal atom such as lithium, sodium or potassium. Examples thereof include alkaline earth metal atoms such as magnesium, calcium and barium, iron, zinc, nickel and copper. Also, R
¹ and R ² are each independently a hydrogen atom, for example, a lower alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, a propyl group and a butyl group (which may be linear or branched) ), For example, a lower alkoxy group having 1 to 4 carbon atoms such as methoxy group, ethoxy group, propoxy group, butoxy group (which may be linear or branched), for example, fluorine atom, chlorine atom, bromine atom , Halogen atom such as iodine atom, aryl group such as phenyl group, naphthyl group, tolyl group, such as vinyl group,
2 carbon atoms such as allyl, isopropenyl and butenyl groups
A lower alkenyl group, a hydroxy group, a nitro group and the like, and R ¹ and R ² may be bonded to each other to form a ring such as an aromatic ring or an alkylene ring.

The compound represented by the general formula (1) is exemplified by JA
It can be easily synthesized according to a method described in a known document such as CS, vol. 81, 2710 (1959). That is, for example, the general formula (2)

[Chemical 3] (In the formula, X, R ¹ and R ² are the same as the above.), And 0.1 to 3 mol, preferably 0.9 to 1.1 mol of the general formula (3) M (OH ) _N (wherein M and n are the same as defined above),
Or a metal body of M, for example, methanol, ethanol,
It can be easily obtained by dissolving it in a solvent such as alcohols such as isopropanol, for example, cyclic ethers such as dioxane and tetrahydrofuran, and drying this under reduced pressure. If necessary, this may be purified by recrystallization or reprecipitation using an organic solvent such as alcohol, hexane, benzene or the like, or a suitable mixture thereof. The compounds represented by the general formula (2) and the general formula (3) used in the above synthetic reaction may be commercially available compounds as they are, or may be appropriately purified before use.

Reagents such as chemiluminescent substances, oxidizing agents, POD or POD-like substances used in the sensitizing method of the present invention may be those used in the chemiluminescence analysis method known per se. Although it can be used without particular limitation, examples of the chemiluminescent substance preferably include luminol or a derivative thereof, isoluminol or a derivative thereof, lophine, lucigenin, and the like, and examples of the oxidizing agent include peroxides. Preference is given to hydrogen, peroxoboric acid or salts thereof, examples of POD include those derived from horseradish, and examples of POD-like substances include heme compounds such as hemoglobin.

The present invention provides a chemiluminescence reaction in a chemiluminescence analysis method known per se, that is, a reaction in which a chemiluminescent substance is reacted with an oxidizing agent and a POD or POD-like substance to generate chemiluminescence. It can be easily carried out by allowing one or more compounds represented by the general formula (1) according to the present invention to coexist as a sensitizer. The concentration of the compound represented by the general formula (1) is slightly different depending on the kind of the compound used, but is usually 1 nM to 1 M, preferably 0.1 to 100 mM, as a concentration in the system that causes chemiluminescence. To be selected.

Also, chemiluminescent substances, oxidants, and PO
The concentration of the D or POD-like substance may be appropriately determined according to the concentration in the reagent solution used in the chemiluminescence analysis method known per se, but the concentration of the chemiluminescent substance is usually 1 μM to 100 mM, preferably From the range of 0.1 to 10 mM, the concentration of the oxidant is usually 0.00003 to 0.3M, preferably 0.0003 to 0.3M.
From the range of M, and the concentration of POD or POD-like substance is usually 100 nU / μl to 10 mU / μl, preferably 1 μU / μl
It is appropriately selected from the range of 1 mU / μl. The pH and temperature during the reaction in the chemiluminescence analysis method utilizing the present invention may be appropriately determined according to the conditions during the reaction in the chemiluminescence analysis method known per se, but the pH is usually 7 to 10, preferably from the range of 8 to 9.5, and the reaction temperature is usually
It is appropriately selected from the range of 0 to 60 ° C, preferably 5 to 30 ° C. In addition, the chemiluminescence analysis reagent solution used in the chemiluminescence analysis method utilizing the present invention contains reagents such as a buffer, a surfactant, an antiseptic, and an enzyme substrate, in addition to the above-mentioned reagents. The types and the amounts used may be appropriately selected and determined according to those in the chemiluminescence analysis method known per se. Examples of the method for measuring the chemiluminescence amount include a method using various photocounters, luminometers, etc., and a method using an X-ray film or various photosensitive films, all the measuring methods usually performed in this field. According to the chemiluminescence analysis method utilizing the present invention, chemiluminescent substances, oxidizing agents, peroxidases or peroxidase-like substances can be detected with higher sensitivity than conventional methods. Can be measured with higher sensitivity. In particular, for example, when quantifying a trace amount component by a chemiluminescence analysis method using a nucleic acid, an antigen, a hapten, an antibody and the like labeled with POD by a method known per se (hereinafter, abbreviated as POD-labeled antibody etc.), When the present invention is used, the POD detection sensitivity can be made higher than that of the conventional method, and as a result, it becomes possible to quantify a target trace component with higher sensitivity than that of the conventional method.

The trace components that can be detected by the chemiluminescence analysis method utilizing the present invention (particularly the method using a POD-labeled antibody) include, for example, hepatitis B contained in biological samples such as serum, plasma and urine. Virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HI)
V), adult T-cell leukemia virus (HTLV), cytomegalovirus (CMV), human papillomavirus (HPV) and other nucleic acids derived from viruses such as human gastric cancer,
Oncogenes derived from human leukemia, human neuroblastoma, etc., such as steroid hormones such as estriol, estradiol, testosterone, thyroid hormones such as thyroglobulin, insulin, glucagon, cortisol, α-fetoprotein, carcinoembryonic antigen (CEA),
Examples thereof include proteins such as ferritin. Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Experimental Examples, Examples and Comparative Examples, but the present invention is not limited thereto.

[0012]

【Example】

Reference example 1. Synthesis of sodium p-iodophenoxide P-iodophenol 10mmol (Wako Pure Chemical Industries, Ltd.)
And sodium hydroxide 10 mmol (Wako Pure Chemical Industries, Ltd.)
And were dissolved in 10 ml of anhydrous methanol, the atmosphere was replaced with nitrogen, and the mixture was concentrated to dryness under reduced pressure for 24 hours to obtain white powder of sodium p-iodophenoxide. Elemental analysis value (C ₆ H ₄ IONa) calculated value (%): C 29.78, H 1.67, Na 9.50. Found (%): C 29.39, H 1.99, Na 9.40.

Reference Example 2. Synthesis of potassium p-bromophenoxide p-bromophenol 10mmol (Wako Pure Chemical Industries, Ltd.)
And 10 mmol of potassium hydroxide were dissolved in 10 ml of anhydrous methanol, and then Reference Example 1. A white powder of potassium p-bromophenoxide was obtained in the same manner as in. Elemental analysis value (C ₆ H ₄ BrOK) calculated value (%): C 34.14, H 1.90, K 18.52. Found (%): C 33.76, H 2.11, K 18.39.

Experimental Example 1. Detection of POD (Chemical luminescence detection reagent) 0.1M tris (hydroxymethyl) aminomethane hydrochloric acid buffer (pH 9.1), luminol (Wako Pure Chemical Industries, Ltd.) 1.5 mM, hydrogen peroxide 0.0
It was dissolved to be 1%, and then sodium p-iodophenoxide (obtained in Reference Example 1) or potassium p-bromophenoxide (obtained in Reference Example 2) was used as a sensitizer. ) Was added to give a predetermined concentration to prepare a reagent solution for chemiluminescence detection. Also, for comparison, p-
A reagent solution for chemiluminescence detection was prepared in the same manner by adding conventional sensitizers (7 kinds) at predetermined concentrations instead of iodophenoxide or p-bromophenoxide. (Sample) Horseradish-derived POD (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water, and 100 nU / μl, 1 μU / μl, 2 μU /
μl, 5 μU / μl, 10 μU / μl, 50 μU / μl, 100 μU / μl, 2
Samples with POD activity of 00 μU / μl, 500 μU / μl or 1 mU / μl were prepared. (Procedure) 45 μl of the specified reagent solution for chemiluminescence detection and 5 μl of the specified sample in the well of a 96-well microtiter plate
After adding and, in contact with the microtiter plate under light-shielding conditions, a photosensitive film (Fuji Photo Film RX-HR)
Was laid and exposed for 10 minutes, then developed and examined for the presence or absence of luminescence. The results are shown in Table 1. In the detection limit section of Table 1, the POD concentration at which luminescence was detected by the above method is shown as the concentration in the sample.

[Table 1] As is clear from the results in Table 1, it is found that the POD activity can be detected with higher sensitivity by using the method of the present invention than by the conventional method.

Example 1. Nucleic acid detection kit Labezyme PODs
Nucleic acid using ET (Wako Pure Chemical Industries, Ltd.) (λ-DN
Detection of A) (POD labeled DNA solution) λ-DNA (manufactured by Wako Pure Chemical Industries, Ltd.)
Was made into a single strand by a conventional method, and then Labeled POD set (manufactured by Wako Pure Chemical Industries, Ltd.) was used to perform labeling with POD according to the standard operating method of the actual product instruction manual to obtain POD-labeled DNA.
A solution was obtained. (Procedure) λ-DNA (manufactured by Wako Pure Chemical Industries, Ltd.) was made into a single strand by a conventional method, and 0, 1, 5, 10, 50, 100, 500 pg each was blotted on a nitrocellulose membrane filter. Then, after performing blocking and prehybridization, it was immersed in a POD-labeled DNA solution to carry out hybridization. Then, the filter was washed with the washing solution in the Labezyme POD set according to the standard operating method described in the instruction manual for the actual product. The filter was 0.15 mM in sodium p-iodophenoxide, 1.5 mM in luminol (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.1 M tris (hydroxymethyl) aminomethane hydrochloride buffer (pH 9.1) containing 0.01% hydrogen peroxide. ), And allowed to stand for about 1 minute. Then, the filter was wrapped in Saran Wrap (registered trademark of Asahi Kasei Co., Ltd.), contacted with an X-ray film, and allowed to stand in a cassette for several minutes at room temperature. Develop the film,
The detection limit of λ-DNA was determined from the results. The results obtained are shown in Table 2.

Comparative Example 1. By the same operation as in Example 1, λ
-A nitrocellulose membrane filter obtained by blotting DNA was reacted with POD-labeled DNA obtained in the same manner as in Example 1 and hybridized, and
The washing solution in the enzyme POD set was used for washing according to the standard operating method described in the instruction manual for the actual product. Then the filter
Staining was performed using the Labezyme POD set staining solution (without sensitizer), and staining was performed according to the standard operating method described in the instruction manual for the actual product, and the detection limit of λ-DNA was determined from the results. The obtained results are also shown in Table 2.

Comparative Example 2. By the same operation as in Example 1, λ
-A nitrocellulose membrane filter obtained by blotting DNA was reacted with POD-labeled DNA obtained in the same manner as in Example 1 and hybridized, and
The washing solution in the enzyme POD set was used for washing according to the standard operating method described in the instruction manual for the actual product. Then, the filter, p-iodophenoxide 0.15 mM, luminol (Wako Pure Chemical Industries, Ltd.) 1.5 mM, and hydrogen peroxide 0.0
It was immersed in 0.1 M tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution (pH 9.1) containing 1% and allowed to stand for about 1 minute.
Then, the filter was wrapped in Saran wrap, contacted with an X-ray film, and allowed to stand in a cassette for several minutes at room temperature. The film was developed, and the detection limit of λ-DNA was determined from the results. The results are also shown in Table 2. As is clear from the results shown in Table 2, it can be seen that the target DNA can be detected with higher sensitivity than the conventional method by using the method of the present invention.

[0018]

Since the conventional sensitizer is poor in water solubility, the sensitizer had to be once dissolved in an organic solvent in order to prepare a reagent solution for chemiluminescence analysis. The test solution contained an organic solvent. Therefore,
In using the test solution, there are cases where the compatibility with an automatic analyzer and problems in waste solution treatment, and further, the organic solvent reduces the enzyme activity in the test solution and reduces the stability of the test solution itself. There was a problem such as, the general formula (1) according to the present invention
Since the compound represented by the formula (1) has a high water solubility, it is not necessary to use an organic solvent when preparing a chemiluminescence analysis reagent solution when using the compound as a sensitizer, which causes such a problem. There is no such thing. Moreover, by using the sensitizing method of the present invention, it becomes possible to detect chemiluminescence with higher sensitivity than in the case of using the conventional method.

[Table 2]

Claims (7)

[Claims] 1. A system for generating chemiluminescence by reacting a chemiluminescent substance with an oxidizing agent and a peroxidase or a peroxidase-like substance is represented by the following general formula (1): [Wherein, X represents a halogen atom, M represents a metal atom, R ¹ and R ² are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, an alkenyl group, a hydroxy group or Represents a nitro group. Also, R
¹ and R ² may combine with each other to form a ring. n represents an integer of 1 to 3. ] The chemiluminescence sensitization method characterized by making one or more types of the compounds shown by these exist. 2. The sensitizing method according to claim 1, wherein M in the general formula (1) is an alkali metal atom or an alkaline earth metal atom. 3. The sensitizing method according to claim 1, wherein M in the general formula (1) is a sodium atom. 4. The sensitizing method according to claim 1, wherein the compound represented by the general formula (1) is sodium 4-iodophenoxide. 5. The sensitizing method according to claim 1, wherein the compound represented by the general formula (1) is sodium 4-bromophenoxide. 6. The sensitizing method according to claim 1, wherein the chemiluminescent substance is luminol or isoluminol. 7. The sensitizing method according to claim 1, wherein the peroxidase-like substance is hemoglobin.