JP2017161349A - Chemiluminescent material supply container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chemiluminescent material supply container which can exclusively supply a chemiluminescent material solution which can be used as a substrate reagent for enzyme immunoassay, has an excellent storage stability, and allows a high sensitive measurement.SOLUTION: The chemiluminescent material supply container includes: at least one storage part for storing a chemiluminescent substrate solution; and at least one storage part for storing a light emission reinforcing solution of the chemiluminescent substrate, the storage parts having a substantially hemispherical inner bottom surfaces and the container transmitting no lights.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a supply container for a chemiluminescent material used for enzyme immunoassay and the like.

  Various chemiluminescent materials are used for enzyme immunoassay. Among these, most used in automatic analyzers are liquid products. Conventionally, the capacity of a supply container is 50 mL to 100 mL, and sometimes 200 mL. The large capacity has the advantage of reducing the frequency of exchanging the chemiluminescent material solution. However, if the chemiluminescent material solution is contaminated, it must be discarded regardless of the remaining amount. Further, when the number of measurements is small, the consumption of the chemiluminescent material solution is also small, and it may be discarded regardless of the remaining amount after a certain period of time after opening. Also, a device in which a set of reagents necessary for enzyme immunoassay is enclosed in an integrated cartridge has been devised (see Patent Document 1). However, the integral cartridge is dependent on the storage stability of reagents other than the chemiluminescent material. For example, even if the storage stability of the chemiluminescent material solution is 1 year, the storage stability of the enzyme-labeled reagent is stable. In the case where the property is 6 months, the effective period of the integrated cartridge is set to 6 months. Therefore, there has been a demand for a supply container in which only the chemiluminescent material solution is independently reduced in size.

JP-A-11-316226

  An object of the present invention is to provide a supply container in which only the chemiluminescent material solution is independent. Another object of the present invention is to provide a chemiluminescent material solution that can be used as a substrate reagent for enzyme immunoassay and has excellent storage stability and enables highly sensitive measurement.

As a result of intensive studies on the above problems, the present inventors have reached the present invention. That is, the present invention is as follows.
(1) one or more storage units containing the chemiluminescent substrate solution;
One or more storage units containing the chemiluminescent substrate luminescence enhancing reagent solution; and
A chemiluminescent material supply container comprising:
The container in which the shape of the inner bottom surface of the housing part is substantially hemispherical and has no light transmittance.
(2) The container according to (1), wherein the container includes one chemiluminescent substrate solution and one luminescence enhancing reagent solution storage unit.

  Hereinafter, the present invention will be described in more detail.

  The supply container of the present invention includes one or more chemiluminescent substrate solution storage units containing a chemiluminescent substrate solution and a luminescence enhancing reagent solution (hereinafter also referred to as an enhancer solution) containing a reagent solution that enhances chemiluminescence of the chemiluminescent substrate. And one or more storage units. The arrangement of the chemiluminescent substrate solution storage unit and the enhancer solution storage unit may be a sequence in which a plurality of storage units are continuous in one row, or a sequence in which a plurality of storage units are continuous in a plurality of rows. Good. However, in consideration of operability on the apparatus, a measurement container having a continuous arrangement of 2 to 20 storage units in one row is preferable, and one chemiluminescent substrate solution storage unit and one enhancer solution storage Particularly preferred is a measuring container in which the parts are aligned in a row and are integrally formed.

  The size of each accommodating portion in the supply container of the present invention may be appropriately selected depending on the measurement scale and the size of the apparatus. As an example, the inner diameter (in the case of a cylindrical type) or one side (in the case of a rectangular tube type) is 1 mm to 20 mm. By the way, it is possible to raise an accommodating portion having a depth of about 1 mm to 50 mm.

The external shape and size of each container in the supply container of the present invention may be the same, or the external shape and / or size may be changed according to the amount of sample (specimen) or reagent.
However, in consideration of the ease of forming the supply container, a measurement container in which a plurality of accommodating portions having the same external shape and size are integrally formed is preferable.

  In the supply container according to the present invention, the inner bottom surface of the storage portion is substantially hemispherical. Thereby, the dead volume of a chemiluminescent substrate solution and an enhancer solution can be reduced compared with a flat bottom.

  Examples of the material of the supply container of the present invention include thermoplastic resins (for example, olefin resins, styrene resins, vinyl resins, carbonate resins, polyester resins, polyamide resins) and glass. However, in view of handling, a thermoplastic resin is preferable as a material. Among thermoplastic resins, olefin homopolymers (homopolymers) or copolymers (copolymers), which are usually used for the production of containers and resin molded products, are particularly preferred. Examples of the particularly preferable materials include low density polyethylene, medium density polyethylene, high density polyethylene, linear low density polyethylene, polypropylene, polybutene-1, poly-4-methylpentene-1, propylene-ethylene copolymer, ionomer, and ethylene. -Acrylic copolymer and ethylene-vinyl acetate copolymer.

  The supply container of the present invention is not light transmissive. For example, a white container in which a white pigment (titanium dioxide, etc.) is kneaded, a gray container in which a white pigment (titanium dioxide, etc.) and a black pigment (carbon black, etc.) are kneaded, or a black pigment (carbon black, etc.) A black container kneaded is preferred. Among these, a gray container and a black container are particularly preferable.

Although it does not specifically limit as a chemiluminescent substrate solution, For example, general formula (I)

で表される１，２−ジオキセタン誘導体（式中、Ｒ^１、Ｒ^２はそれぞれ独立に水素原子、アルキル基又はアリール基である。また、Ｒ^１、Ｒ^２は一体となり、ジオキセタン環にスピロ結合する環式又は多環式有機環基を形成することもできる。Ｒ^３はアルキル基又はアリール基であり、Ｒ^３とＲ^１又はＲ^２とが一体となってジオキセタン環と酸素原子を含む縮合環を形成してもよい。Ａｒは一般式（ＩＩ）

(Wherein, R ¹ and R ² are each independently a hydrogen atom, an alkyl group or an aryl group. Also, R ¹ and R ² are combined together to form a spiro bond to the dioxetane ring.) R ³ is an alkyl group or an aryl group, and R ³ and R ¹ or R ² are combined to form a condensed dioxetane ring and an oxygen atom. Ar may form a ring, Ar is represented by the general formula (II)

であり、Ｒ^４はヒドロキシル基、アルコキシル基、アラルキルオキシ基、−ＯＳｉ（Ｒ^６Ｒ^７Ｒ^８）（ただし、Ｒ^６、Ｒ^７およびＲ^８は互いに独立にアルキル基又はアリール基である。）で表される基、リン酸塩基またはＳ（Ｃ＝Ｏ）Ｒ^９（ただし、Ｒ^９はアルキル基またはアリール基である。）で表される基であり、Ｒ^５は水素原子、ハロゲン原子、アルキル基またはアルコキシル基である。）の溶液を用いることができる。

And R ⁴ is a hydroxyl group, an alkoxyl group, an aralkyloxy group, —OSi (R ⁶ R ⁷ R ⁸ ) (where R ⁶ , R ⁷ and R ⁸ are each independently an alkyl group or an aryl group). Or a phosphate group or a group represented by S (C═O) R ⁹ (wherein R ⁹ is an alkyl group or an aryl group), and R ⁵ is a hydrogen atom, a halogen atom, An alkyl group or an alkoxyl group; ) Solution can be used.

  The alkyl group for the substituent means a linear, branched or cyclic alkyl group having 1 to 20 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl. , Decyl, undecyl, dodecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosanyl and the like, or a group in which these alkyl groups are appropriately branched (for example, an i-propyl group, i- A butyl group, a t-butyl group, etc.) or a cyclic group. These alkyl groups may further have a substituent, for example, a hydroxyl group, an alkoxyl group, an aryl group, or the like may be substituted.

  Examples of the aryl group include aromatic hydrocarbon groups having 6 to 20 carbon atoms such as phenyl and naphthyl groups, and 1 to 5 nitrogen atoms and oxygen atoms in the ring such as furyl, thienyl and pyridyl groups. Or the heteroaryl group which has a sulfur atom etc. can be mentioned. Examples of the alkoxyl group include linear alkoxyl groups having 1 to 20 carbon atoms such as methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, and methoxyethoxyethoxy groups. And 1-5 bonded in a branched or branched form. Examples of the aralkyloxy group include groups having 7 to 20 carbon atoms such as benzyloxy and phenethyloxy groups, and examples of the halogen atom include fluorine, chlorine, bromine and the like.

In addition, examples of the cyclic or polycyclic organic ring group in which R ¹ and R ² are combined and spiro-bonded to the dioxetane ring include an adamantyl group. Examples of the condensed ring in which R ³ and R ¹ or R ² are combined and contain a dioxetane ring and an oxygen atom include a furan ring and a pyran ring. Examples of the phosphate group of R ⁴ include sodium salt, potassium salt and ammonium salt of PO ₄ ³⁻ .

Among such substituents, R ¹ and R ² are preferably linear, branched or cyclic alkyl groups having 1 to 20 carbon atoms, and R ⁴ is a hydroxyl group or phosphoric acid. A base, and R ⁵ is a hydrogen atom or a halogen atom.

  Specific examples of the 1,2-dioxetane derivative represented by the general formula (I) having such a substituent include 3- [3′-methoxyspiro phosphate [adamantane-2,4 ′-[1, 2] Dioxetane] -3′-yl] phenyl, 5-t-butyl-4,4-dimethyl-1- (3′-phosphoryloxy) phenyl-2,6,7-trioxabicyclo [3.2.0 ] Heptane disodium salt, mono- [5- (5-t-butyl-4,4-dimethyl-2,6,7-trioxabicyclo [3.2.0] hept-1-yl) -2 phosphate -Chlorophenyl] ester disodium salt and the like. Among them, 3- [3′-methoxyspiro [adamantane-2,4 ′-[1,2] dioxetane] -3′-yl] phenyl phosphate, 5-t-butyl-4,4-dimethyl-1- (3 '-Phosphoryloxy) phenyl-2,6,7-trioxabicyclo [3.2.0] heptane disodium salt is preferred, 5-t-butyl-4,4-dimethyl-1- (3'-phosphoryloxy ) Phenyl-2,6,7-trioxabicyclo [3.2.0] heptane disodium salt is more preferred.

As the luminescence enhancing reagent solution (enhancer solution), for example, a solution containing a cationic surfactant and a fluorescent substance can be used. For example, cationic surfactants include poly (benzyltributyl) ammonium chloride and general formula (III) contained in commercially available Emerald-II ^™ (manufactured by TROPIX).

（式中、Ｒ^１０，Ｒ^１１，Ｒ^１２およびＲ^１３のうち、一つは炭素数８〜２０のアルキル基または炭素数８〜２０のアリール基であり、他の三つは互いに独立に炭素数１〜８のアルキル基、ベンジル基またはフェニル基である。Ｍは窒素原子またはリン原子を示す。Ｘはハロゲン原子を表す。）で示される第４級アンモニウム塩または第４級ホスホニウム塩をあげることができる。

(In the formula, one of R ¹⁰ , R ¹¹ , R ¹² and R ¹³ is an alkyl group having 8 to 20 carbon atoms or an aryl group having 8 to 20 carbon atoms, and the other three are carbon atoms independently of each other. A quaternary ammonium salt or a quaternary phosphonium salt represented by the following formula: an alkyl group, a benzyl group or a phenyl group of 1 to 8, M represents a nitrogen atom or a phosphorus atom, and X represents a halogen atom. be able to.

  In the general formula (III), the alkyl group having 8 to 20 carbon atoms refers to a linear or branched alkyl group having 8 to 20 carbon atoms, such as octyl, nonyl, decyl, undecyl, dodecyl, Examples thereof include straight-chain groups such as tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosanyl, and groups in which these alkyl groups are appropriately linked. The alkyl group having 1 to 8 carbon atoms refers to a linear or branched alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl and the like. Examples thereof include a linear group or a group in which these alkyl groups are appropriately branched. Examples of the aryl group having 8 to 20 carbon atoms include an aromatic hydrocarbon group having 8 to 20 carbon atoms such as a naphthyl group, or a heteroaryl having 1 to 5 nitrogen atoms, oxygen atoms, or sulfur atoms in the ring. Groups and the like. Examples of the halogen atom include a fluorine atom, a chlorine atom, and a bromine atom.

  Preferable examples of the quaternary ammonium salt represented by the general formula (III) having the above substituent include cetyltrimethylammonium bromide (CTAB), benzalkonium chloride and benzethonium chloride. Preferable examples of the quaternary phosphonium salt include tri-n-butylhexadecylphosphonium bromide, ethyltriphenylphosphonium bromide, triheptylphenylphosphonium bromide and tetradecyltriphenylphosphonium bromide. Of these, tri-n-butylhexadecylphosphonium bromide is highly desirable.

  The fluorescent substance used in the present invention is not particularly limited as long as it has an effect of enhancing the chemiluminescence of the 1,2-dioxetane derivative represented by the general formula (I) together with the cationic surfactant. Fluorescein and fluorescein derivatives (sodium fluorescein, aminofluorescein, fluorescein isothiocyanate (FITC), etc.), body pea (BODIPY) dye, Oregon Green 488, Oregon Green 514, Rhodol green (registered trademark), Alexa Fluor 488 (registered trademark) And hydrazide, 5- (and -6) -carboxy-2 ', 7'-dichlorofluorescein. Among these, fluorescein, sodium fluorescein, and fluorescein isocyanate are preferable, and fluorescein is particularly desirable.

  The supply container for the chemiluminescent material of the present invention has the aforementioned solution. As the solvent, for example, a protic solvent such as water or an aqueous buffer is used. In addition, the concentration of the chemiluminescent substrate and the luminescence enhancing reagent in the solution is not particularly limited, but when used as a substrate for enzyme immunoassay, it is preferable that the concentration range is suitable for use. . In addition, the solution of the chemiluminescent substrate does not substantially contain a luminescence enhancing reagent, and thereby high storage stability of the chemiluminescent substrate can be obtained. On the other hand, a pH adjuster and a preservative such as sodium azide may be appropriately contained in order to ensure storage stability.

  In the present invention, when the chemiluminescent substrate is chemiluminescent with an activator, a luminescence enhancing reagent is added. Thereby, it is possible to obtain a light emission amount equal to or higher than that of the conventional method.

The activator used here may be appropriately selected according to the chemiluminescent substrate. However, when the chemiluminescent substrate is a 1,2-dioxetane derivative represented by the general formula (I), a base, an acid, Those selected from the group consisting of salts, fluorine compounds, enzymes, catalysts and amine compounds are used. This is capable of acting on the group R ⁴ of the 1,2-dioxetane derivative represented by the general formula (I) to be converted into a carbonyl compound via an unstable oxide intermediate and simultaneously generating chemiluminescence. is there. For example, when R ⁴ is a hydroxyl group, the base is used, and R ⁴ is —OSi (R ⁶ R ⁷ R ⁸ ) (wherein R ⁶ , R ⁷ and R ⁸ are each independently an alkyl group or an aryl group). If it is a group represented, tetrabutylammonium fluoride can be suitably used as an activator. When the 1,2-dioxetane derivative represented by the general formula (I) is used as a substrate for enzyme immunoassay, various enzymes such as alkaline phosphatase, arylesterase, acetylcholinesterase, peroxidase and galactosidase are suitable as the activator. Can be used for In particular, when R ⁴ is a phosphate group, alkaline phosphatase can be preferably used.

  According to the present invention, a supply container of a chemiluminescent material having storage stability can be provided.

It is the top view and front view (sectional drawing) of the supply container of a chemiluminescent material provided with the chemiluminescent substrate solution storage part and the light emission enhancement reagent solution (enhancer solution) storage part whose inner bottom face is substantially hemispherical.

  Next, an Example is shown and this invention is demonstrated concretely. However, the present invention is not limited to these examples.

Example 1
The inner bottom surface shown in FIG. 1 has two substantially hemispherical accommodating parts, and the maximum capacity of each accommodating part is 500 μL, and light transmission kneaded with white pigment (titanium dioxide) and black pigment (carbon black). An innocent gray polypropylene container was prepared. As a 1,2-dioxetane derivative, 5-t-butyl-4,4-dimethyl-1- (3′-phosphoryloxy) phenyl-2,6,7-trioxabicyclo [3.2.0] heptane disodium A 50 mM 2-amino-2-methyl-1-propanol buffer solution (pH 10) containing 0.8 mM salt and 0.1% sodium azide was prepared, and 350 μL of the inner bottom surface was placed in a substantially hemispherical container. Dispensed. 500 mM 2-amino-2-containing 0.3 mM fluorescein as a fluorescent substance, 2.0 mM tri-n-butylhexadecylphosphonium bromide as a cationic surfactant, and 0.1% sodium azide A methyl-1-propanol buffer solution (pH 10) was prepared, and 350 μL was dispensed into a housing portion having the other inner bottom surface in a substantially hemispherical shape. Thereafter, the two accommodating portions were sealed with an aluminum seal and stored at 4 ° C. or 20 ° C. for 7 days. During storage, the aluminum seal surface was left as it was as an upper surface.

After 7 days, the aluminum seal was broken, and 25 μL of each solution was taken and mixed, and then background measurement was performed. Further, it was reacted with 10 μL of 50 mM Tris-HCl buffer solution containing 0.005 μg / mL of alkaline phosphatase, 0.1% gelatin hydrolyzate, 1 mM MgCl ₂ , 0.15 M NaCl and 0.05% sodium azide, and its luminescence The amount was measured. Luminometer LB96V (detector: single photon counter; measurement wavelength region: 380 to 630 nm) manufactured by Bertoled Co. was used for luminescence measurement, and the photometric conditions were a mixture of chemiluminescent material and enzyme solution as described above. The light emission amount (count number) for 1 second after a minute was measured.

(Example 2)
The same procedure as in Example 1 was performed, except that the aluminum seal surface was used as the lower surface and the contents were in contact with the aluminum seal surface so as to contact the aluminum seal surface.

(Comparative Example 1)
It was carried out in the same manner as in Example 1 except that a translucent polypropylene container into which no pigment was kneaded was used and stored at 20 ° C. for 3 days.

  The results are shown in Table 1 together with the results of Example 1 and Example 2.

灰色不透明で光透過性の無い小型容器を使用した場合には、保存温度が２０℃にて７日間の保管において、静置（アルミシール面に液が接触しない）と転倒（アルミシール面に液が接触する）の二つの条件（実施例２）において、バックグラウンドに大きな変動はなかった。またアルカリ性ホスファターゼ酵素反応もリファレンスである４℃静置（実施例１）と比較して、４℃転倒で１００．１％（実施例１）、２０℃静置で９８．４％（実施例２）、２０℃転倒で１０１．７％（実施例２）と非常に良好な結果となっている。それに対して顔料を練りこまない半透明で光透過性の有る小型容器を使用した場合には、保存温度が２０℃にて３日間と保管期間が短く、静置（アルミシール面に液が接触しない）条件（比較例１）にもかかわらず、アルカリ性ホスファターゼ酵素反応が９３．７％となる結果であった。

When using a small container that is gray and opaque and does not transmit light, when stored at 20 ° C for 7 days, leave it (no liquid touching the aluminum seal surface) and tipping over (liquid on the aluminum seal surface) In the two conditions (Example 2), the background did not vary greatly. In addition, the alkaline phosphatase enzyme reaction was 100.1% (Example 1) after falling at 4 ° C. and 98.4% when left at 20 ° C. (Example 2), compared to the reference at 4 ° C. (Example 1). ), 201.7 ° C., 101.7% (Example 2), which is a very good result. On the other hand, when using a semi-transparent and light-transmitting small container that does not knead the pigment, the storage temperature is 20 days and the storage period is as short as 3 days. Not) The result was that the alkaline phosphatase enzyme reaction was 93.7% despite the condition (Comparative Example 1).

  From the above results, a chemiluminescent material having storage stability can be provided by using a container that does not transmit light.

1: Chemiluminescent material supply containers 2a, 2b: Supply container opening 3a: Chemiluminescent substrate solution 3b: Luminescence enhancing reagent solution 4: Aluminum seal

Claims (2)

One or more containing parts containing the chemiluminescent substrate solution;
One or more storage units containing the chemiluminescent substrate luminescence enhancing reagent solution; and
A chemiluminescent material supply container comprising:
The container in which the shape of the inner bottom surface of the housing part is substantially hemispherical and has no light transmittance.   The container according to claim 1, wherein the container has one chemiluminescent substrate solution and one luminescence enhancing reagent solution container.

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