JP2008054585A - Chemiluminescent reagent kit for measuring peroxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chemiluminescent reagent kit for measuring a super trace amount of a substance in a living body with high accuracy. <P>SOLUTION: The chemiluminescent reagent kit for measuring a peroxidase characterized as comprising a first liquid containing a fluorine atom-containing surfactant (a), a 2,3-dihydro-1,4-phthalazinedione compound (b) and a chemiluminescent enhancer (c) as essential constituent components and a second liquid containing an oxidizing agent (d) and water (e) as essential constituent components is used. The fluorine atom-containing surfactant (a) is preferably at least one kind selected from the group consisting of a perfluoroalkylamine oxide, a perfluoroalkyl ethylene oxide adduct and a perfluoroalkyl carboxylate. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a chemiluminescent reagent kit for measuring peroxidase.

  Conventionally, in order to further enhance the generated luminescence, a chemiluminescent reagent kit for measuring peroxidase added with a surfactant (Patent Document 1), a chemiluminescent reagent kit for measuring peroxidase added with ovalbumin (Patent Document 2), and fullerene An added chemiluminescent reagent kit for measuring peroxidase (Patent Document 3) is known.

Japanese Patent Application Laid-Open No. 08-261944 JP 2002-323495 A Japanese Patent Laid-Open No. 2003-254773

  However, with the conventional chemiluminescent reagent kit for measuring peroxidase, it has become difficult to accurately measure the analyte in the living body, which is becoming extremely minute in recent years. That is, an object of the present invention is to provide a chemiluminescent reagent kit for measuring ultra-trace substances in a living body with high accuracy.

The chemiluminescent reagent kit for peroxidase measurement of the present invention is characterized by a fluorine atom-containing surfactant (a), a 2,3-dihydro-1,4-phthalazinedione compound (b), and a chemiluminescence enhancer (c). A first liquid as an essential component;
The gist of the present invention is that it comprises a second liquid comprising oxidant (d) and water (e) as essential components.

  By using the chemiluminescence reagent kit for measuring peroxidase of the present invention, it is possible to measure ultra-trace substances in living bodies with high accuracy. In particular, it is possible to measure PSA (human prostate specific antigen) in the serum of a patient after prostatectomy, which is increasingly required in recent years, with high sensitivity. For example, it can contribute to monitoring of postoperative PSA levels.

As the fluorine atom-containing surfactant (a), any surfactant containing a fluorine atom can be used without limitation.
The number of fluorine atoms contained in the fluorine atom-containing surfactant (a) is preferably 7 to 43, more preferably 11 to 39, and particularly preferably 15 to 35.
Examples of the fluorine atom-containing surfactant (a) include surfactants having a perfluoroalkyl group, fluorine atom-containing nonionic surfactant (a1), fluorine atom-containing anionic surfactant (a2), and these Can be used.

Examples of the fluorine atom-containing nonionic surfactant (a1) include perfluoroalkylamine oxides and perfluoroalkylethylene oxide adducts.
The alkyl group may be linear, branched or cyclic, but is preferably linear alkyl or branched alkyl, more preferably linear alkyl (hereinafter the same).

ｅは０〜２０の整数を表し、１〜１８の整数が好ましく、さらに好ましくは２〜１２の整数である。また、ｆは０〜５の整数を表し、１〜４の整数が好ましく、さらに好ましくは１〜２の整数である。また、Ｒは炭素数１〜６のアルキルを表し、メチル、エチル及びプロピルが好ましく、さらに好ましくはメチルである（２つのＲは同じでも異なってもよい）。Ｃは炭素原子、Ｆはフッ素原子、Ｈは水素原子、Ｎは窒素原子、Ｏは酸素原子を表す（以下同様である。）。 The perfluoroalkylamine oxide includes a compound represented by the formula (1).

e represents an integer of 0 to 20, preferably an integer of 1 to 18, and more preferably an integer of 2 to 12. Moreover, f represents the integer of 0-5, the integer of 1-4 is preferable, More preferably, it is the integer of 1-2. R represents alkyl having 1 to 6 carbon atoms, preferably methyl, ethyl and propyl, more preferably methyl (two Rs may be the same or different). C represents a carbon atom, F represents a fluorine atom, H represents a hydrogen atom, N represents a nitrogen atom, and O represents an oxygen atom (the same applies hereinafter).

ｇは０〜２０の整数を表し、１〜１８の整数が好ましく、さらに好ましくは２〜１２の整数である。また、ｈは０〜５の整数を表し、１〜４の整数が好ましく、さらに好ましくは１〜２の整数である。また、ｉは１〜１２の整数を表し、２〜１０の整数が好ましく、さらに好ましくは２〜８の整数である。 The perfluoroalkylethylene oxide adduct includes a compound represented by the formula (2).

g represents the integer of 0-20, the integer of 1-18 is preferable, More preferably, it is the integer of 2-12. H represents an integer of 0 to 5, an integer of 1 to 4 is preferable, and an integer of 1 to 2 is more preferable. Moreover, i represents the integer of 1-12, the integer of 2-10 is preferable, More preferably, it is the integer of 2-8.

ｊは０〜２０の整数を表し、１〜１８の整数が好ましく、さらに好ましくは２〜１２の整数である。また、ｋは０〜５の整数を表し、１〜４の整数が好ましく、さらに好ましくは１〜２の整数である。また、Ｍはアルカリ金属（リチウム、カリウム及びナトリウム等）イオン、アンモニウムイオン、炭素数１〜８のアルキルアンモニウムイオン（メチルアンモニウム、トリメチルアンモニウム、テトラメチルアンモニウム、メチルトリエチルアンモニウム等）を表し、アルカリ金属イオンが好ましく、さらに好ましくはナトリウムイオンである（以下、同様である。）。 Examples of the fluorine atom-containing anionic surfactant (a2) include perfluoroalkyl carboxylates and perfluoroalkyl phosphates.
The perfluoroalkyl carboxylate includes a compound represented by the formula (3).

j represents an integer of 0 to 20, an integer of 1 to 18 is preferable, and an integer of 2 to 12 is more preferable. Moreover, k represents the integer of 0-5, the integer of 1-4 is preferable, More preferably, it is the integer of 1-2. M represents an alkali metal ion (such as lithium, potassium, or sodium), an ammonium ion, or an alkylammonium ion having 1 to 8 carbon atoms (such as methylammonium, trimethylammonium, tetramethylammonium, or methyltriethylammonium). Is more preferable, and sodium ion is more preferable (hereinafter the same).

ｍは０〜２０の整数を表し、１〜１８の整数が好ましく、さらに好ましくは２〜１２の整数である。また、ｎは０〜５の整数を表し、１〜４の整数が好ましく、さらに好ましくは１〜２の整数である。 The perfluoroalkyl phosphate ester includes a compound represented by the formula (4).

m represents an integer of 0 to 20, an integer of 1 to 18 is preferable, and an integer of 2 to 12 is more preferable. Moreover, n represents the integer of 0-5, the integer of 1-4 is preferable, More preferably, it is the integer of 1-2.

  Among these fluorine atom-containing surfactants, perfluoroalkylamine oxides, perfluoroalkylethylene oxide adducts and perfluoroalkyl carboxylates are preferable from the viewpoint of chemiluminescence enhancing effect and storage stability, and more preferably perfluoroalkylcarboxylates. Fluoroalkylamine oxides and perfluoroalkylethylene oxide adducts, particularly preferably perfluoroalkylethylene oxide adducts.

The content of the fluorine atom-containing surfactant (a) is appropriately set depending on the type, measurement method to be applied, measurement conditions, and the like, but the following ranges are preferable from the viewpoints of chemiluminescence enhancing effect and storage stability.
That is, the content (mol%) of the fluorine atom-containing surfactant (a) is such that the fluorine atom-containing surfactant (a), 2,3-dihydro-1,4-phthalazinedione compound (b) and chemiluminescence. Based on the number of moles of the enhancer (c), 3 to 54 is preferable, 5 to 51 is more preferable, and 7 to 48 is particularly preferable.

  The 2,3-dihydro-1,4-phthalazinedione compound (b) is known (for example, JP-A-2-291299, JP-A-10-31915, JP-A-2000-279196, etc.) Compounds and mixtures thereof can be used. Of these, luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI) and metal salts thereof are preferred, and luminol and luminol are more preferred. Of these, particularly preferred is the sodium salt of luminol.

The content of the 2,3-dihydro-1,4-phthalazinedione compound (b) is appropriately set according to the type and measurement method to be applied, measurement conditions, and the like. A range is preferred.
That is, the content (mol%) of the 2,3-dihydro-1,4-phthalazinedione compound (b) is the same as the fluorine atom-containing surfactant (a), 2,3-dihydro-1,4-phthalazine. Based on the number of moles of the dione compound (b) and the chemiluminescence enhancer (c), it is preferably 14 to 95, more preferably 20 to 92, and particularly preferably 26 to 89.

  As the chemiluminescence enhancer (c), known compounds (for example, JP-A-59-500262, JP-A-59-171839, JP-A-2-291299, etc.) and mixtures thereof are used. it can. Of these, phenol is preferred from the viewpoint of chemiluminescence enhancing effect, etc., more preferably P-iodophenol, 4- (cyanomethylthio) phenol and 4-cyanomethylthio-2-chlorophenol, particularly preferably 4- (cyano Methylthio) phenol.

The content of the chemiluminescence enhancer (c) is appropriately set depending on the type, measurement method to be applied, measurement conditions, and the like, but the following ranges are preferable from the viewpoint of the chemiluminescence enhancement effect and the like.
That is, the content (mol%) of the chemiluminescence enhancer (c) is as follows: fluorine atom-containing surfactant (a), 2,3-dihydro-1,4-phthalazinedione compound (b), and chemiluminescence enhancer. Based on the number of moles of (c), 2 to 32 is preferable, 3 to 29 is more preferable, and 4 to 26 is particularly preferable.

  In addition to the fluorine atom-containing surfactant (a), the 2,3-dihydro-1,4-phthalazinedione compound (b) and the chemiluminescence enhancer (c), the first liquid includes a buffer and / or a chelate. Agents and the like.

As the buffer solution, a known buffer solution (for example, JP-A No. 10-31915 or JP-A No. 2003-279489) can be used. Among these, from the viewpoint of chemiluminescence enhancing effect and storage stability, 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer, 2-hydroxy-3- [ 4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid monohydrate / sodium hydroxide buffer and piperazinyl-1,4-bis (2-hydroxy-3-propanesulfonic acid) dihydrate Product / sodium hydroxide buffer, more preferably 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer and 2-hydroxy-3- [4- (2 -Hydroxyethyl) -1-piperazinyl] propanesulfonic acid monohydrate / sodium hydroxide buffer, particularly preferably 3- [4- (2- Dorokishiechiru) -1-piperazinyl] propane sulfonic acid / sodium hydroxide buffer.
The concentration of the buffering agent in these buffers (mM: mmol / liter) is preferably 1 to 500, more preferably 5 to 300, and particularly preferably 10 to 200.

  When the buffer solution is contained, the content (% by weight) is preferably 20 to 10,000, more preferably 100 to 2000, and particularly preferably 200 to 1000, based on the weight of the fluorine atom-containing surfactant (a). It is. Within this range, the chemiluminescence enhancing effect and storage stability are further improved.

  As the chelating agent, known chelating agents (for example, JP-A-9-75099 or JP-A-2003-279489) can be used. Among these, from the viewpoint of chemiluminescence enhancing effect and storage stability, a 4-coordinate chelating agent is preferable, and ethylenediaminetetraacetic acid (EDTA) and its salt (ethylenediaminetetraacetic acid disodium, ethylenediaminetetraacetic acid trisodium, Ethylenediaminetetraacetic acid tetrasodium, ethylenediaminetetraacetic acid dipotassium and ethylenediaminetetraacetic acid tripotassium, etc.), and trans-1,2-diaminocyclohexane-N, N, N ′, N′-tetraacetic acid (CyDTA), particularly preferably ethylenediaminetetraacetic acid Acetic acid (EDTA) and its salts.

  When the chelating agent is contained, the content (% by weight) is preferably 0.001 to 4, more preferably 0.01 to 2, particularly preferably based on the weight of the fluorine atom-containing surfactant (a). Is 0.05-1. Within this range, the chemiluminescence enhancing effect and storage stability are further improved.

The first liquid is preferably alkaline, more preferably pH (20 ° C.) 7 to 11, and particularly preferably pH (20 ° C.) 8 to 10.
The pH is measured according to JIS K0400-12-10: 2000.

  The first liquid is a fluorine atom-containing surfactant (a), a 2,3-dihydro-1,4-phthalazinedione compound (b), a chemiluminescence enhancer (c), and, if necessary, a buffer and / or It can be easily obtained by uniformly mixing the chelating agent.

  As the oxidizing agent (d), known oxidizing agents (for example, described in JP-A-8-261944 and JP-A-2000-279196) and mixtures thereof can be used. Of these, hydrogen peroxide, sodium perborate and potassium perborate are preferable from the viewpoint of storage stability and the like, and hydrogen peroxide is more preferable.

The concentration of the oxidizing agent (d) is appropriately set depending on the type, the applied measurement method, measurement conditions, and the like, but the following ranges are preferable from the viewpoint of the chemiluminescence enhancing effect and the like.
That is, the concentration (mM; mmol / liter) of the oxidizing agent (d) in the second liquid is preferably 0.1 to 50, more preferably 0.5 to 20, and particularly preferably 1 to 10.

  Examples of water (e) include distilled water, reverse osmosis water, and deionized water. Of these, distilled water and deionized water are preferable from the viewpoint of chemiluminescence enhancing effect and storage stability, and more preferably deionized water.

The second liquid can contain a chelating agent and the like in addition to the oxidizing agent (d) and water (e).
When the chelating agent is contained, the content (% by weight) is preferably 0.2 to 100, more preferably 0.5 to 20, particularly preferably 1 to 10 based on the weight of the oxidizing agent (d). It is. Within this range, the chemiluminescence enhancing effect and storage stability are further improved.
The second liquid can be easily obtained by using the oxidizing agent (d) as it is, or adjusting the concentration with water, and if necessary, adding a chelating agent and mixing them uniformly.

The chemiluminescent reagent kit for measuring peroxidase according to the present invention is the same as the chemiluminescent reagent kit known in the art {for example, JP-A-2-291299, JP-A-10-319015, JP-A-2000-279196, etc.). Can be used. The luminescence reaction proceeds by adding and mixing the first solution and the second solution to the specimen containing peroxidase. As an addition method, the first solution is added to the sample containing peroxidase, and then the second solution is added (1). The second solution is added to the sample containing peroxidase, and then the first solution is added. Method (2) for adding, Method (3) for simultaneously adding the first liquid and the second liquid to the specimen containing peroxidase, mixing the first liquid and the second liquid, and then mixing the liquid mixture with the specimen containing peroxidase The method (4) of adding to is mentioned. Among these, from the viewpoints of operability and chemiluminescence enhancement effect, the method (3) in which the first solution and the second solution are simultaneously added to the specimen containing peroxidase is preferable.
The liquid volume ratio between the first liquid and the second liquid is appropriately set depending on the measurement method to be applied, the measurement conditions, and the like, but from the viewpoint of operability and the like, it is preferable to set the same volume.

  The chemiluminescent reagent kit of the present invention can be used for a measurement method using peroxidase; an enzyme immunoassay method; a method for detecting a target nucleic acid in a genome by chemiluminescence using a nucleic acid probe; a Southern blot method and a Northern blot method, Among these, it is suitable for enzyme immunoassay; Southern blot and Northern blot, and is most suitable for enzyme immunoassay.

  The chemiluminescence reagent kit for peroxidase measurement of the present invention can contain a labeled antibody reagent, an antibody-bound solid phase reagent, and the like in addition to the first solution and the second solution. For example, the reagent etc. which are used for the enzyme immunoassay method (JP, 2004-264294, JP, 2004-208693, etc.) using peroxidase can be included.

As the peroxidase used for the peroxidase measurement, ordinary ones {peroxidase extracted from horseradish, microorganisms, milk, leukocytes, etc.} can be used. Of these, horseradish-derived peroxidase.
Note that peroxidase is in the form of a complex bound to a ligand (for example, antigen, antibody, hapten, protein A, avidin, biotin, etc.) used in immunoassay even in a free state. Also good.

  When the chemiluminescence reagent kit for measuring peroxidase of the present invention is used, ultra-trace substances (several pg / mL to several tens pg / mL) in a living body can be measured with high accuracy. Examples of ultra trace substances include PSA (human prostate specific antigen), ProGRP (gastrin releasing peptide precursor), BNP (brain natriuretic peptide) and the like. In particular, regarding PSA, the PSA value of a patient after prostatectomy is used for postoperative follow-up (monitoring), and in recent years, higher sensitivity is required. By using the chemiluminescent reagent kit of the present invention, highly accurate monitoring can be performed, which can contribute to the care of postoperative patients.

EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this. Unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.
<Synthesis Example 1>
_{CF 3 - (CF 2) 3} - (CH 2) 2 -OH { Daikin Chemical Sales Co., Ltd., 2- (perfluorobutyl) ethanol} 100 parts of potassium hydroxide {manufactured by Wako Pure Chemical Industries, Ltd. , Reagent special grade} and 0.5 parts of ethylene oxide and 400 parts of ethylene oxide were reacted at 160 ° C. for 3 hours by a conventional method, and then potassium hydroxide was removed by adsorption (Kyoward treatment) to obtain 8 mol of perfluoroalkylethylene oxide. An adduct (a11) was obtained.

<Synthesis Example 2>
_{CF 3 - (CF 2) 3} - (CH 2) 2 -OH { Daikin Chemical Sales Co., Ltd., 2- (perfluorobutyl) ethanol} 100 parts of water 900 parts of potassium hydroxide {Wako Pure Chemical Industries, (Co., Ltd., reagent special grade) 10 parts were mixed, and then potassium permanganate {Wako Pure Chemical Industries, Ltd., reagent special grade} 5 parts was used, and the mixture was reacted at 120 ° C. for 5 hours by a conventional method. Thereafter, potassium hydroxide, manganese dioxide and the like were removed by adsorption (Kyoward treatment) to obtain potassium perfluoroalkylcarboxylate (a21).

<Synthesis Example 3>
Using 100 parts of perfluoroalkylcarboxylic acid potassium salt (a21) obtained in Synthesis Example 2 and 20 parts of ethanol, 1 part of sulfuric acid {manufactured by Wako Pure Chemical Industries, Ltd., reagent grade} and 50 parts of cyclohexane Was allowed to react at 75 ° C. for 6 hours, and a perfluoroalkylcarboxylic acid ester was obtained by distillation.
Next, 100 parts of the obtained perfluoroalkylcarboxylic acid ester and 20 parts of dimethylamine hydrochloride {manufactured by Wako Pure Chemical Industries, Ltd., purity 98%} are reacted at 30 ° C. for 1 hour by a conventional method. The perfluoroalkylamide was obtained.
Further, using 100 parts of the obtained perfluoroalkylamide and 100 parts of water and 10 parts of hydrogen peroxide water (manufactured by Wako Pure Chemical Industries, Ltd., reagent grade, purity 30%), by a conventional method, 50 ° C., The reaction was carried out for 10 hours to obtain a perfluoroalkylamine oxide compound (a12).

<Synthesis Example 4>
_{CF 3 - (CF 2) 3} - (CH 2) 2 -OH { Daikin Chemical Sales Co., Ltd., 2- (perfluorobutyl) ethanol} 100 parts of phosphorus oxychloride {Nacalai Tesque Co., Ltd., reagent First grade, purity 98%} 70 parts were mixed and reacted at 20 ° C. for 1 hour by a conventional method, and then 100 parts of water was added to obtain perfluoroalkyl phosphoric acid. Next, 100 parts of sodium hydroxide {manufactured by Wako Pure Chemical Industries, Ltd., reagent grade} was added and reacted at 20 ° C. for 1 hour to obtain sodium perfluoroalkylphosphate (a22).

<Synthesis Example 5>
“CF ₃ — (CF ₂ ) ₃ — (CH ₂ ) ₂ —OH” is replaced by “CF ₃ — (CF ₂ ) ₉ — (CH ₂ ) ₂ —OH” manufactured by Daikin Chemicals Sales Co., Ltd. Perfluoroalkylethylene oxide 8-mole adduct (a13) was obtained in the same manner as in Synthesis Example 1 except that it was changed to "fluorodecyl) ethanol}".

<Synthesis Example 6>
“CF ₃ — (CF ₂ ) ₃ — (CH ₂ ) ₂ —OH” is replaced by “CF ₃ — (CF ₂ ) ₉ — (CH ₂ ) ₂ —OH” manufactured by Daikin Chemicals Sales Co., Ltd. Perfluoroalkylcarboxylic acid potassium salt (a23) was obtained in the same manner as in Synthesis Example 2 except that the fluorodecyl) ethanol} was changed.

<Synthesis Example 7>
“CF ₃ — (CF ₂ ) ₃ — (CH ₂ ) ₂ —OH” is replaced by “CF ₃ — (CF ₂ ) ₉ — (CH ₂ ) ₂ —OH” manufactured by Daikin Chemicals Sales Co., Ltd. Synthesis example 3 except that “fluorodecyl) ethanol” was changed, “ethanol 20 parts” was changed to “ethanol 8 parts”, and “dimethylamine 20 parts” was changed to “dimethylamine 8 parts”. In the same manner as described above, a perfluoroalkylamine oxide compound (a14) was obtained.

<Synthesis Example 8>
“CF ₃ — (CF ₂ ) ₃ — (CH ₂ ) ₂ —OH” is replaced by “CF ₃ — (CF ₂ ) ₉ — (CH ₂ ) ₂ —OH” manufactured by Daikin Chemicals Sales Co., Ltd. Perfluoroalkyl phosphate sodium salt (a24) in the same manner as in Synthesis Example 4, except that the compound was changed to "fluorodecyl) ethanol}" and "70 parts of phosphorus oxychloride" was changed to "28 parts of phosphorus oxychloride". )

<Example 1>
The chemiluminescence reagent kit for peroxidase measurement of the present invention was obtained as follows {consisting of the first solution, the second solution, an anti-PSA polyclonal rabbit antibody-binding bead reagent and a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent. }.
1) Preparation of the first liquid 2.8 parts of sodium salt of luminol (b1), 0.56 parts of 4- (cyanomethylthio) phenol (c1), 3 parts of perfluoroalkylethylene oxide 8 mol adduct (a11), 3- First, 1000 parts of [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer (36.2 mM, pH 8.6) were uniformly mixed to prepare the first liquid (A1), It was stored refrigerated (2-10 ° C.) until used for measurement.

2) Preparation of second liquid 0.14 parts of hydrogen peroxide {made by Wako Pure Chemical Industries, Ltd., reagent grade, 30%} is dissolved in 1 L of deionized water to prepare a second liquid for measurement. Stored refrigerated (2-10 ° C.) until used.

3) Preparation of Anti-PSA Polyclonal Rabbit Antibody-Binding Bead Reagent Anti-PSA polyclonal rabbit antibody {manufactured by Dako Cytation Co., Ltd.} was dissolved in 0.1 M carbonate buffer at pH 9 at a concentration of 20 μg / mL. To 50 mL of this solution, 2,000 polystyrene beads having a diameter of 3.2 mm (manufactured by Immunochemical Co., Ltd.) were added and allowed to stand for 48 hours to physically adsorb the anti-PSA polyclonal rabbit antibody to the polystyrene beads.
Thereafter, the solution was removed by suction with an aspirator, and the beads were washed twice with 50 mL of a 0.1% bovine serum albumin-containing phosphate buffer to prepare anti-PSA polyclonal rabbit antibody-bound beads. This anti-PSA polyclonal rabbit antibody-bound bead was again immersed in 50 mL of 0.1% bovine serum albumin-containing phosphate buffer to prepare an anti-PSA polyclonal rabbit antibody-bound bead reagent and stored refrigerated (2 to 10 ° C.). .

4) Preparation of peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent Anti-PSA polyclonal rabbit antibody {manufactured by Dako Cymation Co., Ltd.}, horseradish-derived peroxidase (manufactured by Toyobo Co., Ltd.), literature [S Yoshitake, M. Imagawa, Yi Ishikawa, Etol; Jay. Biochem, Vol. 92 (1982) 1413-1424], a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent was prepared and stored frozen (-25 to -35 ° C).

Next, using the chemiluminescent reagent kit for measuring peroxidase of the present invention, the amount of chemiluminescence was measured as follows, and the results are shown in Table 1 {hereinafter, measured in the same manner, and the results are shown in Table 1. It was shown to. }.
Sample for measurement by mixing 1 μL of standard PSA solution (PSA concentration 100 ng / mL) {manufactured by Sanyo Kasei Kogyo Co., Ltd., trade name: Spherelite PSA calibrator set} and 999 μL of 1% bovine serum albumin-containing phosphate buffer Adjusted. 300 μL of this measurement sample and one bead taken out from the anti-PSA polyclonal rabbit antibody-bound bead reagent using tweezers were reacted (37 ° C.) for 1 hour in a test tube (12 × 75 mm).
Subsequently, after removing the reaction solution from the test tube with an aspirator, 1 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator to obtain reaction beads.
Next, a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent was diluted with a 1% bovine serum albumin-containing phosphate buffer to an antibody concentration of 1 μg / mL to prepare a labeled antibody solution. 300 μL of this labeled antibody solution was added to a test tube containing reaction beads and reacted at 37 ° C. for 1 hour. The reaction solution was removed with an aspirator, 1 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator to obtain washed beads.
The test tube containing the washed beads was set in a luminescence reader BLR-201 type sample holder manufactured by Aroka, and 200 μL of the first solution and 200 μL of the second solution were simultaneously added to the test tube to start the chemiluminescence reaction.
Forty seconds after the start of the chemiluminescence reaction, the amount of luminescence (integrated amount) for 10 seconds was measured, and this integrated amount was defined as the luminescence amount (C) indicating enzyme activity.
Note that the amount of luminescence measured in the same manner using 1 μL of 1% bovine serum albumin-containing phosphate buffer instead of 1 μL of the standard PSA solution was defined as a blank luminescence amount (D). Moreover, the ratio (C / D) which remove | divided the light-emission amount (C) which shows an enzyme activity by the light-emission amount (D) of the blank was made into the measurement sensitivity.

<Example 2>
The chemiluminescent reagent kit for measuring peroxidase of the present invention was obtained in the same manner as in Example 1 except that the “perfluoroalkylethylene oxide 8 mol adduct (a11)” was changed to “perfluoroalkylamine oxide compound (a12)”. The obtained {first solution, second solution, anti-PSA polyclonal rabbit antibody-binding bead reagent and peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent are included. }.

<Example 3>
The chemiluminescence reagent kit for peroxidase measurement of the present invention is the same as in Example 1 except that the “perfluoroalkylethylene oxide 8 mol adduct (a11)” is changed to “perfluoroalkylcarboxylic acid potassium salt (a21)”. {The first solution, the second solution, an anti-PSA polyclonal rabbit antibody-binding bead reagent and a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent. }.

<Example 4>
Except having changed "perfluoroalkyl ethylene oxide 8 mol adduct (a11)" into "perfluoroalkyl phosphate sodium salt (a22)", it carried out similarly to Example 1,
A chemiluminescence reagent kit for measuring peroxidase of the present invention was obtained {consisting of a first solution, a second solution, an anti-PSA polyclonal rabbit antibody-binding bead reagent and a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent. }.

<Example 5>
The chemiluminescent reagent for measuring peroxidase according to the present invention was the same as in Example 1 except that "perfluoroalkylethylene oxide 8 mol adduct (a11)" was changed to "perfluoroalkylethylene oxide 8 mol adduct (a13)". A kit was obtained {consisting of a first solution, a second solution, an anti-PSA polyclonal rabbit antibody-binding bead reagent and a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent. }.

<Example 6>
The chemiluminescent reagent kit for measuring peroxidase of the present invention was obtained in the same manner as in Example 1 except that the “perfluoroalkylethylene oxide 8 mol adduct (a11)” was changed to “perfluoroalkylamine oxide compound (a14)”. The obtained {first solution, second solution, anti-PSA polyclonal rabbit antibody-binding bead reagent and peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent are included. }.

<Example 7>
Chemiluminescent reagent kit for peroxidase measurement of the present invention in the same manner as in Example 1 except that “perfluoroalkylethylene oxide 8 mol adduct (a11)” was changed to “perfluoroalkylcarboxylic acid potassium salt (a23)” {The first solution, the second solution, an anti-PSA polyclonal rabbit antibody-binding bead reagent and a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent. }.

<Example 8>
Chemiluminescent reagent kit for peroxidase measurement of the present invention in the same manner as in Example 1 except that "perfluoroalkylethylene oxide 8 mol adduct (a11)" was changed to "perfluoroalkyl phosphate sodium salt (a24)" {The first solution, the second solution, an anti-PSA polyclonal rabbit antibody-binding bead reagent and a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent. }.

<Comparative example>
2.8 parts of luminol sodium salt, 0.56 parts of 4- (cyanomethylthio) phenol, and 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid / sodium hydroxide buffer (36. 2 mM, pH 8.6) 1000 parts were uniformly mixed to prepare the first liquid (H1), which was stored refrigerated (2-10 ° C.) until used for measurement.
A chemiluminescent reagent kit for comparison peroxidase measurement was obtained in the same manner as in Example 1 except that the first liquid was changed to the first liquid in Example 1. {First liquid, second liquid, anti-PSA It consists of a polyclonal rabbit antibody-bound bead reagent and a peroxidase-labeled anti-PSA polyclonal rabbit antibody reagent. }.

When the chemiluminescence reagent kit for measuring peroxidase of the present invention was used, the amount of luminescence was remarkably higher (high sensitivity) than when a comparative chemiluminescence reagent kit was used. Particularly in Examples 5 to 6, extremely high sensitivity was shown.

Claims (2)

A first liquid comprising a fluorine atom-containing surfactant (a), a 2,3-dihydro-1,4-phthalazinedione compound (b) and a chemiluminescence enhancer (c) as essential components;
A chemiluminescent reagent kit for measuring peroxidase, comprising a second liquid comprising oxidant (d) and water (e) as essential constituents. The peroxidase measurement according to claim 1, wherein the fluorine atom-containing surfactant (a) is at least one selected from the group consisting of perfluoroalkylamine oxides, perfluoroalkylethylene oxide adducts, and perfluoroalkylcarboxylates. Chemiluminescent reagent kit.