JPH1038809A - Chemiluminescent reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To increase total chemiluminescence quantity so as to improve measurement precision by using a chemiluminescent reagent consisting of a solution which contains a specific chemiluminescent substance, a specific sensitizer, and two or more kinds of buffers with specific pKa values and shows a specific pH value. SOLUTION: For a chemiluminescent substance, a luminol group nitrogen- containing heterocyclic compound, which is oxidized under alkaline conditions so as to generate chemiluminescence, such as luminol and a luminol derivative including isoluminol or an acridine group nitrogen-containing heterocyclic compound is available, however, luminol or its derivative is desirable. For a sensitizer, a phenol derivative, a naphthol derivative and the like are available. Two or more kinds of compounds, each of which is provided with a pKa value of 8.0-10.5 at a temperature of 20 deg.C, are selected from an amine group buffer, a Good's buffer, a carbonate buffer and used for buffers. The chemiluminescent reagent solution is prepared so as to exhibit a pH value of 9.0-10.0.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to, for example, serum, plasma, urine,
The present invention relates to a reagent for chemiluminescence used for measuring a trace component in a sample derived from a living body such as a cerebrospinal fluid.

[0002]

BACKGROUND OF THE INVENTION Chemiluminescence-based analysis and measurement of specific components are often used especially for the measurement of trace components due to their high measurement sensitivity. Recently, it has been often practiced to extend the measurement time to further increase the measurement sensitivity and measure the total light emission amount during that time.

However, the amount of light emitted by chemiluminescence is
Since it generally decreases with time, there are many cases where the total light emission amount does not increase so much even if the measurement time is extended, in other words, the measurement sensitivity does not increase so much.

[0004] In some cases, a reagent called a sensitizer is added to a reagent for measurement utilizing chemiluminescence in order to increase the sensitivity of chemiluminescence. The problem also occurred when a reagent to which such a component was added was used.

[0005] Therefore, it is presently desired to develop a reagent for chemiluminescence which can simply and effectively further increase the total amount of chemiluminescence and enable highly sensitive measurement.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object thereof is to provide a simple and effective reagent for chemiluminescence and a measuring method using the same.

[0007]

In order to solve the above problems, the present invention provides (1) a chemiluminescent substance, a sensitizer, and p
A chemiluminescence reaction reagent comprising a solution having a pH of 9.0 to 10.0, comprising two or more buffers having a Ka value of 8.0 to 10.5 at 20 ° C.

Further, the present invention uses (2) a solution having a pH of 9.0 to 10.0 containing a chemiluminescent substance, a sensitizer and two or more buffers having a pKa value of 8.0 to 10.5 at 20 ° C. It is an invention of a method for measuring peroxidase activity, hydrogen peroxide or peroxidase-binding protein, characterized in that:

Furthermore, the present invention relates to (3) a) a chemiluminescent substance, a sensitizer, and two or more buffers having a pKa value of 8.0 to 10.5 at 20 ° C. It is an invention of a reagent kit for a chemiluminescence reaction comprising a combination of a solution and b) a hydrogen peroxide-containing buffer.

Further, the present invention provides (4) a solution having a pH of 8.0 to 10.0 containing a chemiluminescent substance, a sensitizer, and a buffer having a pKa value of 8.0 to 10.5 at 20 ° C .; A reagent kit for a chemiluminescence reaction, comprising a combination of a buffer solution and a solution containing a buffer having a pKa value of 8.0 to 10.5 at 20 ° C.

The present invention further provides (5) a method for enhancing chemiluminescence, which comprises performing a chemiluminescent reaction using the reagent of (1).

That is, the present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, two or more buffers having a pKa value of 8.0 to 10.5 at 20 ° C. It has been found that the total amount of chemiluminescence can be further increased by adjusting the pH to 9.0 to 10.0 as a buffering agent, thereby completing the present invention.

The chemiluminescent substance according to the present invention is not particularly limited as long as it is generally used in this field, and specific examples thereof include, for example, luminol, for example, isoluminol and amino. Ethylisoluminol, aminoethylethylisoluminol, aminopropylisoluminol, aminobutylisoluminol,
Luminol derivatives such as aminohexylethyl isorminol and the like, luminol-based nitrogen-containing heterocyclic compounds that are oxidized under alkaline to produce chemiluminescence, and oxidized chemiluminescence such as acridinium salts and lucigenin. An acridine-based nitrogen-containing heterocyclic compound to be produced may be mentioned, and preferred is luminol or a derivative thereof.
These concentrations may be appropriately selected from the range usually used in this field, and are not particularly limited. Specifically, although the concentration slightly varies depending on the type of the chemiluminescent substance used, the chemiluminescence is usually used. Is appropriately selected from the range of 1 μM to 10 mM, preferably 30 μM to 3 mM, more preferably 100 μM to 1.5 mM.

As the sensitizer according to the present invention, any sensitizers which are usually used in this field can be used and are not particularly limited. For example, p-iodophenol, 4-hydroxycinnamic acid, 4- (4 Phenol derivatives such as' -thiazolyl) phenol, for example, naphthol derivatives such as 1-bromonaphthol, for example, oxazole derivatives such as 4- (4-hydroxyphenyl) oxazole, for example, 4- (4-hydroxyphenyl) Thiazole derivatives such as thiazole, for example, 4-
And imidazole derivatives such as (imidazol-1-yl) phenol. The concentration of these may be appropriately selected from the range usually used in this field, and is not particularly limited. Specifically, although the concentration slightly varies depending on the type of the sensitizer used, it is usually used. The concentration for performing chemiluminescence is 0.1 μM to 10 mM, preferably 100 μM to 5 mM, more preferably 0.2 mM to 2 mM.
It is appropriately selected from the range of mM. These sensitizers may be used in combination of two or more.

The buffer according to the present invention is selected from those usually used in this field and has a pKa value of 8.
It suffices that two or more of those having a ratio of 0 to 10.5, preferably 8.3 to 10.0 are appropriately selected and used. A pKa value of 8.0 to 10.5 at 20 ° C
As a specific example of the buffer which is, for example, a borate, for example, an amine-based buffer such as tris (hydroxymethyl) aminomethane and ethanolamine, for example, 5,5-diethyl barbiturate, for example, N, N- Bis (2-hydroxymethyl) glycine (BICINE), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-
Aminopropanesulfonic acid (CAPSO), N-cyclohexyl-
Good amino acids such as 2-aminoethanesulfonic acid (CHES), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), carbonates and the like, and more preferred are, for example, borate, Tris (hydroxymethyl) aminomethane, 5,5-diethyl barbiturate, N-
Cyclohexyl-3-aminopropanesulfonic acid (CAPS),
Carbonates and the like. These concentrations may be appropriately selected from the range usually used in this field. Specifically, the concentration varies slightly depending on the type of buffer used, but usually 0.05 M to 1.5 M, preferably 0.7 M, as the total concentration of two or more buffers at the time of performing chemiluminescence.
It is appropriately selected from the range of 5M to 1M, more preferably 0.1M to 0.6M. The molar ratio of these two types of buffers is different depending on the combination of the buffers, and may be any ratio that can achieve the object of the present invention, and is not particularly limited.
Usually, it is appropriately selected from the range of 1: 0.3 to 10, preferably 1: 0.5 to 5. In consideration of the effect of enhancing chemiluminescence,
The pKa value of the buffer is preferably 8.3 to 10.0 at 20 ° C.

The chemiluminescent reagent of the present invention is prepared, for example, as follows. That is, the chemiluminescent substance, the sensitizer, and the two or more buffers having a pKa value of 8.0 to 10.5 at 20 ° C. are dissolved so that the concentration at the time of chemiluminescence becomes the above-described concentration. If a solution having a pH of 9.0 to 10.0 is prepared, it becomes the reagent of the present invention.

The reagent for chemiluminescence of the present invention contains a compound which oxidizes a chemiluminescent substance to generate chemiluminescence. Therefore, the reagent is combined with, for example, hydrogen peroxide, perchlorate, oxygen and permanganate. Chemiluminescence can be generated by reacting an oxidizing agent such as iodine or the like with a substance having peroxidase activity such as a peroxidase such as a heme compound such as hemoglobin.

Therefore, the reagent for use in the measurement of peroxidase activity can be obtained by further adding the oxidizing agent as described above to the reagent for chemiluminescence of the present invention.

The origin of the peroxidase which can be measured by such a reagent is not particularly limited. For example, peroxidase derived from plants such as horseradish, pineapple and figs, and derived from microorganisms such as mold and yeast can be used. For example, those derived from animal leukocytes, thyroid gland and the like are preferable. In addition, the peroxidase used for measuring the activity is itself (unmodified),
For example, any of those bound to an antigen, an antibody or the like may be used without any particular limitation.

To measure the peroxidase activity derived from, for example, horseradish using these reagents, the following method may be used. That is, the chemiluminescent substance, the sensitizer, and the two or more buffers having a pKa value of 8.0 to 10.5 at 20 ° C. as described above are adjusted to have the concentrations as described above at the time of chemiluminescence, and an appropriate amount. Oxidizing agents, such as hydrogen peroxide
A solution having a pH of 9.0 to 10.0 dissolved in 0.1 to 10 mM, preferably 1 to 5 mM is used as a luminescent solution. Next, this is mixed with a sample containing peroxidase, and the amount of generated chemiluminescence is measured with a luminometer or the like.
Separately, a luminescence solution is mixed with a standard solution having a predetermined peroxidase activity, and the amount of chemiluminescence generated is measured by a luminometer.
The calibration curve representing the relationship between the peroxidase activity and the amount of chemiluminescence is prepared in advance, and the amount of chemiluminescence of the obtained sample is applied to the calibration curve to determine the peroxidase activity in the sample.

The detection of peroxidase activity bound to an antibody or the like may be performed, for example, as follows.
That is, a sample containing a substance to be measured is blotted on a nitrocellulose membrane and dried. Then, after performing a masking treatment, the antibody is reacted with the substance to be measured. After washing the membrane, peroxidase bound to the antibody capable of binding the antibody is reacted. After washing, the membrane is immersed in the luminescent solution and exposed on a film such as a polaroid film or an X-ray film, and the peroxidase activity can be detected from the resulting luminescent image.

When the reagent for chemiluminescence of the present invention is used in combination with a substance having peroxidase activity as described above, it is possible to measure an oxide such as hydrogen peroxide. The measurement of hydrogen peroxide using such a reagent may be performed, for example, as follows. That is, when the chemiluminescent substance, the sensitizer, and the pKa value
The concentration at the time of chemiluminescence of two or more buffer agents of 8.0 to 10.5 is adjusted to the concentration as described above, and a suitable amount of a substance having peroxidase activity, for example, peroxidase is adjusted to 0.1 to 2 u / ml. PH 9.0 to 10.0
Is used as a luminescent solution. Next, this is mixed with a sample containing hydrogen peroxide, and the amount of generated chemiluminescence is measured with a luminometer or the like. Separately, a luminescence solution is mixed with a standard solution containing a predetermined concentration of hydrogen peroxide, and the amount of generated chemiluminescence is measured with a luminometer or the like, and a calibration curve showing the relationship between the concentration of hydrogen peroxide and the amount of chemiluminescence is obtained. Is prepared in advance, and the concentration of hydrogen peroxide in the sample can be determined by applying the amount of chemiluminescence of the obtained sample to the calibration curve.
In addition, the origin of the hydrogen peroxide to be measured is not particularly limited, but preferably includes, for example, those generated by an oxidase reaction.

The use of the reagent of the present invention makes it possible to measure trace components in a sample derived from a living body such as serum, plasma, urine, and cerebrospinal fluid with high sensitivity and high accuracy. That is,
As described above, since the peroxidase activity and hydrogen peroxide can be measured with high sensitivity and accuracy by using the reagent of the present invention, a conventional enzyme immunoassay using peroxidase as a labeling enzyme or an oxidase reaction was used. If the reagent of the present invention is used in combination with the method for measuring trace components, trace components that can be measured by these known methods are:
It is possible to measure with higher sensitivity and accuracy.

When chemiluminescence is carried out using the reagent of the present invention, the amount of chemiluminescence after the reaction is remarkably increased as compared with the conventional one, and the effect lasts for a long time. , The total light emission amount is significantly increased as compared with the conventional one. When chemiluminescence is carried out using a solution having a pH of 9.0 to 10.0, comprising a chemiluminescent substance, a sensitizer, and at least one of the two or more buffers having a pKa value of 8.3 to 10.5. It was quite surprising that such a phenomenon would occur.

In the present invention, a) a chemiluminescent substance, a sensitizer, and
A reagent kit for a chemiluminescence reaction comprising a combination of a solution having a pH of 9.0 to 10.0 and a buffer containing hydrogen peroxide containing two or more buffers having a pKa value of 8.0 to 10.5 at 20 ° C. It is suitable for measuring peroxidase activity, and the preferred embodiments of its constituent elements are as described above.

A) a solution having a pH of 9.0 to 10.0 containing a) a chemiluminescent substance, a sensitizer, and two or more buffers having a pKa value of 8.0 to 10.5 at 20 ° C .; The hydrogen oxide-containing buffer is usually 1 to 10: 1 to 5, preferably 1 to 4: 1 to 1
The mixture mixed at a ratio of 2 is used as a reagent solution for measuring peroxidase activity.

The concentration of the chemiluminescent substance in the solution a) is usually 2 μM to 100 mM, preferably 60 μM to 40 mM.
M, more preferably 200 μM to 6 mM, and the concentration of the sensitizer is generally 0.2 μM to 100 mM, preferably 200 μM to 200 mM.
μM to 20 mM, more preferably 0.4 μM to 10 mM, and the concentration of the buffer is usually 0.05 to 1.5 M, preferably 0.075 to 1 M as the sum of the concentrations of two or more buffers.
More preferably, it is 0.1 to 0.6M.

As the buffer containing hydrogen peroxide of b), hydrogen peroxide contains 0.2 mM to 100 mM, preferably 2 mM.
PH between 8.3 and 10.0, preferably 8.5
9.59.5 buffer. The buffer used at this time is not particularly limited as long as it is usually used in this field. For example, borates such as amine buffers such as tris (hydroxymethyl) aminomethane and ethanolamine; , 5-diethyl barbiturate, such as N, N-bis (2-hydroxymethyl) glycine (BIC
INE), N-cyclohexyl-3-aminopropanesulfonic acid
(CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-tris (hydroxymethyl) methyl-3-aminopropanesulfone Preferable examples include a good buffer such as an acid (TAPS), a carbonate, and the like, and the use concentration thereof is appropriately selected usually from the range of 0.01 to 1.5 M, preferably 0.05 to 1 M, and more preferably 0.1 to 0.5 M.

The pH of the reagent of a) and the reagent of b) are a)
The pH of a luminescent solution prepared by mixing the reagent of the formula (1) and the reagent of the formula (b) at a predetermined ratio as described above is appropriately selected so as to be 9.0 to 10.0. Further, the molar ratio of the two kinds of buffers in the luminescent solution thus prepared depends on the combination of the buffers, and may be any ratio that can achieve the object of the present invention, and is not particularly limited. Usually, it is appropriately selected from the range of 1: 0.3 to 10, preferably 1: 0.5 to 5.

The p-protein of the present invention contains a chemiluminescent substance, a sensitizer and a buffer having a pKa value of 8.0 to 10.5 at 20 ° C.
H8.0-1.0 solution, hydrogen peroxide containing buffer, p
A chemiluminescence reaction reagent kit comprising a combination with a solution containing a buffer having a Ka value of 8.0 to 10.5 at 20 ° C. is also suitable for measuring peroxidase activity, and is a preferred embodiment of its constituent elements. Etc. are as described above.

The kit contains a chemiluminescent substance, a sensitizer, and a buffer having a pKa value of 8.0 to 10.5 at 20 ° C.
8.0 to 10.0 solution and hydrogen peroxide-containing buffer, pKa
The solution containing a buffer having a value of 8.0 to 10.5 at 20 ° C. is generally 1 to 10: 1 to 5: 1 to 5, preferably 1 to 4: 1 to 1
The mixture mixed at a ratio of 2: 2 is used as a reagent for measuring peroxidase activity.

The concentration of the chemiluminescent substance in the above solution is usually 3 μM to 150 mM, preferably 90 μM to 60 mM.
M, more preferably 300 μM to 9 mM, and the concentration of the sensitizer is usually 0.3 μM to 150 mM, preferably 300 μM to 150 mM.
The concentration is from μM to 30 mM, more preferably from 0.6 μM to 15 mM, and the concentration of the buffer is usually from 0.05 to 1.5 M, preferably from 0.075 to 1 M, more preferably from 0.1 to 0.6 M.
The buffer may be used in combination of two or more kinds.

The buffer containing hydrogen peroxide contains 0.15 mM to 15 mM, preferably 0.75 mM, hydrogen peroxide.
pH of 8.3 to 10.0, preferably containing mM to 7.5 mM, preferably
8.5 to 9.5 buffers. The buffer used at this time is not particularly limited as long as it is usually used in this field. For example, borates such as amine buffers such as tris (hydroxymethyl) aminomethane and ethanolamine; , 5-diethyl barbiturate, such as N, N-bis (2-hydroxymethyl) glycine (BIC
INE), N-cyclohexyl-3-aminopropanesulfonic acid
(CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-tris (hydroxymethyl) methyl-3-aminopropanesulfone Preferable examples include a good buffer such as an acid (TAPS), a carbonate, and the like, and the use concentration thereof is appropriately selected usually from the range of 0.01 to 1.5 M, preferably 0.05 to 1 M, and more preferably 0.1 to 0.5 M.

Further, the above-mentioned pKa value is 8.0 at 20 ° C.
A solution containing a buffer having a pKa value of 20 ° C.
The pH of the buffer is usually 8.5-1.5 M, preferably 0.01-1.5 M, preferably 0.05-1 M, more preferably 0.1-0.5 M.
To 11.5, preferably 9.0 to 11.0.
The buffer may be used in combination of two or more kinds.

The above-mentioned reagent and the reagent
The pH of the luminescent solution prepared by mixing the above-mentioned reagent and the reagent at a predetermined ratio as described above is 9.0 to 9.0.
It is appropriately selected to be 10.0. Further, the molar ratio of the two kinds of buffers in the luminescent solution thus prepared depends on the combination of the buffers, and may be any ratio that can achieve the object of the present invention, and is not particularly limited. Usually, it is appropriately selected from the range of 1: 0.3 to 10, preferably 1: 0.5 to 5. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0036]

【Example】

Embodiment 1 FIG. (Luminescent solution) Tris (hydroxymethyl) aminomethane (pKa = 8.3)
0.1 M, 5,5-diethyl barbituric acid (pKa = 8.06) 0.4
M, a buffer (pH 9.4) containing 0.53 mM of luminol, 2.07 mM of H ₂ O _{2, and} 0.16 mM of 4- (4-hydroxyphenyl) thiazole. Boric acid (pKa = 9.28) 0.1 M, sodium carbonate (pKa = 10.
28) 0.08 M, luminol 0.53 mM, H ₂ O ₂ 2.07 mM, 4-
A buffer (pH 9.4) containing 0.16 mM of (4-hydroxyphenyl) thiazole. Boric acid (pKa = 9.28) 0.1 M, tris (hydroxymethyl) aminomethane (pKa = 8.3) 0.35 M, luminol 0.53
buffer containing 2.07 mM of H ₂ O _{2 and} 0.16 mM of 4- (4-hydroxyphenyl) thiazole (pH 9.4). (Procedure) 1 μl (537.5 pg to 2.1 pg in POD content) of a predetermined dilution of a peroxidase (POD) -labeled anti-mouse immunoglobulin antibody (derived from rabbit, manufactured by Dako) was added to nitrocellulose.
After blotting on a membrane and drying at room temperature, it was immersed in a predetermined luminescent solution. After 1 minute, remove the nitrocellulose membrane, wrap it with a polyvinylidene chloride film, and after 20 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 5 minutes against the X-ray film in a dark room It was exposed. Develop this film,
After fixing, the amount of POD detectable from the light emission image was determined.
Table 1 shows the results.

[0037]

[Table 1] From the results in Table 1, it can be seen that by using the reagent of the present invention, chemiluminescence can be maintained for a long time, and 67.2 pg of POD can be detected even after 6 hours.

Comparative Example 1 (Luminescent solution) Tris (hydroxymethyl) aminomethane (pKa = 8.3)
At a predetermined concentration, luminol 0.53 mM, H ₂ O ₂ 2.07 mM,
Buffer containing 0.16 mM 4- (4-hydroxyphenyl) thiazole (pH 9.4). Boric acid (pKa = 9.28) at specified concentration, luminol 0.53m
M, buffer containing 2.07 mM H ₂ O _{2 and} 0.16 mM 4- (4-hydroxyphenyl) thiazole (pH 9.4). (Operation method) Except for using the above as the luminescent solution,
Using the same reagent as in Example 1, the detectable POD amount was determined by the same operation method. Table 2 shows the results obtained using tris (hydroxymethyl) aminomethane as the buffer.
Table 3 shows the results obtained using boric acid as a buffer. Show.

[0039]

[Table 2] *-: Indicates that detection is not possible.

[0040]

[Table 3] *-: Indicates that detection is not possible. From the results shown in Tables 2 and 3, when only one buffer having a pKa at 20 ° C. of 8.0 to 10.5 was used, chemiluminescence could not be maintained for a long time, and as compared with the present invention. POD
It can be seen that the detection sensitivity of the compound greatly decreases.

Comparative Example 2 (Luminescent solution) Boric acid (pKa = 9.28) 0.1 M, triethanolamine (pKa
(Ka = 10.78) 0.15 M, luminol 0.53 mM, H ₂ O ₂ 2.07
mM, 4- (4-hydroxyphenyl) thiazole 0.16m
Buffer containing M (pH 9.4). Tris (hydroxymethyl) aminomethane (pKa = 8.3)
0.1 M, triethanolamine (pKa = 10.78) 0.15 M,
Luminometer - Le _{0.53mM, H 2 O 2 2.07mM,} 4- (4- hydroxyphenyl) thiazole - buffer containing Le 0.16 mM (p
H9.4). Citric acid (pKa = 6.39) 0.1M, tris (hydroxymethyl) aminomethane (pKa = 8.3) 0.04M, luminol 0.
_{53mM, H 2 O 2 2.07mM,} 4- (4- hydroxyphenyl)
Buffer containing 0.16 mM thiazole (pH 9.4). Boric acid (pKa = 9.28) 0.2M, phosphoric acid (pKa = 7.22) 0.1
M, a buffer (pH 9.4) containing 0.53 mM of luminol, 2.07 mM of H ₂ O _{2, and} 0.16 mM of 4- (4-hydroxyphenyl) thiazole. (Operation method) Except for using the above as the luminescent solution,
Using the same reagent as in Example 1, the detectable POD amount was determined by the same operation method. Table 4 shows the results.

[0042]

[Table 4] *-: Indicates that detection is not possible. From the results in Table 4, it is found that when a buffer having a pKa at 20 ° C. of 8.0 to 10.5 is used in combination with one buffer, the chemiluminescence cannot be maintained for a long time. It can be seen that the POD detection sensitivity is greatly reduced as compared with the present invention.

Comparative Example 3 (Luminescent solution) ε-aminocaproic acid (pKa = 10.98) 0.1 M, triethylamine (pKa = 10.78) 0.02 M, luminol 0.53 mM,
_{H 2 O 2 2.07mM, 4- (} 4- hydroxyphenyl) thiazole -
Buffer containing 0.16 mM (pH 9.4). Citric acid (pKa = 6.39) 0.1 M, triethylamine (pKa
= 10.98) 0.02 M, luminometer - Le 0.53mM, H ₂ O ₂ 2.07m
M, 4- (4-hydroxyphenyl) thiazole 0.16 mM
(PH 9.4). (Operation method) Except for using the above as the luminescent solution,
Using the same reagent as in Example 1, the detectable POD amount was determined by the same operation method. Table 5 shows the results.

[0044]

[Table 5] *-: Indicates that detection is not possible. From the results shown in Table 5, when two kinds of buffers having a pKa at 20 ° C outside the range of 8.0 to 10.5 were used in combination, chemiluminescence could not be maintained for a long time, and the present invention It can be seen that the POD detection sensitivity is significantly reduced in comparison.

Embodiment 2 FIG. (Luminescent solution) Boric acid (pKa = 9.28) 0.1 M, tris (hydroxymethyl) aminomethane (pKa = 8.3) 0.4 M, luminol 0.53
buffer containing 2.07 mM of H ₂ O _{2 and} 0.16 mM of 4- (4-hydroxyphenyl) thiazole (pH 9.4). Boric acid (pKa = 9.28) 0.1 M, luminol 0.53 mM, H ₂
O ₂ 2.07 mM, 4- (4-hydroxyphenyl) thiazole
Buffer containing 0.16 mM (pH 9.4). (Procedure) 300 μl of a predetermined luminescent solution and 1 μl of peroxidase (POD) -labeled anti-rabbit IgG antibody (porcine-derived, Dako, peroxidase content 134 pg / μl) were mixed, and the amount of chemiluminescence after the lapse of a predetermined time ( Kcpm) was measured using a luminometer (BLR-201, manufactured by Aloka). Table 6 shows the results.

[0046]

[Table 6] As is clear from the results in Table 6, when the luminescent solution of the present invention is used, chemiluminescence can be maintained for a long time, but when the conventional luminescent solution is used, It can be seen that the amount of chemiluminescence sharply decreases with time.

[0047]

As described above, the present invention provides a reagent for chemiluminescence used for measuring a trace component in a biological sample such as serum, plasma, urine, and cerebrospinal fluid. By using the present invention, it is possible to easily and effectively increase the amount of total chemiluminescence significantly more than conventional ones. For example, the measurement of trace components in a biological sample can be performed with higher accuracy. This is a great invention that contributes to the industry because it has the effect of obtaining.

Claims (6)

[Claims] 1. A chemiluminescent substance, a sensitizer and a pKa value of 20
PH 9.0 containing two or more buffers that are 8.0 to 10.5 at 0 ° C
A chemiluminescent reagent consisting of a solution of 110.0. 2. A chemiluminescent substance, a sensitizer and a pKa value of 20.
PH 9.0 containing two or more buffers that are 8.0 to 10.5 at 0 ° C
A method for measuring peroxidase activity or hydrogen peroxide, which comprises using a solution of 〜10.0. 3. A chemiluminescent substance, a sensitizer and a pKa value of 20.
PH 9.0 containing two or more buffers that are 8.0 to 10.5 at 0 ° C
A method for measuring a peroxidase-binding protein, characterized by using a solution of 0.1 to 10.0. 4. a) a chemiluminescent substance, a sensitizer and a pKa value
PH 9.0, containing two or more buffers that are 8.0-0.5 at 20 ° C.
A reagent kit for a chemiluminescence reaction comprising a combination of a solution of 0 to 10.0 and b) a buffer containing hydrogen peroxide. 5. A chemiluminescent substance, a sensitizer and a pKa value
A solution having a pH of 8.0 to 10.0 containing a buffer which is 8.0 to 10.5 at 20 ° C., a buffer containing hydrogen peroxide, and a solution having a pKa value of 20 ° C.
8. A reagent kit for a chemiluminescence reaction comprising a combination with a solution containing a buffer agent having a pH of from 0 to 10.5. 6. A method for enhancing chemiluminescence, comprising performing a chemiluminescence reaction using the reagent according to claim 1.