JP2000166595A - Reagent for determining conjugated bilirubin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject reagent for determining the conjugated bilirubin by using a chelating agent, a metal ion or a metal complex having the action to inhibit the reactions of the unconjugated bilirubin and albumen-bonding bilirubin. SOLUTION: In the reagent for determining bilirubin, at least one selected from a chelating agent, for example, nitrilotris (methylenephosphonic acid) trisodium salt (NTPO), ethylenediamine-N,N'-bis(methylenephosphonic acid) 1/2 H2O, a metal ion selected from divalent iron ion, divalent copper ion and the like and a metal complex, for example, disodium cobalt ethylenediaminetetraacetate, having the action to inhibit the reaction with the unconjugated bilirubin and (or) bilirubin covalently bonds to albumen is added to the bilirubin oxidase as the main reaction component whereby the objective reagent is obtained that can determine only the conjugated bilirubin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring reagent and a measuring method capable of separating and quantifying only conjugated bilirubin, and more particularly, to the inhibition of the reaction to unconjugated bilirubin and / or bilirubin covalently bound to albumin. The present invention relates to a reagent for measuring conjugated bilirubin and a measuring method containing one or more reaction inhibitors selected from a specific chelating agent having an action, a metal ion and a metal complex.

[0002]

2. Description of the Related Art Bilirubin in a living body is taken into hepatocytes and conjugated with glucuronic acid, and conjugated bilirubin (B
c) and is excreted through the biliary tract into bile. When the excretion pathway after this conjugation reaction is impaired (hepatotoxic disorder or biliary obstruction), serum conjugated bilirubin increases. From this, the measurement of conjugated bilirubin in serum is useful for the diagnosis and prognosis of hepatobiliary diseases, hepatobiliary diseases including intrahepatic and extrahepatic bile closure, Dubin-Johnson syndrome, hepatocellular disorders, Rotor syndrome and the like. Used for diagnosis. In order to diagnose the increase in conjugated bilirubin, generally, a diazotization method for directly measuring bilirubin, the Malloy-Evelyn method (Malloy, HT, Evelyn, KA: The determination)
of bilirubin with the photometric colorimeter.
J. Biol. Chem., 119: 481-490, 1937) and the like. However, in the diazo reaction, conjugated bilirubin covalently binds to albumin and reacts to delta bilirubin (Bδ) having a longer half-life, so that accurate measurement of conjugated bilirubin cannot be performed. Prognostic diagnosis of pathological disorders and diagnosis of chronic hepatotoxic disorders are inaccurate. Therefore, development of a reagent for measuring conjugated bilirubin (Bc) that does not measure Bδ has been desired.

At present, as a method for determining only conjugated bilirubin (Bc), high performance liquid chromatography (HP
LC) (Lauff, JJ, Kaspe
r, ME and Ambrose, RT: Separation of bilirub
in species in serum and bile by high-performance r
eversed-phase liquid chromatography .J. Chromat
o., 226: 391-402, 1981.) or the method of Adachi (Adac
hi, Y., Inufusa, H., Yamashita, M., Kambe, A., Yama
zaki, K., Sawada, Y., Yamamoto, T .: Clinical appl
ication of serum bilirubin fractionation by simpli
fied liquid chromatography. Clin. Chem., 34: 385-
388, 1988) and a Vitros dry bilirubin BuBc (Wu, TW., Dappen, GM, Sp.) Using a dedicated medical device (Kodak Ektachem Analyzer or Vitros Chemistry System).
ayde, RW, Sundberg, MW, Powers, DM: Th
e Ektachem clinical chemistry slide for simultaneo
us determination of unconjugated and sugarconjugat
ed bilirubin. Clin. Chem. 30: 1304-1309, 1984), but in order to measure conjugated bilirubin (Bc) using these methods, special equipment and dedicated measuring equipment are required. This is necessary, and the cost per test is high.

As a method for measuring conjugated bilirubin by an enzymatic method using bilirubin oxidase, the measurement is carried out by reacting a reagent consisting of an acidic buffer solution having a pH of 5 to 6 containing bilirubin oxidase and an anionic surfactant. Method (Japanese Patent Publication No. 5-9066), a method in which bilirubin oxidase is allowed to act on a sample containing bilirubin in a buffer solution having a pH in the range of 9 to 11 in the presence of an anionic surfactant (Japanese Patent Publication No. 5-68240). And a conjugated bilirubin measurement reagent characterized by containing bilirubin oxidase produced by a strain of Pseudomonasaceae (Japanese Patent Application Laid-Open No. 9-149794). However, in these measurement methods, when the reaction to conjugated bilirubin is made sufficient, there is a problem in that it reacts with some delta bilirubin and a problem that a special raw material is required. Also, among commercially available bilirubin measurement reagents corresponding to commonly used automatic analyzers, certain direct bilirubin measurement reagents do not react with bilirubin covalently bound to albumin, but only measure conjugated bilirubin. (A document distributed at the presentation site of the 35th Annual Meeting of the Japanese Society of Clinical Chemistry (October 6, 1995) "Evaluation of selectivity of bilirubin measurement by enzymatic method and its analysis") However, it has been reported that bilirubin covalently bound to albumin also reacts 17% in this reagent (The 37th Annual Meeting of the Japanese Society of Clinical Chemistry (1997)
Summary of "Differentiation Measurement and Clinical Evaluation of Conjugated Bilirubin"), a discriminative property seems to be insufficient as a reagent for measuring only conjugated bilirubin. It has also been reported that a chelating agent, a metal ion or a metal complex is used in the measurement of the fraction of bilirubin. For example, one or more chelating agents, preferably one or two or more metal ions, are also used. There is a method of directly measuring bilirubin by adding more than one species (Japanese Patent Application Laid-Open No. 9-37795). In this method, bilirubin covalently bonded to albumin is reacted in an effective pH range of pH 3.0 to 5.0. Further, when the pH is 4.0 to 5.0, it is necessary to increase the amount of bilirubin oxidase to be added or to add a reaction accelerator, so that the reaction between albumin and bilirubin covalently bonded is promoted. And it was difficult to measure only conjugated bilirubin.

[0005]

In view of the above circumstances, the present invention relates to a method for optically measuring conjugated bilirubin by the action of bilirubin oxidase, which is useful for bilirubin covalently bonded to unconjugated bilirubin and / or albumin. An object of the present invention is to provide a reagent and a method for selectively suppressing reactivity and accurately quantifying only conjugated bilirubin.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that, in a reagent for measuring bilirubin by acting on bilirubin oxidase, the reagent for bilirubin covalently bonded to unconjugated bilirubin and / or albumin was used. One or two of a reaction inhibitor selected from a chelating agent, a metal ion and a metal complex having a reaction inhibitory action.
It has been found that by adding more than one species, only the reaction to unconjugated bilirubin and / or bilirubin covalently bound to albumin can be suppressed without suppressing the reaction to conjugated bilirubin. Further, the reaction of the reaction inhibitor with unconjugated bilirubin and / or bilirubin covalently bound to albumin to a reagent to which one or more chelating agents of the present invention, which is one of the above reaction inhibitors, is added. The combined use of one or more reaction inhibitory enhancers selected from metal ions and metal complexes having an inhibitory effect enhances the reaction inhibitory effect on unconjugated bilirubin and bilirubin covalently bonded to albumin. I found something to be done.

[0007] The present inventors further provide the following formula (1):

Embedded image (Wherein, R is an alkyl group having 4 or 5 carbon atoms, or 1
Means an alkenylene group having two double bonds and forms a heterocyclic group together with the nitrogen atom to which it is bonded, and the heterocyclic group includes an alkyl group, an amino group, an amide group, a carbamoyl group, a carboxyl group, and a keto group. Group, hydroxyl group, sulfonic acid group,
It may have a substituent such as a phenyl group, and 1 to 3 carbon atoms in the R group may be substituted with another hetero atom such as a nitrogen atom, an oxygen atom, and a sulfur atom. It has been found that the compound having a free radical represented by the formula (2) also enhances the reaction inhibitory effect of the chelating agent, which is the above reaction inhibitor, on the unconjugated bilirubin and / or bilirubin covalently bonded to albumin. Furthermore, they have found that steroid glycosides and steroid glycoside-containing substances are also useful as reaction inhibitory action enhancers.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for optically measuring conjugated bilirubin by the action of bilirubin oxidase, wherein the reagent for measuring bilirubin includes a reagent for measuring unconjugated bilirubin and / or bilirubin covalently bonded to albumin. A specific chelating agent having a reaction suppressing action,
It is an object of the present invention to provide a conjugated bilirubin measurement reagent characterized in that one or more reaction inhibitors selected from metal ions and metal complexes are blended. The present invention also provides a chelating agent for the reaction inhibitor, which is a metal ion, a metal complex, a compound having a free radical (1), a steroid glycoside or a steroid glycoside-containing substance. It is intended to provide a reagent for measuring conjugated bilirubin, which is characterized by blending two or more species.

The chelating agents which are reaction inhibitors used in the present invention include nitrilotris (methylene phosphonic acid) trisodium salt (NTPO), ethylenediamine
N, N'-bis (methylenephosphonic _{acid) · 1 / 2H 2 O (} E
DDPO), ethylenediaminetetrakis (methylenephosphonic acid) (EDTPO), and bis (2-hydroxybenzyl) ethylenediaminediacetic acid (HBED). Examples of the metal ion that is another reaction inhibitor used in the present invention include divalent iron ions, divalent copper ions, and divalent zinc ions.These metal ions are usually sulfuric acid,
It is used in the form of a salt with an inorganic acid such as hydrochloric acid or nitric acid, for example, ferrous sulfate, copper (II) sulfate, zinc (II) sulfate, zinc (II) chloride and the like. As the metal complex of the reaction inhibitor, disodium cobalt ethylenediaminetetraacetate (I
I) (EDTA-2Na-Co (II)), disodium ethylenediaminetetraacetate Mn (II) (EDTA-2
Na-Mn (II)).

[0010] The chelating agent, metal ion and metal complex used as the reaction inhibitor may be used alone or in combination. Preferably, one or more chelating agents are used, or one chelating agent is used. And one or more combinations of metal ions or metal complexes. These reaction inhibitors exhibit a desired effect by being mixed with a generally used conjugated bilirubin measurement reagent, but it is preferable to change the method of compounding according to the type of the reaction inhibitor. That is, the conjugated bilirubin measurement reagent basically consists of a buffer solution (pH 4.4 to 6.0) such as an acetate buffer, MES, or phthalate buffer, and if necessary, may be added with Triton X-100 and Neugen. A first reagent (buffer) for adjusting the pH of the reaction, to which a nonionic surfactant such as EA170 is added, and a buffer (pH 8.0 to 11.0) such as a bicine buffer or a borate buffer.
A bilirubin oxidase is added to the second reagent (enzyme reagent), and among the above reaction inhibitors, a chelating agent or a metal complex may be added to either the first reagent or the second reagent. Preferably, it is used by being mixed with the first reagent solution. On the other hand, when metal ions are added to the first reagent solution, these metal ions have an action of oxidizing bilirubin during measurement, which hinders the measurement, which is not preferable.
Therefore, it is preferable to mix the metal ions in the second reagent solution.

The chelating agent used as the reaction inhibitor may be used alone or in combination of two or more. Among the above chelating agents, NTPO and HBE
D is preferred because of its large effect, but NTPO is particularly preferred. The amount of the chelating agent to be added is not particularly limited as long as the effect of suppressing the reaction of unconjugated bilirubin and / or bilirubin covalently bound to albumin is sufficiently obtained and does not affect the reaction of conjugated bilirubin. However, when the chelating agent is used alone, a desired effect can be obtained if the final concentration in the reaction solution is 5 mM or more. Usually used concentrations are 5 mM to 2
00 mM, preferably 10 mM to 100 mM, more preferably 20 mM to 80 mM. In addition, these chelating agents include metal ions and metal complexes of other reaction inhibitors, metal complexes as reaction inhibitory enhancers, compounds having free radicals (1), steroid glycosides, and substances containing steroid glycosides. When used in combination, the effect of inhibiting the reaction of unconjugated bilirubin and / or bilirubin covalently bound to albumin by the chelating agent is much stronger, so that the amount of the chelating agent added can be reduced, and If the final concentration in the solution is 1 mM or more, a desired effect can be obtained, and the concentration usually used is 1 mM to 100 mM, preferably 1 mM to 80 mM, more preferably 2 mM to 50 mM as the final concentration.
mM.

The metal ions and metal complexes which are other reaction inhibitors used in the present invention can be used alone or in combination of two or more. Among them, divalent copper ions and EDTA-2Na- Co (II), EDTA
-2Na-Mn (II) is more effective when combined with the above-mentioned chelating agent of the present invention. Of these, ED
TA-2Na-Co (II) or EDTA-2Na-
Mn (II) is particularly preferred. The amount of addition of these metal ions and metal complexes is such that the reaction inhibitory effect on unconjugated bilirubin and / or bilirubin covalently bound to albumin is sufficiently obtained and does not affect the reaction on conjugated bilirubin. There is no particular limitation, and a desired effect can be generally obtained when the final concentration in the reaction solution is 1 mM or more. The concentration usually used is 1 to 50 mM as final concentration, preferably 2 to 10 mM. In addition, when combined with the chelating agent of the present invention, the effect of the chelating agent on inhibiting the reaction of unconjugated bilirubin and / or bilirubin covalently bound to albumin is significantly increased, and the final concentration in the reaction solution is 0.1%. 1 mM
With the above, a desired effect can be obtained. The concentration usually used is 0.1 to 50 mM as the final concentration, preferably,
It is 0.2 to 10 mM, more preferably 0.5 to 5 mM.

The metal ion which is one of the reaction inhibitory enhancers having the effect of enhancing the effect of the chelating agent as the reaction inhibitor used in the present invention is divalent manganese ion,
Trivalent manganese ion, divalent cobalt ion, trivalent cerium ion, tetravalent cerium ion and the like, these metal ions are usually sulfuric acid, hydrochloric acid, inorganic acids such as nitric acid or acetoacetic acid and the like Salts with organic acids such as manganese (II) sulfate, copper (II) sulfate, cerium (III) chloride, cerium (IV) sulfate, manganese (I
I) acetyl acetate, manganese (III) acetyl acetate and the like. The metal complex is E
DTA-2Na-Cu (II), EDTA-2Na-F
e (II) and the like. These metal ions and metal complexes can be used alone or in combination of two or more. Of these, divalent manganese is particularly preferred, and manganese (II) sulfate and manganese chloride (I
I), manganese (II) acetylacetonate and the like can be used. The metal ion and the metal complex as the reaction-suppressing action enhancer are usually added to the second test solution among the conjugated bilirubin measurement reagents, and the amount added is
When combined with the chelating agent as the reaction inhibitor of the present invention, a sufficient effect of suppressing the reaction on unconjugated bilirubin and / or bilirubin covalently bound to albumin is obtained, and does not affect the reaction on conjugated bilirubin. The amount is not particularly limited as long as the final concentration in the reaction solution is at least 0.1 mM, and a desired effect can be obtained. The concentration usually used is 0.1 to 50 mM as final concentration, preferably 0.2 to 10 mM, more preferably 0.5 to 5 mM.

Examples of the compound (1) having a free radical which is another reaction inhibitory action enhancer include, for example, 2,2,
6,6-tetramethylpiperidine 1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1
-Oxyl, 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl, 4-carbamoyl-2,
2,6,6-tetramethylpiperidine 1-oxyl, 4-
Amino-2,2,6,6-tetramethylpiperidine 1-oxyl, 4-carboxy-2,2,5,5-tetramethylpyrrolidine 1-oxyl, 3-carbamoyl-2,2,
5,5-tetramethylpyrrolidine 1-oxyl, 4-oxo-2,2,6,6-tetramethylpiperidine 1-oxyl, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline 1- Oxyl, 4-acetamide-2,2,
6,6-tetramethylpiperidine 1-oxyl, 4-
Examples thereof include (2-chloroacetamide) -2,2,6,6-tetramethylpiperidine 1-oxyl, and any compound having a free radical and being water-soluble can be used. The compound (1) having these free radicals is also usually added to the second test solution, and the amount of the compound (1) is sufficient to suppress the reaction to unconjugated bilirubin and / or bilirubin covalently bound to albumin. The amount is not particularly limited as long as the amount does not affect the reaction with the conjugated bilirubin. Generally, the desired effect can be obtained if the final concentration in the reaction solution is 0.01% (the same or less by weight). Can be The concentration normally used is
The final concentration is 0.01 to 1%, preferably 0.02 to 1%.
It is 0.5%, more preferably 0.05 to 0.2%.

[0015] The steroid glycoside which is another reaction inhibitory action enhancer used in the present invention includes digitonin, and the steroid glycoside-containing substance includes saponin. These steroid glycosides or steroid glycoside-containing substances are also usually added to the second test solution, and the added amount can sufficiently obtain a reaction suppressing effect on unconjugated bilirubin and affect the reaction on conjugated bilirubin. The amount is not particularly limited as long as it is not present. Generally, the desired effect can be obtained if the final concentration in the reaction solution is 0.01% or more. The concentration usually used is 0.02 to 2% as a final concentration, preferably 0.02 to 1%, more preferably 0.05 to 0.5%.

Any known enzyme can be used as bilirubin oxidase, for example, myrothesium (Myrotheium).
cium) -derived bilirubin oxidase, shrimp mushroom (Trach
ydermatsumodae) and the like. The amount of bilirubin oxidase to be used is suitably used within a range showing the desired enzyme activity, but is usually 0.02 to 10 units / ml in the reaction solution, preferably 0.02 to 8 units in the conjugated bilirubin. / Ml range.

In order to carry out the measurement using the conjugated bilirubin measuring reagent of the present invention, the reaction is usually carried out under the condition of pH 4.4 to 6.0. At that time, the chelating agent as a reaction inhibitor of the present invention is 5 mM or more, or the metal ion or metal complex is 5 mM or more, or the chelating agent is 1 mM.
A combination of the above and a metal ion or metal complex of 0.1 mM or more, or a metal ion or metal complex 0.1 m or more as a chelating agent of the present invention of 1 mM or more and a reaction suppressing action enhancer.
M or a combination of at least 1 mM of a chelating agent and 0.01% or more of a compound (1) having a free radical, or 1 mM of a chelating agent as a reaction inhibitor
Or a combination of 0.1 mM or more of a metal ion or a metal complex with 0.1 mM or more of a metal ion or a metal complex as a reaction inhibitor, or a chelating agent of 1 mM or more as a reaction inhibitor and a metal ion or a metal complex 0 A combination of at least 1 mM and a compound (1) having a free radical as a reaction-suppressing action-enhancing agent at 0.01% or more, or a chelating agent at 1 mM or more as a reaction-inhibiting action and a metal ion as a reaction-suppressing action-enhancing agent; Enhancement of the reaction inhibitory effect with a combination of at least 0.1 mM of a metal complex and at least 0.01% of a compound having a free radical (1) or at least 1 mM of a chelating agent as a reaction inhibitor and at least 0.1 mM of a metal ion or a metal complex. Compounds having at least 0.1 mM of a metal ion or metal complex as an agent and a free radical (1 0.01% or more is used. By using these additives, only the reaction to unconjugated bilirubin and / or bilirubin covalently bound to albumin is suppressed without suppressing the reaction to conjugated bilirubin. can do.

The reagent for measuring conjugated bilirubin of the present invention comprises:
As described above, it is basically composed of a normal first pH adjustment buffer (buffer) and a second bilirubin oxidase-containing reagent (enzyme reagent).
It is adjusted by adding one or more kinds, preferably one or more kinds of reaction-suppressing action enhancers, and the dosage form is a liquid preparation or a solid form thereof by a conventional method such as freeze-drying. It can also be a preparation, and in such a case, it can be a kit with a solution. Also, if necessary,
The reagent for measuring conjugated bilirubin of the present invention includes a buffer, a preservative, a surfactant, or a reaction accelerator (potassium ferrocyanide, potassium ferricyanide, potassium ferrocyanide or potassium ferricyanide and a thiol compound, or a sulfide compound, a sulfoxide compound, or a thiol compound). Aged product with urea or its derivative (JP-A-9-3
7795)] or other components may be appropriately added or combined.

[0019]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. The basic formulation for conjugated bilirubin measurement, the measurement conditions for bilirubin, and the type of specimen used for examining the effects of the present invention in the examples are shown below. Basic prescription for conjugated bilirubin measurement: The first reagent for conjugated bilirubin measurement is 100 mM acetate buffer (p
H. 5.0) and Triton X-100 0.05% added thereto. As a second reagent for conjugated bilirubin measurement, 10 mM bicine buffer (pH 10.0) was added to bilirubin oxidase (Mirocesium vercaria MT-MT). 1
(U.S.A., Amano Pharmaceutical Co., Ltd.) 4 U / ml. Assay: 240 μl of the first test solution is added to 9 μl of the bilirubin sample, incubated at 37 ° C. for 5 minutes, and then 80 μl of the second test solution is added and incubated for 5 minutes. At this time, the change in absorbance (main wavelength 450 nm / sub-wavelength 546 nm) was measured 5 minutes after the addition of the first reagent solution and 5 minutes after the addition of the second reagent solution, and the absorbance of the previously measured bilirubin standard serum was measured. And the concentration of bilirubin is measured. Specimen type: A conjugated bilirubin specimen was prepared by dissolving ditaurobilirubin (manufactured by Wako Pure Chemical Industries) in 4% bovine serum albumin to a concentration of 7 mg / 100 ml. As a sample of unconjugated bilirubin, a sample prepared by dissolving bilirubin (manufactured by NIST) in 4% bovine serum albumin to a concentration of 8 mg / 100 ml was used. Bilirubin covalently bound to albumin is obtained by adding a solution containing caffeine, Na benzoate and Na acetate to a patient sample of high bilirubin and having a molecular weight of 30,000 or less (conjugated bilirubin and unconjugated bilirubin Was passed through ultrafiltration to prepare serum containing only bilirubin covalently bound to albumin (H. Weihara: Extraction of delta bilirubin and its chemical synthesis. Medical examination 43)
Vol. 7, No. 1253-1255, 1994).

Examples 1 to 6 and Comparative Example 1 When bilirubin is measured using bilirubin oxidase, it is shared with unconjugated bilirubin and / or albumin when a chelating agent or a metal complex shown in Table 1 below is added. The effect of suppressing the reaction on the bound bilirubin was examined. As Comparative Example 1, EDTA-2Na, which is a usual metal chelating agent, was used. The first reagent solution for conjugated bilirubin measurement was prepared so as to contain 50 mM of a chelating agent or a metal complex, and the above-mentioned effect of the chelating agent and the metal complex on the inhibition of the reaction of unconjugated bilirubin and / or bilirubin covalently bound to albumin was measured. Measured by the method and compared. Table 1 shows the results. The determination of the presence or absence of the effect did not affect the reaction to conjugated bilirubin, and the one that suppressed only the reaction to unconjugated bilirubin and / or bilirubin covalently bound to albumin was determined to be effective. (If the measured value in the table is smaller than that of the control, it is determined that the reaction has been suppressed.) (The following examples were similarly determined.)

[0021]

[Table 1]

As shown in Table 1, among the chelating agents and metal complexes, nitrilotris (methylenephosphonic acid) trisodium salt (NTPO), ethylenediamine-N,
N'-bis (methylene phosphonic acid) 1/2 H ₂ O (EDD
PO), ethylenediaminetetrakis (methylenephosphonic acid) (EDTPO), bis (2-hydroxybenzyl) ethylenediaminediacetic acid (HBED), EDTA-
2Na-Co (II), EDTA-2Na-Mn (I
I) was found to have the effect of suppressing the reaction to unconjugated bilirubin and / or to bilirubin covalently bound to albumin without suppressing the reaction to conjugated bilirubin. In particular, the effects of HBED and NTPO were remarkable.

Examples 7 to 9 The metal salt (metal ion) shown in Table 2 below was added to the second reagent solution for measuring conjugated bilirubin so as to contain 5 mM, and the unconjugated bilirubin and / or albumin containing the metal ion was added. The inhibitory effect on the reaction of bilirubin covalently bound with was measured. Table 2 shows the results.

[0024]

[Table 2]

As shown in Table 2, among the metal ions, divalent iron ions, divalent copper ions, divalent zinc ions,
Thus, it was confirmed that the compound had an effect of suppressing the reaction to unconjugated bilirubin and / or to bilirubin covalently bound to albumin without suppressing the reaction to conjugated bilirubin. In particular, the effect of divalent iron ions was remarkable.

Examples 10 to 20 Covalently bound to unconjugated bilirubin and / or albumin when a metal salt (metal ion) or metal complex as a reaction inhibitor was combined with a chelating agent as a reaction inhibitor. The effect of suppressing the reaction to bilirubin was investigated. N in the first reagent solution for conjugated bilirubin measurement
Except that TPO was prepared to contain 40 mM or HBED to 2 mM, and the second reagent solution for conjugated bilirubin measurement was prepared to contain 5 mM of a metal salt or metal complex.
In the same manner as in Examples 1 to 6, the reaction inhibitory effect on unconjugated bilirubin and / or bilirubin covalently bound to albumin was measured. Table 3 shows the results.

[0027]

[Table 3]

As shown in Table 3, divalent copper ion, divalent manganese ion, trivalent manganese ion, divalent cobalt ion, trivalent cerium ion, tetravalent cerium ion, EDTA-2Na-Co (II), EDTA-2
Na-Mn (II), EDTA-2Na-Cu (I
I), EDTA-2Na-Fe (II) or the like has the effect of promoting the reaction inhibitory effect of the chelating agent on unconjugated bilirubin and / or bilirubin covalently bound to albumin without inhibiting the reaction on conjugated bilirubin. It was recognized that. Particularly, the effects of divalent manganese ion and EDTA-2Na-Co (II) were remarkable.

Examples 21 to 30 and Comparative Examples 2 to 4 Non-conjugated bilirubin and / or unconjugated bilirubin when a compound (1) having a free radical as a reaction inhibitor is combined with a chelating agent as a reaction inhibitor. ) The effect of suppressing the reaction to bilirubin covalently bound to albumin was examined. Examples 1 to 6 except that the first test solution for conjugated bilirubin measurement was prepared to contain 40 mM of NTPO, and the second test solution for conjugated bilirubin measurement was prepared to contain 0.1% of compound (1). In the same manner as described above, the reaction inhibitory effect on unconjugated bilirubin and / or bilirubin covalently bound to albumin was measured. In addition, as a comparative example, measurement was similarly performed using a compound having a structure similar to that of the compound (1) but having no free radical (Comparative Examples 2 to 4). Table 4 shows the results.

[0030]

[Table 4]

As shown in Table 4, the compound (1) having a free radical does not inhibit the reaction to the conjugated bilirubin and inhibits the reaction of the unconjugated bilirubin and / or bilirubin covalently bound to albumin by the chelating agent. It was recognized that it had the effect of promoting the effect. On the other hand, 4-hydroxy-2,2,6,6-
It was found that there was no effect on 4-hydroxy-2,2,6,6-tetramethylpiperidine which is a substance similar in structure to tetramethylpiperidine 1-oxyl but having no free radical.

Examples 31 to 33 In the conjugated bilirubin measurement reagent, the reaction inhibitory effect of unconjugated bilirubin when a steroid glycoside or a steroid glycoside-containing substance was combined with a chelating agent as a reaction inhibitor was examined. . In the same manner as in Examples 1 to 6, except that the first test solution for conjugated bilirubin measurement was prepared to contain 40 mM of NTPO, and the test solution was prepared to contain saponin or digitonin at the concentration shown in Table 5. The effect of inhibiting the reaction to unconjugated bilirubin was measured. Regarding digitonin, a case was also examined in which the first test solution for conjugated bilirubin measurement was prepared to contain 40 mM of NTPO, and the second test solution for conjugated bilirubin measurement was prepared to contain digitonin. Table 5 shows the results.

[0033]

[Table 5]

As shown in Table 5, the addition of digitonin and saponin to the first reagent and digitonin to the second reagent have the effect of suppressing the reaction to unconjugated bilirubin without suppressing the reaction to conjugated bilirubin. Was observed.

[0035]

According to the present invention, when optically measuring conjugated bilirubin using bilirubin oxidase, specific bilirubin having an action of suppressing the reaction to unconjugated bilirubin and / or bilirubin covalently bound to albumin is provided. By using one or more reaction inhibitors selected from chelating agents, metal ions and metal complexes, only conjugated bilirubin can be measured. Further, a chelating agent as a reaction inhibitor is combined with one or more reaction inhibitory enhancers selected from metal ions, metal complexes, compounds having free radicals, steroid glycosides and steroid glycoside-containing substances. By doing so, it is possible to enhance the inhibitory effect on the reaction of unconjugated bilirubin and / or bilirubin covalently bound to albumin, to reduce the required amount of the chelating agent, and to reduce the cost of the reagent for measuring conjugated bilirubin. Can be reduced. Further, since these chelating agents, metal ions, metal complexes, compounds having free radicals, and steroid glycosides, and steroid glycoside-containing substances are all stable even when they are in liquid form, they are used as reagents for conjugated bilirubin measurement. It can be used effectively for liquid reagents.

Claims (7)

[Claims] 1. A reagent for measuring bilirubin, which comprises one or more selected from a chelating agent having a reaction inhibiting effect on unconjugated bilirubin and / or bilirubin covalently bound to albumin, a metal ion and a metal complex. A reagent for measuring conjugated bilirubin. 2. The conjugated bilirubin measurement reagent according to claim 1, wherein the main reaction component of the bilirubin measurement reagent is bilirubin oxidase. 3. The reaction of a reagent for measuring bilirubin has a pH of 4.
The reagent for measuring conjugated bilirubin according to claim 1, which is performed under the condition of 4-6.0. 4. The method according to claim 1, wherein the chelating agent is nitrilotris (methylenephosphonic acid) trisodium salt (NTPO), ethylenediamine-N, N′-bis (methylenephosphonic acid) · 1
/ 2H ₂ O (EDDPO), ethylenediamine tetrakis (methylenephosphonic acid) (EDTPO), or bis (2-hydroxybenzyl) ethylenediaminediacetic acetate (HBED), metal ions are divalent iron ions, divalent copper ions, Or a divalent zinc ion wherein the metal complex is disodium cobalt (II) ethylenediaminetetraacetate (E)
DTA-2Na-Co (II)) or disodium manganese (II) ethylenediaminetetraacetate (EDTA-2
Na-Mn (II)).
4. The reagent for measuring conjugated bilirubin according to any one of the above. 5. A divalent manganese ion, a trivalent manganese ion, a divalent cobalt ion, and a trivalent cerium ion in order to enhance the reaction inhibitory effect on unconjugated bilirubin and / or bilirubin covalently bound to albumin. And metal ions selected from tetravalent cerium ions; disodium copper (II) ethylenediaminetetraacetate
(EDTA-2Na-Cu (II)) and disodium iron (II) ethylenediaminetetraacetate (EDTA-2N
a-Fe (II)) and the following formula (1): (Wherein R is an alkylene group having 4 or 5 carbon atoms or 1
Means an alkenylene group having two double bonds, forms a heterocyclic group together with the nitrogen atom to which it is bonded, and the heterocyclic group is an alkyl group, an amino group, an amide group, a carbamoyl group,
It may have a substituent such as a carboxyl group, a keto group, a hydroxyl group, a sulfonic acid group and a phenyl group, and one to three of the carbon atoms in R may be a nitrogen atom, an oxygen atom, a sulfur atom or the like. Wherein at least one kind of a reaction-suppressing action enhancer selected from compounds having a free radical represented by the formula (1) may be substituted. Item 6. The reagent for measuring conjugated bilirubin according to any one of the above items. 6. The method according to claim 1, further comprising one or more steroid glycosides or steroid glycoside-containing substances having an action of enhancing the action of suppressing the reaction of unconjugated bilirubin. 6. The reagent for measuring conjugated bilirubin according to any one of 1 to 5. 7. The reagent according to claim 6, wherein the steroid glycoside is digitonin and the steroid glycoside-containing substance is saponin.