JP2000093200A - Reagent for measuring leucine aminopeptidase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent for measuring leucine aminopeptidase not being influenced by chyle in serum, capable of avoiding the influence of hemolytic hemoglobin. SOLUTION: This reagent comprises a substrate having 400-450 nm absorbance of a coloring compound to be dissociated by the action of leucine aminopeptidase, such as L-leucyl-p-nitroanilide, a nonionic surfactant having 0.005-0.01 w/v% final concentration and a buffer solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring leucine aminopeptidase activity, which is not affected by milky milk in serum and can avoid the influence of hemolyzed hemoglobin.

[0002]

2. Description of the Related Art Serum leucine aminopeptidase (hereinafter abbreviated as LAP) is an enzyme that releases leucine from the N-terminus of a peptide and appears in various types of obstructive jaundice, biliary tract cancer, hepatic parenchyma, etc. As one of the important factors for diagnosing the disease, it has been conventionally measured by various measuring methods. As a method for measuring LAP, L-
Measuring methods using leucyl-p-nitroanilide and the like have been widely used. A color-forming compound in which these enzyme substrates are dissociated by the action of LAP, that is, p-nitroaniline, usually has an absorbance at a wavelength of 400 to 450 nm. When an absorbance measurement is performed at a wavelength in this range, the measurement result is often affected by milky milk in serum.

[0003] Conventionally, surfactants have been used in reagents for measuring enzyme activity by absorbance in order to prevent the solubility of the reagent or the turbidity of the reaction solution. However, it is not uncommon to occasionally introduce unexpected measurement interference or interference. Therefore, non-ionic surfactants that are inexpensive and do not cause protein denaturation have been used as reagents used as clinical diagnostic agents. However, nonionic surfactants are used in the normal concentration range, 0.
When used at concentrations of 02-1.0 w / v%, it has been experienced that degradation due to autoxidation occurs and affects the measured values.

Further, a nonionic surfactant is used in an amount of 400 to 450 n.
In the measurement system for measuring the absorbance in the region of m, there is a problem that as the concentration of the nonionic surfactant increases, the negative influence of hemolyzed hemoglobin present in serum increases.

It is known to use a cationic surfactant or an amphoteric surfactant to avoid the influence of hemolyzed hemoglobin (Japanese Patent Publication No. 8-78). However, when these surfactants are used, if the charge of the protein present during the reaction is negative, the surfactants bind and cause turbidity.

[0006] Further, sodium lauryl sulfate, cetyl sulfate, laurylamine acetate which is one of surfactants,
It is known to use cetylamine hydrochloride (Japanese Patent Publication No. 3-58467). However, the use of these surfactants also results in turbidity.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a reagent for measuring leucine aminopeptidase activity, which is not affected by milky serum in serum and can avoid the influence of hemolyzed hemoglobin.

[0008]

Means for Solving the Problems The present inventors have conducted various studies to solve the above-mentioned problems, and found that the above-mentioned problems can be solved by adding a nonionic surfactant in a smaller amount than before. The present invention has been reached.

That is, according to the present invention, the absorbance of a color-forming compound dissociated by the action of leucine aminopeptidase is 40 or less.
Substrate 0-450 nm, final concentration 0.005-0.01% w / v
A reagent for measuring leucine aminopeptidase activity, comprising a nonionic surfactant and a buffer.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The substrate of the present invention is a compound in which the color-forming compound dissociated by the action of LAP has an absorbance of 400 to 450 nm, such as L-leucyl-p-nitroanilide. P-Nitroaniline dissociated from the compound has a large negative effect, particularly in the measurement of LAP.

The nonionic surfactant used in the present invention is a compound having a hydrophilic portion and a hydrophobic portion, and having a sugar or polyoxyethylene chain in the hydrophilic portion.
Sugar chains include glucoside, thioglucoside, and analogs thereof. Examples of the polyoxyethylene chain include polyoxyethylene, phenyl polyoxyethylene, and polyoxyethylene sorbitan ester. Examples of the hydrophobic moiety (group) include alkyl groups such as decyl, dodecyl, tridecyl, tetradecyl, cetyl, stearyl, oleyl, cetyl-stearyl, heptyl, and decanoyl, p-octylphenyl, p-isooctylphenyl, and p-nonylphenyl. And the like.

Specifically, octyl glycoside, heptyl glycoside, decanoyl-N-methylglucamide, polyoxyethylene decyl ether, polyoxyethylene dodecyl ether, polyoxyethylene tridecyl ether, polyoxyethylene heptamethylhexyl ether, Polyoxyethylene-p-isooctylphenyl ether, polyoxyethylene-p-nonylphenyl ether, polyoxyethylene-pt-octylphenyl ether, polyoxyethylene fatty acid ester, sucrose fatty acid ester, polyoxyethylene sorbitol ester, poly Examples thereof include oxyethylene lauryl ether and polyoxyethylene-sorbitan monolaurate. Among these, nonionic surfactants having an HLB value of 12 to 19, particularly polyoxyethylene lauryl ether (oxyethylene number = 21, HLB = 19.0, trade name BL-21)
), Polyoxyethylene dodecyl ether (oxyethylene number = 23, HLB = 15.3, trade name Brij 35), polyoxyethylene-pt-octylphenyl ether (oxyethylene number = 9,10, HLB = 13.5, trade name) Triton X-100)
Is preferred.

In the present invention, it is necessary that the concentration of the nonionic surfactant is 0.005 to 0.01 w / v% (final concentration) in the reagent. If the content is less than 0.005 w / v%, there is a risk of turbidity in the serum due to the influence of hydrophobic substances such as milk and milk. If the content is more than 0.01 w / v%, the hemolytic hemoglobin present in the sample may be lost. There is a risk of increased impact.

The reagent of the present invention comprises a buffer such as Tris buffer and a substrate such as L-leucyl-p-nitroanilide. Preferably, the final concentration of Tris buffer is 50-100 mM and the final concentration of substrate is 2-10 mM. Further, the reagent is preferably a liquid reagent. The reagent may be a one-part system or a two-part system. In the two-reagent system, a buffer solution containing a nonionic surfactant is added to serum as a first reagent, and then an aqueous solution containing a substrate is added as a second reagent.

In order to measure LAP in serum using the reagent of the present invention, usually, after adding a reagent to a sample, for example, serum, preheating at 37 ° C.
Measure absorbance from 0 to 450 nm. The measuring method is preferably a rate method. The rate method is a method in which a change with time (increase rate) of the absorbance of the coloring compound dissociated from the substrate in the 400 to 450 nm region is determined, and the LAP activity value is calculated from the measurement result. Enzyme activity 1 IU / l is 1 μm per minute
It is defined as the amount of enzyme that catalyzes the reaction of mol / l. To determine the enzyme activity from the change over time in absorbance (E), the change in absorbance per minute (ΔE) ΔE / min) is calculated and further substituted into the following equation to obtain the enzyme activity (IU / l).

[0016]

RV: liquid volume of reagent (solution) used for measurement SV: liquid volume of sample used for measurement ε: extinction coefficient of substance whose absorbance is measured

The enzyme substrate to be used is not particularly limited as long as an arbitrary optimal concentration can be appropriately determined depending on the conditions of the serum as a sample, the type of the substrate to be used, and various factors under other conditions of use. There is no. Preferably 2-10 mM
It is. The preparation of the reagent of the present invention and the method for measuring enzyme activity using the reagent follow conventional techniques.

[0019]

Next, the present invention will be described in detail with reference to examples. Example 1 The following reagents were prepared. First reagent: (1) 100 mM Tris buffer, pH 7.8, 200 mM NaCl No nonionic surfactant (2) 100 mM Tris buffer, pH 7.8, 200 mM NaCl 0.0062% BL-21 (3) 100 mM Tris buffer, pH 7.8, 200 mM NaCl 0.013% BL-21 (4) 100 mM Tris buffer, pH 7.8, 200 mM NaCl 0.13% BL-21 BL-21: polyoxyethylene lauryl ether (oxyethylene number = 21, HLB = 19.0) Second reagent: 12 mM L- Leucyl-p-nitroanilide aqueous solution pH3.0 200mM NaCl

The specimen was prepared by the following method. (A) Serum samples having different hemolytic hemoglobin concentrations 9 volumes of serum of control serum Moni-Trol IX (manufactured by International Reagents) and 0 concentration of aqueous solution containing no hemoglobin or prepared hemolytic hemoglobin (interference check: manufactured by International Reagents) 100
One volume of each of the aqueous solutions of 0, 2000, 3000, 4000, and 5000 mg / dl was mixed, and the hemolytic hemoglobin concentration was 0,
Serum samples were prepared at 100, 200, 300, 400 and 500 mg / dl. (B) Serum samples with different intrafat concentrations 9 volumes of serum of control serum Moni-Trol IX (manufactured by International Reagents) and an aqueous solution containing no intrafat (manufactured by Takeda Pharmaceutical) or prepared intrafat concentrations of 0, 20, and 40 , 60, 80,
One volume of each 100% aqueous solution was mixed to prepare serum samples having intrafat concentrations of 0, 2, 4, 6, 8, and 10%, respectively.

The measuring operation is as follows. Sample 8μ
To this was added 250 μl of the first reagent and preliminarily heated at 37 ° C. for 5 minutes, 125 μl of the second reagent was added, and the mixture was stirred for 2 minutes. Then, the rate of increase in absorbance per unit time at 405 nm (main wavelength) and 700 nm (sub wavelength) was measured. The enzyme activity in each serum was determined based on the rate of increase in the absorbance of the enzyme solution whose LAP activity value was known. The measuring device used was an automatic analyzer (Hitachi 7150).

Using the above reagents, the LAP activity in the above sample was measured according to the above procedure, and the results are shown in Tables 1 and 2. The concentration of the nonionic surfactant in the table is the final concentration after the addition of the first reagent and the second reagent.

[0023]

[Table 1]

[0024]

[Table 2]

FIG. 1 shows the results of Tables 1 and 2. As is clear from FIG. 1, the concentration of BL-21 was 0.005% and 0.01%.
%, It is possible to avoid the influence of milky milk and also the effect of hemolyzed hemoglobin. A BL-21 concentration of 0.1% is affected by haemolysed hemoglobin, even though the effects of chyle can be avoided. Also, if BL-21 is not used, it will be affected by milk

Example 2 In place of BL-21 in Example 1, Brij 35 (polyoxyethylene dodecyl ether, oxyethylene number = 23,
LAP was measured in the same manner using HLB value = 15.3).
The results are shown in Tables 3 and 4. The concentration of the nonionic surfactant in the table is the final concentration after the addition of the first reagent and the second reagent.

[0027]

[Table 3]

[0028]

[Table 4]

FIG. 2 shows the results of Tables 3 and 4.
As is clear from FIG. 2, the concentration of Brij 35 was 0.005%
And 0.01% reagent can avoid the effects of milk
Further, the effects of hemolyzed hemoglobin can be avoided. If the concentration of Brij 35 is 0.1%, hemolytic hemoglobin is affected, even if the effects of chyle can be avoided. Also Br
If you do not use ij 35, you will be affected by milk.

Example 3 In place of BL-21 in Example 1, Triton X-100 (polyoxyethylene-pt-octylphenyl ether,
LAP was measured in the same manner using oxyethylene number = 9,10, HLB value = 13.5). The results are shown in Tables 5 and 6. The concentration of the nonionic surfactant in the table is the final concentration after the addition of the first reagent and the second reagent.

[0031]

[Table 5]

[0032]

[Table 6]

FIG. 3 shows the results of Tables 5 and 6.
As is clear from FIG. 3, the concentration of Triton X-100 was 0.00
With the reagents of 5% and 0.01%, the effects of milky milk can be avoided and the effects of hemolytic hemoglobin can be avoided. When the concentration of Triton X-100 is 0.1%, the effect of hemolytic hemoglobin is affected, even if the effect of milk can be avoided.
If you do not use Triton X-100, you will be affected by milk.

[0034]

According to the present invention, the effect of milky hemoglobin can be avoided and the effect of hemolyzed hemoglobin can be avoided by using a smaller amount of nonionic surfactant than in the prior art at a concentration of 0.005 to 0.01 w / v% (final concentration). Can be avoided. Therefore, LAP can be accurately measured even if the sample is high fat serum.

[Brief description of the drawings]

FIG. 1 is a graph showing the effects of hemolyzed hemoglobin and chyle on increasing BL-21 concentration.

FIG. 2 is a graph showing the effects of hemolysed hemoglobin and chyle upon increasing the concentration of Brij 35.

FIG. 3 is a graph showing the effects of hemolytic hemoglobin and milky vine on increasing the concentration of Triton X-100.

Claims (4)

[Claims] The present invention is characterized in that a color-forming compound dissociated by the action of leucine aminopeptidase contains a substrate having an absorbance of 400 to 450 nm, a nonionic surfactant having a final concentration of 0.005 to 0.01 w / v%, and a buffer. A reagent for measuring leucine aminopeptidase activity. 2. The reagent for measuring leucine aminopeptidase activity according to claim 1, wherein the substrate having an absorbance of 400 to 450 nm of the color-forming compound dissociated by the action of leucine aminopeptidase is L-leucyl-p-nitroanilide. 3. The nonionic surfactant is trade name, BL-21
2. The reagent for measuring leucine aminopeptidase activity according to claim 1, which is Brij 35, or Triton X-100. 4. L-leucyl-p-nitroanilide having a final concentration of 2 to 10 mM, a final concentration of 0.005 to 0.01 w / v%.
A reagent for measuring leucine aminopeptidase activity, comprising a nonionic surfactant selected from the group consisting of BL-21, Brij 35 or TritonX-100 and Tris buffer.