JP2001249132A - Method for stabilizing heme protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for stabilizing heme proteins which is effective in modifying and decomposing the heme proteins, and a preservation solution for use therein. SOLUTION: The method for stabilizing heme proteins includes causing transition metals to coexist in a sample containing the heme proteins. The preservation solution for use in the method is also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing a heme protein, and more particularly to a method for stabilizing a heme protein in a test for a heme protein in feces, urine and blood samples.

[0002]

2. Description of the Related Art In recent years, detection of human hemoglobin (fecal occult blood) in feces caused by bleeding from digestive organs has been widely performed as a method for examining digestive diseases such as colon cancer. This method of detecting human hemoglobin is an immunological method that uses an antibody specific to human hemoglobin instead of the conventional test strip method based on chemical chromogenic reaction, and is a simple test method that does not require dietary restriction. It has been established as.

[0003] As a method for immunologically detecting human hemoglobin, for example, a one-dimensional immunodiffusion method utilizing a precipitation line between an anti-human hemoglobin antibody and a human hemoglobin in a test sample on an agar plate, an anti-human hemoglobin antibody is detected. An agglutination method using prepared latex particles or colloidal gold particles, an enzyme immunization method using an anti-human hemoglobin antibody labeled with an enzyme or a radioactive element, a radioimmunization method, and the like.

[0004] However, human hemoglobin has the property that it gradually denatures in the test solution and its antigenicity decreases. In addition, denaturation of human hemoglobin in a test solution is often accelerated by storage conditions such as storage temperature, and is often decomposed by bacteria and digestive enzymes in feces. As a result of such denaturation / decomposition, the three-dimensional structure of human hemoglobin is destroyed, leading to a decrease in antigenicity.
For this reason, in the immunological method for measuring human hemoglobin, denaturation / decomposition of hemoglobin causes erroneous diagnostic results.

[0005] On the other hand, in a fecal occult blood test, on the other hand, the subject often dissolves feces collected at home or the like in a closed container containing a stool solution for use in the test. In this case, human hemoglobin in feces is often left at a high temperature by being left in a solution for several days or by using transportation means such as mail. In addition, even when a stool is collected by a laboratory, a large amount of time may be required for a test for fecal occult blood since another test is performed. Under such circumstances, as described above, denaturation and degradation of human hemoglobin occur, which hinders accurate measurement.

In order to prevent denaturation / decomposition of human hemoglobin in such a solution, a method of adding a general antibacterial agent such as thimerosal or chlorhexidine (Japanese Patent Application Laid-Open No.
3-271160), a method of adding saccharides (JP-A-63-243756), addition of non-human animal hemoglobin (JP-A-2-296149), addition of non-human animal serum (Japanese Unexamined Patent Publication No.
No. 66) and addition of a lytic enzyme (Japanese Patent Publication No. 5-69466).
Publication), addition of iron protoporphyrin (Japanese Unexamined Patent Publication No.
81227) and the like have been proposed. However, the human hemoglobin stabilization technology described in these publications cannot sufficiently suppress denaturation and degradation of human hemoglobin in a test solution containing feces.

[0007] In addition, a method for stabilizing hemoglobin using ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) has been proposed (JP-A-5-99923). However, as a result of the additional tests by the present inventors, E
It was confirmed that DTA alone could not expect a sufficient stabilizing effect on hemoglobin in feces. For this reason,
The present applicant has proposed a method of adding a water-soluble transition metal complex having a higher stabilizing effect than EDTA alone (JP-A-7-229902). Further, the present applicant has proposed a method for stabilizing hemoglobin in the presence of a ferrocyanide compound (Japanese Patent Application Laid-Open No. H11-118806) and a method for stabilizing hemoglobin in the presence of an enzymatic degradation product of hemoglobin (Japanese Patent Application Laid-Open No. H11-218533). Gazette) has already been proposed.

[0008]

DISCLOSURE OF THE INVENTION The first object of the present invention is to modify the denaturation of a heme protein represented by hemoglobin.
It is an object of the present invention to provide a novel method for stabilizing a heme protein, which is effective against a decomposition action. In particular, it is an object of the present invention to provide a technique for effectively stabilizing hemoglobin coexisting with fecal components having a strong denaturing / degrading action. A second object of the present invention is to provide a heme protein storage solution using a novel heme protein stabilizer.

[0009]

SUMMARY OF THE INVENTION The first object of the present invention is to
The present invention has been achieved by a method for stabilizing a heme protein, which comprises allowing transition metals to coexist in a sample containing the heme protein. The second object of the present invention has been attained by a heme protein storage solution containing transition metals. Hereinafter, the present invention will be described in more detail.

The transition metals used in the present invention can be appropriately selected from known ones. Specific examples thereof include, for example, at least one selected from the group consisting of chromium, molybdenum, tungsten, manganese, iron, cobalt, nickel, copper, zinc, and cadmium.
Species, or salts thereof. Among these transition metals, metals belonging to Group VIa of the periodic table, namely, chromium, molybdenum, and tungsten are preferable, molybdenum is more preferable, and molybdates such as sodium molybdate and potassium molybdate are more preferable.

In the present invention, the transition metal is contained in the hemoprotein-containing sample in a concentration of 0.5 to 100 mM, preferably 1 to 50 mM, more preferably 10 mM. When the concentration of the transition metal is less than 0.5 mM, the stabilizing effect becomes insufficient. On the contrary, when the concentration exceeds 100 mM, the immune reaction is inhibited and the measured value is rather lowered.

Further, in the present invention, the addition of an azide in addition to the above-mentioned transition metals can further stabilize the heme protein in the sample. Specific examples of the azide include, for example, at least one selected from the group consisting of sodium azide, potassium azide, ammonium azide and lithium azide.

The azide is contained in the hemoprotein-containing sample at a concentration of 0.01 to 0.2 w / v%, preferably 0.1 w / v%. When the azide concentration is less than 0.01 w / v%, the stabilizing effect becomes insufficient. On the other hand, among the azides, sodium azide is designated as a virulent substance (decree 40
No. 5: Cabinet order that partially revise the Ordinance for Designating Poisonous and Deleterious Substances), it is not preferable to use more than 0.2 w / v%.

In the present invention, although not particularly required, the effect of stabilizing the heme protein can be further enhanced by adding a known protein protective agent as needed. Examples of such a protein protective agent include inactive proteins represented by albumin and gelatin.

Any albumin can be used, but albumin derived from animal serum or eggs is particularly preferred. Specific examples include cows, horses, goats,
Sheep, pigs, rabbits, and albumins derived from fetal blood or fetal blood of these animals. In addition to the above-mentioned albumin, enzymatically degraded products thereof are also known, but albumin in the present invention may include proteins derived from such albumin.

In addition to the above-mentioned protein protective agents, there are many other known heme protein protective agents such as antibacterial agents for preventing unnecessary propagation of microorganisms and buffers for giving a pH which is advantageous for preservation of heme proteins. Addition of components is also effective. Examples of the antibacterial agent include a lytic enzyme, ethyl benzoate, penicillin, fungison, streptomycin, cephamycin, and a series of non-penicillin-based antibiotics. It is also known that protease inhibitors such as trypsin inhibitors and α2 macroglobulin stabilize heme proteins.

[0017] The pH of the preservation solution of the present invention is set within a range in which the heme protein can be stably retained. Under extreme acid or alkaline conditions, the stability of the heme protein may be impaired, and it is difficult to obtain the protective action of the transition metals. Therefore, a pH in a neutral range is desirable. Specifically, the pH is 5 to 10, preferably about 6 to 8. Appropriate buffers are used to maintain the pH. For example, hydroxyethylpiperazine-2-ethanesulfonic acid (abbreviated as HEPES) or piperazine-bis (2-ethanesulfonic acid)
Good buffers, such as (abbreviated PIPES), provide a pH (6 to 6) that appears to most stabilize the structure of the heme protein.
At the same time as 8), it is also used as a reaction buffer when detecting a heme protein by an immune reaction, and is mentioned as a particularly preferred buffer. In addition, a phosphate buffer, a Tris buffer, a glycine buffer, and the like can also be used.

The method for stabilizing a heme protein of the present invention comprises:
It can be used for detection of heme protein in feces, urine, and blood samples. The heme protein can be appropriately selected from proteins having heme as a component, and examples include hemoglobin, myoglobin, peroxidase, and catalase. Particularly, it is useful for maintaining the antigenicity of a heme protein as an immunological analysis target requiring protection of the antigen structure.

In the present invention, it is possible to provide a preservation solution of hemoclobin containing a transition metal which acts as a stabilizing factor for a heme protein. In this case, examples of the transition metal to be added include at least one selected from the group consisting of chromium, molybdenum, tungsten, manganese, iron, cobalt, nickel, copper, zinc, and cadmium, or salts thereof. Among these transition metals, metals belonging to Group VIa of the periodic table, namely, chromium, molybdenum, and tungsten are preferable, molybdenum is more preferable, and molybdates such as sodium molybdate and potassium molybdate are more preferable.

Further, the preservation solution of the present invention may contain an azide, a protein protecting agent and a buffer, if necessary. The azide, protein protectant, and buffer can be appropriately selected from those described above.

The present invention provides a method for immunologically measuring heme protein to which the above-mentioned technique for stabilizing heme protein is applied. Examples of immunological measurement methods include latex agglutination, colloidal gold agglutination, immunochromatography, and ELISA.
In any of the measurement methods, the coexistence of a transition metal in the heme protein-containing sample protects the antigen activity during storage and suppresses a decrease in the measured value.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION The method for stabilizing a heme protein of the present invention is useful for stabilizing a heme protein which is present in a biological sample and which is to be analyzed. In particular, in a method for measuring a heme protein by an immunological technique using an antibody that recognizes a heme protein, the antigenicity of the heme protein to be measured is highly stabilized. Fecal matter, urine, and blood are known as biological samples from which heme proteins are to be detected. In particular, hemoglobin in feces serves as an indicator of bleeding in the digestive tract, and hemoglobin in urine serves as an indicator of bleeding in the urinary tract.

When the stabilizing method of the present invention is applied to hemoglobin present in a stool sample, it is preferable to add a transition metal to a storage solution for suspending the stool. When detecting hemoglobin in ordinary stool, the stool is suspended in an appropriate preservation solution and, if necessary, filtered to obtain an immunological analysis sample.

[0024]

According to the method for stabilizing a heme protein of the present invention, a protective effect can be obtained even in the presence of a biological component having a strong denaturing / degrading effect on a heme protein, particularly a fecal component. Therefore, it is useful for analyzing biological components, for example, for stabilizing hemoglobin contained in a sample for detecting fecal occult blood. In particular, it contributes to maintaining the antigenicity of a heme protein as an immunological analysis target requiring protection of the antigenic structure. According to the present invention, hemoglobin in a stool sample is effectively stabilized, and prevention of false negative results due to denaturation / decomposition of hemoglobin can be expected.

[0025]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto.

Embodiment 1 FIG. Stabilization effect of hemoglobin by sodium molybdate Sodium molybdate was 0 mM, 10 mM and 50 mM, respectively.
Stock solution containing (50 mM HEPES buffer, pH
7.0, 0.9% NaCl, 0.1% BSA) 2 ml
Then, 10 mg of a normal stool specimen (A) and human hemolyzed blood were added, and the measured value of hemoglobin immediately after the addition and the measured value of hemoglobin when stored at 37 ° C. for 17 hours were compared. The measurement was performed using OC-270R (manufactured by Eiken Chemical Co., Ltd.) and a dedicated reagent (OC-Auto II:
(Eiken Chemical). Table 1 shows the results. As shown in Table 1, 10 mM and 50 mM were added at 37 ° C. as compared with those without sodium molybdate added.
It was confirmed that the hemoglobin stabilizing effect after storage for 17 hours was remarkably high.

[0027]

[Table 1]

Embodiment 2 FIG. Hemoglobin Stabilizing Effect of Sodium Molybdate and Sodium Azide Normal stool samples (B) to (B) were prepared in exactly the same manner as in Example 1 except that 0.1 w / v% of sodium azide was added.
(E) was examined for the stabilizing effect of hemoglobin. The results are shown in Tables 2 to 5. As shown in the results, it can be seen that the addition of sodium azide in addition to molybdenum further improved the effect of stabilizing hemoglobin.

[0029]

[Table 2]

[0030]

[Table 3]

[0031]

[Table 4]

[0032]

[Table 5]

[0033]

Claims (16)

[Claims] (1) A method for stabilizing a heme protein, wherein a transition metal is coexistent in a sample containing the heme protein. 2. The method for stabilizing a heme protein according to claim 1, wherein the concentration of the transition metal is in the range of 0.5 to 100 mM. 3. The transition metal according to claim 1, wherein the transition metal is at least one selected from the group consisting of chromium, molybdenum, tungsten, manganese, iron, cobalt, nickel, copper, zinc and cadmium, or salts thereof. A method for stabilizing a heme protein according to the above. 4. The method for stabilizing a heme protein according to claim 1, wherein the transition metal is a metal belonging to No. VIa of the periodic table. 5. The method according to claim 1, wherein the transition metal is molybdenum or a salt thereof. 6. The method for stabilizing a heme protein according to claim 1, wherein an azide coexists. 7. The method for stabilizing a heme protein according to claim 6, wherein the azide is at least one selected from the group consisting of sodium azide, potassium azide, ammonium azide and lithium azide. 8. The method for stabilizing a heme protein according to claim 1, wherein the heme protein is hemoglobin, myoglobin, peroxidase, or catalase. 9. The method for stabilizing a heme protein according to claim 1, wherein the sample containing the heme protein is stool, urine or blood. 10. A preservation solution of a heme protein, comprising a transition metal. 11. The transition metal according to claim 10, wherein the transition metal is at least one selected from the group consisting of chromium, molybdenum, tungsten, manganese, iron, cobalt, nickel, copper, zinc and cadmium, or salts thereof. Heme protein stock solution. 12. The preservation solution of heme protein according to claim 10, wherein the transition metals are metals belonging to the Periodic Table No. VIa. 13. The preservation solution of heme protein according to claim 10, wherein the transition metal is molybdenum or a salt thereof. 14. A preservation solution of a heme protein according to claim 10, which comprises an azide. 15. The azide is at least one selected from the group consisting of sodium azide, potassium azide, ammonium azide and lithium azide.
A storage solution of the described heme protein. 16. A method for immunologically measuring a heme protein, which comprises reacting a heme protein in a sample containing transition metals with an anti-heme protein antibody.