JP2003194825A - Hemoprotein stabilizing method and preserving solution used therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel hemoprotein stabilizing method effective in the denaturing-decomposing action of a hemoprotein, and a novel hemoprotein preserving solution using a stabilizer of the hemoprotein. <P>SOLUTION: This stabilizing method of the hemoprotein in a specimen and the preserving solution used therefor are characterized by allowing the coexistence of defatted albumin or its modified material in the specimen including the hemoprotein. <P>COPYRIGHT: (C)2003,JPO

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 heme protein test 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 diseases of the digestive system such as colon cancer. This human hemoglobin detection method is an immunological method that uses an antibody specific to human hemoglobin instead of the conventional test strip method based on a chemical color reaction, and is a simple test method that does not require dietary restriction. Has been established as.

As an immunological detection method for human hemoglobin, for example, a one-dimensional immunodiffusion method utilizing an anti-human hemoglobin antibody in an agar plate and a precipitation line between human hemoglobin in a test sample and anti-human hemoglobin antibody is used. Latex agglutination method using prepared latex particles, enzyme immunoassay method using anti-human hemoglobin antibody labeled with enzyme or radioactive element, radioimmunization method, gold colloid aggregation colorimetric method using gold colloid particles sensitized with anti-human hemoglobin antibody Etc.

However, denaturation of human hemoglobin is promoted by storage conditions such as storage temperature, and it is often decomposed by bacteria or enzymes in the sample. As a result of such denaturation / decomposition, the three-dimensional structure of human hemoglobin is destroyed, resulting in a decrease in antigenicity. Therefore, in the immunological method for measuring human hemoglobin, the denaturation / degradation of hemoglobin causes erroneous diagnostic results.

On the other hand, in the fecal occult blood test, it is often the case that the subject himself or herself collects feces at home or the like and dissolves them in a closed container containing a stool lysate for use in the test. In this case, human hemoglobin in the feces is often left in a solution for several days and kept under high temperature by using transportation means such as mail. Also, when collecting stools at an inspection agency,
It may take a lot of time to test fecal occult blood because other tests are performed. Under such circumstances, as described above, denaturation / degradation of human hemoglobin occurs, which hinders accurate measurement.

In order to prevent the denaturation / decomposition of human hemoglobin in such a solution, a method of adding a general antibacterial agent such as thimerosal or chlorhexidine (Japanese Patent Laid-Open No. 6-58242).
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. (JP-A-4-1453
No. 66), addition of lytic enzyme (Japanese Patent Publication No. 5-69466).
Japanese Patent Publication) and addition of iron protoporphyrin (Japanese Patent Application Laid-Open No. 5-2
No. 81227) has been proposed. However, the techniques for stabilizing human hemoglobin described in these publications cannot sufficiently suppress the denaturation / degradation of human hemoglobin in a test liquid containing feces.

In addition to this, a method for stabilizing hemoglobin using ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) has been proposed (Japanese Patent Laid-Open No. 5-99923). However, as a result of the additional test by the inventors, E
It was confirmed that DTA alone cannot be expected to have 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). Furthermore, the present applicant has found that a method for stabilizing hemoglobin coexisting with a ferrocyanine compound (JP-A-11-118806) and a method for stabilizing hemoglobin coexisting with an enzymatic degradation product of hemoglobin (JP-A-11-218533). Patent Publication), a method for stabilizing a heme protein in the presence of transition metals (Japanese Patent Laid-Open No. 2001-2001).
249132), and a method for stabilizing a heme protein in the presence of malic acid and the like (Japanese Patent Application No. 2001-1992).
No. 24) has already been proposed.

[0008]

The first object of the present invention is to denature and modify heme proteins 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 degradation action. In particular, it is an object of the present invention to provide a technique for effectively stabilizing hemoglobin that coexists with fecal components having a strong denaturing / degrading action. A second object of the present invention is to provide a preservative solution of heme protein using a novel heme protein stabilizer.

[0009]

The first object of the present invention is to:
This has been achieved by a method for stabilizing a heme protein in a specimen, which comprises coexisting delipidated albumin (hereinafter referred to as delipidated albumin) or a modified product thereof in a specimen containing a heme protein. The second object of the present invention has been achieved by a storage solution of heme protein characterized by containing delipidated albumin or a modified product thereof. Hereinafter, the present invention will be described in more detail.

The defatted albumin used in the present invention is not particularly limited as long as it is defatted by a conventional method, and may be appropriately selected from known ones. Any degreasing albumin that can be used in the present invention can be used, but albumin derived from animal serum or egg is particularly preferable. Specific examples thereof include albumin derived from blood of cattle, horses, goats, sheep, pigs, rabbits, and pups of these animals or fetuses.

Further, as modified products of albumin, albumin-enzyme-treated products and those chemically bound with polyalkylene glycol are known. The albumin-modified products of the present invention include such albumin products. Proteins derived from can also be included. In addition to albumin derived from these organisms, recombinant albumin in which a gene encoding albumin is recombinantly expressed and mutant albumin due to mutation such as substitution / insertion / deletion of amino acid sequence are also stable in the present invention. Examples of albumin that are effective for the method of chemical conversion.

The binding amount of fatty acid per gram of defatted albumin or its modified product is preferably 100 μEq or less,
Most preferably, it is completely degreased with 0 μEq. When the binding amount of fatty acid exceeds 100 μEq, the stabilizing effect of heme protein is significantly reduced. Albumin is generally 6-7
It contains molecular fatty acids and is stable by binding with fatty acids. Therefore, fatty acids are usually contained in albumin. It is presumed that defatted albumin becomes stronger in reactivity with heme proteins due to the loss of fatty acids and contributes to stabilization of heme.

In the present invention, defatted albumin or a modified product thereof is added to a sample containing heme protein in an amount of 0.
It is made to coexist so that it becomes 01 to 10%, preferably 0.1 to 5%, and more preferably 0.5%. If the concentration of these substances is less than 0.01%, the stabilizing effect becomes insufficient, while if it exceeds 10%, it becomes difficult to dissolve and the immune reaction is inhibited.

Further, in the present invention, by adding organic acids in addition to the above substances, the heme protein in the sample can be further stabilized. Specific examples of this organic acid include at least one selected from the group consisting of malic acid, succinic acid, fumaric acid, glycolic acid, 2-ketoglutaric acid, isocitric acid, lactic acid, pyruvic acid, and oxalacetic acid, or those. The salts of.

These organic acids are 0.1 mM to 2000 m
M, preferably 1 mM to 1000 mM, more preferably 1000 mM. If the concentration of these substances is less than 0.1 mM, the stabilizing effect cannot be improved, and if it exceeds 2000 mM, it becomes difficult to dissolve and the immune reaction is also inhibited.

Furthermore, in the present invention, the heme protein in the sample can be further stabilized by adding an azide in addition to the above organic acid. Specific examples of the azide include at least one selected from the group consisting of sodium azide, potassium azide, ammonium azide and lithium azide.

This azide is contained in the heme protein-containing specimen in an amount of 0.01 to 0.2 w / v%, preferably 0.1 w / v%. If the azide concentration is less than 0.01 w / v%, the stabilizing effect cannot be improved.
On the other hand, of the azides, sodium azide is designated as a poisonous substance (Government Ordinance No. 405: Cabinet Order that partially revises the poisonous and deleterious substance designation order), so 0.2 w / v% It is not preferable to use it over.

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

In addition to the above protein protecting agents, for example, many known heme protein protecting agents such as an antibacterial agent for preventing unnecessary growth of microorganisms and a buffering agent giving a pH advantageous for the storage of heme proteins are known. Addition of ingredients is also effective. Examples of the antibacterial agent include lytic enzymes, ethyl benzoate, penicillin, fungison, streptomycin, cefamycin, and other non-penicillin series antibiotics. It is also known that protease inhibitors such as trypsin inhibitor and α2 macroglobulin stabilize heme proteins.

The pH of the storage solution is set within a range in which heme proteins can be stably retained. Under extremely acidic or alkaline conditions, the stability of the heme protein may be impaired, and a pH in the neutral range is desirable. Specifically, the pH is about 5 to 10, preferably about 6 to 8. A suitable buffer is used to maintain the pH. For example, hydroxyethylpiperazine-2-ethanesulfonic acid (abbreviated as HEPES) or piperazine-bis (2-ethanesulfonic acid)
Good buffering agents such as (abbreviated as PIPES) have the pH (6 to
It is also used as a reaction buffer solution when heme protein is detected by an immunoreaction at the same time as 8) is given, and it is mentioned as a particularly preferable buffer agent. In addition, phosphate buffer, Tris buffer, glycine buffer,
A borate buffer solution or the like can also be used.

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

The present invention can provide a stock solution of heme protein containing defatted albumin or a modified product thereof which acts as a stabilizing factor of heme protein. The defatted albumin or a modified product thereof is most preferably a binding amount of fatty acid of 100 μEq or less per gram, and particularly 0 μEq completely defatted.

Furthermore, the preservation solution of the present invention may contain an organic acid, an azide, a protein protecting agent and a buffering agent, if necessary. The organic acid, azide, protein protecting agent and buffering agent can be appropriately selected and used from those mentioned above.

The present invention can provide a method for immunologically measuring a heme protein, which is an application of the above-mentioned stabilization technology for a heme protein. Examples of the immunological measurement method include a latex agglutination reaction method, a gold colloid agglutination reaction method, an immunochromatography method, and an ELISA method. In any of the measurement methods, coexistence of defatted albumin or a modified product thereof in a heme protein-containing sample protects the antigen activity during storage and suppresses a decrease in the measured value.

The method for stabilizing a heme protein of the present invention comprises:
It is useful for stabilizing the hemoprotein as an analyte in the sample. Particularly, in the method for measuring a heme protein by an immunological method using an antibody that recognizes the heme protein, the antigenicity of the heme protein to be measured is highly stabilized. Feces, urine, and blood are known as samples for detecting heme proteins. In particular, hemoglobin in feces serves as an index of bleeding in the digestive system, and hemoglobin in urine serves as an index of bleeding in the urinary tract.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION When the stabilization method of the present invention is applied to hemoglobin present in a fecal sample, defatted albumin or a modified product thereof may be added to a storage solution in which feces are suspended. . Usually, in the detection of hemoglobin in feces, the feces are suspended in an appropriate storage solution and filtered as needed to obtain a sample for immunological analysis.

[0027]

INDUSTRIAL APPLICABILITY According to the method for stabilizing a heme protein of the present invention, a protective action can be obtained even in the coexistence of a biological component having a strong denaturing / degrading action on the heme protein, particularly a fecal component. Therefore, it is useful for analysis of biological components, for example, stabilization of hemoglobin contained in a sample for the purpose of detecting fecal occult blood. In particular, it contributes to the maintenance of the antigenicity of the heme protein as an immunological analysis target for which protection of the antigen structure is required. The present invention effectively stabilizes hemoglobin in a fecal sample, and can be expected to prevent false negative results due to denaturation / degradation of hemoglobin.

[0028]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1. Stabilizing effect of hemoglobin by defatted BSA Defatted BSA (manufactured by Roche Diagnostics, Inc., trade name: fraction5, fatty acid free) and undefatted BSA as a control are 0.5% each.
Buffer containing (50 mM HEPES buffer, pH
7.0, 0.9% NaCl, 0.1% NaN ₃ ) 2
10 ml of normal stool sample (AC) and human hemolyzed blood were added to ml, and hemoglobin measurement value immediately after addition and 1 at 37 ° C.
The hemoglobin measurement values after storage for 7 hours were compared.
The measurement was carried out using an OC sensor neo (manufactured by Eiken Chemical Co., Ltd.) with a dedicated reagent (OC Hemodia Auto III: manufactured by Eiken Kagaku Co., Ltd.) having a latex agglutination reaction as a principle of measurement. The results are shown in Tables 1 to 3. As shown in the table, it was confirmed that the one to which defatted BSA was added had a significantly higher effect of stabilizing hemoglobin after storage at 37 ° C. for 17 hours, as compared with the one to which non-defatted BSA was added as a control. It was

[0030]

[Table 1]             ────────────────────────────                                  Stool sample A             ────────────────────────────                        0 hours after 37 ° C / 17 hours                         ng / ml ng / ml%             ────────────────────────────             Control BSA 541 306 56.6             Degreasing BSA 578 405 70.1             ────────────────────────────

[0031]

[Table 2]             ────────────────────────────                                  Stool sample B             ────────────────────────────                        0 hours after 37 ° C / 17 hours                         ng / ml ng / ml%             ────────────────────────────             Control BSA 509 250 49.1             Degreasing BSA 508 411 80.9             ────────────────────────────

[0032]

[Table 3]             ────────────────────────────                                  Stool sample C             ────────────────────────────                        0 hours after 37 ° C / 17 hours                         ng / ml ng / ml%             ────────────────────────────             Control BSA 585 389 66.5             Degreasing BSA 580 420 72.4             ────────────────────────────

Example 2. Stabilizing effect of hemoglobin by defatted BSA and organic acid As defatted BSA and organic acid used in Example 1, (3)
Disodium malate (1M), (4) Disodium succinate (1M), (5) Disodium fumarate (1M)
And (2) defatted BSA and (1) 0.5% undefatted BSA as a comparative control (50 mM HEPES buffer, pH 7.0, 0.9%).
NaCl, 0.1% NaN ₃ ) 2 ml, normal stool sample (DF) 10 mg and human hemolyzed blood were added, and hemoglobin measurement value immediately after addition and hemoglobin measurement when stored at 37 ° C. for 17 hours The values were compared. Measurement is OC-NE
It was carried out by using a dedicated reagent (OC-Auto III: manufactured by Eiken Chemical Co., Ltd.) whose measuring principle is latex agglutination reaction using O (manufactured by Eiken Chemical Chemicals). The results are shown in Tables 4-6. As shown in the table,
Compared with (1) non-defatted BSA as a control and (2) defatted BSA, (3) to (5) defatted BSA and an organic acid were added after storage at 37 ° C. for 17 hours. It was confirmed that the stabilizing effect of hemoglobin of (1) was further improved. In particular, it can be seen that the combination of defatted BSA and disodium malate produced a remarkable effect as compared with the non-defatted BSA and the defatted BSA alone.

[0034]

[Table 4]             ────────────────────────────                                  Stool sample D             ────────────────────────────                        0 hours after 37 ° C / 17 hours                         ng / ml ng / ml%             ────────────────────────────             (1) 579 311 53.7             (2) 581 402 69.2             (3) 582 468 80.4             (4) 581 418 71.9             (5) 578 405 70.1             ────────────────────────────

[0035]

[Table 5]             ────────────────────────────                                  Stool sample E             ────────────────────────────                        0 hours after 37 ° C / 17 hours                         ng / ml ng / ml%             ────────────────────────────             (1) 513 158 30.8             (2) 510 318 62.4             (3) 506 491 97.0             (4) 515 438 85.0             (5) 512 392 76.6             ────────────────────────────

[0036]

[Table 6]             ────────────────────────────                                  Stool sample F             ────────────────────────────                        0 hours after 37 ° C / 17 hours                         ng / ml ng / ml%             ────────────────────────────             (1) 588 371 63.1             (2) 590 401 68.0             (3) 593 472 79.6             (4) 597 456 76.4             (5) 587 427 72.7             ────────────────────────────

Claims (16)

[Claims] 1. A method for stabilizing a heme protein in a specimen, which comprises allowing delipidated albumin or a modified product thereof to coexist in a specimen containing a heme protein. 2. The method for stabilizing a heme protein in a sample according to claim 1, wherein the bound amount of fatty acid is 100 μEq or less per gram of defatted albumin or a modified product thereof. 3. The method for stabilizing heme proteins in a sample according to claim 1, wherein the concentration of defatted albumin or its modified product is in the range of 0.01 to 10%. 4. The method for stabilizing a heme protein in a sample according to claim 1, wherein an organic acid coexists. 5. The organic acid is malic acid, succinic acid, fumaric acid,
The method for stabilizing a heme protein in a sample according to claim 4, which is at least one selected from the group consisting of glycolic acid, 2-ketoglutaric acid, isocitric acid, lactic acid, pyruvic acid, and oxalacetic acid, or salts thereof. . 6. The method for stabilizing a heme protein in a sample according to claim 4, wherein the concentration of the organic acid is in the range of 0.1 to 2000 mM. 7. The method for stabilizing a heme protein in a sample according to claim 1, wherein an azide is coexistent. 8. The method for stabilizing a heme protein in a sample according to claim 7, wherein the azide is at least one selected from the group consisting of sodium azide, potassium azide, ammonium azide and lithium azide. 9. The method for stabilizing a heme protein in a sample according to claim 1, wherein the heme protein is hemoglobin, myoglobin, peroxidase, or catalase. 10. The sample containing heme protein is feces,
The method for stabilizing a heme protein in a sample according to claim 1, which is urine or blood. 11. A preservation solution of a heme protein, which contains defatted albumin or a modified product thereof. 12. Defatted albumin or modified product 1 thereof
The storage solution of heme protein according to claim 11, wherein the binding amount of fatty acid per gram is 100 μEq or less. 13. The method according to claim 11, wherein an organic acid coexists.
A stock solution of the hemoprotein according to 2. 14. The organic acid is at least one selected from the group consisting of malic acid, succinic acid, fumaric acid, glycolic acid, 2-ketoglutaric acid, isocitric acid, lactic acid, pyruvic acid, and oxalacetic acid, or a combination thereof. The heme protein preservation solution according to claim 13, which is a salt. 15. The method according to claim 11, further comprising an azide.
Stock solution of the described heme protein. 16. The azide is at least one selected from the group consisting of sodium azide, potassium azide, ammonium azide and lithium azide.
Stock solution of the described heme protein.