JPH11118806A - Stabilization method of hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stabilize hemoglobin contained in a sample, and particularly to keep an antigenic structure of hemoglobin as an object to be immunologically analyzed, by making a ferrocyanic compound coexist in a bio-sample such as feces and urine. SOLUTION: For example, a human feces sample is added and suspended into a dispersion medium of a specific composition containing, for example, sodium ferrocyanide as a ferrocyanic compound, and is preserved under a specific condition, and a concentration of human hemoglobin is measured by the immunological agglutination. When a ratio of a measured value after the preservation to a measured value before the preservation, is regarded as a Hb residual ratio, the residual ratio is remarkably high when the ferrocyanic compound is added, in comparison with the case when it is not added. The similar effect can be also obtained when pottasium ferrocyanide is used. The concentration of the ferrocyanic compound in the dispersion medium is preferably 0.5-100 mM, and an addition in that case, is preferably 0.05-0 mM per 1 mg of faces. By applying the method mentioned above, the pseudo-negative result can be effectively prevented in the detection of the occult blood in the feces or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for stabilizing hemoglobin. Specifically, the present invention relates to a technique for stabilizing hemoglobin in a sample to be detected and hemoglobin as a standard used as a positive control. Detection of hemoglobin contained in urine and feces
Useful for the diagnosis of many diseases. In particular, detection of hemoglobin (fecal occult blood) in feces is important information in diagnosis of digestive diseases such as colorectal cancer. Instead of the test paper method based on the chemical color reaction that has been used for detecting hemoglobin in feces for a long time, detection methods by immunological methods using antibodies against hemoglobin have become widespread in recent years, and dietary restriction is not required Established as a simple inspection method.

[0002]

2. Description of the Related Art In order to detect hemoglobin contained in a sample such as stool, it is necessary to transport the stool sample to a laboratory after collecting the stool. A transport container [1] equipped with a stool collection mechanism and a stool suspension filtration mechanism is used to transport the stool sample. By using this type of container, it is possible to quantitatively collect feces, and the fecal suspension can be easily filtered. The container from which feces are collected is transported to the laboratory by mail or other means.

During transportation, hemoglobin coexists with bacteria and many other components contained in feces. In addition, since it is generally difficult to control the temperature, exposure to unfavorable temperature conditions for storage cannot be avoided. Therefore, hemoglobin during transportation may always be denatured or degraded. Denaturation / decomposition of hemoglobin during transportation leads to erroneous diagnosis results, and therefore it is desired to minimize it.

[0004] In addition to hemoglobin, other proteins and sugars are used for stabilizing proteinaceous substances. Regarding hemoglobin, bovine serum albumin (hereinafter abbreviated as BSA), rabbit serum albumin, or ovalbumin as a protein, and sucrose as a saccharide show a stabilizing effect.
[2] It is known. The use of animal serum has also been reported [3]. However, these general stabilizers have a small effect of stabilizing hemoglobin particularly in a fecal suspension, so that sufficient storage performance cannot be expected. In feces, there are many components that cause denaturation / decomposition of hemoglobin, such as bacteria and proteolytic enzymes, and hemoglobin cannot be sufficiently protected only by proteins and sugars.

[0005] In addition, animal serum is not a purified and pure substance, so that a lot difference is likely to occur in the stabilizing effect. In addition, since it is not possible to deny the possibility that a component that has a denaturing effect on hemoglobin is present in animal serum containing many components, it is hard to say that it is a desirable stabilizer. In a report on attempts to stabilize hemoglobin with bovine serum [3],
It was necessary to add as much serum as 10-20% v / v. Stabilization techniques that require large amounts of expensive animal serum
Economically disadvantageous.

Techniques for stabilizing hemoglobin in fecal suspensions include the addition of lytic enzymes [4], the use of antibacterial compounds and the like [5] [6], the addition of animal hemoglobin [7], protease inhibitors [8], pH control [9], iron protoporphyrin [10], transferrin [11]
Addition of iron-containing proteins such as [15] and peroxidase [12], and addition of sodium fluoride [14] are known. Alternatively, combinations of multiple components [13] have been attempted. These are considered to show the protective effect of hemoglobin by suppressing the effect of bacteria or dispersing the effect on hemoglobin by a compound structurally similar to hemoglobin. However, there is a hemoglobin denaturing / degrading action in feces that cannot be suppressed by these known hemoglobin stabilization techniques. Therefore, the current hemoglobin stabilization technology still has room for improvement.

[0007] In addition, the effect of stabilizing hemoglobin by ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) is known [16]. However, as a result of the additional examination by the present inventors,
It was confirmed that EDTA cannot be expected to have a sufficient stabilizing effect on hemoglobin in feces. Therefore, in feces, which is a very disadvantageous condition for storage, E
It can be said that hemoglobin cannot be stabilized by DTA.

In addition to these hemoglobin stabilizing factors,
The present applicant has newly found a stabilizing effect on hemoglobin of a water-soluble metal complex of a transition metal ion and has filed a patent application [1
7]. Furthermore, a patent application was filed for a blood protein stabilization technique utilizing the fact that antibodies against stool components contribute to stabilization of blood proteins present in feces [18].

[0009]

SUMMARY OF THE INVENTION The first object of the present invention is to provide a novel hemoglobin protective substance. In particular, the most important object of the present invention is to provide a technique for effectively stabilizing hemoglobin which coexists with a fecal component having a strong denaturing / degrading action.

A second object of the present invention is to provide a technique for stabilizing hemoglobin as a sample for analysis. Specifically, it is an object of the present invention to provide a hemoglobin stabilization technique to be detected in a hemoglobin detection method based on an immunological technique using an antibody that recognizes hemoglobin, which is currently the mainstream.

A third object of the present invention is to provide a dispersion medium for fecal sample suspension for immunological analysis using a new hemoglobin stabilizer, and a measurement technique using the same. It is in.

[0012]

SUMMARY OF THE INVENTION The present invention relates to a method for stabilizing hemoglobin by coexisting a ferrocyan compound and a dispersion medium for suspending a stool sample to which this stabilization method is applied. In addition, the present invention provides an immunological measurement method using an antibody recognizing human hemoglobin of human hemoglobin contained in a biological sample, wherein the method is a biological sample stored in the presence of a ferrocyanide compound. I do.

The method for stabilizing hemoglobin of the present invention is useful for stabilizing hemoglobin as an analysis target present in a biological sample. Particularly, in a method for measuring hemoglobin by an immunological technique using an antibody that recognizes hemoglobin, the antigenicity of hemoglobin to be measured is highly stabilized. Feces and urine are known as biological samples from which hemoglobin is to be detected. Hemoglobin in feces serves as an indicator of bleeding in the digestive tract, while bleeding in the urinary tract is suspected when detecting hemoglobin in urine. In particular, it has become common to use immunological techniques with high specificity to distinguish hemoglobin from feces, in order to distinguish it from dietary hemoglobin, and to maintain its antigenicity stably The need is high.

When the stabilizing method of the present invention is applied to hemoglobin present in a stool sample, a ferrocyanide compound is preferably added to a dispersion medium for suspending the stool. When detecting hemoglobin in ordinary stool, the stool is suspended in an appropriate dispersion medium and, if necessary, filtered to obtain an immunological analysis sample. It is advantageous to add a ferrocyan compound to the dispersion medium used at this time. The concentration of the ferrocyan compound in the dispersion medium was 0.5
The amount used is mM or more.

On the other hand, as described above, at present, a simple transport container capable of collecting, transporting, suspending, and filtering feces in one container has been put to practical use. In this type of container,
The dispersing medium is filled at the time of shipment, so that the subject can collect the feces by himself and mail the container to the testing facility. In general, the subject must be unfamiliar with such a task, and therefore must assume a case in which the amount of feces collected cannot be strictly controlled. Also, it is desirable to use an excess amount so that there is no problem even if the hemoglobin protection effect of the ferrocyanide compound is slightly reduced during transportation. In order to bring hemoglobin into contact with the ferrocyanide compound promptly in the dispersion medium, it is desirable to use an excessive amount of hemoglobin in an excess amount. Therefore, for example, assuming a case in which potassium ferrocyanide is used for stabilizing human hemoglobin, the amount of feces collected at the time of design is 1 mg stool, 0.05 to 1 stool.
It is good to add potassium ferrocyanide in 0 mM, more preferably in the range of 0.06 to 1 mM.

Known components can be added to the dispersion medium in addition to the ferrocyan compound which is the protective agent of the present invention.
For example, it is also effective to add a buffer which gives a pH advantageous for preservation of hemoglobin, an antibacterial agent for preventing unnecessary growth of microorganisms, or many known hemoglobin protective components. Because the protective agent of the present invention, the ferrocyanide compound, is presumed to stabilize hemoglobin by a different mechanism of action from known protective components. This is because enhancement of the protective action can be expected. For example, the protective effects of the following components are known.

Examples of the inactive protein having a hemoglobin stabilizing effect include serum albumin derived from humans, cows, rabbits, horses, sheep, goats, etc., and albumin derived from egg whites. Amino acids such as lysine and histidine also have a protective effect on hemoglobin. Antibacterial substances include lytic enzymes [4], azide, ethyl benzoate, penicillin, fungisone [5],
Streptomycin, cephamycin, and a series of non-penicillin antibiotics [6] are known. It is also known that protease inhibitors such as trypsin inhibitors and α2 macroglobulin stabilize hemoglobin [8]. Sodium fluoride [14] or Fe
It has also been reported that a water-soluble metal complex of a transition metal ion such as (III) -EDTA complex [17] has a hemoglobin stabilizing effect. Further, salts for adjusting the ionic strength can be added.

It is also possible to use a deodorant, a fragrance, a dye or the like in combination within a range that does not hinder the analysis and the stabilization of hemoglobin. P
An H indicator can be combined, or an indicator of a component present in normal feces can be added so that the presence or absence and amount of stool can be confirmed. For example, if a diazonium compound that changes color by reacting with bilirubin is added, it can be visually recognized whether a required amount of feces has been collected. Specific examples of the composition of the solution for stabilizing hemoglobin according to the present invention are shown below. Ferrocyanide compound: 0.5 to 100 mM HEPES buffer (pH 7.4): 10 to 500 mM BSA: 0.1 to 5% NaN3: 0.1 to 1%

The pH of the dispersion medium is set within a range in which hemoglobin can be stably maintained. Under extreme acid or alkali conditions, the stability of hemoglobin may be impaired, and it is difficult to obtain the protective action of the ferrocyanide compound. Therefore, a neutral pH is desirable. Specifically, the pH may be 5 to 10, preferably about 6 to 8. Appropriate buffers can be used to maintain the pH. For example, hydroxyethylpiperazine-2-ethanesulfonic acid (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid)
acid, HEPES) and piperazine-bis (2-ethanesulfonic acid) (Piperazine-N, N'-bis (2-e
G such as thanesulfonic acid) and PIPES)
The OOD buffer is particularly preferable because it provides a pH (6 to 8) that seems to stabilize the structure of hemoglobin most, and is also used as a reaction buffer when hemoglobin is detected by an immune reaction. As a buffer. In addition, a phosphate buffer, a Tris buffer, a glycine buffer, or the like can be used.

The method for stabilizing hemoglobin of the present invention can be used for stabilizing hemoglobin in a fecal sample for the purpose of detecting fecal occult blood. Particularly, hemoglobin as an immunological analysis target requiring protection of the antigen structure is useful for maintaining its antigenicity.

The present invention provides a method for immunologically measuring hemoglobin to which the above-mentioned hemoglobin stabilization technique is applied.
As an immunological measurement method, a latex agglutination method,
A colloidal gold agglutination reaction method, an immunochromatography method, an ELISA method and the like can be mentioned. In any of the measurement methods, the coexistence of the ferrocyan compound in the hemoglobin-containing sample protects the antigen activity during storage and suppresses a decrease in the measured value.

It is also possible to construct an agglutination reaction system using only monoclonal antibodies. In order to perform an agglutination reaction using only monoclonal antibodies, it is advantageous to combine a plurality of types that recognize different epitopes on hemoglobin. Hemoglobin has a tetrameric structure in which two α-chains and two β-chains are associated with each other, and in principle, even a single type of monoclonal antibody aggregates. However, since hemoglobin is in an equilibrium state between a dimer structure and a tetramer structure of αβ in a solution, a single monoclonal antibody cannot react with all of them. In addition, since a single monoclonal antibody cannot react if the target epitope is denatured, it is stochastically advantageous to use a combination of a plurality of types of monoclonal antibodies.

[0023]

The ferrocyanide compound of the present invention has an effect of effectively suppressing a decrease in the measured value of hemoglobin during the storage period. In particular, it contains a lot of denaturing / degrading components,
In addition, it shows a sufficient protective effect against hemoglobin in a fecal suspension for which storage conditions are difficult to control. It is not known how ferrocyanide compounds protect hemoglobin by its mechanism of action.

[0024]

According to the hemoglobin stabilizing technique of the present invention,
A protective effect can be obtained even in the presence of a fecal component having a strong denaturing / degrading effect on hemoglobin.
Therefore, it is useful for stabilizing hemoglobin contained in a sample for the purpose of analyzing fecal occult blood. Particularly, hemoglobin as an immunological analysis target requiring protection of the antigen structure contributes to maintaining its antigenicity. 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. The ferrocyan compound required for the present invention is inexpensive and economically advantageous compared to expensive substances such as special enzymes and antibiotics. In addition, since a small amount of a ferrocyanide compound exhibits a high stabilizing effect, it can be said that this is also an economically advantageous stabilizing technique.

Next, the present invention will be described in more detail with reference to examples.

[0026]

【Example】

1. Stabilization of hemoglobin by ferrocyanide compound Five kinds of human stool samples whose hemoglobin amounts were measured in advance were selected from five samples having a hemoglobin amount of 700 ng / ml or more, and a dispersion medium (5 to 100 mM) containing a ferrocyanide compound (0 to 100 mM) was selected.
0 mM HEPES buffer, pH 7.4, 0.1% BSA
Was observed at 37 ° C. for 24 hours. Sodium ferrocyanide (manufactured by Wako Pure Chemical Industries) in a commercially available stool collection container (manufactured by Eiken Chemical)
Two containers were prepared by dispensing 2 ml of a dispersion medium containing 0, 20, 50, and 100 mM, respectively, and the above five types of feces were collected according to the instructions of the container for collecting feces, and were well suspended. The amount of hemoglobin was measured, and the remaining one was stored in a thermostat at 37 ° C., and the amount of hemoglobin was measured 24 hours later.

The above-mentioned stool collection container combines a mechanism capable of filtering and removing the liquid inside by providing a hole in the dropping portion, together with a stool collection mechanism. If it is attached to the container, the stool can be collected quantitatively by the rubbing mechanism. The containers used for the experiments are designed to collect approximately 10 mg of feces.

Measurement of hemoglobin: A hole was made in the dropping portion of the container according to the instructions for the container for stool collection, the container was squeezed, and the fecal suspension was dropped to obtain a filtrate. Using a commercially available reagent for measuring hemoglobin OC-Hemodiaauto II 'Eiken' (manufactured by Eiken Chemical Co., Ltd.), add 300 µl of the reagent to 50 µl of the filtrate, and add
The reaction was performed at 7 ° C. for 3 minutes. The hemoglobin concentration was determined from the change in absorbance at 585 nm based on the immunological agglutination during this time. As a standard, human hemolyzed blood (HEP containing 1000 ng / ml human hemoglobin) attached to the reagent was used.
ES buffer).

The results are shown in Table 1 and FIG. The numbers in the table represent the hemoglobin measured values of the five types of feces after storage at 37 ° C. for 24 hours in terms of% with the measured value at the start of the experiment being 100. FIG. 2 is a line graph showing the effect of the concentration of sodium ferrocyanide. Although the stabilizing effect of hemoglobin decreased as the peak concentration of the ferrocyanide compound increased to 5 mM and became higher, a remarkable stabilizing effect was observed as compared with no addition.

[0030]

[Table 1]

The ferrocyanide compound was replaced with potassium ferrocyanide (manufactured by Wako Pure Chemical Industries, Ltd.), and the number of fecal samples was increased to 10 types.
The stabilizing effect in a low concentration range of 5 mM or less was examined. The measuring method is the same as in 1. The results are shown in Table 2 and FIG. A response curve that almost reached a plateau was obtained at a potassium ferrocyanide concentration of 1.2 mM.

[0032]

[Table 2]

The effects of 1.2 mM potassium ferrocyanide and two other hemoglobin stabilizers were compared. 500 μg of bovine hemoglobin (manufactured by Sigma) and 2 mM of an iron-EDTA complex (Fe (III) -EDTA (manufactured by Dojindo Chemical)) were added to 2 ml of the dispersion medium, respectively.
The hemoglobin residual ratio after 4 hours was measured. Fig. 3 shows the results.
Shown in Although the stabilizing effect varies from feces to feces, the ferrocyanide compound showed a stabilizing effect equal to or higher than that of animal hemoglobin and Fe (III) -EDTA which were conventionally considered to be effective for stability.

References [1] Japanese Utility Model Publication No. 5-175652 [2] JP-A-63-243756 [3] JP-A-4-145366 [4] Japanese Patent Publication No. 5-69466 [5] JP-A-63-271160 [6] JP-A-7-72154 [7] JP-A-2-296149 [8] JP-A-3-279598 [9] JP-A-5-281226 [10] JP-A-5-281227 [11] JP-A-8-29429 [12] 8-29430 [13] JP-A-6-281654 [14] JP-A-7-191026 [15] JP-A-8-262020 [16] JP-A-2-22159 [17] JP-A-7-229902 [18] -302051

[Brief description of the drawings]

FIG. 1. Stabilization effect of hemoglobin by sodium ferrocyanide at high concentration

Fig. 2 Hemoglobin stabilizing effect of potassium ferrocyanide in low concentration range

FIG. 3 Comparison of stability between potassium ferrocyanide and various stabilizers

Claims (10)

[Claims] 1. A method for stabilizing hemoglobin by coexisting a ferrocyanide compound. 2. The method for stabilizing hemoglobin according to claim 1, wherein hemoglobin is present in the fecal suspension, and the dispersion medium of the suspension contains a ferrocyanide compound. 3. The method for stabilizing hemoglobin according to claim 1, wherein hemoglobin is present in urine and a ferrocyanide compound is added to urine. 4. The ferrocyanide compound is sodium ferrocyanide or potassium ferrocyanide.
Method 3 for stabilizing hemoglobin 5. The method for stabilizing hemoglobin according to claim 1, wherein hemoglobin is a component to be analyzed by an immunological technique using an antibody recognizing hemoglobin, and the antigen activity is stabilized. 6. The method for stabilizing hemoglobin according to claim 1, wherein the hemoglobin is human hemoglobin. 7. A ferrocyanide compound concentration of 0.5 to 100.
2. The method for stabilizing hemoglobin according to claim 1, wherein the amount is mM. 8. A dispersion medium for suspending a stool sample to be tested for the presence of hemoglobin, wherein the dispersion medium has a ferrocyanide compound added thereto in advance. 9. The amount of 0.05-1 per mg of feces to be suspended
10. The dispersion medium according to claim 9, wherein 00 mM of a ferrocyanide compound is added. 10. A method for immunologically measuring human hemoglobin contained in a biological sample using an antibody that recognizes hemoglobin, wherein the biological sample stored in the presence of a ferrocyan compound is analyzed. Measuring method