Method for stabilizing a complex between hemoglobin and haptoglobin, and storage solution for storing a specimen containing hemoglobin

Abstract

To stabilize a hemoglobin-haptoglobin complex.SOLUTION: A method for stabilizing a hemoglobin-haptoglobin complex comprises preserving a hemoglobin-haptoglobin complex under the presence of a hemoglobin degradant. With such a method, the hemoglobin-haptoglobin complex can be stabilized.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for stabilizing a complex between hemoglobin and haptoglobin, a preservation solution for preserving a complex between hemoglobin and haptoglobin, a preservation solution for preserving a specimen containing hemoglobin, and a method and kit for detecting hemoglobin in a specimen. [Background technology]

[0002] Detection of blood contained in feces, urine, saliva, etc. is useful for diagnosing many diseases. For example, fecal occult blood tests that detect blood in feces are used in colon cancer screening. As a method for detecting occult blood, an immunological method is known in which hemoglobin contained in occult blood in a specimen such as feces is detected using an anti-hemoglobin antibody. A specimen to be subjected to an occult blood test is usually collected by a subject in a container containing a preservative solution and sent to a testing institution such as a hospital. In many cases, the preservative solution (sample) containing the specimen is stored for several days until it is actually used for testing, and during that time, it is often placed under high temperatures. Hemoglobin is unstable in solution and is particularly prone to denaturation or decomposition under high temperature conditions. If the structure of the epitope or its surrounding site changes due to the denaturation or decomposition of hemoglobin, the antibody will no longer be able to recognize hemoglobin, and therefore the accuracy of hemoglobin detection by the immunological method will decrease.

[0003] In addition, in occult blood tests, automatic analyzers capable of analyzing a large number of samples quickly and accurately are widely used to measure hemoglobin concentration by immunological methods. In general, in measurements using automatic analyzers, changes in the device and changes in the reagents used in the measurements have a large effect on the measurement results, so that the automatic analyzer is periodically calibrated or quality controlled using a calibrator or control containing a substance to be measured of a known concentration. The calibration of the automatic analyzer is performed by measuring a calibrator containing a substance to be measured of a known concentration and creating a calibration curve, and the quality control of the automatic analyzer is performed by measuring a control containing a substance to be measured of a known concentration and checking whether the measured value is within a predetermined range. However, hemoglobin is unstable in a solution, and if the structure of the epitope or its surrounding site changes due to denaturation or decomposition of the hemoglobin contained in the calibrator or control, the antibody will not be able to recognize the hemoglobin, and the calibration and quality control of the automatic analyzer will not be able to be performed accurately, resulting in inaccurate measurement.

[0004] Under such a background, a number of methods have been proposed to stabilize hemoglobin in a sample. For example, a method of adding an antibacterial agent such as thimerosal or chlorhexidine (e.g., Patent Document 1), a method of adding hemoglobin from animals other than humans (e.g., Patent Document 2), a method of adding serum from animals other than humans (e.g., Patent Document 3), a method of adding a glycosidase-type lytic enzyme (e.g., Patent Document 4), a method of adding a water-soluble transition metal complex (e.g., Patent Document 5), a method of adding an enzyme decomposition product of hemoglobin (e.g., Patent Document 6), a method of adding sulfurous acid or disulfurous acid, etc. (e.g., Patent Document 7), a method of adding an organic acid such as malic acid (e.g., Patent Document 8), a method of adding an iminocarboxylic acid (e.g., Patent Document 9), a method of adding glyoxylic acid (e.g., Patent Document 10), a method of adding a haloalkanesulfonic acid (e.g., Patent Document 11), etc. have been proposed.

[0005] However, since hemoglobin is very unstable, even these methods for stabilizing hemoglobin have not yet been able to sufficiently suppress its denaturation or decomposition. On the other hand, a method of adding haptoglobin to stabilize hemoglobin is also known (for example, Patent Document 12). Haptoglobin is present in the blood of a wide variety of animals and is a protein that plays a role in recovering hemoglobin released into the blood due to hemolysis of red blood cells. It is known that haptoglobin rapidly binds to hemoglobin to form a stable hemoglobin-haptoglobin complex (Hb-Hp complex). By adding haptoglobin in advance to a preservation solution or the like to which a specimen such as feces is added, the hemoglobin contained in the specimen can be made into a stable hemoglobin-haptoglobin complex when the specimen is added. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 63-271160 [Patent Document 2] Japanese Patent Application Publication No. 2-296149 [Patent Document 3] Japanese Patent Application Publication No. 4-145366 [Patent Document 4] Special Publication No. 5-69466 [Patent Document 5] Japanese Patent Application Publication No. 7-229902 [Patent Document 6] Japanese Patent Application Publication No. 11-218533 [Patent Document 7] JP 2000-258420 A [Patent Document 8] JP 2003-14768 A [Patent Document 9] JP 2009-097956 A [Patent Document 10] JP 2013-257216 A [Patent Document 11] JP 2016-191580 A [Patent Document 12] Japanese Patent Application Publication No. 10-132824 Summary of the Invention [Problem to be solved by the invention]

[0007] Since there are many bacteria and protease enzymes that cause hemoglobin decomposition in biological samples, particularly in feces, even hemoglobin-haptoglobin complexes may be decomposed. Therefore, an object of the present invention is to stabilize the hemoglobin-haptoglobin complex. [Means for solving the problem]

[0008] The method for stabilizing a hemoglobin-haptoglobin complex according to the present invention includes a step of storing the hemoglobin-haptoglobin complex in the presence of a hemoglobin degradation product. The hemoglobin degradation product may be an enzymatic hemoglobin degradation product. The method may include a step of storing the hemoglobin-haptoglobin complex in a storage solution containing the hemoglobin degradation product, and the concentration of the hemoglobin degradation product in the storage solution may be 0.012 mg / L or more in terms of iron equivalent. The hemoglobin-haptoglobin complex may include a hemoglobin-haptoglobin complex produced by contacting a specimen containing hemoglobin with haptoglobin. The specimen may be feces, saliva, or urine, or may be feces.

[0009] The preservation solution for preserving a complex of hemoglobin and haptoglobin according to the present invention comprises a hemoglobin degradation product, which may further comprise a complex of hemoglobin and haptoglobin and can be used as a calibrator or control.

[0010] A preservation solution for preserving a specimen containing hemoglobin according to the present invention includes haptoglobin and a hemoglobin degradation product. The specimen may be feces, saliva, or urine.

[0011] The hemoglobin degradation product may be an enzymatic degradation product, and the concentration of the hemoglobin degradation product may be 0.012 mg / L or more in terms of iron equivalent.

[0012] The method of the present invention for detecting hemoglobin in a specimen comprises the steps of: obtaining a specimen containing the specimen by adding the specimen to the preservation solution for preserving the specimen containing hemoglobin; and detecting the hemoglobin in the sample by an immunological technique, wherein the hemoglobin in the sample forms a complex with haptoglobin.

[0013] A kit for detecting hemoglobin in a sample according to the present invention comprises the above-mentioned preservation solution for preserving a sample containing hemoglobin, and a reagent containing an anti-hemoglobin antibody. Effect of the Invention

[0014] According to the present invention, it is possible to stabilize the hemoglobin-haptoglobin complex. In other words, according to the present invention, it is possible to suppress the denaturation and decomposition of hemoglobin in the hemoglobin-haptoglobin complex. Therefore, according to the present invention, it is possible to detect hemoglobin in a sample by an immunological method with higher accuracy. In addition, it is possible to provide a calibrator or control with excellent storage stability. [Brief description of the drawings]

[0015] [Figure 1] 1 is a graph showing the effect of addition of hemoglobin degradation products on the recovery rate of hemoglobin-haptoglobin complexes at 37° C. [Diagram 2] 1 is a graph showing the effect of addition of hemoglobin digests on the recovery rate of hemoglobin-haptoglobin complexes at 56° C. [Diagram 3] 1 is a graph showing the effect of addition of hemoglobin digests on hemoglobin recovery at 37° C. [Figure 4]1 is a graph showing the effect of addition of hemoglobin digests on hemoglobin recovery at 56° C. [Diagram 5] 1 is a graph showing the relationship between the concentration of hemoglobin degradation products and the recovery rate of hemoglobin in feces. [Figure 6] 1 is a graph showing the relationship between the concentration of hemoglobin degradation products and the recovery rate of hemoglobin in feces. [Figure 7] 1 is a graph showing the recovery rate of hemoglobin in feces when haptoglobin was added but no hemoglobin degradation product was added. [Figure 8] 1 is a graph showing the recovery rate of hemoglobin when haptoglobin and hemoglobin degradation products were added. [Figure 9] 1 is a graph showing the recovery rate of hemoglobin when haptoglobin and hemoglobin degradation products were not added. [Figure 10] 1 is a graph showing the recovery rate of hemoglobin when no haptoglobin was added and a hemoglobin degradation product was added. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The method of the present invention for stabilizing a hemoglobin-haptoglobin complex comprises storing the hemoglobin-haptoglobin complex in the presence of a hemoglobin degradation product.

[0017] The hemoglobin decomposition product is a fragment of hemoglobin, and examples of the fragmentation method include an enzymatic decomposition method and a chemical decomposition method. The hemoglobin decomposition product is preferably a hemoglobin decomposition product by an enzyme, which has been used conventionally. The enzyme may be a protease such as trypsin, pepsin, or alcalase. The hemoglobin decomposition product may be completely decomposed hemoglobin, partially decomposed hemoglobin, or a mixture thereof. The completely decomposed hemoglobin means a hemoglobin decomposition product obtained when the decomposition reaction by the enzyme is completed, or a hemoglobin decomposition product identical thereto obtained by a chemical decomposition method. The partially decomposed hemoglobin means a hemoglobin decomposition product obtained at any stage before the decomposition reaction by the enzyme is completed, or a hemoglobin decomposition product identical thereto obtained by a chemical decomposition method. The hemoglobin decomposition product is preferably partially decomposed hemoglobin. That is, the hemoglobin degradation product is preferably an enzyme partial degradation product of hemoglobin. Partially decomposed hemoglobin has excellent solubility, and the globin fragment is expected to have an auxiliary stabilizing effect on the hemoglobin-haptoglobin complex. The hemoglobin degradation product preferably contains heme, which is a complex of iron and porphyrin, and contains globin decomposed to an extent that it does not show antigenicity. Furthermore, the hemoglobin degradation product is preferably decomposed to an extent that it does not form a complex with haptoglobin. The origin animal of the hemoglobin degradation product is not limited, and may be, for example, a human or a vertebrate other than a human having hemoglobin, such as a mammal, a bird, or a fish, such as a pig, a cow, a horse, a sheep, a goat, or a rabbit.

[0018] One embodiment of the method includes storing the hemoglobin-haptoglobin complex in a storage solution that includes a hemoglobin decomposition product.

[0019] The storage solution may be a buffer solution containing a Good's buffer such as 2-morpholinoethanesulfonic acid (MES), hydroxyethylpiperazine-2-ethanesulfonic acid (HEPES), piperazine-bis(2-ethanesulfonic acid) (PIPES), or the like, or may be a phosphate buffer, a Tris buffer, a glycine buffer, or the like.

[0020] The concentration of the hemoglobin decomposition product is preferably 0.012 mg / L or more, 0.012 mg / L to 60 mg / L, 0.12 mg / L to 12 mg / L, 1.2 mg / L to 6.3 mg / L, or 1.2 mg / L to 3.6 mg / L in terms of iron equivalent. When the concentration of the hemoglobin decomposition product is 60 mg / L or less in terms of iron equivalent, the viscosity of the preservation solution does not become excessively high, and therefore the concentration of hemoglobin or hemoglobin-haptoglobin complex in the sample is easily measured. In addition, when the concentration of the hemoglobin decomposition product is 60 mg / L or less in terms of iron equivalent, coloration of the preservation solution due to the hemoglobin decomposition product can be suppressed. The iron equivalent refers to the amount of iron atoms (mg Fe / L) contained in the hemoglobin decomposition product. The iron equivalent of the hemoglobin decomposition product can be determined by ortho-phenanthroline colorimetry, atomic absorption spectrometry, or the like.

[0021] The pH of the storage solution may be 5-10, or may be 6-8.

[0022] The storage solution contains sodium azide (NaN 3 Known additives that can be used during storage of hemoglobin, such as antibacterial agents such as erythritol, phosphate buffered saline, pH adjusters, and salts for adjusting ionic strength, may be further added. Antibacterial agents include antibiotics and lytic enzymes. Examples of additives include known components that are known to have an effect of stabilizing hemoglobin, such as amino acids such as lysine and histidine, albumin, protease inhibitors, water-soluble complexes of transition metal ions, and ethylenediaminetetraacetic acid (EDTA). Examples of albumin include serum albumins such as bovine serum albumin (BSA), and albumin derived from egg white (ovalbumin).

[0023] By further adding a hemoglobin-haptoglobin complex of known concentration to the preservation solution having the above composition, the preservation solution can be used as a calibrator or control for detecting or analyzing the hemoglobin-haptoglobin complex. Such a calibrator or control can be stably stored even under high temperature conditions because the hemoglobin-haptoglobin complex is stabilized by hemoglobin degradation products.

[0024] A more specific embodiment of the method includes a step of preserving a hemoglobin-haptoglobin complex formed by contacting a specimen containing hemoglobin with haptoglobin in the above-mentioned preservation solution. The specimen containing hemoglobin may be feces, saliva, or urine. Since feces contains a particularly large amount of bacteria and proteolytic enzymes that cause the decomposition of hemoglobin, the method of the present invention is particularly effective.

[0025] The specimen containing hemoglobin may be contacted with haptoglobin in any manner. Preferably, the specimen containing hemoglobin may be added to the above-mentioned preservation solution further containing haptoglobin. The hemoglobin in the specimen reacts quickly with the haptoglobin in the preservation solution to form a hemoglobin-haptoglobin complex. The hemoglobin-haptoglobin complex can be stably preserved by preserving the specimen as it is in the preservation solution. In other words, according to the above-mentioned method for stabilizing the hemoglobin-haptoglobin complex, the specimen can be preserved while maintaining the structure of the hemoglobin epitope and its surrounding site in the hemoglobin-haptoglobin complex. Therefore, it can be said that the present invention provides a preservation solution for preserving a specimen containing hemoglobin. When hemoglobin forms a complex with haptoglobin, hemoglobin dissociates from a tetramer (α2β2) consisting of two α chains and two β chains into two dimers (αβ); however, this phenomenon does not fall under the category of "decomposition" or "denaturation" in this specification.

[0026] In the present specification, the haptoglobin is not particularly limited as long as it is capable of forming a hemoglobin-haptoglobin complex by complexing with hemoglobin. Since the binding between hemoglobin and haptoglobin has low species specificity, haptoglobin derived from a wide range of species can be used. When the hemoglobin in the sample is human hemoglobin, haptoglobin derived from animals such as humans, horses, pigs, monkeys, dogs, rabbits, and rats can be used. The haptoglobin does not necessarily need to be highly purified.

[0027] The preservation solution for preserving a specimen containing hemoglobin according to the present invention is obtained by further adding haptoglobin to the above-mentioned preservation solution containing a hemoglobin degradation product. The concentration of haptoglobin in the preservation solution varies depending on the amount of the specimen, and is, for example, 0.05 units / L to 50 units / L, 0.1 units / L to 10 units / L, or 0.2 units / L to 2 units / L. Here, 1 unit represents the amount of haptoglobin that binds to 1 mg of hemoglobin. It is preferable that the haptoglobin concentration is adjusted to a concentration sufficient to allow all hemoglobin in the specimen to form a complex with haptoglobin.

[0028] According to the above-mentioned stabilization method or preservation solution, the hemoglobin in the sample can be stabilized in the form of a hemoglobin-haptoglobin complex. In other words, according to the above-mentioned method or preservation solution, the denaturation and decomposition of the hemoglobin in the sample can be suppressed, and thus the structure of the hemoglobin epitope and its surrounding sites can be maintained. Therefore, when the hemoglobin in the sample is detected by an immunological method, it is expected that the accuracy of detection can be improved.

[0029] The method for detecting hemoglobin in a specimen provided by the present invention comprises the steps of: adding the specimen to the preservation solution for preserving the specimen containing hemoglobin to obtain a specimen containing the specimen; and detecting the hemoglobin in the sample by an immunological technique.

[0030] The immunological technique is a method that utilizes an anti-hemoglobin antibody, and any known immunological technique can be used. The immunological technique may be, for example, an immunoagglutination method (e.g., a latex agglutination method or a gold colloid agglutination method), an immunochromatography method, or an ELISA method.

[0031] Hemoglobin in a specimen can be detected, for example, as follows. First, the specimen is collected in a container containing a preservation solution. When hemoglobin is present in the specimen, the hemoglobin forms a hemoglobin-haptoglobin complex. It is not necessary for all hemoglobin in the specimen to form a complex, and the preservation solution (sample) containing the specimen may contain hemoglobin that is not complexed with haptoglobin, but it is preferable that substantially all hemoglobin in the specimen forms a complex with haptoglobin. After storing the specimen in the container for a desired time, the preservation solution containing the specimen is filtered. Then, hemoglobin in the filtrate is detected by a latex agglutination method. More specifically, a reagent containing an anti-hemoglobin antibody having latex particles bound to its surface is added to the filtrate. It is preferable that the anti-hemoglobin antibody is capable of recognizing an epitope of hemoglobin in the hemoglobin-haptoglobin complex and does not cross-react with haptoglobin. If hemoglobin is present in the filtrate, anti-hemoglobin antibodies will recognize the hemoglobin, and the latex particles bound to the antibodies will agglutinate. The change in turbidity due to agglutination is measured, and the hemoglobin concentration in the filtrate is determined from a calibration curve prepared using a calibrator containing hemoglobin-haptoglobin complexes with known hemoglobin concentrations. The concentration of hemoglobin-haptoglobin complexes in the filtrate can also be determined from a calibration curve prepared based on the concentration of the hemoglobin-haptoglobin complexes in the calibrator.

[0032] The present invention also provides a sample that can be used to detect hemoglobin in a specimen. The sample includes a complex of hemoglobin and haptoglobin and a hemoglobin degradation product. More specifically, the sample includes a complex of hemoglobin and haptoglobin formed by haptoglobin and hemoglobin in the specimen, and a hemoglobin degradation product. In such a sample, the complex of hemoglobin and haptoglobin is stabilized, so that hemoglobin in the specimen can be detected with higher accuracy.

[0033] The present invention further provides a kit that can be used when detecting hemoglobin in a sample by the above method. The kit includes the above preservation solution for preserving a sample containing hemoglobin, and a reagent containing an antihemoglobin antibody. There is no limitation on the antihemoglobin antibody, and it may be a polyclonal antibody, a monoclonal antibody, or a fragment of an antihemoglobin antibody capable of recognizing hemoglobin. The antihemoglobin antibody may be bound to a substance necessary for detection, such as latex. The kit may further include any components, such as an instrument and container for collecting a sample, a calibrator, a control, and a solution for diluting the sample. EXAMPLES

[0034] (Test Example 1-1) 50 mM HEPES (pH 7.4), 0.1% BSA, 0.1% NaN 3A storage solution was prepared by adding 0 to 5000 mg / L (iron equivalent: 0 to 60 mg Fe / L) of hemoglobin degradation product (Hb degradation product). As the hemoglobin degradation product, a hemoglobin degradation product derived from pigs using a protease (manufactured by ILS Co., Ltd.) was used. When the hemoglobin degradation product was analyzed by SDS-PAGE, a broad band was observed at a molecular weight position of 3 kDa to 9 kDa. The average molecular weight of the hemoglobin degradation product estimated from the iron content was 4.6 kDa. The hemoglobin degradation product was used after confirming that it was decomposed to such an extent that it did not form a complex with haptoglobin. A hemoglobin-haptoglobin complex (containing about 900 μg / L of hemoglobin and about 0.9 units / L of haptoglobin as components) was added to the storage solution, and the solution was stored at 4, 25, 37, 45, or 56°C for 0, 3, 7, 12, and 20 days. The concentration of Hb-Hp complexes in the stored samples (μg / L) was measured by the latex agglutination method, and was calculated as the hemoglobin content in the Hb-Hp complexes.

[0035] The concentration of Hb-Hp complex was measured using a measuring reagent "OC-Hemodia (registered trademark) Auto S'Eiken'" (manufactured by Eiken Chemical Co., Ltd.) and a measuring device "JCA-BM2250" (manufactured by JEOL Ltd.). The measuring reagent contains anti-human hemoglobin rabbit polyclonal antibody-sensitized latex particles. The measurement conditions for JCA-BM2250 are as follows. Sample volume: 7.0 μL First reagent: 40 μL Second reagent: 20 μL Measurement wavelength: 658nm

[0036] From the measured Hb-Hp complex concentration, the recovery rate (%) of the Hb-Hp complex was calculated based on the concentration of the Hb-Hp complex immediately after the addition of the Hb-Hp complex (i.e., the concentration 0 days after the addition of the Hb-Hp complex). The results are shown in Table 1, Figures 1 and 2. In Table 1, the concentration of the Hb decomposition product is shown in the iron equivalent concentration (mg Fe / L). As shown in these tables and figures, the recovery rate of the Hb-Hp complex was improved by adding the Hb decomposition product. The recovery rate after 20 days of storage at 37°C was 80% or more (Figure 1), and the recovery rate after 3 days of storage at 56°C was 50% or more (Figure 2), and the Hb-Hp complex was highly stable even when stored in a high-temperature environment. The recovery rate (%) also improved depending on the concentration of the hemoglobin decomposition product added. This result showed that the Hb decomposition product stabilized the Hb-Hp complex. Although test results for temperatures of 25°C and 45°C are not shown, results were obtained at these temperatures that allowed the same conclusions to be drawn as above.

[0037] [Table 1]

[0038] (Test Example 1-2) For reference, a test similar to that in Test Example 1-1 was performed by adding hemoglobin to the storage solution instead of the Hb-Hp complex. The results of calculating the hemoglobin recovery rate (%) are shown in Table 2, Figures 3 and 4. As shown in these tables and figures, the recovery rate of hemoglobin was improved by adding the Hb degradation product, but it was lower than that of hemoglobin that formed a complex with haptoglobin (Test Example 1-1). The recovery rate after 20 days of storage at 37°C was a maximum of 35% (Figure 3), and the recovery rate after 3 days of storage at 56°C was 2% or less (Figure 4), indicating that the storage stability of hemoglobin in high-temperature environments was significantly low.

[0039] [Table 2]

[0040] (Test Example 2-1) 50 mM HEPES (pH 6.8), 0.1% BSA, 0.1% NaN 3 A storage solution was prepared by adding 0-1000mg / L (iron equivalent: 0-12mg Fe / L) of hemoglobin degradation product (manufactured by ILS Inc.) and 1 unit / L of haptoglobin. The fecal specimens to which hemoglobin had been added were added to the storage solution so that the fecal concentration was 0.5% by mass, and were stored at 37°C for 0, 7, and 14 days. The hemoglobin concentration (μg / L) in the stored samples was measured by latex agglutination. The fecal specimens were added with hemoglobin in an amount such that the hemoglobin concentration in the samples was approximately 500μg / L.

[0041] The hemoglobin concentration was measured using a measurement reagent "OC-Hemodia (registered trademark) Auto III 'Eiken'" (manufactured by Eiken Chemical Co., Ltd.) and a measurement device "OC Sensor DIANA" (manufactured by Eiken Chemical Co., Ltd.) The measurement reagent contains anti-human hemoglobin rabbit polyclonal antibody-sensitized latex particles.

[0042] From the measured hemoglobin concentration, the recovery rate (%) of hemoglobin in the fecal samples was calculated based on the hemoglobin concentration immediately after the fecal specimen was added to the preservation solution (i.e., the concentration 0 days after the fecal specimen was added). The results are shown in Table 3, Figure 5, and Figure 6. Figure 5 and Figure 6 show the results for fecal sample 1 and fecal sample 2, respectively. In Table 3, the concentration of the Hb degradation product is shown as the iron equivalent concentration (mg Fe / L). As shown in these tables and figures, in both fecal samples, feces 1 and feces 2, the recovery rate of hemoglobin in the sample was improved in a concentration-dependent manner by adding the Hb degradation product. This result showed that the Hb degradation product stabilized hemoglobin. Note that the hemoglobin in the sample exists in the form of an Hb-Hp complex bound to haptoglobin contained in the preservation solution, so the above result means that the Hb degradation product stabilized the Hb-Hp complex.

[0043] [Table 3]

[0044] (Test Example 2-2) The concentration of the hemoglobin decomposition product added to the preservation solution was fixed at 300 mg / L (iron equivalent: 3.6 mg Fe / L), and the test was performed in the same manner as in Test Example 2-1. In addition, as a comparative example, the same test was performed without adding the hemoglobin decomposition product to the preservation solution. The results are shown in Table 4, Figures 7 and 8. Figures 7 and 8 show the results of an example in which the hemoglobin decomposition product was not added and an example in which the hemoglobin decomposition product was added, respectively. As shown in these tables and figures, the recovery rate of hemoglobin in the sample was improved by adding the Hb decomposition product. This result showed that the Hb decomposition product stabilized hemoglobin. Note that the hemoglobin in the sample exists in the form of an Hb-Hp complex bound to haptoglobin contained in the preservation solution, and the above result means that the Hb decomposition product stabilized the Hb-Hp complex.

[0045] [Table 4]

[0046] (Test Example 2-3) For reference, a test similar to that of Test Example 2-2 was performed without adding haptoglobin to the preservation solution. The results are shown in Table 5 and Figures 9 and 10. Figures 9 and 10 respectively show the results of an example in which a hemoglobin degradation product was not added and an example in which a hemoglobin degradation product was added. As shown in these tables and figures, the recovery rate of hemoglobin was lower than that of hemoglobin that formed a complex with haptoglobin (Test Example 2-2). [Table 5]

Claims

1. 1. A method for stabilizing a complex of hemoglobin and haptoglobin, comprising the steps of: The method includes a step of storing a complex of hemoglobin and haptoglobin in the presence of a hemoglobin degradation product, The hemoglobin degradation product contains heme and a globin degradation product, does not form a complex with haptoglobin, and the globin degradation product does not exhibit antigenicity.

2. The method according to claim 1 , wherein the hemoglobin degradation product is an enzymatic hemoglobin degradation product.

3. The method includes a step of storing a complex of hemoglobin and haptoglobin in a storage solution containing a hemoglobin degradation product, The method according to claim 1 or 2, wherein the concentration of the hemoglobin degradation product in the storage solution is 0.012 mg / L or more in terms of iron equivalent.

4. The method according to any one of claims 1 to 3, wherein the complex of hemoglobin and haptoglobin comprises a complex of hemoglobin and haptoglobin produced by contacting a sample containing hemoglobin with haptoglobin.

5. The method according to any one of claims 1 to 3, wherein the complex of hemoglobin and haptoglobin is contained in feces, saliva or urine.

6. A storage solution for storing a complex of hemoglobin and haptoglobin, comprising: A preservation solution comprising a hemoglobin degradation product, the hemoglobin degradation product containing heme and a globin degradation product, the hemoglobin degradation product not forming a complex with haptoglobin, and the globin degradation product not exhibiting antigenicity.

7. A preservation solution for preserving a specimen containing hemoglobin, comprising: Contains haptoglobin and hemoglobin degradation products, the hemoglobin degradation product contains heme and a globin degradation product, does not form a complex with haptoglobin, and the globin degradation product does not exhibit antigenicity; Hemoglobin in the specimen forms a complex with haptoglobin, a preservative solution.

8. 7. The preservation solution of claim 6, further comprising a complex of hemoglobin and haptoglobin, for use as a calibrator or control.

9. The preservation solution according to any one of claims 6 to 8, wherein the hemoglobin degradation product is an enzymatic hemoglobin degradation product.

10. The preservation solution according to any one of claims 6 to 9, wherein the concentration of hemoglobin degradation products is 0.012 mg / L or more in terms of iron equivalent.

11. 1. A method for detecting hemoglobin in a sample, comprising: A step of adding a specimen to the preservation solution according to claim 7 to obtain a sample containing the specimen; detecting hemoglobin in the sample by an immunological technique; Equipped with A method wherein the hemoglobin in the sample is complexed with haptoglobin.

12. 1. A kit for detecting hemoglobin in a sample, comprising: A preservation solution according to claim 7 ; A reagent containing an anti-hemoglobin antibody; Including the kit.

Images