Method for stabilizing a complex of hemoglobin and haptoglobin and preservation solution for preserving a sample containing hemoglobin

Abstract

The method for stabilizing a hemoglobin-haptoglobin complex according to the present invention includes the step of storing the hemoglobin-haptoglobin complex in the presence of a hemoglobin degradation product. According to this method, the hemoglobin-haptoglobin complex can be stabilized.

Description

Technical Field

[0001] The present invention relates to a method for stabilizing a complex of hemoglobin and haptoglobin, a preservation solution for preserving the complex of hemoglobin and haptoglobin, a preservation solution for preserving a sample containing hemoglobin, and a method and kit for detecting hemoglobin in a sample. Background Art

[0002] Detection of blood contained in feces, urine, saliva, etc. is useful for the diagnosis of most diseases. For example, a fecal occult blood test for detecting blood in feces is used for the diagnosis of colorectal cancer. As a method for detecting occult blood, an immunological method is known in which an anti-hemoglobin antibody is used to detect hemoglobin contained in the occult blood in a sample such as feces. A sample for a fecal occult blood test is usually collected from a subject into a container containing a preservation solution and sent to an examination institution such as a hospital. In most cases, the preservation solution (specimen) containing the sample is stored for several days until it is actually subjected to examination, and during this period, it is often placed at a high temperature. Hemoglobin is unstable in solution and is particularly likely to denature or decompose under high temperature conditions. When the structure of an epitope or its peripheral region changes due to denaturation or decomposition of hemoglobin, the antibody cannot recognize hemoglobin, and thus the accuracy of detecting hemoglobin based on the immunological method decreases.

[0003] In addition, in a fecal occult blood test, in the determination of hemoglobin concentration based on an immunological method, an automatic analysis device that can quickly and accurately analyze a large number of specimens is widely used. Generally, in the determination using an automatic analysis device, changes in the device and changes in the reagents used in the determination significantly affect the determination results. Therefore, a calibrator or control containing a measurement target substance with a known concentration is used to regularly correct the automatic analysis device or manage its accuracy. Calibration of the automatic analysis device is performed by measuring a calibrator containing a measurement target substance with a known concentration and creating a calibration curve, and accuracy management of the automatic analysis device is performed by measuring a control containing a measurement target substance with a known concentration and confirming whether the measured value is within a specified range. However, hemoglobin is unstable in solution, and when the structure of an epitope or its peripheral region changes due to denaturation or decomposition of hemoglobin contained in the calibrator or control, the antibody cannot recognize hemoglobin, and accurate calibration and accuracy management of the automatic analysis device cannot be performed, and accurate determination cannot be performed.

[0004] Under such circumstances, various methods have been proposed to stabilize hemoglobin in a sample. For example, methods of adding antibacterial agents such as thimerosal and chlorhexidine (e.g., Patent Document 1), methods of adding hemoglobin of animals other than humans (e.g., Patent Document 2), methods of adding sera of animals other than humans (e.g., Patent Document 3), methods of adding glycosidase-type lysozyme (e.g., Patent Document 4), methods of adding water-soluble transition metal complexes (e.g., Patent Document 5), methods of adding enzymatically decomposed products of hemoglobin (e.g., Patent Document 6), methods of adding sulfurous acid or dithionous acid, etc. (e.g., Patent Document 7), methods of adding organic acids such as malic acid (e.g., Patent Document 8), methods of adding iminocarboxylic acid (e.g., Patent Document 9), methods of adding glyoxylic acid (e.g., Patent Document 10), methods of adding haloalkanesulfonic acid (e.g., Patent Document 11), etc.

[0005] However, hemoglobin is very unstable. Therefore, even these methods for stabilizing hemoglobin still cannot sufficiently inhibit its denaturation or decomposition. On the other hand, a method of adding haptoglobin is also known for stabilizing hemoglobin (e.g., Patent Document 12). Haptoglobin is widely present in the blood 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 pre-adding haptoglobin to a preservation solution or the like to which a sample such as feces will be added, it is possible to form a stable hemoglobin-haptoglobin complex with the hemoglobin contained in the sample when the sample is added.

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 63-271160

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2-296149

[0010] Patent Document 3: Japanese Patent Application Laid-Open No. 4-145366

[0011] Patent Document 4: Japanese Patent Publication No. 5-69466

[0012] Patent Document 5: Japanese Patent Application Laid-Open No. 7-229902

[0013] Patent Document 6: Japanese Patent Application Laid-Open No. 11-218533

[0014] Patent Document 7: Japanese Patent Application Laid-Open No. 2000-258420

[0015] Patent Document 8: Japanese Patent Application Laid-Open No. 2003-14768

[0016] Patent Document 9: Japanese Patent Application Laid-Open No. 2009-097956

[0017] Patent Document 10: Japanese Patent Application Laid-Open No. 2013-257216

[0018] Patent Document 11: Japanese Patent Application Laid-Open No. 2016-191580

[0019] Patent Document 12: Japanese Patent Application Laid-Open No. 10-132824 Summary of the Invention

[0020] Problems to be Solved by the Invention

[0021] There are a large number of bacteria and proteolytic enzymes that cause hemoglobin decomposition in samples derived from organisms, especially feces. Therefore, even hemoglobin-haptoglobin complexes are sometimes decomposed. Accordingly, an object of the present invention is to stabilize hemoglobin-haptoglobin complexes.

[0022] Means for Solving the Problems

[0023] The method for stabilizing a hemoglobin-haptoglobin complex of the present invention includes a step of storing the hemoglobin-haptoglobin complex in the presence of a hemoglobin degradation product. The hemoglobin degradation product can be a hemoglobin degradation product obtained using an enzyme. The above method can include a step of storing the complex of hemoglobin and haptoglobin in a storage solution containing a hemoglobin degradation product, and the concentration of the hemoglobin degradation product in the storage solution can be 0.012 mg / L or more in terms of iron equivalent. The complex of hemoglobin and haptoglobin can include a complex of hemoglobin and haptoglobin generated by contacting a sample containing hemoglobin with haptoglobin. The sample can be feces, saliva, or urine, or can be feces.

[0024] The storage solution for storing the complex of hemoglobin and haptoglobin of the present invention contains a hemoglobin degradation product. The storage solution can further contain the complex of hemoglobin and haptoglobin and can be used as a calibrator or a control.

[0025] The storage solution for storing a sample containing hemoglobin of the present invention contains haptoglobin and a hemoglobin degradation product. The sample can be feces, saliva, or urine.

[0026] The hemoglobin degradation product can be a hemoglobin degradation product obtained using an enzyme. The concentration of the hemoglobin degradation product can be 0.012 mg / L or more in terms of iron equivalent.

[0027] The method for detecting hemoglobin in a test sample of the present invention includes a step of adding a sample to the above-mentioned preservation solution for preserving a sample containing hemoglobin to obtain a test specimen containing the sample, and a step of detecting hemoglobin in the test specimen by an immunological method. Hemoglobin in the test specimen forms a complex with haptoglobin.

[0028] The kit for detecting hemoglobin in a test sample of the present invention contains the above-mentioned preservation solution for preserving a sample containing hemoglobin and a reagent containing an anti-hemoglobin antibody.

[0029] Advantages of the Invention

[0030] 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 inhibit 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 a control with excellent preservation stability. Description of the Drawings

[0031] Figure 1 It is a graph showing the influence of the addition of hemoglobin degradation products on the recovery rate of the hemoglobin-haptoglobin complex at 37°C.

[0032] Figure 2 It is a graph showing the influence of the addition of hemoglobin degradation products on the recovery rate of the hemoglobin-haptoglobin complex at 56°C.

[0033] Figure 3 It is a graph showing the influence of the addition of hemoglobin degradation products on the recovery rate of hemoglobin at 37°C.

[0034] Figure 4 It is a graph showing the influence of the addition of hemoglobin degradation products on the recovery rate of hemoglobin at 56°C.

[0035] Figure 5 It is a graph showing the relationship between the concentration of hemoglobin degradation products and the recovery rate of hemoglobin in feces.

[0036] Figure 6 It is a graph showing the relationship between the concentration of hemoglobin degradation products and the recovery rate of hemoglobin in feces.

[0037] Figure 7 It is a graph showing the recovery rate of hemoglobin in feces in the case where haptoglobin is added and hemoglobin degradation products are not added.

[0038] Figure 8 It is a graph showing the recovery rate of hemoglobin in the case where haptoglobin and hemoglobin degradation products are added.

[0039] Figure 9 This is a graph showing the recovery rate of hemoglobin in the absence of added haptoglobin and hemoglobin degradation products.

[0040] Figure 10 This is a graph showing the recovery rate of hemoglobin in the absence of added haptoglobin but in the presence of hemoglobin degradation products. Detailed implementation mode

[0041] The method for stabilizing a hemoglobin-haptoglobin complex of the present invention includes the step of storing the hemoglobin-haptoglobin complex in the presence of hemoglobin degradation products.

[0042] Hemoglobin degradation products are substances obtained by fragmenting hemoglobin. As methods for fragmenting, methods such as enzymatic decomposition and chemical decomposition can be cited. Hemoglobin degradation products are preferably hemoglobin degradation products obtained using enzymes as used in the past. The enzyme can be a proteolytic enzyme such as trypsin, pepsin, or alkaline protease. Hemoglobin degradation products can be completely decomposed hemoglobin, partially decomposed hemoglobin, or a mixture thereof. Completely decomposed hemoglobin refers to hemoglobin degradation products obtained when the enzymatic decomposition reaction is completed or the same hemoglobin degradation products obtained by chemical decomposition. Partially decomposed hemoglobin refers to hemoglobin degradation products obtained at any stage before the completion of the enzymatic decomposition reaction or the same hemoglobin degradation products obtained by chemical decomposition. As hemoglobin degradation products, partially decomposed hemoglobin is preferred. That is, as hemoglobin degradation products, enzymatic partial decomposition products of hemoglobin are preferred. Partially decomposed hemoglobin has excellent solubility and can be expected to have an auxiliary stabilizing effect on the hemoglobin-haptoglobin complex brought about by globin fragments. Hemoglobin degradation products preferably contain heme as a complex of iron and porphyrin and contain globin that has been decomposed to the extent that it does not show antigenicity. In addition, hemoglobin degradation products are preferably decomposed to the extent that they do not form a complex with haptoglobin. The source animal of hemoglobin degradation products is not limited. For example, it can be a human or a vertebrate other than a human having hemoglobin, or it can be a mammal such as a pig, cow, horse, sheep, goat, rabbit, a bird, or a fish.

[0043] One mode of this method includes the step of storing the hemoglobin-haptoglobin complex in a storage solution containing hemoglobin degradation products.

[0044] The storage solution can be a buffer solution containing Good's buffer such as 2-(N-morpholino)ethanesulfonic acid (MES), N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), piperazine-bis(2-ethanesulfonic acid) (PIPES), or it can be a phosphate buffer, Tris buffer, or glycine buffer, etc.

[0045] The concentration of the hemoglobin degradation 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 degradation product is 60 mg / L or less in terms of iron equivalent, the viscosity of the preservation solution will not be too high. Therefore, it is easy to measure the concentration of hemoglobin or the hemoglobin-haptoglobin complex in the sample. In addition, when the concentration of the hemoglobin degradation product is 60 mg / L or less in terms of iron equivalent, the coloring of the preservation solution caused by the hemoglobin degradation product can be suppressed. The iron equivalent refers to the amount of iron atoms contained in the hemoglobin degradation product (mg Fe / L). The iron equivalent of the hemoglobin degradation product can be determined by methods such as the phenanthroline colorimetric method or atomic absorption spectrophotometry.

[0046] The pH of the preservation solution can be 5 to 10, or can also be 6 to 8.

[0047] In the preservation solution, known additives that can be used during the preservation of hemoglobin, such as antibacterial agents like sodium azide (NaN3), pH regulators, and salts for adjusting ionic strength, can be further added. The antibacterial agents include antibiotics and lysozyme. Examples of the additives also include known components such as amino acids like lysine and histidine, albumin, protease inhibitors, water-soluble complexes of transition metal ions, and ethylenediaminetetraacetic acid (EDTA) that are known to have the effect of stabilizing hemoglobin. Examples of albumin include serum albumin such as bovine serum albumin (BSA) and albumin derived from proteins (ovalbumin).

[0048] By further adding a hemoglobin-haptoglobin complex with a known concentration to the preservation solution having the above composition, the above preservation solution can be used as a calibrator or control for detecting or analyzing the hemoglobin-haptoglobin complex. In such a calibrator or control, the hemoglobin-haptoglobin complex is stabilized by the hemoglobin degradation product, and thus can be stably preserved even under high-temperature conditions.

[0049] A more specific embodiment of this method includes the step of preserving the hemoglobin-haptoglobin complex generated by contacting a sample containing hemoglobin with haptoglobin in the above preservation solution. The sample containing hemoglobin can be feces, saliva, or urine. Since there are a particularly large number of bacteria and proteases that cause hemoglobin degradation in feces, the method of the present invention is particularly effective.

[0050] A sample containing hemoglobin can come into contact with haptoglobin in any manner. Preferably, the sample containing hemoglobin can be added to the above-mentioned preservation solution further containing haptoglobin. Hemoglobin in the sample reacts rapidly with haptoglobin in the preservation solution to form a hemoglobin-haptoglobin complex. Thus, by directly preserving the sample in the preservation solution, the hemoglobin-haptoglobin complex can be stably preserved. In other words, according to the above method for stabilizing the hemoglobin-haptoglobin complex, the sample can be preserved while maintaining the structure of the epitope of hemoglobin and its surrounding regions in the hemoglobin-haptoglobin complex. Therefore, the present invention can also be said to provide a preservation solution for preserving a sample containing hemoglobin. It should be noted that when hemoglobin forms a complex with haptoglobin, hemoglobin dissociates from the tetramer (α2β2) composed of two α-chains and two β-chains into two dimers (αβ), but this phenomenon does not belong to "decomposition" and "denaturation" in this specification.

[0051] In this specification, haptoglobin is not particularly limited as long as it is haptoglobin that complexes with hemoglobin to form a hemoglobin-haptoglobin complex. Since the species specificity of the binding between hemoglobin and haptoglobin is low, haptoglobin from a wide range of species can be used. When the hemoglobin in the sample is human hemoglobin, haptoglobin from animals such as humans, horses, pigs, monkeys, dogs, rabbits, and rats can be used. Haptoglobin does not necessarily need to be highly purified.

[0052] The preservation solution of the present invention for preserving a sample containing hemoglobin is a preservation solution obtained by further adding haptoglobin to the above-mentioned preservation solution containing hemoglobin degradation products. The concentration of haptoglobin in the preservation solution depends on the amount of the sample, for example, it is 0.05 unit / L to 50 unit / L, 0.1 unit / L to 10 unit / L, or 0.2 unit / L to 2 unit / L. Here, 1 unit represents the amount of haptoglobin that binds to 1 mg of hemoglobin. The haptoglobin concentration is preferably adjusted to a concentration sufficient to form a complex between all the hemoglobin in the sample and haptoglobin.

[0053] According to the above stabilization method or preservation solution, hemoglobin in the sample can be stabilized in the form of a hemoglobin-haptoglobin complex. In other words, according to the above method or preservation solution, the denaturation and decomposition of hemoglobin in the sample can be inhibited, and thus the structure of the epitope of hemoglobin and its surrounding regions can be maintained. Therefore, when detecting hemoglobin in the sample by an immunological method, an improvement in the accuracy of detection can be expected.

[0054] The method for detecting hemoglobin in a test sample provided by the present invention comprises the steps of adding a sample to the above-mentioned preservation solution for preserving a sample containing hemoglobin to obtain a specimen containing the sample, and detecting hemoglobin in the specimen by an immunological method.

[0055] The immunological method is a method using an anti-hemoglobin antibody, and a known immunological method can be used. For example, the immunological method can be an immunoprecipitation method (e.g., latex agglutination method or colloidal gold agglutination method), immunochromatography method or ELISA method.

[0056] The detection of hemoglobin in a sample can be carried out, for example, in the following manner. First, the sample is collected into a container containing a preservation solution. In the case where hemoglobin is present in the sample, hemoglobin forms a hemoglobin-haptoglobin complex. It is not necessary for all the hemoglobin in the sample to form a complex, and hemoglobin that has not formed a complex with haptoglobin may be present in the preservation solution (specimen) containing the sample, but it is preferred that substantially all the hemoglobin in the sample forms a complex with haptoglobin. After the sample is preserved in the container for an arbitrary time, the preservation solution containing the sample is filtered. Then, the hemoglobin in the filtrate is detected by the latex agglutination method. More specifically, a reagent containing an anti-hemoglobin antibody with latex particles bound to the surface is added to the filtrate. The anti-hemoglobin antibody preferably can recognize the epitope of hemoglobin in the hemoglobin-haptoglobin complex and does not cross-react with haptoglobin. In the case where hemoglobin is present in the filtrate, the anti-hemoglobin antibody recognizes hemoglobin, and the latex particles bound to the antibody agglutinate. The change in turbidity caused by agglutination is measured, and the hemoglobin concentration in the filtrate is determined using a calibration curve prepared using a calibrator containing a hemoglobin-haptoglobin complex with a known hemoglobin concentration. In addition, the concentration of the hemoglobin-haptoglobin complex in the filtrate can also be determined using a calibration curve prepared based on the concentration of the hemoglobin-haptoglobin complex in the calibrator.

[0057] In addition, the present invention provides a specimen that can be used for detecting hemoglobin in a sample. The specimen contains a complex of hemoglobin and haptoglobin and a hemoglobin degradation product. More specifically, the specimen contains a complex of hemoglobin and haptoglobin generated from haptoglobin and hemoglobin in the sample, and a hemoglobin degradation product. In this specimen, the complex of hemoglobin and haptoglobin is stabilized, and therefore, hemoglobin in the sample can be detected with higher accuracy.

[0058] 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-mentioned preservation solution for preserving the sample containing hemoglobin and a reagent containing an anti-hemoglobin antibody. The anti-hemoglobin antibody is not limited and can be a polyclonal antibody, a monoclonal antibody, or a fragment of an anti-hemoglobin antibody that can recognize hemoglobin. A substance required for detection, such as latex, can be bound to the anti-hemoglobin antibody. The kit can further include any components such as an instrument and a container for collecting the sample, a solution for diluting the calibrator, control, and sample, etc.

[0059] [Examples]

[0060] (Test Example 1-1)

[0061] Prepare a preservation solution added with 50 mM HEPES (pH 7.4), 0.1% BSA, 0.1% NaN3, and hemoglobin degradation product (Hb degradation product) at 0 - 5000 mg / L (0 - 60 mg Fe / L in terms of iron equivalent). As the hemoglobin degradation product, a hemoglobin degradation product derived from pigs obtained using a protease (manufactured by ILS Co., Ltd.) is used. The hemoglobin degradation product is analyzed by SDS-PAGE, and as a result, a broad band is observed at the position of a molecular weight of 3 kDa - 9 kDa. The average molecular weight of the hemoglobin degradation product estimated from the iron content is 4.6 kDa. After confirming that the hemoglobin degradation product is decomposed to the extent that it does not form a complex with haptoglobin, it is used. Add a hemoglobin-haptoglobin complex (containing approximately 900 μg / L of hemoglobin and approximately 0.9 unit / L of haptoglobin as components) to the preservation solution and store it at 4, 25, 37, 45, or 56 °C for 0, 3, 7, 12, and 20 days. The concentration (μg / L) of the Hb-Hp complex in the stored sample is measured by the latex agglutination method. The concentration of the Hb-Hp complex is determined using the content of hemoglobin in the Hb-Hp complex.

[0062] The concentration of the Hb-Hp complex is measured using the measurement reagent "OC-Hemodia (registered trademark) Auto S 'Eiken'" (manufactured by Eiken Chemical Co., Ltd.) and the measurement device "JCA-BM2250" (manufactured by JEOL Ltd.). The above measurement reagent contains latex particles sensitized with a rabbit polyclonal antibody against human hemoglobin.

[0063] The measurement conditions in the JCA-BM2250 are as follows.

[0064] Sample volume: 7.0 μL

[0065] First reagent: 40 μL

[0066] Second reagent: 20 μL

[0067] Measurement wavelength: 658 nm

[0068] Based on the concentration of the measured Hb-Hp complex, with the concentration of the Hb-Hp complex immediately after adding the Hb-Hp complex (i.e., the concentration 0 days after adding the Hb-Hp complex) as the reference, calculate the recovery rate (%) of the Hb-Hp complex. The results are shown in Table 1, Figure 1 and Figure 2 in. In Table 1, the concentration of the Hb degradation product is expressed as the concentration of iron equivalent (mg Fe / L). As shown in these tables and figures, by adding the Hb degradation product, the recovery rate of the Hb-Hp complex is increased. The recovery rate after 20 days of storage at 37 °C is above 80% ( Figure 1 ), the recovery rate after 3 days of storage at 56 °C is above 50% ( Figure 2 ), and high storage stability of the Hb-Hp complex is obtained even during storage in a high-temperature environment. In addition, the recovery rate (%) increases depending on the added concentration of the hemoglobin degradation product. From this result, it is shown that the Hb degradation product stabilizes the Hb-Hp complex. It should be noted that although the test results at temperatures of 25 °C and 45 °C are not shown, results leading to the same conclusion as above were also obtained at these temperatures.

[0069] [Table 1]

[0070]

[0071] *: Concentration of iron equivalent

[0072] **: Content of hemoglobin in the Hb-Hp complex

[0073] A dash in the table indicates that the measurement was not performed.

[0074] (Test Example 1-2)

[0075] For reference, hemoglobin was added to the storage solution instead of the Hb-Hp complex, and the same test as in Test Example 1-1 was performed. The results obtained by calculating the recovery rate (%) of hemoglobin are shown in Table 2, Figure 3 and Figure 4 in. As shown in these tables and figures, by adding the Hb degradation product, the recovery rate of hemoglobin is increased, but it is lower than that of hemoglobin complexed with haptoglobin (Test Example 1-1). The maximum recovery rate after 20 days of storage at 37 °C is 35% ( Figure 3 ), and the recovery rate after 3 days of storage at 56 °C is 2% or less ( Figure 4 ), and the storage stability of hemoglobin during storage in a high-temperature environment is significantly low.

[0076] [Table 2]

[0077]

[0078] *: Concentration of iron equivalent

[0079] (Test Example 2-1)

[0080] Prepare a preservation solution containing 50 mM of HEPES (pH 6.8), 0.1% of BSA, 0.1% of NaN3, hemoglobin degradation product (manufactured by ILS Co., Ltd.) at 0 - 1000 mg / L (0 - 12 mg Fe / L in terms of iron equivalent), and 1 unit / L of haptoglobin. Add the fecal sample with hemoglobin added thereto to the preservation solution so that the fecal concentration is 0.5% by mass, and store at 37°C for 0, 7, and 14 days. Measure the concentration (μg / L) of hemoglobin in the preserved sample by latex agglutination method. Note that the fecal sample is added with an amount of hemoglobin such that the hemoglobin concentration in the sample is approximately 500 μg / L.

[0081] The concentration of hemoglobin is measured using the measurement reagent "OC-Hemodia (registered trademark) Auto III 'Eiken'" (manufactured by Eiken Chemical Co., Ltd.) and the measurement device "OC Sensor DIANA" (manufactured by Eiken Chemical Co., Ltd.). The above measurement reagent contains latex particles sensitized with anti-human hemoglobin rabbit polyclonal antibody.

[0082] Based on the measured concentration of hemoglobin, taking the concentration of hemoglobin immediately after adding the fecal sample to the preservation solution (i.e., the concentration after 0 days from adding the fecal sample) as a reference, calculate the recovery rate (%) of hemoglobin in the fecal sample. The results are shown in Table 3 Figure 5 and Figure 6 as follows. Figure 5 , Figure 6 show the results of fecal sample 1 and fecal sample 2 respectively. In Table 3, the concentration of Hb degradation product is expressed as the concentration of iron equivalent (mg Fe / L). As shown in these tables and figures, for any of the fecal samples in feces 1 and feces 2, by adding Hb degradation product, the recovery rate of hemoglobin in the sample increases in a concentration-dependent manner. From this result, it is shown that Hb degradation product stabilizes hemoglobin. Note that the hemoglobin in the sample exists in the form of Hb-Hp complex bound to haptoglobin contained in the preservation solution. Therefore, the above result means that Hb degradation product stabilizes the Hb-Hp complex.

[0083] [Table 3]

[0084]

[0085] *: Concentration of iron equivalent

[0086] (Test Example 2-2)

[0087] The concentration of the hemoglobin degradation product added to the preservation solution was fixed at 300 mg / L (3.6 mg Fe / L in terms of iron equivalent), and the test was carried out in the same manner as in Test Example 2-1. In addition, as a comparative example, the same test was carried out under the condition that no hemoglobin degradation product was added to the preservation solution. The results are shown in Table 4, Figure 7 and 8 in. Figure 7 、 Figure 8 show the results of the example without adding the hemoglobin degradation product and the example with the added hemoglobin degradation product, respectively. As shown in these tables and figures, the recovery rate of hemoglobin in the sample was improved by adding the Hb degradation product. This result indicates that the Hb degradation product stabilizes hemoglobin. It should be noted that the hemoglobin in the sample exists in the form of the Hb-Hp complex bound to the haptoglobin contained in the preservation solution. Therefore, the above result means that the Hb degradation product stabilizes the Hb-Hp complex.

[0088] [Table 4]

[0089]

[0090] (Test Example 2-3)

[0091] As a reference, the same test as in Test Example 2-2 was carried out under the condition that no haptoglobin was added to the preservation solution. The results are shown in Table 5, Figure 9 and Figure 10 in. Figure 9 、 Figure 10 show the results of the example without adding the hemoglobin degradation product and the example with the added hemoglobin degradation product, respectively. As shown in these tables and figures, the recovery rate of hemoglobin was lower compared to the hemoglobin that formed a complex with haptoglobin (Test Example 2-2).

[0092] [Table 5]

[0093]

Claims

1. A method for stabilizing the complex of hemoglobin and haptoglobin, wherein, it includes the step of storing the complex of hemoglobin and haptoglobin in the presence of a hemoglobin degradation product, the hemoglobin degradation product is a hemoglobin degradation product obtained by using an enzyme, and its molecular weight is 3 - 9 kDa.

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

3. The method according to claim 1 or 2, wherein The complex of hemoglobin and haptoglobin includes the complex of hemoglobin and haptoglobin generated by contacting a sample containing hemoglobin with haptoglobin.

4. The method according to claim 3, wherein, The sample is feces, saliva or urine.

5. Use of a storage solution containing a hemoglobin degradation product in storing the complex of hemoglobin and haptoglobin, wherein, the hemoglobin degradation product is a hemoglobin degradation product obtained by using an enzyme, and its molecular weight is 3 - 9 kDa.

6. The application according to claim 5, wherein The storage solution further contains the complex of hemoglobin and haptoglobin, and this storage solution can be used as a calibrator or a control.

7. The application according to claim 5 or 6, wherein, The concentration of the hemoglobin degradation product is 0.012 mg / L or more in terms of iron equivalent.

8. Use of a hemoglobin degradation product in manufacturing a storage solution for storing a hemoglobin-haptoglobin complex, wherein, the storage solution contains the hemoglobin-haptoglobin complex and the hemoglobin degradation product, the hemoglobin-haptoglobin complex is generated by contacting a sample containing hemoglobin with haptoglobin, the hemoglobin degradation product is a hemoglobin degradation product obtained by using an enzyme, and its molecular weight is 3 - 9 kDa.

9. The application according to claim 8, wherein, The sample is feces, saliva or urine.

10. The application according to claim 8 or 9, wherein, The concentration of the hemoglobin degradation product is 0.012 mg / L or more in terms of iron equivalent.

11. A method for detecting hemoglobin in a sample, which comprises: A step of obtaining a sample-containing test specimen by adding a sample containing hemoglobin to a preservation solution containing haptoglobin and hemoglobin degradation products, wherein, hemoglobin in the sample contacts with haptoglobin to form a hemoglobin-haptoglobin complex, and the hemoglobin-haptoglobin complex is stably stored in the presence of the hemoglobin degradation product; and a step of detecting hemoglobin in the sample by an immunological method, the hemoglobin degradation product is a hemoglobin degradation product obtained by using an enzyme, and its molecular weight is 3 - 9 kDa.

12. The method according to claim 11, wherein, The sample is feces, saliva or urine.

13. Use of a hemoglobin degradation product in manufacturing a kit, wherein, the kit is used in the method for detecting hemoglobin in a sample according to claim 11 or 12, the kit contains the storage solution and a reagent containing an anti-hemoglobin antibody.

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