Method for immunological detection of hemoglobin using deglycosylated haptoglobin

Abstract

A method for immunological detection of hemoglobin, the method comprising a step for performing immunological detection using an anti-hemoglobin antibody in the presence of hemoglobin and haptoglobin, the haptoglobin having the following characteristics (1): (1) at least a portion of an N-linked sugar chain is deleted. The present invention further provides a method for suppressing fluctuations in the detection value of hemoglobin, a method and a solution for stabilizing hemoglobin, a kit for detecting hemoglobin, and the haptoglobin and a method for producing the haptoglobin. According to the present invention, it is possible to detect hemoglobin by suppressing fluctuations in the detection value of hemoglobin due to haptoglobin.

Description

Technical Field

[0001] The present invention relates to an immunological detection method for hemoglobin, and in particular to an immunological detection method using deglycosylated haptoglobin. The present invention also relates to a method for suppressing fluctuations in hemoglobin detection values, a hemoglobin stabilization method and stabilizing solution, a hemoglobin detection kit, the haptoglobin, and a method for producing the same. Background Art

[0002] Detecting blood in samples such as stool, urine, and saliva can help diagnose many diseases. For example, fecal occult blood testing, which detects blood in stool, is used to screen for colorectal cancer. Among known methods for detecting occult blood, one immunological method uses anti-hemoglobin antibodies to detect hemoglobin (sometimes referred to as Hb) in occult blood in samples such as stool.

[0003] Samples for occult blood testing are typically collected from the patient in a container containing a preservative solution and sent to a testing facility such as a hospital. In many cases, the preservative solution containing the sample is stored for several days before being used for testing, often exposed to high temperatures during this period. Hemoglobin is unstable in solution and is particularly susceptible to denaturation or degradation at high temperatures. When hemoglobin denaturation or degradation alters the structure of the epitope or its surrounding area, antibodies are unable to recognize hemoglobin, thereby reducing the accuracy of immunological hemoglobin detection.

[0004] Therefore, in order to stabilize hemoglobin, a method has been used to add haptoglobin (hereinafter sometimes referred to as Hpt) to the sample storage solution (for example, Patent Documents 1 and 2). Haptoglobin is a protein responsible for recovering hemoglobin released into the blood due to hemolysis of red blood cells. It is well known that haptoglobin quickly and irreversibly binds to hemoglobin to form a stable hemoglobin-haptoglobin complex (hereinafter sometimes referred to as Hb-Hpt). When haptoglobin is added to a storage solution, etc. in advance, and the sample is placed in the storage solution, etc., the hemoglobin contained in the sample forms a hemoglobin-haptoglobin complex and is stabilized.

[0005] However, it is known that haptoglobin sometimes causes fluctuations in hemoglobin measurement values ​​(Patent Documents 1 and 3).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 10-132824

[0009] Patent Document 2: International Publication No. 2011 / 058958

[0010] Patent document 3: International Publication No. 2020 / 066722. Summary of the Invention

[0011] Problems to be solved by the invention

[0012] The present invention has been completed in view of the above-mentioned problems, and its purpose is to provide an immunological detection method, a hemoglobin stabilization method and stabilization solution, a hemoglobin immunological detection kit and the haptoglobin, which can suppress the fluctuation of hemoglobin detection value caused by haptoglobin to detect hemoglobin.

[0013] Solutions to the Problem

[0014] The inventors conducted research to address the above-mentioned issues and discovered that using haptoglobin with a specific sugar chain structure can suppress fluctuations in hemoglobin test values, regardless of the specific forms of free hemoglobin and the hemoglobin-haptoglobin complex. This led to the completion of the present invention. Specifically, the present invention is as follows.

[0015] [1] A method for immunological detection of hemoglobin, comprising the following steps:

[0016] Immunological detection is performed using an anti-hemoglobin antibody in the presence of the hemoglobin and haptoglobin, wherein the haptoglobin has the following characteristics (1):

[0017] (1) At least a portion of the N-linked sugar chain is missing.

[0018] [2] The immunological detection method according to [1], wherein the haptoglobin further has the following characteristics (2):

[0019] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0020] [3] The immunological detection method according to [1] or [2], wherein the characteristic (1) is the characteristic of the following (1a):

[0021] (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

[0022] [4] The immunological detection method according to any one of [1] to [3], wherein the haptoglobin does not have an N-linked sugar chain.

[0023] [5] The immunological detection method according to any one of [1] to [3], wherein the N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is deleted is: a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

[0024] [6] The immunological detection method according to any one of [1] to [5], wherein the hemoglobin is contained in feces, blood, serum, plasma, urine, sputum or saliva.

[0025] [7] According to any one of [1] to [6], at least one of the anti-hemoglobin antibodies is carried on an insoluble carrier.

[0026] [8] According to the immunological detection method described in [7], the insoluble carrier is insoluble particles.

[0027] [9] The immunological detection method according to any one of [1] to [8], which is an immunoagglutination method.

[0028]

[10] A method for suppressing fluctuations in hemoglobin test values ​​in immunological hemoglobin testing, comprising the following steps:

[0029] The immunological detection is performed using an anti-hemoglobin antibody in the presence of hemoglobin and haptoglobin, and the method uses haptoglobin having the following characteristics (1) as the haptoglobin:

[0030] (1) At least a portion of the N-linked sugar chain is missing.

[0031]

[11] The method for suppressing fluctuations in hemoglobin detection values ​​according to

[10] , wherein the haptoglobin further has the following characteristic (2):

[0032] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0033]

[12] A method for stabilizing hemoglobin, comprising the following steps:

[0034] Hemoglobin is allowed to coexist with haptoglobin having the following properties (1):

[0035] (1) At least a portion of the N-linked sugar chain is missing.

[0036]

[13] The hemoglobin stabilization method according to

[12] , wherein the haptoglobin further has the following property (2):

[0037] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0038]

[14] The hemoglobin stabilization method according to

[12] or

[13] , wherein the haptoglobin does not have an N-linked sugar chain.

[0039]

[15] The hemoglobin stabilization method according to

[12] or

[13] , wherein the N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is deleted is: a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

[0040]

[16] A hemoglobin stabilization solution comprising haptoglobin,

[0041] The haptoglobin has the following characteristics (1):

[0042] (1) At least a portion of the N-linked sugar chain is missing.

[0043]

[17] The hemoglobin stabilizing solution according to

[16] ,

[0044] The haptoglobin also has the following (2) characteristics:

[0045] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0046]

[18] The hemoglobin stabilization solution according to

[16] or

[17] , wherein the characteristic (1) is the following characteristic (1a):

[0047] (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

[0048]

[19] The hemoglobin stabilization solution according to any one of

[16] to

[18] , wherein the haptoglobin does not have an N-linked sugar chain.

[0049]

[20] The hemoglobin stabilization solution according to any one of

[16] to

[19] , wherein the haptoglobin is a naturally derived haptoglobin treated with peptide-N-glycosidase (PNGase).

[0050]

[21] The hemoglobin stabilizing solution according to any one of

[16] to

[18] , wherein the N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is missing is: a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

[0051]

[22] The hemoglobin stabilizing solution according to any one of

[16] to

[18] and

[21] , wherein the haptoglobin is a naturally derived haptoglobin treated with endo-β-N-acetylglucosaminidase (ENGase).

[0052]

[23] The hemoglobin stabilization solution according to any one of

[16] to

[22] , wherein the hemoglobin is contained in feces, blood, serum, plasma, urine, sputum or saliva.

[0053]

[24] A hemoglobin detection kit for immunological detection of hemoglobin, comprising:

[0054] The hemoglobin stabilizing solution according to any one of

[16] to

[23] ; and

[0055] A reagent comprising an anti-hemoglobin antibody.

[0056]

[25] According to the hemoglobin detection kit described in

[24] , at least one of the anti-hemoglobin antibodies is supported on an insoluble carrier.

[0057]

[26] According to the hemoglobin detection kit described in

[25] , the insoluble carrier is insoluble particles.

[0058]

[27] The hemoglobin detection kit according to any one of

[24] to

[26] , wherein the reagent is a reagent for immunoagglutination method.

[0059]

[28] A haptoglobin having the following properties (1) and (2):

[0060] (1) at least a portion of the N-linked sugar chain is missing; and

[0061] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0062]

[29] The haptoglobin according to

[28] , wherein the property of (1) is the property of (1a) below:

[0063] (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

[0064]

[30] The haptoglobin according to

[28] or

[29] , which does not have an N-linked sugar chain.

[0065]

[31] The haptoglobin according to any one of

[28] to

[30] , wherein the haptoglobin is a haptoglobin obtained by treating a naturally derived haptoglobin with peptide-N-glycosidase (PNGase).

[0066]

[32] The haptoglobin according to

[28] or

[29] , wherein the N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is deleted is: a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

[0067]

[33] The haptoglobin according to any one of

[28] to

[29] and

[32] , wherein the haptoglobin is a haptoglobin obtained by treating a naturally derived haptoglobin with endo-β-N-acetylglucosaminidase (ENGase).

[0068]

[34] A haptoglobin having an N-linked sugar chain, wherein the sugar chain of the N-linked sugar chain is a sugar chain consisting of a single residue N-acetylglucosamine or a sugar chain consisting of only a single residue N-acetylglucosamine and a single residue fucose.

[0069]

[35] A method for producing haptoglobin, characterized by comprising the following steps:

[0070] Glycosidase treatment of natural haptoglobin

[0071] The manufacturing method satisfies the following (a) and (b):

[0072] (a) before the glycosidase treatment, the naturally derived haptoglobin is not subjected to a heat shock above 90° C.;

[0073] (b) During the glycosidase treatment, SDS was not coexisted.

[0074]

[36] The method for producing haptoglobin according to

[35] , wherein the glycosidase is peptide-N-glycosidase (PNGase).

[0075]

[37] The method for producing haptoglobin according to

[36] , wherein the peptide-N-glycosidase is PNGase F.

[0076]

[38] The method for producing haptoglobin according to

[35] , wherein the glycosidase is endo-β-N-acetylglucosaminidase (ENGase).

[0077]

[39] The method for producing haptoglobin according to

[38] , wherein the endo-β-N-acetylglucosaminidase is Endo F2.

[0078] Effects of the Invention

[0079] According to the storage solution containing haptoglobin hemoglobin, the hemoglobin detection kit, the immunological detection method and the haptoglobin of the present invention, fluctuations in hemoglobin detection values ​​caused by haptoglobin can be suppressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Schematic diagram of N-linked sugar chains.

[0081] Figure 2 The amino acid sequence of type 2 human haptoglobin precursor is shown (SEQ ID NO: 1); Figures 2 to 5 In the figure, the underlined part indicates the specific consensus sequence for N-linked glycosylation, and the bold part indicates the asparagine residue that undergoes N-linked glycosylation.

[0082] Figure 3 The amino acid sequence of the human haptoglobin type 1 precursor is shown (SEQ ID NO: 2).

[0083] Figure 4 The amino acid sequence of horse haptoglobin precursor is shown (SEQ ID NO: 3).

[0084] Figure 5 The amino acid sequence of porcine haptoglobin precursor is shown (SEQ ID NO: 4).

[0085] Figure 6 The graph shows the results of detecting various mixed solutions (samples) of free hemoglobin (free-Hb) / hemoglobin-haptoglobin (Hb-Hp) complexes using the immunoagglutination reaction detection reagent prepared in Reference Example 4 and a commercially available colorimetric detection reagent.

[0086] Figure 7 The figure shows electrophoresis (SDS-PAGE) images showing the results of deglycosylation treatment of porcine Hpt, horse Hpt, and human Hpt.

[0087] Figure 8 The figure shows a MALDI-TOF mass spectrum showing the results of deglycosylation treatment of horse Hpt.

[0088] Figure 9The diagram is a diagram of MALDI-TOF mass spectra showing whether PNGase treatment of deglycosylated horse Hpt (dHpt) and untreated horse Hpt causes a change in molecular weight.

[0089] Figure 10 This is a graph showing the results of measuring the amount of free Hb remaining after various Hpt and horse dHpt form complexes with free Hb at various concentrations. DETAILED DESCRIPTION

[0090] Hereinafter, embodiments of the present invention will be described.

[0091] 〔the term〕

[0092] (Hemoglobin)

[0093] Hemoglobin is a protein contained in red blood cells in organisms. It has the property of binding to oxygen molecules and participates in oxygen transport.

[0094] Hemoglobin is a tetrameric structure [α2β2], consisting of two α chains (subunits) composed of 141 amino acids and two β chains (subunits) composed of 146 amino acids. The molecular weight of the α chain is approximately 15,500, and the molecular weight of the β chain is approximately 17,000, for a total molecular weight of approximately 64,500 for hemoglobin as a whole.

[0095] (haptoglobin)

[0096] Haptoglobin is a glycoprotein that specifically binds to hemoglobin to form a hemoglobin-haptoglobin complex.

[0097] The source of the haptoglobin used in this embodiment is not particularly limited, as long as it forms a hemoglobin-haptoglobin complex with hemoglobin. Because the species specificity of hemoglobin-haptoglobin binding is very low, haptoglobin from a variety of species can be used. When the hemoglobin in the sample is human, haptoglobin from animals such as horses, pigs, monkeys, dogs, and cows, including humans, can be used. Recombinant haptoglobin can also be used. Haptoglobin does not necessarily need to be highly purified.

[0098] There are three serotypes of human haptoglobin: type 1-1, type 2-1, and type 2-2. Haptoglobin is composed of α and β chains, with humans having two α chains (α1 and α2) and one β chain.

[0099] Human 1-1 type has the simplest structure, consisting of two α1 chains and two β chains, which can be written as [α1β]2. In human 1-1 type, the α1 chains are connected to the β chains and the α1 chains are connected to each other by SS bonds. Human 2-1 type can be written as [α1β]2[α2β] n, n=0, 1, 2…, human 2-2 type can be recorded as [α2β] m , m=3, 4, 5….

[0100] It is well known that asparagine residues in the β chain of haptoglobin are modified with sugar chains (N-linked sugar chains).

[0101] The molecular weight of the α1 chain is approximately 10,000, the α2 chain is approximately 18,000, and the β chain (sugar-modified) is approximately 39,000. The molecular weight of the entire haptoglobin, for example, type 1-1, is approximately 98,000.

[0102] Haptoglobin is translated into a haptoglobin precursor with a signal sequence, an α chain, and a β chain. When the signal sequence is removed and the main chain (peptide bond) between the α chain and the β chain is further cleaved, a disulfide bond is formed to form a dimer of the α chain and the β chain, thus forming the basic structure of the haptoglobin molecule. When the dimer polymerizes, various haptoglobin molecules ([α1β]2, [α1β]2[α2β] n , [α2β] m wait).

[0103] Haptoglobin is also known in other mammals and birds. For example, it is known that haptoglobin of horses and pigs has the same structure as human type 1-1 (ie, [αβ]2).

[0104] The haptoglobin used in this embodiment may be a molecule having a tetramer or higher, and may be composed of a plurality of dimers of α chains and β chains aggregated together.

[0105] (Hemoglobin-haptoglobin complex)

[0106] When hemoglobin and haptoglobin form a complex, one tetrameric hemoglobin [α2β2] molecule dissociates into two dimerized hemoglobin [αβ] molecules, each of which binds to a haptoglobin. Human type 1-1 haptoglobin has two binding sites for dimeric hemoglobin within the molecule (one on each β chain). Therefore, when human type 1-1 haptoglobin forms a complex with tetrameric hemoglobin [α2β2], two dimerized hemoglobin [αβ] molecules bind to one molecule of human type 1-1 haptoglobin, forming a complex.

[0107] Human 2-1 and 2-2 haptoglobins have two or more β chains in their molecules and can bind to dimeric hemoglobin [αβ] depending on the number of β chains.

[0108] In addition, horse and pig haptoglobin has the same structure as human type 1-1 and can form a complex in which two dimers of hemoglobin [αβ] are bound to one haptoglobin molecule.

[0109] It should be noted that, in this specification, when hemoglobin and haptoglobin form a complex, one molecule of tetrameric hemoglobin [α2β2] dissociates into two molecules of dimer hemoglobin [αβ], and this does not include the denaturation and decomposition of hemoglobin, which is equivalent to the reduction in the storage stability of hemoglobin.

[0110] (N-linked sugar chain)

[0111] The N-linked sugar chain refers to a sugar chain bound to the amide nitrogen atom of the asparagine (Asn) side chain in the consensus sequence of proteins (Asn-X-Ser / Thr: X is any amino acid except Pro).

[0112] N-linked sugar chains generally have a basic skeleton called a core structure. Figure 1 As shown in (i), the core structure has N-acetylglucosamine (GlcNAc) bound to the amide nitrogen atom of Asn, which is then bound to GlcNAc and mannose (Man). Two mannose residues are also branched and bound to the mannose, that is, it has a structure composed of two GlcNAc residues and three Man residues.

[0113] It is known that the aforementioned haptoglobin is an N-linked glycoprotein having an N-linked sugar chain. Examples of N-linked sugar chains possessed by naturally derived haptoglobin are Figure 1 It is shown in (ii)-1 and (ii)-2. It should be noted that "naturally derived haptoglobin" refers to haptoglobin originally expressed by the organism and has nothing to do with transgenics or the like.

[0114] As shown in (ii)-1, the N-linked sugar chains of naturally derived haptoglobin are further supplemented with GlcNAc and galactose (Gal) in that order at the ends of the two Man residues branching from the core structure, and sialic acid is often further added to the Gal.

[0115] In addition, one or more GlcNAc residues contained in an N-linked sugar chain may be further modified with fucose (fucosylated). (ii)-1 shows that the N-linked sugar chain has a single residue of fucose, but it is not clear which GlcNAc residue is bound to it. However, fucosylated GlcNAc residues are usually directly bound to Asn.

[0116] Figure 1(ii)-2 shows that a total of three GlcNAc-Gal-(sialic acid) sugar chains are bound to the two Man residues at the forked ends of the core structure, but it is not clear which of the two Man residues is bound to the two sugar chains. Other than that, everything is the same as (ii)-1. It should be noted that a total of four GlcNAc-Gal-(sialic acid) sugar chains are known to be bound to naturally derived haptoglobin.

[0117] The positions of asparagine residues in haptoglobin precursors and N-linked sugar chains of various animals are shown in Table 1. It should be noted that each amino acid sequence can be obtained through UniProt (https: / / www.uniprot.org / ), and the accession numbers shown in this specification are all from UniProt.

[0118] In humans, there are known human haptoglobin type 1 comprising a signal sequence-α1 chain-β chain and human haptoglobin type 2 comprising a signal sequence-α2 chain-β chain. The combination of these two human haptoglobins expressed constitutes the three serotypes (type 1-1, type 2-1, and type 2-2) described above. Generally, in the case of human haptoglobin type 2 having an amino acid sequence of sequence number 1 (accession number P00738-1, sequence number 1, refer to Figure 2 ), sugar chains are added to asparagine at positions 184, 207, 211, and 241. It should be noted that the type 1 haptoglobin having the amino acid sequence of sequence number 2 is a haptoglobin in which amino acids 38 to 96 are deleted from the type 2 haptoglobin (accession number P00738-2, sequence number 2, refer to Figure 3 ).

[0119] In the horse haptoglobin having the amino acid sequence of sequence number 3 (accession number F6XWM5, sequence number 3, refer to Figure 4 ), sugar chains are added to asparagine at positions 125, 148, 232, and 265.

[0120] In the pig haptoglobin having the amino acid sequence of sequence number 4 (accession number Q8SPS7, sequence number 4, refer to Figure 5 ), sugar chains are added to asparagine at positions 125, 151, 183, and 232.

[0121] Table 1

[0122]

[0123] As shown in the examples described below, hemoglobin forms a complex with haptoglobin to form a hemoglobin-haptoglobin complex, which causes fluctuations in the measured value of hemoglobin concentration.

[0124] In this regard, the use of haptoglobin with at least a portion of its N-linked sugar chains deleted can suppress fluctuations in hemoglobin concentration measurements, particularly lowering the values. This haptoglobin can also inhibit the denaturation and degradation of hemoglobin, thereby stabilizing hemoglobin.

[0125] Deglycosylated haptoglobin

[0126] The haptoglobin according to one embodiment of the present invention has at least a portion of its N-linked sugar chain deleted. Furthermore, it preferably has the storage stability of hemoglobin.

[0127] (Features (1))

[0128] The haptoglobin of this embodiment has the following characteristics (1):

[0129] (1) At least a portion of the N-linked sugar chain is missing.

[0130] In this embodiment, "at least a portion of the N-linked sugar chains are missing" means that at least a portion of the N-linked sugar chains are missing compared to naturally derived haptoglobin. As mentioned above, naturally derived haptoglobin has N-linked sugar chains added.

[0131] The inventors consider the following mechanism as to how the deletion of at least a portion of the sugar chain of haptoglobin suppresses fluctuations in hemoglobin measurement values:

[0132] Hemoglobin detection is typically performed using immunological assays using anti-hemoglobin antibodies. For accurate detection, the hemoglobin epitope that the antibody reacts with must be exposed. When hemoglobin and haptoglobin form a complex, the hemoglobin epitope is hidden by the haptoglobin's N-linked sugar chains, or there may be a physical barrier to antibody binding. Therefore, it is speculated that haptoglobin with missing N-linked sugar chains does not present this barrier, allowing for accurate detection.

[0133] However, the effects of this embodiment are not limited to the above-mentioned mechanism of action.

[0134] For example, the haptoglobin to be evaluated can be treated with peptide-N-glycosidase (PNGase), and the molecular weight of the β chain of the treated haptoglobin can be compared with that of the untreated haptoglobin, thereby evaluating whether a certain haptoglobin has the above-mentioned characteristics (1).

[0135] Among them, it is known that, for example, human type 1-1 haptoglobin ([α1β]2) has a molecular weight increase of approximately 21,000 due to the N-linked sugar chains bound to its β chain. This increase in molecular weight is approximately 10,500 per β chain. Therefore, as a standard, for example, if the difference in molecular weight per β chain between PNGase-treated haptoglobin and untreated haptoglobin is 6,500 or less, the haptoglobin can be said to have characteristic (1). In this case, characteristic (1) can preferably be expressed as follows:

[0136] (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

[0137] When multiple peaks derived from β-chain haptoglobin are detected, the differences in the molecular weights are compared between the peaks with the highest signal intensity.

[0138] The difference in the above molecular weights can be 6500 or less (13000 or less based on one molecule of [αβ]2 of human type 1-1 or horse, etc.), 5000 or less (same as 10000 or less), 2500 (same as 5000 or less), or 1550 (same as 3100 or less).

[0139] It should be noted that when evaluating whether or not characteristic (1a) is present, only the molecular weight of the haptoglobin β chain is evaluated. Therefore, unlike the method described later (method for producing haptoglobin), haptoglobin can be denatured during the peptide-N-glycosidase treatment. Furthermore, the molecular weight can be measured, for example, by TOF MS analysis.

[0140] Regarding the above-mentioned characteristic (1), for a certain N-linked sugar chain, "at least a portion of the N-linked sugar chain is missing" may mean that a portion of the N-linked sugar chain is missing, or the entire N-linked sugar chain is missing (i.e., there is no N-linked sugar chain).

[0141] Furthermore, when a naturally-derived haptoglobin has multiple N-linked sugar chains, the phrase "at least a portion of the N-linked sugar chains is deleted" may refer to a partial or complete deletion of at least one of the N-linked sugar chains possessed by the naturally-derived haptoglobin. Alternatively, a partial or complete deletion of multiple N-linked sugar chains may be sufficient, or a partial or complete deletion of all N-linked sugar chains may be sufficient. It should be noted that a haptoglobin completely lacking all N-linked sugar chains means that the haptoglobin has no N-linked sugar chains at all.

[0142] On the other hand, for example, Figure 1As shown in (iii) to (vi) in FIG, haptoglobin in which a part of the N-linked sugar chain is deleted is shown as "haptoglobin in which a part of the N-linked sugar chain is deleted".

[0143] exist Figure 1 (iii) to (vi) show that a single residue of fucose is added to the remaining N-linked sugar chain. However, depending on the number of GlcNAc residues in the remaining N-linked sugar chain, two or more fucose residues may be added, or no fucose residue may be added.

[0144] (iii) sialic acid and Gal are missing, and GlcNAc is added to the core structure;

[0145] (iv) sialic acid, Gal, and GlcNAc are missing, leaving only the core structure;

[0146] (v) The two Man residues in the branch of the core structure are missing, and the remaining two GlcNAc residues and a single Man residue are left;

[0147] (vi) the three-residue Man and single-residue GlcNAc in the core structure are missing, and the single-residue GlcNAc remains;

[0148] From the perspective of being able to effectively exert the effects of the present embodiment or from the perspective of ease of preparation, the haptoglobin of the present embodiment preferably does not have at least one of the multiple N-linked sugar chains; preferably does not have any N-linked sugar chains; preferably, at least one or all of the multiple N-linked sugar chains are single-residue N-acetylglucosamine, or single-residue N-acetylglucosamine is bound to a single-residue fucose.

[0149] (Feature 2)

[0150] The haptoglobin of this embodiment preferably also has the following characteristics (2):

[0151] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0152] In this embodiment, the "hemoglobin residual rate (%)" can be calculated as:

[0153] (Hemoglobin concentration measured after storage at 37°C for 7 days / hemoglobin concentration measured immediately after adding hemoglobin to the buffer)×100.

[0154] In this specification, haptoglobin having the above-mentioned characteristics (1) or (1) and (2) may also be referred to as "deglycosylated haptoglobin" (hereinafter also referred to as dHpt).

[0155] Haptoglobin having the above-mentioned property (2) has the property of being able to form a complex with hemoglobin and stabilize the hemoglobin. The residual rate of hemoglobin in property (2) is preferably 75% or more, particularly preferably 80% or more.

[0156] It should be noted that haptoglobin is a type of glycoprotein, and there are many reports on examples of sugar chain analysis using various glycosidase treatments. However, in such sugar chain analysis, the glycoproteins containing haptoglobin are generally denatured before glycosidase treatment. Therefore, haptoglobin treated with glycosidases generally does not meet the above-mentioned property (2) for sugar chain analysis.

[0157] In this specification, unless otherwise specified, the amount of haptoglobin is expressed in terms of the amount of substance at a molar equivalent of 1:1 to human hemoglobin.

[0158] As mentioned above, human haptoglobin is known to have types 1-1 ([α1β]2), 2-1 ([α1β]2[α2β] n , n=0, 1, 2...), type 2-2 ([α2β] m , m=3, 4, 5...), in this specification, regardless of type 1-1, type 2-1 and type 2-2, for example, the amount of haptoglobin that can form a complex with 1 μmol of human free hemoglobin is expressed as 1 μmol.

[0159] By expressing the amount of haptoglobin using the amount of substance at a molar equivalent of 1:1 to human hemoglobin, the amount of haptoglobin can be determined regardless of its type, and quantification can be performed even when haptoglobin is contained in a mixture.

[0160] It should be noted that the amount of haptoglobin can be quantified as the amount of human free hemoglobin lost when all the predetermined amount of human free hemoglobin is bound to haptoglobin to form a complex. This can be detected using a free hemoglobin-specific detection reagent. The specific detection method is described in the Examples below.

[0161] [Method for producing deglycosylated haptoglobin]

[0162] For example, by treating a haptoglobin having natural N-linked sugar chains with a glycosidase to remove all or part of the N-linked sugar chains from the haptoglobin, a haptoglobin having the above-mentioned properties (1) or (1) and (2) can be obtained as a haptoglobin in which at least a part of the N-linked sugar chains has been removed by the glycosidase treatment. Alternatively, for example, by modifying the consensus sequence specific for N-linked glycosylation contained in the haptoglobin by genetic engineering methods, a recombinant haptoglobin not modified with N-linked sugar chains can be obtained.

[0163] (Glycosidase treatment)

[0164] In the present embodiment, "haptoglobin having native N-linked sugar chains" refers to a haptoglobin having the same N-linked sugar chains as the naturally derived haptoglobin described above. For example, in addition to naturally derived haptoglobin, recombinant haptoglobin expressed in cultured cells (host cells) can also be used as a haptoglobin having native N-linked sugar chains.

[0165] As naturally derived haptoglobin, haptoglobin derived from various animals such as humans, horses, and pigs can be preferably used.

[0166] Recombinant haptoglobin can be, for example, recombinant haptoglobin obtained by co-expression with complement C1r subcomponent-like protein (C1r-LP) (see Schaer et al. BMC Biotechnology (2018) 18:15).

[0167] From the viewpoints of safety and cost, naturally derived haptoglobin is preferred, and haptoglobin derived from humans, horses, or pigs is particularly preferred.

[0168] Among these, when obtaining a haptoglobin having characteristic (1) that does not have at least one of the multiple N-linked sugar chains (e.g., a haptoglobin that does not have any N-linked sugar chains), the glycosidase used is not particularly limited and may be peptide-N-glycosidase (PNGase). Peptide-N-glycosidase is an enzyme that cleaves the glycosidic bond between a nitrogen atom and a sugar, and cleaves the bond between a GlcNAc residue and an asparagine residue in an N-linked sugar chain. An example of a peptide-N-glycosidase is PNGase F.

[0169] In addition, when obtaining a haptoglobin in which a portion of at least one N-linked sugar chain is deleted, the glycosidase that can be used and the N-linked sugar chain deleted therefrom are not particularly limited, and examples thereof are as follows:

[0170] Sialidase and β-galactosidase: Sialic acid and Gal are missing, and GlcNAc is added to the core structure ( Figure 1 (iii) in the above clause;

[0171] There are also β-N-acetylglucosaminidase (such as β-(1-2,3,4,6)-N-glucosidase): sialic acid, Gal and GlcNAc are missing, leaving only the core structure ( Figure 1 (iv) in the above);

[0172] There are also α-mannosidases (such as α-(1-2,3,6)-mannosidase): the two Man residues in the core structure are missing, and the remaining two GlcNAc residues and a single Man residue ( Figure 1 (v) in the

[0173] Endo-β-N-acetylglucosaminidase (ENGase): The three Man residues and the single GlcNAc residue in the core structure are missing, and the remaining single GlcNAc residue ( Figure 1 (vi) in the

[0174] It should be noted that, as mentioned above, Figure 1 (iii) to (vi) show that a single residue of fucose is added to the remaining N-linked sugar chains, but two or more fucose residues may not be added, or no fucose residue may be added.

[0175] Among the above-mentioned glycosidases, sialidase, β-galactosidase, β-(1-2,3,4,6)-N-glucosidase, and α-(1-2,3,6)-mannosidase are known as exoglycosidases. When using these enzymes, the desired N-linked sugar chains can be obtained by treating the glycosidases in the above-mentioned order. The glycosidases can be treated sequentially, or some or all of them can be treated simultaneously, as long as the above-mentioned order is followed.

[0176] Endo-β-N-acetylglucosaminidase (ENGase) is an endoglycosidase that cleaves the β1-4 bond between GlcNAc residues. This bond is cleaved in the core structure of N-linked sugar chains. The endo-β-N-acetylglucosaminidase is not particularly limited, and examples thereof include Endo S, Endo F3, Endo F2, Endo M, Endo Om, and Endo CC.

[0177] The aforementioned glycosidase is sold by New England Biolabs, Ludger, and the like.

[0178] In addition to characteristic (1), the deglycosylated haptoglobin of this embodiment also preferably has characteristic (2). In order to obtain haptoglobin having this characteristic (2), it is preferred that the haptoglobin is not denatured during the glycosidase treatment.

[0179] More specifically, the method comprises the following steps: treating haptoglobin having a natural N-linked sugar chain (preferably a naturally derived haptoglobin) with a glycosidase;

[0180] Preferably, the following (a) and (b) are satisfied:

[0181] (a) before the glycosidase treatment, the naturally derived haptoglobin is not subjected to a heat shock above 90° C.;

[0182] (b) SDS is not allowed to coexist during the glycosidase treatment.

[0183] As previously mentioned, when sugar chain analysis is performed on glycoproteins including haptoglobin, the glycoproteins are generally denatured before being treated with glycosidases. This is because denaturing the glycoproteins facilitates access to sugar chains by glycosidases, improving reaction efficiency. However, when producing deglycosylated haptoglobin having characteristic (2), it is preferred not to perform denaturation treatment, particularly the treatments (a) and (b) described above.

[0184] (Genetic Engineering Method)

[0185] The method for deleting N-linked sugar chains by genetic engineering methods can be to modify the N-linked glycosylation-specific consensus sequence, thereby expressing recombinant haptoglobin that is not modified with N-linked sugar chains in host cells.

[0186] The haptoglobin before consensus sequence modification may have an amino acid sequence that is identical to a known haptoglobin, for example, any of SEQ ID NOs: 1 to 4, or has an amino acid sequence identity of 80% or more, more preferably 90% or more, and even more preferably 95% or more.

[0187] Specific methods for modifying the consensus sequence (Asn-X-Ser / Thr, where X is any amino acid other than Pro) include: replacing Asn or Ser / Thr within the consensus sequence with other amino acids; deleting one or more amino acids within the consensus sequence; or inserting one or more amino acids within the consensus sequence. Modification (replacement, deletion, or insertion) of the consensus sequence can be performed using conventional genetic engineering methods or equivalent methods. When a haptoglobin has multiple consensus sequences, modification of at least one consensus sequence is sufficient, preferably modifying two or more consensus sequences, and more preferably modifying all consensus sequences.

[0188] As a method for expressing and purifying the modified haptoglobin gene obtained as described above in a host cell, conventionally known methods or methods equivalent thereto can be appropriately used.

[0189] [Hemoglobin stabilization method and stabilizing solution]

[0190] A method for stabilizing hemoglobin according to one embodiment of the present invention includes the step of allowing hemoglobin to coexist with the above-described deglycosylated haptoglobin.

[0191] Furthermore, a hemoglobin stabilization solution according to one embodiment of the present invention contains the above-mentioned deglycosylated haptoglobin.

[0192] Specifically, the haptoglobin (deglycosylated haptoglobin) used in this embodiment has the following characteristics (1):

[0193] (1) At least a portion of the N-linked sugar chain is missing.

[0194] The above characteristic (1) can preferably be expressed as follows:

[0195] (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

[0196] Furthermore, the haptoglobin used in this embodiment preferably has the following property (2):

[0197] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0198] In the present specification, "coexistence" of hemoglobin and deglycosylated haptoglobin includes a state in which hemoglobin and deglycosylated haptoglobin form a complex (hemoglobin-haptoglobin complex).

[0199] The method for making hemoglobin and deglycosylated haptoglobin coexist is not particularly limited. For example, hemoglobin can be dispersed in a solution containing deglycosylated haptoglobin (ie, a stable solution). The term "dispersed" includes both dissolved and suspended.

[0200] The hemoglobin stabilizing solution can be used as a storage solution for storing hemoglobin, a dilution solution for further diluting a sample in which hemoglobin is dispersed in the storage solution, or a reaction solution in a kit for detecting hemoglobin. The hemoglobin stabilizing solution can also be used as a calibrant or control solution for calibration or accuracy control of automated analyzers.

[0201] The hemoglobin to be stabilized may be contained in a sample from a biological body, such as blood, serum, plasma, urine, sputum, lymph, puncture fluid, cerebrospinal fluid, sweat, saliva, gastric juice, lung lavage fluid, or feces.

[0202] The concentration of deglycosylated haptoglobin in the hemoglobin stabilizing solution is not particularly limited, and may be, for example, 0.005 to 500 pmol / mL, 0.05 to 250 pmol / mL, or 0.5 to 50 pmol / mL.

[0203] The hemoglobin stabilizing solution can be any buffer solution that maintains a pH between 5 and 10, preferably between 6 and 8. It can be a buffer solution including Good's buffer such as 2-morpholineethanesulfonic acid (MES), hydroxyethylpiperazine 2-ethanesulfonic acid (HEPES), piperazine-bis(2-ethanesulfonic acid) (PIPES), or a phosphate buffer, Tris buffer or glycine buffer.

[0204] In addition to the deglycosylated haptoglobin described above, the hemoglobin stabilization solution of this embodiment may also contain other components that stabilize hemoglobin. Examples of such components include additives such as antimicrobial agents, pH regulators, salts used to adjust ionic strength, surfactants, aggregation promoters, and known stabilizers used during protein storage. Antimicrobial agents include sodium azide, antibiotics, and lysozyme. Additives include substances known to stabilize proteins, such as amino acids such as histidine and lysine; albumin; protease inhibitors; and chelating agents such as ethylenediaminetetraacetic acid (EDTA).

[0205] [Immunological detection method of hemoglobin]

[0206] The immunological detection method of hemoglobin according to one embodiment of the present invention comprises the following steps:

[0207] Immunological detection was performed using anti-hemoglobin antibodies in the presence of hemoglobin and the above-mentioned deglycosylated haptoglobin.

[0208] The method for allowing hemoglobin and deglycosylated haptoglobin to coexist is not particularly limited, and for example, the methods described in the aforementioned stabilization method and stabilization solution can be used.

[0209] Specifically, the haptoglobin (deglycosylated haptoglobin) used in this embodiment has the following characteristics (1):

[0210] (1) At least a portion of the N-linked sugar chain is missing.

[0211] The above characteristic (1) can preferably be expressed as follows:

[0212] (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

[0213] Furthermore, the haptoglobin used in this embodiment preferably has the following property (2):

[0214] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0215] (Immunological detection method)

[0216] The immunological detection method of hemoglobin of the present embodiment is not particularly limited, as long as it is a method utilizing an antigen-antibody reaction, i.e., an immunological method, for example, it can be an immunological wax method, an immunoagglutination method derived from an immunoturbidimetric method, etc. (e.g., latex agglutination method, colloidal gold agglutination method, etc.); for example, enzyme immunoassays (EIA methods) such as ELISA and bioluminescent enzyme immunoassay; radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA method), electrochemiluminescent immunoassay (ECLEIA method), fluorescent immunoassay, immunochromatography, western blotting, immunoblotting, but is not limited thereto. Among them, the detection method of the present embodiment is preferably used for immunoagglutination method, more preferably for latex agglutination method.

[0217] (Anti-hemoglobin antibodies)

[0218] The type of anti-hemoglobin antibody that can be used in this embodiment is not particularly limited. For example, the animal species from which the antibody is derived is not particularly limited; for example, antibodies from rabbits, goats, mice, rats, horses, sheep, and the like can be used. Polyclonal antibodies, monoclonal antibodies, or fragments thereof (e.g., F(ab')2, Fab, Fab', or Fv) can also be used. These antibodies can be obtained by known methods. For example, polyclonal antibodies can be obtained from the serum of animals immunized against the analyte, while monoclonal antibodies can be obtained by fusion of myeloma cells with the spleen or lymph nodes of animals immunized against the analyte.

[0219] (Insoluble carrier)

[0220] The anti-hemoglobin antibodies used in this embodiment may be at least one type supported on an insoluble carrier, or two or more types may be supported on an insoluble carrier.

[0221] The insoluble carrier is not particularly limited, as long as it can carry the antibody, and can be appropriately selected according to the type of the aforementioned immunological detection method. For example, insoluble carriers, such as plates and porous membranes that can be used for immunological methods, can also include insoluble particles. The insoluble particles can include metal colloid particles such as commonly used gold colloid particles, latex particles, silica particles, magnetic particles, fluorescent particles, red blood cells, etc. The insoluble particles are preferably latex particles, more preferably polystyrene latex particles. The insoluble carrier is preferably granular, and its average particle size is preferably 5 to 1000 nm, more preferably 30 to 500 nm, and more preferably 75 to 350 nm, but is not particularly limited to this range when used.

[0222] Carrying antibody refers to that antibody is fixed on the insoluble carrier surface by physical adsorption or chemical bond.Carrying method (immobilization method) for example can be mixed antibody and insoluble carrier particles, and thereby antibody is fixed on the insoluble carrier particles by antibody physical adsorption on the insoluble carrier particles surface, and this is a kind of known technology.In addition, when using the insoluble carrier particles with amino or carboxyl group on the surface, antibody can be fixed on the insoluble carrier particles surface by the chemical bond that has used glutaraldehyde or carboxylic acid imide reagent.

[0223] The antibody loading capacity is not particularly limited, as long as it is 0.5 to 2000 μg / mg latex, and can be 1 to 1000 μg / mg latex or 2 to 500 μg / mg latex. The antibody loading capacity can be calculated by subtracting the amount of antibody after immobilization from the amount of antibody before immobilization on the insoluble carrier.

[0224] Furthermore, when two or more anti-hemoglobin antibodies are supported on an insoluble carrier, they may be supported on the same insoluble carrier. Furthermore, a mixture of multiple insoluble carriers may be used, wherein one anti-hemoglobin antibody is supported on one insoluble carrier. In this case, the insoluble carriers used to support different types of anti-hemoglobin antibodies may be of the same type or different types, differing in material, particle size, etc.

[0225] (Detection object)

[0226] The immunological detection method of this embodiment is not particularly limited to the test subjects, as long as they can contain hemoglobin. The test subjects may include: blood, serum, plasma, urine, sputum, lymph, puncture fluid, spinal fluid, sweat, saliva, gastric juice, lung lavage fluid, feces, and other biological samples; as well as diluted or suspended samples of these samples.

[0227] Among them, feces, blood, serum, plasma, saliva, urine, and sputum are preferably used as the detection targets containing hemoglobin.

[0228] The haptoglobin of this embodiment can suppress fluctuations in hemoglobin test values ​​caused by haptoglobin, and in particular, can suppress low values. Furthermore, since the haptoglobin can also suppress the denaturation and decomposition of hemoglobin, hemoglobin in stool samples, where the denaturation and decomposition of hemoglobin are particularly pronounced, is preferably used as the detection target.

[0229] (Detection)

[0230] The immunological detection process can appropriately utilize known immunological assays. Detection indicators can be appropriately set based on the immunological method used. For example, in the case of an immunoagglutination assay, the change in turbidity over a predetermined time period can be used, while in the case of an ELISA assay, the coloration and absorbance caused by the labeled antibody can be used. Therefore, known detection methods, such as optical methods, can be used, and commonly available optical detection devices can be used.

[0231] As an example, the immunological detection of hemoglobin can be carried out as follows. First, a sample is added to a container containing a preservation solution as a sample. The sample can be stored in the container for any length of time, or the preservation solution containing the sample can be filtered as a sample. Then, the hemoglobin in the sample is detected by an immunological detection method such as latex agglutination method. More specifically, a reagent containing latex particles with anti-hemoglobin antibodies on the surface is added to the sample. Before adding the reagent containing latex particles, the sample can be diluted with a diluent, or a reaction solution can be added. In this method, the above-mentioned stable solution containing deglycosylated haptoglobin can be any one of a preservation solution, a diluent, and a reaction solution. It is particularly preferred that the preservation solution is the above-mentioned stable solution, and in this case, the diluent and / or the reaction solution may or may not contain deglycosylated haptoglobin.

[0232] When hemoglobin is present in a sample, anti-hemoglobin antibodies recognize it, causing the antibody-carrying latex particles to agglutinate. The change in turbidity caused by agglutination is detected, and the hemoglobin concentration in the sample is determined using a calibration line drawn using a calibrant containing known concentrations of hemoglobin and / or hemoglobin-haptoglobin complex. Alternatively, the hemoglobin concentration in the sample can be determined using a calibration line drawn based on the hemoglobin concentration in the calibrant.

[0233] The immunological detection method of the above embodiment can suppress fluctuations in hemoglobin detection values, especially false low values, caused by haptoglobin by using anti-hemoglobin antibodies to carry out an antigen-antibody reaction for detecting hemoglobin in the presence of the above-mentioned deglycosylated haptoglobin (equivalent to a method for suppressing fluctuations in hemoglobin detection values ​​according to one embodiment of the present invention).

[0234] 〔Hemoglobin detection kit〕

[0235] A kit for immunological detection of hemoglobin according to one embodiment of the present invention comprises: a hemoglobin stabilization solution comprising the above-mentioned deglycosylated haptoglobin; and

[0236] A reagent comprising an anti-hemoglobin antibody.

[0237] The hemoglobin stabilizing solution used in this embodiment can use the aforementioned stabilizing solution.

[0238] Specifically, the haptoglobin used in this embodiment has the following characteristics (1):

[0239] (1) At least a portion of the N-linked sugar chain is missing.

[0240] The above characteristic (1) can preferably be expressed as follows:

[0241] (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

[0242] Furthermore, the haptoglobin used in this embodiment preferably has the following property (2):

[0243] (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

[0244] (Reagent)

[0245] The reagent used in this embodiment is not particularly limited, as long as it contains anti-hemoglobin antibodies and can be used for immunological detection of hemoglobin. It can be a reagent that utilizes the aforementioned method utilizing antigen-antibody reaction, such as immunoagglutination method (e.g., latex agglutination method, gold colloid agglutination method, etc.), ELISA method, immunochromatography method, etc. Among them, the reagent of immunoagglutination method is preferred, and the reagent of latex agglutination method is more preferred.

[0246] The anti-hemoglobin antibody used in this embodiment can be the antibody described in the aforementioned immunological detection method.

[0247] Furthermore, at least one anti-hemoglobin antibody may be supported on an insoluble carrier, or two or more anti-hemoglobin antibodies may be supported on an insoluble carrier. The insoluble carrier may be insoluble particles. The types of insoluble carriers and insoluble particles, and the method for supporting the antibodies are as described above for the immunological detection method.

[0248] (Example of reagent composition)

[0249] The detection reagent of this embodiment can, for example, be composed of a double reagent system consisting of a reagent (first reagent) that does not contain an insoluble carrier and a reagent (second reagent) that contains an insoluble carrier that carries an antibody (antibody-carrying insoluble carrier), or it can be composed of a single reagent system consisting only of a reagent that contains an antibody-carrying insoluble carrier.

[0250] The first reagent can be used to adjust the detection environment, such as as a diluent to adjust the concentration of the analyte or foreign matter in the reaction system or to adjust the reaction rate. The second reagent contains an insoluble carrier that supports the antibody and produces an immune agglutination reaction when mixed with the first reagent and sample. The first and second reagents may appropriately contain pH buffers, salts, surfactants, agglutination promoters, preservatives, etc. The pH during the agglutination reaction is preferably between 5 and 9.

[0251] In addition, the detection reagent is mixed with the sample to obtain a reaction solution. The particle size of the insoluble carrier used and the overall design of the detection system can be combined to appropriately select the concentration of the insoluble carrier in the reaction solution, for example, from 0.0001 mg / mL to 10 mg / mL. The concentration of the insoluble carrier carrying the anti-hemoglobin antibody in the detection reagent can be 0.01 to 5 mg / mL or 0.05 to 1 mg / mL. It should be noted that the concentration of the insoluble carrier in the second reagent can be appropriately adjusted. Since the second reagent will be mixed with the first reagent or sample for dilution during use, the concentration of the insoluble carrier in the second reagent can be appropriately selected according to the dilution multiple, for example, 0.0002 to 20 mg / mL when diluted 2 times for use, and 0.0003 to 30 mg / mL when diluted 3 times for use.

[0252] (Other components)

[0253] In addition to the aforementioned stabilizing solution and detection reagent, the hemoglobin detection kit of this embodiment may also include components such as calibration solutions and control solutions. Furthermore, it may also include components such as instruments and containers for collecting samples. Furthermore, it may also include accessories such as instructions (e.g., printed on paper or plastic and included with the kit, or available for viewing or downloading online), describing instructions for use and / or a standard range of values ​​(standard range) used as a reference for determining hemoglobin positivity and negativity.

[0254] The embodiments described above are intended to facilitate understanding of the present invention, rather than to limit the present invention. Therefore, the elements disclosed in the above embodiments are intended to include all design variations or equivalents within the technical scope of the present invention.

[0255] [Reference Example]

[0256] It should be noted that the free hemoglobin detection reagent used in the examples described below was obtained according to the following reference example.

[0257] Reference Example 1: Preparation of anti-human hemoglobin monoclonal antibody

[0258] (1) Mouse immunization

[0259] Mice were immunized with hemoglobin. After each immunization,125 The antibody titers of mice were measured by RIA using a double antibody method with I-labeled hemoglobin, and mice with high antiserum titers were selected.

[0260] (2) Cell fusion

[0261] Spleens were removed from selected mice and splenocytes were prepared. The prepared splenocytes were fused with mouse myeloma cells using electrofusion, suspended in fusion cell selection medium, and seeded into 96-well microplates.

[0262] (3) Screening of monoclonal antibody-producing cell lines

[0263] After 10 days of cell fusion, the 125 Cells producing anti-human hemoglobin monoclonal antibodies were screened by the double antibody RIA method using I-labeled hemoglobin, and 10 clones were obtained.

[0264] Reference Example 2: Confirmation of the specificity of anti-hemoglobin antibodies

[0265] Human hemoglobin (Eiken Chemical Co., Ltd.) purified from type O human blood was electrophoresed using a precast polyacrylamide gel (ATTO) and an AE-6530 Rapidus Mini Vertical Electrophoresis Tank (ATTO). The resulting protein was transferred to a PVDF membrane using a Transblot SD cell (Bio-Rad).

[0266] Western blotting was performed using the antibody obtained in Reference Example 1 (1 μg / mL) as the primary antibody and an HRP-labeled anti-mouse IgG recognition antibody (rabbit polyclonal antibody, manufactured by Cappel) as the secondary antibody. HRP was illuminated using Western Lightning ECLPro (manufactured by PerkinElmer) to obtain band images. Band images were analyzed using CS Analyzer ver. 3.0 (manufactured by ATTO) in the area microdensitometry measurement mode, where the electrophoretic band brightness was calculated as a cumulative value based on the designated area.

[0267] Next, the cumulative band brightness of the α chain and β chain was calculated based on the western blot band brightness. Note that the band brightness of the α chain was corrected by multiplying the cumulative band value by a coefficient of 0.783 calculated according to the method described below.

[0268] The ratio of the brightness of the bands for each subunit was calculated, assuming the cumulative value of the α chain plus the cumulative value of the β chain was 100%. Antibodies with a cumulative α chain band ratio of 75% or greater were considered hemoglobin α chain-specific antibodies, and antibodies with a cumulative β chain band ratio of 75% or greater were considered hemoglobin β chain-specific antibodies. The results are shown in Table 2.

[0269] To correct for band brightness, human hemoglobin was electrophoresed using a precast polyacrylamide gel (manufactured by ATTO) and an AE-6530 Rapidus mini vertical electrophoresis tank (manufactured by ATTO) in the same manner as described above, followed by CBB staining. The cumulative band brightness of the α and β chains in the resulting electrophoresis images was calculated, and the ratio of the band brightness of each subunit was determined, assuming the cumulative value of the α chain + the cumulative value of the β chain was 100%. The band brightness ratio for the β chain was 43.9%:56.1% for the α chain. The cumulative band value for the α chain was multiplied by a coefficient of 0.783 to normalize the band brightness.

[0270] Table 2

[0271]

[0272] As shown in Table 2, among the antibodies obtained in Reference Example 1, No. 1, 8, 12, and 16, since the ratio of the α chain cumulative value to the sum of the α chain cumulative value and the β chain cumulative value was 75% or more, No. 1, 8, 12, and 16 were considered to be α chain-specific antibodies.

[0273] In contrast, No. 4, 11, 21, 23, 24, and 32 were considered to be β-chain specific antibodies because the ratio of the β-chain cumulative value to the sum of the α-chain cumulative value and the β-chain cumulative value was 75% or more.

[0274] Reference Example 3: Reactivity of Monoclonal Antibodies

[0275] Reactivity to free hemoglobin and reactivity to the hemoglobin-haptoglobin complex were evaluated using the antibodies obtained in Reference Example 1. Each anti-human hemoglobin monoclonal antibody was immobilized on polystyrene latex particles according to the following method, and the degree of aggregation was compared when reacting with samples containing hemoglobin or the hemoglobin-haptoglobin complex.

[0276] (1) Immobilization of monoclonal antibodies on polystyrene latex particles

[0277] Antibodies were immobilized on polystyrene latex particles using known techniques. Anti-hemoglobin monoclonal antibodies were mixed with polystyrene latex particles (200 nm in diameter) and immobilized separately. Anti-hemoglobin monoclonal antibodies were loaded onto the surface of the polystyrene latex particles to prepare antibody-loaded polystyrene latex particle solutions.

[0278] (2) Sample preparation

[0279] A hemoglobin-haptoglobin complex sample was prepared by mixing 32.3 pmol / mL hemoglobin and an equal molar amount of 32.3 pmol / mL haptoglobin in 50 mM HEPES buffer (pH 7.4). A hemoglobin-only sample was prepared by mixing 16.1 pmol / mL hemoglobin in 50 mM HEPES buffer (pH 7.4).

[0280] (3) Latex agglutination detection method

[0281] Agglutination reaction was performed using the wells of a 96-well flat-bottom microplate. Specifically, 100 μL of 50 mM HEPES buffer (pH 7.4) was injected into each well of the microplate, and after adding 50 μL of a polystyrene latex particle solution immobilized with each antibody, 30 μL of the sample prepared in (2) was added. The absorbance was measured at a wavelength of 660 nm using an absorbance microplate reader (Tecan, Japan) 10 seconds after adding the sample and 5 minutes and 10 seconds after adding the sample, and the difference was used as the agglutination index. Furthermore, the ratio of the agglutination reactivity to hemoglobin and the agglutination reactivity to the hemoglobin-haptoglobin complex was also calculated.

[0282] The results are shown in Table 3.

[0283] Table 3

[0284]

[0285] As shown in Table 3, the combination of the hemoglobin α chain-specific antibody and the hemoglobin β chain-specific antibody was confirmed to react specifically with free hemoglobin. The reactivity with the hemoglobin-haptoglobin complex was less than 15% of the reactivity with free hemoglobin.

[0286] Reference Example 4: Free Hemoglobin Detection Reagent

[0287] Using the antibody obtained in Reference Example 1, an immunoagglutination reaction detection reagent was prepared.

[0288] Reagents 1 and 2 were prepared as detection reagents. A 50 mM HEPES buffer (pH 7.4) was used as the first reagent. An immunoagglutination reaction detection reagent was prepared as the second reagent by mixing the polystyrene latexes supporting each anti-hemoglobin monoclonal antibody (anti-Hb antibody) described in Reference Example 2 and adjusting the latex concentration to 1.0 mg / mL.

[0289] Anti-hemoglobin monoclonal antibodies were mixed with polystyrene latex particles (average particle size 100 nm) and loaded onto the surface of the polystyrene latex particles to prepare an antibody-loaded insoluble carrier. In this reagent, No. 16 was used as an α-chain-specific anti-hemoglobin monoclonal antibody, and No. 11 was used as a β-chain-specific anti-hemoglobin monoclonal antibody.

[0290] Reference Example 5: Comparison with other detection methods

[0291] The assay was compared using samples containing a mixture of free hemoglobin and hemoglobin-haptoglobin complex.

[0292] (1) Preparation of purified free hemoglobin and hemoglobin-haptoglobin complex

[0293] The commonly used reference standard material for total human hemoglobin, JCCRM912-3H (developed by the Institute of Laboratory Medicine Standard Materials), was diluted to 2.0 mg / mL using normal saline.

[0294] Separately, 1 mg of freeze-dried haptoglobin (manufactured by SIGMA-ALDRICH) derived from human pooled plasma was dissolved in 303 μL of physiological saline to prepare a haptoglobin solution (3.3 mg / mL).

[0295] (2) Mixture of free hemoglobin and hemoglobin-haptoglobin complex

[0296] Free hemoglobin (free-Hb) was prepared as a constant at 2.0 mg / mL, and haptoglobin (Hpt) solutions were mixed with it to prepare a 3.3 mg / mL solution according to the volumes listed in Table 4. Solutions (samples) with varying mixing ratios were prepared. Note that "free-Complex" in Table 4 represents the molar ratio of free hemoglobin to the hemoglobin-haptoglobin complex.

[0297] Table 4

[0298] free-Complex 0:1 1:3 1:1 3:1 1:0 free-Hb (2.0 mg / mL) 200 μL 200 μL 200 μL 200 μL 200 μL Hpt (3.3 mg / mL) 200 μL 150μL 100 μL 50μL 0μL Normal saline 0μL 50μL 100 μL 150μL 200 μL Total 400 μL 400 μL 400 μL 400 μL 400 μL Free-Hb concentration (mg / mL) 0.00 0.25 0.50 0.75 1.00

[0299] (3) Detection of free hemoglobin / hemoglobin-haptoglobin complex mixture

[0300] A commercially available hemoglobin colorimetric detection reagent was used for comparison with the immunoagglutination reaction detection reagent prepared in Reference Example 4.

[0301] The hemoglobin colorimetric detection reagent is a reagent that quantifies the amount of hemoglobin by dissolving the red blood cell membrane with sodium dodecyl sulfate and detecting the absorbance of the dissolved hemoglobin. However, the hemolysis step is omitted in this test. Each mixed solution (sample) of the free hemoglobin (free-Hb) / hemoglobin-haptoglobin (Hb-Hp) complex prepared in (2) above is mixed with the hemoglobin colorimetric detection reagent and detected at 540 nm using a spectrophotometer (manufactured by Shimadzu Corporation, UV-1900).

[0302] In addition, each mixed solution (sample) prepared in the above (2) was diluted 50 times with physiological saline, and the immunoagglutination reaction detection reagent (Latex reagent) prepared in Reference Example 4 was detected using an automatic biochemical analyzer JCA-BM6070 under the following conditions.

[0303] Sample volume: 1.0 μL; Reagent 1: 50 μL; Reagent 2: 50 μL; Detection wavelength: 658 nm.

[0304] The results are shown in Table 5 and Figure 6 Shown in.

[0305] Table 5

[0306] Free-Hb concentration (mg / mL) 0.000 0.250 0.500 0.750 1.000 Colorimetric assay 1.036 0.990 0.984 0.977 0.971 Latex reagent 0.065 0.387 0.544 0.706 1.010

[0307] Even if the ratio of free hemoglobin to the hemoglobin-haptoglobin complex changes, the detection value of the commercially available hemoglobin colorimetric quantitative reagent remains unchanged, making it impossible to distinguish and detect the two. In contrast, the immunoagglutination reaction detection reagent (latex reagent) in Reference Example 4 is not affected by the amount of hemoglobin-haptoglobin complex, and the detection value obtained is proportional to the free hemoglobin concentration.

[0308] The above results indicate that the latex reagent in Reference Example 4 can specifically detect free hemoglobin. This indicates that the combination of a hemoglobin α chain-specific antibody and a hemoglobin β chain-specific antibody can specifically detect free hemoglobin.

[0309] Example

[0310] Hereinafter, the present invention will be described in further detail by showing Preparation Examples, Test Examples, etc. However, the present invention is not limited to any of the following Preparation Examples, Test Examples, etc.

[0311] Preparation Example 1: Preparation of deglycosylated haptoglobin (dHpt)

[0312] (1) Naturally derived haptoglobin

[0313] As natural-source haptoglobin (Hpt), human Hpt (type 1-1), horse Hpt, and porcine Hpt purified from human serum, horse serum, and porcine serum, respectively, were used.

[0314] (2) ENGase treatment

[0315] 64 U of Endo F2 (endo-β-N-acetylglucosaminidase F2, manufactured by New England Biolabs) was added to each 20 μg of Hpt, and the mixture was treated in 40 μL of GlycoBufer 1 (5 mM CaCl2 / 50 mM sodium acetate, pH 5.5) (manufactured by New England Biolabs) at 37°C for 5 days to obtain ENGase-treated Hpt (dHpt).

[0316] (3) PNGase processing

[0317] 4000 U of PNGase F (peptide-N-glycosidase F, manufactured by New England Biolabs) was added to each 20 μg of Hpt, and the mixture was treated in 40 μL of GlycoBufer 2 (50 mM sodium phosphate buffer, pH 7.5) (manufactured by New England Biolabs) at 37°C for 5 days to obtain PNGase-treated Hpt (dHpt).

[0318] Test Example 1: Confirmation of Deglycosylation Treatment (SDS-PAGE)

[0319] As samples, various Hpts (dHpt) treated with ENGase and PNGase obtained in Preparation Example 1 and various Hpts not treated with glycosidase as a control group were used.

[0320] 8 μL of 200 μg / mL Hpt solution and 2 μL of 5× SDS-PAGE Sample Buffer (5% SDS, 25% glycerol, 0.05% bromophenol blue, 0.5 M Tris / HCl, pH 6.8) were mixed to prepare the electrophoresis sample.

[0321] 10 μL of the resulting electrophoresis sample was applied to a polyacrylamide gel (e-PAGEL HR 7.5%, manufactured by ATTO). Electrophoresis was performed using a Rapidus mini vertical electrophoresis tank and a Power Station Gibli I (both manufactured by ATTO) at a constant current of 60 mA for 60 minutes. It should be noted that the running buffer used was a solution of Tris-glycine-SDS buffer (manufactured by Takara Bio) dissolved as specified.

[0322] After the electrophoresis, the cells were stained with TaKaRa CBB Protein Safe Stain (manufactured by Takara Bio) for 30 minutes and then destained with purified water.

[0323] The results are Figure 7 Shown in.

[0324] like Figure 7 As shown, in any of porcine Hpt, horse Hpt and human Hpt, the molecular weight of Hpt treated with ENGase or PNGase was lower than that of the sample not treated with glycosidase, demonstrating that deglycosylation had occurred.

[0325] Detection Example 2: Confirmation of Deglycosylation Treatment (MALDI-TOF MS)

[0326] As samples, the ENGase-treated and PNGase-treated horse Hpt (dHpt) obtained in Preparation Example 1 and the glycosidase-untreated horse Hpt as a control group were used.

[0327] 1 μL of 200 μg / mL horse Hpt was mixed with an equal volume of matrix (2',4'-dihydroxyacetophenone (DHAP) saturated with 100% acetonitrile) and 1 μL was dripped onto a Big Anchor target plate. After drying, the sample was analyzed using a MALDI-TOF MS (ultraflextreme, Bruker) (detection conditions: linear positive ion mode).

[0328] The results are Figure 8 Shown in.

[0329] like Figure 8 As shown in Figure 3, the molecular weight of Hpt after ENGase or PNGase treatment was lower than that of the sample not treated with glycosidase, indicating that it had been deglycosylated.

[0330] It should be noted that the horse Hpt used in this test has an [αβ]2 structure, that is, there are two β chains in one molecule.

[0331] Detection Example 3: Confirmation of Deglycosylation Treatment (MALDI-TOF MS, Reducing PNGase Treatment)

[0332] As samples, the ENGase-treated horse Hpt (dHpt) obtained in Preparation Example 1 and the glycosidase-untreated horse Hpt as a control group were used.

[0333] 25 μg of each horse Hpt were reduced in 5 μL of lysis buffer (0.5% SDS, 0.5 mol / L Tris-HCl (pH 8.6), 0.75% 2-mercaptoethanol (added as needed), manufactured by Takara Bio) by heating at 100°C for 3 minutes. NP-40 was added to the reduced sample to a final concentration of 1%, followed by 1 mU of PNGase F (peptide-N-glycosidase F, manufactured by Takara Bio), and the sample was treated with purified water in a total volume of 25 μL at 37°C for 24 hours.

[0334] For horse dHpt and glycosidase-untreated horse Hpt, samples were prepared that had been reduced and then treated with PNGase, as well as samples that had been reduced but not treated with PNGase. The resulting samples were analyzed using MALDI-TOF MS in the same manner as in Test Example 2. It should be noted that because haptoglobin was reduced in this assay, the α and β chains were detected as separate molecules.

[0335] The results are Figure 9 Shown in.

[0336] like Figure 9 As shown, the molecular weight of the β chain in (a) unglycosidase-treated horse Hpt is approximately 36,000, while the molecular weight of the β chain in (b) PNGase-treated haptoglobin is approximately 27,000, indicating a lower molecular weight. On the other hand, the molecular weight of the β chain in (c) horse dHpt deglycosylated with ENGase and (d) horse dHpt further treated with PNGase showed little difference. This indicates that the difference in β chain molecular weight between deglycosylated haptoglobin and haptoglobin treated with PNGase is less than 6,500.

[0337] This indicates that by comparing the molecular weight of the haptoglobin β chain with or without PNGase treatment, it is possible to assess whether the haptoglobin has undergone deglycosylation treatment.

[0338] Test Example 4: Quantification of Haptoglobin Using Free Hemoglobin Reagent

[0339] (1) Sample preparation

[0340] A 10.5 mg / mL solution of purified hemoglobin (Hb) purified from human blood was diluted with a diluent solution (0.1% BSA / 0.9% NaCl / 0.1% NaN 3 / 50 mM HEPES, pH 7.0) to prepare an approximately 120 μg / mL Hb solution.

[0341] Various Hpt solutions were prepared by diluting the stock solutions of purified horse Hpt, purified human Hpt, purified porcine Hpt, and ENGase-treated horse Hpt (horse dHpt) to 2% (vol / vol) with a diluting solution.

[0342] A sample was prepared by mixing 200 μL of the Hb solution and 200 μL of the Hpt solution. The resulting sample contained 59.4 μg / mL (921 pmol / mL) of Hb, representing 1% (vol / vol) of the Hpt in the original solution. The Hb content in this sample was determined by assaying a mixture of 200 μL of the Hb solution and 200 μL of the dilution solution according to the method described below.

[0343] Furthermore, a series of samples with different Hpt concentrations were prepared in the same manner as above, except that the Hpt concentrations in the samples were changed to those shown in Table 6A. In addition, samples with the addition of dilution solution but without Hpt were prepared as a control group.

[0344] (2) Detection

[0345] The free hemoglobin content in these samples was determined using the free hemoglobin detection reagent described in Reference Example 4. The determination was performed using an automated biochemical analyzer JCA-BM6070 / C (BioMajesty 6070G) (manufactured by JEOL) under the following conditions:

[0346] Analytical sample volume: 5.0 μL; First reagent: 50 μL; Second reagent: 50 μL; Detection wavelength: 658 nm; Dilution factor: 5 times.

[0347] ※Analytical sample: The sample is diluted with physiological saline at a specified dilution factor.

[0348] The test results are Figure 10 and shown in Table 6A. Based on the test results, an approximate straight line represented by the following formula was obtained:

[0349] Y=aX+b.

[0350] Y: free Hb concentration (μg / mL); a: slope; X: Hpt concentration (% (vol / vol)); b: intercept.

[0351] Then, the amount of Hpt stock solution added to the sample was calculated when the free Hb concentration was 0 μg / mL, and the Hpt concentration in the Hpt stock solution was calculated (Table 6B).

[0352] The amount of Hpt at a free Hb concentration of 0 μg / mL refers to the amount of Hpt that would have resulted from the disappearance of free Hb due to the binding of all Hb in the sample to Hpt, forming an Hb-Hpt complex. Therefore, even in mixtures with moderate Hpt content, Hpt can be quantified as a molar equivalent relative to human Hb.

[0353] The results are Figure 10 , as shown in Table 6A and Table 6B.

[0354] Table 6A

[0355]

[0356] Table 6B

[0357]

[0358] like Figure 10 As shown in Tables 6A and 6B, horse dHpt also retained the ability to bind to free Hb, similar to various Hpts that were not deglycosylated, and the Hpt concentration could be quantified as a molar equivalent to human Hb.

[0359] Test Example 5: Hb stability test (no feces)

[0360] (1) Sample preparation

[0361] An 80 μg / mL purified human Hb solution was diluted with a diluent solution (0.1% BSA / 0.9% NaCl / 0.1% NaN 3 / 50 mM HEPES, pH 7.0) to prepare an 800 ng / mL (12.4 pmol / mL) Hb solution.

[0362] Hpt solutions were prepared by diluting the above-mentioned dilution solution with purified porcine Hpt (undeglycosylated), ENGase-treated porcine Hpt, and PNGase-treated porcine Hpt. It should be noted that the Hpt concentrations (in mol equivalents relative to human Hb) of these Hpt solutions were calculated according to the method described in Test Example 4, and the concentrations were adjusted accordingly.

[0363] 250 μL of the above Hb solution and 250 μL of the above Hpt solution were mixed to prepare a sample. The resulting sample was 500 μL and contained 200 ng (3.1 pmol) of Hb and 3.1 pmol of each Hpt.

[0364] In the above sample, 3.1 pmol of Hpt is sufficient to form an Hb-Hpt complex with all 200 ng (3.1 pmol) of Hb in the sample. This amount of Hpt (3.1 pmol) is 1 Unit in this assay, and samples were prepared by adding 1.0, 0.5, 0.25, and 0.125 Units of Hpt, respectively.

[0365] The samples of equine Hpt were prepared in the same manner as the various Hpt of pig.

[0366] In addition, human Hpt samples were prepared in the same manner as for various porcine Hpt samples, and samples to which 2 units of Hpt were added were also prepared.

[0367] Furthermore, a sample to which a dilution solution was added but Hpt was not added (no Hpt addition) was prepared as a control group.

[0368] (2) Detection

[0369] Use Hb detection reagent (OC-Hemodia (R) The Hb levels of these samples were measured using an AutoIII 'Eiken', manufactured by Eiken Chemical Co., Ltd. and a fully automated fecal occult blood immunochemical analyzer (OC sensor DIANA, manufactured by Eiken Chemical Co., Ltd.). The values ​​here are initial values.

[0370] The samples with initial detection values ​​were stored at 37°C for 7 and 14 days, and then the Hb values ​​were detected again.

[0371] The initial value was compared with the initial value of the sample without Hpt addition, and the "ratio to the value without Hpt addition" was calculated to evaluate the fluctuation of Hb detection value caused by Hpt addition.

[0372] Ratio to no Hpt addition (%) = (concentration of hemoglobin in samples with different amounts of haptoglobin added / concentration of sample without haptoglobin added) × 100

[0373] Furthermore, the measured values ​​after 7 and 14 days of storage were compared with their respective initial values, and the "hemoglobin residual rate" was calculated according to the following formula to evaluate the Hb stabilization effect achieved by the addition of Hpt.

[0374] Residual rate of hemoglobin (%) = (hemoglobin concentration measured after 7 days or 14 days at 37°C / hemoglobin concentration measured immediately after mixing hemoglobin with the diluent and haptoglobin (initial value)) × 100

[0375] The results are shown in Tables 7 to 9.

[0376] Table 7

[0377]

[0378] Table 8

[0379]

[0380] Table 9

[0381]

[0382] As shown in each A in Tables 7 to 9, each initial value is compared with the initial value of the sample without adding Hpt. Adding Hpt that has not been deglycosylated will reduce the Hb detection value, and the degree of reduction in the detection value will also increase when the amount of Hpt added increases.

[0383] Compared with Hpt that has not been deglycosylated, the decrease in Hb detection value of Hpt treated with ENGase or PNGase was inhibited, and the inhibitory effect was particularly higher for Hpt treated with ENGase.

[0384] The above trends can be confirmed in all porcine Hpt, equine Hpt, and human Hpt.

[0385] Furthermore, as shown in Tables 7 to 9, each B shows that Hpt treated with ENGase or PNGase stabilized Hb in a Hpt concentration-dependent manner to the same extent as Hpt that was not deglycosylated.

[0386] Note that, in samples containing 1 Unit of Hpt (ie, 500 μL of a sample containing 3.1 pmol of Hpt and Hb), the Hb residual rate was 70% or higher after storage at 37°C for 7 days.

[0387] The above results indicate that deglycosylation of Hpt can maintain the Hb stabilization effect while suppressing the fluctuation of Hb detection value.

[0388] Test Example 6: Hb stability test (with feces)

[0389] (1) Sample preparation

[0390] An 80 μg / mL purified human Hb solution (manufactured by Eiken Chemical Co., Ltd.) was diluted with a diluent solution (0.1% BSA / 0.9% NaCl / 0.1% NaN 3 / 50 mM HEPES, pH 7.0) to prepare a 1200 ng / mL Hb solution.

[0391] Furthermore, Hpt solutions were prepared by diluting the above-mentioned dilution solution with stock solutions of purified porcine Hpt (not deglycosylated), ENGase-treated porcine Hpt, and PNGase-treated porcine Hpt (Hpt concentrations (mol equivalent to human Hb) were calculated for each).

[0392] Furthermore, human feces were dissolved in the above dilution solution to prepare a 20% (wt / vol) feces solution.

[0393] A sample was prepared by mixing 250 μL of the above Hb solution, 237.5 μL of the Hpt solution, and 12.5 μL of a 20% feces solution. The resulting sample, 500 μL, contained 300 ng (4.65 pmol) of Hb, 4.65 pmol of various Hpts, and 0.5% feces.

[0394] The amount of Hpt (4.65 pmol) in the above sample is 1 Unit in this test. In this test, samples with 1.0, 0.5, and 0.25 Unit of Hpt added were prepared.

[0395] In addition, each sample of horse Hpt and human Hpt was also prepared in the same manner as that of pig Hpt.

[0396] Furthermore, a sample to which a dilution solution was added but Hpt was not added (no Hpt addition) was prepared as a control group.

[0397] (2) Detection

[0398] Use Hb detection reagent (OC-Hemodia (R) The Hb levels of these samples were measured using an AutoIII 'Eiken', manufactured by Eiken Chemical Co., Ltd.) and a fully automated fecal occult blood immunochemical analyzer (OC sensor DIANA, manufactured by Eiken Chemical Co., Ltd.). The values ​​here are initial values.

[0399] The samples with initial detection values ​​were stored at 37°C for 7 and 14 days, and then the Hb values ​​were detected again.

[0400] The initial values ​​were compared with those of the sample without Hpt addition, and the fluctuation of Hb detection values ​​caused by the addition of Hpt was evaluated.

[0401] Furthermore, the Hb stabilization effect achieved by the addition of Hpt was evaluated by comparing the measured values ​​after 7 and 14 days of storage with the respective initial values.

[0402] The results are shown in Tables 10 to 12.

[0403] Table 10

[0404]

[0405] Table 11

[0406]

[0407] Table 12

[0408]

[0409] As shown in Tables 10 to 12, even when the test sample contained feces, the same trend as when there was no feces was observed.

[0410] That is, the addition of Hpt that had not been deglycosylated decreased the Hb detection value (initial value) in a concentration-dependent manner, but this decrease was suppressed by Hpt that had been treated with ENGase or PNGase.

[0411] Furthermore, Hpt stabilized Hb in an Hpt concentration-dependent manner after ENGase and PNGase treatment.

[0412] The above trends can be confirmed in all porcine Hpt, equine Hpt, and human Hpt.

Claims

1. An immunological detection method for hemoglobin, comprising the following steps: In the case where the hemoglobin and haptoglobin coexist, immunological detection is performed using anti-hemoglobin antibodies, The haptoglobin has the following characteristics (1): (1) At least a portion of the N-linked sugar chain is missing.

2. The immunological detection method according to claim 1, wherein The haptoglobin further has the following (2) characteristics: (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

3. The immunological detection method according to claim 1 or 2, wherein The characteristic (1) is the characteristic of the following (1a): (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

4. The immunological detection method according to claim 1 or 2, wherein The haptoglobin has no N-linked sugar chains.

5. The immunological detection method according to claim 1 or 2, wherein The N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is deleted is a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

6. The immunological detection method according to claim 1 or 2, wherein: The hemoglobin is contained in feces, blood, serum, plasma, urine, sputum or saliva.

7. The immunological detection method according to claim 1 or 2, wherein: At least one of the anti-hemoglobin antibodies is supported on an insoluble carrier.

8. The immunological detection method according to claim 7, wherein The insoluble carrier is insoluble particles.

9. The immunological detection method according to claim 1 or 2, wherein: The immunological detection method is an immunoagglutination method.

10. A method for suppressing fluctuations in hemoglobin detection values, A method for suppressing fluctuations in hemoglobin detection values ​​in an immunological test of hemoglobin comprises the following steps: In the case where the hemoglobin and haptoglobin coexist, immunological detection is performed using anti-hemoglobin antibodies, The method uses a haptoglobin having the following properties (1) as the haptoglobin: (1) At least a portion of the N-linked sugar chain is missing.

11. The method for suppressing fluctuations in hemoglobin detection values ​​according to claim 10, wherein: The haptoglobin further has the following (2) characteristics: (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

12. A method for stabilizing hemoglobin, comprising the following steps: Hemoglobin is allowed to coexist with haptoglobin having the following properties (1): (1) At least a portion of the N-linked sugar chain is missing.

13. The method for stabilizing hemoglobin according to claim 12, wherein: The haptoglobin further has the following (2) characteristics: (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

14. The method for stabilizing hemoglobin according to claim 12 or 13, wherein: The haptoglobin has no N-linked sugar chains.

15. The method for stabilizing hemoglobin according to claim 12 or 13, wherein: The N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is deleted is a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

16. A hemoglobin stabilization solution comprising haptoglobin, The haptoglobin has the following characteristics (1): (1) At least a portion of the N-linked sugar chain is missing.

17. The hemoglobin stabilizing solution according to claim 16, wherein The haptoglobin further has the following (2) characteristics: (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

18. The hemoglobin stabilizing solution according to claim 16 or 17, wherein The characteristic (1) is the characteristic of the following (1a): (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

19. The hemoglobin stabilizing solution according to claim 16 or 17, wherein The haptoglobin has no N-linked sugar chains.

20. The hemoglobin stabilizing solution according to claim 16 or 17, wherein The haptoglobin is a naturally derived haptoglobin treated with peptide-N-glycosidase (PNGase).

21. The hemoglobin stabilizing solution according to claim 16 or 17, wherein The N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is deleted is a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

22. The hemoglobin stabilizing solution according to claim 16 or 17, wherein The haptoglobin is a haptoglobin obtained by treating the haptoglobin from natural sources with endo-β-N-acetylglucosaminidase (ENGase).

23. The hemoglobin stabilizing solution according to claim 16 or 17, wherein The hemoglobin is contained in feces, blood, serum, plasma, urine, sputum or saliva.

24. A hemoglobin detection kit for immunological detection of hemoglobin, comprising: The hemoglobin stabilizing solution according to claim 16 or 17; as well as A reagent comprising an anti-hemoglobin antibody.

25. The hemoglobin detection kit according to claim 24, wherein: At least one of the anti-hemoglobin antibodies is supported on an insoluble carrier.

26. The hemoglobin detection kit according to claim 25, wherein The insoluble carrier is insoluble particles.

27. The hemoglobin detection kit according to claim 24, wherein: The reagent is a reagent for immunoagglutination method.

28. A haptoglobin having the following properties (1) and (2): (1) at least a portion of the N-linked sugar chain is missing; and (2) After 3.1 pmol of hemoglobin is stored in 500 μL of a buffer solution containing 3.1 pmol of the haptoglobin at 37° C. for 7 days, the residual rate of the hemoglobin is 70% or more.

29. The haptoglobin according to claim 28, wherein The characteristics of (1) are the characteristics of (1a) below: (1a) The difference between the molecular weight of the β chain in the haptoglobin treated with peptide-N-glycosidase (PNGase) and the molecular weight of the β chain in the haptoglobin not treated with the PNGase is 6500 or less.

30. The haptoglobin according to claim 28 or 29, wherein The haptoglobin has no N-linked sugar chains.

31. The haptoglobin according to claim 28 or 29, wherein The haptoglobin is a naturally derived haptoglobin treated with peptide-N-glycosidase (PNGase).

32. The haptoglobin according to claim 28 or 29, wherein The N-linked sugar chain present in the haptoglobin in which at least a portion of the N-linked sugar chain is deleted is a single residue N-acetylglucosamine, or a combination of a single residue N-acetylglucosamine and a single residue fucose.

33. The haptoglobin according to claim 28 or 29, wherein The haptoglobin is a haptoglobin obtained by treating the haptoglobin from natural sources with endo-β-N-acetylglucosaminidase (ENGase).

34. A haptoglobin having an N-linked sugar chain, wherein the sugar chain of the N-linked sugar chain is a single residue N-acetylglucosamine, or a sugar chain consisting solely of a single residue N-acetylglucosamine and a single residue fucose.

35. A method for producing haptoglobin, characterized in that: The process includes the following steps: Glycosidase treatment of natural haptoglobin The manufacturing method satisfies the following (a) and (b): (a) before the glycosidase treatment, the naturally derived haptoglobin is not subjected to a heat shock above 90° C.; (b) SDS is not allowed to coexist during the glycosidase treatment.

36. The method for producing haptoglobin according to claim 35, wherein The glycosidase is peptide-N-glycosidase (PNGase).

37. The method for producing haptoglobin according to claim 36, wherein The peptide-N-glycosidase is PNGaseF.

38. The method for producing haptoglobin according to claim 35, wherein The glycosidase is endo-β-N-acetylglucosaminidase (ENGase).

39. The method for producing haptoglobin according to claim 38, wherein The endo-β-N-acetylglucosaminidase is Endo F2.

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