Detection method for hepatitis e virus infection, antigen polypeptide for hepatitis e virus infection detection, and kit

Abstract

A method for detecting hepatitis E virus infection, comprising a detection step of detecting anti-hepatitis E virus antibody in a sample using at least one polypeptide selected from the group consisting of (a) to (c) as an antigen polypeptide. (a) a polypeptide consisting of an amino acid sequence of 28 to 175 of any one of the amino acid sequences recited in SEQ ID NOs: 1 to 4, and having a total length of 200 or less of the amino acid sequence recited in the SEQ ID NO; (b) a polypeptide consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of 28 to 175 of any one of the amino acid sequences recited in SEQ ID NOs: 1 to 4, and having a total length of 200 or less of the amino acid sequence recited in the SEQ ID NO; and (c) a polypeptide consisting of an amino acid sequence having 80% or more homology with the amino acid sequence of 28 to 175 of any one of the amino acid sequences recited in SEQ ID NOs: 1 to 4, and having a length of 200 or less of the amino acid sequence recited in the SEQ ID NO.

Description

Technical Field

[0001] This invention relates to a method for detecting hepatitis E virus infection, an antigenic peptide for detecting hepatitis E virus infection using the method, and a kit. Background Technology

[0002] Hepatitis E is an acute or severe form of hepatitis caused by the hepatitis E virus (HEV). Its mortality rate is reportedly about 10 times higher than that of hepatitis A, and there are reports of a 20% mortality rate among pregnant women. Hepatitis E is known to be prevalent primarily in India, Asia, Africa, and Central and South America. Even in other regions, with the increasing diversification of diets in recent years, there have been reports of cases strongly suspected to be caused by the ingestion of uncooked meat contaminated with HEV (e.g., pork, wild boar, and deer meat). Furthermore, sporadic cases of post-transfusion infection and hepatitis reactivation have been observed, thus increasing the need for testing for HEV infection in individuals.

[0003] Previously, HEV infection in subjects was detected by PCR to detect the HEV gene in the subject's serum. However, the HEV gene in serum can only be detected within a limited period of about 2 weeks to 1 month after HEV infection, making detection outside this period difficult.

[0004] Furthermore, due to the low concentration of HEV secreted by infected individuals or animals, direct detection of HEV using anti-hepatitis E virus antibodies (anti-HEV antibodies) is difficult. Therefore, the mainstream clinical method currently used is to indirectly detect HEV infection by detecting anti-HEV antibodies in subjects using HEV-based synthetic antigen peptides or recombinant antigens. Anti-HEV antibodies, for example, for IgA or IgM types, persist for approximately 2–5 months after hepatitis E virus infection.

[0005] As a method for detecting anti-HEV antibodies, and the reagents used therein, there are commercially available kits for detecting IgA-type anti-HEV antibodies using a two-step ELISA kit that uses a plate with a solid-phase recombinant HEV antigen peptide and labeled anti-human IgA antibodies. Non-Patent Literature 1 describes the detection of anti-HEV antibodies using the amino acid positions 111 to 660 encoded by the ORF2 (DDBJ / GenBank / EMBL Accession No.: AB082545) of the HEV gene of genotype 4 as the recombinant HEV antigen peptide.

[0006] In addition, there are commercially available kits for detecting IgM-type anti-HEV antibodies using a two-step ELISA kit that uses a solid-phase plate labeled with recombinant HEV antigen peptides and anti-human IgM antibodies. Japanese Patent Publication No. 2004-525613 (Patent Document 1) describes a kit for diagnosing HEV infection, which contains a polypeptide fragment from position 394 to 606 of the amino acid sequence encoded by ORF2 (DDBJ Accession No.: D11092) of the HEV gene corresponding to genotype 1.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Publication No. 2004-525613

[0010] Non-patent literature

[0011] Non-patent literature 1: Mizuo H. et al., J. Clin. Microbopl., Vol. 40, No. 9, 2002, pp. 3209-3218 Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] However, the inventors further validated the hepatitis E virus infection detection kit currently used clinically in Japan and confirmed that several samples could not be detected using the existing kits described above. They found that the detection sensitivity of anti-HEV antibodies using these kits was not yet sufficient.

[0014] The present invention was made in view of the problems discovered by the inventors above, and aims to provide a detection method for hepatitis E virus infection that can detect anti-hepatitis E virus antibodies with higher sensitivity than existing methods, an antigen peptide for hepatitis E virus infection detection and a kit for using the method.

[0015] Methods for solving problems

[0016] The inventors have discovered that in a method for indirectly detecting HEV infection, specifically by detecting anti-HEV antibodies using HEV antigens, the E2s domain located at the C-terminus of the amino acid encoded by ORF2 of the HEV capsid protein is used as the HEV antigenic peptide. Furthermore, the length of this peptide is shortened to less than 200 residues, thereby enabling high-sensitivity detection even for samples where anti-HEV antibodies cannot be detected using existing kits. This invention is thus completed. The existing kits use amino acid positions 111-660 of the ORF2 of the HEV genotype 4 gene (DDBJ / GenBank / EMBL Accession No.: AB082545) as the HEV antigenic peptide, and kits use amino acid positions 394-606 of the ORF2 of the HEV genotype 1 gene (DDBJ Accession No.: D11092) as the HEV antigenic peptide. The invention derived from this insight is as follows. [1]

[0018] The method for detecting hepatitis E virus infection includes a detection step of using at least one polypeptide selected from (1a) to (1c), (2a) to (2c), (3a) to (3c), and (4a) to (4c) below as an antigenic polypeptide to detect anti-hepatitis E virus antibodies in a sample.

[0019] (1a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 1, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 1.

[0020] (1b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence comprising amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 1 and the total length is less than 200 residues in the amino acid sequence described in Serial No. 1.

[0021] (1c) A polypeptide consisting of an amino acid sequence having more than 80% homology to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 1, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 1.

[0022] (2a) A polypeptide consisting of amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 2, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 2.

[0023] (2b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence comprising amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 2, and whose total length is less than 200 residues in the amino acid sequence described in Serial No. 2.

[0024] (2c) A polypeptide consisting of an amino acid sequence having more than 80% homology to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 2, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 2.

[0025] (3a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 3, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 3.

[0026] (3b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence containing amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 3 and having a total length of less than 200 residues in the amino acid sequence described in Serial No. 3.

[0027] (3c) A polypeptide consisting of an amino acid sequence having more than 80% homology to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 3, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 3.

[0028] (4a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 4, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 4.

[0029] (4b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence containing amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 4 and whose total length is less than 200 residues in the amino acid sequence described in Serial No. 4.

[0030] (4c) A polypeptide consisting of an amino acid sequence having more than 80% homology with an amino acid sequence containing amino acids 28 to 175 of the amino acid sequence described in Serial No. 4 and having a full length of less than 200 residues of the amino acid sequence described in Serial No. 4. [2]

[0032] According to the detection method for hepatitis E virus infection described in [1], the antigen polypeptide is a combination of at least one polypeptide selected from (3a) to (3c) and at least one polypeptide selected from (1a) to (1c), (2a) to (2c), and (4a) to (4c). [3]

[0034] According to the detection method for hepatitis E virus infection described in [1] or [2], the antigenic polypeptide is a dimer composed of any one polypeptide selected from (3a) to (3c) and any one polypeptide selected from (1a) to (1c), (2a) to (2c), and (4a) to (4c). [4]

[0036] According to any one of the methods for detecting hepatitis E virus infection in [1] to [3], the isotype of the anti-hepatitis E virus antibody is IgM. [5]

[0038] According to any one of the methods for detecting hepatitis E virus infection described in [1] to [4],

[0039] The detection process involves bringing the sample into contact with the trap and the marker.

[0040] The trapping body comprises a water-insoluble carrier and a probe molecule capable of binding to anti-hepatitis E virus antibodies, and

[0041] The marker comprises a marker substance and the antigenic polypeptide. [6]

[0043] The antigenic polypeptide for detecting hepatitis E virus infection is an antigenic polypeptide used in any of the methods for detecting hepatitis E virus infection described in [1] to [5], and is selected from at least one polypeptide selected from (1a) to (1c), (2a) to (2c), (3a) to (3c), and (4a) to (4c).

[0044] (1a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 1, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 1.

[0045] (1b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence comprising amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 1 and the total length is less than 200 residues in the amino acid sequence described in Serial No. 1.

[0046] (1c) A polypeptide consisting of an amino acid sequence having more than 80% homology to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 1, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 1.

[0047] (2a) A polypeptide consisting of amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 2, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 2.

[0048] (2b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence comprising amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 2, and whose total length is less than 200 residues in the amino acid sequence described in Serial No. 2.

[0049] (2c) A polypeptide consisting of an amino acid sequence having more than 80% homology to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 2, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 2.

[0050] (3a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 3, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 3.

[0051] (3b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence containing amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 3 and having a total length of less than 200 residues in the amino acid sequence described in Serial No. 3.

[0052] (3c) A polypeptide consisting of an amino acid sequence having more than 80% homology to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 3, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 3.

[0053] (4a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 4, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 4.

[0054] (4b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence containing amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 4 and whose total length is less than 200 residues in the amino acid sequence described in Serial No. 4.

[0055] (4c) A polypeptide consisting of an amino acid sequence having more than 80% homology with an amino acid sequence containing amino acids 28 to 175 of the amino acid sequence described in Serial No. 4 and having a full length of less than 200 residues of the amino acid sequence described in Serial No. 4. [7]

[0057] The antigenic polypeptide for detecting hepatitis E virus infection according to [6] is a combination of at least one polypeptide selected from (3a) to (3c) and at least one polypeptide selected from (1a) to (1c), (2a) to (2c) and (4a) to (4c). [8]

[0059] The antigenic polypeptide for detecting hepatitis E virus infection according to [6] or [7] is a dimer composed of any one polypeptide selected from (3a) to (3c) and any one polypeptide selected from (1a) to (1c), (2a) to (2c), and (4a) to (4c). [9]

[0061] The antigenic polypeptide for detecting hepatitis E virus infection according to any one of [6] to [8] is a labeled marker.

[10]

[0063] The kit for detecting hepatitis E virus infection is a kit for detecting hepatitis E virus infection according to any one of [1] to [5], and contains the antigen polypeptide according to any one of [6] to [9].

[11]

[0065] The hepatitis E virus infection detection kit according to

[10] further includes a trap having a non-water-soluble carrier and a probe molecule that can bind to anti-hepatitis E virus antibodies.

[0066] The effects of the invention

[0067] According to the present invention, a method for detecting hepatitis E virus infection with higher sensitivity than existing methods, an antigen peptide for detecting hepatitis E virus infection using the method, and a kit are provided. Attached Figure Description

[0068] Figure 1 It is a diagram showing the amino acid sequences of genotypes 1 to 4 (Gen1 to Gen4) recorded in sequence numbers 1 to 4.

[0069] Figure 2These are electrophoretic images obtained by performing SDS-polyacrylamide gel electrophoresis on the solutions or precipitates obtained in each purification stage of Hybrid-E2s (G3 / G1-E2s).

[0070] Figure 3A These are electrophoretic images obtained by performing SDS-polyacrylamide gel electrophoresis on solutions or precipitates obtained in each purification stage of Hybrid-E2s (G3 / G4-E2s).

[0071] Figure 3B These are electrophoretic images obtained by performing SDS-polyacrylamide gel electrophoresis on solutions or precipitates obtained in each purification stage of Hybrid-E2s (G3 / G4-E2s). Detailed Implementation

[0072] The present invention will now be described in detail through its preferred embodiments.

[0073] <Methods for detecting hepatitis E virus infection>

[0074] The method for detecting hepatitis E virus infection of the present invention includes methods selected from (a) to (c) below:

[0075] (a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in any one of sequence numbers 1 to 4, and having a total length of less than 200 residues of the amino acid sequence described in that sequence number.

[0076] (b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence comprising amino acids at positions 28 to 175 of the amino acid sequence contained in any of the sequences 1 to 4, and whose total length is less than 200 residues in the amino acid sequence contained in that sequence number.

[0077] (c) A polypeptide consisting of an amino acid sequence having more than 80% homology to an amino acid sequence containing amino acids at positions 28 to 175 of any of the amino acid sequences described in any of the sequences 1 to 4, and having a total length of less than 200 residues of the amino acid sequence described in that sequence number.

[0078] A method for detecting antibodies against hepatitis E virus in a sample by using at least one polypeptide as an antigenic polypeptide, thereby detecting the presence or absence of hepatitis E virus infection in the sample.

[0079] Hepatitis E virus

[0080] In this invention, the presence of hepatitis E virus (HEV) is indirectly detected by detecting antibodies against HEV in a sample, i.e., anti-HEV antibodies. More specifically, the presence of HEV is detected in the sample, preferably in a subject from the same sample. Hepatitis E virus (HEV) is a small, unenveloped, spherical particle (diameter: 27–34 nm), and four genotypes (genotypes 1–4) are currently known. Its genome is a single-stranded RNA of approximately 7200 bases, containing three ORFs (open reading frames: ORF1, ORF2, and ORF3). ORF1 encodes non-structural proteins such as helicases and RNA polymerases, ORF2 encodes capsid proteins, and ORF3 encodes phosphorylated proteins consisting of 113 or 114 amino acid residues. ORF2 typically encodes a polypeptide consisting of a 660-residue amino acid sequence. Amino acids 129–606 of this polypeptide constitute the capsid protein, which, starting from its N-terminus, possesses an S domain (typically amino acids 129–319) forming the viral capsid; an M domain (M: typically amino acids 320–455) forming dimers, trimers, or pentamers; and a P domain (P: typically amino acids 456–606) forming a protrusion. Furthermore, it has been reported that, typically, the E2s domain, composed of amino acids 459–606 of the amino acid sequence encoded by ORF2, forms a dimer within the P domain, playing a crucial role in viral host recognition (Li, S. et al., PLos Pathog. 5, e1000537, 2009). In addition, in this invention, when "position" and "number" are displayed in the amino acid sequence, they indicate the number of amino acid residues starting from the N-terminus.

[0081] [Anti-hepatitis E virus antibody]

[0082] In this invention, the anti-hepatitis E virus antibody (referred to as "anti-HEV antibody" as appropriate in this specification) is an antibody capable of specifically binding to the hepatitis E virus, more specifically, an antibody produced in the body of the subject from whom the sample is derived. As the anti-HEV antibody involved in this invention, it may not be a complete antibody or may be an antibody fragment, as long as it can recognize the following antigenic polypeptide.

[0083] The isotype of the anti-HEV antibody involved in this invention is not particularly limited and can be any of IgG, IgM, IgA, IgE, and IgD, or a combination of two or more of them. Furthermore, each of the isotypes can be any subtype. For example, in the case of human infection with HEV, IgM appears first, followed by IgA, and persists in the serum for several months (up to about 5 months). By using them as targets, detection in the early stages of infection can be performed. Additionally, IgG appears slightly later and persists for a longer period, thus enabling detection of past HEV infections. From the viewpoint of enabling earlier detection when using sandwich methods (e.g., sandwich ELISA), IgM is preferred. For example, from the viewpoint of enabling more detailed analysis of specific infection stages, a combination of IgM and IgG is preferred, but not limited to these.

[0084] [Subject]

[0085] In this invention, the term "subject" refers to the human being from whom the sample for the hepatitis E virus infection detection method of this invention originates, and also refers to the object to which the hepatitis E virus testing method of this invention is performed. Examples of subjects include humans or mammals other than humans (pigs, wild boars, cattle, deer, etc.), with humans being preferred. The subjects involved in this invention are not particularly limited; they can be healthy individuals who are not infected with HEV and have not developed hepatitis E, or individuals infected with HEV but who have not developed hepatitis E. Furthermore, for specific reasons such as the stage of illness, they can also be patients known to be infected with HEV or patients who have previously developed hepatitis E.

[0086] [Sample]

[0087] In this invention, the term "sample" refers to the object of the detection method for hepatitis E virus infection of this invention. The sample is not particularly limited to any sample in which the anti-HEV antibody can be present. Generally, examples include blood samples such as serum, plasma, and whole blood collected from the subject or other subjects; body fluid samples other than blood such as urine, feces, sputum, saliva, sweat, cerebrospinal fluid, digestive fluid, semen, lymph, and ascites; mucosal samples such as oral mucosa, pharyngeal mucosa, and intestinal mucosa; and various biopsy samples. Among these, blood samples are preferred as the sample involved in this invention, and serum or plasma are more preferred.

[0088] These samples may be diluted or suspended with a diluent as needed. Examples of such diluents include phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, phthalate buffer, etc. Furthermore, the samples involved in this invention may have undergone appropriate pretreatment as needed. Examples of such pretreatment include pulverization, freezing, heating, concentration, fractionation, desalting, etc.; addition of pH adjusters, stabilizers, preservatives, antiseptics, surfactants, etc.; purification, etc., and may be one or a combination of two or more of these methods.

[0089] [Antigen polypeptide]

[0090] The method of the present invention includes a detection step of detecting anti-HEV antibodies in a sample by using at least one polypeptide selected from (1a) to (1c), (2a) to (2c), (3a) to (3c), and (4a) to (4c) as an antigenic polypeptide. In this invention, "detection" includes not only acquiring the presence and amount of anti-HEV antibodies in the sample as a signal, but also quantifying or semi-quantitating the detected anti-HEV antibodies based on the signal amount.

[0091] In this invention, the antigenic polypeptide used in the detection of anti-HEV antibodies is typically (a) a polypeptide consisting of amino acids comprising positions 28 to 175 of the amino acid sequence described in any one of sequence numbers 1 to 4, and having a total length of less than 200 residues in the amino acid sequence described in that sequence number, i.e., selected from...

[0092] (1a) A polypeptide consisting of amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 1, and an amino acid sequence of less than 200 residues in length.

[0093] (2a) A polypeptide consisting of amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 2, and of a total length of less than 200 residues of the amino acid sequence described in Serial No. 2.

[0094] (3a) A polypeptide comprising amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 3, and an amino acid sequence of less than 200 residues in length of the amino acid sequence described in Serial No. 3, and

[0095] (4a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 4, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 4.

[0096] At least one polypeptide (hereinafter referred to as “antigen polypeptide (a)” as appropriate).

[0097] The amino acid sequence recorded in sequence number 1 is the amino acid sequence of positions 432-660 of the ORF2 of genotype 1 HEV (DDBJ Accession No.: D11092), the amino acid sequence recorded in sequence number 2 is the amino acid sequence of positions 432-660 of the ORF2 of genotype 2 HEV (DDBJ Accession No.: M74506), the amino acid sequence recorded in sequence number 3 is the amino acid sequence of positions 432-660 of the ORF2 of genotype 3 HEV (GenBank Accession No.: AB481229), and the amino acid sequence recorded in sequence number 4 is the amino acid sequence of positions 432-660 of the ORF2 of genotype 4 HEV (GenBank Accession No.: AB481227).

[0098] Figure 1 The amino acid sequences described by sequence numbers 1 to 4 are displayed as Gen1 to Gen4, respectively. Furthermore, the identity between amino acid positions 21 to 186 in each amino acid sequence is shown in Table 1 below. Figure 1 As shown in Table 1, there is high amino acid identity among genotypes 1 through 4.

[0099] Table 1

[0100]

[0101] The amino acids at positions 28 to 175 of the amino acid sequences described in Serial Numbers 1 to 4 typically correspond to amino acids at positions 459 to 606 (E2s domain) of the amino acid sequence encoded by the ORF2 of the HEV gene. As the amino acid sequences to be included in the antigenic polypeptide (a), each of the amino acid sequences described in Serial Numbers 1 to 4 is preferably at positions 21 to 175, more preferably at positions 21 to 186, and even more preferably at positions 14 to 186. Furthermore, when the antigenic polypeptide involved in this invention is a dimer (heterodimer) as described below, when it is a combination of any one polypeptide selected from (3a) to (3c) and any one polypeptide selected from (4a) to (4c) (antigen polypeptide (4)), from the viewpoint of making it easier to purify the heterodimer, as the amino acid sequence that needs to be included in one of the polypeptides (preferably antigen polypeptide (4)), it is preferable to be positions 14 to 175 of the amino acid sequence recorded in sequence numbers 3 and 4 (preferably sequence number 4), and more preferably positions 14 to 186.

[0102] The length of the antigenic polypeptide (a) involved in this invention needs to be less than 200 residues in the amino acid sequence described in any one of serial numbers 1 to 4. This allows for the detection of anti-HEV antibodies with higher sensitivity than existing methods. The length of the antigenic polypeptide (a) is preferably less than 173 residues, more preferably less than 166 residues.

[0103] Furthermore, in nature, mutations in the amino acid sequence encoded by a nucleotide sequence can occur. Therefore, with current technology, anyone skilled in the art, for example, having obtained the amino acid sequence information or the nucleotide sequence information encoding the antigenic polypeptide (a), can use known site-directed mutagenesis methods to alter its sequence and prepare a modified version of the antigenic polypeptide (a).

[0104] Therefore, the antigenic polypeptide schemes involved in this invention further include (b) a polypeptide composed of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence comprising amino acids at positions 28 to 175 of the amino acid sequence described in any one of the sequences 1 to 4, and whose total length is less than 200 residues in the amino acid sequence described in that sequence number; that is, a polypeptide selected from...

[0105] (1b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence containing amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 1, and whose total length is less than 200 residues in the amino acid sequence described in Serial No. 1.

[0106] (2b) A polypeptide consisting of an amino acid sequence comprising substitutions, deletions, insertions, and / or additions of one or more amino acids in an amino acid sequence containing amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 2, and having a total length of less than 200 residues in the amino acid sequence described in Serial No. 2.

[0107] (3b) A polypeptide consisting of an amino acid sequence comprising substitutions, deletions, insertions, and / or additions of one or more amino acids in an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 3, and an amino acid sequence of less than 200 residues in length.

[0108] (4b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added in an amino acid sequence containing amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 4 and whose total length is less than 200 residues in the amino acid sequence described in Serial No. 4.

[0109] At least one polypeptide (hereinafter referred to as “antigen polypeptide (b)” as appropriate).

[0110] The term "amino acid sequence in which one or more amino acids are replaced, deleted, inserted, and / or added" refers to an amino acid sequence in which amino acid residues are replaced, deleted, inserted, and / or added, or an amino acid sequence in which two or more of these methods are combined. Furthermore, "multiple" means an integer of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. The term "one or more amino acid residues" preferably refers to one or more but less than 10 amino acid residues, more preferably one or more but less than 5, further preferably one or more but less than 3, and particularly preferably less than 2.

[0111] Furthermore, with the current level of technology, anyone skilled in the art, for example, having obtained the amino acid sequence information of the antigenic polypeptide (a), can obtain homologous genes from other viruses based on the nucleotide sequence encoding it, using hybridization techniques (Southern, EM, J. Mol. Biol., 98, 1975, pp. 503-517), polymerase chain reaction (PCR) techniques (Saiki, RK et al., Science, 230, 1985, pp. 1350-1354, Saiki, RK et al., Science, 239, 1988, pp. 487-491).

[0112] For example, polypeptides encoded by homologous genes obtained by such methods typically have high homology and identity with the amino acid sequence of the antigenic polypeptide (a). Therefore, the antigenic polypeptide schemes involved in this invention also include (c) polypeptides composed of amino acid sequences having at least 80% homology (preferably identity) with amino acid sequences containing amino acid positions 28-175 of the amino acid sequence described in any one of sequence numbers 1-4, and whose total length is less than 200 residues of the amino acid sequence described in that sequence number; that is, polypeptides selected from...

[0113] (1c) A polypeptide composed of an amino acid sequence having at least 80% homology (preferably identical) to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 1, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 1.

[0114] (2c) A polypeptide composed of an amino acid sequence having more than 80% homology (preferably identical) to an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 2, and having a total length of less than 200 residues in the amino acid sequence described in Serial No. 2.

[0115] (3c) A polypeptide consisting of an amino acid sequence having at least 80% homology (preferably identity) with an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 3, and an amino acid sequence having a total length of less than 200 residues of the amino acid sequence described in Serial No. 3.

[0116] (4c) A polypeptide consisting of an amino acid sequence having more than 80% homology (preferably identity) with an amino acid sequence containing amino acids from positions 28 to 175 of the amino acid sequence described in Serial No. 4, and having a total length of less than 200 residues of the amino acid sequence described in Serial No. 4.

[0117] At least one polypeptide (hereinafter referred to as “antigen polypeptide (c)” as appropriate).

[0118] Furthermore, when discussing amino acid sequence homology, if the amino acid sequence of the comparison object is aligned with the amino acid sequence of the target object using amino acid sequence analysis software, the amino acid in the same column as the amino acid of the comparison object can be the same amino acid as the amino acid of the comparison object, or it can be an amino acid with the same properties as the amino acid of the comparison object. When discussing amino acid sequence identity, the amino acid in the same column as the amino acid of the comparison object is the same amino acid as the amino acid of the comparison object. Groups of amino acids with similar properties are well known in the technical field to which this invention pertains. For example, they can be classified as acidic amino acids (aspartic acid and glutamic acid); basic amino acids (lysine / arginine / histidine); and among neutral amino acids, amino acids with hydrocarbon chains (glycine / alanine / valine / leucine / isoleucine / proline), amino acids with hydroxyl groups (serine / threonine), sulfur-containing amino acids (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine / tyrosine / tryptophan).

[0119] The homology and identity of such amino acid sequences can be determined, for example, using the BLASTP procedure (Altschul et al., J. Mol. Biol., 215, 1990, pp. 403-410). Furthermore, the homology with the amino acid sequence of the antigenic polypeptide (a) should be 80% or more, but preferably 85%, 90%, or 95% or more (e.g., 96%, 97%, 98%, or 99%). More preferably, the identity should be 80% or more, and more preferably 85%, 90%, or 95% or more (e.g., 96%, 97%, 98%, or 99%).

[0120] In this invention, from the viewpoints of being able to detect more samples containing anti-HEV antibodies (e.g., being able to detect more positive samples) and having higher detection sensitivity (e.g., being able to detect even low amounts of anti-HEV antibodies), at least one polypeptide selected from (3a) to (3c) (hereinafter referred to as "antigen polypeptide (3)" as appropriate) is preferred. It is known that HEV contains genotypes 1 to 4 as described above, but existing hepatitis E virus infection detection kits use antigen polypeptides derived from genotypes 1 and 4. In contrast, although HEV detected in Japan so far consists of genotypes 1, 3, and 4, and genotype 4 has also been sporadically detected in Hokkaido, genotype 3 is the most prevalent. Therefore, it is considered that existing kits produce false negatives in a considerable number of samples. In contrast, the inventors hypothesize that by using the antigen polypeptide (3) derived from genotype 3 HEV as the HEV antigen, the frequency of such false negatives can be reduced.

[0121] Furthermore, regarding the antigenic polypeptide involved in this invention, from the viewpoints of being able to detect more samples containing anti-HEV antibodies (being able to detect more positive samples) and further improving detection sensitivity (being able to detect even with lower amounts of anti-HEV antibodies), the combination of the antigenic polypeptide (3) with at least one polypeptide selected from (1a) to (1c), (2a) to (2c), and (4a) to (4c) is further preferred, and the combination of the antigenic polypeptide (3) with at least one polypeptide selected from (1a) to (1c) and (4a) to (4c) is even more preferred. The combination of the antigenic polypeptide (3) with at least one polypeptide selected from (1a) to (1c) is particularly preferred. Thus, in addition to reducing the frequency of false negatives by using the aforementioned antigenic polypeptide (3) as the HEV antigen, a more comprehensive detection of diverse HEV genotypes is also possible. The inventors further discovered that the peptides in this combination work synergistically, and surprisingly, they can detect anti-HEV antibodies with high sensitivity even for samples where one of the peptides alone cannot detect them or has low sensitivity.

[0122] Furthermore, the combination scheme of the antigenic polypeptides is not particularly limited, and combinations of one or more of the antigenic polypeptides (3) with one or more of the antigenic polypeptides (1a) to (1c), (2a) to (2c), and (4a) to (4c) can form a multimer of dimer or more. In this invention, as a combination scheme, it is particularly preferred that any one polypeptide selected from (3a) to (3c) forms a dimer (heterodimer) with any one polypeptide selected from (1a) to (1c), (2a) to (2c), and (4a) to (4c). More preferably, it is preferred that any one polypeptide selected from (3a) to (3c) forms a dimer (heterodimer) with any one polypeptide selected from (1a) to (1c) and (4a) to (4c). Even more preferably, it is preferred that any one polypeptide selected from (3a) to (3c) forms a dimer (heterodimer) with any one polypeptide selected from (1a) to (1c). As described below, the inventors have discovered that the antigenic polypeptide involved in this invention is easy to purify as a heterodimer. Furthermore, the synergistic effect produced by the combination of the two polypeptides in the heterodimer is higher. That is, even for samples where one of the polypeptides alone cannot detect anti-HEV antibodies or has low sensitivity, anti-HEV antibodies can still be detected, thereby increasing the detection sensitivity.

[0123] [Methods for manufacturing antigenic peptides]

[0124] The antigenic polypeptide of this invention can be obtained using known methods or methods thereof. For example, it can be obtained by a method including the steps of culturing a host cell infused with at least one of a nucleotide encoding the antigenic polypeptide and a vector containing the nucleotide, and collecting the polypeptide expressed in the host cell. More specifically, for example, firstly, RNA encoding the antigenic polypeptide is obtained from HEV using conventional methods, and cDNA is synthesized. The cDNA may, for example, be synthetic DNA chemically synthesized based on the amino acid sequence of the antigenic polypeptide of this invention. Next, a DNA fragment encoding the antigenic polypeptide or an expression vector containing it is prepared from the cDNA, introduced into a host cell to obtain a transformant, and by culturing the transformant, the antigenic polypeptide of this invention is expressed, and can be obtained from the culture as a recombinant polypeptide.

[0125] As a method for obtaining nucleotides encoding the antigenic polypeptide involved in this invention from HEV, examples include: preparing a cDNA library by ligating cDNA synthesized based on RNA extracted from HEV with a vector such as a plasmid vector, phage vector, granular vector, BAC vector, or PAC vector; amplifying a DNA fragment containing the target nucleotide sequence using primers based on the nucleotide sequence encoding the antigenic polypeptide; and ligating it with an appropriate vector as needed.

[0126] The expression vector is a vector capable of replicating within a host cell and containing the polypeptide encoded by the nucleotide sequence in a state suitable for expression within the host cell. This expression vector can essentially be constructed as a self-replicating vector, i.e., existing as an independent entity outside the chromosome, whose replication is independent of chromosome replication, such as a plasmid (e.g., pBR322). Alternatively, when introduced into a host cell, the expression vector can also essentially be constructed as a phage DNA (e.g., λ phage) integrated into the host cell's genome and replicated along with the chromosome incorporating the expression vector.

[0127] The steps and methods for constructing the expression vector can appropriately employ known methods or methods based thereon. For example, to insert a DNA fragment containing the target nucleotide sequence into the expression vector, methods such as cleaving the DNA fragment with a suitable restriction enzyme and ligating it to a suitable restriction enzyme site or multiple cloning site of a plasmid can be used. In order to actually introduce the expression vector into a host cell to express the antigenic polypeptide, the expression vector preferably contains, in addition to the nucleotide sequence encoding the antigenic polypeptide, a nucleotide sequence controlling its expression (e.g., a sequence encoding a promoter, terminator, etc.), an inducible nucleotide sequence other than the nucleotide sequence controlling the expression (e.g., a lactose operon, etc.), and a gene marker sequence for selecting cells (e.g., a sequence of a drug resistance gene).

[0128] The host cell is not particularly limited, but is preferably a microorganism, such as filamentous bacteria, yeast, Escherichia coli, actinomycetes, lactic acid bacteria, etc. The host cell may, as needed, be a cell that has been transformed in a specific functional deficit manner or a mutant. The method for introducing the DNA fragment or expression vector into these host cells can be a known method or a method based thereon, such as heat shock method, electroporation method, protoplast method, lithium acetate method.

[0129] The antigenic polypeptide can be collected from its culture (e.g., cultured microbial cells) by culturing a transformant in which the DNA fragment or expression vector has been introduced into the host cell using a suitable culture medium. As for the culture conditions of the transformant, for example, the culture conditions of the host cell can be used. Anyone skilled in the art can appropriately adjust the temperature, the presence or absence of air, the oxygen concentration, the carbon dioxide concentration, the pH of the culture medium, the culture temperature, the culture time, and the humidity, depending on the type of host cell and the culture medium used.

[0130] As a method for collecting the antigenic polypeptide from the culture, a known method or a method based thereon can be appropriately employed. For example, the antigenic polypeptide can be expressed in host cells (e.g., *E. coli*), and after the transformation culture is completed, the cultured cells are lysed, and an insoluble fraction obtained by centrifugation, filtration, etc., is obtained as a crude purified product. Furthermore, the insoluble fraction is washed as needed, for example, soluble by denaturing agents such as urea or guanidine hydrochloride, and purified by salting out, dialysis, organic solvent precipitation, membrane separation, or chromatographic separation, alone or in combination of two or more methods. Alternatively, the antigenic polypeptide with a purification tag (e.g., HIS tag, trpE tag, etc.) can be expressed in host cells (e.g., *E. coli*), and the crude extract can be passed through a protein purification column (e.g., Ni column, affinity column, etc.) to purify it by dissolving the tagged protein. One or more of these purification methods can be used.

[0131] The antigenic polypeptides involved in this invention can be expressed, for example, by adding a HIS tag, thereby facilitating purification using a Ni column. Alternatively, expression can be achieved by adding a trpE tag, thereby increasing expression efficiency and allowing purification using an affinity column with an anti-trpE antibody. More specifically, as one method for purifying the antigenic polypeptides involved in this invention, the crude purified product obtained by adding the HIS tag is first washed as needed and then made soluble. A solution containing, for example, 4-8 M urea is preferred as the solution used for soluble purification. Next, dialyzing is performed with PBS (pH 7.3), and after capturing the supernatant after dialysis on a Ni column, the product is washed as needed and dissolved with imidazole, thereby obtaining the purified antigenic polypeptide as the dissolution product. The dissolution product can be dialyzed again as needed.

[0132] Furthermore, when the antigenic polypeptide involved in this invention is the aforementioned dimer (heterodimer), as one method of its manufacturing, for example, firstly, an HIS tag is added to one polypeptide (polypeptide A) constituting the target heterodimer to induce its expression, while the other polypeptide (polypeptide B) is expressed without adding an HIS tag. Then, they are made soluble separately using the same method as described above. As the solution used for this soluble process, a solution containing, for example, 4-8 M urea is preferred. Next, they are mixed and dialyzed. As the solution used for this dialysis, PBS (pH 7.3) is preferred, for example. Thus, most of the monomer and homodimer of the HIS-tagged polypeptide A are recovered as precipitate, but by forming a heterodimer with the HIS-untagged polypeptide B, it can be soluble and recovered as supernatant. The inventors speculate that this is because polypeptide B exerts a molecular chaperone effect on polypeptide A. Next, by capturing the supernatant with a Ni column, monomers and homodimers of polypeptide B without the HIS tag are removed by fractionation. Therefore, by dissolving the protein captured by the Ni column, heterodimers of polypeptide A and polypeptide B can be obtained. An imidazole solution, for example, can be used as the solution for this dissolution.

[0133] [Inspection Process]

[0134] In this invention, the detection method for detecting anti-HEV antibodies using the aforementioned antigenic peptide is not particularly limited to any method capable of detecting anti-HEV antibodies; existing known methods, methods based thereon, or combinations thereof can be appropriately employed. Examples include, for instance, immunological techniques such as sandwich assays, competitive assays, and immunoturbidimetric assays, and detection methods based on their principles; there is no particular limitation. In such detection methods, for example, methods using microplates, particles, etc., as carriers, such as ELISA, digital ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescent immunoassay), ECLIA (electrochemical immunoassay), and RIA (radioimmunoassay), can generally be used; immunochromatography; surface plasmon resonance analysis; and detection methods utilizing fluorescence resonance energy transfer, etc.

[0135] From the viewpoint of constructing a measurement system with higher sensitivity and specificity, the sandwich method is preferred as the detection method in this invention. Hereinafter, a more specific scheme will be described using the sandwich method as an example for the detection steps involved in this invention.

[0136] When using the sandwich method described above, examples of sandwich methods according to the present invention can be listed.

[0137] The detection process involves bringing the sample into contact with the trap and the marker.

[0138] The capture body comprises a water-insoluble carrier and a probe molecule capable of binding to an anti-HEV antibody, and the label comprises a scheme of a labeling substance and the antigen peptide (at least one of the peptides) (hereinafter referred to as "Scheme 1") as appropriate; and

[0139] The detection process involves bringing the sample into contact with the trap and the marker.

[0140] The capture body comprises a water-insoluble carrier and the antigenic polypeptide, and

[0141] The label comprises a labeling substance and a probe molecule capable of binding to an anti-HEV antibody (hereinafter referred to as "Scheme 2" as appropriate). From the viewpoint of further reducing nonspecific reactions, Scheme 1 is preferred.

[0142] In the sandwich method, a probe molecule (hereinafter referred to as a "probe molecule" as appropriate) capable of binding to the anti-HEV antibody and at least one of the polypeptides (hereinafter referred to as an "antigen polypeptide") may be present in either the capture body or the label body, but one may be contained in the capture body and the other in the label body. This allows the anti-HEV antibody in the sample to be captured by the capture body and labeled by the label body, thus enabling the detection of the anti-HEV antibody with high accuracy and ease by detecting the signal derived from the labeled substance.

[0143] The detected signal can be semi-quantitatively or quantitatively determined as needed to represent the amount of anti-HEV antibody. The "signal" includes, depending on the labeling substance, color development (chromogenicity), extinction, reflected light, luminescence, fluorescence, radiation generated by radioactive isotopes, etc., including those that can be confirmed by the naked eye and those that can be confirmed by detection methods / devices adapted to the type of signal. In this invention, the amount of anti-HEV antibody can be quantified using a standard curve or similar method employing a standard sample; however, from a simpler and faster perspective, the signal quantity can also be directly treated as the amount of anti-HEV antibody in this invention.

[0144] Examples of such sandwich methods include two-step forward sandwich methods (a method that sequentially reacts the trap with the anti-HEV antibody in the sample, and the anti-HEV antibody bound to the trap reacts with the label), reverse sandwich methods (a method that pre-reacts the label with the anti-HEV antibody in the sample, and then reacts the resulting complex with the trap), and one-step methods (a method that simultaneously reacts the anti-HEV antibody, the trap, and the label in the sample in one step). These methods can be used arbitrarily.

[0145] For example, in the forward sandwich method of Scheme 1 or Scheme 2, firstly, the sample is contacted with the trapping body, and the trapping body captures the anti-HEV antibody via the binding of the probe molecule (or antigenic peptide) of the trapping body (1st reaction: trapping step). Next, the anti-HEV antibody captured by the trapping body is contacted with the label, and labeling is achieved via the binding of the antigenic peptide (or probe molecule) of the label to the anti-HEV antibody (2nd reaction: labeling step). Through this reaction, a complex comprising the trapping body, anti-HEV antibody, and label is formed. After washing away the unbound sample and label as needed (washing step), the signal originating from the label is detected by a method adapted to the specified labeling substance (detection step).

[0146] [Non-water-soluble carrier]

[0147] The water-insoluble carrier primarily carries the probe molecules or the antigen peptides, functioning as a solid-phase carrier, and is a water-insoluble substance. In this invention, "water-insoluble substance" refers to a substance that is insoluble in water at room temperature and pressure (its solubility relative to water is less than 0.001 g / mL, preferably less than 0.0001 g / mL, and the same applies below).

[0148] As the material for such a water-insoluble carrier, materials commonly used in immunoassays and the assays based thereon can be used, without particular limitation. Examples include at least one selected from polymers (polystyrene, (meth)acrylate, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, glass, latex, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, cobalt oxide, nickel ferrite, etc.). Furthermore, the material for the aforementioned water-insoluble carrier can be a composite of these substances, or a composite of these substances with other substances. For example, it can be an organic-inorganic composite material (e.g., magnetic particles, etc.) composed of at least one organic polymer selected from the aforementioned polymers, gelatin, and latex, and at least one metal compound selected from iron oxide (spinel ferrite, etc.), cobalt oxide, and nickel ferrite. Furthermore, the non-water-soluble carrier can be a non-water-soluble carrier whose surface has been modified with active groups such as carboxyl, epoxy, p-toluenesulfonyl, amino, hydroxy, isothiocyanate, isocyanate, azide, aldehyde, carbonate, allyl, aminooxy, maleimide, and thiol.

[0149] Furthermore, in this invention, the shape of the water-insoluble carrier is not particularly limited; for example, it can be any material such as a plate, fiber, membrane, or particle. Existing known or commercially available water-insoluble carriers can be used as such carriers.

[0150] [Probe molecules that can bind to anti-HEV antibodies]

[0151] Examples of probe molecules capable of binding to anti-HEV antibodies include, for example, antigens; antibodies; binding proteins such as protein A, protein G, and protein L; avidin proteins such as avidin D and streptavidin; lectins; galactagogues; glycan receptors; and immunoreceptors. The "antibody" includes not only complete antibodies but also antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, single-chain antibodies, bispecific antibodies, etc.) and low-molecular-weight antibodies formed by binding the variable region of an antibody. Preferably, the probe molecule is an antibody capable of binding to anti-HEV antibodies. Such antibodies can be selected, for example, from anti-IgG antibodies, anti-IgM antibodies, anti-IgA antibodies, anti-IgE antibodies, and anti-IgD antibodies, depending on the isotype of the target anti-HEV antibody being detected, or combinations of two or more of these. Antibodies suitable for these isotypes can be prepared using existing known methods, or commercially available antibodies can be used.

[0152] [Marked substances]

[0153] As the labeling substance, any substance used as a labeling substance in known immunoassay methods and methods thereof may be used without particular limitation. Examples include enzymes; luminescent substances such as acridinium ester derivatives; fluorescent substances such as europium; and fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE). 125 Radioactive substances of class I; low molecular weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrobenzene (DNP); digoxigenin (DIG), which may be one or a combination of two or more of these. For example, when an enzyme is used as the labeling substance, various signals can be detected by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc., as the substrate. Examples of such enzymes include, but are not limited to, peroxidase (POD: horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.

[0154] 〔Capture body〕

[0155] The label is a complex comprising the water-insoluble carrier and the probe molecule or the antigenic polypeptide, wherein the water-insoluble carrier binds directly or indirectly to the probe molecule or the antigenic polypeptide.

[0156] The amount of the probe molecule or antigenic polypeptide in the capture body is not particularly limited and can be appropriately adjusted to suit their ease of binding to the anti-HEV antibody, for example, when the insoluble carrier is a plate, the amount can be adjusted relative to its surface area of ​​1 cm².2 The mass (the total of two or more combinations) is preferably 0.01 to 1.5 μg.

[0157] The capture body can be manufactured by binding the probe molecule or the antigenic polypeptide to the water-insoluble carrier. As a manufacturing method, existing known methods or methods based thereon can be suitable for directly binding the probe molecule or the antigenic polypeptide (hereinafter referred to as "supported carrier") to the water-insoluble carrier, or indirectly binding it.

[0158] In the case of direct bonding, for example, as the insoluble carrier and / or the supported carrier, a carrier having active groups such as carboxyl, epoxy, p-toluenesulfonyl, amino, hydroxyl, isothiocyanate, isocyanate, azide, aldehyde, carbonate, allyl, aminooxy, maleimide, thiol, or pyridyl disulfide can be used, or, as needed, such active groups can be applied to bind them, thereby directly fixing the supported carrier to the insoluble carrier. Alternatively, if the insoluble carrier is a plate, the supported carrier can be coated onto the plate, sealed with sodium caseinate, bovine serum albumin (BSA), etc., as needed, and then dried, thereby directly fixing the supported carrier to the insoluble carrier.

[0159] In the case of indirect binding, for example, the supported carrier can be indirectly immobilized on the water-insoluble carrier by binding via polyhistidine, polyethylene glycol, oligopeptides containing cysteine ​​and / or lysine, or linkers bound to the supported carrier. The linkers are not particularly limited and can be exemplified by secondary antibodies, protein G, protein A, photolytically cleavable linkers capable of binding to the supported carrier (preferably the probe molecule), or linker molecules having the active group (e.g., hydrazine salts, acylhydrazides). Alternatively, the supported carrier (preferably the probe molecule) can be modified in some way, and a substance capturing the modified portion can be immobilized on the water-insoluble carrier, thereby immobilizing the supported carrier on the water-insoluble carrier. For example, biotin can be cited as a representative example of the modified portion, and streptoavidin can be cited as a representative example of the substance capturing the modified portion, but these are not limited to these.

[0160] The ratio of the water-insoluble carrier to the supported carrier for these reactions can be suitably selected in a manner that achieves a preferred range of the ratio in the trapping agent described above. Furthermore, if necessary, to prevent non-specific adsorption to the supported carrier and the water-insoluble carrier, the water-insoluble carrier can be blocked with a suitable blocking agent (e.g., bovine serum albumin, gelatin, etc.). Moreover, when the supported carrier is the probe molecule, a commercially available trapping agent can be suitable.

[0161] [Marker body]

[0162] The label is a complex comprising the labeling substance and the probe molecule or the antigenic polypeptide, wherein the labeling substance binds directly or indirectly to the probe molecule or the antigenic polypeptide. The content of the labeling substance, the probe molecule, or the antigenic polypeptide in the label is not particularly limited and can be appropriately adjusted according to the type of labeling substance, etc.

[0163] The label can be manufactured by binding the labeling substance to the probe molecule or the antigenic polypeptide. As a manufacturing method, existing known methods or methods based thereon can be appropriately employed, allowing the labeling substance to bind directly or indirectly to the probe molecule or the antigenic polypeptide. Methods similar to those described in the capture body can be listed as binding methods for these methods.

[0164] (Capture steps)

[0165] In the capture step, the method of contacting the anti-HEV antibody with the capture body is not particularly limited, and existing known methods or methods based thereon can be used. For example, if the water-insoluble carrier is a plate, a method of injecting the sample into it (supported carrier immobilized plate) can be listed. If the water-insoluble carrier is a particle, a method of adding the capture body (supported carrier immobilized particle) to the sample can be listed.

[0166] In the reaction between the sample and the trapping body during the trapping step, the amount of the sample is not particularly limited and can be adjusted appropriately according to the type and concentration of the sample. Furthermore, the conditions for the trapping step are not particularly limited and can be adjusted appropriately. For example, the step can be carried out at room temperature to 45°C, preferably 20 to 37°C, at a pH of approximately 6.0 to 9.0, preferably at a pH of 7.0 to 8.0, for approximately 30 to 90 minutes, preferably approximately 60 minutes, but is not limited to these conditions.

[0167] (Marking steps)

[0168] In the reaction between the marker and the anti-HEV antibody in the labeling step, the content (final concentration) of the marker in the reaction solution containing the marker and the anti-HEV antibody (the total concentration if there is a combination of two or more markers) is not particularly limited and is appropriately adjusted according to the type and concentration of the sample, and is therefore not particularly limited. From the viewpoint that excessive use may produce a high background signal, for example, it is preferably 0.01 to 10 μg / mL, more preferably 0.1 to 5 μg / mL, and even more preferably 0.2 to 1 μg / mL.

[0169] In addition, other conditions for the labeling step are not particularly limited and can be appropriately adjusted. For example, the step can be carried out at room temperature to 37°C, preferably 20 to 37°C, pH 5.0 to 7.0, preferably 5.5 to 6.5 for about 30 to 90 minutes, preferably about 60 minutes, but is not limited to these conditions.

[0170] (Washing steps)

[0171] The sandwich method, following the capture step and / or the labeling step, preferably further includes a washing step to separate the anti-HEV antibody bound to the capture body and other impurities not bound to the capture body (i.e., uncaptured). The method for removing the impurities is not particularly limited; existing known methods or methods based thereon can be suitable. Examples include, for instance, removing the liquid phase (supernatant) from the plate when the capture body is the supported carrier solidified plate, and removing the liquid phase (supernatant) by recovering the particles by centrifugation or magnetic attraction when the capture body is the supported carrier solidified particles. Additionally, the washing step may involve repeating the injection and removal of washing solution as needed. Examples of wash solutions include, for instance, known neutral (preferably pH 6.0 to 9.0) buffer solutions (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, benzyltoluidine buffer, N,N-dihydroxyethylglycine buffer, glycine buffer, etc.). Additionally, stabilizing proteins such as BSA and surfactants may be added.

[0172] When the sandwich method is used as the detection step involved in this invention, the sample can be diluted with a diluent as described above. However, if the trapping body is the solidified particles of the supported carrier, it can be used in suspension in a particle suspension medium (particle liquid). Furthermore, other reaction buffers can be appropriately added to the reaction system of the sample and the trapping body and / or the label. These particle suspension media and reaction buffers are not particularly limited; for example, known buffers (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, benzyltoluidine buffer, N,N-dihydroxyethylglycine buffer, glycine buffer, etc.) can be listed independently. Additionally, stabilizing proteins such as BSA and serum can be added independently.

[0173] (Testing steps)

[0174] In the detection step, a signal is detected based on the labeled substance. For example, if the labeled substance is an enzyme, a signal is generated by adding a chromogenic substrate or a luminescent substrate corresponding to the enzyme to cause a reaction (e.g., color development, luminescence). Thus, the presence or absence of anti-HEV antibodies in the sample is detected by the presence or absence of the signal. Furthermore, if anti-HEV antibodies are present, the amount of anti-HEV antibodies in the sample can be obtained as a signal quantity. Additionally, the amount of anti-HEV antibodies in the sample can be checked as needed by comparing the signal quantity with that in a standard sample.

[0175] <Methods for testing for hepatitis E virus>

[0176] According to the method for detecting hepatitis E virus infection of the present invention, the presence or absence of anti-HEV antibodies detected in the above-described detection steps can be used as an indicator to detect whether the sample is infected with hepatitis E virus, preferably to detect whether the subject from the same sample is infected with hepatitis E virus. Furthermore, by using the amount of anti-HEV antibodies detected in the above-described detection steps as an indicator, information about the degree of hepatitis E virus infection (infection period, severity of hepatitis E) in the sample, preferably in the subject from the same sample, can be obtained. Therefore, the present invention also provides a method for testing for hepatitis E virus (hereinafter, as applicable, simply referred to as "testing method"), including a step of detecting anti-hepatitis E virus antibodies in a sample from a subject using the method for detecting hepatitis E virus infection of the present invention.

[0177] As a specific example of the inspection method of the present invention, the following solutions can be listed:

[0178] The method includes a detection procedure for detecting anti-HEV antibodies in a sample derived from a test subject using the antigenic peptide, and a determination procedure for using the detected anti-HEV antibodies as an indicator to determine whether the test subject is infected with HEV.

[0179] The method includes a detection procedure for detecting anti-HEV antibodies in a sample from the subject using the antigenic peptide, and a selection procedure (screening method) for selecting subjects who are predicted to have hepatitis E or who are highly likely to develop hepatitis E, using the detected anti-HEV antibodies as an indicator.

[0180] The procedure includes a detection step of using the antigenic peptide to detect anti-HEV antibodies in a sample from the subject, and an identification step of using the detected anti-HEV antibodies as an indicator to identify whether the subject has hepatitis E.

[0181] In each of the aforementioned testing methods, for the determination step, selection step, and identification step, as long as even a trace amount of anti-HEV antibody is detected in the sample during the detection step, the subject from whom the sample originated can be classified as having a high probability of being infected with hepatitis E virus, having hepatitis E or developing hepatitis E, or having hepatitis E (i.e., being positive). However, it is preferable to determine, select, or identify based on the amount of anti-HEV antibody detected. For example, the amount of anti-HEV antibody detected in the detection step can be compared with a pre-set cutoff value, and subjects whose anti-HEV antibody levels are higher than the cutoff value can be classified as having a high probability of being infected with hepatitis E virus, having hepatitis E or developing hepatitis E, or having hepatitis E.

[0182] The "cutoff value" is a predetermined value that serves as a benchmark for determining the amount of anti-HEV antibodies, representing the boundary value used to distinguish between the positive and negative groups. Such a cutoff value is appropriately set according to the purpose of the testing method according to the present invention, the method for detecting anti-HEV antibodies, the nature of the subject and sample, dilution conditions, etc., and is therefore not particularly limited. For example, by setting the cutoff value to a relatively low value, the detection sensitivity can be improved, that is, a wider range of subjects suspected of being positive can be collected to some extent; on the other hand, by setting the cutoff value to a relatively high value, the various testing methods can be performed with higher accuracy.

[0183] According to the examination method of the present invention, information can be provided to determine a treatment strategy specific to hepatitis E, distinguishing it from other types of hepatitis. Furthermore, by targeting individuals with a high probability of having (including reactivation) hepatitis E, early treatment intervention can be achieved. Moreover, as an example of the examination method of the present invention, a detection step including detecting anti-HEV antibodies in a sample derived from the subject using the antigenic polypeptide, and a prediction step using the detected anti-HEV antibodies as an indicator to predict the HEV infection stage of the subject, for example, by appropriately selecting anti-IgG antibodies, anti-IgM antibodies, and anti-IgA antibodies as the probe molecules, the HEV infection stage of the subject can be predicted from the isotype of the anti-HEV antibodies. Furthermore, the examination method of the present invention is a method to assist in the diagnosis of hepatitis E performed by physicians, or a method to provide information for the diagnosis of hepatitis E performed by physicians.

[0184] <Antigen peptides and kits for detecting hepatitis E virus infection>

[0185] The antigenic polypeptide for detecting hepatitis E virus infection of the present invention is an antigenic polypeptide used in the above-described method for detecting hepatitis E virus infection of the present invention, and is selected from at least one polypeptide selected from (1a) to (1c), (2a) to (2c), (3a) to (3c), and (4a) to (4c). Preferred embodiments of the antigenic polypeptide of the present invention are also included, as described above. Furthermore, the kit for detecting hepatitis E virus infection of the present invention is a kit used in the above-described method for detecting hepatitis E virus infection of the present invention, and contains the antigenic polypeptide of the present invention.

[0186] The antigenic polypeptide included in the kit of the present invention may consist solely of the antigenic polypeptide (including a combination of two or more polypeptides) or may be a composition containing the antigenic polypeptide. The composition may further contain other components, which are not particularly limited and may include, for example, sterile water, physiological saline, surfactants, preservatives, etc., and may be one of these or a combination of two or more.

[0187] As a kit of the present invention, it is preferable to further include the trap having the water-insoluble carrier and the probe molecule. In this case, the antigen polypeptide is preferably the label having been labeled with the labeling substance. Furthermore, as a kit of the present invention, it is also preferable to further include the label having the labeling substance and the probe molecule. In this case, the antigen polypeptide is preferably the trap supported on the water-insoluble carrier. The trap and the label include their preferred embodiments as described above. In addition, in this case, the trap and the label can each independently be in solid (powder) form or in liquid form dissolved or dispersed in a buffer solution (for example, a particle dispersion in the case of the trap). In the case of liquid form, the concentration of the trap and the label in each solution is not particularly limited, but each is preferably, for example, 0.01 to 10 μg / mL, more preferably 0.1 to 5.0 μg / mL, and even more preferably 0.2 to 1.0 μg / mL.

[0188] As a kit of the present invention, it may further include the components typically found in conventional immunoassay methods such as ELISA, CLEIA, and immunochromatography, and methods based thereon. For example, when the sandwich method described above is used as the principle of the detection method involved in the present invention, it may further include at least one selected from the following: magnetic beads or plates for immobilizing the supported carrier; the labeled substance; the standard samples (each concentration); control reagents; the particle suspension medium; the reaction buffer; and the washing solution. Furthermore, when the labeled substance is an enzyme, it may further include substrates and reaction stop solutions required for the detection / quantification of the labeled substance.

[0189] Furthermore, the kit of the present invention may, as needed, include the diluent, a pretreatment solution for sample pretreatment, a dilution cassette, a reaction stop solution or neutralization solution for pretreatment, etc. Additionally, when immunochromatography is used as the sandwich method, it may further include a device containing a band carrying the trap and / or the label. This device may include other components suitable for immunochromatography, such as a developing buffer and an absorption buffer. Furthermore, the kit of the present invention may further include an instruction manual for the kit.

[0190] Example

[0191] The present invention will now be described in more detail based on embodiments and comparative examples, but the present invention is not limited to the following embodiments.

[0192] (Preparation Example 1) Cloning of the E2s (G3E2s) gene from genotype 3 HEV

[0193] (1-1) Cloning of HEV gene of genotype 3 using RT-PCR

[0194] For genotype 3 HEV (HEVG3, GenBank Accession No.: AB481229), the HEV gene was cloned by RT-PCR. Specifically, first, 100 μL of HEVG3 sample (HEVG3: 10...) was used... 4 RNA (HEV-RNA) was extracted and purified using the High Pure ViralRNA Kit (manufactured by Luxu). 11 μL of purified HEV-RNA, 1 μL of reverse primer G3AS1 (2 μM, sequence number: 5), and 1 μL of dNTPs (10 mM each) were dispensed into 0.5 mL tubes and heated at 65°C for 5 minutes, then rapidly cooled on ice. Next, 1 μL of RNase inhibitor (manufactured by Takarazuchi), 1 μL of 100 mM DTT, 4 μL of 5×SSIV buffer (manufactured by Invistron), and 1 μL of reverse transcriptase SSIV (manufactured by Invistron) were added to each tube to prepare a 20 μL reaction solution. The reaction solution was incubated at 50°C for 10 minutes, followed by heating at 80°C for 10 minutes to inactivate the enzymes, yielding a cDNA synthesis reaction solution containing cDNA.

[0195] Nested PCR is used to improve the sensitivity and specificity of DNA amplification detection. Specifically, a first-step PCR is performed using two primers, followed by a second-step PCR using two primers located inside the DNA sequence of the PCR product. The first-step PCR uses primer G3S1 (accession number: 6) and the reverse primer G3AS1, while the second-step PCR uses primer G3S2 (accession number: 7) and primer G3AS2 (accession number: 8).

[0196] The reaction solution for the first-step PCR consisted of 5 μL of the cDNA synthesis reaction solution, 2 μL each of the two primers (50 pmol each) for the first-step PCR, 10 μL of KAPATaq Extra HS RM+dye (manufactured by KAPA Biosystems), and 3 μL of sterile water, for a total volume of 20 μL. The first-step PCR reaction was performed by heating at 95°C for 3 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes.

[0197] The reaction solution for the second step PCR consisted of 2 μL of the first step PCR reaction end solution, 2 μL each of the two types of primers (50 pmol each) for the second step PCR, 9 μL of KAPATaq Extra HS RM+dye, and 7 μL of sterile water, for a total volume of 20 μL. The reaction was carried out under the same conditions as the first step PCR reaction.

[0198] 10 μL of the 2nd step PCR reaction end solution was subjected to agarose gel electrophoresis. The DNA fragment, which was specifically amplified to a length of about 5 kbp, was recovered from the agarose gel using the QIAquick Gel Extraction Kit (manufactured by Kiagen Co., Ltd.) and cloned into pGEM-T Easy (manufactured by Promega Co., Ltd.) to obtain pGEM-HEV3 containing the nucleotide sequence of ORF2 of the HEV gene of genotype 3.

[0199] (1-2) Cloning of the E2s (G3E2s) gene of genotype 3 into an expression vector

[0200] Using pGEM-HEV3 as a template, PCR was performed using primers G3E2s-his452-S (Sequence No.: 9) and G3E2s-617-AS (Sequence No.: 10) to amplify a DNA fragment encoding G3E2s (approximately 530 bp). The primers were designed to sequentially add the EcoRI restriction enzyme recognition sequence and the HIS tag to the 5' end of the amplified DNA fragment, starting from the 5' end, and to sequentially add the stop codon and the BamHI restriction enzyme recognition sequence to the 3' end, starting from the 5' end. Using these two restriction enzymes, the amplified DNA fragment was cloned into the EcoRI-BamHI site of the pAT-trp-trpE expression vector (the E. coli trpE protein expression vector, the vector described in Japanese Patent No. 3217600), and the nucleotide sequence pAT-trp-trpE-his-G3E2s (with HIS tag, trpE) containing the full-length 166 amino acid residues (G3E2s) from position 21 to 186 of the amino acid sequence recorded in sequence number 3 of the ORF2 of the HEV gene of genotype 3 was obtained.

[0201] In addition, PCR was performed using primers without the HIS tag: primer G3E2s-452-S (sequence number: 11) and primer G3E2s-617-AS, to amplify a DNA fragment encoding G3E2s (approximately 510 bp). The primers were designed to add the EcoRI restriction enzyme recognition sequence to the 5' end of the amplified DNA fragment, and to add a stop codon and the BamHI restriction enzyme recognition sequence sequentially from the 5' end to the 3' end. Using these two restriction enzymes, the amplified DNA fragment was cloned into the EcoRI-BamHI site of the pAT-trp-trpE expression vector, obtaining the nucleotide sequence pAT-trp-trpE-G3E2s (without the HIS tag, with trpE) containing the full-length 166 amino acids (G3E2s) encoded by sequence number 3 in the ORF2 of the HEV gene of genotype 3.

[0202] (Preparation Example 2) Cloning of the E2s gene from other HEV genotypes

[0203] (2-1) Cloning of the E2s (G1E2s) gene of genotype 1 into an expression vector

[0204] For genotype 1 HEV (HEVG1), the nucleotide sequence of its ORF2 and the amino acid sequence it encodes were obtained from the DNA Data Bank of Japan (DDBJ) (DDBJ Accession No.: D11092). Nucleotides encoding amino acids 394–617 of the obtained amino acid sequence (ORF2) (G1E2s 394–617, sequence shown in Serial No. 12) were synthesized by Eurofin Genemics to obtain pEX-A-HEV1 containing the nucleotide sequence described in Serial No. 12.

[0205] Using pEX-A-HEV1 as a template, PCR was performed using primers G1E2s-his452-S (serial number: 13) and G1E2s-617-AS (serial number: 14) to amplify a DNA fragment encoding G1E2s (approximately 530 bp). The primers were designed to sequentially add the EcoRI restriction enzyme recognition sequence and the HIS tag to the 5' end of the amplified DNA fragment, starting from the 5' end, and to sequentially add the stop codon and the BamHI restriction enzyme recognition sequence to the 3' end, starting from the 5' end. Using these two restriction enzymes, the amplified DNA fragment was cloned into the EcoRI-BamHI site of the pAT-trp-trpE expression vector to obtain the nucleotide sequence pAT-trp-trpE-his-G1E2s (with HIS tag and trpE) containing the full-length 166 amino acid residues (G1E2s) from position 21 to 186 of the amino acid sequence recorded in sequence number 1 of the ORF2 of the HEV gene of genotype 1.

[0206] (2-2) Cloning of HEV gene of genotype 4 using RT-PCR

[0207] For HEV genotype 4 (HEVG4, GenBank Accession No.: AB481227), cloning was performed by RT-PCR in the same manner as in (1-1). Specifically, first, 100 μL of HEVG4 sample (HEVG4: 10 4RNA (HEV-RNA) was extracted and purified using the High PureViral RNA Kit (manufactured by Luxu). 11 μL of purified HEV-RNA, 1 μL of reverse primer G4AS1 (2 μM, sequence number: 15), and 1 μL of dNTPs (10 mM each) were dispensed into 0.5 mL tubes, and then heated at 65°C for 5 minutes, followed by rapid cooling on ice. Next, 1 μL of RNase inhibitor (manufactured by Takarazuchi), 1 μL of 100 mM DTT, 4 μL of 5×SSIV buffer (manufactured by Invistron), and 1 μL of reverse transcriptase SSIV (manufactured by Invistron) were added to each tube to prepare a 20 μL reaction solution. The reaction solution was incubated at 50°C for 10 minutes, followed by heating at 80°C for 10 minutes to inactivate the enzyme, yielding a cDNA synthesis reaction solution containing cDNA.

[0208] The first step PCR used primer G4S1 (serial number: 16) and the reverse primer G4AS1, and the second step PCR used primer G4S2 (serial number: 17) and primer G4AS2 (serial number: 18).

[0209] The reaction solution for the first-step PCR consisted of 5 μL of the cDNA synthesis reaction solution, 2 μL each of the two types of primers (50 pmol each) for the first-step PCR, 10 μL of KAPATaq Extra HS RM+dye (manufactured by KAPA Biosystems), and 3 μL of sterile water, for a total volume of 20 μL. The first-step PCR reaction was performed by heating at 95°C for 3 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes.

[0210] The reaction solution for the second step PCR consisted of 2 μL of the first step PCR reaction end solution, 2 μL each of the two types of primers (50 pmol each) for the second step PCR, 9 μL of KAPATaq Extra HS RM+dye, and 7 μL of sterile water, for a total volume of 20 μL. The reaction was carried out under the same conditions as the first step PCR reaction.

[0211] 10 μL of the 2nd step PCR reaction end solution was subjected to agarose gel electrophoresis. The DNA fragment, which was specifically amplified to a length of about 5 kbp, was recovered from the agarose gel using the QIAquick Gel Extraction Kit (manufactured by Kiagen Co., Ltd.) and cloned into pGEM-T Easy (manufactured by Promega Co., Ltd.) to obtain pGEM-HEV4, which contains the nucleotide sequence of ORF2 of the HEV gene of genotype 4.

[0212] (2-3) Cloning of the E2s (G4E2s) gene of genotype 4 into an expression vector

[0213] Using pGEM-HEV4 as a template, PCR was performed using primers G4E2s-his445-S (Sequence No.: 19) and G4E2s-606-AS (Sequence No.: 20) to amplify a DNA fragment encoding G4E2s (approximately 530 bp). The primers were designed to sequentially add the EcoRI restriction enzyme recognition sequence and the HIS tag to the 5' end of the amplified DNA fragment, starting from the 5' end, and to sequentially add the stop codon and the BamHI restriction enzyme recognition sequence to the 3' end, starting from the 5' end. Using these two restriction enzymes, the amplified DNA fragment was cloned into the EcoRI-BamHI site of the pAT-trp-trpE expression vector to obtain the nucleotide sequence pAT-trp-trpE-his-G4E2s (with HIS tag and trpE) containing the full-length 162 amino acid residues (G4E2s) from position 14 to 175 of the amino acid sequence recorded in sequence number 4 of the ORF2 of the HEV gene of genotype 4.

[0214] (Preparation Example 3) Expression and purification of polypeptide (E2s)

[0215] Using the four expression plasmids prepared in Preparation Examples 1-2 above, four polypeptides: trpE-his-G3E2s, trpE-G3E2s, trpE-his-G1E2s, and trpE-his-G4E2s were expressed and purified. Specifically, firstly, *E. coli* XL1-Blue prepared in Preparation Examples 1-2 with each of the four expression plasmids was inoculated into 40 mL of LB medium containing 50 μg / mL ampicillin and cultured overnight at 37°C. This 40 mL culture was then further inoculated into 4 L of M9-CA medium (0.6% Na2HPO4, 0.5% KH2PO4, 0.5% NaCl, 0.1% NH4Cl, 0.1 mM CaCl2, 2 mM MgSO4, 0.5% casein amino acids, 0.2% glucose) containing 50 μg / mL ampicillin and cultured at 37°C. When OD600 = 0.3, indoleacrylic acid was added to achieve a final concentration of 40 mg / L, and the culture was further incubated for 16 hours. The culture was then centrifuged, and approximately 12 g of bacterial cells were collected.

[0216] Next, 60 mL of lysozyme buffer (50 mM Tris-HCl (pH 8.0), 30 mM NaCl, 5 mM EDTA) was added to the obtained bacterial cells for suspension, and 12 mg of lysozyme (manufactured by Biochemical Industrial Co., Ltd.) was added and the mixture was reacted at 37°C for 1 hour. After the reaction, the E. coli membrane was broken by sonication for 150 seconds (1), and the supernatant was removed by centrifugation at 15000 rpm for 30 minutes at 4°C (2) to obtain an insoluble fraction containing the fusion peptide of the polypeptide encoded by each target nucleotide sequence and trpE.

[0217] Next, 55 mL of a washing buffer containing a surfactant (25 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.1% NP-40) was added to the insoluble fraction for resuspension. The supernatant was removed by centrifugation at 15,000 rpm for 30 minutes at 4°C (3) to obtain a washed insoluble fraction. Then, 55 mL of washing buffer (25 mM Tris-HCl (pH 8.0), 5 mM EDTA) was added to the insoluble fraction for resuspension. The supernatant was removed by centrifugation at 15,000 rpm for 30 minutes at 4°C (4) to obtain a rewashed insoluble fraction. Next, 55 mL of a soluble buffer (4 M urea, 25 mM Tris-HCl (pH 8.0), 5 mM EDTA) was added to the rewashed insoluble fraction to obtain a soluble product (5) that solubles the fusion peptide.

[0218] Next, the soluble substance was placed in a dialysis bag (MWCO: 10kD, manufactured by Special Co., Ltd.) and dialyzed overnight at 4°C with stirring relative to PBS (10mM sodium phosphate buffer (pH 7.3) containing 0.15M NaCl) in a 3L volumetric beaker. A white precipitate was confirmed in the dialysis bag. The dialyzed sample was centrifuged at 15000 rpm for 30 minutes at 4°C to remove the precipitate (7). The supernatant was filtered through a 0.22μm filter membrane to obtain the crude purified product (6).

[0219] The crude purified extracts of the three HIS-tagged fusion peptides were further loaded onto a Ni column (His GraviTrap, manufactured by GE Health) for capture, then dissolved in 500 mM imidazole. The resulting extracts were dialyzed against PBS to obtain the purified extracts.

[0220] (Preparation Example 4) Preparation of Hybrid-E2s

[0221] The two polypeptides, trpE-his-G1E2s and trpE-his-G4E2s, prepared in Preparation Example 3, produced a large amount of precipitate during dialysis after being dissolved in the soluble buffer. Therefore, they can be purified as heterodimers by mixing them separately with trpE-G3E2s, which do not readily produce precipitates. For example, trpE-G3E2s, which are soluble in the soluble buffer from the ultrasonic fragments (1) of E. coli, can be mixed with trpE-his-G1E2s or trpE-his-G4E2s and then purified as their heterodimers (Hybrid-E2s) by dialysis.

[0222] (4-1) Preparation of Hybrid-E2s (G3 / G1-E2s)

[0223] First, the soluble trpE-his-G1E2s (G1E2s-4MUrea(5)) and the soluble trpE-G3E2s (G3E2s-4MUrea(5)) obtained in Preparation Example 3 were mixed at an equal concentration (1:1) to obtain a mixture (Hyb-4MUreamix(5)). Next, the mixture was dialyzed overnight at 4°C relative to PBS, as in Preparation Example 3. The dialyzed sample was centrifuged at 15000 rpm for 30 minutes at 4°C to remove the precipitate (Hyb-4M-dia-ppt(7)) and the supernatant was recovered (Hyb-4M-dia-sup(6)). Next, the recovered supernatant was loaded onto a Ni column (His GraviTrap, manufactured by GE Health). At this point, the pass-through level (Hyb-Ni-FT(8)) is trpE-G3E2s without the HIS tag (hereinafter referred to as... Figure 2 Lane 8 (III)). Next, after washing with column wash buffer (PBS) (by fractionation: Hyb-Ni-Wash (9)), the extract obtained by dissolution with 500 mM imidazole was dialyzed against PBS to obtain a purified product (Hyb-Ni-Elute (10)).

[0224] For molecular weight markers (MWM) and solutions or precipitates obtained at each purification stage (5)–(10), the precipitates were soluble in PBS containing 6M urea and subjected to SDS-polyacrylamide gel electrophoresis. The resulting electrophoretic images are shown below. Figure 2 . Figure 2The combined electrophoresis results show the precipitate (G1E2s-4M-dia-ppt(7)) formed during the separate dialysis of trpE-his-G1E2s, which was soluble in a soluble buffer (containing 4M urea) in Preparation Example 3. Since trpE-his-G1E2s does not form heterodimers but exists alone, most of it is thus removed as precipitate by the dialysis. Figure 2 Lane 3(I)), and thus the purified product obtained from the supernatant of the dialysis ( Figure 2 Lane 10(IV) contains heterodimers of trpE-G3E2s and trpE-his-G1E2s (Hybrid-E2s(G3 / G1-E2s)) and dimers of trpE-his-G1E2s (homodimers), but since, as mentioned above, most of trpE-his-G1E2s are removed as precipitates during dialysis, it can be said that it is mostly Hybrid-E2s. Furthermore, if trpE-his-G1E2s are mixed with trpE-G3E2s for dialysis, trpE-G3E2s exert a molecular chaperone effect relative to trpE-his-G1E2s through their contact and dimer formation.

[0225] (4-2) Preparation of Hybrid-E2s (G3 / G4-E2s)

[0226] Except for the soluble form of trpE-his-G1E2s (G1E2s-4MUrea(5)) which was used instead of the soluble form of trpE-his-G4E2s (G4E2s-4MUrea(5)) obtained in Preparation Example 3, the purified product was obtained in the same manner as in Preparation Example 4-1.

[0227] For molecular weight markers (MWM) and solutions or precipitates obtained in each purification stage, the precipitates were soluble in PBS containing 6M urea and subjected to SDS-polyacrylamide gel electrophoresis. The resulting electrophoretic images are shown below. Figure 3A and Figure 3B .exist Figure 3AIn the image, starting from the left side facing the photograph, the following are images: molecular weight marker (MWM), ultrasonically broken up E. coli trpE-his-G4E2s (G4E2s-cell(1)) and its centrifuged supernatant (G4E2s-sup(2)), centrifuged supernatant washed with a surfactant-containing wash solution (G4E2s-NP40(3)) and its rewashed centrifuged supernatant (G4E2s-Wash(4)), and images prepared with soluble buffer (2M, 4M, 6M, or 4M urea, 25mM Tris-HCl (pH 8.0), 5mM...). The soluble products after EDTA soluble solution (G4E2s-2M, 4M, 6M, 4MUrera(5)), the crude purified product after dialysis of the soluble ...

[0228] exist Figure 3B In the image, starting from the left side facing the photograph, the following are the molecular weight marker (MWM), the Hyb-dia-sup (6), the Hyb-dia-ppt (7), the soluble product of trpE-his-G4E2s soluble in a soluble buffer containing 4M urea and the soluble product of trpE-G3E2s soluble in a soluble buffer containing 4M urea (Hyb-4M urea mix (5)), the Ni column pass fraction of the supernatant after dialysis of the mixture (Hyb-Ni-FT (8)), the pass fraction during Ni column washing (Hyb-Ni-Wash (9)), the purified Ni column eluent (Hyb-Ni-Elute (10)), and twice the volume of lanes 19-21 respectively.

[0229] If trpE-his-G4E2s are dialyzed separately, most of them are recovered as precipitate. Figure 3ALanes 10-11). Thus, similar to the combination of trpE-G3E2s and trpE-his-G1E2s, the purified product contains heterodimers of trpE-G3E2s and trpE-his-G4E2s (Hybrid-E2s(G3 / G4-E2s)) and dimers of trpE-his-G4E2s (homodimers), but it can be said that it is mostly Hybrid-E2s.

[0230] (Preparation Example 5) Preparation of monoclonal antibodies recognizing G1E2s and G3E2s

[0231] (5-1) Hybridoma Production

[0232] To prepare monoclonal antibodies recognizing trpE-his-G1E2s and trpE-his-G3E2s as antigenic peptides, mice were immunized as follows: 25–100 μg of a mixed antigen obtained by mixing equal volumes of trpE-his-G1E2s and trpE-his-G3E2s purified in Preparation Example 3, along with TiterMax Gold (manufactured by TiterMax) as an adjuvant, was administered intraperitoneally to BALB / c mice. Two weeks later, a booster immunization was performed, and approximately two weeks later, a solution of the mixed antigen dissolved in PBS was administered intravenously via the tail vein as the final immunization.

[0233] On day 3 post-final immunization, the spleen was aseptically removed from the mice. The spleen was then crushed into single cells using scissors and a metal mesh, and washed three times with RPMI-1640 medium. Log-proliferating mouse myeloma cell line SP2 / 0-Ag14 was washed three times with RPMI-1640 medium and then mixed with the spleen cells at a 1:5 cell ratio. After centrifugation at 200×g for 5 minutes, the supernatant was removed. While slowly mixing the precipitated cell clumps, 1 mL of RPMI-1640 medium containing 50% polyethylene glycol (PEG) 4000 (Merk) was slowly added, followed by 10 mL of RPMI-1640 medium to induce cell confluence.

[0234] The resulting fusion cells were centrifuged at 200×g for 5 minutes to remove PEG, then suspended in RPMI-1640 medium containing 10% fetal bovine serum and hypoxanthine / aminopterin / thymine (HAT), and seeded into 96-well cell culture plates. After culturing for approximately 10 days to allow hybridoma proliferation, clones producing monoclonal antibodies specifically reacting with the mixed antigen were identified by ELISA, thus obtaining hybridomas reacting with the mixed antigen. Monoclonalization was performed using limiting dilution to establish six target hybridoma lines (A505, A519, A906, A907, A924, and A926).

[0235] The established hybridoma lines were cultured in serum-free medium (Hybridoma-SFM, GIBCO) at 37°C and 5% CO2 for 2–3 weeks. The culture medium was then subjected to affinity chromatography using a protein A sephorose column to purify the monoclonal antibodies (A505, A519, A924, and A926) produced in the culture medium.

[0236] Next, the ELISA reactivity was evaluated using trpE-his-G1E2s (G1E2s antigen) and trpE-his-G3E2s (G3E2s antigen) as antigenic peptides, respectively. First, the purified antigenic peptides from Preparation Example 3 were diluted to a final concentration of 0.5 μg / mL with PBS (10 mM sodium phosphate buffer containing 0.15 M NaCl (pH 7.3)), and 100 μL was dispensed into each well of a 96-well microplate (manufactured by Nunk). After incubating overnight at 4°C, each well was washed twice with 0.35 mL of PBS. Then, 0.35 mL of blocking buffer (PBS containing 0.5% casein-Na) was added to each well, and the plate was incubated at room temperature for 3 hours. After removing the blocking buffer, the microplate was vacuum-dried to prepare the ELISA plate, and stored at 4°C until use.

[0237] Next, 100 μL of antibody reaction solution containing 0.2 μg / mL of each monoclonal antibody obtained above was added to each well of the ELISA plate in reaction buffer 1 (0.2 M NaCl, 1% BSA, 100 mM sodium phosphate buffer containing 0.07% casein-Na (pH 7.3)). After incubating at room temperature for 60 minutes, the plate was washed five times with PBS-T (PBS containing 0.05% Tween 20). Then, labeled body fluid containing POD (peroxidase)-labeled anti-mouse IgG antibody (manufactured by JACAN Iminolithus) in reaction buffer 1 was added to each well, and the plate was incubated at room temperature for 60 minutes, followed by washing five times with PBS-T. Next, 100 μL of the mixed solution from the ELISA POD substrate TMB kit (manufactured by Nakalitesk) was added to each well, and the reaction was allowed to proceed for 30 minutes at room temperature in the dark. After the reaction, 100 μL of 2N sulfuric acid solution was added to each well. The absorbance at 630 nm (OD630) was used as a control, and the absorbance at 450 nm (OD450) was measured to evaluate the ELISA reactivity.

[0238] Table 2 below shows the ELISA reactivity results when G1E2s and G3E2s antigens were used as antigenic peptides, respectively. In Table 2, "-" indicates no reactivity (OD450: less than 0.500), "+" indicates adequate reactivity (OD450: greater than 1.000 and less than 3.000), and "++" indicates strong reactivity (OD450: greater than 3.000). As shown in Table 2, when G1E2s was used as the antigenic peptide, reactivity was confirmed for all peptides except A505. On the other hand, when G3E2s was used as the antigenic peptide, reactivity was confirmed for A505, A519, A924, and A926.

[0239] (5-2) Obtaining the isotype of the monoclonal antibody

[0240] The hybridoma lines established in (5-1) were cultured in serum-free medium (Hybridoma-SFM, GIBCO) at 37°C and 5% CO2 for 2–3 weeks. The culture medium was then subjected to affinity chromatography using a protein A sephorose column to purify the monoclonal antibodies (A505, A519, A924, and A926) produced in the culture medium. Next, the subtypes of the purified monoclonal antibodies were identified using an isotyping kit (Zymed) for each anti-mouse Ig isotype antibody. The results are summarized in Table 2 below. As shown in Table 2, all isotypes were confirmed to be IgG, except for A505, which was subtype IgG2b; all others were subtype IgG1.

[0241] Table 2

[0242]

[0243] (Preparation Example 6) Preparation of Biotinylated E2s Antigen and Biotinylated Monoclonal Antibody

[0244] To biotinylate the G1E2s antigen, G3E2s antigen, Hybrid-E2s antigen, and each monoclonal antibody, biotinylate reagent (Sulfo-NHS-LC-Biotin, manufactured by Sulfo) was used to modify each antigen / antibody with biotin. Specifically, firstly, 1 mg of biotin reagent was dissolved in 180 μL of ultrapure water to prepare a 10 mM biotin solution just before use. Next, 1 mg each of the following prepared antibodies, prepared in Preparation Example 3: trpE-his-G1E2s (G1E2s antigen) and trpE-his-G3E2s (G3E2s antigen), prepared in Preparation Example 4: Hybrid-E2s (G3 / G1-E2s) (Hybrid-E2s antigen, Hyb-Ag), and prepared in Preparation Example 5, dissolved in 1 mL of PBS, were mixed with 66.7 μL of the 10 mM biotin solution and incubated at room temperature for 30 minutes. Then, the unreacted biotin reagent was desalted with PBS using PD-10 to purify the biotinylated E2s antigens (biotinylated E2s antigens) and monoclonal antibodies (biotinylated antibodies).

[0245] (Experimental Example 1) Evaluation of Hybrid-E2s (G3 / G1-E2s)

[0246] In Table 2, sandwich ELISAs were performed using monoclonal antibodies A505 (non-reactive to G1E2s antigen) and A907 (non-reactive to G3E2s antigen), monoclonal antibodies A906 (non-reactive to G3E2s antigen) and A924 (reactive to both G1E2s and G3E2s antigens), monoclonal antibodies A926 (reactive to both G1E2s and G3E2s antigens) and A907 (non-reactive to G3E2s antigen), and monoclonal antibodies A924 and A926 (reactive to both G1E2s and G3E2s antigens). The Hybrid-E2s (G3 / G1-E2s) (Hybrid-E2s antigen) prepared in Preparation Example 4 were evaluated using monoclonal antibodies A924 and A926 (reactive to both G1E2s and G3E2s antigens).

[0247] First, similar to Example 1 below, monoclonal antibodies A907 and A924 were immobilized in 96-well microplates (0.5 μg per well). Next, 190 μL of the reaction buffer solution and 10 μL of trpE-his-G1E2s (G1E2s antigen), trpE-his-G3E2s (G3E2s antigen), or the Hybrid-E2s antigen prepared in Preparation Example 4 (final antigen concentration 0.1 μg / mL) were added to each well, and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction, each well was washed 5 times with 0.35 mL of washing buffer (PBS containing 0.05% Tween 20). Then, 100 μL of the labeled body solution containing each biotinylated monoclonal antibody (0.2 μg / mL) prepared in Preparation Example 6, as shown in Table 3 below, was added to each well of the plate, and the plate was incubated at room temperature for 1 hour. Next, after washing five times with the washing buffer, 100 μL of the labeled body solution prepared by diluting POD-labeled streptavidin (500 units / mL) (SA-POD, manufactured by Lux) 5000 times was added to each well, and the plate was incubated at room temperature for 30 minutes. After five washes, 100 μL of the mixed solution from the ELISA POD substrate TMB kit (Nacalitesk) was added, and the plate was incubated at room temperature in the dark for 30 minutes. After the reaction, 100 μL of 2N sulfuric acid solution was added to each well. The absorbance at 630 nm (OD630) was used as a control, and the absorbance at 450 nm (OD450) was measured. The values ​​calculated using the formula OD450 - OD630 were used as the measured values, as shown in Table 3 below. Table 3 also shows the S / N value for each measured value (the value obtained by dividing each measured value by the OD450 - OD630 value without the antigen (blank)). In the "(Reactivity)" column of Table 3, "-" indicates no reactivity (measured value: less than 0.100), "+" indicates adequate reactivity (measured value: greater than 0.100 and less than 1.000), and "++" indicates strong reactivity (measured value: greater than 1.000).

[0248] Table 3

[0249]

[0250] As shown in Table 3, in either the sandwich ELISA of monoclonal antibodies A505 and A907, which react specifically with the G3E2s antigen only, or the sandwich ELISA of monoclonal antibodies A906 and A924, which react specifically with the G1E2s antigen only, the Hybrid-E2s antigen was reacted. Therefore, it was confirmed that the Hybrid-E2s antigen is composed of G3E2s antigen and G1E2s antigen.

[0251] (Experimental Example 2) Confirmation of the reactivity of various assay systems for HEV positive / negative samples

[0252] (Example 1) IgM Measurement System

[0253] Anti-human IgM antibodies, generated using commercially available or known methods, were diluted to a final concentration of 5 μg / mL with PBS (10 mM sodium phosphate buffer containing 0.15 M NaCl (pH 7.3)). 100 μL was aliquoted into each well of a 96-well microplate (manufactured by Nunk). The plates were then incubated overnight at 4°C, followed by washing each well twice with 0.35 mL of PBS. Next, 0.35 mL of blocking buffer (PBS containing 0.5% casein-Na) was added to each well, and the plates were incubated at room temperature for another 3 hours. After removing the blocking buffer, the microplates were vacuum-dried to prepare an anti-human IgM antibody immobilized plate, which was stored at 4°C until use.

[0254] Using this microplate, the HEV IgM antibody titer was determined in a sample group (16 samples) consisting of human plasma containing plasma from patients with hepatitis E, purchased from TRINA. Specifically, 90 μL of reaction buffer 2 containing 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA, 2% BSA, 20 ppm KLG, and 2% gelatin, and 10 μL of each sample diluted 10-fold with the reaction buffer 2 were added to the anti-human IgM antibody immobilized plate, and the plate was allowed to react at room temperature for 1 hour. After the reaction, each well was washed 5 times with 0.35 mL of washing buffer (PBS containing 0.05% Tween 20). Then, 100 μL of each of the three biotinylated E2s antigens (1 mg / mL) prepared in Preparation Example 6, diluted 5000 times (dilution buffer: reaction buffer 2), was added to each well of the anti-human IgM antibody immobilized plate, and the plate was incubated at room temperature for 1 hour. Next, after washing with the washing buffer, 100 μL of labeled body fluid prepared by diluting alkaline phosphatase (ALP) streptavidin (0.2 mg / mL) (SA-ALP, manufactured by Samo) 5000 times was added to each well, and the plate was incubated at room temperature for 30 minutes. Next, after washing with the washing solution, 100 μL of Lumipars (registered trademark) substrate solution (manufactured by Fuji Rebio) containing AMPPD (3-(2'-spiroadamantine)-4-methoxy-4-(3'-phosphoryloxy)benzene-1,2-dioxane-2-sodium salt) was added, and the mixture was allowed to react at room temperature in the dark for 20 minutes. After the reaction, the luminescence intensity (counting) of the light emitted by the decomposition of AMPPD through the catalysis of ALP, which has a maximum absorption at a wavelength of 463 nm, was measured using a Lumipars (registered trademark) L-2400 (manufactured by Fuji Rebio), and this was taken as the determination result (HEV IgM antibody titer in the sample). The determination results for each sample are shown in Table 4 below. In addition, the results shown in Table 4 represent the average of two determinations.

[0255] (Comparative Example 1) IgM Measurement System

[0256] Using a commercially available anti-HEV-IgM ELISA kit that employs amino acid positions 394-606 encoded by the ORF2 (DDBJ Accession No.: D11092) of the HEV gene of genotype 1 as a recombinant HEV antigenic peptide, the same sample group as in Example 1 was measured by ELISA using a plate immobilized with anti-IgM antibody as the trapping medium and the recombinant HEV antigenic peptide labeled with POD as the labeling medium. For the color development produced by free TMBZ oxide in the reaction of POD with hydrogen peroxide and tetramethylbenzidine (TMBZ) substrate, the absorbance at 450 nm (OD450) was measured as a control, using the absorbance at 630 nm (OD630) as a control. The value calculated using the formula: OD450 - OD630, was taken as the HEV IgM antibody titer in the sample.

[0257] (Comparative Example 2) IgA Measurement System

[0258] Using a commercially available anti-HEV-IgA ELISA kit that employs amino acid positions 111-660 encoded by the ORF2 (DDBJ / GenBank / EMBL Accession No.: AB082545) of the HEV gene of genotype 4 as a recombinant HEV antigenic peptide, the same sample group as in Example 1 was measured by ELISA using a plate immobilized with the recombinant HEV antigenic peptide as the trap and POD-labeled anti-human IgA antibody as the label. For the colorimetric reaction of free TMBZ oxide produced by the reaction of POD with hydrogen peroxide and tetramethylbenzidine (TMBZ) substrate, the absorbance at 450 nm (OD450) was measured as a control, using the absorbance at 630 nm (OD630) as a control. The value calculated using the formula: OD450 - OD630, was taken as the HEV IgA antibody titer in the sample.

[0259] The results obtained in Example 1 (3 types) and Comparative Examples 1-2 are summarized in Table 4 below. The cutoff value for Comparative Example 1 was calculated using the following formula: (mean value of negative control (samples without IgM anti-HEV antibodies)) + 0.26. The cutoff value for Comparative Example 2 was calculated using the following formula: (mean value of positive control (samples containing IgA anti-HEV antibodies) - mean value of negative control (samples without IgA anti-HEV antibodies)) × 0.5. The cutoff value for Example 1 was calculated using the mean value of a sample group consisting of human plasma (negative samples: n = 40) purchased from TRINA that did not contain plasma from hepatitis patients + 8 SD. In Example 1 of Table 4, "-" indicates negative (below the cutoff value), "+" indicates positive (above the cutoff value and less than the cutoff value × 3), "++" indicates strong positive (cutoff value × 3 or more and less than the cutoff value × 6), "++++" indicates very strong positive (cutoff value × 6 or more and less than the cutoff value × 30), and "++++" indicates extremely strong positive (cutoff value × 30 or more) (the same applies to Example 2 in Table 5 below). In Comparative Examples 1 and 2 of Table 4, "-" indicates negative (below the cutoff value), "+" indicates positive (above the cutoff value and less than the cutoff value × 3), and "++" indicates strong positive (cutoff value × 3 or more) (the same applies to Comparative Examples 1 and 2 in Table 5 below).

[0260] Table 4

[0261]

[0262] In Table 4, samples No. 3, No. 5, and No. 16 were HEV infection-negative samples. The other samples showed a reaction on any assay system. Firstly, compared to the IgM assay system, the assay system of Example 1, utilizing G1E2s antigen, G3E2s antigen, or Hybrid-E2s antigen, showed higher reactivity in any sample compared to the assay system of Comparative Example 1. In particular, for the six samples No. 4, No. 8, No. 10, No. 12, No. 13, and No. 14, any of the samples from Example 1 were positive, while those from Comparative Example 1 were negative (false negatives).

[0263] Furthermore, even in Example 1, when the Hybrid-E2s antigen, as a heterodimer of G1E2s antigen or G3E2s antigen, was used compared to the case where G1E2s antigen or G3E2s antigen was used alone, high reactivity was shown for samples that were negative for at least one antigen alone (No. 10), and reactivity was also shown for tests that were negative for any antigen alone (No. 14), thereby confirming the synergistic effect of detecting positive samples that could not be detected by the antigen peptide alone.

[0264] Next, when compared with the IgA assay system, the assay system of Example 1, which utilized the G1E2s antigen, G3E2s antigen, or Hybrid-E2s antigen, showed higher reactivity than the assay system of Comparative Example 2 in any sample. In particular, for the eight samples No. 4, No. 7, No. 8, No. 10-12, No. 14, and No. 15, all samples from Example 1 were positive, while those from Comparative Example 2 were negative (false negatives). Since IgA is produced by a different immune mechanism than IgM, direct comparison is difficult, suggesting an intermediate reactivity between IgM and IgG.

[0265] (Example 2) IgG Assay System

[0266] The trpE-his-G3E2s (G3E2s antigen) prepared in Preparation Example 3 was diluted with PBS to a final concentration of 0.5 μg / mL, and 100 μL was dispensed into each well of a 96-well microplate. The microplate was then incubated overnight at 4°C, followed by washing each well twice with 0.35 mL of PBS. Then, 0.35 mL of the same blocking buffer as in Example 1 was added to each well, and the plate was incubated at room temperature for 3 hours. After removing the blocking buffer, the microplate was vacuum-dried to prepare a G3E2s antigen immobilized plate, which was stored at 4°C until use.

[0267] Using this microplate, the HEV IgG antibody titer in the samples was determined for the same sample group as in Example 1. Specifically, 290 μL of the reaction buffer and 10 μL of each sample diluted 10-fold with the reaction buffer 2 were added to the G3E2s antigen-immobilized plate, and the plate was incubated at room temperature for 1 hour. After the reaction, each well was washed 5 times with 0.35 mL of the same washing buffer as in Example 1. Then, 100 μL of a 5000-fold dilution of POD-labeled anti-human IgG antibody (1 mg / mL) (dilution buffer: reaction buffer 2) was added to each well, and the plate was incubated at room temperature for 1 hour. Next, after washing 5 times with the washing buffer, 100 μL of a mixture of ELISA POD substrate TMB kit (manufactured by Nakalaitest) was added to each well, and the plate was incubated at room temperature / in the dark for 30 minutes. After the reaction, 100 μL of 2N sulfuric acid solution was added to each well. The absorbance at a wavelength of 630 nm (OD630) was used as a control, and the absorbance at a wavelength of 450 nm (OD450) was measured. The value calculated by the following formula: OD450-OD630 was taken as the HEV IgG antibody titer in the sample.

[0268] The results obtained in Example 2 are shown in Table 5 below. The cutoff value for Example 2 was compared with the average value of a sample group (negative samples: n = 40) consisting of human plasma from patients without hepatitis E, purchased from TRINA, as a control, and was approximately 10 times (0.400) in terms of S / N ratio. The results of Comparative Examples 1-2 for each sample are shown in Table 5.

[0269] Table 5

[0270]

[0271] Similar to the results shown in Table 4, samples No. 3, No. 5, and No. 16 were HEV-negative, and HEV-IgG negative samples were confirmed in samples No. 10 and No. 14. Other samples showed a reaction in any assay system. As shown in Table 5, in the IgG assay system, the assay system of Example 2, which utilized the G3E2s antigen, showed higher reactivity compared to the assay system of Comparative Example 1. Furthermore, similar to Example 2, it also showed higher reactivity compared to the assay system of Comparative Example 2, which used the antigen as a trap.

[0272] Industry availability

[0273] According to the present invention, a detection method for hepatitis E virus infection with high sensitivity compared to existing methods, an antigen peptide for detecting hepatitis E virus infection and a kit for use in the detection method are provided.

[0274] Sequence List Free Text

[0275] Serial Number: 5

[0276] <223> Reverse primer G3AS1

[0277] Serial Number: 6

[0278] <223> Primer G3S1

[0279] Serial Number: 7

[0280] <223> Primer G3S2

[0281] Serial Number: 8

[0282] <223> Primer G3AS2

[0283] Serial Number: 9

[0284] <223> Primer G3E2s-his452-S

[0285] Serial Number: 10

[0286] <223> Primer G3E2s-617-AS

[0287] Serial Number: 11

[0288] <223> Primer G3E2s-452-S

[0289] Serial Number: 12

[0290] <223> G1E2s 394-617

[0291] Serial Number: 13

[0292] <223> Primer G1E2s-his452-S

[0293] Serial Number: 14

[0294] <223> Primer G1E2s-617-AS

[0295] Serial Number: 15

[0296] <223> Reverse primer G4AS1

[0297] Serial Number: 16

[0298] <223> Primer G4S1

[0299] Serial Number: 17

[0300] <223> Primer G4S2

[0301] Serial Number: 18

[0302] <223> Primer G4AS2

[0303] Serial Number: 19

[0304] <223> Primer G4E2s-his445-S

[0305] Serial Number: 20

[0306] <223> Primer G4E2s-606-AS

Claims

1. The use of an antigenic polypeptide in the manufacture of a kit for detecting hepatitis E virus infection, wherein the antigenic polypeptide is a dimer composed of a polypeptide of (3a) below and any one polypeptide selected from (1a), (2a) and (4a) below. The hepatitis E virus infection detection includes a detection step of using the antigenic polypeptide to detect anti-hepatitis E virus antibodies in the sample. (1a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 1, and having a total length of less than 200 residues of the amino acid sequence described in Serial No.

1. (2a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 2, and whose total length is less than 200 residues of the amino acid sequence described in Serial No.

2. (3a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in sequence number 3, and having a total length of less than 200 residues of the amino acid sequence described in sequence number 3. (4a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in sequence number 4, and having a total length of less than 200 residues of the amino acid sequence described in sequence number 4.

2. In accordance with the use described in claim 1, the isotype of the anti-hepatitis E virus antibody is IgM.

3. The use according to claim 1, The detection process involves bringing the sample into contact with the trap and the marker. The trapping body comprises a water-insoluble carrier and a probe molecule capable of binding to anti-hepatitis E virus antibodies, and The marker comprises a marker substance and the antigenic polypeptide.

4. The antigenic polypeptide for detecting hepatitis E virus infection is a dimer composed of the polypeptide described in (3a) below and any one polypeptide selected from (1a), (2a) and (4a) below. (1a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 1, and having a total length of less than 200 residues of the amino acid sequence described in Serial No.

1. (2a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in Serial No. 2, and whose total length is less than 200 residues of the amino acid sequence described in Serial No.

2. (3a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in sequence number 3, and having a total length of less than 200 residues of the amino acid sequence described in sequence number 3. (4a) A polypeptide consisting of amino acids from position 28 to 175 of the amino acid sequence described in sequence number 4, and having a total length of less than 200 residues of the amino acid sequence described in sequence number 4.

5. The antigenic polypeptide according to claim 4 is a labeled polypeptide.

6. A kit for detecting hepatitis E virus infection, comprising the antigenic polypeptide as described in claim 4 or 5.

7. The hepatitis E virus infection detection kit according to claim 6 further comprises a trap having a non-water-soluble carrier and a probe molecule capable of binding to anti-hepatitis E virus antibodies.

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